A 3D model of a human epiblast reveals BMP4-driven symmetry breaking


Breaking the anterior–posterior symmetry in mammals occurs at gastrulation. Much of the signalling network underlying this process has been elucidated in the mouse; however, there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro three-dimensional model of a human epiblast whose size, cell polarity and gene expression are similar to a day 10 human epiblast. A defined dose of BMP4 spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial-to-mesenchymal transition. We show that WNT signalling and its inhibitor DKK1 play key roles in this process downstream of BMP4. Our work demonstrates that a model human epiblast can break axial symmetry despite the absence of asymmetry in the initial signal and of extra-embryonic tissues or maternal cues. Our three-dimensional model is an assay for the molecular events underlying human axial symmetry breaking.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: In vitro 3D model of a pre-gastrulation human epiblast.
Fig. 2: Concentration-dependent patterning by BMP4.
Fig. 3: Breaking AP symmetry in a 3D model of a human epiblast.
Fig. 4: Signs of the primitive streak and EMT.
Fig. 5: Dynamics of AP symmetry breaking, EMT and SOX17 activation.
Fig. 6: Cells in the 3D model of the epiblast respond uniformly to the initial BMP4 signalling.
Fig. 7: Molecular mechanism of symmetry breaking in the 3D model of the epiblast.

Data availability

The numerical source data for all figures have been provided as Supplementary Table 2. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

The code used for cell segmentation in this work is available from the corresponding author on request.


  1. 1.

    Benazeraf, B. & Pourquie, O. Formation and segmentation of the vertebrate body axis. Annu. Rev. Cell Dev. Biol. 29, 1–26 (2013).

    CAS  Article  Google Scholar 

  2. 2.

    Stern, C. Gastrulation: From Cells To Embryo (Cold Spring Harbor Press, 2004).

  3. 3.

    O’Rahilly, R. & Muller, F. Developmental stages in human embryos: revised and new measurements. Cells Tissues Organs 192, 73–84 (2010).

    Article  Google Scholar 

  4. 4.

    Arnold, S. J. & Robertson, E. J. Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat. Rev. Mol. Cell Biol. 10, 91–103 (2009).

    CAS  Article  Google Scholar 

  5. 5.

    Migeotte, I., Omelchenko, T., Hall, A. & Anderson, K. V. Rac1-dependent collective cell migration is required for specification of the anterior-posterior body axis of the mouse. PLoS Biol. 8, e1000442 (2010).

    Article  Google Scholar 

  6. 6.

    Nowotschin, S. et al. The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo. Genes Dev. 27, 997–1002 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Mukhopadhyay, M. et al. Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse. Dev. Cell 1, 423–434 (2001).

    CAS  Article  Google Scholar 

  8. 8.

    ten Berge, D. et al. Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3, 508–518 (2008).

    Article  Google Scholar 

  9. 9.

    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 

  10. 10.

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

    CAS  Article  Google Scholar 

  11. 11.

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

    Article  Google Scholar 

  12. 12.

    Harrison, S. E., Sozen, B., Christodoulou, N., Kyprianou, C. & Zernicka-Goetz, M. Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro. Science 356, eaal1810 (2017).

  13. 13.

    Sozen, B. et al. Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures. Nat. Cell Biol. 20, 979–989 (2018).

    CAS  Article  Google Scholar 

  14. 14.

    Zhang, S. P. et al. Implantation initiation of self-assembled embryolike structures generated using three types of mouse blastocyst-derived stem cells. Nat. Comm. 10, 496 (2019).

    Article  Google Scholar 

  15. 15.

    National Research Council and Institute of Medicine. Final Report of the National Academies’ Human Embryonic Stem Cell Research Advisory Committee and 2010 Amendments to the National Academies’ Guidelines for Human Embryonic Stem Cell Research (National Academies Press, 2010).

  16. 16.

    Shao, Y. et al. A pluripotent stem cell-based model for post-implantation human amniotic sac development. Nat. Commun. 8, 208 (2017).

    Article  Google Scholar 

  17. 17.

    Shao, Y. et al. Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche. Nat. Mater. 16, 419–425 (2016).

    Article  Google Scholar 

  18. 18.

    Ben-Haim, N. et al. The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4. Dev. Cell 11, 313–323 (2006).

    CAS  Article  Google Scholar 

  19. 19.

    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 

  20. 20.

    Lee, L. H. et al. Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages. Stem Cell Res. 2, 155–162 (2009).

    CAS  Article  Google Scholar 

  21. 21.

    Deglincerti, A. et al. Self-organization of the in vitro attached human embryo. Nature 533, 251–254 (2016).

    CAS  Article  Google Scholar 

  22. 22.

    Shahbazi, M. N. et al. Self-organization of the human embryo in the absence of maternal tissues. Nat. Cell Biol. 18, 700–708 (2016).

    CAS  Article  Google Scholar 

  23. 23.

    Taniguchi, K. et al. Lumen formation is an intrinsic property of isolated human pluripotent stem cells. Stem Cell Rep. 5, 954–962 (2015).

    CAS  Article  Google Scholar 

  24. 24.

    Lei, Y. & Schaffer, D. V. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. Proc. Natl Acad. Sci. USA 110, E5039–E5048 (2013).

    CAS  Article  Google Scholar 

  25. 25.

    Alakpa, E. V. et al. Tunable supramolecular hydrogels for selection of lineage-guiding metabolites in stem cell cultures. Chem 1, 298–319 (2016).

    CAS  Article  Google Scholar 

  26. 26.

    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).

    CAS  Article  Google Scholar 

  27. 27.

    Nakamura, T. et al. Single-cell transcriptome of early embryos and cultured embryonic stem cells of cynomolgus monkeys. Sci. Data 4, 170067 (2017).

    CAS  Article  Google Scholar 

  28. 28.

    Sasaki, K. et al. The germ cell fate of cynomolgus monkeys is specified in the nascent amnion. Dev. Cell 39, 169–185 (2016).

    CAS  Article  Google Scholar 

  29. 29.

    Amita, M. et al. Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4. Proc. Natl Acad. Sci. USA 110, E1212–E1221 (2013).

    CAS  Article  Google Scholar 

  30. 30.

    Carver, E. A., Jiang, R., Lan, Y., Oram, K. F. & Gridley, T. The mouse Snail gene encodes a key regulator of the epithelial–mesenchymal transition. Mol. Cell. Biol. 21, 8184–8188 (2001).

    CAS  Article  Google Scholar 

  31. 31.

    Cano, A. et al. The transcription factor Snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nat. Cell Biol. 2, 76–83 (2000).

    CAS  Article  Google Scholar 

  32. 32.

    Ullmann, U. et al. Epithelial–mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions. Mol. Hum. Reprod. 13, 21–32 (2007).

    CAS  Article  Google Scholar 

  33. 33.

    Williams, M., Burdsal, C., Periasamy, A., Lewandoski, M. & Sutherland, A. Mouse primitive streak forms in situ by initiation of epithelial to mesenchymal transition without migration of a cell population. Dev. Dyn. 241, 270–283 (2012).

    Article  Google Scholar 

  34. 34.

    Etoc, F. et al. A balance between secreted inhibitors and edge sensing controls gastruloid self-organization. Dev. Cell 39, 302–315 (2016).

    CAS  Article  Google Scholar 

  35. 35.

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

  36. 36.

    Martyn, I., Brivanlou, A. H. & Siggia, E. D. A wave of WNT signaling balanced by secreted inhibitors controls primitive streak formation in micropattern colonies of human embryonic stem cells. Development 146, dev172791 (2019).

  37. 37.

    Sasai, Y., Eiraku, M. & Suga, H. In vitro organogenesis in three dimensions: self-organising stem cells. Development 139, 4111–4121 (2012).

    CAS  Article  Google Scholar 

  38. 38.

    Srinivas, S., Rodriguez, T., Clements, M., Smith, J. C. & Beddington, R. S. Active cell migration drives the unilateral movements of the anterior visceral endoderm. Development 131, 1157–1164 (2004).

    CAS  Article  Google Scholar 

  39. 39.

    Perea-Gomez, A. et al. Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks. Dev. Cell 3, 745–756 (2002).

    CAS  Article  Google Scholar 

  40. 40.

    Yoon, Y. et al. Extra-embryonic Wnt3 regulates the establishment of the primitive streak in mice. Dev. Biol. 403, 80–88 (2015).

    CAS  Article  Google Scholar 

  41. 41.

    Peng, G. et al. Spatial transcriptome for the molecular annotation of lineage fates and cell identity in mid-gastrula mouse embryo. Dev. Cell 36, 681–697 (2016).

    CAS  Article  Google Scholar 

  42. 42.

    Idkowiak, J., Weisheit, G., Plitzner, J. & Viebahn, C. Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo. Dev. Genes Evol. 214, 591–605 (2004).

    Article  Google Scholar 

  43. 43.

    Meinhardt, H. Models of biological pattern formation: from elementary steps to the organization of embryonic axes. Curr. Top. Dev. Biol. 81, 1–63 (2008).

    Article  Google Scholar 

  44. 44.

    Simunovic, M., Brivanlou, A. H. & Siggia, E. D. BMP4-induced symmetry breaking in a 3D model of the human epiblast. Protoc. Exch. https://doi.org/10.21203/rs.2.9730/v1 (2019).

Download references


M.S. is a Junior Fellow of the Simons Society of Fellows. We were supported by the NIH grant nos R01 HD080699 and R01 GM101653 (to A.H.B. and E.D.S), and NSF PHY grant no. 1502151 (to E.D.S). Imaging was performed at The Rockefeller University Bio-Imaging Resource Center. We thank S. Morgani (MSKCC) and the members of the Siggia and Brivanlou laboratories for their helpful discussions and for critically reading the manuscript.

Author information




M.S., A.H.B, and E.D.S. concieved the study. M.S. performed the majority of the experiments and concieved and performed the analysis. J.J.M. wrote the cell segmentation and data analysis codes and helped analyze the data. F.E. contributed with the RUES2 NOGGIN KO cell line. E.R. contributed with the RUES2-GLR cell line. F.E. and A.R. conducted the filter experiments for FACS sorting and RT-qPCR shown in Fig. 7a–c and Supplementary Fig. 7c. A.Y. contributed with the SMAD1 reporter line and helped carry out the live-imaging experiment in Fig. 6. G.C. contributed with human embryo data. D.S.Y conducted the filter experiments and RT-qPCR in Supplementary Fig. 7h. I.M. contributed with the RUES2-GLR DKK KO and the RUES2 CERBERUS 1 and LEFTY1 double KO cell lines. M.S., A.H.B, and E.D.S. wrote the manuscript. All authors contributed with the critical reading of the manuscript.

Corresponding authors

Correspondence to Ali H. Brivanlou or Eric D. Siggia.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Integrated supplementary information

Supplementary Figure 1 Supplementary analysis of an in vitro 3D model of a pre-gastrulation human epiblast.

(ab) Spontaneous differentiation of spherical hESC epithelia grown in pure Matrigel from single cells. (a) Shown are IF of pluripotency markers of a colony grown in Matrigel (Mgel) for three days and in Matrigel and LDN (Mgel/LDN) for five days. Plots quantify the pluripotency loss where each colony is binned based on the fraction of its cells expressing the pluripotency marker (n = 31 independent colonies for Mgel; n = 28 independent colonies for Mgel/LDN). The spontaneous differentiation of Matrigel-grown hESC colonies is BMP4-dependent, since colonies remain pluripotent in the presence of the BMP inhibitor LDN. (b) After five days in Matrigel, cells lose structural integrity and appear to invade into the surrounding gel (representative of 12 independent colonies). Shown are two different examples. Top right shows a detail in the dotted rectangle 15 μm displaced in z from the image on the left, where these nuclei are in focus. (c) Day-10 in vitro attached human embryo. Left: DAPI of an entire embryo; right: magnified epiblast (NANOG+ and OCT4+) and primitive endoderm (GATA6+). Shown is a different example from Fig. 1 and representative of 4 embryos. (d) Additional data on human epiblast model (spherical hESC epithelia grown in hydrogel/Matrigel mix). Pluripotency markers in the human epiblast model grown for 3 (left) or 4 days (right). Left example shows a z-slice and the entire z-stack in DAPI. Both differ from Fig. 1 and are based on 51 epiblast models from the main figure. (e) Surface markers z-stacks in a human epiblast model. Shown is the same example as in Fig. 1. All scale bars, 20 μm. (f) Schematics of the RUES2-GLR line, expressing mCitrine-SOX2 (mCit-SOX2), mCerulean-BRACHYURY (mCer-BRA), and tdTomato-SOX17 (tdTom-SOX17). (gh) Live-cell imaging of the human epiblast models made up of RUES2-GLR cells under pluripotency conditions, starting ~ 48 h after seeding. The quantification shown is for the single examples shown here. Imaging frequency: 1 h−1. Shown examples are different from the one in Fig. 1 and representative of 39 model epiblasts that remained SOX2+ out of 48 total imaged independent model epiblasts. See also Supplementary Video 1. All scale bars, 20 μm. Numerical source data are available in Supplementary Table 2.

Supplementary Figure 2 Patterning of a human epiblast model by high BMP4 concentration.

(a) Population fractions of colonies at various BMP4 concentrations from IF staining and from live-cell imaging data, presented separately. The statistics was deduced from seven independent experiments for 1 ng/ml (four IF stained, three imaged live), from two independent experiments for 5 ng/ml (one IF stained, one imaged live), and from three independent experiments for 0.1 ng/ml (one IF stained, two imaged live). The data for each BMP4 concentration from independently run experiments was pooled, IF and live imaging separately, and the n indicated in each pie chart represents the number of total individual 3D colonies. (b) BRA and SOX2 intensity change of the majority population under pluripotency conditions and with BMP4 for 2 days at different concentrations. From left to right: n = 38, n = 8, and n = 23 individual 3D colonies. Thick line is mean, grey line is s.e.m. (c) Live-cell imaging of a human epiblast model stimulated with 0.1 ng/ml BMP4 added at time zero, representative of the majority population from 13 imaged 3D colonies. See also Supplementary Video 2. (d) IF staining of epiblast models stimulated for 2 days with 0.1 ng/ml BMP4, showing epiblast markers (representative of the majority population of 82 3D colonies). (e) Live-cell imaging of a model human epiblast stimulated with 5 ng/ml BMP4 added at time zero, representative of majority population from 28 imaged 3D colonies. See also Supplementary Video 3. (f) Left: IF of representative colonies after high (5 or 10 ng/ml) BMP4 dose for two days, showing the expression of germ layer and extra-embryonic markers (top, representative of 20 3D colonies; bottom, 30 3D colonies). Right: population fractions of colonies 2 days after 5 ng/ml BMP4 in terms of differentiation markers (n = 30 individual 3D colonies).

Supplementary Figure 3 Breaking the AP symmetry: further examples and analyses.

(a) Symmetry breaking was observed in two tested chemically different hydrogels (PEG-based, n = 66 BRA+/SOX2+ individual 3D colonies; and Fmoc-based, representative of 21 BRA+/SOX2+ individual 3D colonies). Shown are examples from IF confocal slices and the single-cell IF quantification. Both examples are different from the ones in Fig. 3. (b) 3D single-cell segmentation of the example in Fig. 3a where the 3D image is rendered by drawing a sphere centred at the centroid of each segmented cell. Spheres are colour-coded based on their BRA or SOX2 IF intensity. (c) %BRA+ cells as a function of colony radius (n = 87 BRA+/SOX2+ individual 3D colonies, the same population as in Fig. 3). (d) Histogram of OCT4 expression levels for three different cell populations in symmetry broken colonies (n = 87, the same population as in Fig. 3). (e) Symmetry-broken colonies stimulated with 1 ng/ml BMP4 do not show appreciable GATA3 expression (7 individual 3D colonies with the BRA/SOX2/GATA3 combination and 8 with the BRA/GATA3 combination using an alternative (rabbit) GATA3 antibody). (f) Additional examples of symmetry-broken 3D colonies, with various σ values indicated below in the σ distribution. The colonies have been rotated so that the dipole moment points up relative to a common origin for all the colonies. All dipole moments were divided by \(R_{{\mathrm{gyr}}}^3\) to compare different size colonies and, when rendering the images, multiplied by 50 for clarity. (g) IF staining, dipole quantification, and population analysis of BMP4-differentiated 3D colonies composed of an alternative hESC line, RUES1, with 1 ng/ml BMP4 for 2 d. 28 individual 3D colonies were imaged regardless of BRA/SOX2 expression and used to generate the population fraction pie chart. Dipole quantification was done on the BRA+/SOX2+ subpopulation as with all data with RUES2, in this case on n = 15 individual colonies.

Supplementary Figure 4 Molecular signatures of symmetry breaking and EMT.

(a-b) Further examples of SNAIL expression, co-stained with SOX2 and NANOG or N-CAD (11 individual 3D colonies with the SNAIL/SOX2 combination, of which 9 were also co-stained for NANOG). (c) Further examples of COLL IV downregulation. Left: thinning of COLL IV, co-expressed with SAIL and N-CAD; right: partial disappearance of COLL IV in the SNAIL+ and N-CAD+ region. The example on the right has prominent EMT marked by cells leaving the tissue (5 individual 3D colonies with the SNAIL/COLL IV/N-CAD combination). (d) Further example of E-CAD downregulation and N-CAD expression in the BRA-rich region (3 individual 3D colonies with the BRA/E-CAD/N-CAD combination). All scale bars, 20 μm.

Supplementary Figure 5 Dynamics of patterning at 1 ng/ml BMP4.

(ab) Symmetry-breaking and expression quantification for the pure SOX2+ (n = 23 individual 3D colonies imaged live) and pure BRA+ (n = 13 individual 3D colonies imaged live) population. Cell segmentation and analysis done on two out of three independent live imaging experiments. (a) Volume-uncorrected AP dipole for the SOX2+/BRA− and the BRA+/SOX2− population, compared to the BRA+/SOX2+ population. Only the BRA+/SOX2+ population shows dipole activation. (b) Protein expression analysis, measured as BRA+ or SOX2+ voxel number, N+, normalized by the maximum number of expressing voxels of individual colony, N+max. The SOX2+ voxel number initially increases due to growth of the colony and finally declines to background. Red line copied from data in Fig. 5b for comparison, thick lines are mean, shadows are s.e.m. (c) Full trajectory of live imaging of symmetry breaking of the example shown in Fig. 5a.

Supplementary Figure 6 Dynamics of symmetry breaking and SOX17 activation.

(a) Live-cell imaging of 3D model epiblasts composed of the RUES2-GLR line in all three channels, showing the activation of SOX17 (observed in 10 out of 11 BRA+/SOX2+ individual 3D colonies imaged live, 19 total imaged colonies in all three channels). Showing a different example from Fig. 5. Arrows point to SOX17+ cells. Bottom row is an overlay of all three markers above. Confocal slices among different channels are different, but same for each channel. (b) IF staining for SOX17 48h after 1 ng/ml BMP4 treatment, showing two different examples from the partial BRA+ population. There is sporadic SOX17 activation seen in examples with prominent EMT (SOX17 observed in 6 out of 8 of imaged 3D colonies with the BRA/SOX17 combination). Shown either confocal slice, partial, or full maximum z projections for clarity, so to show all SOX17+ cells. All scale bars, 20 μm.

Supplementary Figure 7 Further evidence for the mechanism of symmetry breaking in the model epiblast.

(a) Z-stack of epiblast model composed of the SMAD1 reporter line 16 h after stimulation with 10 ng/ml BMP4 (from the same sample as in Fig. 6 with 16 individual 3D colonies). (b) IF staining 2 days after 1 ng/ml BMP4, showing examples with partial BRA expression and a heterogeneous pSMAD1 expression, with no apparent correlation between pSMAD1 and BRA expression. Plot shows no strong correlation between pSMAD1 and BRA IF intensity levels in partial BRA+ examples (n = 6 imaged 3D colonies showing partial BRA expression). (c) FACS sorting dot plot of dissociated RUES2-GLR epithelium grown on filters under pluripotency conditions (left) and after 24 h of 10 ng/ml BMP4 (centre). Right: gene expression analysis from Fig. 7b, c of sorted cells compared (in log2 fold change) to untreated cells. The measurement was done by pooling n = 10 individual samples (separate filters) and compared to n = 5 untreated samples, thus the variance was not measured. (d) Monitoring pluripotency maintenance of epiblast models made from the RUES2-GLR DKK1 KO line. Pie chart shows end-point patterning statistics. Snapshots show an example of live-cell imaging of the majority population under pluripotency conditions (n = 12 3D colonies for the majority population). Scale bar, 20 μm. (e) Patterning statistics and examples of WT RUES2 epiblast models stimulated with 2 μM IWP2 or 2 µM IWR1-endo along with 1 ng/ml BMP4 for 2 days. For each case, a representative example of a colony from the majority population is shown (that is, no differentiation). In both cases, n = 39 3D colonies were imaged. Scale bar, 20 μm. (f) Patterning of filter cultures, comparing RUES2 WT (3 independent experiments) and RUES2 DKK1 KO (2 independent experiments), stimulated for 24 h with 10 ng/ml BMP4 from the bottom compartment. Scale bar, 200 μm. (g) Filter colony stimulated with 1 ng/ml from the bottom compartment for 24 h (2 independent experiments). (h) Gene expression analysis from bulk cells at 4 h and 24 h, of filters stimulated with 10 ng/ml from the bottom compartment (n = 3 independent experiments, box centred at mean, error is s.e.m., each dot represents a mean of three technical replicas). (i) Differentiation of RUES2 NOGGIN KO model epiblasts with 1 ng/ml BMP4 for 2 days. Left: end-point statistics; right: IF stain of an example 3D colony from the majority population (total n = 19 imaged 3D colonies). Scale bar, 20 μm. (j) Checking for NODAL influence on symmetry breaking. Shown are stimulation with 1 ng/ml BMP4 for 2 days of RUES2 LEFTY1 & CERBERUS 1 (CER1) KO epiblast models (top) and the WT with the NODAL inhibitor SB (bottom). Pie charts show end-point statistics (n represents total imaged 3D colonies) and AP dipole quantification (the measured and the random population are statistically different, P = 0.008 calculated with the two-sided Mann-Whitney U test; n = 6 and n = 20 segmented BRA+/SOX2+ colonies for the LEFTY1/CER1 KO and WT with SB cases, respectively); right: IF stains of symmetry-broken 3D colonies. All scale bars, 20 μm.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and Supplementary Table titles/legends

Reporting Summary

Supplementary Table 1

A list of primary and secondary antibodies used.

Supplementary Table 2

Numerical source data for figures.

Supplementary Video 1

Growth of RUES2-GLR model epiblasts under pluripotency conditions.

Supplementary Video 2

Live-cell imaging of epiblast models under low BMP4 concentration (0.1 ng/ml).

Supplementary Video 3

Live-cell imaging of epiblast models under high BMP4 concentration (5 ng/ml).

Supplementary Video 4

Live-cell imaging of epiblast models at 1 ng/ml BMP4.

Supplementary Video 5

Live-cell imaging of epiblast models at 1 ng/ml BMP4.

Supplementary Video 6

Live-cell imaging of epiblast models at 1 ng/ml BMP4 showing SOX17 activation following symmetry breaking.

Supplementary Video 7

Live-cell imaging of epiblast models composed of RUES2 with fluorescently tagged SMAD1.

Supplementary Video 8

Live-cell imaging of epiblast models stimulated with 1 ng/ml BMP4, comparing the WT (RUES2-GLR) with RUES2-GLR DKK1 KO lines.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Simunovic, M., Metzger, J.J., Etoc, F. et al. A 3D model of a human epiblast reveals BMP4-driven symmetry breaking. Nat Cell Biol 21, 900–910 (2019). https://doi.org/10.1038/s41556-019-0349-7

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing