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

Nature Cell Biology volume 17, pages 397–408 (2015)Cite this article

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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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Figure 1: Light-sheet microscopy set-up to study gastrulation in chicken embryos.
Figure 2: Tissue dynamics using PIV analysis.
Figure 3: Analysis of strain rates during streak formation.
Figure 4: Analysis of cell behaviours controlling gastrulation.
Figure 5: Spatiotemporal localization of phosphorylated Mlc (pMlc) cables.
Figure 6: Effect of inhibition of conventional and non-conventional myosins on streak formation.
Figure 7: Effects of siRNA-mediated myosin I, II and V downregulation.
Figure 8: Model of the forces and cell behaviours controlling streak formation.

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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).

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Author notes

  1. Emil Rozbicki, Manli Chuai and Antti I. Karjalainen: These authors contributed equally to this work.

Authors and 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.

Corresponding authors

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

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Streak formation, expansion contraction dynamics and cell behaviour of a different sample embryo.

(a) Images showing the formation of the streak on top images of the embryo as a function of time. (b) Time sequence of the expansion/contraction map. (c) Time sequence of the isotropic and anisotropic strain rates. Symbols and meaning are the same as for the embryo as shown in fig 3a. White scale bar indicates size in panels a, b and c is 200 μm. In panel c the white scale bar also indicates a strain rate of 10−4/sec, as well as a domain displacement speed of 4 μm/min.

Supplementary Figure 2 Alignment of contraction expansion maps of 9 embryos.

This result of averaging of the contraction expansion maps of nine embryos. The embryos were aligned and synchronised according to developmental time as described in detail in methods. The main features such as the initiation of contraction in the central mesendoderm followed by outward expansion of this contraction domain followed by streak formation and deformation of the area in front of the streak appear highly conserved. The black scale bar in the first image is 200 μm.

Supplementary Figure 3 Changes in cell volume and cross section area during early development.

(a) Changes in cellular cross-sectional area over time. The average cell diameters were calculated from the segmented images in small domains and colour coded as indicated in the legend. Initially the average cross-sectional area of cells is smallest in the area pellucida. Over time the average cross-sectional area is decreasing in all the regions in the embryo. Size of each coloured square is initially 6,600 μm2. The white scale bar is 200 μm. (b) Relative average cell volume of rounded cells prior to cytokinesis (red line) and relative average apical cell diameter (green line) in the posterior domain (B) in the sickle region shown in a. It is seen that cell volume stays essentially constant, but that cross-sectional area decreases considerably. (c) Same volume and cross-sectional area measurements for the anterior domain (C) shown in panel a.

Supplementary Figure 4 Frequency of cell divisions and alignment of cell division axes.

(a) Number of cell divisions in the four domains shown in figure 4a as a function of time, showing that cell doubling times in all domains are similar (∼6 h). (b) Number of cell divisions as a function of time of the embryo shown in C. (c) Histograms showing the distributions of the cell division axes occurring in domains in different parts of the embryo. Initially the cell divisions axes in the posterior parts of the embryo are aligned perpendicular to the midline of the embryo (0–4.3 h). From 4.4 h of development onwards this polarity is gradually lost. All histograms contain the total number of divisions occurring over a 2.1 h period in the domain. Scale bar is 200 μm.

Supplementary Figure 5 Alignment of polarised cells in the embryo.

(a) Alignment of cells with a shape eccentricity of >0.87 in various domains before and during streak formation. The percentages show how many cells in each domain have eccentricity >0.87. It is evident that all ready at the beginning of development before the onset to motion (1 h) cells in the posterior Area Opaca are aligned perpendicular to the direction of the forming streak and that the number of polarised cells increase during the onset of motion (4.2 h) after which their fraction and orientation decreases again (7.3 h). Scale bar in each panel is 200 μm. (b) Tissue texture tensor (red bars). The tissue texture tensor is calculated from centres of mass of all cells in a given domain as described in detail in methods. The length and spatial orientation of the tensors shows that cells in the posterior embryo are initially aligned along their long axis. With the onset of streak formation the cells become smaller and the alignment becomes less pronounced supporting the data shown in fig S3a, S5a. White scale bar is 200 μm. The yellow scale bar corresponds to the tensor magnitude of 200 μm2 and a domain speed 4 μm/min.

Supplementary Figure 6 Effects of inhibitors on development and Myosin light chain phosphorylation.

(a) Left panels phosphorylated Myosin light chain (green) and actin staining (red) for a control embryo incubated for 6 h in-ovo followed by 2 h in EC culture, middle panels, an embryo incubated for 6 hours in-ovo followed by 2 h treatment with 50 μM of the Rho kinase inhibitor H1152 in EC culture, resulting in a partial inhibition of Myosin regulatory light chain phosphorylation, right panels an embryo incubated for 6 hours in-ovo followed by 2 h treatment with 50 μM of the Blebbistatin in EC culture showing only very limited inhibition of Myosin light chain phosphorylation. (b) Upper panel, embryo developed for 16 h in the presence of 50 μM Blebbistatin showing a streak indistinguishable in shape from control embryos. Lower panel an embryo developed in the presence of 50 μM, H1152 showing a characteristic short wide streak. (c) Myosin Ib (green) and actin (red) staining in the posterior epiblast in an embryo incubated for 6 h in ovo, followed by either 2 h in EC culture, or 2 h in EC culture containing 5uM PCP. (c) Myosin Va (green) and actin (red) staining in the posterior epiblast in an embryo incubated for 6 h in ovo, followed by either 2 h in EC culture, or 2 h in EC culture containing 5uM PCP. The results show are representative for several experiments Blebbistatin (control n = 11 embryos/10 streaks/3 experiments, Blebbistatin 10 streaks/12 embryos/3 experiments), HH1152 experiments (control n = 35 streaks/38 embryos/5 experiments, HH1152 n = 28 streaks/33 embryos/5 experiments). White Scale bars in a,c,d, 25 μm. White scale bar in b 1 mm.

Supplementary Figure 7 Phospho Myosin light chain localization in selective junctions is not reflected in Par3, PDZ RhoGef (AHRGef11) or phospho-Ezrin localization.

(a) Phospho-Myosin regulatory light chain shows only partial co-localization with Myosin IIb, (similar results for IIa are not shown), suggesting that only a fraction of Myosin IIb is assembled into mini-filaments in apical junctions. (b) Phospho-Myosin light chain is only found in selective junctions, which are organised in a super cellular pattern in mesendoderm cells. (c) Par3, PDZRhoGef (AHRgef11) and phospho-Ezrin do not show an asymmetric cellular localization in mesendoderm cells. The figures are representative of at least 2 experiments per antibody typically between 10 embryos /experiment. White scale bar 25 μm.

Supplementary information

Supplementary Information

Supplementary Information (PDF 4480 kb)

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. (AVI 47301 kb)

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. (AVI 34865 kb)

Example of cell ingression.

Cell ingression as described in text and shown in Fig. 1G. (AVI 4141 kb)

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. (AVI 2922 kb)

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. (AVI 15109 kb)

Deformation visualised on a dynamic grid.

Colours indicate contraction (shades of blue) and expansion (shades of red). (AVI 27070 kb)

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. (AVI 35980 kb)

Averaged contraction expansion maps for 9 embryos.

Video shows full data set of the embryos shown in figure S2. (AVI 2554 kb)

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. (AVI 31134 kb)

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. (AVI 6875 kb)

Another example of contracting apical junctions.

Conditions as described in legend to Video S10. (AVI 17615 kb)

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. (AVI 46686 kb)

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. (AVI 37667 kb)

Dynamics changes in the Expansion/Contraction map for Myosin I inhibitor treatment experiment. (AVI 14655 kb)

Immediate inhibition of junctional contraction after addition of 5 μM pentachloropseudilin. (AVI 9995 kb)

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. (AVI 32900 kb)

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Rozbicki, E., Chuai, M., Karjalainen, A. et al. Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation. Nat Cell Biol 17, 397–408 (2015). https://doi.org/10.1038/ncb3138

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