During gastrulation, cell types from all three germ layers are specified and the basic body plan is established1. However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single-cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems2. Here, we report single-cell transcriptomic characterization of >20,000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain substructure, including three distinct signatures in early foregut cells. Pseudo-space ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterization of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. Thus, this comprehensive single-cell map can be exploited to reveal previously unrecognized pathways that contribute to tissue development.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Kaufman, M. & Bard, J. The Anatomical Basis of Mouse Development. (Academic Press, San Diego, 1999).
Wang, Y. & Navin, N. E. Advances and applications of single-cell sequencing technologies. Mol. Cell 58, 598–609 (2015).
Grapin-Botton, A. in StemBook (ed. The Stem Cell Research Community) (Stembook, 2008); http://www.stembook.org/node/524
Inoue-Yokoo, T., Tani, K. & Sugiyama, D. Mesodermal and hematopoietic differentiation from ES and iPS cells. Stem Cell Rev. 9, 422–434 (2013).
Tang, K., Peng, G., Qiao, Y., Song, L. & Jing, N. Intrinsic regulations in neural fate commitment. Dev. Growth Differ. 57, 109–120 (2015).
Spence, J. R., Lauf, R. & Shroyer, N. F. Vertebrate intestinal endoderm development. Dev. Dyn. 240, 501–520 (2011).
Haghverdi, L., Buttner, M., Wolf, F. A., Buettner, F. & Theis, F. J. Diffusion pseudotime robustly reconstructs lineage branching. Nat. Methods 13, 845–848 (2016).
Yap, C., Goh, H. N., Familari, M., Rathjen, P. D. & Rathjen, J. The formation of proximal and distal definitive endoderm populations in culture requires p38 MAPK activity. J. Cell Sci. 127, 2204–2216 (2014).
Hou, J. et al. A systematic screen for genes expressed in definitive endoderm by Serial Analysis of Gene Expression (SAGE). BMC Dev. Biol. 7, 92 (2007).
Si-Tayeb, K., Lemaigre, F. P. & Duncan, S. A. Organogenesis and development of the liver. Dev. Cell 18, 175–189 (2010).
Becker, M. B., Zulch, A., Bosse, A. & Gruss, P. Irx1 and Irx2 expression in early lung development. Mech. Dev. 106, 155–158 (2001).
Mou, H. et al. Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs. Cell Stem Cell 10, 385–397 (2012).
Franklin, V. et al. Regionalisation of the endoderm progenitors and morphogenesis of the gut portals of the mouse embryo. Mech. Dev. 125, 587–600 (2008).
Andoniadou, C. L. et al. Lack of the murine homeobox gene Hesx1 leads to a posterior transformation of the anterior forebrain. Development 134, 1499–1508 (2007).
Scialdone, A. et al. Computational assignment of cell-cycle stage from single-cell transcriptome data. Methods 85, 54–61 (2015).
Oates, A. C., Morelli, L. G. & Ares, S. Patterning embryos with oscillations: structure, function and dynamics of the vertebrate segmentation clock. Development 139, 625–639 (2012).
Dunwoodie, S. L., Rodriguez, T. A. & Beddington, R. S. Msg1 and Mrg1, founding members of a gene family, show distinct patterns of gene expression during mouse embryogenesis. Mech. Dev. 72, 27–40 (1998).
Plisov, S. et al. Cited1 is a bifunctional transcriptional cofactor that regulates early nephronic patterning. J. Am. Soc. Nephrol. 16, 1632–1644 (2005).
Dahmann, C., Oates, A. C. & Brand, M. Boundary formation and maintenance in tissue development. Nat. Rev. Genet. 12, 43–55 (2011).
De Val, S. & Black, B. L. Transcriptional control of endothelial cell development. Dev. Cell 16, 180–195 (2009).
Scotti, M. & Kmita, M. Recruitment of 5′ Hoxa genes in the allantois is essential for proper extra-embryonic function in placental mammals. Development 139, 731–739 (2012).
Drake, C. J. & Fleming, P. A. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 95, 1671–1679 (2000).
Lee, L. K. et al. LYVE1 marks the divergence of yolk sac definitive hemogenic endothelium from the primitive erythroid lineage. Cell Rep. 17, 2286–2298 (2016).
McGrath, K. E. et al. Distinct sources of hematopoietic progenitors emerge before HSCs and provide functional blood cells in the mammalian embryo. Cell Rep. 11, 1892–1904 (2015).
Scialdone, A. et al. Resolving early mesoderm diversification through single-cell expression profiling. Nature 535, 289–293 (2016).
Hayashi, K., de Sousa Lopes, S. M. & Surani, M. A. Germ cell specification in mice. Science 316, 394–396 (2007).
Slukvin, I. Generating human hematopoietic stem cells in vitro—exploring endothelial to hematopoietic transition as a portal for stemness acquisition. FEBS Lett. 590, 4126–4143 (2016).
Thambyrajah, R. et al. New insights into the regulation by RUNX1 and GFI1(s) proteins of the endothelial to hematopoietic transition generating primordial hematopoietic cells. Cell Cycle 15, 2108–2114 (2016).
Jiang, X. et al. Let-7 microRNA-dependent control of leukotriene signaling regulates the transition of hematopoietic niche in mice. Nat. Commun. 8, 128 (2017).
Li, P. et al. Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment. Nature 523, 468–471 (2015).
North, T. E. et al. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature 447, 1007–1011 (2007).
Cutler, C. et al. Prostaglandin-modulated umbilical cord blood hematopoietic stem cell transplantation. Blood 122, 3074–3081 (2013).
Lun, A. T., Bach, K. & Marioni, J. C. Pooling across cells to normalize single-cell RNA sequencing data with many zero counts. Genome Biol. 17, 75 (2016).
Lun, A. T., McCarthy, D. J. & Marioni, J. C. A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor. F1000Res. 5, 2122 (2016).
Kolodziejczyk, A. A. et al. Single cell RNA-sequencing of pluripotent states unlocks modular transcriptional variation. Cell Stem Cell 17, 471–485 (2015).
Rtsne: T-distributed stochastic neighbor embedding using Barnes–Hut implementation. R package version 0.11 (Krijthe, J., 2015); https://github.com/jkrijthe/Rtsne
Langfelder, P., Zhang, B. & Horvath, S. Defining clusters from a hierarchical cluster tree: the Dynamic Tree Cut package for R. Bioinformatics 24, 719–720 (2008).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Angerer, P. et al. destiny: diffusion maps for large-scale single-cell data in R. Bioinformatics 32, 1241–1243 (2016).
Chlis, N. K., Wolf, F. A. & Theis, F. J. Model-based branching point detection in single-cell data by K-branches clustering. Bioinformatics 33, 3211–3219 (2017).
cluster: Cluster analysis basics and extensions. R package version 2.0.5 (Maechler, M., Rousseeuw, P., Struyf, A., Hubert, M. & Hornik, K., 2016).
Hannan, N. R., Segeritz, C. P., Touboul, T. & Vallier, L. Production of hepatocyte-like cells from human pluripotent stem cells. Nat. Protoc. 8, 430–437 (2013).
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
We thank A. Lun for help with the Cell Ranger tool and the CRUK Cambridge Institute Genomics and Bioinformatics Cores for supporting the DNA sequencing and demultiplexing of the data. Research in the authors’ laboratories is supported by the MRC, CRUK, Bloodwise, the Leukemia and Lymphoma Society, NIH-NIDDK, the Sanger-EBI Single Cell Centre and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, and by core funding from Cancer Research UK and the European Molecular Biology Laboratory. W.J. is a Wellcome Trust Clinical Research Fellow. B.P.-S is funded by the Wellcome Trust 4 Year PhD programme in Stem Cell Biology and Medicine and the University of Cambridge. A.S. is supported by the Sanger-EBI Single Cell Centre. L.V. is supported by the ERC starting grant Relieve-IMDs. This work was funded as part of the Wellcome Trust Strategic Award 105031/D/14/Z “Tracing early mammalian lineage decisions by single cell genomics” awarded to W. Reik, S. Teichmann, J.N., B.D.S., T. Voet, S.S., L.V., B.G. and J.C.M.
Supplementary Figures 1–5 and Supplementary References.
The different samples contribute equally to each of the 20 identified subpopulations.
Differential expression between the germ layers.
Differential expression between three foregut subpopulations.
Transcriptomes of the primordial germ cells.
Dissection strategy used to validate the oscillating genes in the presomitic mesoderm.
About this article
Nature Cell Biology (2019)
Nature Biotechnology (2019)