Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration

Abstract

Intestinal organoids capture essential features of the intestinal epithelium such as crypt folding, cellular compartmentalization and collective movements. Each of these processes and their coordination require patterned forces that are at present unknown. Here we map three-dimensional cellular forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the extracellular matrix and folds through apical constriction, whereas the transit amplifying zone pulls the extracellular matrix and elongates through basal constriction. The size of the stem cell compartment depends on the extracellular-matrix stiffness and endogenous cellular forces. Computational modelling reveals that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. Finally, cells are pulled out of the crypt along a gradient of increasing tension. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal epithelium.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Tractions exerted by intestinal organoids define mechanical compartments.
Fig. 2: A stiffness-independent normal traction folds the crypt.
Fig. 3: The size of the stem cell compartment decreases with substrate rigidity.
Fig. 4: The crypt folds under tension generated by myosin II.
Fig. 5: Crypts fold through apical constriction.
Fig. 6: Co-evolution of cell fate and tissue mechanics during de novo crypt formation.
Fig. 7: Cell morphology and cytoskeletal organization of the intestinal epithelium in vivo.
Fig. 8: Cells are dragged out of the crypt towards the villus-like domain.

Data availability

The data that support the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

MATLAB analysis procedures and the code implementing the 3D vertex model calculations are available from the corresponding authors on reasonable request.

References

  1. Chacón-Martínez, C. A., Koester, J. & Wickström, S. A. Signaling in the stem cell niche: regulating cell fate, function and plasticity. Development 145, dev165399 (2018).

    PubMed  Article  CAS  Google Scholar 

  2. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).

    CAS  PubMed  Article  Google Scholar 

  3. Ritsma, L. et al. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging. Nature 507, 362–365 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    CAS  PubMed  Article  Google Scholar 

  5. Shyer, A. E. et al. Villification: how the gut gets its villi. Science 342, 212–218 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. Hughes, A. J. et al. Engineered tissue folding by mechanical compaction of the mesenchyme. Dev. Cell 44, 165–178 (2018).

    CAS  PubMed  Article  Google Scholar 

  7. Walton, K. D. et al. Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi. Proc. Natl Acad. Sci. USA 109, 15817–15822 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Hannezo, E., Prost, J. & Joanny, J. F. Instabilities of monolayered epithelia: shape and structure of villi and crypts. Phys. Rev. Lett. 107, 078104 (2011).

    CAS  PubMed  Article  Google Scholar 

  9. Almet, A. A., Maini, P. K., Moulton, D. E. & Byrne, H. M. Modeling perspectives on the intestinal crypt, a canonical system for growth, mechanics, and remodeling. Curr. Opin. Biomed. Eng. 15, 32–39 (2020).

    Article  Google Scholar 

  10. Tozluoǧlu, M. et al. Planar differential growth rates initiate precise fold positions in complex epithelia. Dev. Cell 51, 299–312 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  11. Drasdo, D. Buckling instabilities of one-layered growing tissues. Phys. Rev. Lett. 84, 4244–4247 (2000).

    CAS  PubMed  Article  Google Scholar 

  12. Chung, S. Y., Kim, S. & Andrew, D. J. Uncoupling apical constriction from tissue invagination. eLife 6, e22235 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  13. Sanchez-Corrales, Y. E., Blanchard, G. B. & Röper, K. Radially patterned cell behaviours during tube budding from an epithelium. eLife 7, e35717 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  14. Sumigray, K. D., Terwilliger, M. & Lechler, T. Morphogenesis and compartmentalization of the intestinal crypt. Dev. Cell 45, 183–197 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Martin, A. C. & Goldstein, B. Apical constriction: themes and variations on a cellular mechanism driving morphogenesis. Development 141, 1987–1998 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Sui, L. et al. Differential lateral and basal tension drive folding of Drosophila wing discs through two distinct mechanisms. Nat. Commun. 9, 4620 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  17. Nelson, C. M. et al. Microfluidic chest cavities reveal that transmural pressure controls the rate of lung development. Development 144, 4328–4335 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Latorre, E. et al. Active superelasticity in three-dimensional epithelia of controlled shape. Nature 563, 203–208 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    CAS  PubMed  Article  Google Scholar 

  20. Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–110 (2011).

    PubMed  Article  CAS  Google Scholar 

  21. Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).

    CAS  PubMed  Article  Google Scholar 

  22. Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).

    CAS  PubMed  Article  Google Scholar 

  23. McKinley, K. L. et al. Cellular aspect ratio and cell division mechanics underlie the patterning of cell progeny in diverse mammalian epithelia. eLife 7, e36739 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  24. Broguiere, N. et al. Growth of epithelial organoids in a defined hydrogel. Adv. Mater. 30, e1801621 (2018).

  25. Pérez-González, C. et al. Active wetting of epithelial tissues. Nat. Phys. 15, 79–88 (2019).

    PubMed  Article  CAS  Google Scholar 

  26. Thorne, C. A. et al. Enteroid monolayers reveal an autonomous WNT and BMP circuit controlling intestinal epithelial growth and organization. Dev. Cell 44, 624–633 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Altay, G. et al. Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function. Sci. Rep. 9, 10140 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Liu, Y., Qi, Z., Li, X., Du, Y. & Chen, Y. G. Monolayer culture of intestinal epithelium sustains Lgr5+ intestinal stem cells. Cell Disco. 4, 32 (2018).

    Article  CAS  Google Scholar 

  29. Kasendra, M. et al. Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids. Sci. Rep. 8, 2871 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  30. Verhulsel, M. et al. Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions. Lab Chip 21, 365–377 (2021).

    CAS  PubMed  Article  Google Scholar 

  31. Nikolaev, M. et al. Homeostatic mini-intestines through scaffold-guided organoid morphogenesis. Nature 585, 574–578 (2020).

    PubMed  Article  CAS  Google Scholar 

  32. Maskarinec, S. A., Franck, C., Tirrell, D. A. & Ravichandran, G. Quantifying cellular traction forces in three dimensions. Proc. Natl Acad. Sci. USA 106, 22108–22113 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Trushko, A. et al. Buckling of an epithelium growing under spherical confinement. Dev. Cell 54, 655–668 (2020).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  34. Merkel, M. & Manning, M. L. A geometrically controlled rigidity transition in a model for confluent 3D tissues. New J. Phys. 20, 022002 (2018).

  35. Alt, S., Ganguly, P. & Salbreux, G. Vertex models: from cell mechanics to tissue morphogenesis. Philos. Trans. R. Soc. B 372, 20150520 (2017).

  36. Hannezo, E., Prost, J. & Joanny, J. F. Theory of epithelial sheet morphology in three dimensions. Proc. Natl Acad. Sci. USA 111, 27–32 (2014).

    CAS  PubMed  Article  Google Scholar 

  37. Van Lidth de Jeude, J. F., Vermeulen, J. L. M., Montenegro-Miranda, P. S., Van den Brink, G. R. & Heijmans, J. A protocol for lentiviral transduction and downstream analysis of intestinal organoids. J. Vis. Exp. 98, e52531 (2015).

    Google Scholar 

  38. Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).

    CAS  PubMed  Article  Google Scholar 

  39. Rodríguez-Colman, M. J. et al. Interplay between metabolic identities in the intestinal crypt supports stem cell function. Nature 543, 424–427 (2017).

    PubMed  Article  CAS  Google Scholar 

  40. Rupprecht, J. F. et al. Geometric constraints alter cell arrangements within curved epithelial tissues. Mol. Biol. Cell 28, 3582–3594 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Krndija, D. et al. Active cell migration is critical for steady-state epithelial turnover in the gut. Science 365, 705–710 (2019).

    CAS  PubMed  Article  Google Scholar 

  42. Parker, A. et al. Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi. FASEB J. 31, 636–649 (2017).

    CAS  PubMed  Article  Google Scholar 

  43. Clevers, H. The intestinal crypt, a prototype stem cell compartment. Cell 154, 274–284 (2013).

    CAS  PubMed  Article  Google Scholar 

  44. Tambe, D. T. et al. Collective cell guidance by cooperative intercellular forces. Nat. Mater. 10, 469–475 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Trepat, X. et al. Physical forces during collective cell migration. Nat. Phys. 5, 426–430 (2009).

    CAS  Article  Google Scholar 

  46. Du Roure, O. et al. Force mapping in epithelial cell migration. Proc. Natl Acad. Sci. USA 102, 2390–2395 (2005).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  47. Theveneau, E. et al. Chase-and-run between adjacent cell populations promotes directional collective migration. Nat. Cell Biol. 15, 763–772 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    CAS  Article  PubMed  Google Scholar 

  49. Chan, C. J. et al. Hydraulic control of mammalian embryo size and cell fate. Nature 571, 112–116 (2019).

    CAS  PubMed  Article  Google Scholar 

  50. Nelson, C. M., VanDuijn, M. M., Inman, J. L., Fletcher, D. A. & Bissell, M. J. Tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures. Science 314, 298–300 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K. & Chen, C. S. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev. Cell 6, 483–496 (2004).

    CAS  PubMed  Article  Google Scholar 

  52. Legant, W. R. et al. Measurement of mechanical tractions exerted by cells in three-dimensional matrices. Nat. Methods 7, 969–971 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. Yang, Q. et al. Cell fate coordinates mechano-osmotic forces in intestinal crypt morphogenesis. Nat. Cell Biol. https://doi.org/10.1038/s41556-021-00700-2 (2021).

  54. Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    CAS  PubMed  Article  Google Scholar 

  55. Riedl, J. et al. Lifeact mice for studying F-actin dynamics. Nat. Methods 7, 168–169 (2010).

    CAS  PubMed  Article  Google Scholar 

  56. Zhang, Y. et al. Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A. Blood 119, 238–250 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. Edelstein, A. D. et al. Advanced methods of microscope control using μManager software. J. Biol. Methods 1, e10 (2014).

    PubMed  Article  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  59. de Reuille, P. B. et al. MorphoGraphX: a platform for quantifying morphogenesis in 4D. eLife 4, 05864 (2015).

    Google Scholar 

Download references

Acknowledgements

We thank J. Barbazán for assistance with laser ablations and discussions; E. Latorre for support with image segmentation and traction software; F. e. Marjou, M. A. Ziane, C. Cortina and X. Hernando for their support and training on organoid culture and in vivo procedures; and A. Marín, N. R. Chahare, T. Golde, J. Abenza, A. Ouzeri and all of the members of the Roca-Cusachs, Vignjevic, Arroyo and Trepat laboratories for their discussions and support. Funding: the authors are funded by the Spanish Ministry for Science, Innovation and Universities MICCINN/FEDER (grant nos PGC2018-099645-B-I00 to X.T. and PID2019-110949GB-I00 to M.A.), the Generalitat de Catalunya (Agaur; grant nos SGR-2017-01602 to X.T., 2017-SGR-1278 to M.A. and 2017-SGR-698 to E.B.; the CERCA Programme and ‘ICREA Academia’ award to M.A. and P.R.-C.), the European Research Council (grant nos Adv-883739 to X.T., CoG-681434 to M.A. and CoG-772487 to D.M.V.), the European Union’s Horizon 2020 research and innovation programme grant agreement no. H2020-FETPROACT-01-2016-731957 and the Marie Skłodowska–Curie grant agreement nos 797621 to M.G.-G. and 792028 to F.G., ‘La Caixa’ Foundation (grant no. LCF/PR/HR20/52400004 to X.T. and P.R.-C., ID 100010434; fellowships LCF/BQ/DR19/11740013 to G.C., LCF/BQ/DE14/10320008 to C.P.-G. and 01/16/FLC to A.A.-V.), the Spanish Ministry of Health (grant no. SAF2017-86782-R to E.B.), Fundació la Marató de TV3 (project 201903-30-31-32 to X.T. and E.B.). IBEC, IRB and CIMNE are recipients of a Severo Ochoa Award of Excellence from the MINECO.

Author information

Authors and Affiliations

Authors

Contributions

C.P.-G. and G.C. performed all of the experiments, except for some of the immunostainings, which were performed by N.C., A.M. and V.R.G. C.P.-G., G.C., M.M. and M.G.-G. developed analysis software and analysed data. M.M. performed the image segmentation. D.K. and A.G.C. contributed in vivo myosin data. F.G., S.K. and M.A. implemented the computational model. A.A.-V., P.R.-C. and E.B. contributed technical expertise, materials and discussion. C.P.-G., D.M.V., M.A. and X.T. conceived the project. D.M.V., M.A. and X.T. supervised the project. C.P.-G., G.C., M.A. and X.T. wrote the manuscript.

Corresponding authors

Correspondence to Danijela Matic Vignjevic, Marino Arroyo or Xavier Trepat.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Cell Biology thanks Timothy Saunders, Benjamin Simons and Christian Dahmann for their contribution to the peer review of this work. Peer reviewer reports are available.

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

Extended data

Extended Data Fig. 1 Organoid monolayers capture key physiological features of the intestinal epithelium.

a, Organoid monolayers expressing Lgr5–eGFP-IRES-CreERT2 stained for GFP and F-actin (phalloidin). Representative image of 2 experiments. b, Organoid monolayers stained for Zonula occludens 1 (ZO-1). Top: Average intensity projections of the 10 most basal (Top left) or apical (Top right) planes of the monolayer. Bottom: lateral view of the monolayer. Representative image of 2 experiments. c, Snapshots of an extrusion event happening at the villus-like region. The extruding cell is indicated with the arrowhead in the orthogonal views (bottom row). The neighbours of the extruding cell are highlighted in colours in the top views of the monolayer (middle row). The lateral views correspond to the midplane indicated with a white dashed line at their corresponding top views (Top row). Representative of 9 extrusions from 3 experiments. d, Snapshots of a cell that exhibits retrograde flow due to division. The contour of the cell of interest is delineated in red. The track of this cell is delineated in yellow. The contour of the second daughter cell that appears after the division event is delineated in green. The first and last snapshots (0 min and 160 min) correspond to basal planes of the crypt (Z = 0 µm and 2 µm, respectively). Because division occurs apically, the second and third snapshots (48 min and 52 min), correspond to an apical plane where division is better observed (Z = 12 µm). 5 crypts from 3 experiments. e, Spontaneous formation of pressurized domes in the villus-like domain. Top left: medial view of an organoid monolayer expressing Lifeact–eGFP. The dashed orange line defines the regions where the monolayer has delaminated to form a pressurized dome (orange arrowheads in bottom panel). Top right: 3D traction map of the same crypt. Yellow vectors represent components tangential to the substrate and the colour map represents the component normal to the substrate. Horizontal cyan line indicates y-axis position of the lateral XZ view (bottom). Bottom: lateral view of the organoid monolayers. Yellow vectors represent tractions. Representative of 3 experiments. Scale vector, 300 Pa. All scale bars, 20 μm. Stiffness of all the gels, 5 kPa.

Extended Data Fig. 2 Crypts fold through actomyosin-driven apical constriction.

a, Top row: 3D tractions of the crypts treated with the indicated concentration of blebbistatin (Bleb). Yellow vectors represent components tangential to the substrate and the colour map represents the component normal to the substrate. Bottom row: lateral views of the organoids along the crypt midline. Representative images from 3 independent experiments. Yellow vectors represent tractions. Scale bar, 20 μm. Scale vector, 200 Pa. b-c, Crypt indentation (b), and normal traction (c) as a function of the distance to the crypt centre for crypts treated with the indicated concentrations of blebbistatin for 3 h. Data are represented as mean ± s.e.m. of n = 18 (DMSO), 24 (0.5 µM), 19 (1.5 µM) 22 (5 µM) and 20 (15 µM) crypts from 3 independent experiments. d, Olfm4 immunostaining in membrane–tdTomato organoids at baseline conditions and 11 h after blebbistatin removal. Representative images from 2 independent experiments. Scale bar, 20 μm. e, Radial distribution of the apical (blue) and basal (red) F-actin intensity as a function of the distance to the crypt centre on 0.7 kPa substrates. Scale bars, 20 μm. Data are presented as mean ± s.d. of n = 12 crypts from 3 independent experiments. f, Bottom: Recoil velocity maps immediately after ablation of two crypts along the red lines on 0.7 kPa substrates. Left: a cut inside the TA. Right: a cut outside the TA. Scale vector, 1.5 μm/s. Top: the two crypts before ablation. Representative images from 3 independent experiments. Scale bar, 20 μm. g, Radial recoil velocity as a function of distance to crypt centre for cuts between stem cell compartment and transit amplifying zone (green) and between transit amplifying zone and villus-like domain (blue) on 0.7 kPa substrates. Data are represented as mean ± s.d. of n = 14 (cut Stem / TA) and 10 (cut TA / Villus-like) crypts from 3 independent experiments. h, Representative kymographs of circumferentially averaged radial velocity as a function of the distance to the crypt centre on 0.7 kPa (Top) and 5 kPa (bottom) substrates. Left: cut inside TA; right: Cut outside TA. The dashed black line indicates the time and position of the cut. Negative velocities point towards crypt centre.

Source data

Extended Data Fig. 3 Apicobasal distribution of F-actin and myosin in organoid monolayers.

a-b, Apical, medial and basal projections of F-Actin (Phalloidin, a) and myosin IIA–eGFP (b). The stem cell compartment (Stem), the Transit amplifying zone (TA) and the villus-like domain (villus-like) are zoomed in the regions of the monolayer indicated with the respective colours. Representative images from 4 (a) and 2 (b) independent experiments. Scale bars, 20 µm. Stiffness of the gel, 5 kPa.

Extended Data Fig. 4 Morphometric analysis of the different cell types in the crypt.

a, Top, front and side 3D renders of a segmented stem cell (left), Paneth cell (centre) and transit amplifying cell (right). b-c, Cell area (b) and aspect ratio (c) along the apicobasal axis of stem (green), Paneth (red) and TA (blue) cells on rigid (left, 15 kPa) and soft (right, 0.7 kPa) gels. n = 190 (stem cells), n = 21 (Paneth cells); n = 218 (transit amplifying cells) for 15 kPa gels. n = 596 (stem cells); n = 52 (Paneth cells); n = 301 (transit amplifying cells) for 0.7 kPa gels. n = 3 crypts per stiffness from 2 (0.7 kPa) and 3 (15kPa) independent experiments. Data are represented as mean ± s.e.m. d, Top: Apical and basal area of Paneth cells as a function of the distance to the crypt centre on stiff (left, 15 kPa) and soft (right, 0.7 kPa) substrates. The boundary between the stem cell compartment and the transit amplifying zone is indicated in all the plots with a dashed vertical line. Bottom: Apicobasal tilt (left) and basal aspect ratio (right) of Paneth cells as a function of the distance to the crypt centre on stiff (red, 15 kPa) and soft (blue, 0.7 kPa) substrates. n = 3 crypts per stiffness from 2 (0.7 kPa) and 3 (15kPa) independent experiments. From centre to edge bins, n = 3, 7 and 10 cells for 15 kPa gels and n = 11, 25 and 15 cells for 0.7 kPa gels. Data are represented as mean ± s.e.m.

Source data

Extended Data Fig. 5 Effect of myosin inhibition on organoid cell shape.

a-b, 3D segmentation of a crypt on 15 kPa gels under baseline conditions (a) and the same crypt after 3 h treatment with 15 µM of blebbistatin (b). Top: medial view. Bottom: lateral view. Representative images of 2 independent experiments. Scale bar, 20 µm. c-d, Apicobasal tilt (c) and basal aspect ratio (d) as a function of the distance to crypt centre on rigid substrates (15 kPa) before and after blebbistatin. Vertical dashed line indicates the boundary between the stem cell compartment and the transit amplifying zone. From centre to edge bins, n = 42, 72, 150 and 232 cells for baseline crypt and 39, 82, 131 and 209 for blebbistatin treatment. Data from 2 independent experiments.

Source data

Extended Data Fig. 6 3D computational vertex model and simulation protocol.

a, Discretization of the tissue: the thick lines denote the intersection between cellular faces and the thin lines the triangulation of the cell surfaces. b, Pattern of apical, basal and lateral surface tensions prescribed in the initial regular cell monolayer. c, Equilibration of the initial regular monolayer with patterned surface tensions on a rigid substrate, where basal nodes are constrained to a plane but can slide horizontally. Initial state (i), equilibrated state (ii), and different view of equilibrated state with basal cell outline (iii). d, Coupling with a deformable substrate, modelled computationally with a tetrahedral mesh discretizing a hyperelastic block (i). The equilibrated crypt on a rigid substrate (c-ii) is further equilibrated on the deformable substrate (d-ii,iii).

Extended Data Fig. 7 Simulation of crypt normal tractions.

a, Pattern of apical, basal and lateral surface tensions prescribed in the initial regular cell monolayer. b, Maps of basal normal traction. (i) Raw normal tractions at the basal plane featuring sub-cellular fine-scale details. To compare with experimental averages, we filtered these tractions with a Gaussian filter with standard deviation of 6 µm, (ii). c, Computational model of the deformed crypt on a soft hyperelastic substrate. d, Crypt folding for two models with different basal tension profiles on soft and rigid substrates.

Extended Data Fig. 8

F-Actin and myosin IIA co-evolution during de novo crypt formation. a-b, Apical and basal projections of F-Actin (Phalloidin, a) and myosin IIA–eGFP (b) of crypts at the indicated timepoints (2 days, 3 days, 4 days). The crypts are the same as in Fig. 6a. Representative images of 2 independent experiments c-d, Quantification of apical and basal myosin IIA intensity (c) and apical/basal myosin IIA ratio (d) at the Olfm4 positive and Olfm4 negative regions for the indicated timepoints. n = 14 (2d), 12 (3d) and 14 (4d) crypts from 2 independent experiments. For all the graphs, data is represented as Mean ± s.d. All Scale bars, 20 μm. Stiffness of the gel, 5 kPa. * (p < 0.05) ***(p < 0.001). Statistical significance was defined by a Kruskal–Wallis followed by a Dunn’s multiple-comparison test (c: basal Olfm4+ and d) and one-way ANOVA followed by a Tukey multiple-comparison test (c: apical Olfm4 + , apical Olfm4- and basal Olfm4-). Only the statistical comparison between 2d and 4d is shown.

Source data

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2, Supplementary references.

Reporting Summary

Peer Review Information

Supplementary Table 1

Supplementary Table 1. Different recipes used for the PAA mix

Supplementary Video 1

Spreading of intestinal crypts. Spreading of intestinal crypts on 5 kPa PAA gels coated with collagen I and laminin 1. Over the course of three days, a 2D monolayer with crypt-like and villus-like domains is established. The membrane is labelled with tdTomato. Total duration, 45 h.

Supplementary Video 2

Crypt dynamics. Time-lapse of a basal (left) and apical (right) plane of the organoid monolayer. Multiple cell divisions can be observed at the crypt (centre). Cells migrate collectively out of the crypt. The membrane is labelled with tdTomato. Total duration, 6 h 32 min.

Supplementary Video 3

Tracking of single cells in their journey out of the crypt. One cell (yellow) starts at the stem cell compartment and enters the transit amplifying zone, acquiring an elongated shape. Another cell (light blue) at the transit amplifying zone divides and enters in the villus-like domain. Some cells (dark blues, light blue and green) enter the villus-like domain and accelerate, separating from their followers. The membrane is labelled with tdTomato. Total duration, 6 h 32 min.

Supplementary Video 4

Division, migration, extrusion and doming in organoid monolayers. Time-lapse of a medial (left) and apical (right) plane of an organoid monolayer. A crypt in the centre exhibits multiple cell divisions and radial migration into the villus-like domain. Multiple epithelial domes appear at 1.20–2 h (top right) and 8.40 h (left). Cells are constantly extruded in the villus-like domain. The membrane is labelled with tdTomato. Total duration, 14 h.

Supplementary Video 5

Cell extrusion event in the villus-like region. Time-lapse of a top (top) and lateral (bottom) view of the extrusion of a cell in the villus-like region. At t = 32 min, the cell begins to be extruded from the monolayer. At t = 80 min, the cell is completely expelled. Total duration, 96 min.

Supplementary Video 6

A stem cell repositions at the crypt centre following division. Time-lapse of a stem cell that moves towards the crypt centre following division. Crypt basal plane (left; z = 2 µm). The red area indicates the segmentation of the cell of interest. The yellow line indicates the track of the cell. Note that the cell divides in the apical plane and thus is not always visible in this basal plane. Time-lapse of the crypt following the z-plane where the cell can be better visualized (right). At t = 200 min, the video pauses transiently and the focus is progressively changed to the apical-most plane of the monolayer to follow cell rounding. After division, the cell reintegrates close to the centre of the crypt and is again visible in the basal plane. The membrane is labelled with tdTomato. Total duration, 400 min.

Supplementary Video 7

Blebbistatin abrogates crypt forces reversibly. Organoid monolayer treated with blebbistatin (t = 90 min) and allowed to recover after washing out the drug (t = 5 h). The addition of blebbistatin triggers a rapid reduction of the crypt forces. After washout, the crypt recovers its characteristic traction pattern. At t = 10 h, an epithelial dome is formed. The membrane is labelled with tdTomato. Total duration, 15 h. 3D traction map of the crypt (top). The yellow vectors represent components tangential to the substrate and the colour map represents the component normal to the substrate. Lateral view of the organoid monolayer (bottom). The yellow vectors represent tractions.

Supplementary Video 8

Laser ablation between the stem cell compartment and the transit amplifying zone on a 5 kPa substrate. Membrane labelling (left) and velocity field (right) of organoid monolayers before and after ablation. The red line indicates the ablated region. The ablation (t = 10 s) induces a radial recoil of the monolayer. The membrane is labelled with tdTomato. Total duration, 20 s.

Supplementary Video 9

Laser ablation between the transit amplifying zone and the villus-like domain on a 5 kPa substrate. Membrane labelling (left) and velocity field (right) of organoid monolayers before and after ablation (red line). Ablation (t = 10 s) induces a radial recoil of the monolayer. The membrane is labelled with tdTomato. Total duration, 20 s.

Supplementary Video 10

Laser ablation between the stem cell compartment and the transit amplifying zone on a 0.7 kPa substrate. Membrane labelling (left) and velocity field (right) of organoid monolayers before and after ablation. The red line indicates the ablated region. The ablation (t = 10 s) induces a radial recoil of the monolayer. The membrane is labelled with tdTomato. Total duration, 20 s.

Supplementary Video 11

Laser ablation between the transit amplifying zone and the villus-like domain on a 0.7 kPa substrate. Membrane labelling (left) and velocity field (right) of organoid monolayers before and after ablation (red line). Ablation (t = 10 s) induces a radial recoil of the monolayer. The membrane is labelled with tdTomato. Total duration, 20 s.

Supplementary Video 12

Three-dimensional single-cell segmentation of a crypt. Example of a segmented crypt seeded on a 0.7 kPa substrate. At the beginning of the video, all of the segmented cells are visible in the 3D rendering. For visualization purposes, a stem cell (green), Paneth cell (red) and transit amplifying cell (blue) are highlighted. Note the different morphologies of each cell type. The stem cell is apically constricted. The Paneth cell has a larger volume and exhibits an apical expansion, probably reflecting its mucus compartment, and the transit amplifying cell is basally elongated and curved.

Supplementary Video 13

Cell shapes are heterogeneous. Example of five 3D rendered segmented stem cells (top), Paneth cells (centre) and transit amplifying cells (bottom). Note that even if each cell type exhibits characteristic morphometric properties, there is a remarkable variability in shape.

Supplementary Video 14

Cell velocity and traction maps of an intestinal monolayer on a 5 kPa substrate. Cell velocities (left) and 3D tractions (right) of an organoid monolayer. The cell velocity fluctuates in time and space but shows a general pattern of radial movement away from the crypt. The traction forces are roughly constant in time. The yellow vectors represent components tangential to the substrate and the colour map represents the component normal to the substrate. The membrane is labelled with tdTomato. Total duration, 6 h 08 min.

Supplementary Video 15

Cell velocity and traction maps of an intestinal monolayer on a 0.7 kPa substrate. Cell velocities (left) and 3D tractions (right) of an organoid monolayer. The cell velocity fluctuates in time and space but shows a general pattern of radial movement away from the crypt. Traction forces are roughly constant in time. The yellow vectors represent components tangential to the substrate and the colour map represents the component normal to the substrate. The membrane is labelled with tdTomato. Due to pronounced crypt folding, for visualization purposes, images are projections along the crypt medial plane. Total duration, 6 h 24 min.

Supplementary Video 16

Radial laser ablation of a crypt on a 5 kPa substrate. Membrane labelling (left) and velocity field (right) of an organoid monolayer before and after ablation (red line). Radial ablation of the monolayer at t = 10 s induces tissue recoil mainly in the direction parallel to the cut. The parallel velocity changes sign at the boundary between the crypt and the villus-like domain. The membrane is labelled with tdTomato. The red line indicates the ablated area. The dashed yellow line indicates the crypt contour. Total time, 20 s.

Supplementary Video 17

Radial laser ablation of a crypt on a 0.7 kPa substrate. Membrane labelling (left) and velocity field (right) of an organoid monolayer before and after ablation (red line). Radial ablation of the monolayer at t = 10 s induces tissue recoil both perpendicular and parallel to the cut. The parallel velocity changes sign at the boundary between the crypt and the villus-like domain. The membrane is labelled with tdTomato. The red line indicates the ablated area. The dashed yellow line indicates crypt contour. Total time, 20 s.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pérez-González, C., Ceada, G., Greco, F. et al. Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration. Nat Cell Biol 23, 745–757 (2021). https://doi.org/10.1038/s41556-021-00699-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-021-00699-6

Further reading

Search

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