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Crumbs2 promotes cell ingression during the epithelial-to-mesenchymal transition at gastrulation

Nature Cell Biology volume 18, pages 12811291 (2016) | Download Citation


During gastrulation of the mouse embryo, individual cells ingress in an apparently stochastic pattern during the epithelial-to-mesenchymal transition (EMT). Here we define a critical role of the apical protein Crumbs2 (CRB2) in the gastrulation EMT. Static and live imaging show that ingressing cells in Crumbs2 mutant embryos become trapped at the primitive streak, where they continue to express the epiblast transcription factor SOX2 and retain thin E-cadherin-containing connections to the epiblast surface that trap them at the streak. CRB2 is distributed in a complex anisotropic pattern on apical cell edges, and the level of CRB2 on a cell edge is inversely correlated with the level of myosin IIB. The data suggest that the distributions of CRB2 and myosin IIB define which cells will ingress, and we propose that cells with high apical CRB2 are basally extruded from the epiblast by neighbouring cells with high levels of apical myosin.

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We thank the MSKCC Molecular Cytology and Mouse Genetics Core Facilities for valuable technical support. We thank the Hadjantonakis laboratory for the X-linked GFP and GFP-GPI strains. We thank M. Lewandoski for Brachyury-Cre mice. We thank J. Zallen, A.-K. Hadjantonakis, A. Hall, I. Migeotte, H. Kakkar and members of the Anderson laboratory for their helpful suggestions. The work was supported by NIH R37 HD03455 to K.V.A. and the MSKCC Cancer Center Support Grant (P30 CA008748).

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Author notes

    • Nitya Ramkumar

    Present address: MRC Laboratory of Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK.


  1. Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA

    • Nitya Ramkumar
    •  & Kathryn V. Anderson
  2. Program in Biochemistry and Structural Biology, Cell and Developmental Biology, and Molecular Biology, Weill Cornell Graduate School of Medical Sciences, Cornell University, 1300 York Avenue, New York, New York 10065, USA

    • Nitya Ramkumar
  3. Cell Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA

    • Tatiana Omelchenko
  4. The Hospital for Sick Children, Arthur and Sonia Labatt Brain Tumor Research Center and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1X8, Canada

    • Nancy F. Silva-Gagliardi
    •  & C. Jane McGlade
  5. Department of Ophthalmology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands

    • Jan Wijnholds


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N.R. carried out the experiments; T.O. participated in and analysed the live imaging experiments. N.F.S.-G., C.J.M. and J.W. provided essential reagents and advice. N.R. and K.V.A. designed experiments, analysed the data and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Kathryn V. Anderson.

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  1. 1.

    Wild-type GFP streak cell translocates from the apical to the basal side of the epiblast.

    The epiblast cells randomly expressing GFP in the mouse embryo at E7.5 are imaged from the primitive streak side. A 3D rendered surface (yellow) is built for visualization of the individual cell shape changes. During ingression basal protrusions are formed, the cell body translocates basally and the cell leaves the epiblast in less than 2 h. Note apical process retraction. Time is h:min. Scale bar, 10 μm.

  2. 2.

    Another wild-type GFP streak cell ingresses from the apical to the basal side of the epiblast.

    The cell with basal protrusions constricts its apical membrane, moves toward basal plane and exits the epiblast. Time is h:min. Scale bar, 6 μm.

  3. 3.

    Cell shape dynamics of wild-type GFP streak cell during ingression.

    The ingressing cell shape highlighted by the 3D rendered surface is highly dynamic. During translocation, the initial cigar-shaped cell body changes to more discoid shape. Multiple protrusions accompanied the ingression. Time is h:min. Scale bar, 10 μm.

  4. 4.

    Mutant GFP streak cell does not translocate from the apical to the basal side of the epiblast.

    The epiblast cells randomly expressing GFP in the mouse mutant embryo at E7.5 were imaged from the primitive streak side. A 3D rendered cell surface shows the bottle-like shape of a Crumbs2−/− cell. During the imaging time, basal protrusions are formed and the cell body is located basally, but the cell does not leave the epiblast within 2 h. Note apical process does not retract. Time is h:min. Scale bar, 10 μm.

  5. 5.

    Another mutant GFP streak cell does not ingress from the apical side of the epiblast.

    During the time-lapse observation a 3D rendered cell (yellow) maintains its bottle-like shape. Note failure of the apical surface of the cell to retract highlighted by black plane in the upper part of the box. Time is h:min. Scale bar, 10 μm.

  6. 6.

    A group of Crumbs2 null mutant streak cells fail to ingress remaining attached to the apical side of the epiblast.

    Bottle-like shape of Crumbs2 null mutant cell with long thin apical extensions at the streak is persistent within multiple cells. Crumbs2 mutant cells accumulate at the streak. Note first frames of the video show cells without rendered cell surfaces (green). Time is h:min. Scale bar, 15 μm.

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