Abstract
Aggregates of stem cells can break symmetry and self-organize into embryo-like structures with complex morphologies and gene expression patterns. Mechanisms including reaction-diffusion Turing patterns and cell sorting have been proposed to explain symmetry breaking but distinguishing between these candidate mechanisms of self-organization requires identifying which early asymmetries evolve into subsequent tissue patterns and cell fates. Here we use synthetic âsignal-recordingâ gene circuits to trace the evolution of signalling patterns in gastruloids, three-dimensional stem cell aggregates that form an anteriorâposterior axis and structures resembling the mammalian primitive streak and tailbud. We find that cell sorting rearranges patchy domains of Wnt activity into a single pole that defines the gastruloid anteriorâposterior axis. We also trace the emergence of Wnt domains to earlier heterogeneity in Nodal activity even before Wnt activity is detectable. Our study defines a mechanism through which aggregates of stem cells can form a patterning axis even in the absence of external spatial cues.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 /Â 30Â days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The scRNAseq data generated in this study are available through the NCBI Gene Expression Omnibus (accession number GSE274389). Plasmids used to construct signalling-reporter and signalling-recorder cell lines are available from Addgene (plasmids 225522â225531). Source data are provided with this paper. All other materials (for example, cell lines) are available on request.
Code availability
The scRNAseq data were processed and analysed using the publicly available Single-Cell Analysis in Python (scanpy) software package version 1.10 and gastruloid images were processed using custom MATLAB code (MATLAB 2024a). All code is available on the authorsâ GitHub repository (https://github.com/toettchlab/McNamara2024).
References
Bardot, E. S. & Hadjantonakis, A.-K. Mouse gastrulation: coordination of tissue patterning, specification and diversification of cell fate. Mech. Dev. 163, 103617 (2020).
Gregor, T., Wieschaus, E. F., McGregor, A. P., Bialek, W. & Tank, D. W. Stability and nuclear dynamics of the bicoid morphogen gradient. Cell 130, 141â152 (2007).
Lord, N. D., Carte, A. N., Abitua, P. B. & Schier, A. F. The pattern of nodal morphogen signaling is shaped by co-receptor expression. eLife 10, e54894 (2021).
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, 3894â3906 (2017).
Beccari, L. et al. Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids. Nature 562, 272â276 (2018).
Veenvliet, J. V. et al. Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites. Science 370, eaba4937 (2020).
Miao, Y. et al. Reconstruction and deconstruction of human somitogenesis in vitro. Nature 614, 500â508 (2023).
Ishihara, K. & Tanaka, E. M. Spontaneous symmetry breaking and pattern formation of organoids. Curr. Opin. Syst. Biol. 11, 123â128 (2018).
van den Brink, S. C. et al. Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. Development 141, 4231â4242 (2014).
Scoones, J. C., Banerjee, D. S. & Banerjee, S. Size-regulated symmetry breaking in reaction-diffusion models of developmental transitions. Cells 9, 1646 (2020).
Sozen, B., Cornwall-Scoones, J. & Zernicka-Goetz, M. The dynamics of morphogenesis in stem cell-based embryology: novel insights for symmetry breaking. Dev. Biol. 474, 82â90 (2021).
Simunovic, M. et al. A 3D model of a human epiblast reveals BMP4-driven symmetry breaking. Nat. Cell Biol. 21, 900â910 (2019).
Sick, S., Reinker, S., Timmer, J. & Schlake, T. WNT and DKK determine hair follicle spacing through a reaction-diffusion mechanism. Science 314, 1447â1450 (2006).
Raspopovic, J., Marcon, L., Russo, L. & Sharpe, J. Digit patterning is controlled by a BmpâSox9âWnt Turing network modulated by morphogen gradients. Science 345, 566â570 (2014).
Glover, J. D. et al. The developmental basis of fingerprint pattern formation and variation. Cell 186, 940â956 (2023).
Hashmi, A. et al. Cell-state transitions and collective cell movement generate an endoderm-like region in gastruloids. eLife 11, e59371 (2022).
Srivastava, V. et al. Configurational entropy is an intrinsic driver of tissue structural heterogeneity. Preprint at bioRxiv https://doi.org/10.1101/2023.07.01.546933 (2023).
Shyer, A. E. et al. Emergent cellular self-organization and mechanosensation initiate follicle pattern in the avian skin. Science 357, 811â815 (2017).
Palmquist, K. H. et al. Reciprocal cellâECM dynamics generate supracellular fluidity underlying spontaneous follicle patterning. Cell 185, 1960â1973 (2022).
Kondo, S., Watanabe, M. & Miyazawa, S. Studies of Turing pattern formation in zebrafish skin. Philos. Trans. R. Soc. A 379, 20200274 (2021).
Korinek, V. et al. Constitutive transcriptional activation by a β-cateninâTcf complex in APCâ/â colon carcinoma. Science 275, 1784â1787 (1997).
Suppinger, S. et al. Multimodal characterization of murine gastruloid development. Cell Stem Cell https://doi.org/10.1016/j.stem.2023.04.018 (2023).
Serup, P. et al. Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice. Dis. Model. Mech. 5, 956â966 (2012).
Morgani, S. M., Metzger, J. J., Nichols, J., Siggia, E. D. & Hadjantonakis, A.-K. Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning. eLife 7, e32839 (2018).
Grosswendt, S. et al. Epigenetic regulator function through mouse gastrulation. Nature 584, 102â108 (2020).
van den Brink, S. C. et al. Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids. Nature 582, 405â409 (2020).
Merle, M., Friedman, L., Chureau, C., Shoushtarizadeh, A. & Gregor, T. Precise and scalable self-organization in mammalian pseudo-embryos. Nat. Struct. Mol. Biol. 31, 896â902 (2024).
Underhill, E. J. & Toettcher, J. E. Control of gastruloid patterning and morphogenesis by the Erk and Akt signaling pathways. Development 150, dev201663 (2023).
Anand, G. M. et al. Controlling organoid symmetry breaking uncovers an excitable system underlying human axial elongation. Cell 186, 497â512 (2023).
Chal, J. & Pourquié, O. Making muscle: skeletal myogenesis in vivo and in vitro. Development 144, 2104â2122 (2017).
Wang, X. et al. The development of highly potent inhibitors for porcupine. J. Med. Chem. 56, 2700â2704 (2013).
Steinberg, M. S. On the mechanism of tissue reconstruction by dissociated cells. I. Population kinetics, differential adhesiveness, and the absence of directed migration. Proc. Natl Acad. Sci. USA 48, 1577â1582 (1962).
Toda, S., Blauch, L. R., Tang, S. K. Y., Morsut, L. & Lim, W. A. Programming self-organizing multicellular structures with synthetic cellâcell signaling. Science 361, 156â162 (2018).
Stevens, A. J. et al. Programming multicellular assembly with synthetic cell adhesion molecules. Nature 614, 144â152 (2023).
Tsai, T. Y.-C. et al. An adhesion code ensures robust pattern formation during tissue morphogenesis. Science 370, 113â116 (2020).
Redmer, T. et al. Eâcadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming. EMBO Rep. 12, 720â726 (2011).
Soncin, F. et al. E-cadherin acts as a regulator of transcripts associated with a wide range of cellular processes in mouse embryonic stem cells. PLoS ONE 6, e21463 (2011).
Xu, P.-F., Houssin, N., Ferri-Lagneau, K. F., Thisse, B. & Thisse, C. Construction of a vertebrate embryo from two opposing morphogen gradients. Science 344, 87â89 (2014).
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, 4989â4998 (2019).
Camacho-Aguilar, E., Yoon, S., Ortiz-Salazar, M. A. & Warmflash, A. Combinatorial interpretation of BMP and WNT controls the decision between primitive streak and extraembryonic fates. Cell Syst. 15, 445â461 (2024).
Mayran, A. et al. Cadherins modulate the self-organizing potential of gastruloids. Preprint at bioRxiv https://doi.org/10.1101/2023.11.22.568291 (2023).
Dingare, C., Cao, D., Yang, J. J., Sozen, B. & Steventon, B. Mannose controls mesoderm specification and symmetry breaking in mouse gastruloids. Dev. Cell 59, 1523â1537 (2024).
Arias, A. M., Marikawa, Y. & Moris, N. Gastruloids: pluripotent stem cell models of mammalian gastrulation and embryo engineering. Dev. Biol. 488, 35â46 (2022).
Pinheiro, D., Kardos, R., Hannezo, Ã. & Heisenberg, C.-P. Morphogen gradient orchestrates pattern-preserving tissue morphogenesis via motility-driven unjamming. Nat. Phys. 18, 1482â1493 (2022).
Schüle, K. M. et al. Eomes restricts Brachyury functions at the onset of mouse gastrulation. Dev. Cell 58, 1627â1642 (2023).
Anlas, K. & Trivedi, V. Studying evolution of the primary body axis in vivo and in vitro. eLife 10, e69066 (2021).
Petersen, C. P. & Reddien, P. W. Wnt signaling and the polarity of the primary body axis. Cell 139, 1056â1068 (2009).
Petersen, C. P. & Reddien, P. W. Polarized notum activation at wounds inhibits Wnt function to promote planarian head regeneration. Science 332, 852â855 (2011).
Feinberg, S., Roure, A., Piron, J. & Darras, S. Anteroâposterior ectoderm patterning by canonical Wnt signaling during ascidian development. PLoS Genet. 15, e1008054 (2019).
Wang, R., Steele, R. E. & Collins, E.-M. S. Wnt signaling determines body axis polarity in regenerating Hydra tissue fragments. Dev. Biol. 467, 88â94 (2020).
Lovas, J. R. & Yuste, R. Dissociation and reaggregation of Hydra vulgaris for studies of self-organization. STAR Protoc. 3, 101504 (2022).
Ferenc, J. et al. Mechanical oscillations orchestrate axial patterning through Wnt activation in Hydra. Sci. Adv. 7, eabj6897 (2021).
Amadei, G. et al. Embryo model completes gastrulation to neurulation and organogenesis. Nature 610, 143â153 (2022).
Tarazi, S. et al. Post-gastrulation synthetic embryos generated ex utero from mouse naive ESCs. Cell 185, 3290â3306 (2022).
Bhattarai-Kline, S. et al. Recording gene expression order in DNA by CRISPR addition of retron barcodes. Nature 608, 217â225 (2022).
Choi, J. et al. A time-resolved, multi-symbol molecular recorder via sequential genome editing. Nature 608, 98â107 (2022).
Thielicke, W. & Sonntag, R. Particle image velocimetry for MATLAB: accuracy and enhanced algorithms in PIVlab. J. Open Res. Softw. 9, 12 (2021).
Acknowledgements
The authors thank all members of the Toettcher laboratory, particularly E. Underhill, for their insightful comments and suggestions. We also thank A. Martinez-Arias for helpful discussions and feedback on the manuscript, K. Zaret for sharing reporter plasmid constructs, A. Lin for advice on single-cell sequencing and the Lewis Sigler Institute Genomics Core Facility team (W. Wang, J. Miller and J. A. Volmar) for technical support. Figure illustrations in Figures 1a, 2a, 4a, 6a,d,f,i and 7a,i were created in part using BioRender. This work was supported by the Lewis Sigler Scholars programme and the NSF Center for the Physics of Biological Function (grant number PHY1734030) to H.M.M., NIH (grant numbers T32GM007388 and F31HD113443) to S.C.S., NIH (grant number R35GM138167) to B.A., and NSF RECODE (grant number 2134935), NIH (grant numbers U01DK127429 and R01GM144362) and a Vallee Scholars award to J.E.T.
Author information
Authors and Affiliations
Contributions
Conceptualization: H.M.M., M.M.C. and J.E.T. Methodology: H.M.M., S.C.S., M.M.C. and J.E.T. Investigation: H.M.M. Funding: H.M.M. and J.E.T. Writing and editing: H.M.M. and J.E.T. Supervision: J.E.T. and B.A.
Corresponding authors
Ethics declarations
Competing interests
J.E.T. is a scientific advisor for Prolific Machines and Nereid Therapeutics. B.A. is an advisory board member with options for Arbor Biotechnologies and Tessera Therapeutics, and holds equity in Celsius Therapeutics. H.M.M. is a cofounder and scientific advisor for C16 Biosciences. The remaining authors declare no conflicts of interest.
Peer review
Peer review information
Nature Cell Biology thanks Takashi Hiiragi, Prisca Liberali and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisherâs note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Benchmarking the Wnt reporter during gastruloid development.
(A) Characterization of off-kinetics of destabilized iRFP reporter of Wnt activity (PTCF/LEF-iRFP-PEST). Shaded region indicates mean ± standard deviation. (B) Experimental protocol for gastruloid growth with âLIF onlyâ seed culture media. (C) Patterns of Wnt activity in gastruloids grown according to protocol in B. Wnt activity is heterogeneous both immediately before (tâ=â48âhaa) and immediately after (tâ=â72 haa) CHIR treatment. Scale bar = 200âµm. (D) Representative images of Wnt activity patterns at different time points during polarization. Maximum-intensity projections are shown. Scale bar = 200 μm. (E) Representative data illustrating how single cells were gated for analysis from flow cytometry data (see Methods). (F) Definition of threshold to define Wnt-active versus Wnt-inactive cell populations (Fig. 1f) based on separation of activity distributions before (tâ=â48 haa) and after (tâ=â72 haa) CHIR stimulation. (G) Schematic of quantification of ÎCOM polarization metric (Fig. 1g).
Extended Data Fig. 2 Characterizing signal-recorder gene circuits.
(A) Recording efficiency in Wnt-Recorder cells depends on doxycycline concentration (2iâ+âLIF medium, 24âh recording window). (B) Recording efficiency in Wnt-Recorder cells depends on the concentration of recombinant Wnt3a ligand (200ângâmlâ1 doxycycline, 24âh recording window). Shaded regions in (A-B) indicate meanâ±âstandard deviation (3 biological replicates per condition). (C) Flow cytometry histograms corresponding to recorder-ligand crosstalk measurement in Fig. 2e. Recording windows were 6âh for Wnt Recorder and Nodal Recorder, and 3âh for BMP recorder. All ligand concentrations (including dox) were 200ângâmlâ1. (D) BMP-Recorder labelling is sensitive to BMP inhibition with LDN-193189.
Extended Data Fig. 3 Measuring positional information in signal-recorded gastruloids.
(A) Illustration of image processing pipeline. Maximum-intensity projections of gastruloids are segmented, skeletonized, and then binned to assign an AâP coordinate to each pixel (Methods). (B) Left: Example quantification of average AâP profiles of both GFP and DsRed expression within a single gastruloid (tdoxâ=â114âh, tfâ=â144âh). Pixel intensities were separately normalized relative to maximum image intensities in each channel. Right: relative fraction of GFP labelling across the AâP axis within the same gastruloid (Methods). (C) Overlay of relative fraction of GFP labelling in individual gastruloids for the same recording condition, along with the mean profile for this condition. (D) Simultaneous measurement of the final Wnt activity pattern and recorded Wnt activity signal within the same gastruloid. Maximum-intensity projection is shown. Scale bar = 200âμm. (E) Kymograph of the data presented in Fig. 3b to visualize different Wnt dynamics corresponding to different spatial positions. Following the emergence of an anterior domain at tdoxâ=â96âh, Wnt signalling becomes progressively more restricted to the posterior domain. (F) Integrated Wnt signalling activity between tâ=â96 and tâ=â134âh shows a linear âtemporal gradientâ associated with AâP fate. Shaded regions indicate meanâ±âstandard deviation as a function of AâP position.
Extended Data Fig. 4 Single-cell sequencing of 120âhaa Wnt-recorded gastruloids.
(A) Comparison of gene expression between Leiden clusters identified in gastruloids at tseqâ=â120âh and annotated cell types from a reference atlas gene expression during mouse gastrulation2. Size of dots indicates the number of shared marker genes; colour indicates the binomial likelihood of having at least this many marker genes if compared to a random choice of marker genes (Methods). (B) Single-cell expression levels of reference genes associated with gastrulation and axial morphogenesis. (C) Illustration of reference gene organization across the anteriorâposterior axis during axial elongation and somitogenesis. The organization of reference gene expression (Extended Data Fig. 4b) aligns with inferred histories of Wnt activity (Fig. 3c). (D) Final Wnt activity distribution across cells as measured by PTCF/LEF-rtTA expression. (E) Top: individual cells in dataset separated according to Wnt-recording condition. Bottom: embedding density of cells according to Wnt-recording condition. (F) Relative contributions of GFP-positive and GFP-negative cells to different Leiden clusters, normalized according to total cells recovered in both conditions (Methods).
Extended Data Fig. 5 Exploring Gastruloid reaggregation.
(A) Computer simulation of signal-recording and dissociation/reaggregation experiment in the case of a reaction-diffusion Turing patterning circuit without cell rearrangements. Cell states are labelled according to signalling activity during an intermediate state, then cells locations are randomly shuffled (simulating dissociation), and continued to evolve according to the same reaction-diffusion equations. (B) Quantification of final AâP profiles of recording simulations under reaction-diffusion assumptions, averaged over nâ=â100 independent trials. Because a ânewâ pole forms following reaggregation, recorded cells remain evenly distributed across the AâP axis. (C) Representative imaging of cell-sorting polarization following bulk reaggregation. nâ=â20 of 48 samples recovered show a single pole of Wnt-recorded cells (left). nâ=â28 of 48 show at least two poles of Wnt activity (right). Smaller âsatelliteâ clusters were not quantified. Maximum-intensity projections are shown. Scale barâ=â100âμm. (D) Quantification of AâP axis length for reaggregated samples by flow cytometric sorting vs âbulkâ reaggregation by pipetting cells into wells. Error bars indicate meanâ±âstandard deviation. (E) Analysis of differential motility in polarized gastruloids. Left, Middle images: beginning and endpoint images of interval used to calculate motility. Right: average motility map, calculated using particle image velocimetry (PIV) on the nuclear iRFP channel. Motility was calculated for each frame, and then averaged to generate average motility maps (Methods). (F) AâP motility profiles for each of three biological replicates, calculated by averaging motility maps along the AâP axis. (G) Overall average AâP motility profile for all samples analysed. Shaded area represents meanâ±âstandard deviation at each AâP position.
Extended Data Fig. 6 Gene expression patterns in 96âhaa Wnt-reporter gastruloids.
(A) Comparison of gene expression between Leiden clusters identified in gastruloids at tseqâ=â96âh and annotated cell types from a reference atlas gene expression during mouse gastrulation (Methods). Size of dots indicates the number of shared marker genes; colour indicates the binomial likelihood of having at least this many marker genes if compared to a random choice of marker genes (Methods). (B) Expression of signalling-associated genes within the scRNAseq dataset at tseqâ=â96âh. (C) Expression of retinoic acid biosynthesis enzyme Aldh1a2 in the Wnt-inactive cell population. Maximum-intensity projections show, scale bar = 50âµm. (D-E) Positive feedback is required to maintain Wnt activity following cessation of CHIR treatment at 72âhaa. (D) Flow cytometry measurements of single cell Wnt activity levels in dissociated gastruloids at 96âhaa. Treatment with the inhibitor of Wnt secretion IWP-2 from 72âhaa-onwards converts majority of cells to the Wnt-off population. (E) Imaging of Wnt activity patterns of intact gastruloids at 96 haa, with and without IWP-2 treatment. Maximum-intensity projections are shown. Scale bar = 50âµm.
Extended Data Fig. 7 Role of cell adhesion in gastruloid self-organization.
(A) Single-cell differential expression of additional genes associated with cellâcell adhesion at 96âhaa. (B) Doxycycline-dependent expression of Cdh1 within the iCdh1 cell line. Maximum-intensity projections shown, scale bar = 10âµm. (C) Doxycycline-dependent expression of Pcdh19 with the iPcdh19 cell line. Maximum-intensity projections shown, scale bar = 10âµm. Clones were chosen for similar expression levels upon induction. (D-E) Average radial concentrations of cell lines in mosaic spheroids with and without doxycycline treatment (related to Fig. 6h). Nâ=â84 spheroids measured (Supplementary Table 3). Shaded regions indicate meanâ±âstandard deviation. (F) Representative images of iCdh1/iPcdh19 mosaic spheroids. Maximum-intensity projections shown, scale bar = 100âµm. (G) Histogram of polarization alignment in fused gastruloids generated by fusing two wild-type spheroids, with and without doxycycline. (H) Quantification of average polarization alignment of fused spheroids in different conditions (related to Fig. 6l, Extended Data Fig. 7g). Nâ=â58 gastruloids total (Supplementary Table 3). Conditions compared via two-sided t-test. P-values: pâ=â0.54, pâ=â0.00012. The asterisks (***) indicate pâ<â0.001.
Extended Data Fig. 8 Recording Nodal/BMP signalling during gastruloid self-organization.
(A) Measurement of Nodal activity (PAR8-mCherry-PEST) at different time points during gastruloid developmentâ=â. Maximum-intensity projections are shown, scale bar = 50âµm. (B) Measurement BMP activity (PIBRE4-mCherry-PEST) at different time points during gastruloid development. Maximum-intensity projections are shown, scale bar = 50âµm. (C) Nodal activity levels in 48âhaa gastruloids as a function of initial aggregate size. Nodal activity at 48âhaa requires aggregation, and monotonically increases with aggregate size. Adherent cells were measured after 48âhaa in equivalent media (N2B27) for comparison. (D) Recordings of early Nodal activity traced to tFâ=â120âhaa. (E) Recordings of early BMP activity traced to tFâ=â120âhaa. Shaded regions for C-D indicate standard deviation as a function of AâP position. Nâ=â116 gastruloids measured total (Supplementary Table 3). (F) Representative image of spatial distribution of cell fates with recorded Nodal activity from 69-72âhaa, imaged at 120âhaa. Top: medial slice. Bottom: Maximum-intensity projection. Scale bar = 200âµm. (G) Representative images of cell fates with recorded BMP activity from 69-72âhaa (top) and from 81-84âhaa (bottom). Scale barâ=â200âµm. Maximum-intensity projections are shown for both recording time points. (H) Simultaneous recording of early Nodal activity (recorded from 45-48âhaa) and reporting of later instantaneous Wnt activity (imaged at 106âhaa) in the same gastruloid using an engineered clonal cell line. Maximum-intensity projections of three separate representative gastruloids are shown. Scale bar = 100âµm. (I) Spatial distribution of early Nodal activity (recorded from 45-48âhaa) traced to tfâ=â120 haa, plotted alongside final Wnt activity. Normalized profiles are shown for comparison. Shaded region represents mean ± standard error of the mean for Nodal recording, and mean ± standard deviation for Wnt reporting. Nodal recording data from (H), plotted without normalization. Recording density varies approximately twofold along the AâP axis. Nâ=â15 gastruloids measured.
Extended Data Fig. 9 Perturbing Nodal/BMP signalling during gastruloid self-organization.
(A) Flow cytometry measurements of single-cell Wnt activity levels with and without Activin A pretreatment, measured at tâ=â72âh. (B) Flow cytometry measurements of single-cell Wnt activity levels with and without BMP4 pretreatment, measured at tâ=â72âh (left) and tâ=â96âh (right). (C) Dynamics of Nodal activity during gastruloid development, measured by flow cytometry of pooled and dissociated gastruloids. Substantial Nodal activity is detectable from 36-60, preceding CHIR stimulation at 48 and subsequently decaying to negligible levels by 72âh. (D) Nodal signalling levels in adherent cells treated for 48âh with different concentrations of recombinant Activin A (in N2B27). Dotted line is the mean gastruloid Nodal activity level at 48âhaa under standard conditions (that is, initial aggregate of nâ=â200âcells) plotted for comparison. (E) Effect of a transient ActA treatment (100ângâmlâ1, applied from 45-48âhaa) on Wnt activity levels at 96. Transient ActA treatment continues to cause an enrichment in Wnt-off cells at 96âhaa, even when duration of treatment is reduced. (F) Co-treatment of adherent Wnt reporter cells with BMP4 affects responses to Wnt3a treatment. Whereas moderate BMP4 (25ângâmlâ1) treatment facilitated the Wnt response (middle), higher BMP4 (125ângâmlâ1) treatment suppressed the Wnt response.
Extended Data Fig. 10 Phenotypic consequences of Nodal perturbation.
A-B). Quantification of anteroposterior morphology. (A) Gastruloid morphologies were segmented, binned according to nearest AâP axis coordinate (Methods, Extended Data Fig. 3), and then divided into anterior and posterior compartments based on the midline of the AâP axis (that is, without reference to Wnt activity). (B) Comparison of difference in area between anterior and posterior compartments under different Activin treatment conditions (that is, for samples analysed in Fig. 7g, h). 100ângâmlâ1 Activin A treatment causes a morphological enlargement of the anterior compartment. Nâ=â42 gastruloids measured (Supplementary Table 3). Statistical comparisons made via two-sided t-test; two asterisks (**) indicate pââ¤â0.01. P-values were: control and 10ângâmlâ1 Activin, pâ=â0.84; control and 100ângâmlâ1 Activin, pâ=â0.010; 10ângâmlâ1 Activin and 100ângâmlâ1 Activin, pâ=â0.0091. (C) Recording in the Nodal Recording cells is abolished with treatment with the Nodal inhibitor SB-431542. Activity was recorded for 3âh in 100ângâmlâ1 doxycycline. (D) Endogenous early Nodal activity in gastruloids is abolished by Nodal inhibition from 24-48âhaa. (Left) Nodal activity was recorded from 45-48âhaa, with 100ângâmlâ1 doxycycline, and measured at 68âhaa. (Right) Early Nodal inhibition (from 24-48âhaa) also abolished the formation of Wnt activity patches during symmetry breaking at 96 haa.
Supplementary information
Supplementary Tables 1â3
Supplementary Table 1. Plasmids used in this study. Supplementary Table 2. Cell lines used in this study. Supplementary Table 3. Number of gastruloids analysed for each experiment.
Supplementary Video 1
Live imaging of cellular rearrangements during formation and coalescence of Wnt-correlated domains in one gastruloid, separated by fluorescent channel. Scale bar, 100âµm.
Supplementary Video 2
Live imaging of cellular rearrangements during formation and coalescence of Wnt-correlated domains in multiple gastruloids. Arrows indicate emergence of a Wnt-excluded anterior domain. Scale bar, 100âµm.
Supplementary Video 3
Live imaging of cellular rearrangements during formation and coalescence of Wnt-correlated domains in multiple gastruloids, without matrigel embedding. Scale bar, 100âµm.
Supplementary Video 4
Live imaging of cell motility (imaged with an H2BâiRFP nuclear marker) following gastruloid polarization. Scale bar, 100âµm.
Source data
Source Data
Numerical source data for Figs. 1â7 and Extended Data Figs. 1â10.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
McNamara, H.M., Solley, S.C., Adamson, B. et al. Recording morphogen signals reveals mechanisms underlying gastruloid symmetry breaking. Nat Cell Biol (2024). https://doi.org/10.1038/s41556-024-01521-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41556-024-01521-9