EphB2 and EphB4 receptors forward signaling promotes SDF-1-induced endothelial cell chemotaxis and branching remodeling.

The complex molecular mechanisms that drive endothelial cell movement and the formation of new vessels are poorly understood and require further investigation. Eph receptor tyrosine kinases and their membrane-anchored ephrin ligands regulate cell movements mostly by cell-cell contact, whereas the G-protein-coupled receptor CXCR4 and its unique SDF-1 chemokine ligand regulate cell movement mostly through soluble gradients. By using biochemical and functional approaches, we investigated how ephrinB and SDF-1 orchestrate endothelial cell movement and morphogenesis into capillary-like structures. We describe how endogenous EphB2 and EphB4 signaling are required for the formation of extracellular matrix-dependent capillary-like structures in primary human endothelial cells. We further demonstrate that EphB2 and EphB4 activation enhance SDF-1-induced signaling and chemotaxis that are also required for extracellular matrix-dependent endothelial cell clustering. These results support a model in which SDF-1 gradients first promote endothelial cell clustering and then EphB2 and EphB4 critically contribute to subsequent cell movement and alignment into cord-like structures. This study reveals a requirement for endogenous Eph signaling in endothelial cell morphogenic processes, uncovers a novel link between EphB forward signaling and SDF-1-induced signaling, and demonstrates a mechanism for cooperative regulation of endothelial cell movement.

EphB2 regulates axonal growth at the midline in the developing auditory brainstem.

Eph receptors play important roles in axon guidance at the midline. In the auditory system, growth of axons across the midline is an important determinant of auditory function. The avian cochlear nucleus, n. magnocellularis (NM), makes bilateral projections to its target, n. laminaris (NL). We examined the time course of NM axon growth toward the midline, the expression of Eph proteins at the midline during this growth, and the effects of Eph receptor misexpression on axonal growth across the midline. We found that NM axons reach the midline at E4. At this age, EphB receptors are expressed at the ventral floor plate. Expression extends dorsally to the ventricular zone beginning at E5. NM axons thus grow across the midline at a time when EphB receptor expression levels are low. Overexpression of EphB2 at E2 resulted in misrouted axons that deflected away from transfected midline cells. This effect was observed when midline cells were transfected but not when NM cells alone were transfected, suggesting that EphB2 acts non-cell autonomously and through reverse signaling. These data suggest an inhibitory role for midline Eph receptors, in which low levels permit axon growth and subsequently high levels prohibit growth after axons have crossed the midline.

Differential expression of Eph receptors and ephrins in the cochlear ganglion and eighth cranial nerve of the chick embryo.

The cochleovestibular ganglion of the chick differentiates at early embryonic stages as VIIIth nerve axons enter the brainstem. The tonotopic organization of the auditory portion of the VIIIth nerve can be discerned at the time axons initially reach their brainstem targets. The mechanisms underlying this early organization are not known. Eph receptor tyrosine kinases and their ligands, the ephrins, have a demonstrated role in guiding axons to topographically appropriate locations in other areas of the nervous system. In order to begin to test whether Eph proteins have a similar role in the auditory system, we investigated the tonotopic expression of several Eph receptors and ephrins in the VIIIth nerve during embryonic ages corresponding to the initial innervation of the auditory brainstem. Expression patterns of EphA4, EphB2, EphB5, ephrin-A2, and ephrin-B1 were evaluated immunohistochemically at embryonic days 4 through 10. Protein expression was observed in the cochlear ganglion and VIIIth nerve axons at these ages. EphB5, ephrin-A2, and ephrin-B1 were expressed throughout the nerve. EphA4 and EphB2 had complementary expression patterns within the nerve, with EphA4 expression higher in the dorsolateral part of the nerve and EphB2 expression higher in the ventromedial part of the nerve. These regions may correspond to auditory and vestibular components, respectively. Moreover, EphA4 expression was higher toward the low-frequency region of both the centrally and peripherally projecting branches of cochlear ganglion cells. Regional variation of Eph protein expression may influence the target selection and topography of developing VIIIth nerve projections.

Novel expression gradients of Eph-like receptor tyrosine kinases in the developing chick retina.

Eph-like receptor tyrosine kinases have recently been identified as critical components in the development of the retinotectal system. Complementary gradients of receptors and ligands in the retina and tectum, and within the retina itself, have previously been described. Here, we present a novel centroperipheral gradient of expression for one member of this family of receptors, Cek9, suggesting that retinal patterning and axon guidance during the establishment of retinotectal projections may involve coordinate mapping along three axes. Furthermore, we show matching gradients of two cytoplasmic kinases, compatible with their putative involvement in the intracellular signaling pathways used by these receptors in the retina. We also demonstrate a dorsal to ventral expression gradient for Cek11, an Eck-like receptor, the Eph subclass previously suggested to specify positional information along the temporonasal axis.

The N-terminal globular domain of Eph receptors is sufficient for ligand binding and receptor signaling.

The Eph family of receptor protein-tyrosine kinases (RTKs) have recently been implicated in patterning and wiring events in the developing nervous system. Eph receptors are unique among other RTKs in that they fall into two large subclasses that show distinct ligand specificities and for the fact that they themselves might function as 'ligands', thereby activating bidirectional signaling. To gain insight into the mechanisms of ligand-receptor interaction, we have mapped the ligand binding domain in Eph receptors. By using a series of deletion and domain substitution mutants, we now report that an N-terminal globular domain of the Nuk/Cek5 receptor is the ligand binding domain of the transmembrane ligand Lerk2. Using focus formation assays, we show that the Cek5 globular domain is sufficient to confer Lerk2-dependent transforming activity on the Cek9 orphan receptor. Extending our binding studies to other members of both subclasses of receptors, it became apparent that the same domain is used for binding of both transmembrane and glycosylphosphatidyl-anchored ligands. Our studies have determined the first structural elements involved in ligand-receptor interaction and will allow more fine-tuned genetic experiments to elucidate the mechanism of action of these important guidance molecules.

Developmental expression and distinctive tyrosine phosphorylation of the Eph-related receptor tyrosine kinase Cek9.

Cek9 is a receptor tyrosine kinase of the Eph subfamily for which only a partial cDNA sequence was known (Sajjadi, F.G., and E.B. Pasquale. 1993. Oncogene. 8:1807-1813). We have obtained the entire cDNA sequence and identified a variant form of Cek9 that lacks a signal peptide. We subsequently examined the spatio-temporal expression and tyrosine phosphorylation of Cek9 in the chicken embryo by using specific antibodies. At embryonic day 2, Cek9 immunoreactivity is concentrated in the eye, the brain, the posterior region of the neural tube, and the most recently formed somites. Later in development, Cek9 expression is widespread but particularly prominent in neural tissues. In the developing visual system, Cek9 is highly concentrated in areas containing retinal ganglion cell axons, suggesting a role in regulating their outgrowth to the optic tectum. Unlike other Eph-related receptors, Cek9 is substantially phosphorylated on tyrosine in many tissues at various developmental stages. Since autophosphorylation of receptor protein-tyrosine kinases typically correlates with increased enzymatic activity, this suggests that Cek9 plays an active role in embryonic signal transduction pathways.

Similarities and differences in the way transmembrane-type ligands interact with the Elk subclass of Eph receptors.

The Eph family of receptor tyrosine kinases and their cell surface bound ligands have been implicated in a number of developmental processes, including axon pathfinding and fasciculation, as well as patterning in the central nervous system. To better understand the complex signaling events taking place, we have undertaken a comparative analysis of ligand-receptor interactions between a subset of ligands, those that are tethered to the cell surface via a transmembrane domain, and a subset of Eph receptors, the so-called Elk subclass. Based on binding characteristics, receptor autophosphorylation, and cellular transformation assays, we find that the transmembrane-type ligands Lerk2 and Elf2 have common and specific receptors within the Elk subclass of receptors. The common receptors Cek10 and Elk bind and signal in response to Lerk2 and Elf2, whereas the Myk1 receptor is specific for Elf2. Elf2, however, fails to signal through Cek5 in a cellular transformation assay, suggesting that Lerk2 may be the preferred Cek5 ligand in vivo. A recently identified third transmembrane-type ligand, Elf3, specifically, but weakly, binds Cek10 and only induces focus formation when activated by C-terminal truncation. This suggests that the physiological Elf3 receptor may have yet to be identified. Knowledge regarding functional ligand-receptor interactions as presented in this study will be important for the design and interpretation of in vivo experiments, e.g., loss-of-function studies in transgenic mice.