Abstract

The physical forces that drive morphogenesis are not well characterized in vivo, especially among vertebrates. In the early limb bud, dorsal and ventral ectoderm converge to form the apical ectodermal ridge (AER), although the underlying mechanisms are unclear. By live imaging mouse embryos, we show that prospective AER progenitors intercalate at the dorsoventral boundary and that ectoderm remodels by concomitant cell division and neighbour exchange. Mesodermal expansion and ectodermal tension together generate a dorsoventrally biased stress pattern that orients ectodermal remodelling. Polarized distribution of cortical actin reflects this stress pattern in a β-catenin- and Fgfr2-dependent manner. Intercalation of AER progenitors generates a tensile gradient that reorients resolution of multicellular rosettes on adjacent surfaces, a process facilitated by β-catenin-dependent attachment of cortex to membrane. Therefore, feedback between tissue stress pattern and cell intercalations remodels mammalian ectoderm.

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Acknowledgements

We thank J. Zallen, Y. Bellaïche, C-P. Heisenberg, J. Gros and C-c. Hui for critical review of the manuscript. This work was financially supported by a March of Dimes Birth Defects Foundation Grant 1-FY10-366, and a Canadian Institutes of Health Research Grant MOP-126115 (S.H.).

Author information

Author notes

    • Kimberly Lau
    •  & Hirotaka Tao

    These authors contributed equally to this work.

Affiliations

  1. Program in Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, Toronto M5G 1X8, Canada

    • Kimberly Lau
    • , Hirotaka Tao
    • , Kendra Sturgeon
    • , Natalie Sorfazlian
    • , Savo Lazic
    • , Jeffrey T. A. Burrows
    • , Danyi Li
    • , Steven Deimling
    • , Brian Ciruna
    • , Ian Scott
    •  & Sevan Hopyan
  2. Department of Mechanical and Industrial Engineering, University of Toronto, Toronto M5S 3G8, Canada

    • Haijiao Liu
    • , Jun Wen
    • , Craig Simmons
    •  & Yu Sun
  3. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto M5S 3G9, Canada

    • Haijiao Liu
    • , Craig Simmons
    • , Rodrigo Fernandez-Gonzalez
    •  & Yu Sun
  4. Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada

    • Savo Lazic
    • , Danyi Li
    • , Brian Ciruna
    • , Ian Scott
    •  & Sevan Hopyan
  5. Mouse Imaging Centre, Hospital for Sick Children, Toronto Centre for Phenogenomics, Toronto M5T 3H7, Canada

    • Michael D. Wong
    •  & R. Mark Henkelman
  6. Department of Medical Biophysics, University of Toronto, Toronto M5T 3H7, Canada

    • Michael D. Wong
    •  & R. Mark Henkelman
  7. Program in Molecular Biology, School of Medicine, University of Colorado, Aurora, Colorado 80045, USA

    • Trevor Williams
  8. Developmental Biology Program, Sloan-Kettering Institute, New York 10065, USA

    • Anna-Katerina Hadjantonakis
  9. Cell and Systems Biology, University of Toronto, Toronto M5G 3G5, Canada

    • Rodrigo Fernandez-Gonzalez
  10. Division of Orthopaedic Surgery, Hospital for Sick Children and University of Toronto, Toronto M5G 1X8, Canada

    • Sevan Hopyan

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Contributions

K.L., H.T. and S.H. designed the experiments. H.T., H.L., C.S. and Y.S. performed and analysed AFM experiments. J.W. performed FEM. K.L., H.T., K.S. and S.H. performed and analysed live imaging and immunofluorescence experiments. N.S. performed cell neighbour analysis. K.L., K.S., S.L., J.T.A.B. and I.S. performed and analysed injection experiments. M.D.W. and R.M.H. performed OPT experiments. D.L. analysed cell division plane and rosette remodelling data. S.D. performed measurements of lateral plate ectoderm. S.H. and B.C. performed zebrafish experiments. T.W. provided Crect mice. A-K.H. provided CAG::myr–Venus and Tcf/Lef::H2B–Venus mice. K.L. and R.F-G. performed and analysed laser ablation experiments. R.F-G. provided SIESTA software and MATLAB scripts. S.H. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sevan Hopyan.

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Videos

  1. 1.

    DV movement of Tcf/Lef::H2B-Venus-positive cells in ventral ectoderm of a 23 som. stage limb bud.

    Shown is a lateral view of the posterior half of the limb bud. Anterior is to the left, dorsal is down. Time course is 165 min, shown in 5 fps. Evidence of convergence by intercalation can be seen, as can DV-biased cell division planes. Over a longer time period (nearly 1 day), these cells will progressively crowd just ventral to the DV midline to form the AER.

  2. 2.

    Tcf/Lef::H2B-Venus-positive cells at 23 som. stage intercalate just ventral to the dorsoventral boundary (in our estimation).

    Anterior is to the left; dorsal is down. Time course is 165 min, shown in 5 fps.

  3. 3.

    Low magnifcation time lapse movie demonstrating interdigitation of Tcf/Lef::H2B-Venus-positive cells (precipitated by division of the light blue cell) near the DV midline of a 20 som. stage limb bud.

    Anterior is to the left, dorsal is downward, and ventral is upward. Time course is 355 min, shown in 5 fps.

  4. 4.

    Wild type 20 som. myr-Venus transgenic embryo with a dorsally biased view of anterior half of limb field (left half) and lateral plate ectoderm (right half).

    Polygonal, anisotropic cell shapes dominate, and multiple examples of cell division and cell neighbour exchange are apparent. Anterior is to the right and dorsal is downward with the DV midline 2/3 up from the base of the visible field.

  5. 5.

    NSC23766 treatment of 22 som. stage myr-Venus embryo demonstrates transition from elongated cell shapes to more isotropic shapes.

    Diminished cell neighbour exchange is also apparent. Time course is 75 min, shown in 5 fps.

  6. 6.

    Protrusive membrane activity of ectodermal cells in 22 som. limb bud expressing myr-Venus in a mosaic fashion.

    Yellow arrowhead indicates membrane protrusion. To the left, a relatively dark cell intercalates among two bright cells. Anterior is to the left and dorsal is downward. Time course is 180 min, shown in 5 fps.

  7. 7.

    Relaxation of principal stress vectors during transient 300 s viscoelastic simulation for 17 som. stage limb bud initiation based on mesodermal growth.

    All principal stresses are relaxed until the material property reaches the asymptote. Same relaxation applies to the stress field (not shown).

  8. 8.

    Daughter cells (blue) in mouse embryonic ectoderm (20 som.) frequently sever the interface between them immediately following division and precipitate neighbour exchange.

    Anterior is left; dorsal, down. Time course is 149 min, shown in 5 fps.

  9. 9.

    A daughter cell (blue) intercalates between neighbouring cells following division (20 som.).

    Anterior is left; dorsal, down. Time course is 149 min, shown in 5 fps.

  10. 10.

    A daughter cell (pink) precipitates rosette formation by intercalating between cells and contributes to the central apex (20 som.).

    Anterior is left; dorsal, down. Time course is 83 min, shown in 5 fps.

  11. 11.

    A large multicellular rosette resolves into two rows of cells by dissolving its central apex and generating new intercellular junctions (20 som.).

    Anterior is left; dorsal, down. Time course is 123 min, shown in 5 fps.

  12. 12.

    Relaxation of the stress field during transient 300 s viscoelastic simulation for 22 som. stage pre-AER limb bud due to mesodermal growth.

  13. 13.

    Relaxation of the stress field during transient 300 s viscoelastic simulation for 22 som. stage pre-AER limb bud due to an ectodermal pulling force at the DV midline.

  14. 14.

    Precise laser ablation of a single AP interface (that is parallel to the DV axis) results in relatively fast initial recoil of adjacent apices along the DV axis (21 som.).

    Anterior is left. Time course is 132 sec, shown in 5 fps.

  15. 15.

    Precise laser ablation of a single DV interface (that is parallel to the AP axis) results in relatively slow initial recoil of adjacent apices along the AP axis (21 som.).

    Dorsal is left. Time course is 132 sec, shown in 5 fps.

  16. 16.

    Laser ablation of multiple interfaces between the coloured rosette and the prospective AER (to the right) results in resolution of the rosette along the AP axis (up/down) rather than toward the AER (21 som.).

    Anterior is up; dorsal is left. Time course is 192 sec, shown in 5 fps.

  17. 17.

    βcatf/f;Crect mutant daughter cells maintain a long interface following division. Anterior is left; dorsal, down (20 som.).

    Time course is 135 min, shown in 5 fps.

  18. 18.

    βcatf/f;Crect mutant rosette resolves with minimal angular change of its long axis.

    Anterior is left; dorsal, down (20 som.). Time course is 140 min, shown in 5 fps.

  19. 19.

    Live R26R::Venus-Actin reporter in vivo shows oscillatory cortical contractions in wild type embryo.

    Anterior is left; dorsal, down. Time course is 120 min, shown in 5 fps.

  20. 20.

    Live R26R::Venus-Actin reporter in vivo shows diminished amplitude and rate of change of oscillatory cortical contractions in βcatf/f;Crect mutant embryo (21 som.).

    Anterior is left; dorsal, down. Time course is 120 min, shown in 5 fps.

  21. 21.

    Tcf/Lef::H2B-Venus positive cells in Fgfr2f/f;Crect mutant limb bud ectoderm move along the DV axis, albeit less efficiently than in WT (21 som.).

    Anterior is left; dorsal, down. Time course is 120 min, shown in 5 fps.

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https://doi.org/10.1038/ncb3156

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