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Supplementary Figure 4 | Effect of manipulations of myosin activity, cadherin levels, cadherin isoforms and ephrin-Eph signaling on separation. (a) Effect of combined manipulations on separation of mesoderm explants from ectoderm BCR. EphA4 depletion in the mesoderm inhibited separation by about 50%. Combining EphA4 depletion with C-cadherin overexpression (Cad) did not reinforce the phenotype, despite the fact that the latter manipulation should dampen adhesive differences between the two tissues. Similarly, inhibition of separation by ephrin depletion in the BCR was not increased by simultaneous cadherin depletion (CcadMO), another condition that should level adhesive differences. (b) Induction of ectoderm-ectoderm separation. Endogenous mesoderm explants were used as a positive control (red). Stimulation of ephrin-Eph signaling, either by expression of ephrinB2 and EphA4, which are normally expressed in the mesoderm, or by treatment of explants with soluble ephrin-Fc yielded the strongest separation. Decreasing cadherin levels (CcadMO), decreasing respectively increasing tension by myosin depletion (MHCMO) or caRho expression, or substituting C- cadherin with E- or N-cadherin all caused some degree of separation. However, in all cases, separation was at least as strong when the same manipulation was performed simultaneously on the explants and on the BCR, indicating that the effect was not due to tissue differences, unlike predicted by DAH, DITH or SAH. Graphs show mean values, error bars s.d. The numbers on top corresponds to the number of explants that remained separated/total explants (10 explants per independent replicate). Individual comparisons to control mesoderm, control ectoderm, or eprhinB2+EphA4 (respectively red, blue and purple 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 |