Abstract

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1−/− chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

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Data availability

Raw sequencing data are available on ArrayExpress with the following accessions: Atlas: E-MTAB-6967; Smart-seq2 endothelial cells: E-MTAB-6970; Tal1−/− chimaeras: E-MTAB-7325; wild-type chimaeras: E-MTAB-7324. Processed data may be downloaded following the instructions at https://github.com/MarioniLab/EmbryoTimecourse2018. Gene Expression Omnibus (GEO) accession GSE87038 was used to support the annotation of myeloid cells (see Methods). All code is available upon request, and at https://github.com/MarioniLab/EmbryoTimecourse2018. Our atlas can be explored at https://marionilab.cruk.cam.ac.uk/MouseGastrulation2018/. All other data are available from the corresponding authors on reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank W. Mansfield for blastocyst injections; A. T. L. Lun and F. Hamey for discussions concerning the analysis; T. L. Hamilton for technical support in embryo collection; S. Kinston and K. Jones for technical assistance; the Flow Cytometry Core Facility at CIMR for cell sorting; the CRUK-CI genomics core for the chimaera scRNA-seq 10x libraries and for letting us use the 10x Chromium after hours; the Wellcome Sanger Institute DNA Pipelines Operations for sequencing; and K. Hadjantonakis for sharing the Ttr::cre mouse line. Research in the authors’ laboratories is supported by Wellcome, the MRC, CRUK, Bloodwise, and NIH-NIDDK; by core support grants from Wellcome to the Cambridge Institute for Medical Research and Wellcome-MRC Cambridge Stem Cell Institute; and by core funding from CRUK and the European Molecular Biology Laboratory. B.P.-S. and D.L.L.H. are funded by the Wellcome 4-Year PhD Programme in Stem Cell Biology and Medicine and the University of Cambridge; D.L.L.H. is also funded by the Cambridge Commonwealth European and International Trust. J.A.G. is funded by the Wellcome Mathematical Genomics and Medicine Programme at the University of Cambridge (109081/Z/15/A). C.G. is funded by the Swedish Research Council (2017-06278, administered by Sahlgrenska Cancer Center, University of Gothenburg). This work was funded as part of a Wellcome Strategic Award (105031/Z/14/Z) awarded to W.R., B.G., J.C.M., J.N., L. Vallier, S.S., B.D.S., S. Teichmann, and T. Voet; by a Wellcome grant (108438/Z/15) awarded to J.C.M. and S.S., and by a BBSRC grant (BBS/E/B/000C0421) awarded to W.R.

Reviewer information

Nature thanks Peter Sims, Patrick Tam and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Blanca Pijuan-Sala, Jonathan A. Griffiths, Carolina Guibentif

Affiliations

  1. Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK

    • Blanca Pijuan-Sala
    • , Carolina Guibentif
    • , Wajid Jawaid
    • , Fernando J. Calero-Nieto
    •  & Berthold Göttgens
  2. Wellcome-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

    • Blanca Pijuan-Sala
    • , Carolina Guibentif
    • , Wajid Jawaid
    • , Fernando J. Calero-Nieto
    • , Carla Mulas
    • , Debbie Lee Lian Ho
    • , Benjamin D. Simons
    • , Jennifer Nichols
    •  & Berthold Göttgens
  3. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK

    • Jonathan A. Griffiths
    • , Tom W. Hiscock
    • , Ximena Ibarra-Soria
    •  & John C. Marioni
  4. The Wellcome/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    • Tom W. Hiscock
    •  & Benjamin D. Simons
  5. Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, UK

    • Richard C. V. Tyser
    •  & Shankar Srinivas
  6. Epigenetics Programme, Babraham Institute, Cambridge, UK

    • Wolf Reik
  7. Centre for Trophoblast Research, University of Cambridge, Cambridge, UK

    • Wolf Reik
  8. Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, UK

    • Wolf Reik
    •  & John C. Marioni
  9. Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK

    • Benjamin D. Simons
  10. EMBL-European Bioinformatics Institute, Wellcome Genome Campus, Cambridge, UK

    • John C. Marioni

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Contributions

B.P.-S., W.J., F.J.C.-N., C.M. and J.N. generated the atlas dataset. C.G. designed and executed the chimaera dataset generation and associated experiments. D.L.L.H. assisted in the generation of the Tal1−/− ES cell line. J.A.G. performed pre-processing, low-level analyses, batch correction, clustering, and global visualization of the atlas and chimaera datasets, and designed the associated website. B.P.-S. curated the clustering and evaluated the connectivity between cell types. B.P.-S. and C.G. annotated atlas cell types. J.A.G. and C.G. analysed atlas endoderm. B.P.-S. assisted in the endoderm analyses by generating force-directed layouts and inferring trajectories using graph abstraction as an alternative approach. R.C.V.T. performed Ttr::cre embryo imaging experiments. B.P.-S. analysed atlas haemato-endothelium and performed associated experiments and analyses. J.A.G. mapped chimaera cells to the atlas. B.P.-S. and C.G. analysed the effects of Tal1−/−. T.W.H. contributed to the mapping and analysis of chimaeras. X.I.-S. provided advice on bioinformatics analysis. W.R., S.S., B.D.S., J.N., J.C.M., and B.G. supervized the study. B.P.-S., J.A.G., C.G., T.W.H., J.C.M., and B.G. wrote the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to John C. Marioni or Berthold Göttgens.

Extended data figures and tables

  1. Extended Data Fig. 1 Embryo images.

    Representative images of embryos collected at the time points indicated. Scale bars, 0.25 mm.

  2. Extended Data Fig. 2 Data quality control.

    a, Left, estimated number of cells present in a single mouse embryo at each time point. Points are values measured previously24; line is an ordinary least squares regression fit. Right, number of cells captured in this study compared with the estimated number of cells in the embryo from the left panel. b, Violin plots illustrating the number of detected genes (top) and unique molecular identifiers (UMIs; bottom) per cell per sample. Sample 11 failed quality control and is therefore not shown. Sample details are provided in Supplementary Table 1. c, UMAP highlighting additional cells identified when a reduced UMI threshold of 1,000 was considered. Additional cells are shown in black. Cells from the atlas are shown in the colour corresponding to their cell type (Fig. 1c). Note that all additional cells are present alongside cells from the atlas: no new cell types are found. d, UMAP plot as shown in Fig. 1c, with cells coloured by biological replicate, showing consistency between samples collected at the same time point. e, Heat map showing the mean gene expression of diagnostic markers (y axis) for each cell type (x axis). Genes are row-normalized. NMP, neuromesodermal progenitors; PGC, primordial germ cells.

  3. Extended Data Fig. 3 Lineage progression.

    a, UMAP plot as shown in Fig. 1c, coloured by the density of each cell in gene expression space; brighter coloured regions (towards yellow) are more densely sampled and darker regions (towards blue) are more sparsely sampled, relative to other regions in the atlas. Values shown are log2(density + 1). b, Box plots summarizing the density per cell type. Values shown are log2(density + 1). c, UMAP plots as shown in Fig. 1c, highlighting cells from each sampled time point and therefore illustrating the transcriptional progression along developmental time. d, Results of atlas stability testing (see Methods). y axis: ratio of the standard deviation of cell-type frequency to the mean cell-type frequency at different degrees of downsampling. Note that when the atlas is downsampled to less than half of its full size (50,000 cells), the standard deviation remains less than 10% of the mean for all cell types. x-axis is log-transformed. e, f, Abstracted graphs, which quantify the degree of similarity between the identified clusters to represent the underlying biological structure of the dataset. Nodes correspond to the annotated cell types, and edges reflect the confidence of adjacency between clusters (thicker edges indicate higher confidence). Node sizes increase as a function of the number of cells within each cluster. Nodes in e are coloured and numbered according to the legend shown in Fig. 1c. Nodes in f show the frequency of cells from each time point, excluding two samples of mixed time-point embryos.

  4. Extended Data Fig. 4 Endoderm convergence.

    a, Schematic representing the process of definitive endoderm intercalation following gastrulation, and subsequent gut maturation. Adapted from a previous publication9. bg, Gene expression levels of Ttr (b), Mixl1 (c), Nkx2-5 (d), Pyy (e), Nepn (f), and Cdx2 (g), overlaid on the Fig. 2a force-directed graph. h, Diffusion map of cells selected for transport map construction; cells selected as termini for pulling mass backward through the transport maps are coloured. i, Results of pushing mass forward through the transport maps are shown on the force-directed layout. j, Violin plots showing expression levels of Trap1a and Rhox5 in all cell-types of the full atlas. k, Dorsal view of a whole-mount fluorescence image of a Ttr::cre; R26R::YFP embryo at E8.5. Green, YFP; red, phalloidin. Arrowhead denotes the increased Ttr–YFP staining in the posterior region of the gut. Scale bar, 300 µm.

  5. Extended Data Fig. 5 Endoderm trajectories.

    a, Top, graph abstraction of the endoderm landscape after fine sub-clustering as an alternative method to resolve which cells should be part of the visceral endoderm (VE)–hindgut 1 trajectory or the definitive endoderm (DE)–hindgut 2 trajectory (supporting transport maps; see Methods). Edges along the VE–hindgut 1 trajectory are highlighted in yellow (nodes 1–4; yellow numbers). Edges along the DE–hindgut 2 trajectory are highlighted in orange (nodes 1–12; orange numbers). Bottom, graph abstraction with the subset of nodes related to the DE–hindgut 2 trajectory to resolve the origin of cluster 4 (between 5 and 6 in the top panel). Resulting DE–hindgut 2 trajectory includes clusters 1–4 and 6–9. The right-hand panels overlay information about the composition of each cluster by developmental stage. b, Force-directed graph coloured by partition-based graph abstraction (PAGA) trajectories. Note that this independent approach for trajectory identification reaches very similar results to those inferred by the transport maps in Fig. 2h. HG1, hindgut 1; HG2, hindgut 2. c, Gene-normalized dynamics of all clusters found along the VE–hindgut 1 and the DE–hindgut 2 trajectories (x axis: DPT along the trajectory; y axis: normalized expression levels). The black line is the mean fitted expression level across all genes in each cluster; the grey shading indicates the standard deviation along the trend across all genes in the cluster; the pink area highlights intercalation process; and the blue area highlights gut maturation steps. Vertical dashed lines correspond to additional stages in the process, deduced from the changes in gene expression trends. Points below the plots are the DPT coordinates of cells from each time point coloured according to time point as in Fig. 2b (from E6.5 in red to E8.5 in blue). d, Gene-normalized dynamics of VE genes along the VE–hindgut 1 trajectory, indicating VE maturation before the intercalation stage. Plot design is as in c. e, Left, Venn diagram of genes that were upregulated during the intercalation process in both VE–hindgut 1 (in clusters 3, 5, 8, and 11) and DE–hindgut 2 (in clusters 4, 6, 7, 8, and 9) trajectories. The overlapping fraction was enriched in genes that are a signature of epithelial remodelling (top 20 genes are listed). Right, gene-normalized dynamics of illustrative genes (Pcna, Epcam, Slc2a1, Vim, Crb3, and Cadm1) along the trajectories. f, Left, Venn diagram of genes that were upregulated after the intercalation process in both trajectories (VE–hindgut 1: clusters 1, 2, 5, and 10; DE–hindgut 2: clusters 1, 3, 5, and 10). The overlapping fraction was enriched in genes that encode transcription factors (TFs), including a large subset of homeodomain proteins (genes are listed). Right, gene-normalized dynamics of Hox and Cdx genes along the trajectories. g, Gene-normalized dynamics of transcription factors that were upregulated specifically in the VE–hindgut 1 trajectory during endoderm intercalation. Points below the x axis in dg are as in c.

  6. Extended Data Fig. 6 Blood development.

    a, Diagram illustrating the two waves of embryonic blood development. At E6.5, gastrulation begins. Previous work using transplantation assays has shown that the proximo-posterior epiblast cells closest to the primitive streak at this stage (red) mainly give rise to primitive erythroid cells in the yolk sac, whereas the epiblast cells located in the middle of the embryo at E6.5 but closer to the primitive streak at a later stage are enriched for endothelial progenitors56. At E7.5, blood islands are apparent (zoomed box of primitive blood wave), where primitive erythroid cells are surrounded by endothelium. At around E8.25, some endothelial cells (haemogenic endothelium) undergo an endothelial-to-haematopoietic transition and become EMPs, which migrate to the fetal liver and give rise to definitive erythrocytes. Adapted from a previous study38. b, Force directed layout of Fig. 3a coloured by original clusters from Fig. 1c. c, Gene expression levels of Cdh5 and Pecam1, overlaid on the graph abstraction visualization from Fig. 3b. d, Experimental design to isolate FLK1+ cells from yolk sac, allantois, and embryo proper for Smart-seq2 scRNA-seq. e, Representative image of an embryo collected for the transcriptional analysis of endothelial cells from the yolk sac, allantois, and embryo proper. f, Sorting strategy of FLK1+ cells from the yolk sac, embryo proper, and allantois on live cells (DAPI). x axis: FLK1 intensity. y axis: DAPI intensity. g, Evidence to support myeloid annotation of the myeloid cell cluster in Fig. 3. Haemato-endothelial cells from Fig. 3a were mapped to a published dataset54 that profiled haematopoietic cells collected at E9.5, E10.5, and E11.5 from different organs. Bar charts show the fraction of atlas cells in the myeloid cell cluster mapped to the clusters defined in figure 8 of the previous study54. h, Representative images of the dissected regions collected to study the location of CSF1R+CD16/32+ cells. Scale bars, 0.25 mm. i, Flow cytometry plots indicating the frequency of CSF1R+CD16/32+ cells in each embryonic region. Two biological replicates were performed for this experiment: with pools of 12 and 13 embryos, respectively. Plots illustrate one biological replicate.

  7. Extended Data Fig. 7 Analysis of Tal1−/− chimaeras.

    a, Left, representative chimaera embryo obtained at E8.5 (left: brightfield; right: tdTomato fluorescence; scale bar, 0.25 mm). Right, flow cytometry plot with tdTomato fluorescence distribution and sorting gates. b, Histograms showing the UMI counts for the tdTomato construct in both tdTomato and tdTomato+ fractions in the Tal1−/− into wild-type experiment (see Methods). c, d, Control mapping results of an E8.0 biological replicate that was removed and mapped back to the atlas. c, Heat map showing the fraction of cells of each labelled cell type that mapped to each cell type in the reference atlas. Numbers above columns indicate the number of cells in each category. Of these cells, 89.4% correctly mapped to their annotated cell type. d, Histogram showing the fraction of cells from the E8.0 biological replicate that mapped to each time point in the reference: 29.2% of cells mapped to the correct time point, and 83.1% of cells mapped within one time point (that is, 6 hours) in either direction. e, Scatter plot comparing the percentage of tdTomato+ cells against tdTomato for each cell type in both Tal1−/− into wild-type (WT; left) and wild-type into wild-type (right) experiments. Black arrowheads indicate extra-embryonic tissues; white arrowheads indicate haematopoietic tissues. f, Force-directed graph of blood-related lineages from the atlas (Fig. 3), coloured by Tal1 expression levels. Darker colouring shows higher expression. g, Flow cytometry analysis of E8.5 Tal1−/− into wild-type chimaeras, showing the complete depletion of the haematopoietic markers CD41 and CD45 (top panels), as well as of the CD71+ Ter119+ erythroid fraction (bottom panels) in Tal1−/− tdTomato+ cells (right panels). h, UMAP plots of wild-type into wild-type experiment, showing balanced contributions to all embryonic lineages. i, Flow cytometry analysis of wild-type into wild-type chimaeras, showing balanced contributions to the haematopoietic lineage from both tdTomato+ and tdTomato cells at E9.5 (representative of 2 individual embryos).

  8. Extended Data Fig. 8 Transcriptional effects of disruption caused by Tal1.

    a, Heat map illustrating the row-normalized expression of genes that were upregulated in EC3-mapped Tal1−/− cells when compared with their closest neighbours in the atlas (labelled ‘EC3’) and EC3-mapped wild-type chimaera cells (labelled ‘WT’). Genes Gm45123 and Fam212a are also known as 5430431A17Rik and Inka1, respectively. b, UMAP plots as in Fig. 1c, showing the expression of Tdo2, Plagl1, and Pcolce.

Supplementary information

  1. Supplementary Note

    This file contains the full legends for Supplementary Tables 1-6.

  2. Reporting Summary

  3. Supplementary Table 1

    Embryo collection information.

  4. Supplementary Table 2

    Dynamically expressed genes along the trajectory from visceral and embryonic endoderm to hindgut.

  5. Supplementary Table 3

    Cell numbers for chimaera haemato-endothelial cell abundance analysis.

  6. Supplementary Table 4

    Metadata for all atlas cells that passed QC.

  7. Supplementary Table 5

    Metadata for chimaera cells from Tal1-/- into wildtype that passed QC.

  8. Supplementary Table 6

    Metadata for chimaera cells from wildtype into wildtype that passed QC.

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DOI

https://doi.org/10.1038/s41586-019-0933-9

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