Characterization of ephrin–Eph specificity.

<p>(A) Identification of functional cognate receptors. The in vitro separation assay was performed using two explants from the same tissue (ectoderm–ectoderm or mesoderm–mesoderm). Under normal conditions these explants mix completely. Separation could be induced when these explants were exposed to soluble Fc fragments corresponding to ephrins or Ephs normally enriched in the other tissue (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001955#pbio-1001955-g001" target="_blank">Figure 1C</a>), thus mimicking the endogenous asymmetric ephrin/Eph expression. The endogenous functional partners were then identified by depleting single candidate ephrins/Ephs in the receiving explant and determining which of them was required for ectopic separation. Note that in this set of experiments, Fc fragments were present during the assay, ensuring that both explants were continuously exposed. Separation can also be induced by treating only one explant, although the penetrance is lower <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001955#pbio.1001955-Rohani1" target="_blank">[4]</a>. (A) Separation of ectoderm explants was induced by soluble Fc fragments corresponding to “mesoderm-specific” ephrinB2, but not ephrinB1, already endogenously enriched in the ectoderm. EphrinB2-Fc–induced separation was strongly inhibited by EphB4MO and more weakly by EphB2MO. (A′) Mesoderm aggregates were tested on large mesoderm layers artificially produced in the animal pole of the embryo (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001955#s4" target="_blank">Materials and Methods</a>). Separation was induced by Fc fragments of “ectoderm-specific” ephrinB3, but not ephrinB2. Ephrin3-induced separation was reversed by EphA4MO but not by EphB4MO (red arrow). (A″) Ectopic separation between ectoderm explants was induced specifically by “mesodermal” EphA4 but not by “ectodermal” EphB4. Separation was inhibited only by ephrinB3MO (red arrow). (A″′) Examples of mixing of ectoderm explants incubated with control Fc (arrows) and of separation of ectoderm explants treated with EphA4-Fc (arrowheads). (B) Biochemical analysis of differential activation of Eph receptors by ephrin ligands. Ectoderm explants were treated with 40 nM ephrinB1, B2, B3 Fc fragments or with control anti-Fc antibodies for 1 h, and then lysed. Endogenous EphB2 and EphB4, and EphA4 were immunoprecipitated and analyzed by immunoblotting for phospho-tyrosine and for total Eph levels. In the case of EphA4, which is only expressed at low levels in the ectoderm, EphA4-GFP was ectopically expressed. EphA4 was strongly phosphorylated in response to both ephrinB2 and ephrinB3, but not ephrinB1, which gave levels similar to negative controls. EphB2 responded strongly to ephrinB2, and only weakly to ephrinB1. EphB4 was highly phosphorylated in response to ephrinB2, whereas activation by ephrinB1 or ephrinB3 was negligible. IgG, control immunoprecipitation with nonimmune IgGs. (C) Summary of the main ectoderm and mesoderm-enriched ephrins/Ephs and of the preferred functional interactions.