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Proliferation-driven mechanical compression induces signalling centre formation during mammalian organ development

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Abstract

Localized sources of morphogens, called signalling centres, play a fundamental role in coordinating tissue growth and cell fate specification during organogenesis. However, how these signalling centres are established in tissues during embryonic development is still unclear. Here we show that the main signalling centre orchestrating development of rodent incisors, the enamel knot (EK), is specified by a cell proliferation-driven buildup in compressive stresses (mechanical pressure) in the tissue. Direct mechanical measurements indicate that the stresses generated by cell proliferation are resisted by the surrounding tissue, creating a circular pattern of mechanical anisotropy with a region of high compressive stress at its centre that becomes the EK. Pharmacological inhibition of proliferation reduces stresses and suppresses EK formation, and application of external pressure in proliferation-inhibited conditions rescues the formation of the EK. Mechanical information is relayed intracellularly through YAP protein localization, which is cytoplasmic in the region of compressive stress that establishes the EK and nuclear in the stretched anisotropic cells that resist the pressure buildup around the EK. Together, our data identify a new role for proliferation-driven mechanical compression in the specification of a model signalling centre during mammalian organ development.

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Fig. 1: Emergence of a circular pattern of structural anisotropy in the embryonic mouse incisor.
Fig. 2: Circular anisotropy pattern arises from mechanical resistance in the tissue.
Fig. 3: Circular mechanical and structural anisotropy depend on cell proliferation in the incisor during EK formation.
Fig. 4: Proliferation-driven tissue pressure regulates EK gene expression in the murine incisor.
Fig. 5: αE-catenin is necessary to create mechanical tissue anisotropy and for EK formation.
Fig. 6: Piezo does not affect EK formation.
Fig. 7: YAP transduces mechanical information to establish the EK in the murine incisor.

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Data availability

The data supporting the findings of this study are all available within the article. All other data supporting the findings of this study are available from the corresponding authors. Source data are provided with this paper.

Code availability

The 3D images of oil microdroplet injected tooth buds were processed using the STRESS code developed at the Campas lab27 (https://github.com/campaslab/STRESS). No other custom codes were used.

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Acknowledgements

We thank D. Cuylear, P. Marangoni, A. Rathnayake, B. Hoehn and A. Cortez for technical support, E. Sletten (University of California Los Angeles) for sharing custom-made fluorinated rhodamine dyes and Klein and Campas laboratory members for helpful discussions. We acknowledge the staff within the Biological Imaging Development CoLab at UCSF Parnassus Heights, especially K. Marchuk and J. Eichorst, for their training and support in using the Nikon A1r and the NIS Elements software. We also thank the Laboratory Animal Resource Center, UCSF for assistance with animal care. Funding has been obtained from the National Institute of Dental and Craniofacial Research grant R01-DE027620 (O.D.K. and O.C.), R35-DE026602 (O.D.K.) and the Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy – EXC 2068 – 390729961– Cluster of Excellence Physics of Life of TU Dresden (O.C.).

Author information

Author notes

  1. These authors contributed equally: Neha Pincha Shroff, Pengfei Xu.

Authors and Affiliations

  1. Department of Orofacial Sciences and Program in Craniofacial Biology, University of California, San Francisco, CA, USA

    Neha Pincha Shroff, Pengfei Xu, Tingsheng Yu Drennon & Ophir D. Klein

  2. Department of Mechanical Engineering, University of California, Santa Barbara, CA, USA

    Sangwoo Kim, Elijah R. Shelton, Ben J. Gross, Yucen Liu & Otger Campàs

  3. Institute of Mechanical Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Sangwoo Kim

  4. Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA

    Carlos O. Gomez & Otger Campàs

  5. School of Dentistry, University of California Los Angeles, Los Angeles, CA, USA

    Qianlin Ye & Jimmy K. Hu

  6. Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA

    Qianlin Ye & Jimmy K. Hu

  7. Centre for Craniofacial Regeneration and Biology, King’s College London, London, UK

    Jeremy B. A. Green

  8. Cluster of Excellence Physics of Life, TU Dresden, Dresden, Germany

    Otger Campàs

  9. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Otger Campàs

  10. Center for Systems Biology Dresden, Dresden, Germany

    Otger Campàs

  11. Department of Pediatrics, Cedars-Sinai Guerin Children’s, Los Angeles, CA, USA

    Ophir D. Klein

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  1. Neha Pincha Shroff
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Contributions

O.C., O.D.K., N.P.S. and P.X. designed the experiments. N.P.S. and P.X. performed all experiments. S.K. performed the analysis of tissue anisotropy. E.R.S. and B.J.G. adapted the STRESS code to analyse stresses. Q.Y. collected the Piezo mutant embryos. Y.L. prepared and calibrated the oil droplets. T.Y.D. and C.O.G. helped in training and initial trials. C.O.G. did the Imaris analysis. J.K.H. participated in conceptualization of the project. J.B.A.G. and J.K.H. provided critical feedback and helped design experiments. N.P.S., P.X., O.C. and O.D.K. wrote the paper, with input from J.K.H. and J.B.A.G. O.C. and O.D.K. supervised the project. All authors edited the paper.

Corresponding authors

Correspondence to Otger Campàs or Ophir D. Klein.

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Nature Cell Biology thanks Thomas Diekwisch, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 A concentric cellular arrangement develops around the EK in the embryonic mouse incisor.

(a) Analysis of 2.5D nuclear orientation in representative images using OrientationJ in 3 confocal slices 15 µm apart in Z from a E13.5 incisor. (n = 1) (b) 3D analysis of nuclear orientation in representative images using Imaris nuclear segmentation of top, middle and bottom planes of a 52 µm thick Z-stack from a E13 incisor. (n = 1) (c) Average quantification of nuclear anisotropy in E11.5 through E14.5 incisor epithelia as shown in Fig. 1f using OrientationJ analysis (n = 4; Methods). (d) Average quantification of cytoskeletal anisotropy in E12.5 and E14.5 incisor epithelia as shown in Fig. 1g using OrientationJ analysis (n = 3). (e) Representative tissue anisotropy analysis (anisotropy in actin spatial distribution) of E13.5 Phalloidin stained bud and the overlay of the E13.5 Shh RNAscope image (from Fig. 1d) on the map. Scale bar, 25 µm. Data are represented as mean ± s.e.m. n represents number of embryos; one incisor measured per embryo.

Extended Data Fig. 2 OrientationJ and Epyseg analysis of incisors in 2D.

(a-b) OrientationJ and Epyseg analysis of representative (a) E12 and (b) E13.5 K14Cre;R26mTmG/mTmG incisors with the spatial averaging of the Epyseg analysis on the right (n = 1 per stage). White arrows indicate the low coherence region. Representative 2D central plane shown. Scale bar, 25 µm. n represents number of embryos; one incisor measured per embryo.

Extended Data Fig. 3 Inhibition of acto-myosin activity does not interfere with anisotropic stress mediated development of EK formation.

(a) Schematic depicting the dissection, agarose embedding, injection of oil microdroplets, and imaging of the incisor. The image of the embryo and the microscope have been obtained from Biorender.com. (b) Outline of E13.5 incisor with reference points 1–3 illustrating the marker locations on the border and point 4 illustrating the oil droplet for vector field and microdroplet orientation analysis. (c) Anisotropy analysis of the live E12 and E13.5 murine incisor. (E12: n = 6 and E13.5: n = 6). (d) Representative E14.5 incisor epithelium separated from the mesenchyme after 45 mins dispase treatment. (n = 3) (e) EdU and E-Cad immunostaining after a 1 h EdU pulse chase in E12 murine incisors cultured with DMSO or blebbistatin for 40 h. Quantification of total EdU positive cells in each field per condition for all incisors. (Control: n = 7; Blebbistatin: n = 7) (f) Whole mount in situ of Shh and E-Cad immunostaining in E12 incisors cultured for 40 h with DMSO or Y27632 (ROCK inhibitor). (DMSO: n = 5; Y27632: n = 5). Scale bar, 25 µm. Dashed line outlines the incisor. Representative 2D central plane shown in all images. Data are represented as mean ± s.e.m. Statistical analysis was done using the unpaired two-tailed Student’s T test (assuming unequal variance) with Welch’s correction for e. n represents number of embryos; one incisor measured per embryo.

Source data

Extended Data Fig. 4 Inhibiting cell proliferation in the embryonic murine incisor interferes with incisor growth and anisotropic stress development.

(a) Measured proliferation profiles from the centre in the epithelium at E11.5, 12.5, 14.5 and 15.5. (Mean values plotted from n = 3 per stage). (b) EdU and eGFP or BrdU and E-Cad immunostaining after a 1 h EdU or BrdU pulse chase in E12 murine incisors cultured with DMSO or Aphidicolin for 7 h (Top panel) and 40 h (Bottom panel). (n = 6 per condition) (c) Morphology of E12 incisors at 0 h and 40 h after culture with DMSO or aphidicolin. Dashed line outlines the incisor in (b, c). (Ctrl: n = 4, 6 and Aphi: n = 4, 6 for 0 and 40 hours respectively) (d) Quantification of the epithelial area (outlined in yellow dotted line) from all the buds represented in c. (n values in c). (e, f) Anisotropy analysis of the live E12 murine incisors cultured for (e) 7 h or (f) 30 h with DMSO or aphidicolin. (7 h – n = 5; 30 h - n = 3) (g) Representation of droplet orientation and cell anisotropy (orientation) maps (reproduced from Fig. 3c, c’) and quantification of droplet orientation with reference to the regional cell orientation in live 7 h DMSO or aphidicolin treated incisors. Compilation of average data from 3–6 h after injection. Orientation angle values for DMSO treated incisors reproduced from Fig. 2f E12 data. (Ctrl: n = 15 in LC and n = 14 in HC; Aphi: n = 13 in LC and n = 15 in HC) LC, low coherence region; HC, high coherence region. (h) Quantification of the tissue stress anisotropy measured by the oil droplets in the LC region of E12 incisors cultured for 7 hours with DMSO or aphidicolin (Ctrl: n = 15 from 2 h; Aphi: n = 13). Dashed line outlines the incisor. Scale bars, 25 µm. epi, epithelium; mes, mesenchyme. Representative 2D central plane shown in all images. Data are represented as mean ± s.e.m. Statistical analysis was done using 2 way ANOVA for d, unpaired two-tailed Student’s T test (assuming unequal variance) with Welch’s correction for g and 2 tailed Mann-Whitney test for h. Source data are available for all plots. n represents number of embryos; one incisor measured per embryo.

Source data

Extended Data Fig. 5 Cell proliferation increases compressive stress and area of the embryonic murine incisor.

(a) Whole mount in situ of Shh and E-Cad immunostaining in E12 incisors cultured for 40 h with DMSO or Mitomycin C. (n = 4 per condition) (b) BrdU-EdU double labelling in the E13.5 murine incisor after sequential pulse chase with EdU (1 h) – BrdU (15 mins). (n = 5) (c) Morphology of E12 incisors at 40 h after culture in DMSO or aphidicolin or aphidicolin washed out after 16 h. (Ctrl: n = 6; Aphi: n = 4; Aphi wash: n = 6) (d) Quantification of the area from all the buds represented in (a). (e) Whole mount in situ of Shh and E-Cad immunostaining in E13 incisors cultured for 40 h with DMSO or aphidicolin. (n = 3 per condition). Dashed line outlines the incisor. Scale bar, 25 µm. epi, epithelium; mes, mesenchyme. Representative 2D central plane shown in all images. Data are represented as mean ± s.e.m. Statistical analysis was done using the 2 tailed Mann-Whitney test for d. n represents number of embryos; one incisor measured per embryo.

Source data

Extended Data Fig. 6 Dextran-induced compression regulates incisor area and EK localization.

(a) Images showing the morphology of E12 incisor buds treated with DMSO or aphidicolin for 40 h with varying concentrations of dextran (30 mg/ml and 60 mg/ml). (b) Quantification of the area (outlined in yellow dashed lines) from all the buds represented in (a) (Ctrl: n = 3, 5 and 4 and Aphi: n = 5, 5 and 5 for Dextran 0, 30 and 60 mg/ml respectively). (c) EdU and E-Cad immunostaining after a 2 hour EdU chase in E12 incisors cultured for 40 h with DMSO or dextran (30 mg/ml). Quantification of EdU positive cells from these and incisors cultured in DMSO, or 30, 60, 80 mg/ml dextran. (n = 6, 5, 4 and 4 respectively) (d) Representative images of E12 mandible explants cultured for 40 h with DMSO or aphidicolin + dextran (30 mg/ml) (n = 6 and 5 respectively). Explants are outlined in red with the center marked by a red dot. Quantification of the relative distance of the EK from the center of the tissue in E12 incisors cultured with DMSO or aphidicolin with 30 or 60 mg/ml dextran. (n = 6, 5, 5 and 5 respectively) (e) Quantification of relative area of Shh expression to incisor area in E12 incisors cultured with DMSO or aphidicolin with 30 or 60 mg/ml dextran. (n = 7, 6, 6 and 5 respectively). Dashed line outlines the incisor. epi, epithelium; mes, mesenchyme. Scale bar, 25 µm. Representative 2D central plane shown in all images. Data are represented as mean ± s.e.m. Statistical analysis was done using the 2 tailed Mann-Whitney test for b and 1 way ANOVA with Bonferroni’s correction for multiple comparisons for c, d and e. n represents number of embryos; one incisor measured per embryo.

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Extended Data Fig. 7 The expression patterns of EK markers Bmp4 and Wnt10a are affected by compressive stress.

(a, b, c) RNAscope for (a) Pax9, (b) Bmp4 and (c) Wnt10a in E12 incisors after 40 h culture with DMSO, aphidicolin throughout, aphidicolin washed out after 16 h (Aphi wash) or aphidicolin + 30 mg/ml dextran throughout (Dxt 30 mg/ml + Aphi) (n = 3). Dashed line outlines the incisor. epi, epithelium; mes, mesenchyme. Representative 2D central plane shown in all images. Scale bar, 25 mm. n represents number of embryos; one incisor measured per embryo.

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Combined single statistical source data excel file for all plots in the manuscript main and extended data figures with clearly named tabs for each figure/extended data figure item.

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Shroff, N.P., Xu, P., Kim, S. et al. Proliferation-driven mechanical compression induces signalling centre formation during mammalian organ development. Nat Cell Biol 26, 519–529 (2024). https://doi.org/10.1038/s41556-024-01380-4

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