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Reelin in neuronal migration It has long been known that the secreted factor Reelin is crucial for proper neuronal migration during the development of the cortex. However, the mechanistic actions leading to a Reelin response are less well known. Acker-Palmer and colleagues reveal that the axonal guidance cue ephrin B2 acts as a co-factor with Reelin receptors to initiate signalling cascades important to produce the proper lamination of the cortex. This additional role for ephrin B2 expands the functions for this developmental protein beyond proper axonal targeting to cell migration. Coordinated migration of neurons in the developing and adult brain is essential for its proper function. The secreted glycoprotein Reelin (also known as RELN) guides migration of neurons by binding to two lipoprotein receptors, the very-low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2, also known as LRP8) 1 . Loss of Reelin function in humans results in the severe developmental disorder lissencephaly 2 and it has also been associated with other neurological disorders such as epilepsy, schizophrenia and Alzheimer’s disease 3 . The molecular mechanisms by which Reelin activates its receptors and controls cellular functions are largely unknown. Here we show that the neuronal guidance cues ephrin B proteins are essential for Reelin signalling during the development of laminated structures in the brain. We show that ephrin Bs genetically interact with Reelin. Notably, compound mouse mutants ( Reln +/− ; Efnb3 −/− or Reln +/− ; Efnb2 −/− ) and triple ephrin B1, B2, B3 knockouts show neuronal migration defects that recapitulate the ones observed in the neocortex, hippocampus and cerebellum of the reeler mouse. Mechanistically, we show that Reelin binds to the extracellular domain of ephrin Bs, which associate at the membrane with VLDLR and ApoER2 in neurons. Clustering of ephrin Bs leads to the recruitment and phosphorylation of Dab1 which is necessary for Reelin signalling. Conversely, loss of function of ephrin Bs severely impairs Reelin-induced Dab1 phosphorylation. Importantly, activation of ephrin Bs can rescue the reeler neuronal migration defects in the absence of Reelin protein. Together, our results identify ephrin Bs as essential components of the Reelin receptor/signalling pathway to control neuronal migration during the development of the nervous system.

Cedar virus (CedV) is a bat-borne henipavirus related to Nipah virus (NiV) and Hendra virus (HeV), zoonotic agents of fatal human disease. CedV receptor-binding protein (G) shares only ∼30% sequence identity with those of NiV and HeV, although they can all use ephrin-B2 as an entry receptor. We demonstrate that CedV also enters cells through additional B- and A-class ephrins (ephrin-B1, ephrin-A2, and ephrin-A5) and report the crystal structure of the CedV G ectodomain alone and in complex with ephrin-B1 or ephrin-B2. The CedV G receptor-binding site is structurally distinct from other henipaviruses, underlying its capability to accommodate additional ephrin receptors. We also show that CedV can enter cells through mouse ephrin-A1 but not human ephrin-A1, which differ by 1 residue in the key contact region. This is evidence of species specific ephrin receptor usage by a henipavirus, and implicates additional ephrin receptors in potential zoonotic transmission.

Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4 . EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4 . Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function. While biallelic mutations of the SLC26A4 gene cause non-syndromic hearing loss with enlarged vestibular aqueducts or Pendred syndrome, a considerable number of patients carry mono-allelic mutations. Here the authors identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4.

Evidence implicating Eph receptor tyrosine kinases and their ephrin ligands (that together make up the ‘Eph system’) in cancer development and progression has been accumulating since the discovery of the first Eph receptor approximately 35 years ago. Advances in the past decade and a half have considerably increased the understanding of Eph receptor–ephrin signalling mechanisms in cancer and have uncovered intriguing new roles in cancer progression and drug resistance. This Review focuses mainly on these more recent developments. I provide an update on the different mechanisms of Eph receptor–ephrin-mediated cell–cell communication and cell autonomous signalling, as well as on the interplay of the Eph system with other signalling systems. I further discuss recent advances in elucidating how the Eph system controls tumour expansion, invasiveness and metastasis, supports cancer stem cells, and drives therapy resistance. In addition to functioning within cancer cells, the Eph system also mediates the reciprocal communication between cancer cells and cells of the tumour microenvironment. The involvement of the Eph system in tumour angiogenesis is well established, but recent findings also demonstrate roles in immune cells, cancer-associated fibroblasts and the extracellular matrix. Lastly, I discuss strategies under evaluation for therapeutic targeting of Eph receptors–ephrins in cancer and conclude with an outlook on promising future research directions. This Review by Elena B. Pasquale outlines the current understanding of Eph receptor–ephrin signalling mechanisms in cancer progression and therapy resistance, and also details therapeutic strategies for targeting the Eph system as a novel cancer therapy and for improving the efficacy of conventional cancer therapies.

Eph receptor tyrosine kinases and their ephrin ligands regulate cell navigation during normal and oncogenic development. Signaling of Ephs is initiated in a multistep process leading to the assembly of higher-order signaling clusters that set off bidirectional signaling in interacting cells. However, the structural and mechanistic details of this assembly remained undefined. Here we present high-resolution structures of the complete EphA2 ectodomain and complexes with ephrin-A1 and A5 as the base unit of an Eph cluster. The structures reveal an elongated architecture with novel Eph/Eph interactions, both within and outside of the Eph ligand-binding domain, that suggest the molecular mechanism underlying Eph/ephrin clustering. Structure-function analysis, by using site-directed mutagenesis and cell-based signaling assays, confirms the importance of the identified oligomerization interfaces for Eph clustering.

Ephrin-B2/VEGF in angiogenesis control Ephrin-B ligands are well known as axon guidance molecules. Ephrin-B2 is also known to play a role in angiogenic remodelling. Two studies now show that signalling through ephrin-B2 controls vessel sprouting. Mechanistically, ephrin-B2 seems to function in part by regulating VEGFR internalization and signalling. The finding suggests that blocking ephrin-B2 signalling may be an alternative approach to blocking VEGFR function in angiogenesis. The protein ephrin-B2 is known to be upregulated during angiogenesis — the growth of new blood vessels — but its precise function has been unclear. Here it is shown that signalling through ephrin-B2 controls vessel sprouting. Mechanistically, ephrin-B2 seems to function in part by regulating the internalization of vascular endothelial growth factor receptors (VEGFRs). The results indicate that blocking ephrin-B2 signalling might be an alternative to blocking VEGFR function to disrupt angiogenesis in tumours. In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions 1 , 2 , 3 . Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

Chemical synapses contain specialized pre- and postsynaptic structures that regulate synaptic transmission and plasticity. EphB receptor tyrosine kinases are important molecular components in this process. Previously, EphB receptors were shown to act postsynaptically, whereas their transmembrane ligands, the ephrinBs, were presumed to act presynaptically. Here we show that in mouse hippocampal CA1 neurons, the Eph/ephrin system is used in an inverted manner: ephrinBs are predominantly localized postsynaptically and are required for synaptic plasticity. We further demonstrate that EphA4, a candidate receptor, is also critically involved in long-term plasticity independent of its cytoplasmic domain, suggesting that ephrinBs are the active signaling partner. This work raises the intriguing possibility that depending on the type of synapse, Eph/ephrins can be involved in activity-dependent plasticity in converse ways, with ephrinBs on the pre- or the postsynaptic side.

Ephrins and Eph receptors are involved in the establishment of vertebrate tissue boundaries. The complexity of the system is puzzling, however in many instances, tissues express multiple ephrins and Ephs on both sides of the boundary, a situation that should in principle cause repulsion between cells within each tissue. Although co-expression of ephrins and Eph receptors is widespread in embryonic tissues, neurons, and cancer cells, it is still unresolved how the respective signals are integrated into a coherent output. We present a simple explanation for the confinement of repulsion to the tissue interface: Using the dorsal ectoderm-mesoderm boundary of the Xenopus embryo as a model, we identify selective functional interactions between ephrin-Eph pairs that are expressed in partial complementary patterns. The combined repulsive signals add up to be strongest across the boundary, where they reach sufficient intensity to trigger cell detachments. The process can be largely explained using a simple model based exclusively on relative ephrin and Eph concentrations and binding affinities. We generalize these findings for the ventral ectoderm-mesoderm boundary and the notochord boundary, both of which appear to function on the same principles. These results provide a paradigm for how developmental systems may integrate multiple cues to generate discrete local outcomes.

Summary The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3K[delta], p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1-/[Delta] mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1-/[increment] mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

Ephrin-B2/VEGF in angiogenesis control Ephrin-B ligands are well known as axon guidance molecules. Ephrin-B2 is also known to play a role in angiogenic remodelling. Two studies now show that signalling through ephrin-B2 controls vessel sprouting. Mechanistically, ephrin-B2 seems to function in part by regulating VEGFR internalization and signalling. The finding suggests that blocking ephrin-B2 signalling may be an alternative approach to blocking VEGFR function in angiogenesis. The protein ephrin-B2 is known to be upregulated during angiogenesis — the growth of new blood vessels — but its precise function has been unclear. Here it is shown that signalling through ephrin-B2 controls vessel sprouting. Mechanistically, ephrin-B2 seems to function in part by regulating the internalization of vascular endothelial growth factor receptors (VEGFRs). The results indicate that blocking ephrin-B2 signalling might be an alternative to blocking VEGFR function to disrupt angiogenesis in tumours. The formation and guidance of specialized endothelial tip cells is essential for both developmental and pathological angiogenesis 1 . Notch-1 signalling regulates the generation of tip cells, which respond to gradients of vascular endothelial growth factor (VEGF-A) 2 . The molecular cues and signalling pathways that control the guidance of tip cells are poorly understood. Bidirectional signalling by Eph receptors and ephrin ligands represents one of the most important guidance cues involved in axon path finding 3 . Here we show that ephrin-B2 reverse signalling involving PDZ interactions regulates endothelial tip cell guidance to control angiogenic sprouting and branching in physiological and pathological angiogenesis. In vivo , ephrin-B2 PDZ-signalling-deficient mice (ephrin-B2ΔV) exhibit a reduced number of tip cells with fewer filopodial extensions at the vascular front in the mouse retina. In pathological settings, impaired PDZ signalling decreases tumour vascularization and growth. Mechanistically, we show that ephrin-B2 controls VEGF receptor (VEGFR)-2 internalization and signalling. Importantly, internalization of VEGFR2 is necessary for activation and downstream signalling of the receptor and is required for VEGF-induced tip cell filopodial extension. Together, our results suggest that ephrin-B2 at the tip cell filopodia regulates the proper spatial activation of VEGFR2 endocytosis and signalling to direct filopodial extension. Blocking ephrin-B2 reverse signalling may be an attractive alternative or combinatorial anti-angiogenic therapy strategy to disrupt VEGFR2 function in tumour angiogenesis.