Mechanisms of Cross-talk Between EphrinB and Alternate Signaling Pathways

Division Of Basic Sciences - Nci

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Abstract

During normal development progenitor cells of many tissues undergo progressive restriction of pluripotency, epithelial-to- mesenchymal transition, proliferation, migration, and differentiation. Most, if not all, of these events involve modifications of cell-cell and cell-matrix adhesion, and abnormal modifications of these adhesion systems are often associated with the formation of tumors. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are frequently over-expressed in a wide variety of cancers, including breast, small-cell lung and gastrointestinal cancers, melanomas, and neuroblastomas. Using the Xenopus embryonic system, we have demonstrated that signaling mediated by the intracellular domain of ephrinB affects cell-cell adhesion, and that this activity can be modulated by interaction with an activated FGF receptor. The transmembrane ephrinB1 protein is a bi-directional signaling molecule that signals through its cytoplasmic domain to promote cellular movements into the eye field, whereas activation of the fibroblast growth factor receptor (FGFR) represses these movements and retinal fate. In Xenopus embryos, ephrinB plays a role in retinal progenitor cell movement into the eye field through an interaction with the scaffold protein Dishevelled (Dsh). We recently identified a signaling complex that includes ephrinB2, a transmembrane ligand for Eph receptors, and non-canonical Wnt signaling molecules that we term the WERDS complex, consisting of Wnt4, EphrinB2, Ror2, Dsh2, and Shroom3. This signaling complex instructs a critical cell shape change known as apical constriction, that is a driving force for neural tube closure during development. We used mass spectrometry of immune-complexes to identify proteins associated with ephrinB2 in the neural tube due to our previous finding that the regulation of ephrinB2 protein levels is necessary for normal neural tube closure (Ji et al Nat Comm 2014). In our current study, we use a blend of biochemistry, live and fixed cell imaging, gain-of-function and loss-of-function along with rescue experiments using wild-type and mutant constructs in vivo to provide mechanistic insight into the instructive cue responsible for proper neural tube closure. These experiments led us to the discovery of the WERDS signaling complex that is responsible for this major morphogenetic process. Briefly, we found that Wnt4 binds to Ror2, a non-canonical Wnt receptor that plays a substantive role in Robinow syndrome, and enhances the interaction of ephrinB2 with this forming complex through Dishevelled, the main scaffold of Wnt signaling. These interactions in turn recruit Shroom3, an adaptor protein critical for neural tube formation, which activates Rho-associated kinase (Rock) to cause contraction of the actin-myosin networks required for neural tube closure. We believe that these findings provide the profound understanding of how cross-talk occurs between two seemingly separate major signal transduction pathways, Eph/ephrin and Wnt, to coordinate an important morphogenetic event, neural tube formation.