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Cell fate coordinates mechano-osmotic forces in intestinal crypt formation

Abstract

Intestinal organoids derived from single cells undergo complex crypt–villus patterning and morphogenesis. However, the nature and coordination of the underlying forces remains poorly characterized. Here, using light-sheet microscopy and large-scale imaging quantification, we demonstrate that crypt formation coincides with a stark reduction in lumen volume. We develop a 3D biophysical model to computationally screen different mechanical scenarios of crypt morphogenesis. Combining this with live-imaging data and multiple mechanical perturbations, we show that actomyosin-driven crypt apical contraction and villus basal tension work synergistically with lumen volume reduction to drive crypt morphogenesis, and demonstrate the existence of a critical point in differential tensions above which crypt morphology becomes robust to volume changes. Finally, we identified a sodium/glucose cotransporter that is specific to differentiated enterocytes that modulates lumen volume reduction through cell swelling in the villus region. Together, our study uncovers the cellular basis of how cell fate modulates osmotic and actomyosin forces to coordinate robust morphogenesis.

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Fig. 1: Crypt morphogenesis during intestinal organoid development.
Fig. 2: Region-specific spontaneous curvature drives crypt morphogenesis.
Fig. 3: Myosin patterns in SCs and enterocytes determine region-specific spontaneous curvature.
Fig. 4: Region-specific localization of cell junctions.
Fig. 5: Lumen volume reduction promotes crypt budding.
Fig. 6: Coordination of lumen volume reduction and region-specific spontaneous curvature promotes crypt morphogenesis.
Fig. 7: Enterocytes control lumen volume reduction through SGLT1.

Data availability

Sequencing data supporting the findings of this study have been deposited in the Gene Expression Omnibus under the accession code GSE115956. All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

Codes generated and analysed during the current study are available at GitHub (https://github.com/fmi-basel/glib-nature_cell_biology2021-materials.git).

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Acknowledgements

We acknowledge the members of the Lennon-Duménil laboratory for sharing the mouse line of Myh9-GFP. We are grateful to the members of the Liberali laboratory and the FMI facilities for their support. We thank E. Tagliavini for IT support; L. Gelman for assistance and training; S. Bichet and A. Bogucki for helping with histology of mouse tissues; H. Kohler for fluorescence-activated cell sorting; G. Q. G. de Medeiros for maintenance of light-sheet microscopy; M. G. Stadler for scRNA-seq analysis; G. Gay for discussions on the 3D vertex model; the members of the Liberali laboratory, C. P. Heisenberg and C. Tsiairis for reading and providing feedback on the manuscript. Funding: Q.Y. is supported by a Postdoc fellowship from Peter und Taul Engelhorn Stiftung (PTES). This work received funding from the European Research Council (ERC) under the EU Horizon 2020 research and Innovation Programme Grant Agreement no. 758617 (to P.L.), the Swiss National Foundation (SNF) (POOP3_157531, to P.L.) and from the ERC under the EU Horizon 2020 Research and Innovation Program Grant Agreements 851288 (to E.H.) and the Austrian Science Fund (FWF) (P31639, to E.H.).

Author information

Affiliations

Authors

Contributions

P.L. and Q.Y. conceived the project and designed the experiments. Q.Y. performed and analysed the experiments. E.H. proposed the physical theory. S.-L.X. designed the physical model, performed the simulations. C.J.C. supported the micropipette aspiration. M.R. and D.V. assisted with the image processing and quantifications. F.M.G. generated organoid lines. C.J.C. and T.H. helped to design the micropipette aspiration and mosaic experiments. Q.Y. wrote the first version of the manuscript, S.-L.X. wrote the first version of the Supplementary Information, and P.L., E.H. and Q.Y. supervised the study. P.L. and E.H. helped to design the project and finalize the manuscript.

Corresponding authors

Correspondence to Qiutan Yang or Edouard Hannezo or Prisca Liberali.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks the anonymous reviewers 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 Cell and tissue quantification.

a, Example of selecting crypt and villus region for quantification of single-cell apical and basal size in bulged organoid. Upper panel, from left to right: Z-projection of ZO-1 and Lyz staining in bulged organoid, selected apical segmentation with Lyz signal in 3D and ZO-1 signal (white) projected in 3D apical side in villus from xy view, in villus from yz view, in crypt from xy view and in crypt from yz view. Lower panel, from left to right: selected basal segmentation with Lyz signal in 3D and ZO-1 signal (white) project in 3D basal side in villus from xy view, outline of villus basal segmentation from xy view, crypt basal segmentation from xy view, outline of crypt basal segmentation from xy view and yz view. Colour of heatmap indicates the size of single-cell apical or basal membranes. b-c′, Tissue compaction along crypt-villus axis in the development of intestinal organoid and in vivo tissue. b, Representative images of bulged and budded organoids with CD44 and DAPI staining. b′, Plot of cell density in crypt (red dashed double arrowhead line) and villus (blue dashed double arrowhead line) tissue as indicated in B. Two-tailed t-test for bulged crypt (n = 22) and bulged villus (n = 22), p < 10−5; for budded crypt (n = 19) and budded villus (n = 19), P < 10−11. c, Representative images of immunostaining on the section of mouse intestine at the age of P1 and P11. c′, Plot of cell density in crypt (red dashed double arrowhead line) and villus (blue dashed double arrowhead line) regions as indicated in C. Two-tailed t-test for P1 crypt (n = 32) and P1 villus (n = 31), p < 10−10; for P11 crypt (n = 40) and P11 villus (n = 44), P < 10−23. Experiments and imaging analysis in A, and experiments in B-C were repeated at least three times independently with similar results. Scale bars, 20 µm. Violin plot lines in B′ and C′ denote quartile for each group.

Source data

Extended Data Fig. 2 Sensitivity analysis for how model parameters affect crypt morphology.

a–c, Schematic of the model and morphometric parameters used (see Supplementary Note). d, Phase diagrams of crypt morphologies with varying volumes v and spontaneous curvature of crypt γc, for different values of in-plane contraction α (left to right: 1, 0.6, and 1.5). e-h, Evolution of thickness ratio hc/hv and radius ratio Rc/Rv during the inflation of an organoid (crypt size φ = 0.2, shape factor \(\tilde \kappa _0 = 10\)): increasing α (γc = 0.1) (panel E) and γc (α = 1, 1.5, and 0.6) (panels F-H). i, Influence of cell swelling on crypt morphology (degree of crypt opening) with varied crypt size φ (α = 1.5, γc = −0.02), α (γc = −0.1, φ = 0.5), and γc (α = 1, φ = 0.5). j, Morphological evolution during the inflation of an organoid (α = 1.5, γc = −0.02) with swollen villus cells (vev = 5). k, Influence of spontaneous curvature of villus γv on crypt morphology (φ = 0.2) with varied γc (α = 1) and α (γc = −0.08). l, Influence of γv on the evolution of hc/hv and Rc/Rv during the inflation of an organoid with α is respectively 1 and 1.5 (γc = −0.1, φ = 0.2).

Extended Data Fig. 3 Comparison between numerical solutions of the full model and analytical scaling laws.

a-a″, Three possible mechanical scenarios that could drive crypt morphogenesis. Stable organoid configuration is calculated by minimizing the energy F (α,γc,γv, φ,v, Λ), which depends on a few key parameters (see Supplementary Note): α, ratio of in-plane contractions in crypt and villus regions; γc, spontaneous curvature crypt region; γv, spontaneous curvature of villus region. φ, relative size of the crypt region; v, normalized lumen volume; Λ, potential line tension along the crypt/villus boundary. a, crypt budding driven by smaller crypt in-plane contraction α, leading to thinner crypts (decreased epithelial thickness ratio hc/hv and decreased radius of curvature Rc/Rv). a′, crypt budding driven by spontaneous curvature γc, leading to thicker crypts (increased hc/hv and decreased Rc/Rv). a″, organoid budding driven by line boundary tension, leading to constant thickness (constant hc/hv and decreased Rc/Rv). The width and location of the green lines indicate the strength and distribution of the driving forces in each model. b, Radius ratio (Rc/Rv) from six experimental samples with the corresponding model fit (see Supplementary Note). c-d, All six samples of bulged organoids can be collapsed via hc/hv vs. time (C) or Rc/Rv vs. time (D). e-f, Comparison of numerical (symbol) and analytic (line) results to verify that, thickness ratio hc/hv and radius ratio Rc/Rv respectively depends on in-plane contraction α (with crypt size φ = 0.05) and coupled parameter φ−1γc(with α = 1.15) for a bulged organoid (normalized volume v = 5) (panel E), hc/hv and Rc/Rv depend on parameter u for a budded organoid (φ = 0.2) (panel F), and analytic results also fit well with numerical results for the inflation of a bulged organoid (α = 1.15, γc = −0.025, φ = 0.05) or a budded organoid (u = 6, φ = 0.2). g, Phase diagram of crypt morphologies of an organoid (φ = 0.2, \(\tilde \kappa _0 = 10\)) under infinite volume expansion (v = 108) (see Supplementary Note), with varying spontaneous curvature γc and in-plane contraction α. h, Influence of normalized volume of a crypt cell vec (top) and that of a villus cell vev (bottom) on hc/hv and Rc/Rv of a budded organoid (u = 6, φ = 0.2).

Source data

Extended Data Fig. 4 Actomyosin drives and maintains crypt budding.

a-a′, Supplementary to Fig. 2d. a, Montage images across the regions of laser cutting and opening with surrounded LifeAct-GFP signal. Dashed lines outline the size of opening. a′, Plot for distance of openings after cutting. b, Staining of phosphorylated Myosin light chain (pMLC) in budded organoid with maximum z-projection (upper panel) and single section (lower panel). c, Day4 LifeAct-GFP organoids in Control (n = 96), Blebbistatin (n = 188) or Cytocalasin D (n = 45) cultures. Left images, maximum z-projection of LifeAct-GFP; right images, merged maximum z-projection of LifeAct-GFP with Lyz and DAPI staining. Plots, quantification of organoid eccentricity and volume ratio (lumen vs total). d, Representative time-lapse recording of organoid expressing Myh-9-GFP during crypt morphogenesis. Upper images, middle sections. Lower images, maximum z-projections. e, Membrane-targeted GFP (mG) in organoid before bulging (n = 12), bulged (n = 20) and budded (n = 22) organoids in the middle section (images); quantification for ratios of mG intensity in cells (box plots). f, Representative time-lapse recording of budded LifeAct-GFP (white) organoid treated with DMSO control, Blebbistatin or Aphidicolin (left images), and corresponding plot for aspect ratio of the organoids (right plot, numbers of recordings in bracket). Solid lines represent average values, shadow regions standard deviations. g-g′, Merged images for Myh-9-GFP and ZO-1 staining in wild-type/Myh-9-GFP (G, left image) or in myh-9±/Myh-9-GFP (G′, left image) mosaic organoid with zoom-in areas (G and G′, right image) from yellow dashed rectangles. White dashed line outlines the morphology of Myh-9-GFP cells. g″, quantification on percentage of basal constriction in Myh-9-GFP cells next to wild-type (n = 62) or myh-9± (n = 79) cells Arrows: red (crypt), blue (villus), white (G and G′, basal domain of Myh-9-GFP cells). P-values are calculated from two-tailed t-test. Box-plot elements show 25% (Q1, upper bounds), 50% (median, black lines within the boxes) and 75% (Q3, lower bounds) quartiles, and whiskers denote 1.5× the interquartile range (maxima: Q3 + 1.5× (Q3-Q1); minima: Q1-1.5× (Q3-Q1)) with outliers (rhombuses). Scale bars: B (20µm), C-D, F, G-G′ left (50µm), G -G′ right zoom-in (10µm). Images in A-G are representative of three independent experiments. Data in G″ are pulled from two independent experiments.

Source data

Extended Data Fig. 5 In vivo tissue-specific localization of Claudin2 and ZO-1.

In vivo staining of Claudin2 (A) and ZO-1 (B) in mouse small intestine at P1, P2, P5, P7, P11, P12, P13, P14, P15, P16, P17 and 6-month adult stages. Stainings were repeated at least three times independently with similar results. Yellow dashed rectangles in B indicate zoom-in regions of villi randomly selected from the same field of views in the presented images. Scale bars: A (100 µm), B (100 µm, zoom-in, 20 µm).

Extended Data Fig. 6 Sensitivity analysis of model results as a function of crypt apical tension and osmotic changes.

a, Phase diagram of crypt morphologies (\(\tilde \kappa _0 = 6\), φ = 0.2) as a function of normalized crypt apical tension m and villus cell volume vev, showing the villus cell swelling favors crypt budding synergistically with crypt apical tension. b, Influence of crypt size φ and shape factor \(\tilde \kappa _0\) on critical values of m (with vev = 1, that is no cell swelling) and vev (with m = 2, that is constant crypt apical tension) for organoid budding. c-e, Influence of preferential proliferation of crypt cells, characterized as crypt size φg, on crypt morphology, with normalized organoid volume v = 1 (panel C) or 1.2 (panel E). After rescaling volume v, crypt morphologies with preferential crypt growth (solid line) are close to those without preferential growth (dash line) (panel D, see Supplementary Note). f-h, Dependence of thickness ratio hc/hv and radius ratio Rc/Rv on normalized crypt apical tension m (vev = 1), with varied \(\tilde \kappa _0\) (φ = 0.2) (panel F) and φ (\(\tilde \kappa _0 = 6\)) (panel G), and on villus cell volume vev (panel H), with varied m (\(\tilde \kappa _0 = 6\), φ = 0.2). i, Evolution of hc/hv and Rc/Rv during organoid inflation with varied m (\(\tilde \kappa _0 = 6\), φ = 0.2, vev = 1). j, Phase diagram of crypt morphologies upon infinite volume expansion (v = 108) (showing three possible phases: fully closed, partially opening, or fully closed with vanishing apical surface), with varying \(\tilde \kappa _0\) and m (φ = 0.2).

Extended Data Fig. 7 The impact of lumen volume on organoid morphology.

a-b″, Fitting for lumen inflation experiments with theoretical model. Images, microinjection of bulged (A) and budded (B) organoids for lumen inflation. Fittings (A′ and B′), fitting experimental measurements with its predictive model based on epithelial thickness ratio (hc/hv), organoid radius ratio (Rc/Rv) and lumen volume (v) change. Plots (A″ and B″), measurements of hc/hv and Rc/Rv in bulged (A″, n = 7 biologically independent organoids) and budded (B″, n = 12 biologically independent organoids) organoids. P-values are calculated from two-tailed Paired Student’s t-test. c-c′, Evolution of morphometric parameters in bulged (C) or budded (C′) organoids during volume inflation, induced by PGE treatment (n = 3 biologically independent organoids) and microinjection (n = 3 biologically independent organoids), can be collapsed via Rc/Rv vs. v and hc/hv vs. v. Scale bars, 50 µm.

Source data

Extended Data Fig. 8 Regulation of lumen volume by enterocytes and the membrane transporters.

a, Representative images of DMSO-treated control (n = 96), CHIR- (n = 145) and IWP2- (n = 78) treated organoids (left panels), and corresponding quantification on eccentricity and lumen ratio (right panels). b, Segmentation (Seg.) of single-cell volume based on β-Catenin signal in bulged and budded organoids (left images), violin plot for the corresponding quantification of single-cell volume (left plot), swarm plot for the lumen ratio of each organoids (right plot). Two-tailed t-test for cells in crypt (n = 385) vs. villus (n = 218) in bulged organoid (p = 0.311), crypt (n = 551) vs. villus (n = 769) in budded organoids (p < 10−71), and bulged villus vs. budded villus (p < 10−75); for lumen ratio bulged (n = 8) vs. budded (n = 6) (p < 10−4). c, Immunostaining of β-Catenin in the section of mouse intestinal tissue at P1 and P11 stages, segmentation (seg.) of single-cell area, zoom-in crypt (red dashed rectangles) and villus (blue dashed rectangles) areas in β-Catenin staining image overlapping with single-cell segmentations (left images), violin plot for the quantification of single-cell areas (left plot), swarm plot for the quantification of distance between villi (right plot). Two-tailed t-test for cells in P1 crypts (n = 54) vs. villi (n = 156) (p < 10−14), in P11 crypts (n = 82) vs. villi (n = 159) (p < 10−14), and cells in P1 villi vs. P11 villi (p < 10−15); for distance between villi, P1 (n = 32) vs. P11 (n = 30) (p < 10−7). d, TSNE-based visualizations of single-cell RNA sequencing data indicate mRNA expressions of aquaporins, atp1a1 and atp1b1. e, Day4 organoid treated with DMSO control, Ouabain and CuSO4 (left images), quantification on the eccentricity and lumen ratio of control (n = 155), Ouabain (n = 146), CuSO4 (n = 192) and Sotagliflozin (n = 119) (right plots). P-values in box plots (A, E) are calculated from two-tailed t-test. Box plot elements show 25% (Q1, upper bounds), 50% (median, black lines within the boxes) and 75% (Q3, lower bounds) quartiles, and whiskers denote 1.5× the interquartile range (maxima: Q3 + 1.5× (Q3-Q1); minima: Q1-1.5× (Q3-Q1)) with outliers (rhombuses). Violin plot lines (B, C) denote quartile for each group. Scale bars: A-B (50 µm), C (left, 50 µm, zoom-in,10 µm), E (20 µm). Images in A, B, C and E are representative of > 3 independent experiments.

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Extended Data Fig. 9 Piezo channels do not strongly regulate crypt budding.

a, TSNE-based visualizations of single-cell RNA sequencing data indicate the maker gene expression for crypt (left panel), villus (middle panel) and piezo1 (right panel). b, Piezo1 and DAPI staining in budded organoid. Piezo1 is detected in few single cells. Staining was repeated at least three times independently with similar results. c–e, Inhibition of Piezo1 (GdCl3 and GsMtx4) did not cause any defect in crypt morphogenesis, while activation of Piezo1 (Yoda-1) leads to slight increased lumen volume and reduced eccentricity. Reduced enterocytes (indicated by AdlB signal) are detected in Yoda-1-treated organoids. c, Representative images of Day 4 organoids treated in DMSO-control, GdCl3, GsMtx4 or Yoda-1 condition with AdlB, CD44 and DAPI staining in maximum z-projection. d-e, Corresponding box-plot quantifications of eccentricity (D) and lumen ratio (E) for organoids from C. Experiment (C-E) was repeated three times independently with similar results. Sample numbers: DMSO-control (n = 94 organoids), GdCl3 (n = 143 organoids), GsMtx4 (n = 68 organoids) and Yoda-1 (n = 71 organoids). P-values are calculated from two-tailed t-test. Box-plot elements show 25% (Q1, upper bounds), 50% (median, black lines within the boxes) and 75% (Q3, lower bounds) quartiles, and whiskers denote 1.5× the interquartile range (maxima: Q3 + 1.5× (Q3-Q1); minima: Q1-1.5× (Q3-Q1)) with outliers (rhombuses). Scale bars, 25 µm.

Source data

Extended Data Fig. 10 ECM remodelling is nonessential for crypt budding.

a, Representative images of Day 4 organoids treated by DMSO as control, or broad-spectrum inhibitors of matrix metalloproteinases (GM6001 and Marimastat) with Laminin staining for Matrigel and DAPI staining for cell nuclei in maximum z-projection. Experiment was repeated > 3 times independently with similar results. b, Corresponding box-plot quantifications of eccentricity for organoids from A. Experiments were repeated three times independently with similar results. Sample numbers: DMSO-control (n = 265 organoids), GM6001 (n = 102 organoids), and Marimastat (n = 94 organoids). P-values are calculated from two-tailed t-test. Box-plot elements show 25% (Q1, upper bounds), 50% (median, black lines within the boxes) and 75% (Q3, lower bounds) quartiles, and whiskers denote 1.5× the interquartile range (maxima: Q3 + 1.5× (Q3-Q1); minima: Q1-1.5× (Q3-Q1)). Scale bars, 50 µm.

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Supplementary Information

Supplementary Note: theoretical note of 3D vertex modelling on crypt morphogenesis.

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Supplementary Video 1

Representative light-sheet time-lapse recording of crypt morphogenesis. A full stack of an organoid expressing LifeAct–GFP was acquired every 10 min for 16 h 30 min from day 3 until budding. Left, single plane intersecting the middle of the organoid. Middle, maximum z-projection. Right, dynamic plots of organoid eccentricity, normalized lumen, tissue and total volume over time. The experiments were repeated with at least five independent recordings. Scale bar, 20 µm.

Supplementary Video 2

Representative light-sheet time-lapse recording of Myh9–GFP expression in an organoid during crypt morphogenesis. A full stack of an organoid expressing Myh9–GFP was acquired every 10 min for 16 h 30 min from day 3 until budding. Left, single plane intersecting the middle of the organoid. Right, maximum z-projection. The experiments were repeated at least three times. Scale bar, 50 µm.

Supplementary Video 3

Representative light-sheet time-lapse recording of budded organoid treated with DMSO. A full stack of budded organoid expressing LifeAct–GFP was acquired every 10 min from day 4, treated with 10 µM DMSO, cultured and recorded for 10 h. The experiments were repeated at least three times. Scale bar, 50 µm.

Supplementary Video 4

Representative light-sheet time-lapse recording of budded organoid treated with aphidicolin. A full stack of budded organoid expressing LifeAct–GFP was acquired every 10 min from day 4.The organoid was treated with 0.6 µM aphidicolin, cultured and recorded for 10 h. The experiments were repeated at least three times. Scale bar, 50 µm.

Supplementary Video 5

Representative light-sheet time-lapse recording of budded organoid treated with blebbistatin. A full stack of budded organoid expressing LifeAct–GFP was acquired every 10 min from day 4, treated with 7.5 µM blebbistatin, cultured and recorded for 10 h. The experiments were repeated at least three times. Scale bar, 50 µm.

Supplementary Video 6

Representative time-lapse recording of PGE induced inflation. Full stacks of bulged and budded organoids expressing LifeAct–GFP were acquired every 3 min immediately after treatment with 0.5 µM PGE for less than 30 min. Left, maximum z-projection. Right, single plane intersecting the middle of the organoid. The experiments were repeated at least three times. Scale bar, 20 µm.

Supplementary Video 7

Representative time-lapse recording of microinjection into the organoid lumen for inflation. Bright-field time-lapse recoding of bulged and budded organoids under microinjection. The experiments were repeated at least three times. Scale bar, 50 µm.

Supplementary Video 8

Representative light-sheet time-lapse recording of budded organoid treated with PGE, or sequentially with PGE and blebbistatin. A full stack of a budded organoid expressing LifeAct–GFP was acquired every 20 min from day 4. Top, single middle-plane (top left) and maximum z-projection (top right) of the organoid that was treated with 0.5 µM PGE for 13 h 20 min. Bottom, single middle-plane (bottom left) and maximum z-projection (bottom right) of the organoid that was treated with 0.5 µM PGE for 3 h 20 min, then 7.5 µM blebbistatin was added and recorded for another 10 h. The experiments were repeated at least three times. Scale bar, 50 µm.

Supplementary Video 9

Representative light-sheet time-lapse recording of enterocyst from day 3. A full stack of an enterocyst expressing LifeAct–GFP was acquired every 10 min from day 3 for 16 h 30 min. Left, single plane intersecting the middle of the organoid. Middle, maximum z-projection. Right, dynamic plots of organoid eccentricity, normalized lumen, tissue and total volume over time. The experiments were repeated with at least five independent recordings. Scale bar, 20 µm.

Supplementary Video 10

Representative light-sheet time-lapse recording of an organoid treated with CHIR from day 3. A full stack of an organoid expressing LifeAct–GFP was acquired every 10 min from day 3 for 16 h 30 min while being treated with 3 µM CHIR from day 3. Left, single plane intersecting the middle of the organoid. Middle, maximum z-projection. Right, dynamic plots of organoid eccentricity, normalized lumen, tissue and total volume over time. The experiments were repeated with at least three independent recordings. Scale bar, 20 µm.

Supplementary Table 1

Supplementary Table 1. Antibodies used in organoid immunostaining.

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Yang, Q., Xue, SL., Chan, C.J. et al. Cell fate coordinates mechano-osmotic forces in intestinal crypt formation. Nat Cell Biol 23, 733–744 (2021). https://doi.org/10.1038/s41556-021-00700-2

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