An in vitro model of early anteroposterior organization during human development

Abstract

The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids—three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Structure and morphology of human gastruloids.
Fig. 2: Dynamic polarization of cell types associated with three germ layers in human gastruloids.
Fig. 3: Transcriptomic anteroposterior organization of human gastruloids.
Fig. 4: Comparative elements of early embryogenesis.

Data availability

All RNA-sequencing datasets produced in this study are deposited in the Gene Expression Omnibus (GEO) under accession code GSE123187. Source data Source data are provided with this paper.

Code availability

Code is available at https://github.com/vikas-trivedi/HumanGastruloids_Fluorescence, https://github.com/anna-alemany/humanGastruloids_tomoseq and https://github.com/naomi-moris/humanGastruloids_shapeDescriptors.

References

  1. 1.

    Solnica-Krezel, L. & Sepich, D. S. Gastrulation: making and shaping germ layers. Annu. Rev. Cell Dev. Biol. 28, 687–717 (2012).

    CAS  Article  Google Scholar 

  2. 2.

    Hyun, I., Wilkerson, A. & Johnston, J. Embryology policy: revisit the 14-day rule. Nature 533, 169–171 (2016).

    ADS  CAS  Article  Google Scholar 

  3. 3.

    O’Rahilly, R. & Müller, F. Developmental stages in human embryos (Carnegie Institution of Washington, 1987).

  4. 4.

    Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    ADS  CAS  Article  Google Scholar 

  5. 5.

    Zhang, X. et al. Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell 7, 90–100 (2010).

    CAS  Article  Google Scholar 

  6. 6.

    Barry, C. et al. Species-specific developmental timing is maintained by pluripotent stem cells ex utero. Dev. Biol. 423, 101–110 (2017).

    CAS  Article  Google Scholar 

  7. 7.

    Loh, K. M. et al. Mapping the pairwise choices leading from pluripotency to human bone, heart, and other mesoderm cell types. Cell 166, 451–467 (2016).

    CAS  Article  Google Scholar 

  8. 8.

    Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat. Methods 11, 847–854 (2014).

    CAS  Article  Google Scholar 

  9. 9.

    Beccari, L. et al. Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids. Nature 562, 272–276 (2018).

    ADS  CAS  Article  Google Scholar 

  10. 10.

    van den Brink, S. C. et al. Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. Development 141, 4231–4242 (2014).

    Article  Google Scholar 

  11. 11.

    Davidson, K. C. et al. Wnt/β-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc. Natl Acad. Sci. USA 109, 4485–4490 (2012).

    ADS  CAS  Article  Google Scholar 

  12. 12.

    Allison, T. F. et al. Identification and single-cell functional characterization of an endodermally biased pluripotent substate in human embryonic stem cells. Stem Cell Reports 10, 1895–1907 (2018).

    CAS  Article  Google Scholar 

  13. 13.

    Martyn, I., Kanno, T. Y., Ruzo, A., Siggia, E. D. & Brivanlou, A. H. Self-organization of a human organizer by combined Wnt and Nodal signalling. Nature 558, 132–135 (2018).

    ADS  CAS  Article  Google Scholar 

  14. 14.

    Massey, J. et al. Synergy with TGFβ ligands switches WNT pathway dynamics from transient to sustained during human pluripotent cell differentiation. Proc. Natl Acad. Sci. USA 116, 4989–4998 (2019).

    CAS  Article  Google Scholar 

  15. 15.

    Piersma, A. H., Hessel, E. V. & Staal, Y. C. Retinoic acid in developmental toxicology: teratogen, morphogen and biomarker. Reprod. Toxicol. 72, 53–61 (2017).

    CAS  Article  Google Scholar 

  16. 16.

    Junker, J. P. et al. Genome-wide RNA tomography in the zebrafish embryo. Cell 159, 662–675 (2014).

    CAS  Article  Google Scholar 

  17. 17.

    Wymeersch, F. J. et al. Transcriptionally dynamic progenitor populations organised around a stable niche drive axial patterning. Development 146, dev168161 (2019).

    Article  Google Scholar 

  18. 18.

    Vega-Hernández, M., Kovacs, A., De Langhe, S. & Ornitz, D. M. FGF10/FGFR2b signaling is essential for cardiac fibroblast development and growth of the myocardium. Development 138, 3331–3340 (2011).

    Article  Google Scholar 

  19. 19.

    Watanabe, Y. et al. Fibroblast growth factor 10 gene regulation in the second heart field by Tbx1, Nkx2-5, and Islet1 reveals a genetic switch for down-regulation in the myocardium. Proc. Natl Acad. Sci. USA 109, 18273–18280 (2012).

    ADS  CAS  Article  Google Scholar 

  20. 20.

    Yoney, A. et al. WNT signaling memory is required for ACTIVIN to function as a morphogen in human gastruloids. eLife 7, e38279 (2018).

    Article  Google Scholar 

  21. 21.

    Dunn, N. R., Vincent, S. D., Oxburgh, L., Robertson, E. J. & Bikoff, E. K. Combinatorial activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse embryo. Development 131, 1717–1728 (2004).

    CAS  Article  Google Scholar 

  22. 22.

    Juan, H. & Hamada, H. Roles of nodal-lefty regulatory loops in embryonic patterning of vertebrates. Genes Cells 6, 923–930 (2001).

    CAS  Article  Google Scholar 

  23. 23.

    Kelly, R. G., Buckingham, M. E. & Moorman, A. F. Heart fields and cardiac morphogenesis. Cold Spring Harb. Perspect. Med. 4, a015750 (2014).

    Article  Google Scholar 

  24. 24.

    Wilson, V., Olivera-Martinez, I. & Storey, K. G. Stem cells, signals and vertebrate body axis extension. Development 136, 1591–1604 (2009).

    CAS  Article  Google Scholar 

  25. 25.

    Koch, F. et al. Antagonistic activities of Sox2 and Brachyury control the fate choice of neuro-mesodermal progenitors. Dev. Cell 42, 514–526 (2017).

    CAS  Article  Google Scholar 

  26. 26.

    Diaz-Cuadros, M. et al. In vitro characterization of the human segmentation clock. Nature 580, 113–118 (2020).

    CAS  Article  Google Scholar 

  27. 27.

    Canham, M. A. et al. The molecular karyotype of 25 clinical-grade human embryonic stem cell lines. Sci. Rep. 5, 17258 (2015).

    ADS  CAS  Article  Google Scholar 

  28. 28.

    Moris, N. et al. Generating human gastruloids from human embryonic stem cells. Protoc. Exch. https://doi.org/10.21203/rs.3.pex-812/v1 (2020).

  29. 29.

    Baillie-Johnson, P., van den Brink, S. C., Balayo, T., Turner, D. A. & Martinez Arias, A. Generation of aggregates of mouse embryonic stem cells that show symmetry breaking, polarization and emergent collective behaviour in vitro. J. Vis. Exp. 105, e53252 (2015).

    Google Scholar 

  30. 30.

    Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).

    CAS  Article  Google Scholar 

  31. 31.

    Susaki, E. A. et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat. Protoc. 10, 1709–1727 (2015).

    CAS  Article  Google Scholar 

  32. 32.

    Turner, D. A. et al. Wnt/β-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor in ensembles of mouse embryonic stem cells. Development 141, 4243–4253 (2014).

    CAS  Article  Google Scholar 

  33. 33.

    Turner, D. A. et al. Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids. Development 144, 3894–3906 (2017).

    CAS  Article  Google Scholar 

  34. 34.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Article  Google Scholar 

  35. 35.

    Bradski, G. The OpenCV library. Dr. Dobb’s: The World of Software Development https://www.drdobbs.com/open-source/the-opencv-library/184404319 (2000).

  36. 36.

    Kruse, F., Junker, J. P., van Oudenaarden, A. & Bakkers, J. Tomo-seq: a method to obtain genome-wide expression data with spatial resolution. Methods Cell Biol. 135, 299–307 (2016).

    CAS  Article  Google Scholar 

  37. 37.

    van den Brink, S. C. et al. Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids. Nature (2020).

  38. 38.

    Muraro, M. J. et al. A single-cell transcriptome atlas of the human pancreas. Cell Syst. 3, 385–394 (2016).

    CAS  Article  Google Scholar 

  39. 39.

    Grün, D., Kester, L. & van Oudenaarden, A. Validation of noise models for single-cell transcriptomics. Nat. Methods 11, 637–640 (2014).

    Article  Google Scholar 

  40. 40.

    Huang, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    CAS  Article  Google Scholar 

  41. 41.

    Klopfenstein, D. V. et al. GOATOOLS: a Python library for gene ontology analyses. Sci. Rep. 8, 10872 (2018).

    ADS  CAS  Article  Google Scholar 

  42. 42.

    Theiler, K. The House Mouse: Atlas of Embryonic Development 2nd edn (Springer-Verlag, 1989).

  43. 43.

    Sonnen, K. F. et al. Modulation of phase shift between Wnt and Notch signalling oscillations controls mesoderm segmentation. Cell 172, 1079–1090 (2018).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank V. Trivedi for his help writing the dynamic fluorescent reporter analysis code; D. Turner and A. Baranowski for their help developing the image analysis code; K. Muller from the Cambridge Advance Imaging Centre (CAIC) for help with scanning electron microscopy; the Utrecht Sequencing Facility for sequencing; A. Ebbing, J. Vivié and M. Betist for the robotized tomo-seq protocol; and members of the Martinez Arias and van Oudenaarden laboratories, as well as B. Steventon, P. Rugg-Gunn, M. Lütolf, M. Johnson and N. Hopwood, for discussions over the course of this work. This work was supported by funds from the Newton Trust (INT16.24b), Leverhulme Trust (RPG-2018-356) and MRC (MR/R017190/1) to A.M.A., N.M., S.G. and T.B. and a European Research Council Advanced Grant (ERC-AdG 742225-IntScOmics), a Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) TOP award (NWO-CW 714.016.001) and the Foundation for Fundamental Research on Matter, financially supported by NWO (FOM-14NOISE01), to S.C.v.d.B., A.A. and A.v.O. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. It was also supported by a Constance Work Junior Research Fellowship from Newnham College, Cambridge to N.M. and an Erasmus+ grant to K.A. and J.S.

Author information

Affiliations

Authors

Contributions

N.M. and K.A. designed, optimized and performed the human gastruloid experiments, with help from T.B. and A.M.A., and the A.v.O. laboratory independently replicated the entire process. N.M. and S.C.v.d.B. embedded the human gastruloids and S.C.v.d.B. performed sectioning and tomo-seq preparation. A.A. analysed transcriptomic datasets, with analysis input from N.M., S.C.v.d.B., A.v.O. and A.M.A. N.M. created embryonic illustrations. J.S. designed and made in situ probes and J.S. and S.G. performed in situ hybridizations. A.M.A. and A.v.O. supervised research. N.M and A.M.A. wrote the manuscript with considerable input from all authors. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Naomi Moris or Alexander van Oudenaarden or Alfonso Martinez Arias.

Ethics declarations

Competing interests

This work is the subject of a patent application (PCT/GB2019/052670) filed by Cambridge Enterprise on behalf of the University of Cambridge (international filing date 23/09/2019; published 26/03/2020), covering the generation and use of human gastruloids. The inventors are N.M. and A.M.A. The remaining authors declare no competing interests.

Additional information

Peer review information Nature thanks Jianping Fu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data figures and tables

Extended Data Fig. 1 Optimization of the Chiron pre-treatment and morphological variability.

a, Aggregation of single RUES2-GLR cells after pre-treatment with Chiron, showing either a single aggregate (left) or the presence of transient ‘satellite’ aggregates (right). These typically merge within 10 h (n = 38 gastruloids from n = 2 experiments). b, Schematic of the protocol without Chiron pre-treatment, but with aggregation in Chiron and ROCK inhibitor medium. c, Gastruloids made from the RUES2-GLR line without Chiron pre-treatment at 24 h, 48 h and 72 h after aggregation. Three examples are shown that are representative of five independent experiments for each time point (n = 415 gastruloids), with all three fluorescent reporters (SOX2–mCitrine, SOX17–tdTomato and BRA–mCerulean; left) and without SOX2–mCitrine (right). Scale bars, 100 μm. d, Examples of reporter patterning in differential morphological classes, as assessed by automated segmentation providing gastruloid outline boundaries (yellow line indicates the boundary used for quantifications). Three gastruloids per category are shown that are representative of seven independent experiments (n = 374 gastruloids). See Methods for details of the classification method. Scale bars, 100 μm. e, Cell-line-dependent optimization of Chiron conditions. Human gastruloids were derived from the MasterShef7 cell line (left; two examples shown) or the RUES2-GLR cell line (right; three examples shown). Red boxes indicate the concentrations at which gastruloids were deemed to be optimally elongated, and the resultant conditions for subsequent gastruloid derivation. Representative examples are shown from three independent experiments. Scale bars, 100 μm.

Extended Data Fig. 2 Effect of Chiron pre-treatment on human ES cells.

a, Gene expression in adherent RUES2-GLR cells after 24 h of Chiron pre-treatment (ChiPT) compared to cells that were not pre-treated (Nutristem alone; NoPT), as assessed by quantitative PCR with reverse transcription. Averages from five biological replicates are shown; bars, mean average; points, technical averages for each experimental replicate. *P < 0.05, **P < 0.01, NS, not significant (P > 0.05) (Welch two-sided, two-sample t-test). See Methods, Source Data. b, Immunostaining of adherent colonies of RUES2-GLR cells that were not pre-treated (top) or that were pre-treated with Chiron for 24 h (bottom) for Brachyury (BRA), E-cadherin and N-cadherin. Dashed boxes show the position of enlarged regions. One representative example is shown from two independent experiments. Scale bars, 100 μm. c, Quantified expression from immunostaining of RUES2-GLR cells as shown in b. The whole image was used to generate these data. d, Profiles of membrane localization of E-cadherin and N-cadherin from immunostaining of RUES2-GLR cells as shown in b. Source data

Extended Data Fig. 3 Establishing axial patterning in human gastruloids.

a, Immunofluorescence imaging of a RUES2-GLR human gastruloid at 24 h. Confocal sections (top) and mean projection (bottom) of the gastruloid. One representative example is shown from two experiments (n = 12 gastruloids). Scale bars, 100 μm. b, MasterShef7 human gastruloids at 72 h after aggregation, showing BRA, SOX2 and N-cadherin (CDH2) localization. Three representative examples are shown, from three experiments (n = 13 gastruloids). Scale bars, 100 μm. c, Projection of immunofluorescently labelled RUES2-GLR human gastruloids at 24 h, 48 h and 72 h with GATA6 (magenta) and CDX2 (yellow) staining. Six representative gastruloids are shown for each time point. Numbers, aspect ratio. Representative examples are shown from two independent experiments (n = 63 gastruloids). Scale bars, 30 μm. d, Scatter plot of the co-expression of GATA6 and CDX2 per cell, across the three time points. Blue points, co-expression over threshold; grey points, expression below threshold. The number of gastruloids in each plot is shown (n). e, Relative axial expression of GATA6 (magenta) and CDX2 (yellow) along the anteroposterior axis. The number of gastruloids in each plot is shown (n). f, Relative axial expression of GATA6 (magenta) and CDX2 (yellow) along the anteroposterior axis, stratified by aspect ratio (as determined using a manual axial patterning quantification; see Methods for details). Thick lines, mean; thin lines, individual gastruloids. The number of gastruloids in each plot is shown (n). Representative images of gastruloid elongation classifications are shown in c. g, Progressive polarization and restriction of GATA6–GFP fluorescence to the anterior pole of S4-GATA6-GFP human gastruloids. One representative example is shown (n = 17 gastruloids). Scale bars, 100 μm. Source data

Extended Data Fig. 4 Disrupting axial patterning in human gastruloids.

a, RUES2-GLR cell aggregates after pre-treatment (PT) with WNT3A instead of Chiron for 24 h. Representative examples are shown (n = 281 gastruloids). b, RUES2-GLR cell aggregates after pre-treatment with BMP4 for 24 h. Representative examples are shown (n = 187 gastruloids). c, Application of a BMP inhibitor, LDN193189 (LDN; left) or tankyrase inhibitor, XAV-939 (XAV; right) during 24-h pre-treatment of RUES2-GLR cells. Representative examples are shown (n = 85 gastruloids). d, Application of a Nodal signalling inhibitor, SB43 (SB43) during 24-h pre-treatment of RUES2-GLR cells. ad, Two representative examples are shown for each condition (three independent experiments). Dark green boxes indicate pre-treatment in Nutristem; teal boxes indicate the aggregation medium composition (E6 and ROCK inhibitor). Scale bars,100 μm. e, Addition of retinoid acid (right) or DMSO (left) to RUES2-GLR human gastruloids for each day of aggregate development. Schematic of the protocol (top) and imaging results (bottom). See Methods for experimental details. Representative examples are shown from four independent experiments (n = 159 gastruloids). Scale bars,100 μm. f, Confocal imaging of axial patterning defects in 72-h RUES2-GLR human gastruloids treated with retinoic acid. Representative examples are shown from three independent experiments (n = 25 gastruloids). Scale bars, 100 μm.

Extended Data Fig. 5 Spatial transcriptomics by tomo-seq identifies clusters of gene expression.

a, Quantification of the number of genes (left) and number of unique transcripts (right) detectable in each section along the anteroposterior axis of 72-h RUES2-GLR human gastruloids pre-treated with Chiron. Blue bars, sections above the threshold used for downstream tomo-seq analysis; grey bars, sections below the threshold (see Methods for details). Two replicates are shown. b, Average expression patterns along the anteroposterior axis of all genes detected in each cluster. Clusters correspond to those in Fig. 3c and Supplementary Data 1. Lines, mean; shading, s.d. for the set of genes within each cluster (n = 2 gastruloids). c, Selection of gene traces along the anteroposterior axis for both gastruloids. Blue and green lines, expression values for replicates 1 and 2, respectively. Source data

Extended Data Fig. 6 Transcriptional profiles and anteroposterior localization in human gastruloids.

a, Normalized expression of anterior neural genes in human gastruloids. b, Total expression (log10-transformed) of each HOX gene across all sections of gastruloid 1 (top) and gastruloid 2 (bottom), for all four clusters (HOXA, HOXB, HOXC and HOXD). White boxes indicate that a gene is not present in the human genome. n = 2 gastruloids (a, b). c, Expression of FOXA2 in the posterior end of 72-h Chiron-pre-treated RUES2-GLR gastruloids. Three representative examples are shown (n = 8 gastruloids). Scale bars, 100 μm. d, Expression of ligands of the BMP (top) and WNT (bottom) signalling pathways. Red box indicates genes with a particularly strong anteroposterior localization bias (n = 2 gastruloids). e, Maximum projection confocal images of SMAD1-RFP;H2B-mCitrine human gastruloids at 72 h. Three representative examples are shown. Insets, magnified views of the regions shown in red dashed boxes. Representative examples are shown (n = 19 gastruloids; two independent experiments). Scale bars; 40 μm or 50 μm (indicated on image). f, Processing to separate the nuclear and cytoplasmic component of the SMAD1–RFP signal (left; see Methods for details), and resultant quantification of the normalized nuclear:cytoplasmic ratio of SMAD1–RFP along the anteroposterior axis (right; each point represents a cell). Three representative examples are shown; two independent experiments. The scale of the images is the same as e. g, Immunostaining of LEF1 and BRA expression in 96-h RUES2-GLR human gastruloids. LEF1 is localized in a gradient primarily in the posterior portion of the gastruloids. Two representative examples are shown from three independent experiments (n = 10 gastruloids). Scale bars, 100 μm. h, Immunostaining of WNT3A and BRA expression in 72-h RUES2-GLR human gastruloids, showing a magnified view of the posterior end. Max. proj., maximum projection. One representative example is shown (n = 8 gastruloids; two independent experiments). Scale bars, 50 μm. i, Localized expression of genes that are related to Nodal signalling towards the posterior of Chiron-pre-treated human gastruloids by tomo-seq (n = 2 gastruloids). Source data

Extended Data Fig. 7 Perturbation of Nodal signalling in human gastruloids.

a, Schematic representation of the protocol used to generate Chiron and SB43 (Chi + SB43)-pre-treated human gastruloids. See Methods for details. b, Representative examples of the dynamic development of Chi + SB43-pre-treated RUES2-GLR gastruloids (three experiments). Colours indicate reporter fluorescence as indicated in Fig. 2a. Scale bars, 100 μm. c, In situ hybridization against BRA and SOX2 mRNA in 96-h Chi + SB43-pre-treated gastruloids. Four representative examples are shown for each gene. d, Wide-field imaging of the two 120-h Chi + SB43-pre-treated RUES2-GLR gastruloids used for tomo-seq. Scale bars, 100 μm. e, Venn diagram showing the number of reproducibly localized genes in the Chiron-pre-treated (Chi hGld, green) and Chi + SB43-pre-treated (Chi + SB43 hGld, yellow) human gastruloids. Numbers indicate counts of genes and percentage values in brackets indicate the proportion of the full figure. See Source Data. f, Differentially expressed genes between Chiron-pre-treated and Chi + SB43-pre-treated gastruloids (total expression). See Methods, Supplementary Data 6, Source Data. g, Gene expression patterns detected in an averaged Chiron-pre-treated gastruloid and an averaged Chi + SB43-pre-treated gastruloid. Grey and black bands show the hierarchical clustering of gene expression; blue and red bands indicate selective reproducibility between replicates from one or other pre-treatment conditions (red, Chi + SB43 only; blue, Chiron only; grey; both); dark red box, cluster for which expression is lost after SB43 pre-treatment (cluster 4); white rows, no expression detected. n = 2 Chiron-pre-treated and n = 2 Chi + SB43-pre-treated gastruloids (ag). See Methods, Supplementary Data 5, Source Data. Source data

Extended Data Fig. 8 Transcriptional profiles of gastruloids exposed to Nodal inhibition before aggregation.

a, Quantification of the number of genes (left) and number of unique transcripts (right) detectable in each section along the anteroposterior axis of 120-h Chi + SB43-pre-treated RUES2-GLR gastruloids. Blue bars, sections above the threshold used for downstream tomo-seq analysis; grey bars, sections below the threshold (see Methods for details). Two replicates are shown. b, Significantly reproducible gene expression patterns of individual replicates of Chi + SB43-pre-treated human gastruloids (left) and the average gastruloid (right) along the anteroposterior axis. See Supplementary Data 4, Source Data. c, Average expression pattern of genes from each cluster shown in b. Lines, mean; shading, s.d. for the set of genes within each cluster. d, Detection of expression for markers of all three germ layers. White rows indicate that no expression was detected for that gene. See Supplementary Data 7, Source Data. e, Gene expression traces along the anteroposterior axis of the four human gastruloids (grey lines, Chiron pre-treatment; blue lines, Chi + SB43 pre-treatment; solid lines, replicate 1; dashed lines, replicate 2). n = 2 gastruloids (ae). Source data

Extended Data Fig. 9 Unique transcriptional profiles of mouse and human gastruloids.

a, Venn diagram showing the number of common reproducibly localized genes in Chiron-pre-treated human gastruloids (Chi hGld, green), Chi + SB43-pre-treated human gastruloids (Chi + SB43 hGld, yellow) and mouse gastruloids (mGld, blue). Numbers indicate counts of genes and percentage values in brackets indicate the proportion of the full figure. b, Unique reproducibly localized gene expression in mouse gastruloids, not detected in Chiron-pre-treated human gastruloids. c, Unique reproducibly localized gene expression in Chiron-pre-treated human gastruloids, not detected in mouse gastruloids. d, Genes with reproducibly localized expression in mouse gastruloids and that are expressed, but not reproducibly localized, in Chiron-pre-treated human gastruloids. e, Genes with reproducibly localized expression in Chiron-pre-treated human gastruloids and that are expressed, but not reproducibly localized, in mouse gastruloids. n = 2 human gastruloids, n = 3 mouse gastruloids (ae). See Supplementary Data 9, Source Data. Source data

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1 | Primer Sequences for RT-qPCR experiments.

Supplementary Table

Supplementary Table 2 | Primer Sequences for In Situ Hybridisation experiments.

Supplementary Data

Supplementary Data 1 | Clusters of reproducible gene expression from TOMO-sequencing of Chiron pre-treated Human Gastruloids. ID column includes ENSEMBL ID, Gene name and Chromosome position. For each gene, the assignment to a cluster using Hierarchical Clustering methods is given. Relates to Fig. 3c-d and Extended Data Fig. 5b.

Supplementary Data

Supplementary Data 2 | Germ Layer Representation in Chiron pre-treated Human Gastruloids. Selection of genes from all three germ layers which are represented along the anterioposterior (AP) axis of the human gastruloids. Localisation of each gene along the axis is shown in Figure 3e. Relates to Fig. 3e.

Supplementary Data

Supplementary Data 3 | Gene Ontology enrichment per TOMO-sequencing cluster of Chiron pre-treated Human Gastruloids. Each sheet of the file corresponds to a different Hierarchical Cluster (cl) and contains the list of enriched Biological Process Gene Ontology (GO) terms for that cluster. GO enrichment was performed using DAVID Functional Annotation tool using the human genome as a background. Adjustment for multiple comparisons was performed, and is reported as Bonferroni, Benjamini and FDR values. Relates to Fig. 3c-d.

Supplementary Data

Supplementary Data 4 | Clusters of reproducible gene expression from TOMO-sequencing of Chiron + SB43 pre-treated Human Gastruloids. ID column includes ENSEMBL ID, Gene name and Chromosome position. For each gene, the assignment to a cluster uses a self-organizing map (som) method, followed by hierarchical clustering (hcl). See Methods for details. Relates to Extended Data Fig. 8b-c.

Supplementary Data

Supplementary Data 5 | Clusters of gene expression from TOMO-sequencing of Chiron + SB43 pre-treated Human Gastruloids compared to Chiron pre-treated Human Gastruloids. ID column includes ENSEMBL ID, Gene name and Chromosome position. For each gene, the assignment to a cluster uses a self-organizing map (som) method, followed by hierarchical clustering (hcl). See Methods for details. Relates to Extended Data Fig. 7g.

Supplementary Data

Supplementary Data 6 | Differential gene expression from TOMO-sequencing between Chiron and Chiron + SB43 pre-treated Human Gastruloids. ID column includes ENSEMBL ID, Gene name and Chromosome position. For each gene, the total expression level as the sum of each section is shown for both conditions (total-Chi and total-SB) as well as the mean level of expression in both systems (total-mean). The Log2 fold change in expression (log2FC) and mu (log2mu) are calculated alongside the probabilities (prob-Chi and prob-SB) total number (N-Chi and N-SB). The resultant P values are calculated using a Binomial test with the alternative condition used as a background (PvalChi and PvalSB). n = 2 Chiron pre-treated gastruloids and 2 Chiron + SB43 pre-treated gastruloids. Relates to Extended Data Fig. 7f.

Supplementary Data

Supplementary Data 7 | Germ Layer Representation in Chrion and Chiron + SB43 pre-treated Human Gastruloids. ID column includes ENSEMBL ID, Gene name and Chromosome position. For each gene, the level of expression across all sections of the average gastruloid are shown. The two sheets correspond to the Chiron pre-treaed human gastruloids and the Chiron and SB43 pre-treated human gastruloids, respectively. Relates to Extended Data Fig. 8d.

Supplementary Data

Supplementary Data 8 | Clusters of gene expression from TOMO-sequencing of Chiron pre-treated Human Gastruloids and Mouse Gastruloids, including GO term enrichment. ID column includes ENSEMBL ID, Gene name and Chromosome position. For each gene, the assignment to a cluster uses a self-organizing map (som) method, followed by hierarchical clustering (hcl). See Methods for details. Each subsequent sheet shows the results of Gene Ontology (GO) term enrichment analysis, per cluster. Relates to Fig. 4g.

Supplementary Data

Supplementary Data 9 | Clusters of unique and specific gene expression from TOMO-sequencing of Chiron pre-treated Human Gastruloids and Mouse Gastruloids. ID column includes ENSEMBL ID, Gene name and Chromosome position. For each gene, the assignment to a cluster uses a self-organizing map (som) method, followed by hierarchical clustering (hcl). See Methods for details. Relates to Extended Data Fig. 9.

Video 1

| Live-cell imaging of the S4-GATA6 human ES cell line, showing GATA6-GFP expression, from 24 – 35 hours. Representative example of n = 25 gastruloids, from 2 independent experiments.

Video 2

| Live-cell imaging of the RUES2-GLR human ES cell line, showing BRA-mCerulean, SOX17-tdTomato and SOX2-mCitrine expression, from 0 – 24 hours. Three representative gastruloids are shown. n = 2 independent experiments.

Video 3

| Live-cell imaging of the RUES2-GLR human ES cell line, showing BRA-mCerulean, SOX17-tdTomato and SOX2-mCitrine expression, from 4 – 61 hours. Multiple individual live-imaging movies of the same gastruloid have been stitched together to give a longer time-course. Note that the objective used changes from 10x (4-12h) to 20x (22-49h) and 10x (52-61h). n = 3 independent experiments.

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Moris, N., Anlas, K., van den Brink, S.C. et al. An in vitro model of early anteroposterior organization during human development. Nature 582, 410–415 (2020). https://doi.org/10.1038/s41586-020-2383-9

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.