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A single-cell transcriptome atlas profiles early organogenesis in human embryos

Nature Cell Biology volume 25, pages 604–615 (2023)Cite this article

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Abstract

The early window of human embryogenesis is largely a black box for developmental biologists. Here we probed the cellular diversity of 4–6 week human embryos when essentially all organs are just laid out. On the basis of over 180,000 single-cell transcriptomes, we generated a comprehensive atlas of 313 clusters in 18 developmental systems, which were annotated with a collection of ontology and markers from 157 publications. Together with spatial transcriptome on embryonic sections, we characterized the molecule and spatial architecture of previously unappreciated cell types. Combined with data from other vertebrates, the rich information shed light on spatial patterning of axes, systemic temporal regulation of developmental progression and potential human-specific regulation. Our study provides a compendium of early progenitor cells of human organs, which can serve as the root of lineage analysis in organogenesis.

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Fig. 1: Single-cell transcriptome atlas of early human embryo.
Fig. 2: ST of a CS13 embryo.
Fig. 3: Spatial domains in limb bud.
Fig. 4: Spatial patterning of neural tube.
Fig. 5: Systemic temporal regulation in vertebrate embryogenesis.
Fig. 6: Systemic data integration.

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Data availability

Sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE157329. An online depository for cell types and gene expression (developed with R package VisCello103 v1.1.2) is available at https://heoa.shinyapps.io/base/. Previously published data that were re-analysed here are from NCBI GSE119945 (single-nucleus RNA-seq in whole mouse embryos2), NCBI PRJNA637987 (scRNA-seq in mouse brain28), ArrayExpress E-MTAB-7320 (scRNA-seq in mouse neural tube26), ENCODE project available at https://cells.ucsc.edu/mouse-limb/10x/200120_10x.h5ad (scRNA-seq in mouse limb bud27), CNGB CNP0001543 (ST in mouse embryos29), NCBI GSE171892 (scRNA-seq in human neural tube63), NCBI GSE107618 (scRNA-seq in human retina9), NCBI GSE156793 (single-nucleus RNA-seq in human fetus7). For published datasets used in LIN28A analysis, see Supplementary Table 3. Mouse in situ database EMBRYS is available at https://www.embrys.jp/embrys/html/MainMenu.html. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

R scripts for analysis and figures are available at https://heoa.shinyapps.io/code/. All of the R packages used are available online. For the complete list of R packages and version numbers, see Supplementary Note.

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Acknowledgements

We thank R. Sheth, H. Etchevers, H. Zhang, S. Teichmann, C. Zhang, D. Huangfu, A. Joyner, L. Studer and G. Lin for discussions. We thank L. Xiao for website design. This work is supported by Funding Project of National Key Research and Development Program of China (2018YFD0900604), Natural Science Foundation of China (41676119 and 41476120) and Fundamental Research Funds for the Central Universities from the Ocean University of China to W.S. Z.B. is supported by funding from Memorial Sloan Kettering Cancer Center. T.Z. is a visiting student at Memorial Sloan Kettering Cancer Center sponsored by the China Scholarship Council.

Author information

Author notes

  1. These authors contributed equally: Yichi Xu, Tengjiao Zhang.

Authors and Affiliations

  1. Developmental Biology Program, Sloan Kettering Institute, New York, NY, USA

    Yichi Xu, Tengjiao Zhang & Zhirong Bao

  2. Institute for Regenerative Medicine, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China

    Tengjiao Zhang

  3. Traditional Chinese Medicine Hospital of Kunshan, Suzhou, China

    Qin Zhou, Yifang Xue & Lihui Wang

  4. Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, China

    Mengzhu Hu, Yao Qi & Weiyang Shi

  5. School of Pharmacy, Fudan University, Shanghai, China

    Yuxiao Nie

  6. Institute for Regenerative Medicine, Shanghai East Hospital, Tongji University, Shanghai, China

    Weiyang Shi

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Contributions

W.S. and Z.B. conceived the project. T.Z., M.H., Y.Q., L.W., Q.Z. and Y.X. collected the embryos and generated the atlas dataset. Y.X., T.Z. and Y.N. performed data processing, clustering and all the bioinformatics analysis. Y.X. created the atlas website. W.S., Z.B., Y.X. and T.Z. wrote the manuscript.

Corresponding authors

Correspondence to Zhirong Bao or Weiyang Shi.

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Extended data

Extended Data Fig. 1 Embryos and data quality.

a, Images of embryos and the number of high-quality cells in each sample. Schematic diagrams (lower panel) showing the territory of embryo dissection parts. b, Histograms showing the distribution of total UMIs and gene numbers per cell. c, Distribution of XIST (female) versus RSP4Y1 (male) expression from randomly sampled 1,000 cells per embryo. d, UMIs of hemoglobin genes (HBA1, HBA2, HBE1, HBG1, HBG2, HBZ) against total UMIs. Each dot denotes a cell. Cells with high linear slope (above the green line) was identified as erythroid cells (red) (see Methods). Black dots denote cells that were not identified as erythroid cells. e, CopyKAT18, designed for 3’ or 5’ scRNA-seq with sparse coverage, was used to estimate copy number aberration in 7 embryos with default setting. 500 cells were randomly sampled in each embryo. Log2 ratios of copy number of segmentations (220 kb of size) were shown by embryo (left) and by genomic position (right).

Source data

Extended Data Fig. 2 Annotation of cell types.

Expression of selected known markers for cell types collapsed by unique terms in annotation (For all markers, see Supplementary Table 1). Cell types in limb and neural tube are detailed in Fig. 3 and Fig. 4, respectively. Dendrogram of cell types was defined according to the lineage relationship based on annotations. The third level of dendrogram shows 18 developmental systems and number of clusters in each system. Dot size represents the fraction of expressing cells (UMIs, unique molecular identifiers > 0) for a given gene (n = 7 embryos) and dot color shows developmental system. EP, epidermis; SN, sensory neuron; IM, intermediate mesoderm; soLPM, somatic LPM; spLPM, splanchic LPM; PGC, primordial germ cell; PA, pharyngeal arch; ZLI, zona limitans intrathalamica; AER, apical ectodermal ridge; NMP, neuro-mesodermal progenitor; a/pPSM, anterior/posterior presomitic mesoderm.

Extended Data Fig. 3 Iterative clustering and quality control.

a, The expression of markers in first round of clustering (solid lines) and second round of clustering (dashed lines) in brain. Markers that support second round of clustering were shown. Two color bars on top denote clustering results of first round and second round, respectively. b, The expression of markers relative to boundaries of one round of clustering and second round of clustering in endothelium. Convention follows panel a. c, The number of clusters resulted from a series of resolution ‘r’ and PCs in the clustering of spinal neuron. d, The pairwise ARI between clusters resulted from different resolution ‘r’ in spinal neuron. e, The mean ARI of clusters in testing resolution ‘r’ and PCs (Methods). Each dot denotes a super-cluster. f, Cross-validation on clustering by scPred21. The first column shows the AUROC (area under receiver operating curve) of testing the identity of developmental systems (each red dot is a system). Other columns show the AUROC of testing ‘Celltype_annotation’ and ‘Final_annotation’ (Supplementary Table 1) within each system (expect PGC) in red and blue dots, respective (Methods, n = 3~59 cell types). Testing of randomly shuffled identity was served as control in each column (grey). The center line denotes the median, while the box contains the 25th to 75th percentiles. The whiskers mark 1.5x interquartile range. g, Batch effects of embryos, technical replicates, cell cycle phase, and total UMIs estimated by the entropy of mixing100 (Methods, n = 1,700 randomly sampled cells). ‘+Ctrl’, cluster identities as batch. ‘-Ctrl’, randomly assigned batch. Boxplots are defined as in panel f. h, UMAP of all cells colored by embryos, technical replicates, cell cycle phase, total UMIs, and developmental systems in each embryo. The missing parts in embryos 03, 06, and 07 are due to 4 libraries that did not pass quality control (Methods).

Extended Data Fig. 4 Dissection parts, embryonic stages, and DEGs of cell types.

a, The origin of dissection parts of cells in each cluster ordered by system. See Supplementary Table 1 for the order of clusters. EP, epidermis; SN, sensory neuron; IM, intermediate mesoderm; LPM, lateral plate mesoderm; PGC, primordial germ cell; misc., miscellaneous. b, Stage distribution of cells in each cluster ordered by system. * denotes clusters missing cells from CS13-14, defined by total number of cells > 50, ratio of cells from CS12 embryo > 0.05, number of cells from CS12 embryo > 5, ratio of cells from CS15-16 embryo > 0.05, number of cells from CS15-16 embryo > 5, ratio of cells from CS13-14 embryo < 0.05, and number of cells from CS13-14 embryo < 5. c, Number of DEGs per cell type/cluster for each developmental system (see Fig. 1 for convention). The center line denotes the median, while the box contains the 25th to 75th percentiles. The whiskers mark 1.5x interquartile range. n = 3~59 cell types except PGC. d, The expression of ligands in DEGs of 9 signaling centers in human (top) and their expression in mouse cell types from published data26,27,28 (bottom) (Methods, n = 7 embryos). Asterisks denote ligands that are also expressed in the corresponding cell types in mouse. ANR, anterior neural ridge; MHB, mid-hindbrain boundary; ZLI, zona limitans intrathalamica; FP.b/s, floor plate (brain, spinal cord); RP.b/s, roof plate (brain, spinal cord); AER, apical ectodermal ridge; ZPA, zone of polarizing activity. e, The number of ligands that are expressed or not in mouse by signaling center. For each signaling center, left bar shows known ligands and right bar shows additional ligands.

Extended Data Fig. 5 Quality control of spatial transcriptome.

a, The distribution of total UMIs and gene numbers per spot. b, The distribution of total UMIs and gene numbers on each section. c, The co-clustering of 2 human sections and 2 published mouse sections29 on the spot level (Methods). Cluster identities of spots are indicated by color and number on each tissue section. d, The correlation of spatial neighborhood of each cluster between human and mouse. The spatial neighborhood for a cluster on a section was defined as the proportions of clusters in its neighbors. The neighbors of a cluster were defined as the union of nearest 6 spots on the section of each spot in this cluster, excluding spots from the same cluster. Human section 1 was compared to mouse E2S1 and human section 2 was compared to mouse E1S2. The color and number of each cluster correspond to panel c. e, The proportion of 5 recognizable structures on H&E staining of section 1 in deconvolution. f, The proportion of structures that are difficult to be recognized only by H&E staining on section 1 (first row) and section 2 (second row) in deconvolution. Five structures are shown in each section as examples.

Source data

Extended Data Fig. 6 Deconvolution, head mesoderm, and signaling interaction in spatial transcriptome.

a, The deconvolution result of selected cell types on section 1. From left to right, second heart field (SHF) along with pharynx arches (PA) and heart, sclerotome, arterial and vascular endothelium. Arrows in the panel of SHF denote the detection of SHF in PA 1/2, PA 3/4, and heart. b, The UMAP of cells in head mesoderm in scRNA-seq colored by cell type/cluster. c, The average z-score of DEGs of the 5 undefined cell types in each spot of section 1. The window of ST is the same with that in Fig. 2e. Only top DEGs from scRNA-seq shown in Fig. 2e were used. d, The comparison of detection of cell types between scRNA-seq and ST. Each dot is a cell type in head mesoderm and red dots are the 5 undefined cell types. Pearson’s correlation was calculated on the detection of 5 undefined cell types between scRNA-seq and section 1 (mainly mesodermal tissues), between scRNA-seq and section 2 (mainly ectodermal tissues). To be consistent on developmental stage between scRNA-seq and ST, only cells from CS13-CS14 were counted in scRNA-seq. e, The pipeline of inferring signaling interaction by scRNA-seq and spatial transcriptome (Methods). f, Examples of known signaling interactions identified by integrating scRNA-seq and ST. For all significant interactions, see Supplementary Table 1. Top left, the -log10 adjusted p value of selected signaling interactons on section 2. Each row is a pair of cell types and each column is a pair of ligand-receptor. Bottom left, the proportion of cell types involved in these interactions in ST. Right panel, the expression of ligands and receptors in these interaction by pathway. g, The expression of genes in ST involved in signaling interaction shown in Fig. 2g. First row is ligand and second row is receptor.

Extended Data Fig. 7 Spatial domains in limb mesenchymal cells.

a, UMAP and RNA velocity analysis of human forelimb at CS12 and CS15-16 (see Fig. 3 for conventions). b, Batch effect of embryo estimated by entropy of mixing at CS13-14 and CS15-16 (Methods, Mann-Whitney U test p < 2.2 × 10−16, n = 100 randomly sampled cells). The center line denotes the median, while the box contains the 25th to 75th percentiles. The whiskers mark 1.5x interquartile range. c, Normalized UMIs of top DEGs of domain m in domain m by embryo. d, UMAP of mouse forelimb at E10.5 and E12 by reclustering a published dataset27 (Supplementary Table 2). e, The expression of marker genes in each domain from human scRNA-seq (red), mouse scRNA-seq (blue), and mouse in situ data (black) at three stages. Mouse in situ data in each domain were manually classified to strong (large dot), weak (small dot) and no expression (no dot) based on in situ images. Domain m was not identified in E12 data of mouse. HOX genes (black boxes) show developmental stages are aligned between human and mouse datasets. f, The expression of specific markers of domain m on UMAP in human and mouse. Dashed circles, domain m. g, Left panel, UMAP co-embedding of CS15-16 of human and E12 of mouse colored by species and domains (see panel a for conventions). Right panel, the average percentage of mouse cells in k-nearest neighbors (KNN) of human cells in each domain in co-embedding space (Methods, mean +/− SEM, number of cells indicated above each bar). The first bar (background) denotes the percentage of mouse cells in total cells of human and mouse. Asterisks denote significant p values by Mann-Whitney U test (domain a, 3 × 10−7; domain b, 5 × 10−7; domain m, 9 × 10−9). In all panels, n = 1, 3, 3 embryos at CS12, CS13-14, and CS15-16, respectively.

Extended Data Fig. 8 Neural tube patterning.

a, LncRNAs in AP patterning. Upper panel, Monocle analysis shown in pseudo value and cell type (see Fig. 4d for conventions). Middle panel, schematic diagram of the genomic location of the 5 lncRNAs identified as AP related genes in neural tube. Lower-left panel, the expression pattern of 5 lncRNAs (solid lines, see left bar of the heatmap for color legend) and nearby HOX genes (dashed lines) along the AP axis on section 2 of ST. Lower-right panel, the division between hindbrain and spinal cord on H&E staining of section 2, and the correlation on gene expression between lncRNAs and HOX genes along AP axis of section 2 (Methods). b, The comparison of expression of canonical markers for cell types in neural progenitors (upper panel) and neurons (lower panel) between human and mouse (n = 7 and 6 embryos, respectively). Box denotes the difference of MSX2 between human and mouse in neural progenitors. c, The comparison of expression of PAX7 and NKX6-2 between human and mouse in neural progenitors (n = 7, 3, and 6 embryos, respectively). Boxes denote human specific expression that are consistent between two datasets of human. d, The expression of markers (dp1 marker MSX1 and dp4 marker ASCL1, log2 scaled) that distinguish dp1 and dp4 cells in individual cells in human (our dataset and Rayon’s dataset) and mouse datasets. Dot size shows MSX2 expression. Dashed boxes denote most confident dp4 cells (ASCL1 > 0 and MSX1 = 0), which are shown in Fig. 4f. e, The expression of MSX2 in dp2-4 in each human embryo in our data (mean + /- SEM). Each dot denotes a cell (n = 34~209 cells). The number on top is mean of normalized UMIs in each embryo. Note the trunk sample of Emb.07 did not pass quality control so that no cell is from Emb.07.

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Extended Data Fig. 9 LIN28A in vertebrate embryogenesis.

a, Expression pattern of 4 groups of TFs in zebrafish and frog. Each group of TFs were identified as TFs that are highly expressed in the corresponding stage. Numbers above curves indicate number of TFs in each group. The width of each curve represents 2 standard deviations within each group of TFs. The black lines show the expression of LIN28A in each species. b, Pairwise correlation of timepoints between species by homologous TFs. Correlation is scaled by row (Methods). Black line denotes the alignment of timepoints between species by dynamic time warping. The yellow asterisks denote the match of timepoints from a previous study74. c, Numbers of systemically up- (red) and down-regulated (blue) genes from stage 1 to stage 2 (labeled as 1→2) and from stage 3 to stage 4 (labeled as 3→4). No transcriptome data is available for stages 1 and 2 in human. d, Expression dynamics of genes in cell cycle, mRNA splicing and translation pathways that are positively correlated to LIN28A in zebrafish and frog.

Extended Data Fig. 10 Match of cell types between embryo and fetus.

The mutual best match (red edge), best match (black edge) and 2nd-best match (grey edge) in our dataset for each cell type in Cao’s dataset by Slingshot (also see Supplementary Table 6). Cell types are colored by developmental system (see Fig. 1 for conventions). The thickness of lines is inversely proportional to z-score across distances between a cell type in Cao’s dataset and all cell types in our dataset. The lineage relationship between cell types within our dataset (orange edge) was determined by annotation.

Supplementary information

Supplementary Information

Supplementary Note.

Reporting Summary

Supplementary Tables

Supplementary Table 1. Cell types and DEGs. Supplementary Table 2. The comparison between human and mouse scRNA-seq (whole embryo and limb). Supplementary Table 3. LIN28A in vertebrate embryogenesis. Supplementary Table 4. Systemically changing genes in vertebrate embryogenesis. Supplementary Table 5. Enriched pathways in systemically changing genes. Supplementary Table 6. Data integration in human embryos.

Source data

Source Data Fig. 1

The colours and coordinates of UMAP.

Source Data Fig. 2

The proportions of cell types at each spot in ST (pie chart).

Source Data Fig. 3

Mean of normalized UMIs and fraction of cells with UMI >0 (dot plot).

Source Data Fig. 4

The value of each dot in Fig. 4f.

Source Data Fig. 5

The value of each dot in Fig. 5a.

Source Data Fig. 6

The meta-data of cells in Fig. 6.

Source Data Extended Data Fig./Table 1

The copy number ratio at each genomic location (e).

Source Data Extended Data Fig./Table 5

The co-clustering of ST in human and mouse.

Source Data Extended Data Fig./Table 8

The value of each dot in e.

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Xu, Y., Zhang, T., Zhou, Q. et al. A single-cell transcriptome atlas profiles early organogenesis in human embryos. Nat Cell Biol 25, 604–615 (2023). https://doi.org/10.1038/s41556-023-01108-w

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