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Fig. 4. Impact of adhesive and contractile difference on ectoderm–mesoderm separation. a Scheme of the separation assay. Embryos injected at the 2 cell-stage were dissected at the early gastrula stage. Explants were laid on a BCR, and the number of explants remaining separated were scored. b Maintenance of ectoderm–mesoderm separation. Cell–cell adhesion was levelled by C-cadherin depletion in the ectoderm (morpholino injection, CcadMO), or overexpression in the mesoderm (mRNA injection, Ccad). Differences in contractility were levelled by depletion of MHC2A and B in the ectoderm (MHCMO) or by expression of constitutively active RhoA (mRNA injection, caRho) in the mesoderm. Ephrin-Eph signalling was inhibited by depletion of ephrinB1 (eB1MO) or EphA4 (A4MO), respectively, in the ectoderm or the mesoderm. c Example of the assay using a blastocoel roof induced to mesoderm as substrate (IMBCR). Ectoderm explants remain separated, whereas mesoderm explants sink into the IMBCR. d Induction of separation between mesoderm explants and IMBCR. Differences in cell–cell adhesion were imposed by C-cadherin overexpression (Ccad) and differences in contractility by expression of caRho. Ephrin-Eph signalling was stimulated by expression of ephrinB1 and B3 and simultaneous depletion of EphA4. Alternatively, signalling was activated at the surface of the mesoderm explants using preclustered ephrinB3-Fc soluble fragments (eB-Fc). Differences in cadherin expression were created by C-cadherin depletion (CcadMO) and its replacement with E-cadherin on one side and with N-cadherin on the other side. Ectoderm explants were used as a positive control (blue column). In an additional control, myosin activity was increased in both explant and substrate (caRho). The numbers on top corresponds to the number of explants that remained separated/total explants (10 explants per independent replicate). Graphs show mean values, error bars s.d. Individual comparisons to control mesoderm (red asterisks) were done using one-sided Student’s t-test 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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