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. 2010 Nov 1;191(3):645–659. doi: 10.1083/jcb.201004074

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© 2010 Maghzal et al.

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Figure 1. — Identification of EpCAM as inhibitor of the ectoderm–mesoderm boundary. (A) Diagram of an early Xenopus gastrula (stage 10.5). Curved arrow indicates direction of mesoderm involution. (A′) Test for disruption of the ectoderm–mesoderm boundary (Brachet’s cleft) by dorsal injection of mRNA (green). (B and B′) Sagittal views of the dorsal region of embryos injected with control GFP mRNA (B) or EpCAM mRNA (B′). The boundary (arrowheads) is disrupted in the posterior region (arrow) of EpCAM-overexpressing embryos. Cryosections were stained with anti–C-cadherin antibody. Note that exposure has been increased for controls to match the staining intensity of EpCAM-expressing samples. For proper comparison of C-cadherin levels, see Fig. 3. (C) Ubiquitous expression of endogenous EpCAM in all three germ layers, ectoderm (ecto), mesoderm (meso), and endoderm (endo) of the early gastrula. Arrowheads point to Brachet’s cleft. (C′–C′′) Detail of ectoderm from cryosections of control MO (COMO) and EpCAM MO-injected embryos stained with anti-EpCAM antibody, demonstrating antibody specificity. (D–F) Reconstituted boundaries made of dissected wild-type mesoderm sandwiched between two injected BCRs, analyzed by cryosectioning and immunofluorescence. (D) Diagram of the assay. (E–E′′) Examples of a control boundary (E) and of the irregular interfaces observed between wild-type mesoderm and EpCAM-overexpressing BCR (E′, E′′). Membrane GFP was coexpressed as tracer. Cell contours were visualized using an anti–β-catenin (red) and injected ectodermal cells with anti-GFP ab (green). (F) Quantification of boundary straightness (see Materials and methods), represented in box plots (50% of the data are within the box, the median is represented by a horizontal line, the whiskers indicate the maximum and minimum value, without outliers, and the single dots the outliers). 1 corresponds to a perfectly rectilinear boundary, high values to convoluted lines, reflecting tissue mixing. EpCAM and its ΔE mutant caused significant mixing compared with controls (P < 0.001, Tukey-HSD test). Numbers on top represent number of fields/number of sandwiches (from three independent experiments).