Mechanisms of Cross-talk Between EphrinB and Alternate Signaling Pathways

在正常发育期间，许多组织的祖细胞经历多能性、上皮-间充质转化、增殖、迁移和分化的进行性限制。这些事件中的大多数（如果不是全部）涉及细胞-细胞和细胞-基质粘附的修饰，并且这些粘附系统的异常修饰通常与肿瘤的形成相关。受体酪氨酸激酶的Eph家族及其配体肝配蛋白在多种癌症中经常过表达，包括乳腺癌、小细胞肺癌和胃肠癌、黑色素瘤和神经母细胞瘤。使用非洲爪蟾胚胎系统，我们已经证明，ephrinB的细胞内结构域介导的信号传导影响细胞-细胞粘附，并且这种活性可以通过与活化的FGF受体的相互作用来调节。跨膜ephrinB 1蛋白是一种双向信号分子，通过其胞质结构域发出信号以促进细胞运动进入眼区，而成纤维细胞生长因子受体（FGFR）的激活抑制这些运动和视网膜命运。在非洲爪蟾胚胎中，ephrinB通过与支架蛋白Dishevelled（Dsh）的相互作用在视网膜祖细胞移动到眼区中发挥作用。我们最近发现了一种信号复合物，包括ephrinB 2，Eph受体的跨膜配体和非经典Wnt信号分子，我们称之为WERDS复合物，由Wnt 4，EphrinB 2，Ror 2，Dsh 2和Shroom 3组成。这种信号复合物指示称为顶端收缩的关键细胞形状变化，这是发育期间神经管闭合的驱动力。我们使用免疫复合物的质谱法来鉴定神经管中与ephrinB 2相关的蛋白质，因为我们之前发现ephrinB 2蛋白水平的调节对于正常神经管闭合是必要的（Ji et al Nat Comm 2014）。在我们目前的研究中，我们使用生物化学，活细胞和固定细胞成像，功能获得和功能丧失沿着与救援实验一起使用野生型和突变体的构建体在体内提供机制洞察的指导性线索负责适当的神经管关闭。这些实验使我们发现了负责这一主要形态发生过程的WERDS信号复合体。简而言之，我们发现Wnt 4与Ror 2结合，Ror 2是一种在Robinow综合征中起实质性作用的非经典Wnt受体，并通过Wnt信号传导的主要支架Dishevelled增强ephrinB 2与该形成复合物的相互作用。这些相互作用反过来又招募Shroom 3，这是一种对神经管形成至关重要的衔接蛋白，它激活Rho相关激酶（Rock），导致神经管闭合所需的肌动蛋白-肌球蛋白网络收缩。我们相信，这些发现提供了对两个看似独立的主要信号转导途径Eph/ephrin和Wnt之间如何发生串扰的深刻理解，以协调重要的形态发生事件，神经管形成。

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.