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Supplementary Figure 5 | Effect of targeted manipulations on the endogenous ectoderm- mesoderm boundary. Animal or dorsal-equatorial injections at the 4 to 8-cell stage allowed to preferentially target either the ectoderm or the mesoderm, respectively. Injected cells were marked by expression of membrane-targeted GFP. Sagittal cryosections from gastrula embryos were immunostained for GFP and β-catenin, used to visualize cell outlines and analyze the morphology of the boundary. Note that this analysis is less unambiguous than the explant-based assay, because the appearance of the boundary requires normal gastrulation movements, in particular mesoderm involution. Thus a boundary phenotype may not necessarily be due to inhibition of the process of separation per se, but to other gastrulation defects. Interfering with adhesion or myosin activity are likely conditions that may have such global effects. This is also the reason why we routinely perform the explant separation assay using mesoderm from the early gastrula dorsal lip, i.e. just before the start of involution, thus insuring that the exact same region is tested, irrespective of potential later defects in mesoderm migration. (a-h) Representative examples. Arrowheads point to the boundary. Arrows point to boundary irregularities/defects. The normal boundary is never perfectly smooth, but displays sparse indents (a,e, arrows), consistent with temporary phases of reattachment during the cycles of ephrin- Eph-dependent repulsion. Ephrin/Eph depletion led to boundary defects that ranged from partial mixing (b) to its complete absence (f). Dampening adhesive differences either by cadherin depletion in the ectoderm or its overexpression in the mesoderm had little effect on the boundary (c,g). Likewise, the boundary would still form in embryos with myosin depleted-ectoderm or with caRho-expressing mesoderm (d,h). The only instances where cadherin or myosin manipulations appeared to impact on the boundary were those where both ectoderm and mesoderm tissues had been simultaneously targeted, indicating that these defects were not related to DAH/DITH. Panel c shows an example where the upper part of the boundary is intact and corresponds to a region where GFP, marker for cadherin MO injection, is mostly found in the ectoderm, while in the lower part both ectoderm and mesoderm have received cadherin MO, and the boundary is completely absent. (i-l) Details of boundaries. Asterisks in k and l mark cells with different degrees of rounded shape reflecting loss of cell-cell adhesion. (m) Example of caRho-expressing embryo with severe gastrulation defects. caRho-expressing mesoderm has failed altogether to involute. Ecto, ectoderm; AME, anterior mesendoderm; AxM, axial mesoderm; bl, blastocoel. (n) Summary of boundary phenotypes, scored in categories of increasing severity. Partially mixed (part mixed) indicates that portions of the boundary were lost while others appeared intact (e.g. panel b). Numbers of embryos are indicated in the columns. Embryos in which both tissues had GFP signal and embryos showing obvious involution defects as in panel m were not included. Image published in: Canty L et al. (2017) © The Author(s) 2017. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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