Mechanisms of Cross-talk Between EphrinB and Alternate Signaling Pathways

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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 Drg1 (Developmentally regulated GTP binding protein 1) as a new but requisite interactor and regulator of Dishevelled (Dvl) for proper ciliogenesis. Dvl is a central scaffold that mediates both canonical and non-canonical Wnt signaling and can decisively orchestrate various developmental and cellular processes. One of these processes is ciliogenesis, and while Dvl is known to be critical for apical docking and planar polarization of basal bodies in ciliated epithelial cells, little mechanistic insight into the players and their roles have been gleaned since this original discovery. We previously reported the identification of Drg1, a little studied GTP-binding protein 1, that interacts with Dvl and localizes it to the basal body region and we provided critical in vivo evidence that a Drg1/Dvl interaction plays a critical role in ciliogenesis. We now show that Ccdc108, a protein linked to male infertility, has an evolutionarily conserved requirement in motile multiciliation. Using Xenopus laevis embryos, Ccdc108 is shown to be required for the migration and docking of basal bodies to the apical membrane in epidermal multiciliated cells (MCCs). We demonstrate that Ccdc108 interacts with the IFTB complex, and governs the centriolar recruitment of IFT while IFT licenses the targeting of Ccdc108 to the cilium. Moreover, Ccdc108 is required for the centriolar recruitment of Drg1 and activated RhoA, factors that help establish the apical actin network in MCCs. Finally, we show that CEP97, which is known as a negative regulator of primary cilia formation, interacts with dual specificity tyrosine phosphorylation regulated kinase 1A (Dyrk1a) to modulate multiciliogenesis. We show that Dyrk1a phosphorylates CEP97, which in turn promotes the recruitment of Polo-like kinase 1 (Plk1), which is a critical regulator of MCC maturation that functions to enhance centriole disengagement in cooperation with the enzyme Separase.Thus, our study reveals that Dyrk1a and CEP97 coordinate with Plk1 to promote Separase function to properly form multicilia in vertebrate MCCs. Our findings on the collaborative role of CEP97 and Dyrk1a in multiciliation may add to our existing knowledge regarding patients with trisomy 21 and therapeutic approaches for the respiratory diseases in patients with ciliopathy and Down syndrome.