Article | Published:

Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation

Nature Cell Biology volume 17, pages 397408 (2015) | Download Citation

Abstract

Primitive streak formation in the chick embryo involves large-scale highly coordinated flows of more than 100,000 cells in the epiblast. These large-scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combination of light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical–basal axis followed by ingression, and asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localization and direction of which correlate strongly with the appearance of active myosin II cables in aligned apical junctions in neighbouring cells. Use of class specific myosin inhibitors and gene-specific knockdown shows that apical contraction and intercalation are myosin II dependent and also reveal critical roles for myosin I and myosin V family members in the assembly of junctional myosin II cables.

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Acknowledgements

We thank E. Stelzer and P. Keller for advice on the construction of LSMs, and A. Sherman, F. Thomson, M. Hutchison and R. Mitchell for support in generation and breeding of the transgenic chick line and supplying the fertilized transgenic eggs. This work was supported by BBSRC (BB/E011276/1) to H.M.S. and C.J.W., (BB/G015082/1) and to C.J.W. and M.P.M., Institute Strategic Grant funding to H.M.S., Wellcome Trust (094131/Z/10/Z) to C.J.W. RNA sequencing was carried out by Edinburgh Genomics, The University of Edinburgh. Edinburgh Genomics is partly supported through core grants from NERC (R8/H10/56), MRC (MR/K001744/1) and BBSRC (BB/J004243/1).

Author information

Author notes

    • Emil Rozbicki
    • , Manli Chuai
    •  & Antti I. Karjalainen

    These authors contributed equally to this work.

Affiliations

  1. Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK

    • Emil Rozbicki
    • , Manli Chuai
    • , Antti I. Karjalainen
    •  & Cornelis J. Weijer
  2. The Roslin Institute, Royal (Dick) School of Veterinary Sciences, University of Edinburgh, Edinburgh EH25 9RG, UK

    • Feifei Song
    •  & Helen M. Sang
  3. Department Chemie, Technische Universität, Dresden, Bergstrasse 66, 01069 Dresden, Germany

    • René Martin
    •  & Hans-Joachim Knölker
  4. Division of Physics, School of Engineering, Physics and Mathematics, University of Dundee, Dundee DD1 4HN, UK

    • Michael P. MacDonald
  5. Institute for Medical Science and Technology, School of Medicine, University of Dundee, Dundee DD2 1FD, UK

    • Michael P. MacDonald

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Contributions

E.R. built the LSM hardware and software, performed experiments and PIV-based data analysis. M.C. performed the biological and myosin perturbation experiments. A.I.K. developed the cell-based image analysis software and analysed experiments. F.S. and H.M.S. developed the Myr-GFP embryos. R.M. and H-J.K. developed and produced the myosin inhibitors. C.J.W. and M.P.M. conceived the design and use of the LSM in the investigation of chick development.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Helen M. Sang or Michael P. MacDonald or Cornelis J. Weijer.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Information

    Supplementary Information

Videos

  1. 1.

    Development from EGXXII to HH3.

    The Image shown is a Z plane constructed from 2500 slices, 2560x400 voxels taken at 1.88 um intervals, every 2.5 minutes. Small inserts show sequences at full resolution from the regions marked in colours in the main image. The data represent 286 time points of one Z plane through the middle of the embryo shown in Fig 1C, taken every 2.5 minutes. The data of this experiment as well as some of the results of the calculations can be seen at full resolution at DOI http://dx.doi.org/10.15132/10000100, they can be viewed at full resolution without downloading the primary data the Omero webbrowser. In the viewing option of the web browser the zoom can be selected as well as the data quality. By selecting the “Show ROIs” option in the bottom of the Viewing option panel the outline of the anterior middle and posterior streak can be seen as overlays over the data.

  2. 2.

    Cross section of images taken from the embryo shown in Video 1 through the developing primitive streak.

    The upper panel shows the full extent of the embryo, the lower panel shows a section around the streak tip at full resolution.

  3. 3.

    Example of cell ingression.

    Cell ingression as described in text and shown in Fig. 1G.

  4. 4.

    Example of cell division.

    Example of a cell division. Note the fact that after division the daughter cells are separated by some of the neighbour cells resulting effectively in an intercalation event, which is observed frequently.

  5. 5.

    Fate mapping of streak forming area.

    Red dots indicate the outline of the streak, blue dots indicate the area pellucida of the experiment shown in Video S1.

  6. 6.

    Deformation visualised on a dynamic grid.

    Colours indicate contraction (shades of blue) and expansion (shades of red).

  7. 7.

    Another example of streak formation, PIV based tissue dynamics and strain rate analysis.

    First and second panels illustrate primitive streak formation using the same notation as in figures 2A and 2B, respectively. The last panel illustrates strain rate tensor as in figure 3A. Each panel shows 10 hours on development.

  8. 8.

    Averaged contraction expansion maps for 9 embryos.

    Video shows full data set of the embryos shown in figure S2.

  9. 9.

    Tissue strain rate tensor changes during streak formation.

    Video illustrates the full temporal data set of the isotropic and anisotropic strain rate changes shown in figure 3A and calculated as described in methods.

  10. 10.

    Sequential contraction of apical junctions.

    Sequential contraction of junctions coloured red bring two cells (initially 3 cells apart) indicated by red and blue dots together. The cells are taken from within the sickle area.

  11. 11.

    Another example of contracting apical junctions.

    Conditions as described in legend to Video S10.

  12. 12.

    Cell based analysis of tissue deformation.

    Left hand panel shows the isotropic (circles) and anisotropic strain rate tensor (blue bars). Right hand panel shows the combined cell deformation (green bars) and cell rearrangement tensor (blue bars) calculated as described in detail in methods. Video illustrates the full time course of data illustrated in figure 4G.

  13. 13.

    Development after addition of 5 μM pentachloropseudilin.

    Embryos were allowed to develop for 2 h after which time 5 μM pentachloropseudilin was added. As can be seen this results in an initial relaxation followed by a loss of adhesion of the outer cells to the vitelline membrane and a contraction of the embryo.

  14. 14.

    Dynamics changes in the Expansion/Contraction map for Myosin I inhibitor treatment experiment.

  15. 15.

    Immediate inhibition of junctional contraction after addition of 5 μM pentachloropseudilin.

  16. 16.

    Streak formation is inhibited following Myh9/Myh10 (Myosin IIa/IIb) siRNA transfection.

    Initially the embryo starts to develop normal vortex flows however from 0 h after transfection the embryo starts to show severe aberrations in its development. The tissue flows stop and the surface of the embryo starts to buckle and show irregular contractions.

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