BIOLOGICAL OVERVIEW

Ephrin/Eph signaling is crucial for axonal pathfinding in vertebrates and invertebrates. An examination of the role of Drosophila Ephrin and its receptor Eph receptor tyrosine kinase (Eph) in embryonic central nervous system (CNS) development shows that Drosophila Ephrin is a transmembrane ephrin with a unique N terminus and an ephrinB-like cytoplasmic tail. Ephrin binds and interacts with Eph, the Drosophila Eph-like receptor, and Ephrin and Eph are confined to different neuronal compartments. Loss of Ephrin or Eph causes the abberant exit of interneuronal axons from the CNS, whereas ectopic expression of Ephrin halts axonal growth. It is proposed that the longitudinal tracts in the Drosophila CNS are molded by a repulsive outer border of Ephrin expression (Bossing, 2002).

During the development of the ventral nerve cord in Drosophila, interneurons form two major axon tracts to reach their targets. Most interneurons project across the midline of the developing CNS to form commissural tracts. After the axons have crossed the midline, they turn and join the longitudinal tracts that run from anterior to posterior, and in parallel to the midline. Commissure formation depends on two conserved signaling pathways: axons are attracted towards the midline by proteins of the Netrin family (see Netrin-A and Netrin-B), and are repelled from the midline by the Slit proteins. Both classes of molecules are secreted by cells at the midline. The decision whether axons will cross or not depends on the balance between these two opposing signals (Bossing, 2002 and references therein).

Less is known about the formation of the longitudinal tracts. The extension of longitudinal axons in the vertebrate spinal cord seems to be defined by two barriers: an inner border defined by the chemorepellent Slit, B class ephrin and semaphorins and an outer border defined by chemorepellents of the Semaphorin family, B class ephrins and BMPs. These repellents squeeze axons out of the cortical layer and force them to grow along a narrow, chemorepellent-free corridor. In contrast to the vertebrate spinal cord, it is believed that in Drosophila longitudinal axons are kept inside the CNS by attractive fasciculation cues. When axons reach a specific distance from the ventral midline, the repulsive activity of Slit decreases and axons can fasciculate with existing pathways to turn and grow in parallel to the midline. The pre-existence of 'labelled pathways' inside more mature connectives clearly favors this model for guidance of axons during late embryogenesis (Bossing, 2002 and references therein).

In Drosophila, these 'labelled pathways' are laid out during germband retraction, about 9 hours after fertilization. In response to repulsion by Slit, the first neurons to extend their axons project away from the midline. When the growth cones reach the outer border of the CNS, they turn to the anterior or posterior. The behavior of these growth cones suggests that, as in vertebrates, the connectives are initially molded by two repulsive forces: an inner, medial border defined by Slit and an outer, lateral border defined by a second, unknown repellent (Bossing, 2002).

In addition to Slits and Semaphorins, a third major class of axonal repellents is conserved throughout the animal kingdom, the ephrin family. Ephrins are ligands of the Eph receptors, the largest family of receptor tyrosine kinases in vertebrates. Ligands and receptors are grouped into A and B classes. Ligands of the A class are tethered to the cell membrane by a GPI anchor. B class ligands have a transmembrane domain and a short cytoplasmic tail (Bossing, 2002).

Ephrin/Eph signaling is important for a diverse array of developmental processes such as topographic mapping of retinal axons onto the tectum in chick embryos, synaptic remodelling in the adult brain and vasculogenesis. Ephrin/Eph signaling is cell contact mediated and depends on the clustering of receptors and their ligands. Multimerization activates the kinase activity of the receptor and leads to the phosphorylation of tyrosine, serine and threonine residues in the receptor's cytoplasmic tail. The phosphorylated residues permit the binding of a battery of downstream effectors. Eph receptor activation can trigger the depolymerization of actin in growth cones and can modify integrin based cell adhesion (Bossing, 2002 and references therein).

Interaction between ephrins and Eph receptors can also activate the ligand. Tyrosine residues in the cytoplasmic tail of B class ephrins become phosphorylated upon binding to Eph receptors. Cell culture experiments suggest that B class ephrins are clustered into membrane microdomains (rafts) and signal back to the ligand-expressing cell by recruitment of PDZ binding proteins, serine/threonine kinases and SH2/SH3 adaptor proteins. Such bidirectional signaling is important for the formation of the corpus callosum in the vertebrate brain, to prevent the formation of gap junctions and to preclude cell intermingling between rhombomeres in the hindbrain. The targets and components of the pathway triggered by ephrin activation are not clearly defined yet, but modification of cytoskeletal components and cell adhesion seems to be the main output (Bossing, 2002 and references therein).

The role of ephrins in axon repulsion in vertebrates and the identification of an Eph-like receptor expressed on the longitudinal axons in Drosophila embryos, prompted a search for an ephrin ortholog in Drosophila. A single Drosophila ephrin ortholog has been identifed. The homology of Drosophila Ephrin to other ephrins is restricted to the ephrin domain and the most C-terminal amino acids, which form a 'B-like' cytoplasmic tail. Structural analysis confirms that Drosophila Ephrin is a transmembrane protein with a cytoplasmic tail. In contrast to all other ephrins, Drosophila Ephrin has no obvious signal peptide and is cleaved at the N terminus. Ephrin is broadly expressed in neurons and localized on neuronal cell bodies but absent from axons. Conversely, Eph, the Drosophila Eph-like kinase, is localized on all interneuronal axons and is absent from cell bodies. Inactivation of Ephrin and Eph by RNAi results in the fusion or loss of commissures and breaks in the connectives. Analysis of single axons shows that the loss of Ephrin or Eph causes the aberrant exit of interneurons from the CNS. Ectopic expression of Ephrin in single glial cells or in all midline cells prevents axon extension. These phenotypes rely on Ephrin/Eph interaction. Ephrin binds to Eph in cell culture and repulsion by Ephrin can be overcome by lowering the level of Eph expression. These results indicate that signaling between Ephrin and Eph creates repulsive barriers that border the commissures and connectives of the embryonic CNS (Bossing, 2002).

Drosophila Ephrin is the first transmembrane ephrin described in invertebrates. Structural analysis indicates that Ephrin, which is cleaved at the N terminus, is composed of an extracellular ephrin domain, a transmembrane domain and a cytoplasmic tail with homology to B class ephrins. Ephrin is found on neuronal cell bodies outlining the presumptive axonal tracts. In contrast, Eph, the Drosophila Eph-like receptor, is found only on interneuronal axons. Ephrin binds to Eph and signaling between Eph and Ephrin is able to block axon extension. Axonal repulsion by Ephrin/Eph signaling plays a role in the separation of commissures. In addition, Ephrin/Eph signaling prevents the abnormal exit of interneuronal axons from the CNS. It is proposed that Ephrin/Eph signaling is essential for the formation of the longitudinal tracts by delimiting the extension of interneuronal axons to the inside of the CNS (Bossing, 2002).

Ephrin signaling in vertebrates is mediated by two classes of receptors and ligands, A and B. In contrast, invertebrates appear to use a single ancestral ephrin-Eph signaling pathway. Four ephrins have been identified in C. elegans. Although these have a B-like receptor binding domain, they are all attached to the membrane by a GPI anchor, a feature characteristic of the A class ephrins (Chin-Sang, 1999). Similarly, the structure of Ephrin does not directly allow it to be classified as an A class or a B class ephrin. The receptor binding domain/ephrin domain shows the same degree of homology to both classes of vertebrate ephrins. As in vertebrates, the ephrin domain in Ephrin is extracellular. In contrast to vertebrate ephrins, the domain is preceded by a stretch of 200 aa with no homology to any other ephrin (Bossing, 2002).

This unusual N terminus has no obvious signal sequence but is essential for the membrane localization of Ephrin. The N terminus also contains sequences needed for posttranslational control of Ephrin. Interestingly, the N terminus is cleaved. The cleavage depends on the full length molecule: neither a truncation containing the N terminus and the ephrin domain (EphrinDeltaCterm) nor a fusion of the N terminus to GFP is cleaved. The function of this cleavage is unclear and it is not known if this cleavage occurs in all Ephrin-expressing cells. The cleavage could be necessary to create an additional membrane anchor at the N terminus by opening up the predicted myristoylation site (aa85-90) in the N terminus. Another possibility is that like Hedgehog ligands, cleavage of Ephrin is needed to attach a cholesterol anchor (Bossing, 2002).

The C terminus of Ephrin encodes two closely spaced predicted transmembrane domains. Consistent with this prediction, it is found that the C terminus of Ephrin is essential to anchor the molecule to the membrane. Currently it is not known which of the predicted transmembrane domains is used. The last predicted transmembrane domain is followed by 30aa, of which the last 19aa show homology to the cytoplasmic tail of B class ephrins. The tyrosine residue identified as a major in vivo phosphorylation target in vertebrate B class ephrins (Kalo, 2001) is conserved. The existence of a cytoplasmic tail in Drosophila Ephrin was confirmed by proteinase K treatment and antibody staining against a C-terminal GFP tag (Bossing, 2002).

In vertebrates, Eph receptors and ephrin B ligands are both able to transduce extracellular signals. The phenotypes caused by ectopic expression of Ephrin or by the loss of Ephrin and Eph appear to be restricted to axonal pathfinding. The localization of Eph on axons, appears to imply that Eph is the receptor and Ephrin only acts as a ligand. However, there are indications that Ephrin may also be able to transduce signals. The tyrosine involved in signal transduction by B class ephrins is conserved in Ephrin, and Ephrin can be immunoprecipitated from embryonic lysates using anti-phosphotyrosine. The function of Ephrin signaling might be obscured by the strong axonal phenotype. For example, Ephrin expression could play a role in the regulation of cell adhesion. In Ephrin and Eph RNAi-treated embryos, the embryonic CNS appears flat with a ragged outline. In contrast, the loss of other major components involved in axonal pathfinding, i.e. Commissureless, Roundabout or Slit, does not affect the shape of the CNS (Bossing, 2002).

Ephrin is expressed by motor neurons and interneurons, whereas the expression of Eph is confined to interneurons. The results show that Eph expression on interneurons restricts their axons to the CNS. It is tempting to speculate that the absence of Eph on motorneuronal axons, which have to project out of the CNS, might be essential to allow these axons to cross over the Ephrin barrier at the border of the connectives (Bossing, 2002).

The expression of Eph and Ephrin is restricted to different subcellular compartments, although their RNA expression most likely overlaps. Ephrin is restricted to neuronal cell bodies, whereas Eph is confined to axons. Eph RNA appears not to be transported into axons, hence the differential sorting of the two components of ephrin signaling occurs at the protein level. Ephrin and Eph bind each other and the separation of the two proteins may be essential to prevent a cell autonomous activation of signaling. Indeed, strong overexpression of Ephrin in interneuronal mosaic clones results in axonal accumulation of Ephrin and interferes with axonal pathfinding. Recently, it has been shown that overlapping expression of ephrinA5 and its receptor EphA4 in retinal axons can desensitize the growth cone, allowing the axons to pass over Eph concentrations that repel axons that are not desensitized (Hornberger, 1999). Although desensitization might play a role in pathfinding of a minority of axons in the embryonic CNS of Drosophila, the results suggest that the correct targeting of most axons depends on the exclusion of Ephrin from axons (Bossing, 2002).

Ephrin/Eph signaling in Drosophila is involved in midline guidance, as are ephrins in vertebrates and C. elegans. Ectopic expression of Ephrin in all midline cells prevents commissural axons from crossing the midline, supporting a role of Ephrin/Eph signaling in axon repulsion. The loss of Ephrin and Eph function results in the fusion of commissures. Ephrin is expressed in midline and non-midline cells located between the forming commissures in each segment. Ephrin may act as a repulsive force that is needed for the separation of commissures. Therefore, this loss of function phenotype can be explained by the loss of these repulsive barriers (Bossing, 2002).

Ephrin/Eph signaling is bi-functional and can promote adhesion as well as repulsion. This bi-functionality is especially striking in midline guidance. During the development of the spinal cord in mouse, the interaction of EphA4 on corticospinal axons with ephrinB3 at the midline of the neural tube prevents the midline crossing of collaterals (Yokoyama, 2001). In contrast, the expression of ephrinB2 on commissural axons and EphA4 on neurons at the anterior commissure of the brain is essential for the midline crossing of these axons. The vab-1/Eph receptor in C. elegans is needed on sensory axons for the ventral attraction towards the nerve ring, but it also functions as a repellent to prevent axonal crossover at the ventral midline. A similar mechanism might apply for the formation of commissures in Drosophila, where loss of Ephrin or Eph can result in the loss of commissures. It may be that Ephrin at the midline is a permissive substrate to which growth cones of commissural axons can adhere, to be channelled towards the entry of the commissures. In vertebrates, the repulsive activity of ephrin/Eph signaling can be transformed into adhesion by the expression of different splice forms of an Eph receptor, by preventing the Kuzbanian-dependent cleavage of ephrins (Hattori, 2000) or by alternating the degree of receptor activation. It has been shown that adhesion of vertebrate cells in culture depends on the level of signaling by the EphB1 receptor. Low to medium level activation of the EphB1 receptor by ephrinB1 promotes adhesion, while high level activation blocks adhesion (Huynh-Do, 1999). No different splice forms of the Eph mRNA have been reported, but the Kuzbanian cleavage site is conserved in the ephrin domain of Ephrin. The observation that either loss of Ephrin or gain of Ephrin in midline cells can result in a loss of commissures, seems to indicate that the level of receptor activation at the midline is critical to distinguish between adhesion and repulsion (Bossing, 2002).

The role of Ephrin expression at the midline differs from that of Slit, the second chemorepellent expressed in midline cells. In the loss of Slit, axons linger at the midline, whereas in the loss of Ephrin, axons do not aberrantly enter the midline. Ectopic Ephrin/Eph expression at the midline does not repel axons through the upregulation of Slit/Robo1 signaling (Bossing, 2002).

The loss of Ephrin or Eph causes breaks in the connectives. Examination of the projection pattern of primary axons in the loss of Ephrin and Eph explains the breaks in the connectives. Some of the first neurons to extend their axons in the CNS are the MP1 and MP2 neurons. The axons of MP1 and MP2 neurons first project mediolaterally, away from the midline, until they nearly reach the outer border of the developing CNS. At the outer border they stop and turn to extend longitudinally. In the loss of Ephrin and Eph, the MP2 neurons do not turn but exit the CNS. It seems likely that an aberrant exit of interneurons can result in breaks in the connectives. The expression of Ephrin along the outside of the CNS seems to form a repulsive barrier confining the extension of interneuronal axons to the inside of the CNS. The repulsive capacity of Ephrin on longitudinal axons is evident because of the ectopic expression of Ephrin in longitudinal glia cells that is able to halt axonal growth along the connectives (Bossing, 2002).

These results led to the proposal of the following model for the formation of the connectives in the embryonic CNS of Drosophila. Repulsion by Ephrin at the outer border of the CNS and by Slit at the midline limits the extension of primary longitudinal axons to within the CNS. Restricting the first interneuronal axons to inside the CNS ensures that axon fascicles are in the correct place to enable selective fasciculation and axonal spacing in late embryogenesis. In late embryogenesis, when the number of axons increases, repulsion by Ephrin/Eph signaling might well be restricted to the most lateral axons. This mechanism is similar to the establishment of the longitudinal tracts in vertebrates. During development of the vertebrate CNS, the medial repulsive border is defined by expression of Slits, Semaphorins and B class ephrins in the floorplate and the ventral spinal cord. The outer repulsive border is formed by B class ephrins, Semaphorins and BMPs in the dorsal spinal cord (Bossing, 2002).

Ephrin/Eph signaling plays a role in many important processes during vertebrate development. The number of receptors and ligands and their functional redundancy hinders the elucidation of the underlying signaling pathways. C. elegans and Drosophila use an ancestral signaling pathway, although many of the functions of ephrin/Eph signaling are conserved. This raises the possibility of identifying the downstream components of the pathway in genetically accessible model organisms. C. elegans has one Eph receptor and four ephrins; this shows functional redundancy. Drosophila has only one ephrin and one Eph receptor. As with the Hedgehog, WNT and Ras signaling pathways, Drosophila might once again be a helpful tool to unravel an evolutionarily conserved signaling mechanism (Bossing, 2002).

GENE STRUCTURE

cDNA clone length - 2363

Bases in 5' UTR - 193

Exons - 4

Bases in 3' UTR - 211

PROTEIN STRUCTURE

Amino Acids - 652

Structural Domains

Drosophila Ephrin (GenBank accession number AF216287) shows overall low similarity to other members of the ephrin family (8% similarity to human ephrinB1, 7% similarity to the C. elegans ephrin vab-2 and only 3% to human ephrinA1). However, Drosophila Ephrin shows significant homology in its central domain to the extracellular domain of ephrins. The ephrin domain in Drosophila Ephrin is as homologous to A ephrins (41% to human ephrin A1) as to B ephrins (42% to human ephrin B1) with a slightly higher homology to ephrins from C. elegans (46% to vab-2) (Bossing, 2002).

The sequence for Ephrin is compiled from four ESTs provided by the BDGP and three full length cDNAs generated by independent PCR reactions using an embryonic cDNA pool as a template. There is no evidence for alternate transcripts other than an alternative polyA site in one of the four ESTs, which shortens the common 3' untranslated region by about 300 bp. In situ hybridization to salivary glands maps Ephrin to position 102C on the fourth chromosome. The gene comprises four exons and three introns. The ephrin domain is encoded by the second exon and the beginning of the third exon (Bossing, 2002).

Ephrin encodes a predicted protein of 652 amino acids. Homology to other ephrins is restricted to the ephrin domain and the C terminus. In vertebrates the most C-terminal sequence differs between A and B class ephrins. A-class ephrins end with a hydrophobic stretch of amino acids. B-class ephrins have a highly conserved and hydrophilic C terminus encoding at least five tyrosines that are phosphorylated upon interaction with Eph receptors in cell culture. Ephrin B1 in the chicken retina is primarily phosphorylated in vivo at the tyrosine residue at position –3 from the C terminus. The predicted cytoplasmic tail of Ephrin is hydrophilic and shows sequence homology to B class ephrins. In addition, the tyrosine at position –3 from the C terminus is conserved (Bossing, 2002).

The C terminus of the cytoplasmic tail of B class ephrins and most Eph recepters forms a PDZ binding domain. A PDZ binding consensus is also present at the C terminus of Eph, the Eph-like receptor. In contrast, the cytoplasmic tail of Drosophila Ephrin does not contain a consensus PDZ binding domain (Bossing, 2002).

date revised: 10 September 2002

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