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

Nature Cell Biology volume 18, pages 1281–1291 (2016)Cite this article

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Abstract

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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Figure 1: Crumbs2 is required for mammalian gastrulation.
Figure 2: Crumbs2 promotes cell ingression at the primitive streak.
Figure 3: Live imaging defines defects in cell ingression in Crumbs2 mutants.
Figure 4: Streak-autonomous and non-cell-autonomous activities of Crumbs2.
Figure 5: CRB2 enrichment at the primitive streak.
Figure 6: CRB2 regulates the distribution of myosin IIB.
Figure 7: CRB2 is required for epiblast integrity only when cells delaminate at the streak.

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Acknowledgements

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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  1. Nitya Ramkumar

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

Authors and Affiliations

  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. Arthur and Sonia Labatt Brain Tumor Research Center and Department of Medical Biophysics, The Hospital for Sick Children, 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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Contributions

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.

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Integrated supplementary information

Supplementary Figure 1 Expression of Crumbs2 RNA and CRB2 protein in the epiblast.

(A) Whole mount in situ hybridization for Crumbs2 RNA expression in a wild-type E7.5 embryo, n = 6 wild type embryos. (B,C) Transverse section through the wild-type embryo at E7.5 (B) and E7.75 (C) after in situ hybridization for Crumbs2, showing its apical localization and posterior enrichment in the epiblast. Apical enrichment of Crumbs RNA has also been seen in Drosophila follicle cells54. (D,D ′) Single optical section from whole mount E6.75 embryo immunostained for CRB2 (green) and E-cadherin (red), showing the posterior enrichment of CRB2 (D) and (D ′) its sub-cellular localization apical to E-cadherin in a higher magnification view of the boxed region in (D), n = 7 wild type embryos. CRB2 was localized to an apical domain of the epiblast that overlapped with apical F-actin and in caps apical to the adherens junctions (see also Fig. 6b, b′). Neither Crumbs2 RNA nor CRB2 protein was detected in the wild-type endoderm or mesoderm. Scale bars A–C, 75 μm; D,D ′, 50 μm. The images shown here are representative.

Supplementary Figure 2 Distinct gastrulation defects in Crumbs2 and Rac1 mutants.

(A) Single optical transverse sections of E8.0 wild type and Crumbs2 mutant embryos immunostained for Laminin (green) and E-cadherin (red), showing broader streak (marked by the break in Laminin) and accumulation of E-cadherin positive cells in the mutant streak, n ≥ 15 mutant embryos. (B) Single optical section from transverse section of E7.75 wild-type and Rac1 epiblast-specific deletion embryos stained for SNAIL1, Phalloidin and Laminin, showing the accumulation of SNAIL1-expressing cells in the Rac1 conditional primitive streak, n = 3 mutant embryos. Scale bars A, 50 μm; B, 20 μm.

Supplementary Figure 3 Mesoderm defects in epiblast-specific deletion of Crumbs2.

(A) In situ hybridization shows the expression of Brachyury, Meox1 and Uncx4.1 at E8.5, n ≥ 3 embryos per genotype. Brachyury expression shows the reduced number of axial mesoderm cells in the discontinuous midline of the mutant. Meox1 expression shows reduction in paraxial mesoderm in the mutant. Striped Uncx4.1 expression shows that somitogenesis clock is active although the mutants do not form normal somites. (B) The progression of Brachyury (T) expression in the axial mesoderm, visualized by whole mount immunostaining, n ≥ 3 mutant embryos per stage. The T expression pattern appears relatively normal at E7.5; after that time it appears that the axial mesoderm extends in the anterior-posterior axis but new cells fail to be added to the axial mesoderm, leading to a thin, broken line of Brachyury-positive cells by E8.5. Scale bar A, 150 μm; B, 110 μm. The images shown here are representative.

Supplementary Figure 4 Crumbs2 chimeric embryos.

(A) Whole mount image of a low contribution chimera that resembled an E8.5 wild-type embryo, n = 20 chimeric embryos. (B) Whole mount image of a high contribution chimera that recapitulated the phenotype of Crumbs2 mutants, n = 9 chimeric embryos. Percentage chimerism was determined in sections. (C) En face view of the anterior epiblast/early neural epithelium of chimeric embryos at E8.5 immunostained for CRB2 (red) and β-catenin (gray), n = 5 chimeric embryos. Mutant cells are GFP-positive (green). Arrows point to the lack of CRB2 expression in wild-type cells at the edges shared with mutant cells, whereas β-catenin expression is maintained at these edges. Scale bar in A,B, 150 μm; C, 10 μm. The images shown here are representative.

Supplementary Figure 5 Geometry and distribution of apical CRB2 in cell edges of the primitive streak region.

Histograms of cell shape distributions at the primitive streak. (A) Cells at the primitive streak have a variety of cell shapes; the most common cells have 4–6 edges. (B) Percentage of the polygons with 3, 4, 5 or 6 edges, and the number of their edges enriched with CRB2, documenting the nature of its anisotropic distribution at the streak. n = 3 embryos, ∼50 cells per embryo; total = 176 cells.

Supplementary Figure 6 Localization of PatJ, a Crumbs complex protein, Crumbs2 and Myosin IIB in the early embryo.

(A) Extended projection en face view of immunostaining for PatJ and ZO1, n ≥ 5 wild type embryos. In the streak region of an E8.0 wild-type embryo. PatJ is distributed anisotropically on cell edges, with some strong apical puncta, and a complex pattern of enrichment at cell edges, similar to the pattern of CRB2 expression at the streak (Fig. 7c). (B) PatJ is not detectable at the streak of Crumbs2 or Poglut1wsnp mutants, n ≥ 3 mutant embryos per genotype. (C,D) Reciprocal localization of Crumb2 and Myosin IIB in the early embryo and in the epiblast adjacent to the streak. (C) Extended projection view of wild-type streak at early bud stage (∼E7.0). Note the striking mulitcellular rosette with high Myosin IIB at the vertex (arrow), which could have surrounded an extruded cell. Also note cells with constricted apical surfaces with high CRB2 surrounded by high myosin (yellow arrows). (D) CRB2 (red) and Myosin IIB (green) are anisotropically distributed in the side epiblast of wild type (B) at E8.5. Proximal is up, n ≥ 5 wild type embryos per stage (C,D). Scale bar in A,B, 25 μm; C,D, 20 μm. The images shown here are representative.

Supplementary Figure 7 Crumbs2 phenotypes in the E7.75-E8.0 anterior epiblast.

(A) Transverse sections through E8.0 wild-type and Crumbs2 embryos immunostained for pHH3 (mitotic cells) and acetylated-tubulin. Anterior is to the left. Arrows point to examples of non-apical pHH3 positive nuclei. The mitotic indices were not significantly different (WT = 9.924 ± 0.9685 (mean ± s.e.m.), n = 3 embryos (2 sections/embryo) and Crumbs2 mutant = 7.002 ± 0.5897 (mean ± s.e.m.), n = 3 embryos (2–3 sections/embryo), P-value = 0.02, not significant), although the percentage of non-apical mitoses (arrows) was significantly higher in the mutants; non-apical mitosis in wild type = 2.6% and Crumbs2 mutants = 23.5%. Scale bar, 40 μm. (B) Transverse sections through the epiblast of E7.75 wild type and Crumbs2−/−mutant embryos immunostained for aPKCλ and Par3 (green), n = 3 embryos per genotype (2–3 sections per embryo); apical markers are localized correctly in the mutants despite the thicker epiblast layer (DAPI, blue). Scale bars, A, 40 μm; B, 10 μm. The images shown here are representative.

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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. (MOV 4161 kb)

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. (MOV 2943 kb)

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. (MOV 4447 kb)

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. (MOV 4621 kb)

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. (MOV 3630 kb)

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. (MOV 5644 kb)

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Ramkumar, N., Omelchenko, T., Silva-Gagliardi, N. et al. Crumbs2 promotes cell ingression during the epithelial-to-mesenchymal transition at gastrulation. Nat Cell Biol 18, 1281–1291 (2016). https://doi.org/10.1038/ncb3442

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