Ephrin

InteractiveFly: GeneBrief

Gene name - Ephrin

Synonyms -

Cytological map position - 102C4

Function - ligand

Keywords - CNS, axon guidance

Symbol - Ephrin

FlyBase ID: FBgn0040324

Genetic map position -

Classification - ephrin

Cellular location - surface

NCBI link: Entrez Gene

Ephrin orthologs: Biolitmine

Recent literature

Liu, L., Tian, Y., Zhang, X. Y., Zhang, X., Li, T., Xie, W. and Han, J. (2017). Neurexin restricts axonal branching in columns by promoting Ephrin clustering. Dev Cell 41(1): 94-106. PubMed ID: 28366281
Summary:
Columnar restriction of neurites is critical for forming nonoverlapping receptive fields and preserving spatial sensory information from the periphery in both vertebrate and invertebrate nervous systems, but the underlying molecular mechanisms remain largely unknown. This study demonstrates that Drosophila homolog of α-neurexin (DNrx) plays an essential role in columnar restriction during L4 axon branching. Depletion of DNrx from L4 neurons resulted in misprojection of L4 axonal branches into neighboring columns due to impaired Ephrin clustering. The proper Ephrin clustering requires its interaction with the intracellular region of DNrx. Furthermore, it was found that Drosophila neuroligin 4 (DNlg4) in Tm2 neurons binds to DNrx and initiates DNrx clustering in L4 neurons, which subsequently induces Ephrin clustering. This study demonstrates that DNrx promotes ephrin clustering and reveals that ephrin/Eph signaling from adjacent L4 neurons restricts axonal branches of L4 neurons in columns.

Franco, M. and Carmena, A. (2019). Eph signaling controls mitotic spindle orientation and cell proliferation in neuroepithelial cells. J Cell Biol. PubMed ID: 30808706
Summary:
Mitotic spindle orientation must be tightly regulated during development and adult tissue homeostasis. It determines cell-fate specification and tissue architecture during asymmetric and symmetric cell division, respectively. This study uncovered a novel role for Ephrin-Eph intercellular signaling in controlling mitotic spindle alignment in Drosophila optic lobe neuroepithelial cells through aPKC activity-dependent myosin II regulation. Conserved core components of the mitotic spindle orientation machinery, including Discs Large1, Mud/NuMA, and Canoe/Afadin, mislocalize in dividing Eph mutant neuroepithelial cells and produce spindle alignment defects in these cells when they are down-regulated. In addition, the loss of Eph leads to a Rho signaling-dependent activation of the PI3K-Akt1 pathway, enhancing cell proliferation within this neuroepithelium. Hence, Eph signaling is a novel extrinsic mechanism that regulates both spindle orientation and cell proliferation in the Drosophila optic lobe neuroepithelium. Similar mechanisms could operate in other Drosophila and vertebrate epithelia.

: 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).

: REGULATION
Reph, a regulator of Eph receptor expression in the Drosophila melanogaster optic lobe

: DEVELOPMENTAL BIOLOGY

: EFFECTS OF MUTATION
Both Ephrin and Eph map to the fourth chromosome, for which it is very difficult to obtain and maintain mutants by classical genetic techniques. For this reason RNAi was used to inhibit expression. RNAi has rapidly become an accepted technique for generating mutant phenotypes. In test injections of dsEphrin RNA only two out of nine injected embryos show a nearly complete loss of Ephrin, while the remainder retains about 20%-50% of wild-type expression. Therefore, it was not expected that Ephrin RNAi would lead to a mutant phenotype in all injected embryos nor that all segments per embryo would be affected. Indeed, only 65% (13/20) of embryos injected with dsEphrin showed an aberrant phenotype and in total 39% (77/200) of all segments are affected. In four injected embryos all segments were affected. The phenotypes include fused commissures, loss of commissures and breaks in the connectives. Although Ephrin RNAi impedes commissure formation, it does not interfere with the differentiation of midline glia. Injection with dsCFP or buffer does not reduce Ephrin expression but occasionally results in phenotypes similar to dsEphrin injections. However, only 30% (5/15) of dsCFP injected embryos and 23% (4/17) of buffer injected embryos show a phenotype. The number of affected segments is reduced to 6% (9/148, dsCFP) or 8% (13/167, buffer) (Bossing, 2002).
Eph RNAi also results in fused commissures, loss of commissures and breaks in the connectives. The phenotype of Eph RNAi is more severe than for Ephrin RNAi. 80% (12/15) of all embryos had a phenotype and in total 69% (98/142) of all segments were affected. In five embryos all segments were abnormal. The difference in the strength of phenotype could either indicate that additional ligands besides Ephrin signal through Eph or the difference might be caused by the efficiency of RNAi, which varies between different genes (Bossing, 2002).
RNAi against Ephrin and Eph results in the fusion or loss of commissures and breaks in the connectives. Using a general axon marker, the origin of these phenotypes is not clear. Therefore the behaviour of single axons was followed in RNAi-treated embryos. The Gal4 line CY27 primarily drives expression of UAS-taumGFP6 in 2 interneurons per hemisegment, the vMP2 and dMP2 neuron. The MP2 neurons are among the first neurons to extend their axons along the connectives. In differentiated embryos the projections of these neurons form a tight fascicle which extends close and in parallel to the midline. Loss of Ephrin or Eph causes the axons of the MP2 neurons to project aberrantly out of the CNS. In 75% of embryos (15/20, Ephrin RNAi) and 82% of embryos (14/17, Eph RNAi), MP2 axons exiting the CNS were found. In the GAL4 line CY27, additional interneurons (i.e., UMI neurons) start to express GFP in late embryogenesis. No attempt was made to examine these weak projections in detail but it was noticed that many of these interneuronal axons also project out of the CNS. Therefore, signalling between Ephrin and Eph plays a role in confining interneuronal axons to the connectives (Bossing, 2002).
In vertebrates activation of Eph receptors in axonal growth cones is able to repel axons. It was speculated that despite the structural differences between vertebrate ephrins and Ephrin, the repulsive ability of Ephrin/Eph signalling might be conserved. Ephrin expression along the outer edge of the connectives and between the commissures could create barriers preventing axon extension. Absence of these barriers would be expected to result in fusion of commissures and the exit of interneuronal axons from the CNS, as was observed in RNAi experiments. To test whether Ephrin can act as an axonal repellent, Ephrin was ectopically expressed (Bossing, 2002).
Only 4-6 out of about 20 midline neurons express Ephrin. Ectopic expression of Ephrin in all midline cells (sim-GAL4) causes fusion, severe thinning or loss of commissures without affecting midline glial cell differentiation. Single cell labelling of neural precursors reveals that ectopic Ephrin in midline cells is able to prevent the midline crossing of axons. In all clones with contralateral axons, the axons are stalled at the midline. Ectopic midline Ephrin does not affect the extension of ipsilateral axons immediately adjacent to the midline or the determination of midline neurons (judged by the expression of Engrailed, Futsch and Odd-skipped) (Bossing, 2002).
Axonal repulsion by Slit, secreted from midline cells, is one of the major mechanisms controlling axons crossing the midline. It is possible that Ephrin expression at the midline exerts its repulsive effect by upregulating the expression or secretion of Slit. To test if repulsion by Ephrin depends on Slit, Ephrin was expressed ectopically in the midline of embryos mutant for Slit and Robo1, one of the receptors for Slit. Expression of Ephrin in slit/robo double mutants forces axons out of the midline. Therefore, Ephrin/Eph signalling at the ventral midline can act independently of Slit and Robo1 (Bossing, 2002).
Ephrin is expressed in nearly all neurons but not in the longitudinal glia that enwrap the connectives. Ephrin-expressing longitudinal glia were generated by injecting UAS-Ephrin plasmids into the syncytial blastoderm of GAL4MZ1580 embryos. When longitudinal glial cells express Ephrin, breaks are observed in the connectives. The breaks are always located near the glial cell. No breaks are observed when neurons express Ephrin. GFP-expressing longitudinal glial cells also do not disrupt axon extension (UAS-tau-mGFP6 plasmid). In summary, ectopic expression of Ephrin blocks axon extension (Bossing, 2002).
Activation of Eph on axons may be the reason that axons stall at Ephrin-expressing midline cells. In that case lowering the level of Eph activation by reducing Eph expression might allow these axons to overcome this repulsion and restore the commissures. To test this hypothesis Eph expression was lowered by Eph RNAi. Embryos with ectopic midline expression of Ephrin show a strong phenotype. Only 2% (2/100) of all segments have wild-type commissures and embryos are never found in which all segments have normal commissures. Injection of dsEph RNA rescues the commissures. In 30% (8/27) of all injected embryos all segments were restored to wild type. In contrast, in all embryos injected with buffer or dsGFP, segments are found with fused or absent commissures, indicating that ectopic Ephrin is still able to repel axons. In dsEph-injected embryos 33% of all segments have wild-type commissures, whereas in control-injected embryos even fewer segments show normal commissures (Bossing, 2002).
Presumably, it is possible to rescue the commissures with Eph RNAi because dsEph-injected embryos do not always show a loss or fusion of commissures. 20% of injected embryos and 31% of all segments have no phenotype. In the rescued embryos, Eph expression might be lowered enough to overcome the repulsion by ectopic midline Ephrin but not low enough to result in fused or lost commissures (Bossing, 2002).

EVOLUTIONARY HOMOLOGS

Ephrins: Interaction with ligands

The Eph-related family of receptor tyrosine kinases consists of at least 13 members, several of which display distinctive expression patterns in the developing and adult nervous system. In order to study functional interactions between B61-related ligands and Eph-related receptors, chimeric receptors, containing an Eph-related ectodomain and the cytoplasmic domain of the TrkB neurotrophin receptor were constructed. Expression and activation of such chimeric receptors in NIH 3T3 cells induce transformation in focus formation assays. Membrane-bound LERK2 ligand signals through three different Eph-related receptors: Cek5, Cek10 and Elk. LERK2, however, fails to interact functionally with the Cek9 receptor. Quantitative analysis including binding assays indicates that Cek10 is the preferred LERK2 receptor. Preliminary mutagenesis of the LERK2 protein suggests a negative regulatory role for its cytoplasmic domain in LERK2 signaling (Brambilla, 1995).

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, a comparative analysis has been undertaken 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, it has been found 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, binds Cek10 specifically, but weakly, 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 (Brambilla, 1996).

Many Eph-related receptor tyrosine kinases, and each of their numerous membrane-bound ligands, can be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan (Gale, 1996).

The Eph receptors are the largest known family of receptor protein tyrosine kinases; they play important roles, along with their ligands (ephrins), in the neural development, angiogenesis, and vascular network assembly. Ephrin-A2, -A3 and -A5 bind to, and activate the EphA8 receptor tyrosine kinase. An examination was performed to see if there are other additional ephrin ligands interacting with the EphA8 receptor tyrosine kinase expressed in NIH3T3 fibroblasts. For this purpose, chimeric ephrin-A1, -A4, -B1, -B2 or -B3 ligands were constructed consisting of the Fc portion of human IgG fused to ephrin ligand carboxyl-terminus. Both ephrin-A1 and ephrin-A4 chimeric ligands efficiently bind to the EphA8 receptor expressed in NIH3T3 fibroblasts, whereas the transmembrane ligands including ephrin-B1, -B2 and -B3 do not. Both the EphA8-TrkB chimeric receptor and the EphA8 receptor expressed in NIH3T3 fibroblasts are efficiently tyrosine-phosphorylated upon stimulating with epthin-A1 or -A4 but none of transmembrane ephrin-B proteins. These results strongly indicate that the EphA8 receptor functions exclusively as an glycosyl phosphatidylinositol (GPI)-linked ephrin ligand-dependent receptor protein tyrosine kinase (Choi, 1999).

The Eph family is thought to exert its function through the complementary expression of receptors and ligands. EphA receptors colocalize on retinal ganglion cell (RGC) axons with EphA ligands, which are expressed in a high-nasal-to-low-temporal pattern. In the stripe assay, only temporal axons are normally sensitive for repellent axon guidance cues of the caudal tectum. However, overexpression of ephrinA ligands on temporal axons abolishes this sensitivity, whereas treatment with PI-PLC both removes ephrinA ligands from retinal axons and induces a striped outgrowth of formerly insensitive nasal axons. In vivo, retinal overexpression of ephrinA2 leads to topographic targeting errors of temporal axons. These data suggest that differential ligand expression on retinal axons is a major determinant of topographic targeting in the retinotectal projection (Hornberger, 1999).

Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. EphB1 regulates cell attachment by discriminating the density at which ephrin-B1 is displayed on a reconstituted surface. EphB1-ephrin-B1 engagement does not promote cell attachment through mechanical tethering, but does activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen is mediated through alphavbeta3 integrin. EphB1 transfection confers ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants fail to stimulate ephrin-B1 dependent attachment. These findings led to the proposal that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment (Huynh-Do, 1999).

Eph receptor tyrosine kinases and their ligands (ephrins) are highly conserved protein families implicated in patterning events during development, particularly in the nervous system. In a number of functional studies, strict conservation of structure and function across distantly related vertebrate species has been confirmed. Soluble human EphA3 (HEK) exerts a dominant negative effect on somite formation and axial organization during zebrafish embryogenesis: this observation has been used to probe receptor function. Based on exon structure, the extracellular region of the EphA3 receptor has been dissected into evolutionarily conserved subdomains; kinetic BIAcore analysis, mRNA injection into zebrafish embryos, and receptor transphosphorylation analysis were all used to study the function of these domains. Ligand binding is restricted to the N-terminal region encoded by exon III, and an independent, C-terminal receptor-dimerization domain is identified. Recombinant proteins encoding either region in isolation can function as receptor antagonists in zebrafish. A two-step mechanism for Eph receptor activation with distinct ligand binding and ligand-independent receptor-receptor oligomerization events is proposed (Lackmann, 1998).

Contact-mediated axon repulsion by ephrins raises an unresolved question: these cell surface ligands form a high-affinity multivalent complex with their receptors present on axons, yet rather than being bound, axons can be rapidly repelled. Ephrin-A2 forms a stable complex with the metalloprotease Kuzbanian, involving interactions outside the cleavage region and the protease domain. Eph receptor binding triggers ephrin-A2 cleavage in a localized reaction specific to the cognate ligand. A cleavage-inhibiting mutation in ephrin-A2 delays axon withdrawal. These studies reveal mechanisms for protease recognition and control of cell surface proteins, and, for ephrin-A2, they may provide a means for efficient axon detachment and termination of signaling (Hattori, 2000).

'Reverse' signaling through Ephrins

Receptor tyrosine kinases of the EPH class have been implicated in the control of axon guidance and fasciculation, in regulating cell migration, and in defining compartments in the developing embryo. Efficient activation of EPH receptors generally requires that their ligands be anchored to the cell surface, either through a transmembrane (TM) region or a glycosyl phosphatidylinositol (GPI) group. These observations have suggested that EPH receptors can transduce signals initiated by direct cell-cell interaction. Genetic analysis of Nuk, a murine EPH receptor that binds TM ligands, has raised the possibility that these ligands might themselves have a signaling function. Consistent with this, the three known TM ligands have a highly conserved cytoplasmic region, with multiple potential sites for tyrosine phosphorylation. Challenging cells that express the TM ligands Elk-L or Htk-L with the clustered ectodomain of Nuk induces phosphorylation of the ligands on tyrosine, a process that can be mimicked both in vitro and in vivo by an activated Src tyrosine kinase. Co-culture of cells expressing a TM ligand with cells expressing Nuk leads to tyrosine phosphorylation of both the ligand and Nuk. These results suggest that the TM ligands are associated with a tyrosine kinase, and are inducibly phosphorylated upon binding Nuk, in a fashion reminiscent of cytokine receptors. Furthermore, TM ligands, as well as Nuk, are phosphorylated on tyrosine in mouse embryos, indicating that this is a physiological process. EPH receptors and their TM ligands therefore mediate bidirectional cell signaling (Holland, 1996).

Axonal pathfinding in the nervous system is mediated in part by cell-to-cell signaling events involving members of the Eph receptor tyrosine kinase (RTK) family and their membrane-bound ligands. Genetic evidence suggests that transmembrane ligands may transduce signals in the developing embryo. The cytoplasmic domain of the transmembrane ligand Lerk2 becomes phosphorylated on tyrosine residues after contact with the Nuk/Cek5 receptor ectodomain; this suggests that Lerk2 has receptorlike intrinsic signaling potential. Moreover, Lerk2 is an in vivo substrate for the platelet-derived growth factor receptor, which suggests crosstalk between Lerk2 signaling and signaling cascades activated by tyrosine kinases. It is proposed that transmembrane ligands of Eph receptors act not only as conventional RTK ligands but also as receptorlike signaling molecules (Bruckner, 1997).

Transmembrane ephrinB proteins have important functions during embryonic patterning as ligands for Eph receptor tyrosine kinases and presumably as signal-transducing receptor-like molecules. Consistent with 'reverse' signaling, ephrinB1 is localized in sphingo-lipid/cholesterol-enriched raft microdomains, platforms for the localized concentration and activation of signaling molecules. Glutamate receptor-interacting protein (GRIP) and a highly related protein, termed GRIP2, are recruited into these rafts through association with the C-terminal PDZ target site of ephrinB1. Stimulation of ephrinB1 with soluble EphB2 receptor ectodomain causes the formation of large raft patches that also contain GRIP proteins. Moreover, a GRIP-associated serine/threonine kinase activity is recruited into ephrinB1-GRIP complexes. These findings suggest that GRIP proteins provide a scaffold for the assembly of a multiprotein signaling complex downstream of ephrinB ligands (Bruckner, 1999).

Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encode polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies have demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bind ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells (Lin, 1999).

Eph proteins are receptors with tyrosine-kinase activity which, with their ephrin ligands, mediate contact-dependent cell interactions that are implicated in the repulsion mechanisms that guide migrating cells andneuronal growth cones to specific destinations. Ephrin-B proteins have conserved cytoplasmic tyrosine residues that are phosphorylated upon interaction with an EphB receptor, and may transduce signals that regulate a cellular response. Because Eph receptors and ephrins have complementary expression in many tissues during embryogenesis, bidirectional activation of Eph receptors and ephrin-B proteins could occur at interfaces of their expression domains, for example at segment boundaries in the vertebrate hindbrain. Previous work has implicated Eph receptors and ephrin-B proteins in the restriction of cell intermingling between hindbrain segments. An analysis was carried out to see whether complementary expression of Eph receptors and ephrins restricts cell intermingling, and whether this requires bidirectional or unidirectional signaling. Bidirectional but not unidirectional signaling restricts the intermingling of adjacent cell populations, whereas unidirectional activation is sufficient to restrict cell communication through gap junctions. These results reveal that Eph receptors and ephrins regulate two aspects of cell behavior that can stabilize a distinct identity of adjacent cell populations (Mellitzer, 1999).

Transmembrane B ephrins and their Eph receptors signal bidirectionally. However, neither the cell biological effects nor signal transduction mechanisms of the reverse signal are well understood. A cytoplasmic protein, PDZ-RGS3, is described that binds B ephrins through a PDZ domain, and has a regulator of heterotrimeric G protein signaling (RGS) domain. PDZ-RGS3 can mediate signaling from the ephrin-B cytoplasmic tail. SDF-1, a chemokine with a G protein-coupled receptor, and BDNF, both act as a chemoattractants for cerebellar granule cells, with SDF-1 action being selectively inhibited by soluble EphB receptor. This study reveals a pathway that links reverse signaling to cellular guidance, uncovers a novel mode of control for G proteins, and demonstrates a mechanism for selective regulation of responsiveness to neuronal guidance cues (Lu, 2001).

To investigate reverse signaling at a molecular level, a screen was performed for proteins that bind the B ephrin cytoplasmic domain, leading to identification of PDZ-RGS3 in a yeast two-hybrid assay. In situ hybridization shows a close overlap of expression patterns for PDZ-RGS3 with any one of the three known B ephrins in several parts of the nervous system. Taken together, these results indicate that PDZ-RGS3 is a genuine biological interaction partner of B ephrins (Lu, 2001).

PDZ domains are known to bind to a short conserved motif at the C terminus of many membrane proteins. A sequence fitting this motif is found at the C terminus of all known B ephrins, and the PDZ domain of PDZ-RGS3 binds the ephrin-B C terminus. Tyrosine residues are found in the binding motif (YYKV-carboxy terminus) suggesting potential control of binding by phosphorylation, although no evidence of this was seen, and the interaction did not appear to be regulated by EphB receptor binding. The presence of an RGS domain suggests PDZ-RGS3 might interact with downstream effector pathways. Accordingly, a Xenopus embryo cell dissociation assay PDZ-RGS3 mediates effects of the B ephrin cytoplasmic tail, in a manner dependent on both its PDZ and RGS domains. The involvement of the RGS domain in signaling, as well as the cerebellar expression of ephrins, led the authors to test cerebellar granule cells for an effect of reverse signaling on the action of the chemokine SDF-1, which acts through a GPCR. Soluble EphB2-Fc selectively regulates the guidance response to SDF-1, and this regulation is blocked by a truncated version of PDZ-RGS3 lacking the RGS domain (Lu, 2001).

Regarding the mechanism for signal transduction across the cell membrane, as with other PDZ proteins that bind B ephrins, the association with PDZ-RGS3 is seen constitutively, and does not appear to be modulated by treating cells with soluble EphB2-Fc. This suggests regulated association between B ephrin and PDZ-RGS3 is not a likely mechanism of signal transduction. An alternative could be regulation of clustering or subcellular localization. It is known that EphB2-Fc can cluster B ephrins and associated PDZ proteins into membrane rafts. Heterotrimeric G proteins have also been localized to rafts. Therefore, one model could be that Eph receptor binding clusters B ephrins into rafts, or other subcellular structures, and this could bring associated PDZ-RGS3 into proximity with the appropriate G proteins, resulting in inhibition of their activity. It is worth noting that not only the PDZ binding motif, but at least 33 amino acids of the B ephrin cytoplasmic tail are strongly conserved, and it is likely that additional protein interactions play a role in signaling, either through independent pathways or in collaboration with PDZ-RGS3 (Lu, 2001).

At the level of cell biological effects, these results show that reverse signaling induced by Eph receptor can regulate cellular guidance. Specifically, soluble EphB2-Fc selectively inhibits SDF-1 chemoattraction of cultured cerebellar granule neurons. Although reverse signaling through B ephrins has been investigated more extensively, soluble EphA receptors can affect adhesion in cell lines, and it will be interesting to see if this may reflect similar developmental functions or signaling pathways. The observations on the regulation of cerebellar granule cell guidance by EphB2-Fc, SDF-1, and BDNF led to a model for control of cell migration in cerebellar development. In principle, these observations could also fit with other developmental functions proposed for B ephrin reverse signaling in blood vessel formation, rhombomere compartmentation, and axon pathway selection -- all involving regulation of migration or morphogenesis (Lu, 2001).

The inward migration of cerebellar granule cells from the EGL is one of the best-characterized models of neuronal migration. The genetic demonstration that SDF-1 and its receptor CXCR4 are required for normal granule cell migration provided the first evidence of chemokines as regulators of neural development. Specifically, the phenotype of premature granule cell migration, taken together with the embryonic expression of SDF-1 in the pia mater overlying the cerebellum, suggest a model where SDF-1 would prevent premature inward migration of cerebellar granule cells by chemoattracting them toward the pia. The results support this model: SDF-1 expression occurs in the pia at postnatal stages that span the onset of granule cell migration, and SDF-1 acts as a chemoattractant for cultured cerebellar granule cells. Reverse signaling induced by soluble EphB2-Fc can inhibit the effect of SDF-1 on cerebellar granule cells. This provides functional evidence for an effect of ephrin signaling on cerebellar granule cells. A developmental role for the interaction of these signaling pathways is supported by the correlated expression of ephrin-B2, SDF-1, and their receptors during cerebellar development (Lu, 2001).

Bidirectional signals mediated by membrane-anchored ephrins and Eph receptor tyrosine kinases have important functions in cell-cell recognition events, including those that occur during axon pathfinding and hindbrain segmentation. The reverse signal that is transduced into B-ephrin-expressing cells is thought to involve tyrosine phosphorylation of the signal's short, conserved carboxy-terminal cytoplasmic domain. The Src-homology-2 (SH2) domain proteins that associate with activated tyrosine-phosphorylated B-subclass ephrins have not been identified, nor has a defined cellular response to reverse signals been described. The SH2/SH3 domain adaptor protein Grb4 binds to the cytoplasmic domain of B ephrins in a phosphotyrosine-dependent manner. In response to B-ephrin reverse signaling, cells increase FAK catalytic activity, redistribute paxillin, lose focal adhesions, round up, and disassemble F-actin-containing stress fibers. These cellular responses can be blocked in a dominant-negative fashion by expression of the isolated Grb4 SH2 domain. The Grb4 SH3 domains bind a unique set of other proteins that are implicated in cytoskeletal regulation, including the Cbl-associated protein (CAP/ponsin), the Abl-interacting protein-1 (Abi-1), dynamin, PAK1, hnRNPK and axin. These data provide a biochemical pathway whereby cytoskeletal regulators are recruited to Eph-ephrin bidirectional signaling complexes (Cowan, 2001).

EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, three tyrosines of ephrin-B1 have been identified as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ. Furthermore, many of the tyrosines of EphB2 identified as phosphorylation sites in 293 cells are also phosphorylated in retinal tissue. These data underline the complexity of ephrin-Eph bidirectional signaling by implicating many tyrosine phosphorylation sites of the ligand-receptor complex (Kalo, 2001).

Ephrins are cell surface-associated ligands for Eph receptors and are important regulators of morphogenic processes such as axon guidance and angiogenesis. Transmembrane ephrinB ligands act as 'receptor-like' signaling molecules, in part mediated by tyrosine phosphorylation and by engagement with PDZ domain proteins. However, the underlying cell biology and signaling mechanisms are poorly understood. Src family kinases (SFKs) are positive regulators of ephrinB phosphorylation and phosphotyrosine-mediated reverse signaling. EphB receptor engagement of ephrinB causes rapid recruitment of SFKs to ephrinB expression domains and transient SFK activation. With delayed kinetics, ephrinB ligands recruit the cytoplasmic PDZ domain containing protein tyrosine phosphatase PTP-BL and are dephosphorylated. These data suggest the presence of a switch mechanism that allows a shift from phosphotyrosine/SFK-dependent signaling to PDZ-dependent signaling (Palmer, 2002).

A first example of ephrinB signaling via PDZ domain proteins has been shown for cerebellar granule cells, which require the PDZ-RGS3 protein to respond to chemoattractants signaling through heterotrimeric G proteins. PTP-BL contains five PDZ domains, which interact with other cytoplasmic proteins including a GTPase-activating protein (GAP) with specificity for the Ras-like GTPase Rho. Rho GTPases are important regulators of actin cytoskeleton dynamics in response to external stimuli, and ephrinB-mediated axon guidance is thought to involve rapid changes in actin filament organization. Moreover, SFKs are known to regulate cell morphology, adhesion, and migration by association with and phosphorylation of focal adhesion kinase (FAK) and p190 RhoGAP. FAK is a downstream phosphorylation target of ephrinB reverse signaling. Mice deficient in p190 RhoGAP exhibit a lack of the anterior commissure, a phenotype associated with a lack of ephrinB reverse signaling in ephB2-/- mice. Thus, it will be interesting to analyze the role of the Src/p190 RhoGAP pathway and how these proteins contribute to both phosphotyrosine-dependent and PDZ domain-dependent signaling linked to rearrangements of the actin cytoskeleton downstream of ephrinB ligands (Palmer, 2002).

Eph receptors and ephrin ligands are key players in many developmental processes including embryo patterning, angiogenesis, and axon guidance. Eph/ephrin interactions lead to the generation of a bidirectional signal, in which both the Eph receptors and the ephrins activate downstream signaling cascades simultaneously. To understand the role of ephrin-B1 and the importance of ephrin-B1-induced reverse signaling during embryonic development, mouse lines carrying mutations in the efnb1 gene were generated. Complete ablation of ephrin-B1 results in perinatal lethality associated with a range of phenotypes, including defects in neural crest cell (NCC)-derived tissues, incomplete body wall closure, and abnormal skeletal patterning. Conditional deletion of ephrin-B1 demonstrated that ephrin-B1 acts autonomously in NCCs, and controls their migration. Last, a mutation in the PDZ binding domain indicates that ephrin-B1-induced reverse signaling is required in NCCs. These results demonstrate that ephrin-B1 acts both as a ligand and as a receptor in a tissue-specific manner during embryogenesis (Davy, 2004).

This work suggests that the role of ephrin-B1 in NCCs is to control directional migration toward target tissues, in agreement with known functions of ephrin/Eph signaling. In the mutant embryos, NCCs exhibited a wandering behavior, which has also been reported for other mutations, in particular for Twist homozygous mutants. The early lethality of the homozygous Twist mutants does not permit an assessment of whether the migration defects would affect similar NCC-derived structures as ephrin-B1 mutation. However, it has been reported that heterozygous Twist mutants present craniofacial defects reminiscent of defects seen in ephrin-B1^null mutants. The migration defects observed in the ephrin-B1^null animals do not provoke defects in formation and differentiation of cranial ganglia. Mutant embryos presented defects in nerve fasciculation and branching that might contribute to the perinatal lethality. A role in axon fasciculation has been described for EphA/ephrinA members previously (Davy, 2004).

What could be the molecular mechanisms by which ephrin-B1 regulates cell migration? Ephrin-B1 might function in part as a ligand to regulate NCC migration via activation of Eph receptors. Alternatively, ephrin-B1 also controls NCCs migration as a receptor, presumably by activating a signaling cascade involving a PDZ-containing protein. One candidate that might act downstream of ephrin-B1 in NCCs is the PDZ-containing protein PDZ-RGS3 that has been shown to be an effector of ephrin-B1 in regulating the migration of cerebellar granule cells. Preliminary data indicate that PDZ-RGS3 is indeed expressed in branchial arches (Davy, 2004).

In conclusion, this work demonstrates that ephrin-B1 plays an important role in different tissues during embryogenesis and that reverse signaling is an essential component of ephrin-B1 function. At the cellular level ephrin-B1 seems to regulate adhesion/migration processes. Interestingly, the results suggest that a reverse signaling cascade is required downstream of ephrin-B1 in a tissue-specific manner. Further studies will be necessary to clarify the role of ephrin-B1 in each tissue and to identify the specific effectors of ephrin-B1-induced reverse signaling in these tissues (Davy, 2004).

The number of cells in an organ is regulated by mitogens and trophic factors that impinge on intrinsic determinants of proliferation and apoptosis. This study reports on the identification of an additional mechanism to control cell number in the brain: EphA7 induces ephrin-A2 reverse signaling, which negatively regulates neural progenitor cell proliferation. Cells in the neural stem cell niche in the adult brain proliferate more and have a shorter cell cycle in mice lacking ephrin-A2. The increased progenitor proliferation is accompanied by a higher number of cells in the olfactory bulb. Disrupting the interaction between ephrin-A2 and EphA7 in the adult brain of wild-type mice disinhibits proliferation and results in increased neurogenesis. The identification of ephrin-A2 and EphA7 as negative regulators of progenitor cell proliferation reveals a novel mechanism to control cell numbers in the brain (Holmberg, 2005).

Mutations in the ephrin-B1 gene result in craniofrontonasal syndrome (CFNS) in humans, a congenital disorder that includes a wide range of craniofacial, skeletal, and neurological malformations. In addition to the ability of ephrin-B1 to forward signal through its cognate EphB tyrosine kinase receptors, ephrin-B1 can also act as a receptor and transduce a reverse signal by either PDZ-dependent or phosphorylation-dependent mechanisms. To investigate how ephrin-B1 acts to influence development and congenital disease, mice were generated harboring a series of targeted point mutations in the ephrin-B1 gene that independently ablate specific reverse signaling pathways, while maintaining forward signaling capacity. Both PDZ and phosphorylation-dependent reverse signaling by ephrin-B1 are dispensable for craniofacial and skeletal development, whereas PDZ-dependent reverse signaling by ephrin-B1 is critical for the formation of a major commissural axon tract, the corpus callosum. Ephrin-B1 is strongly expressed within axons of the corpus callosum, and reverse signaling acts autonomously in cortical axons to mediate an avoidance response to its signaling partner EphB2. These results demonstrate the importance of PDZ-dependent reverse signaling for a subset of Ephrin-B1 developmental roles in vivo (Bush, 2009).

Inhibitory interneurons control the flow of information and synchronization in the cerebral cortex at the circuit level. During embryonic development, multiple subtypes of cortical interneurons are generated in different regions of the ventral telencephalon, such as the medial and caudal ganglionic eminence (MGE and CGE), as well as the preoptic area (POA). These neurons then migrate over long distances towards their cortical target areas. Diverse families of diffusible and cell-bound signaling molecules, including the Eph/ephrin system, regulate and orchestrate interneuron migration. Ephrin A3 and A5, for instance, are expressed at the borders of the pathway of MGE-derived interneurons and prevent these cells from entering inappropriate regions via EphA4 forward signaling. This study found that MGE-derived interneurons, in addition to EphA4, also express ephrin A and B ligands, suggesting Eph/ephrin forward and reverse signaling in the same cell. In vitro and in vivo approaches showed that EphA4-induced reverse signaling in MGE-derived interneurons promotes their migration and that this effect is mediated by ephrin A2 ligands. In EphA4 mutant mice, as well as after ephrin A2 knockdown using in utero electroporation, delayed interneuron migration was found at embryonic stages. Thus, besides functions in guiding MGE-derived interneurons to the cortex through forward signaling, this study describes a novel role of the ephrins in driving these neurons to their target via reverse signaling (Steinecke, 2014).

Cytoplasmic interactions of Ephrins

Eph tyrosine kinase receptors and their membrane-bound ligands, ephrins, are presumed to regulate cell-cell interactions. The major consequence of bidirectional activation of Eph receptors and ephrin ligands is cell repulsion. In this study, Xenopus Dishevelled (Xdsh) is found to form a complex with Eph receptors and ephrin-B ligands and mediate the cell repulsion induced by Eph and ephrin. In vitro re-aggregation assays with Xenopus animal cap explants revealed that co-expression of a dominant-negative mutant of Xdsh affects the sorting of cells expressing EphB2 and those expressing ephrin-B1. Co-expression of Xdsh induces the activation of RhoA and Rho kinase in the EphB2-overexpressing cells and in the cells expressing EphB2-stimulated ephrin-B1. Therefore, Xdsh mediates both forward and reverse signaling of EphB2 and ephrin-B1, leading to the activation of RhoA and its effector protein Rho kinase. The inhibition of RhoA activity in animal caps significantly prevents the EphB2- and ephrin-B1-mediated cell sorting. It is proposed that Xdsh, which is expressed in various tissues, is involved in EphB and ephrin-B signaling related to regulation of cell repulsion via modification of RhoA activity (Tanaka, 2003).

Proteolysis of ephrins

Rhomboid-1 is a serine protease that cleaves the membrane domain of the Drosophila EGF-family protein, Spitz, to release a soluble growth factor. Several vertebrate rhomboid-like proteins have been identified, although their substrates and functions remain unknown. The human rhomboid, RHBDL2, cleaves the membrane domain of Drosophila Spitz when the proteins are co-expressed in mammalian cells. However, the membrane domains of several mammalian EGF-family proteins were not cleaved by RHBDL2, suggesting that the endogenous targets of the human protease are not EGF-related factors. The amino acid sequence at the luminal face of the membrane domain of a substrate protein determines whether it is cleaved by RHBDL2. Based on this finding, B-type ephrins are predicted as potential RHBDL2 substrates. One of these, ephrinB3, was cleaved so efficiently by the protease that little ephrinB3 was detected on the surface of cells co-expressing RHBDL2. These results raise the possibility that RHBDL2-mediated proteolytic processing may regulate intercellular interactions between ephrinB3 and eph receptors (Pascall, 2004).

Regulation of Ephrin expression

Although Apc is well characterized as a tumor-suppressor gene in the intestine, the precise mechanism of this suppression remains to be defined. Using a novel inducible Ahcre transgenic line in conjunction with a loxP-flanked Apc allele, loss of Apc is shown to acutely activate Wnt signaling through the nuclear accumulation of ß-catenin. Coincidentally, it perturbs differentiation, migration, proliferation, and apoptosis, such that Apc-deficient cells maintain a 'crypt progenitor-like' phenotype. Critically, a series of Wnt target molecules has been confirmed in an in vivo setting, and a series of new candidate targets has been identified within the same setting (Sansom, 2004).

ß-catenin levels were examined within the Cre⁺Apc^fl/fl tissue at day 5. There was no increase in total ß-catenin in the Cre⁺Apc^fl/fl samples. However, levels of dephosphorylated ß-catenin were moderately elevated and, crucially, ß-catenin relocalized to the nuclei in the Cre⁺Apc^fl/fl tissue. To more precisely define the time scale of nuclear relocalization, immunohistochemical analyses were performed at days 1, 2, 3, and 4 following induction of the cre recombinase. This analysis showed that relocalization occurred at day 3, and this was coincident with the observed onset of changes in morphology, proliferation, and apoptosis (Sansom, 2004).

To test whether nuclear ß-catenin was activating transcription of its known target genes, microarray analysis was performed using the affymetrix U74A chip. RNA samples were derived from sibling Cre⁺Apc^fl/fl and Cre⁺Apc^+/+ mice given four daily injections of ß-napthoflavone and killed at days 4 and 5. Of the 100 most significantly up-regulated genes, 10 have been associated with Wnt signaling (either directly or through arrays that had examined targets of the ß-catenin/TCF4 complex). Of the comparable genes up-regulated at day 5, 45 of 47 showed increases, of which 36 were in excess of twofold. These 36 included c-Myc, CD44, Tiam 1, Sema3c, and EphB3, all of which were confirmed changes at day 5. These data are, therefore, consistent with the notion that these are important early changes following nuclear relocalization of ß-catenin. Use of the larger chip set also revealed up-regulation of other Wnt target genes at day 4, including Sox17 and Axin2 (Sansom, 2004).

To validate the results obtained from the microarray analysis, the expression pattern of a subset of dysregulated genes was examined. Up-regulation of CD44, C-Myc, laminin gamma2, EphB2, and EphB3 was confirmed immunohistochemically in the Apc-deficient tissue. Expression of the EphrinB2 ligand, which is normally restricted to the top of the crypts and villi, was reduced in concordance with the reduction in villus differentiation in the Cre⁺Apc^fl/fl tissue. These data therefore confirm, in an in vivo setting, many of the targets of Wnt signaling that have been implicated from in vitro studies. These include up-regulation of CD44, c-Myc, MMP-7 (matrilysin), gamma-2 laminin, Sema3c (confirmed by RT-PCR) Ets-2, EphB2, EphB3, and GPR49. The array analysis also indicates up-regulation of a series of genes that either interact with CD44 or are targets of CD44. These include MMP-7, TIAM1, FGF4 and its receptor, and TASR-2. The up-regulation of TIAM1 is particularly interesting, since TIAM1 has been shown to mediate Ras signaling. Indeed, mice deficient in TIAM1 are resistant to Ras-induced skin tumors (Sansom, 2004).

Deficiency of EphB3 has been shown to lead to abnormal Paneth cell positioning in the crypt. Wnt-mediated up-regulation of EphB3 yields a similar Paneth cell phenotype, confirming a pivotal role for the EphB/ephrinB mutual repulsion system in defining crypt-villus architecture. These results are also consistent with the notion that Apc mutant cells express the same genetic program as cells at positions 1-2 of the crypt, with notable increases in EphB3, MMP7, and Pla2g2a being characteristic of both Paneth cells and the Apc-deficient cells described in this study (Sansom, 2004).

In summary, Apc has been shown to be a critical determinant of cell fate in the murine small intestinal epithelium. Acute activation of Wnt signaling immediately produces many of the phenotypes associated with early colorectal lesions: failed differentiation, increased proliferation, and aberrant migration. Within a short time scale, multiple processes are affected: interactions with the cellular matrix, interactions with the basement membrane, increased proliferation, and failure of positional cues (EphB/ephrinB) (Sansom, 2004).

Pathfinding of retinal ganglion cell (RGC) axons at the midline optic chiasm determine whether RGCs project to ipsilateral or contralateral brain visual centers, critical for binocular vision. Using Isl2^tau-lacZ knockin mice, it has been shown that the LIM-homeodomain transcription factor Isl2 marks only contralaterally projecting RGCs. The transcription factor Zic2 and guidance receptor EphB1, required by RGCs to project ipsilaterally, colocalize in RGCs distinct from Isl2 RGCs in the ventral-temporal crescent (VTC), the source of ipsilateral projections. Isl2 knockout mice have an increased ipsilateral projection originating from significantly more RGCs limited to the VTC. Isl2 knockouts also have increased Zic2 and EphB1 expression and significantly more Zic2 RGCs in the VTC. It is concluded that Isl2 specifies RGC laterality by repressing an ipsilateral pathfinding program unique to VTC RGCs and involving Zic2 and EphB1. This genetic hierarchy controls binocular vision by regulating the magnitude and source of ipsilateral projections and reveals unique retinal domains (Pak, 2004).

These findings indicate that Isl2 normally represses Zic2 expression in RGCs in the VTC and either directly represses EphB1 expression or indirectly through repression of Zic2 and that the increased ipsilateral projection in Isl2-null mice is due to a loss of this repression and upregulation of Zic2 and EphB1. This model is consistent with several pieces of data. (1) Regarding the timing of expression of Isl2 and Zic2 in VTC RGCs, the onset of Isl2 expression in VTC RGCs is similar to that of Zic2: weak Isl2 expression is detected in the VTC as early as E13.5, and moderate levels of Isl2 expression are evident by E14.5, the age when Zic2 expression in VTC RGCs is first detected. (2) Zic2 and EphB1 colocalize in a subset of RGCs distinct from Isl2 RGCs. (3) Increased expression of Zic2 and EphB1 and a significant increase in Zic2-positive RGCs are found in the VTC of Isl2-null retina. (4) The laterality phenotype of Isl2-null mice complements that of Zic2^kd/kd and EphB1 mutants (Pak, 2004).

Ephrin-Eph signalling drives asymmetric division in Ciona embryos

Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. This study uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. The segregation of notochord and neural fates is an autonomous property of the mother cell, and the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. These cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. It has been demonstrated that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. A model is proposed whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification (Picco, 2007).

Mesodermal tissues arise from diverse cell lineages and molecular strategies in the Ciona embryo. For example, the notochord and mesenchyme are induced by FGF/MAPK signaling, whereas the tail muscles are specified autonomously by the localized determinant, Macho-1. A unique mesoderm lineage, the trunk lateral cells, develop from a single pair of endomesoderm cells, the A6.3 blastomeres, which form part of the anterior endoderm, hematopoietic mesoderm and muscle derivatives. MAPK signaling is active in the endoderm descendants of A6.3, but is absent from the mesoderm lineage. Inhibition of MAPK signaling results in expanded expression of mesoderm marker genes and loss of endoderm markers, whereas ectopic MAPK activation produces the opposite phenotype: the transformation of mesoderm into endoderm. Evidence is presented that a specific Ephrin signaling molecule, Ci-ephrin-Ad, is required to establish asymmetric MAPK signaling in the endomesoderm. Reducing Ci-ephrin-Ad activity via morpholino injection results in ectopic MAPK signaling and conversion of the mesoderm lineage into endoderm. Conversely, misexpression of Ci-ephrin-Ad in the endoderm induces ectopic activation of mesodermal marker genes. These results extend recent observations regarding the role of Ephrin signaling in the establishment of asymmetric cell fates in the Ciona notochord and neural tube (Shi, 2008).

This study presents evidence that competition between Eprhin and FGF signaling is important for the asymmetric specification of endoderm and mesoderm lineages from a common endomesoderm progenitor cell, the A6.3 blastomere. A similar mechanism was recently invoked to account for the asymmetric specification of the notochord and nerve cord from common A6.2 and A6.4 progenitors (Picco, 2007). In both cases, a localized Ephrin-Ad signal produced by the primitive ectoderm (the animal blastomeres) competes with FGF9 signals from the primitive gut. Those cells in extended contact with the ectoderm lack MAPK activation, whereas those cells in contact with the endoderm experience MAPK activation and follow a different fate. It is conceivable that this interplay of Ephrin and FGF signaling is used in other systems to produce asymmetric cell fates (Shi, 2008).

Ephrins have been implicated in a variety of cellular processes, including axonal guidance, repulsive cell-cell interactions, and adhesion. Different Ephrin family members can activate or inhibit RTK signaling in different cellular contexts. The present study, along with the recent analysis of Ci-Bra regulation (Picco, 2007), suggest that Ci-ephrin-Ad functions as a localized inhibitor of FGF signaling to produce asymmetric cell fates in Ciona. The presumptive endoderm/endomesoderm produces a localized source of FGF9/16/20, which induces the specification of diverse mesoderm lineages, including the notochord and mesenchyme. Evidence is presented that Ephrin also controls the subdivision of the A6.3 endomesoderm (Shi, 2008).

A model is presented for the specification of the A7.6 blastomere. Previous studies have shown that Nodal is essential for the expression of several A7.6 marker genes, including Hand-like (also known as NoTrlc), FGF8 and Delta-like. Nodal is expressed in the A6.3 blastomere of 32-cell embryos, as well as in the other progenitors of the endoderm. FGF/MAPK signaling is also active in the A6.3 at this stage, as judged by anti-dpERK staining. Ephrin-Ad produced by b6.5 (and other animal blastomeres) inhibits MAPK in A7.6, thereby permitting Nodal to activate the A7.6 group genes. Nodal signaling in A7.6 might be reinforced by Nodal expression in the b6.5 lineage. Thus, the inhibition of FGF signaling by Eprhin-Ad, along with augmented levels of Nodal signal, might be responsible for the activation of A7.6 group genes. However, evidence is presented that Nodal in b6.5 is not essential for A7.6 group gene expression. Instead, it would appear that the combination of endogenous Nodal in the A6.3 progenitor, along with the localized inhibition of MAPK in A7.6 by Eprhin-Ad, is the decisive determinant of A7.6 specification (Shi, 2008).

Inhibition of MAPK signaling via drug treatment or ectopic expression of Ephrin-Ad leads to misexpression of A7.6 marker genes in the anterior endoderm, where Nodal is normally inactive owing to FGF/MAPK signaling. Posterior endoderm cells also contain Nodal but fail to express A7.6 marker genes upon inhibition of MAPK. This might reflect the restricted distribution of additional activators required for A7.6 gene expression. For example, Hand-like is activated by the combination of Nodal signaling and the FoxA transcription factor. FoxA expression is restricted to the anterior endoderm, dorsal mesoderm and future CNS floorplate, but is absent from the posterior endoderm. This is consistent with the result of ectopic Hand-like and Delta-like activation in the A-line neural lineage by expression of the FoxD::Nodal transgene (Shi, 2008).

A7.6 expresses a number of localized determinants, including two crucial signaling molecules, FGF8 and Delta-like. A7.6 is located in a strategically important position within the vegetal hemisphere. It contacts components of all three germ layers: the endoderm, ectoderm and mesenchyme. The Delta-like ligand expressed in A7.6 induces the secondary notochord lineage via Notch signaling, and also induces the lateralmost neural fate. Similarly, FGF8 expression is required for maintaining the primary notochord fate. Because these signaling pathways require either direct cell-cell contact (Notch) or act over one or two cell diameters (FGF), it is crucial to activate the expression of Delta-like and FGF8 to A7.6, but not in its sibling A7.5 endoderm blastomere. The activities of three pathways, Ephrin, MAPK and Nodal signaling, are employed to achieve this precise asymmetric cell-fate specification event (Shi, 2008).

Recent phylogenetic analysis suggests that tunicates (e.g., Ciona) are the closest living relatives of the vertebrates. As a result, it is possible that vertebrates employ a mechanism for the specification and subdivision of the endomesoderm that is similar to the one used in Ciona. The A6.3 endomesoderm cell is established by the action of a localized maternal determinant, β-Catenin, which activates the expression of multiple signaling molecules including Nodal and FGF9. Nodal is required to activate A7.6-specific genes such as Hand-like, FGF8 and Delta-like. The failure of Nodal to activate A7.6 group genes in the endoderm is due to MAPK signaling. FGF signaling either directly or indirectly inhibits Nodal. As a result, Nodal signaling is blocked in A6.3, but is activated in A7.6 owing to the localized inhibition of FGF signaling by Eprhin-Ad (Shi, 2008).

Most or all metazoan embryos possess a transient endomesoderm that generates specific mesodermal derivatives. In vertebrates, the presumptive endomesoderm gives rise to blood, heart and muscle. Formation of the vertebrate endomesoderm depends on TGF-β signaling molecules such as Xnrs in Xenopus and Squint and Cyclops (Nodal-related 1 and 2, respectively; ZFIN) in zebrafish. The subsequent subdivision of the endomesoderm is not clearly understood, but might depend on FGF signaling. It remains to be seen if competitive interactions between Nodal (or some other TGF-β signaling molecule) and FGF lead to the subdivision of endomesoderm in vertebrate embryos (Shi, 2008).

Ephrin-B2 regulates endothelial cell morphology and motility independently of Eph-receptor binding

The transmembrane protein ephrin-B2 regulates angiogenesis, i.e. the formation of new blood vessels through endothelial sprouting, proliferation and remodeling processes. In addition to essential roles in the embryonic vasculature, ephrin-B2 expression is upregulated in the adult at sites of neovascularization, such as tumors and wounds. Ephrins are known to bind Eph receptor family tyrosine kinases on neighboring cells and trigger bidirectional signal transduction downstream of both interacting molecules. This study shows that ephrin-B2 dynamically modulates the motility and cellular morphology of isolated endothelial cells. Even in the absence of Eph-receptor binding, ephrin-B2 stimulates repeated cycling between actomyosin-dependent cell contraction and spreading episodes, which requires the presence of the C-terminal PDZ motif. These results show that ephrin-B2 is a potent regulator of endothelial cell behavior, and indicate that the control of cell migration and angiogenesis by ephrins might involve both receptor-dependent and receptor-independent activities (Bochenek, 2010).

Activation of endogenous ephrin-B2 or overexpression of ephrin-B2 in endothelial cells revealed that cells undergo retraction and membrane blebbing followed by membrane protrusion. Overexpressed ephrin-B2 appears not to be visibly clustered at the cell surface, nor is it concentrated in vesicles inside the expressing cells. This might suggest that overexpressed ephrin-B2 is only weakly clustered at the cell surface and that this is sufficient for its autoactivation and subsequent stimulation of cycles of membrane retraction and cell expansion. How ephrin-B2 becomes activated is unclear, but EphB receptors have been shown to become autoactivated and autophosphorylated by overexpression. The difference in cell behavior between endothelial cells on the one hand and MDCKs and NIH3T3s on the other, after increased ephrin-B activation, might be due to expression of different complements of downstream effectors involved in ephrin-B2 signaling, expressed by these cells. It has been demonstrated that focal adhesion formation and membrane ruffling can be controlled, in smooth muscle cells or in human aortic endothelial cells, via Eph-ephrin signaling, through the Crk-p130 (CAS)-Rac pathway. This pathway, for example, may not be activated in NIH3T3 and MDCK cells by upregulated ephrin-B2 expression (Bochenek, 2010).

Balancing of ephrin/Eph forward and reverse signaling as the driving force of adaptive topographic mapping

The retinotectal projection, which topographically maps retinal axons onto the tectum of the midbrain, is an ideal model system with which to investigate the molecular genetics of embryonic brain wiring. Corroborating Sperry's seminal hypothesis, ephrin/Eph counter-gradients on both retina and tectum were found to represent matching chemospecificity markers. Intriguingly, however, it has never been possible to reconstitute topographically appropriate fiber growth in vitro with these cues. Moreover, experimentally derived molecular mechanisms have failed to provide explanations as to why the mapping adapts to grossly diverse targets in some experiments, while displaying strict point-to-point specificity in others. In vitro, ephrin-A/EphA forward, as well as reverse, signaling mediate differential repulsion to retinal fibers, instead of providing topographic guidance. It is argued that those responses are indicative of ephrin-A and EphA being members of a guidance system that requires two counteracting cues per axis. Experimentally, it was demonstrate by introducing novel double-cue stripe assays that the simultaneous presence of both cues indeed suffices to elicit topographically appropriate guidance. The peculiar mechanism, which uses forward and reverse signaling through a single receptor/ligand combination, entails fiber/fiber interactions. It is therefore proposed to extend Sperry's model to include ephrin-A/EphA-based fiber/fiber chemospecificity, eventually out-competing fiber/target interactions. By computational simulation, it was shown that this model is consistent with stripe assay results. More importantly, however, it not only accounts for classical in vivo evidence of point-to-point and adaptive topographic mapping, but also for the map duplication found in retinal EphA knock-in mice. Nonetheless, it is based on a single constraint of topographic growth cone navigation: the balancing of ephrin-A/EphA forward and reverse signaling (Gebhardt, 2012).

Evidence for rigid mapping originates from classical regeneration studies carried out by Roger Sperry. When the optic nerve is transected and a part of the retina is deleted in adult teleosts or amphibians, the regenerating partial projection selectively targets its original destination, ignoring termination sites vacated by the deletion. Recent experiments in the zebrafish indicate that the leading tips of individual RGC axonal arbors map topographically even in the absence of any other retinal fibers. This evidence corroborates Sperry's hypothesis (Sperry, 1963), which postulates genetically encoded matching labels on fiber terminals and target sites [fiber/target (FT) chemospecificity]. Indeed, tectal cell membranes convey graded repulsive signals (a<p) to retinal fibers in vitro owing to the expression of ephrin-As sensed through counter-graded retinal EphA receptors. Eventually, individual ephrin-A/EphA expression patterns were found to sum up to counter-gradients along both the retinal n/t and the tectal a/p axis. Ephrin-A/EphA forward, as well as reverse, signaling exert repulsive actions on RGC growth cones in vitro and genetic deletions in mice suggest corresponding roles in vivo. Surprisingly, however, it has never been possible to reconstitute fully topographically appropriate growth of RGC fibers in vitro using the identified guidance molecules. Thus, in choice assays, in which retinal fibers are confronted with alternating stripes of ephrin-A and a neutral substrate (here called single-cue stripe assay), nasals never decide properly. The reasons for this failure have remained puzzling (Gebhardt, 2012 and references therein).

The concept of rigid chemospecificity was first challenged by other seminal regeneration experiments, which instead promoted the concept of adaptive mapping. When a regenerating half-retina innervates a full tectum, terminals only initially occupy their proper tectal half. After longer regeneration periods, the projection spreads to cover the whole target evenly (expansion). Conversely, the projection of a full retina properly scales to a half-tectum (compression) (. When a nasal half-retina is forced to innervate an emptied anterior half-tectum (mismatch), a normally oriented half-map develops on the foreign field. If in a similar experiment the occupied p-tectum is left in place when the a-tectum is de-afferentiated, a second nasal population again forms a half-map on the foreign anterior target, but now with reversed orientation (polarity reversal). These results have usually been taken to indicate guidance mechanisms based on fiber/fiber (FF) interactions, the molecular underpinnings of which, however, are unknown. Recently, FF interactions have gained renewed interest. When, in a scattered half-population of RGCs, a constant amount of EphA is added by transgenic expression, a map duplication occurs, in which knock-in terminals occupying the a-tectum displace wild-type terminals posteriorly (Gebhardt, 2012 and references therein).

These seemingly conflicting bodies of evidence have been accompanied by numerous attempts at computational modeling. Although many models addressed certain aspects, at least four were successful in conceptually reconciling rigid and adaptive mapping evidences. Neither of them, however, rigorously relies on experimentally observed ephrin-A/EphA-based guidance mechanisms. Significantly, none of them has successfully addressed the seminal results of stripe assay experiments (Gebhardt, 2012).

In this study, by introduction of novel receptor/ligand (double-cue) stripe assays, fabricated by a combination of microfluidic network and contact-printing techniques, the reconstitute was accomplished of topographically appropriate guidance of RGC growth cones in vitro, revealing that the simultaneous presence of forward and reverse FT signaling is sufficient for rigid topographic growth decisions. To additionally cover adaptive mapping, it is suggest that Sperry's model be extended to include FF chemospecificity, also based on ephrin-A/EphA bidirectional signaling. A comprehensive model is presented for the guidance of topographically projecting axons that is essentially based on the single constraint of balancing ephrin-A/EphA forward and reverse signaling. In computational simulations, it successfully replicates evidences for both, rigid and adaptive mapping (Gebhardt, 2012).

Developmental expression Ephrins

Recent studies have implicated Eph-related receptor tyrosine kinases and their membrane-bound ligands in restricting or stimulating the movement of cells and axons. Members of these large families of receptors and ligands fall into two major binding specificity classes, in which the GPI-anchored subgroup of ligands can each bind to all members of a subgroup of receptors, whereas the transmembrane ligands interact with a distinct subgroup of receptors. Analysis of expression patterns is therefore important in order to understand which receptor-ligand interactions occur in vivo. Mouse orthologs of five members of the ligand family have been cloned and their developmental expression has been analysed in detail, both in comparison with one other, and with the receptor specificity class with which they interact. B61, AL-1/RAGS, LERK4, and ELF-1, members of the GPI-anchored subgroup of ligands, have both distinct and overlapping aspects to their expression in early mesoderm, somites, and branchial arches; in complex, dynamic patterns in the limb, and in spatial domains and specific neurons in the CNS. Similarly, Elk-L is expressed in hindbrain segments, the roof plate, and floor plate, overlapping in these locations the expression of other transmembrane ligands, but Elk-L has distinct expression in somites. The expression domains of ligands are complementary to those of the corresponding receptors in a number of tissues, including the midbrain, hindbrain, and differentiating limbs, consistent with potential roles in restricting cell movement. There are some overlaps in the expression of receptors and ligands, for example, in somites and the early limb. Taken together with previous studies showing that Eph-related receptors also have distinct but overlapping expression patterns, these data indicate that each ligand may have stage- and tissue-specific interactions with an individual member or multiple members of the receptor family (Flenniken, 1996).

Neuron-target interaction is a key feature in the establishment of neuronal networks. However, the underlying mechanism remains unclear. At the time of target innervation, Bsk, an eph family receptor, is expressed at high levels in several brain regions, including the hippocampus, olfactory bulb, and retina. To study whether the ligands are expressed in the target tissues, the expression of Bsk ligands was examined using a ligand-affinity probe, Bsk-AP, which consisted of the extracellular domain of Bsk fused in frame with a human placental alkaline phosphatase. These analyses show that the ligands are expressed at high levels in the developing septum, hypothalamus, olfactory neural epithelium, and tectum. In situ hybridization studies reveal that at least three different factors are responsible for the Bsk-AP binding. In the septum, Elf-1, Lerk3 (Eff-2), and AL-1/Lerk7 are transcribed. In the hypothalamus, AL-1/Lerk7 is the ligand detected by Bsk-AP. In the olfactory system, high levels of Lerk3 are detected in the sensory neurons. Both Elf-1 and AL-1/Lerk7 are present in the tectum. These ligand-positive areas are known to be anatomically connected to Bsk-expressing regions. These observations strongly suggest that Bsk and the ligands participate in neuron-target interactions in multiple systems and provide support for their involvement in topographic projection (Zhang, 1996).

The task of organizing the vast array of longitudinal axons that constitute the ventral and lateral funiculi, which project alongside or near the vertebrate midline of the developing vertebrate spinal cord, is likely to be relegated to a number of discrete repellent guidance cues that act upon axons emanating from multiple populations of functionally distinct interneurons. The Eph family of receptor tyrosine kinases are particularly good candidates since they interact exclusively with membrane-associated ligands, exhibit dynamic and spatially restricted expression patterns in the developing central nervous system, and together with the ephrins, mediate axonal patterning and pathway selection through contact-dependent repulsion in a variety of neural systems. Given their transmembrane structure, B-class ephrins are especially suited to function as highly localized, contact-dependent repellents. Although previous studies have documented a role for these proteins in repulsive axon guidance, the function of the transmembrane ephrins in the spinal cord proper are presently unknown. mRNA encoding all three transmembrane ligands is expressed in the floor plate during commissural axon pathfinding. Furthermore, B-class ephrin protein is tightly localized to the lateral floor plate margins, in immediate proximity to longitudinal fiber tracts formed by decussated commissural axons. Strikingly, expression of B-class Eph receptors is detected on only those segments of commissural axons that have crossed the floor plate and turned into the longitudinal axis. It is proposed that the onset of EphB receptor expression subsequent to midline crossing plays an important role in maintaining the longitudinal trajectory of commissural axons through a repulsive interaction with transmembrane ephrins situated at the lateral floor plate boundaries. In support of this interpretation, it is shown that all three B-class ephrins can induce the collapse of a subset of commissural growth cones in vitro. This is the first identification of a membrane-associated factor that directly promotes the collapse of vertebrate commissural growth cones (Imondi, 2000).

To identify molecules involved in neurogenesis, monoclonal antibodies were raised against embryonic day 12.5 mouse telencephalon. One antibody, monoclonal antibody 25H11, stains predominantly the ventricular zone of the anterior and lateral telencephalon. Purification of the 25H11 antigen, a 47 kDa integral membrane protein, from mouse telencephali reveals its identity with ephrin B1. Ephrin B1 appears at the onset of neocortical neurogenesis, being first expressed in neuron-generating neuroepithelial cells and rapidly thereafter in virtually all neuroepithelial cells. Expression of ephrin B1 persists through the period of neocortical neurogenesis and is downregulated thereafter. Ephrin B1 is present on the ventricular as well as basolateral plasma membrane of neuroepithelial cells and exhibits a ventricular-high to pial-low gradient across the ventricular zone. Expression of ephrin B1 is also detected on radial glial cells, extending all the way to their pial endfeet, and on neurons in the mantle/intermediate zone but not in the cortical plate. These results suggest that ephrin B1, presumably via ephrin-Eph receptor signaling, has a role in neurogenesis. Given the ventricular-to-pial gradient of ephrin B1 on the neuroepithelial cell surface and its known role in cell migration in other systems mediated by its repulsive properties, it is proposed that ephrin B1 may be involved in the migration of newborn neurons out from the ventricular zone toward the neocortex (Stuckmann, 2001).

Development of the tectum and the cerebellum is induced by a reciprocal inductive signaling between their respective primordia, the midbrain and the midbrain/hindbrain boundary (MHB). It is of interest to identify molecules that function in and downstream of this reciprocal signaling. Overexpression of LIM domain of the transcription factor Islet-3 (LIM ^Isl-3) leads to inhibition of this reciprocal signaling and to resultant defects in tectal and cerebellar development. Genes were sought that may be either up- or down-regulated by overexpression of LIM^Isl-3 by comparing the gene expression profiles in the midbrain and the MHB of normal embryos and embryos in which Islet-3 function is repressed, using a combination of ordered differential display and whole-mount in situ hybridization. Among genes identified in this search, two cDNA fragments encode Wnt1 and FGF8, which are already known to be essential for the reciprocal signaling between the midbrain and the MHB, confirming the effectiveness of this strategy. Four other partial cDNA clones were identified that were specifically expressed around the MHB, ten cDNAs specifically expressed in the tectum, and three cDNAs expressed in neural crest cells, including those derived from the midbrain level. The ephrin-A3 gene is specifically expressed in posterior tectum in a gradient that decreases anteriorly. Although ephrin-A2 and ephrin-A5 have been reported to be expressed in the corresponding region in mouse embryos (the superior/inferior colliculi), mouse ephrin-A3 is not expressed prominently in this region, suggesting that the role of ephrin-A3 in brain development may have been altered in the process of brain evolution (Hirate, 2001).

In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. Here it has been shown that ß-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3-/- mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. It is concluded that in the intestinal epithelium ß-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system (Batlle, 2002).

Chick brain factor 1 (CBF1), a nasal retina-specific winged-helix transcription factor, is known to prescribe the nasal specificity that leads to the formation of the precise retinotectal map, especially along the anteroposterior (AP) axis. However, its downstream topographic genes and the molecular mechanisms by which CBF1 controls the expression of them have not been elucidated. Misexpression of CBF1 represses the expression of EphA3 and CBF2, and induces that of SOHo1, GH6, ephrin A2 and ephrin A5. CBF1 controls ephrin A5 by a DNA binding-dependent mechanism, ephrin A2 by a DNA binding-independent mechanism, and CBF2, SOHo1, GH6 and EphA3 by dual mechanisms. BMP2 expression begins double-gradiently (varying in both naso-temporal and ventral-dorsal axes) in the retina from E5 in a complementary pattern to Ventroptin expression. Ventroptin antagonizes BMP2 as well as BMP4. CBF1 interferes in BMP2 signaling and thereby induces expression of ephrin A2. These data suggest that CBF1 is located at the top of the gene cascade for the regional specification along the nasotemporal (NT) axis in the retina and distinct BMP signals play pivotal roles in the topographic projection along both axes (Takahashi, 2003).

Ephrins and axonal pathfinding

The transmembrane (TM) subfamily of Eph ligands and their receptors have been implicated in axon pathfinding and in pattern formation during embryogenesis. These functions are thought to involve repulsive interactions but this has not been demonstrated directly. A growth cone collapse assay has been used to determine if the TM ligands Lerk2 and HtkL have repellant guidance activity. Lerk2, but not HtkL, is a collapsing factor for a subset of embryonic cortical neurons. Analysis of the effects of Lerk2 on both the morphology and the cytoskeleton of cortical neurons suggests a mechanism of action different from that of AL-1, a GPI-linked Eph ligand having similar repellant activity. Treatment with Lerk2 disrupts the organization of both the actin cytoskeleton and the microtubules and induces the formation of swellings in the center of the growth cone and along the axon. Measurement of the relative F-actin concentrations in the neurites and soma indicate that F-actin levels in the neurites decrease while those in the soma increase, with the net F-actin content of the neuron remaining unchanged. In contrast, prolonged treatment with AL-1 leads to a net loss of F-actin, consistent with the hypothesis that AL-1 acts by perturbing actin polymerization. These results provide evidence that the ectodomain of Lerk2 functions as a repellant guidance cue and show that, despite overlapping specificities in vitro, the biological activities of related ligands are not necessarily overlapping. Further, TM and GPI-linked Eph ligands appear to exert repellant activity by different mechanisms, opening up the possibility that they may have different effects on growth cones in vivo (Meima, 1997).

During the past few years, evidence has accumulated that activation of EphA kinases on the axons of retinal ganglion cells by ephrins-A on cells of the optic tectum (superior colliculus) plays a critical role in mapping the rostrocaudal axis of the retina onto the corresponding axis of the tectum. The similarities between topographic maps in the motor and visual systems has suggested that similar molecular mechanisms might underlie them. Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Subfamily A ephrins are implicated in this topographic mapping because: (1) developing muscles express all five of the ephrin-A genes; (2) rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5; (3) the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles; (4) topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms. If the conclusion that ephrins are directly involved in nerve-muscle matching is accepted, new questions arise about how they act. These can be divided into issues of mapping and synaptogenesis. In terms of mapping, one plausible model is that muscle fibers express different levels or combinations of ephrins in accordance with their positions and that motor axons express different levels or combinations of Eph kinases (the likely ephrin receptors) in accordance with their positions. For example, levels of expression might be graded along the rostrocaudal axis, as appears to be the case in the retinotectal system. Alternatively, patterns of ephrin and Eph kinase expression might describe a more complex combinatorial code, as suggested by the discrete identities of motor pools and muscles. Indeed, whereas Eph kinase and ephrin levels are graded along the rostrocaudal axis in the visual system, there is currently no evidence for graded expression of ligands or receptors in the motor system. Another possiblity is that interactions between ephrins and their receptors are permissive rather than instructive, enabling positional information to be transmitted by other molecules. It is important to emphasize that the data do not distinguish permissive versus instructive roles for ephrins (Feng, 2000).

In the embryonic visual system, EphA receptors are expressed on both temporal and nasal retinal ganglion cell axons. Only the temporal axons, however, are sensitive to the low concentrations of ephrin-A ligands found in the anterior optic tectum. The poor responsiveness of nasal axons to ephrin-A ligands, which allows them to traverse the anterior tectum and reach their targets in the posterior tectum, has been attributed to constitutive activation of the EphA4 receptor expressed in these axons. EphA4 is highly expressed throughout the retina, but is preferentially phosphorylated on tyrosine (activated) in nasal retina. In a screen for EphA4 ligands expressed in chicken embryonic retina, a novel ephrin, ephrin-A6, has been identified. Like ephrin-A5, ephrin-A6 has high affinity for EphA4 and activates this receptor in cultured retinal cells. In the embryonic day 8 (E8) chicken visual system, ephrin-A6 is predominantly expressed in the nasal retina and ephrin-A5 in the posterior tectum. Thus, ephrin-A6 has the properties of a ligand that activates the EphA4 receptor in nasal retinal cells. Ephrin-A6 binds with high affinity to several other EphA receptors as well and causes growth cone collapse in retinal explants, demonstrating that it can elicit biological responses in retinal neurons. Ephrin-A6 expression is high at E6 and E8, when retinal axons grow to their tectal targets, and gradually declines at later developmental stages. The asymmetric distribution of ephrin-A6 in retinal cells, and the time course of its expression, suggest that this new ephrin plays a role in the establishment of visual system topography (Menzel, 2001).

The role of the Eph family of receptor tyrosine kinases and their ligands in the establishment of the vomeronasal projection in the mouse has been investigated. The data show intriguing differential expression patterns of ephrin-A5 on vomeronasal axons and of EphA6 in the accessory olfactory bulb (AOB), such that axons with high ligand concentration project onto regions of the AOB with high receptor concentration and vice versa. These data suggest a mechanism for development of this projection that is the opposite of the repellent interaction between Eph receptors and ligands observed in other systems. In support of this idea, when given the choice of whether to grow on lanes containing EphA-Fc/laminin or Fc/laminin protein (in the stripe assay), vomeronasal axons prefer to grow on EphA-Fc/ laminin. Analysis of ephrin-A5 mutant mice reveal a disturbance of the topographic targeting of vomeronasal axons to the AOB. In summary, these data, which are derived from in vitro and in vivo experiments, indicate an important role of the EphA family in setting up the vomeronasal projection (Knoll, 2001a).

Growing axons follow highly stereotypical pathways, guided by a variety of attractive and repulsive cues, before establishing specific connections with distant targets. A particularly well-known example that illustrates the complexity of axonal migration pathways involves the axonal projections of motor neurons located in the motor cortex. These projections take a complex route during which they first cross the midline, then form the corticospinal tract, and ultimately connect with motor neurons in the contralateral side of the spinal cord. These obligatory contralateral connections account for why one side of the brain controls movement on the opposing side of the body. The netrins and slits provide well-known midline signals that regulate axonal crossings at the midline. A member of the ephrin family, ephrin-B3, also plays a key role at the midline to regulate axonal crossing. In particular, ephrin-B3 acts as the midline barrier that prevents corticospinal tract projections from recrossing when they enter the spinal gray matter. In ephrin-B3-/- mice, corticospinal tract projections freely recross in the spinal gray matter, such that the motor cortex on one side of the brain now provides bilateral input to the spinal cord. This neuroanatomical abnormality in ephrin-B3-/- mice correlates with loss of unilateral motor control, yielding mice that simultaneously move their right and left limbs and thus have a peculiar hopping gait quite unlike the alternate step gait displayed by normal mice. The corticospinal and walking defects in ephrin-B3-/- mice resemble those recently reported for mice lacking the EphA4 receptor, which binds ephrin-B3 as well as other ephrins, suggesting that the binding of EphA4-bearing axonal processes to ephrin-B3 at the midline provides the repulsive signal that prevents corticospinal tract projections from recrossing the midline in the developing spinal cord (Kullander, 2001).

To investigate Eph-ephrin bidirectional signaling, a series of mutations were generated in the ephrin-B3 locus. The absence of both forward and reverse signaling results in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract is defective since axons fail to respect the midline bound high-fidelity connection between the motor cortex and contralateral and ipsilateral motor neuron populations. A second mutation that expresses a truncated ephrin-pathfinding protein lacking Ephrin-B3's cytoplasmic domain does not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal cortico-spinal neurons as their fibers pathfind down the contralateral spinal cord. These data indicate ephrin-B3 functions as a midline-anchored repellent to stimulate forward signaling in EphA4-expressing axons (Yokoyama, 2001).

In both invertebrate and lower vertebrate species, decussated commissural axons travel away from the midline and assume positions within distinct longitudinal tracts. In the developing chick and mouse spinal cord, most dorsally situated commissural neuron populations extend axons across the ventral midline and through the ventral white matter along an arcuate trajectory on the contralateral side of the floor plate. Within the dorsal (chick) and intermediate (mouse) marginal zone, commissural axons turn at a conserved boundary of transmembrane ephrin expression, adjacent to which they form a discrete ascending fiber tract. In vitro perturbation of endogenous EphB-ephrinB interactions results in the failure of commissural axons to turn at the appropriate dorsoventral position on the contralateral side of the spinal cord; consequently, axons inappropriately invade more dorsal regions of B-class ephrin expression in the dorsal spinal cord. Taken together, these observations suggest that B-class ephrins act locally during a late phase of commissural axon pathfinding to specify the dorsoventral position at which decussated commissural axons turn into the longitudinal axis (Imondi, 2001).

Ascending sensory information reaches primary sensory cortical areas via thalamic relay neurons that are organized into modality-specific compartments or nuclei. Although the sensory relay nuclei of the thalamus show consistent modality-specific segregation of afferents, in a wild-type mouse strain it has been shown that the visual pathway can be surgically 'rewired' so as to induce permanent retinal innervation of auditory thalamic cell groups. Applying the same rewiring paradigm to a transgenic mouse lacking the EphA receptor family ligands ephrin-A2 and ephrin-A5 results in more extensive rewiring than in the wild-type strain. Ephrin-A2 and ephrin-A5 define a distinct border between visual and auditory thalamus. In the absence of this ephrin-A2/A5 border and after rewiring surgery, retinal afferents are better able to invade and innervate the deafferented auditory thalamus. These data suggest that signals that induce retinal axons to innervate the denervated auditory thalamus may compete with barriers, such as the ephrins, that serve to contain retinal axons within the normal target. The present findings thus show that the targeting of retinothalamic projections can be surgically manipulated in the mouse and that such plasticity can be controlled by proteins known to regulate topographic mapping (Lyckman, 2001).

The development of connections between thalamic afferents and their cortical target cells occurs in a highly precise manner. Thalamic axons enter the cortex through deep cortical layers, then stop their growth in layer 4 and elaborate terminal arbors specifically within this layer. The mechanisms that underlie target layer recognition for thalamocortical projections are not known. The growth patterns have been compared of thalamic explants cultured on membrane substrates purified from cortical layer 4, the main recipient layer for thalamic axons, and cortical layer 5, a non-target layer. Thalamic axons exhibit a reduced growth rate and an increased branching density on their appropriate target membranes compared with non-target substrate. When confronted with alternating stripes of both membrane substrates, thalamic axons grow preferentially on their target membrane stripes. Enzymatic treatment of cortical membranes has revealed that growth, branching and guidance of thalamic axons are independently regulated by attractive and repulsive cues differentially expressed in distinct cortical layers. These results indicate that multiple membrane-associated molecules collectively contribute to the laminar targeting of thalamic afferents. Furthermore, it was found that interfering with the function of Eph tyrosine kinase receptors and their ligands (ephrins) abolishes the preferential branching of thalamic axons on their target membranes, and that recombinant ephrin-A5 ligand elicites a branch-promoting activity on thalamic axons. It is concluded that interactions between Eph receptors and ephrins mediate branch formation of thalamic axons and thereby may play a role in the establishment of layer-specific thalamocortical connections (Mann, 2002a).

Neural maps in the vertebrate central nervous system often show discontinuously segregated, domain-to-domain patterns. However, the molecular mechanism that establishes such maps is not well understood. In the chicken olivocerebellar system, EphA receptors and ephrin-As are expressed with distinct levels and combinations in mapping domains. When ephrin-A2 is retrovirally overexpressed in the cerebellum, the olivocerebellar map is disrupted, excluding axons with high receptor activity from ectopic expression domains. Conversely, overexpression of a truncated EphA3 receptor in the cerebellum reduces endogenous ligand activity to undetectable levels and causes aberrant mapping, with high receptor axons invading high ligand domains. In vitro, ephrin-A2 inhibits outgrowth of inferior olive axons in a region-specific manner. These results suggest that Eph receptors and ephrins constitute domain-specific positional information, and the spatially accurate receptor-ligand interaction is essential to guide inferior olive axons to their correct target domains (Nishida, 2002).

Olfactory sensory neurons expressing a given odorant receptor (OR) project with precision to specific glomeruli in the olfactory bulb, generating a topographic map. Neurons expressing different ORs express different levels of ephrin-A protein on their axons. Moreover, alterations in the level of ephrin-A alter the glomerular map. Deletion of the ephrin-A5 and ephrin-A3 genes posteriorizes the glomerular locations for neurons expressing either the P2 or SR1 receptor, whereas overexpression of ephrin-A5 in P2 neurons results in an anterior shift in their glomeruli. Thus the ephrin-As are differentially expressed in distinct subpopulations of neurons and are likely to participate, along with the ORs, as one of a complement of guidance receptors governing the targeting of like axons to precise locations in the olfactory bulb (Cutforth, 2003).

The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, in vivo evidence is provided that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. These results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas (Dufour, 2003).

Molecular mechanisms generating the topographic organization of corticothalamic (CT) circuits, which comprise more than three-quarters of the synaptic inputs onto sensory relay neurons, and their interdependence with thalamocortical (TC) axon development, are unknown. Using in utero electroporation-mediated gene transfer, EphA7-mediated signaling on neocortical axons was shown to control the within-nucleus topography of CT projections in the thalamus. Notably, CT axons that misexpress EphA7 do not shift the relative positioning of their pathway within the subcortical telencephalon (ST), indicating that they do not depend upon EphA7/ephrin-A signaling in the ST for establishing this topography. Moreover, misexpression of cortical EphA7 results in disrupted topography of CT projections, but unchanged inter- and intra-areal topography of TC projections. These results support a model in which EphA/ephrin-A signaling controls independently the precision with which CT and TC projections develop, yet is essential for establishing their topographic reciprocity (Torii, 2005).

How can TC and CT projections achieve their reciprocal organization without interdependent controls? Based on the present results and studies suggesting that the intra-areal topography of TC projections is regulated by an EphA gradient in thalamic nuclei and an ephrin-A gradient in target cortical areas, a model is proposed in which complementary gradients of EphA and ephrin-A activity, located between as well as within specific cortical areas and their corresponding thalamic nuclei, produce reciprocal signaling between these structures. In this model, however, the EphA/ephrin-A signaling controls the topography of TC and CT projections independently, yet establishes essential functional topographic reciprocity (Torii, 2005).

In this model, functional gradients of EphA receptors and ephrin-A ligands are required to be complementary both between and within each reciprocal target. Specific expression patterns of EphAs and ephrin-As appear before CT and TC axons reach their targets. Together with recent evidence for intrinsic regulation of EphA7 and ephrin-A5 expression within the neocortex by transcription factors or morphogens, gradients of EphAs and ephrin-As are likely established by intrinsic regulation within the neocortex and DT independently. Complementary patterns might be achieved by reciprocal transcriptional regulation for each by the same transcription factor. For example, in dorsoventral patterning of the retina, Tbx5 enhances transcription of ephrin-Bs and represses EphBs. In contrast, Vax2 enhances the transcription of EphBs and represses ephrin-Bs. In addition to transcriptional regulation, repressive modulation of EphA function by coexpressed ephrin-A, as shown on retinal ganglion cell axons, might sharpen their functional complementarity where their expression overlaps. Reciprocal and topographic neural connections are found in many other systems throughout the brain. It will be important to determine whether this independent, but reciprocal, EphA/ephrin-A signaling model is applicable generally to other systems (Torii, 2005).

The cadherin Celsr3, homolog of Drosophila Flamingo, regulates the directional growth and targeting of axons in the CNS, but whether it acts in collaboration with or in parallel to other guidance cues is unknown. Furthermore, the function of Celsr3 in the peripheral nervous system is still largely unexplored. This study shows that Celsr3 mediates pathfinding of motor axons innervating the hindlimb. In mice, Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall near the branch point. Mutant axons respond to repulsive ephrinA-EphA forward signaling and glial cell-derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor alpha1 (GFRalpha1) and Frizzled3 (Fzd3). The function of Celsr3 is Fzd3 dependent but Vangl2 independent. These results provide evidence that the Celsr3-Fzd3 pathway interacts with EphA-ephrinA reverse signaling to guide motor axons in the hindlimb (Chai, 2014).

Ephrins and targeting of retinal fibers

Visual connections to the mammalian forebrain are known to be patterned by neural activity, but it remains unknown whether the map topography of such higher sensory projections depends on axon guidance labels. Complementary expression and binding are shown for the receptor EphA5 in mouse retina and its ligands ephrin-A2 and ephrin-A5 in multiple retinal targets, including the major forebrain target, the dorsal lateral geniculate nucleus (dLGN). These ligands can act in vitro as topographically specific repellents for mammalian retinal axons and are necessary for normal dLGN mapping in vivo. The results suggest a general and economic modular mechanism for brain mapping whereby a projecting field is mapped onto multiple targets by repeated use of the same labels. They also indicate the nature of a coordinate system for the mapping of sensory connections to the forebrain (Feldheim, 1999).

In the retinotectal projection, the Eph receptor tyrosine kinase ligands ephrinA2 and ephrinA5 are differentially expressed, not only in the tectum, but also in a high-nasal-to-low-temporal pattern in the retina. Recently, retrovirally driven overexpression of ephrinA2 on retinal axons leads to topographic targeting errors of temporal axons in that they overshoot their normal termination zones in the rostral tectum and project onto the mid- and caudal-tectum. The behavior of nasal axons, however, is only marginally affected. Overexpression of ephrinA5 affects the topographic targeting behavior of both temporal and nasal axons. These data reinforce the idea that differential ligand expression on retinal axons contributes to topographic targeting in the retinotectal projection. Additionally, it has been found that ectopic expression of ephrinA2 and ephrinA5 frequently leads to pathfinding errors at the chiasm, resulting in an increased stable ipsilateral projection (Dutting, 1999).

The Eph family is thought to exert its function through the complementary expression of receptors and ligands. Here, EphA receptors colocalize on retinal ganglion cell (RGC) axons with EphA ligands, which are expressed in a high-nasal-to-low-temporal pattern. In the stripe assay, only temporal axons are normally sensitive for repellent axon guidance cues of the caudal tectum. However, overexpression of ephrinA ligands on temporal axons abolishes this sensitivity, whereas treatment with PI-PLC both removes ephrinA ligands from retinal axons and induces a striped outgrowth of formerly insensitive nasal axons. In vivo, retinal overexpression of ephrinA2 leads to topographic targeting errors of temporal axons. These data suggest that differential ligand expression on retinal axons is a major determinant of topographic targeting in the retinotectal projection (Hornberger, 1999).

Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinal axonal projection to the tectum or to its mammalian equivalent, the superior colliculus (SC). In this paper, gene disruptions for both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes (Feldheim, 2000).

Currently, a leading model is that both attractant and repellent gradients may be prespecified within the target. Each axon then identifies its correct termination zone as the point where repellent and attractant forces cancel out. In this model, wild-type nasal axons map to posterior SC because they are attracted there. A prediction of this model is that in the ephrin-A2-/-; ephrin-A5-/- double mutant, axons would tend to map more posteriorly than normal, or in the case of extreme nasal axons might show no shift. This appears to be contrary to the results obtained in this study, especially considering that the opposite shifts in the nasal and temporal axons are of similar magnitudes. For similar reasons these results do not seem consistent with a variant of the counterbalanced gradient model, where a posterior to anterior ephrin gradient would be balanced by a prespecified repellent in an anterior to posterior gradient. An alternative possibility that could perhaps be reconciled with these results would be for ephrins in the target to act as either repellents or attractants at different concentrations, although there is currently no direct evidence to support such an effect from axon guidance assays (Feldheim, 2000 and references therein).

As an alternative to models with two counterbalanced gradients prespecified in the target, these results could be explained by a model involving a repellent gradient of ephrins, in combination with axon-axon competition. This competition could be for limiting positive factors in the target or could also involve direct axon-axon interactions. A model of this type has been suggested in an analysis of the LGN in ephrin-A5-/- mice, and the model now receives strong support from the more comprehensive analysis in the SC presented here. The repulsion/competition model would account for normal mapping as follows. Temporal axons would be unable to terminate in the posterior SC, because they are repelled by the ephrins, so they would be forced to arborize in the anterior SC. Nasal axons are less sensitive to ephrin repulsion and so would be able to terminate in the posterior SC. In the anterior SC, nasal axons face greater competition for limiting amounts of permissive factor(s), so they prefer to avoid this competition and arborize only in the posterior SC (Feldheim, 2000).

Incorporating competition in the model can explain several aspects of the data. (1) Nasal axons shift anteriorly in the ephrin-A5-/- single mutant (although this could be explained by the axon sensitivity model outlined above), and shift even further to the anterior in the ephrin-A2-/-; ephrin-A5-/- double mutant. According to the competition model, posterior repellents are removed, so axons from temporal or central retina are now able to compete more effectively in posterior SC; nasal axons therefore face increased competition in the posterior SC, so they lose their strong preference for this region and spread out into more anterior regions. The model thus seems to fit well with the opposite and quantitatively similar shifts of axons from temporal and nasal extremes of the retina. Labelings at intermediate retinal positions are more difficult to characterize because of the lack of fixed landmarks but, consistent with the competition model, such labelings revealed multiple arborizations, seemingly shifted in both anterior and posterior directions. (2) Axons are not respecified to a specific ectopic position. Instead, arborizations are scattered over an abnormally broad zone in the mutants, including both normal and abnormal regions. This is the result predicted by the competition model: as the topographically specific repellents are removed, axons would spread into abnormal regions, but there is no reason for them to disfavor the correct region. (3) Even in the ephrin-A2-/-; ephrin-A5-/- mutant, retinal axons fill the entire SC and axons from both nasal and temporal extremes of the retina form connections within the target. Models that involve a strict matching of values in the projecting and target field would predict that the mutant should have unmatched areas, whereas the competition model predicts that the entire projecting and target fields should still match up (Feldheim, 2000).

In the mammalian visual system, retinal axons undergo temporal and spatial rearrangements as they project bilaterally to targets on the brain. Retinal axons cross the neuraxis to form the optic chiasm on the hypothalamus in a position defined by overlapping domains of regulatory gene expression. However, the downstream molecules that direct these processes remain largely unknown. A novel in vitro paradigm was used to study possible roles of the Eph family of receptor tyrosine kinases in chiasm formation. In vivo, Eph receptors and their ligands distribute in complex patterns in the retina and hypothalamus. In vitro, retinal axons are inhibited by reaggregates of isolated hypothalamic, but not dorsal diencephalic or cerebellar cells. Furthermore, temporal retinal neurites are more inhibited than nasal neurites by hypothalamic cells. Addition of soluble EphA5-Fc to block Eph 'A' subclass interactions decreases both the inhibition and the differential response of retinal neurites by hypothalamic reaggregates. These data show that isolated hypothalamic cells elicit specific, position-dependent inhibitory responses from retinal neurites in culture. Moreover, these responses are mediated, in part, by Eph interactions. Together with the in vivo distributions, these data suggest possible roles for Eph family members in directing retinal axon growth and/or reorganization during optic chiasm formation (Marcus, 2000).

Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. The distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon has been examined during the period when visual connections are being established. Both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor-ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph-ephrin molecules is observed. A similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway (Marin, 2001).

The idea has been put forward that molecules and mechanisms acting during development are re-used during regeneration in the adult, for example in response to traumatic injury in order to re-establish the functional integrity of neuronal circuits. Members of the Eph family of receptor tyrosine kinases and their 'ligands', the ephrins, play a prominent role during development of the retinocollicular projection in rodents, where EphA receptors and ephrin-As are expressed in gradients in both the retina and the superior colliculi (SC). Whether EphA family members are also expressed or re-expressed in the adult after optic nerve lesion was examined, since the presence of axon guidance information is an important prerequisite for a topographically appropriate re-connection by retinal ganglion cell (RGC) axons. This analysis was encouraged by results showing that RGC axons do not exert guidance preferences in response to membranes from adult unlesioned SC, but in response to membranes from the adult deafferented SC. A graded expression pattern of ephrin-As was found in the SC both before and after deafferentation, which is remarkably similar to those found during development. EphA receptor levels are reduced in the SC after deafferentation and the expression patterns of the EphB family are not changed. In particular, the presence of a graded ephrin-A expression in the deafferented SC suggests that -- if robust regeneration of RGC axons can be achieved -- topographic guidance information as a likely requirement for a functionally successful re-establishment of the retinocollicular projection is available (Knoll, 2001b).

The retinotectal projection is the predominant model for studying molecular mechanisms controlling development of topographic axonal connections. Analyses of topographic mapping of retinal ganglion cell (RGC) axons in chick optic tectum indicate that a primary role for guidance molecules is to regulate topographic branching along RGC axons, a process that imposes unique requirements on the molecular control of map development. Topographically appropriate connections are established exclusively by branches that form along the axon shaft. Initially, RGC axons overshoot their appropriate termination zone (TZ) along the anterior-posterior (A-P) tectal axis; temporal axons overshoot the greatest distance and nasal axons the least: this correlates with the nonlinear increasing A-P gradient of ephrin-A repellents. In contrast, branches form along the shaft of RGC axons with substantial A-P topographic specificity. Topography is enhanced through the preferential arborization of appropriately positioned branches and elimination of ectopic branches. Using a membrane stripe assay and time-lapse microscopy, it has been shown that branches form de novo along retinal axons. Temporal axons preferentially branch on their topographically appropriate anterior tectal membranes. After the addition of soluble EphA3-Fc, which blocks ephrin-A function, temporal axons branch equally on anterior and posterior tectal membranes, indicating that the level of ephrin-As in posterior tectum is sufficient to inhibit temporal axon branching and generate branching specificity in vitro. These findings indicate that topographic branch formation and arborization along RGC axons are critical events in retinotectal mapping. Ephrin-As inhibit branching along RGC axons posterior to their correct TZ, but alone cannot account for topographic branching and must cooperate with other molecular activities to generate appropriate mapping along the A-P tectal axis (Yates, 2001).

Ephrin-B and EphB are distributed in matching dorsoventral gradients in the embryonic Xenopus visual system with retinal axons bearing high levels of ligand (dorsal axons) projecting to tectal regions with high receptor expression (ventral regions). In vitro stripe assays show that dorsal retinal axons prefer to grow on EphB receptor stripes supporting an attractive guidance mechanism. In vivo disruption of EphB/ephrin-B function by application of exogenous EphB or expression of dominant-negative ephrin-B ligand in dorsal retinal axons causes these axons to shift dorsally in the tectum, while misexpression of wild-type ephrin-B in ventral axons causes them to shift ventrally. These dorsoventral targeting errors are consistent with the hypothesis that an attractive mechanism that requires ephrin-B cytoplasmic domain is critical for retinotectal mapping in this axis (Mann, 2002b).

The retinotectal map is the best characterized model system to study how axons respond to guidance cues during the formation of the nervous system. The critical event in forming this map is topographic-specific axon branching. To elucidate the in vivo role of the repulsive cue ephrin-A5 in this event, chromophore-assisted laser inactivation (CALI) was used to generate acute loss of ephrin-A5 function in localized areas of the posterior tectum of chick embryos in ovo and the resulting changes of retinal projections were analyzed during initial outgrowth (E11) and when retinal axons arborize in the deep layers in the tectum (E12). Ephrin-A5 functions to restrict initial axon outgrowth at E11. At E12, CALI of ephrin-A5 did not affect the extent of axon outgrowth on the tectal surface but instead caused ectopic arborization posterior to the topographically correct site in deeper layers of the tectum. This shows that ephrin-A5 restricts arborization during this critical process for developing the retinotopic map. CALI provides an approach to inactivate in vivo function in higher vertebrates with high temporal and spatial specificity that may have wide application (Sakurai, 2002).

The EphB receptor ligand, ephrin-B1, may act bifunctionally as both a branch repellent and attractant to control the unique mechanisms in mapping the dorsal-ventral (DV) retinal axis along the lateral-medial (LM) axis of the optic tectum. EphB receptors are expressed in a low to high DV gradient by retinal ganglion cells (RGCs), and ephrin-B1 is expressed in a low to high LM gradient in the tectum. RGC axons lack DV ordering along the LM tectal axis, but directionally extend interstitial branches that establish retinotopically ordered arbors. Recent studies show that ephrin-B1 acts as an attractant in DV mapping and in controlling directional branch extension. Modeling indicates that proper DV mapping requires that this attractant activity cooperates with a repellent activity in a gradient that mimics ephrin-B1. Ectopic domains of high, graded ephrin-B1 expression created by retroviral transfection repel interstitial branches of RGC axons and redirect their extension along the LM tectal axis, away from their proper termination zones (TZs). In contrast, the primary RGC axons are unaffected and extend through the ectopic domains of ephrin-B1 and arborize at the topographically correct site. However, when the location of a TZ is coincident with ectopic domains of ephrin-B1, the domains appear to inhibit arborization and shape the distribution of arbors. These findings indicate that ephrin-B1 selectively controls, through either attraction or repulsion, the directional extension and arborization of interstitial branches extended by RGC axons arising from the same DV position: branches that arise from axons positioned lateral to the correct TZ are attracted up the gradient of ephrin-B1 and branches that arise from axons positioned medial to the same TZ are repelled down the ephrin-B1 gradient. Alternatively, EphB receptor signaling may act as a 'ligand-density sensor' and titrate signaling pathways that promote branch extension toward an optimal ephrin-B1 concentration found at the TZ; branches located either medial or lateral to the TZ would encounter a gradient of increasingly favored attachment in the direction of the TZ (McLaughlin, 2003).

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, play important roles during development of the nervous system. Frequently they exert their functions through a repellent mechanism, so that, for example, an axon expressing an Eph receptor does not invade a territory in which an ephrin is expressed. Eph receptor activation requires membrane-associated ligands. This feature discriminates ephrins from other molecules that sculpt the nervous system such as netrins, slits and class 3 semaphorins, which are secreted molecules. While the ability of secreted molecules to guide axons, i.e., to change their growth direction, is well established in vitro, little is known about this for the membrane-bound ephrins. Using Xenopus laevis retinal axons the properties were investigated of substratum-bound and (artificially) soluble forms of ephrin-A5 (ephrin-A5-Fc) to guide axons. When immobilized in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from the chick. The analysis of growth cone behavior in a gradient of soluble ephrin-A5 in the 'turning assay' reveals a substratum-dependent reaction of Xenopus retinal axons. On fibronectin, a repulsive response is observed, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations, indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, these data indicate that ephrin-As are able to guide axons in immobilized bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown (Wein, 2003).

In animals with binocular vision, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. Ephrin-Bs in the chiasm region direct the divergence of retinal axons through the selective repulsion of a subset of RGCs that express EphB1. Ephrin-B2 is expressed in radial glia at the mouse chiasm midline as the ipsilateral projection is generated and is selectively inhibitory to axons from ventrotemporal (VT) retina, where ipsilaterally projecting RGCs reside. Moreover, blocking ephrin-B2 function in vitro rescues the inhibitory effect of chiasm cells and eliminates the ipsilateral projection in the semiintact mouse visual system. A receptor for ephrin-B2, EphB1, is found exclusively in regions of retina that give rise to the ipsilateral projection. EphB1 null mice exhibit a dramatically reduced ipsilateral projection, suggesting that this receptor contributes to the formation of the ipsilateral retinal projection, most likely through its repulsive interaction with ephrin-B2. This study provides the first direct evidence that ephrin-B2 is more inhibitory to ipsilateral than contralateral retinal axons and demonstrates that ephrin-B2 is necessary for the ipsilateral projection to form. Using in situ hybridization and analyses of mutant mice, EphB1, a receptor for ephrin-B2, is shown to be expressed specifically in regions of the retina that give rise to the ipsilateral projection, and in mice lacking EphB1. Together these data identify ephrin-B2 and EphB1 as key players in retinal axon divergence and suggest that the function of B-class Ephs and ephrins in patterning binocular vision is conserved between species (Williams, 2003).

Ephrin-As act as retinal topographic mapping labels, but the molecular basis for two key aspects of mapping remains unclear. (1) Although mapping is believed to require balanced opposing forces, ephrin-As have been reported to be retinal axon repellents, and the counterbalanced force has not been molecularly identified. (2) Although graded responsiveness across the retina is required for smooth mapping, a sharp discontinuity has instead been reported. An axon growth assay has been developed to systematically vary both retinal position and ephrin concentration and test responses quantitatively. Responses varied continuously with retinal position, fulfilling the requirement for smooth mapping. Ephrin-A2 inhibits growth at high concentrations but promoted growth at lower concentrations. Moreover, the concentration producing a transition from promotion to inhibition varied topographically with retinal position. These results lead directly to a mapping model where position within a concentration gradient may be specified at the neutral point between growth promotion and inhibition (Hansen, 2004).

A quantitative axon outgrowth assay has been developed that allows both retinal position and ephrin concentration to be varied systematically. This outgrowth assay, unlike the stripe assay, is not a growth cone steering assay. However, anterior-posterior mapping primarily involves the regulation of the final extent of axon growth across the tectum/SC rather than growth cone steering. Moreover, ephrins are known to regulate several types of mapping-related axon growth response, including growth cone steering, collateral branching, and extent of outgrowth. While the output of this assay (or any other assay) is not taken as precisely matching the biology of normal mapping, the system used in this study provides a quantitative and controllable test of the growth response of retinal axons to added ephrins and displays topographic specificity (Hansen, 2004).

Initial experiments that tested the response to tectal membranes found that both nasal and temporal axons show selectivity; both grow preferentially on anterior membranes, with a stronger preference being shown by temporal axons. This finding differs from the in vitro stripe assay, where axons from the entire nasal side of the retina were reported to be unresponsive to posterior tectal membranes. The results here can therefore provide a resolution to the previously puzzling question of how axons across the nasal half of the retina are mapped if they are unresponsive to posterior tectal labels (Hansen, 2004).

In subsequent experiments that tested responses to ephrins, the results show that responsiveness across the retinal N-T axis does not fit a two-step discontinuous model and instead appear to be smoothly graded. It is not clear why previous assays detected a sharp cutoff between nasal and temporal axons. One possibility is a species difference between the mouse axons that were used in this study versus the chick axons that were used in earlier studies. In chick, normal in vivo mapping involves an initial phase with a simple nasal versus temporal discrimination, before the full graded map develops, and the in vitro assays might reflect this initial phase. An alternative explanation is suggested by the finding that, at the highest ephrin-A2 concentrations that were tested in this study (comparable to ephrin-A levels in posterior tectum), outgrowth across the retina did not appear to be graded and instead fit a two-step model of responsiveness, with a sharp cutoff between the two halves of the retina. This result may help explain why previous studies have shown a nasal versus temporal discontinuity if, as seems likely, those studies used a growth substrate with ephrin activity that was similar to or higher than the 100% ephrin-A2 substrate. Whatever the reason for the discontinuity in previous studies, the graded responsiveness that was seen here fits the predictions of the chemoaffinity theory, as required to form a smooth topographic map (Hansen, 2004).

The results show that the shape of the responsiveness curve along the N-T axis of the retina differs, depending on whether the axons were tested with ephrin-A5 or ephrin-A2. In response to ephrin-A5, retinal positions showed a continuous monotonic variation in response, from high outgrowth at the nasal extreme to low outgrowth at the temporal extreme. In contrast, ephrin-A2 elicits a biphasic response curve, with maximum outgrowth from axons midway across the nasal half of the retina, decreasing in both nasal and temporal directions. These responsiveness profiles in the retina show an interesting correspondence with the expression profiles in the superior colliculus (SC). Ephrin-A5 expression increases in a monotonic gradient from the anterior to the posterior SC, whereas ephrin-A2 shows a biphasic distribution with a high point midway across the posterior half of the SC, decreasing in both anterior and posterior directions. Thus, the retinal position that gave the highest outgrowth on each ligand maps to the SC region with the highest concentration of that particular ligand (Hansen, 2004).

In terms of the overall significance for mapping, the distinctive gradients of ephrin-A2 and ephrin-A5 could be interpreted by two models. One model could be that ephrin-A2 plays no role in mapping the far posterior SC and declines there simply because it is not needed. This model could be consistent with genetic studies that observed map disruptions in the far posterior SC in ephrin-A5 but not ephrin-A2 gene-targeted mice. However, those studies would not necessarily have detected a more subtle ephrin-A2 phenotype. An alternative model is that ephrin-A2 may make some contribution as a reverse-orientation mapping gradient in the far posterior SC. This second model could be consistent with proposals by Sperry that in mammals the mapping labels are not likely to be simple orthogonal gradients and were predicted to have a central-to-peripheral radial component (Hansen, 2004).

Regarding the molecular basis for the difference in response curves to ephrin-A2 and ephrin-A5, presumably the two ligands are differentially recognized by specific Eph receptors. This idea appears to be consistent with genetic evidence suggesting that a preferential functional relationship exists between specific ligand-receptor pairs, such as ephrin-A5 and EphA5. Such preferential relationships could arise from differential binding affinities, which have been observed among the ephrin-A ligands and EphA receptors. Although binding interactions within ephrin and Eph receptor subfamilies are relatively promiscuous, preferential quantitative aspects of the interactions may be functionally important, especially for a quantitative process such as mapping (Hansen, 2004).

Ephrin-As and -Bs have been shown in a number of assay systems to have either repellent/inhibitory effects or attractant/adhesive effects. In some cases, positive and negative effects have been seen in a single biological system. Transient growth promotion followed by axon fragmentation, has been observed when hippocampal axons are grown on ephrin-A-expressing fibroblast monolayers. These observations suggest a potential relevance to hippocamposeptal development, although they did not lead to a model to specify map position, since both positive and negative actions were highest on axons from the same side of the projecting area. The current study found no evidence that the growth-promoting effect was transient, and a possible explanation for the transient effect that was seen in the co-culture system could be a continuing rise in expression during long-term culture of fibroblast monolayers. Another study has reported that soluble ephrin-As can be either attractant or repellent for retinal axons, depending on the substrate upon which the axons are growing. While very interesting as a mechanism for axon regulation, this again does not lead to any obvious model to specify position within a topographic map (Hansen, 2004).

Another example is provided by the ephrin-Bs in dorsoventral retinotectal/retinocollicular mapping. Expression patterns initially led to the prediction that the interaction must be attractant. Consistent with this prediction, in vitro assays, in vivo overexpression, and dominant-negative experiments in Xenopus, as well as gene knockout analysis in mouse, all concluded that ephrin-B/EphB interactions do indeed have attractant effects. However, a more recent chick overexpression study found repellent effects, with patches of ectopic ephrin-B1 always being avoided by retinal axons. The observation of attraction in some assays and repulsion in other assays led to a suggestion that normal mapping by ephrin-Bs might involve a transition between attraction and repulsion. However, such a transition has not actually been observed for ephrin-Bs, and ephrin-As are still assumed to act in retinotectal position specification only by repulsion (Hansen, 2004).

The initial goal in developing a quantitative assay for axon outgrowth was to test for graded responses, and it was expected that ephrin-As would only have inhibitory effects on retinal axons. However, the results showed that ephrin-A2 could also promote retinal axon growth. There was no indication that either the positive or negative responses were transient, with outgrowth following a similar time course at all concentrations that were tested (Hansen, 2004).

The positive and negative effects that were observed in this study have three crucial features in relation to map specification: (1) they are concentration dependent, with a transition between positive and negative depending on the ephrin-A2 concentration; (2) whether the response is positive or negative is also dependent on retinal position, and (3) the direction of these two dependences fits the orientation of the retinotectal map, with appropriate topographic specificity (Hansen, 2004).

Based on these results, the following model is proposed for topographic map development. Axon growth would be promoted by low ephrin-A concentrations anterior to the topographically correct position and inhibited by higher ephrin-A concentrations posterior to the correct position. Each axon would ultimately form a termination zone at the neutral point between these positive and negative influences. The transition point between positive and negative effects would vary according to retinal position, occurring at higher ephrin-A concentrations for nasal axons, which terminate posteriorly, and lower ephrin-A concentrations for temporal axons, which terminate anteriorly. The result would be the formation of a topographic map. It is proposed that axons originating from different positions across the retina have different sensitivities to ephrin, presumably due to the graded distribution of EphA receptors in the retina, so that the neutral inflection point between positive and negative effects in the tectum/SC varies with retinal position. The result is the production of a smooth topographic map (Hansen, 2004).

During development of the retinocollicular projection in mouse, retinal axons initially overshoot their future termination zones (TZs) in the superior colliculus (SC). The formation of TZs is initiated by interstitial branching at topographically appropriate positions. Ephrin-As are expressed in a decreasing posterior-to-anterior gradient in the SC, and they suppress branching posterior to future TZs. This study investigates the role of an EphA7 gradient in the SC, which has the reverse orientation compared to the ephrin-A gradient. In EphA7 mutant mice the retinocollicular map is disrupted, with nasal and temporal axons forming additional or extended TZs, respectively. In vitro, retinal axons are repelled from growing on EphA7-containing stripes. These data support the idea that EphA7 is involved in suppressing branching anterior to future TZs. These findings suggest that opposing ephrin-A and EphA gradients are required for the proper development of the retinocollicular projection (Rashid, 2005).

Engrailed transcription factors regulate the expression of guidance cues that pattern retinal axon terminals in the dorsal midbrain. They also act directly to guide axon growth in vitro. This study shows that an extracellular En gradient exists in the tectum along the anterior-posterior axis. Neutralizing extracellular Engrailed in vivo, with genetically encoded secreted single-chain antihomeoprotein antibodies expressed in the tectum, causes temporal axons to map aberrantly to the posterior tectum in chick and Xenopus. Furthermore, posterior membranes from wild-type tecta incubated with anti-Engrailed antibodies or posterior membranes from Engrailed-1 knockout mice exhibit diminished repulsive activity for temporal axons. Since EphrinAs play a major role in anterior-posterior mapping, tests were performed to see whether Engrailed cooperates with EphrinA5 in vitro. It was found that Engrailed restores full repulsion to axons given subthreshold doses of EphrinA5. Collectively, these results indicate that extracellular Engrailed contributes to retinotectal mapping in vivo by modulating the sensitivity of growth cones to EphrinA (Wizenmann, 2009).

Ephrins guide the formation of functional maps in the visual cortex

Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act to organize thalamic inputs. This study mapped the visual cortex (V1) in mice deficient for ephrin-A2, -A3, and -A5 functionally [ephrin-A triple knock out (TKO)], using intrinsic signal optical imaging and microelectrode recording, and structurally, by anatomical tracing of thalamocortical projections. V1 is shifted medially, rotated, and compressed and its internal organization is degraded. Expressing ephrin-A5 ectopically by in utero electroporation in the lateral cortex shifts the map of V1 medially, and expression within V1 disrupts its internal organization. These findings indicate that interactions between gradients of EphA/ephrin-A in the cortex guide map formation, but that factors other than redundant ephrin-As are responsible for the remnant map. Together with earlier work on the retinogeniculate map, the current findings show that the same molecular interactions may operate at successive stages of the visual pathway to organize maps (Cang, 2005).

The visual system has been a long-standing model in the study of topographic map development. EphA receptors and ephrin-A ligands have been shown to be necessary for the development of topographic maps from the retina to two of its subcortical targets, the superior colliculus (SC) in the midbrain and dorsal lateral geniculate nucleus (dLGN) in the thalamus. The dLGN then projects to the visual cortex. In this study, it was found that the topographic mapping of geniculocortical projections is disrupted both structurally and functionally in an ephrin-A TKO. The expression patterns of EphA receptors and ephrin-A ligands in the geniculocortical projection are consistent with EphA/ephrin-A interactions acting in thalamocortical (TC) projections in the same manner as that proposed for EphA/ephrin-A interactions in subcortical mapping. In the cases in which ephrin-A ligand was successfully misexpressed within the developing visual cortex, the functional topographic maps were defective, consistent with ephrin-A ligands acting as molecular labels in the cortex. Together, these experiments suggest that the same mapping mechanisms are used at subcortical and cortical stages, acting in each projection and target area to maintain the topographic order of visual information as it is transferred to the next stage (Cang, 2005).

Ephrins and the segmentation of the hindbrain

Described here is the isolation and characterization of two zebrafish Eph receptor ligand cDNAs that have been called zfEphL3 and zfEphL4. These genes are expressed in the presumptive midbrain of developing embryos from 6 somites. By 24 hours, L3 is expressed throughout the midbrain including the region of the presumptive tectum, whereas L4 is strongly expressed in the midbrain caudal to the presumptive tectum. At later stages of development L3 is expressed in a graded fashion throughout the tectum and L4 is maintained at its posterior margin. Growth cone collapse and pathway selection assays demonstrate that both these proteins have a collapse activity for retinal ganglion cells. When faced with a choice of substrate on which to grow, temporal axons from chick retinal ganglion cells selectively avoid membranes from Cos cells transfected with L3, whereas nasal axons do not. Both temporal and nasal axons avoid membranes from Cos cells transfected with L4. The expression patterns, together with the functional data, suggest that although both ligands may be able to guide retinal ganglion cells axons in vitro, they have different roles in the guidance of retinotectal projections in vivo. The expression of L3 is consistent with a role in the guidance of retinal ganglion cells to their targets on the tectum, whereas that of L4 suggests a role in delineating the posterior boundary of the optic tectum (Brennan, 1997).

Rhombomeres are segmental units of the developing vertebrate hindbrain that underlie the reiterated organization of cranial neural crest migration and neuronal differentiation. valentino (val), a zebrafish homolog of the mouse bzip transcription factor-encoding gene, kreisler (potential Drosophila homolog: CG10034), is required for segment boundary formation caudal to rhombomere 4 (r4). val is normally expressed in r5/6 and is required for cells to contribute to this region. In val minus mutants, rX, a region one rhombomere in length and of mixed identity, lies between r4 and r7. While a number of genes involved in establishing rhombomeric identity are known, it is still largely unclear how segmental integrity is established and boundaries are formed. Members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins, are candidates for functioning in rhombomere boundary formation. Indeed, expression of the receptor ephB4a coincides with val in r5/6, while ephrin-B2a, which encodes a ligand for EphB4a, is expressed in r4 and r7, complementary to the domain of val expression (Cooke, 2001).

In val minus embryos, ephB4a expression is downregulated and ephrin-B2a expression is upregulated between r4 and r7, indicating that Val is normally required to establish the mutually exclusive expression domains of these two genes. Juxtaposition of ephB4a-expressing cells and ephrin-B2a-expressing cells in the hindbrain leads to boundary formation. Loss of the normal spatial regulation of eph/ephrin expression in val mutants correlates not only with absence of boundaries but also with the inability of mutant cells to contribute to wild-type r5/6. Using a genetic mosaic approach, it has been shown that spatially inappropriate Eph signaling underlies the repulsion of val minus cells from r5/6. It is proposed that Val controls eph expression and that interactions between EphB4a and Ephrin-B2a mediate cell sorting and boundary formation in the segmenting caudal hindbrain (Cooke, 2001).

Hox proteins drive cell segregation and non-autonomous apical remodelling during hindbrain segmentation

Hox genes encode a conserved family of homeodomain transcription factors regulating development along the major body axis. During embryogenesis, Hox proteins are expressed in segment-specific patterns and control numerous different segment-specific cell fates. It has been unclear, however, whether Hox proteins drive the epithelial cell segregation mechanism that is thought to initiate the segmentation process. This study investigated the role of vertebrate Hox proteins during the partitioning of the developing hindbrain into lineage-restricted units called rhombomeres. Loss-of-function mutants and ectopic expression assays reveal that Hoxb4 and its paralogue Hoxd4 are necessary and sufficient for cell segregation, and for the most caudal rhombomere boundary (r6/r7). Hox4 proteins regulate Eph/ephrins and other cell-surface proteins, and can function in a non-cell-autonomous manner to induce apical cell enlargement on both sides of their expression border. Similarly, other Hox proteins expressed at more rostral rhombomere interfaces can also regulate Eph/ephrins, induce apical remodelling and drive cell segregation in ectopic expression assays. However, Krox20, a key segmentation factor expressed in odd rhombomeres (r3 and r5), can largely override Hox proteins at the level of regulation of a cell surface target, Epha4. This study suggests that most, if not all, Hox proteins share a common potential to induce cell segregation but in some contexts this is masked or modulated by other transcription factors (Prin, 2014).

Astrocytic Ephrin-B1 Controls Excitatory-Inhibitory Balance in Developing Hippocampus

Astrocytes are implicated in synapse formation and elimination, which are associated with developmental refinements of neuronal circuits. Astrocyte dysfunctions are also linked to synapse pathologies associated with neurodevelopmental disorders and neurodegenerative diseases. Although several astrocyte-derived secreted factors are implicated in synaptogenesis, the role of contact-mediated glial-neuronal interactions in synapse formation and elimination during development is still unknown. This study examined whether the loss or overexpression of the membrane-bound ephrin-B1 (see Drosophila Ephrin) in astrocytes during postnatal day (P) 14-28 period would affect synapse formation and maturation in the developing hippocampus. Enhanced excitation of CA1 pyramidal neurons in astrocyte-specific ephrin-B1 KO male mice, which coincided with a greater vGlut1/PSD95 colocalization, higher dendritic spine density, and enhanced evoked AMPAR and NMDAR EPSCs. In contrast, EPSCs were reduced in CA1 neurons neighboring ephrin-B1-overexpressing astrocytes. Overexpression of ephrin-B1 in astrocytes during P14-28 developmental period also facilitated evoked IPSCs in CA1 neurons, while evoked IPSCs and miniature IPSC amplitude were reduced following astrocytic ephrin-B1 loss. Lower numbers of parvalbumin-expressing cells and a reduction in the inhibitory VGAT/gephyrin-positive synaptic sites on CA1 neurons in the stratum pyramidale and stratum oriens layers of KO hippocampus may contribute to reduced inhibition and higher excitation. Finally, dysregulation of excitatory/inhibitory balance in KO male mice is most likely responsible for impaired sociability observed in these mice. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits (Nguyen, 2020).

Ephrins and neural crest patterning

During vertebrate head development, neural crest cells migrate from hindbrain segments to specific branchial arches, where they differentiate into distinct patterns of skeletal structures. The rostrocaudal identity of branchial neural crest cells appears to be specified prior to migration, so it is important that they are targeted to the correct destination. In Xenopus embryos, branchial neural crest cells segregate into four streams that are adjacent during early stages of migration. It is not known what restricts the intermingling of these migrating cell populations and targets them to specific branchial arches. Xenopus EphA4 and EphB1 are expressed in migrating neural crest cells and mesoderm of the third arch, and third plus fourth arches, respectively. The ephrin-B2 ligand, which interacts with these receptors, is expressed in the adjacent second arch neural crest and mesoderm. Using truncated receptors, it has been shown that the inhibition of EphA4/EphB1 function leads to abnormal migration of third arch neural crest cells into second and fourth arch territories. Furthermore, ectopic activation of these receptors by overexpression of ephrin-B2 leads to scattering of third arch neural crest cells into adjacent regions. Similar disruptions occur when the expression of ephrin-B2 or truncated receptors is targeted to the neural crest. These data indicate that the complementary expression of EphA4/EphB1 receptors and ephrin-B2 is involved in restricting the intermingling of third and second arch neural crest and in targeting third arch neural crest to the correct destination. Together with previous work showing that Eph receptors and ligands mediate neuronal growth cone repulsion, these findings suggest that similar mechanisms are used for neural crest and axon pathfinding (Smith, 1997).

In the trunk of avian embryos, neural crest migration through the somites is segmental, with neural crest cells entering the rostral half of each somitic sclerotome but avoiding the caudal half. Little is known about the molecular nature of the cues (intrinsic to the somites) that are responsible for this segmental migration of neural crest cells. Eph-related receptor tyrosine kinases and their ligands are essential for the segmental migration of avian trunk neural crest cells through the somites. EphB3 localizes to the rostral half-sclerotome, including the neural crest, and the ligand ephrin-B1 has a complementary pattern of expression in the caudal half-sclerotome. To test the functional significance of this striking asymmetry, soluble ligand ephrin-B1 was added to interfere with receptor function in either whole trunk explants or neural crest cells cultured on alternating stripes of ephrin-B1 versus fibronection. Neural crest cells in vitro avoid migrating on lanes of immobilized ephrin-B1; the addition of soluble ephrin-B1 blocks this inhibition. Similarly, in whole trunk explants, the metameric pattern of neural crest migration is disrupted by the addition of soluble ephrin-B1, allowing entry of neural crest cells into caudal portions of the sclerotome. Thus, both in vivo and in vitro, the addition of soluble ephrin-B1 results in a loss of the metameric migratory pattern and a disorganization of neural crest cell movement. These results demonstrate that Eph-family receptor tyrosine kinases and their transmembrane ligands are involved in interactions between neural crest and sclerotomal cells, mediating an inhibitory activity necessary to constrain neural precursors to specific territories in the developing nervous system (Krull, 1997).

Little is known about the mechanisms that direct neural crest cells to the appropriate migratory pathways. This study set out to determine how neural crest cells that are specified as neurons and glial cells migrate only ventrally and are prevented from migrating dorsolaterally into the skin, whereas neural crest cells specified as melanoblasts are directed into the dorsolateral pathway. Eph receptors and their ephrin ligands have been shown to be essential for migration of many cell types during embryonic development. Consequently, it was asked if ephrin-B proteins participate in the guidance of melanoblasts along the dorsolateral pathway, and prevent early migratory neural crest cells from invading the dorsolateral pathway. Using Fc fusion proteins, the expression of ephrin-B ligands was detected in the dorsolateral pathway at the stage when neural crest cells are migrating ventrally. Furthermore, ephrins block dorsolateral migration of early-migrating neural crest cells because when the Eph-ephrin interactions are disrupted by addition of soluble ephrin-B ligand to trunk explants, early neural crest cells migrate inappropriately into the dorsolateral pathway. Surprisingly, the ephrin-B ligands continue to be expressed along the dorsolateral pathway during melanoblast migration. RT-PCR analysis, in situ hybridization, and cell surface-labelling of neural crest cell cultures demonstrate that melanoblasts express several EphB receptors. In adhesion assays, engagement of ephrin-B ligands to EphB receptors increases melanoblast attachment to fibronectin. Cell migration assays demonstrate that ephrin-B ligands stimulate the migration of melanoblasts. Furthermore, when Eph signaling is disrupted in vivo, melanoblasts are prevented from migrating dorsolaterally, suggesting ephrin-B ligands promote the dorsolateral migration of melanoblasts. Thus, transmembrane ephrins act as bifunctional guidance cues: they first repel early migratory neural crest cells from the dorsolateral path, and then later stimulate the migration of melanoblasts into this pathway. The mechanisms by which ephrins regulate repulsion or attraction in neural crest cells are unknown. One possibility is that the cellular response involves signaling to the actin cytoskeleton, potentially involving the activation of the Cdc42/Rac family of GTPases. In support of this hypothesis, the adhesion of early migratory cells to an ephrin-B-derivatized substratum has been shown to result in cell rounding and disruption of the actin cytoskeleton, whereas plating of melanoblasts on an ephrin-B substratum induces the formation of microspikes filled with F-actin (Santiago, 2002).

The segmental pattern of neural-crest-derived sympathetic ganglia arises as a direct result of signals that restrict neural crest cell migratory streams through rostral somite halves. The spatiotemporal pattern of chick sympathetic ganglia formation is a two-phase process. Neural crest cells migrate laterally to the dorsal aorta, then surprisingly spread out in the longitudinal direction, before sorting into discrete ganglia. This study investigated the function of two families of molecules that are thought to regulate cell sorting and aggregation. By blocking Eph/ephrins or N-cadherin function, changes were measured in neural crest cell migratory behaviors that lead to alterations in sympathetic ganglia formation using a sagittal slice explant culture and 3D confocal time-lapse imaging. The results demonstrate that local inhibitory interactions within inter-ganglionic regions, mediated by Eph/ephrins, and adhesive cell-cell contacts at ganglia sites, mediated by N-cadherin, coordinate to sculpt discrete sympathetic ganglia (Kasemeier-Kulesa, 2006).

Heterozygous loss of Twist1 function causes coronal synostosis in both mice and humans. In mice this phenotype is associated with a defect in the neural crest-mesoderm boundary within the coronal suture, as well as with a reduction in the expression of ephrin A2 (Efna2), ephrin A4 (Efna4) and EphA4 in the coronal suture. Mutations in human EFNA4 are a cause of non-syndromic coronal synostosis. This study investigated the cellular mechanisms by which Twist1, acting through Eph-ephrin signaling, regulates coronal suture development. EphA4 mutant mice exhibit defects in the coronal suture and neural crest-mesoderm boundary that phenocopy those of Twist1^+/- mice. Further, it was demonstrated that Twist1 and EphA4 interact genetically: EphA4 expression in the coronal suture is reduced in Twist1 mutants, and compound Twist1-EphA4 heterozygotes have suture defects of greater severity than those of individual heterozygotes. Thus, EphA4 is a Twist1 effector in coronal suture development. Finally, by DiI labeling of migratory osteogenic precursor cells that contribute to the frontal and parietal bones, it was shown that Twist1 and EphA4 are required for the exclusion of such cells from the coronal suture. It is suggested that the failure of this process in Twist1 and EphA4 mutants is the cause of craniosynostosis (Ting, 2009).

Other roles of Ephrins in brain development

The hippocampus and septum play central roles in one of the most important spheres of brain function: learning and memory. Although their topographic connections have been known for two decades and topography may be critical for cognitive functions, the basis for hippocamposeptal topographic projection is unknown. Elf-1, a membrane-bound eph family ligand, is a candidate molecular tag for the genesis of the hippocamposeptal topographic projection. Elf-1 is expressed in an increasing gradient from dorsal to ventral septum. Furthermore, Elf-1 selectively allows growth of neurites from topographically appropriate lateral hippocampal neurons, while inhibiting neurite outgrowth by medial hippocampal neurons. Complementary to the expression of Elf-1, an eph family receptor, Bsk, is expressed in the hippocampus in a lateral to medial gradient, consistent with a function as a receptor for Elf-1. Further, Elf-1 specifically binds Bsk, eliciting tyrosine kinase activity. It is concluded that the Elf-1/Bsk ligand-receptor pair exhibits traits of a chemoaffinity system for the organization of hippocamposeptal topographic projections (Gao, 1996).

Neuronal connections are arranged topographically such that the spatial organization of neurons is preserved by their termini in the targets. During the development of topographic projections, axons initially explore areas much wider than the final targets, and mistargeted axons are pruned later. The molecules regulating these processes are not known. The ligands of the Eph family tyrosine kinase receptors may regulate both the initial outgrowth and the subsequent pruning of axons. In the presence of ephrins, the outgrowth and branching of the receptor-positive hippocampal axons are enhanced. However, these axons are induced later to degenerate. These observations suggest that the ephrins and their receptors may regulate topographic map formation by stimulating axonal arborization and by pruning mistargeted axons (Gao, 1999).

Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens/olfactory tubercle, respectively, constituting mesostriatal and mesolimbic pathways. The molecular signals that confer target specificity of different dopaminergic neurons are not known. EphB1 and ephrin-B2, a receptor and ligand of the Eph family, are candidate guidance molecules for the development of these distinct pathways. EphB1 and ephrin-B2 are expressed in complementary patterns in the midbrain dopaminergic neurons and their targets, and the ligand specifically inhibits the growth of neurites and induces the cell loss of substantia nigra, but not ventral tegmental, dopaminergic neurons. These studies suggest that the ligand-receptor pair may contribute to the establishment of distinct neural pathways by selectively inhibiting the neurite outgrowth and cell survival of mistargeted neurons. Ephrin-B2 expression is upregulated by cocaine and amphetamine in adult mice, suggesting that ephrin-B2/EphB1 interaction may play a role in drug-induced plasticity in adults as well (Yue, 1999).

Eph tyrosine kinase receptors and their membrane-bound ephrin ligands mediate cell interactions and participate in several developmental processes. Ligand binding to an Eph receptor results in tyrosine phosphorylation of the kinase domain, and repulsion of axonal growth cones and migrating cells. A subpopulation of ephrin-A5 null mice display neural tube defects resembling anencephaly in man. This is caused by the failure of the neural folds to fuse in the dorsal midline, suggesting that ephrin-A5, in addition to its involvement in cell repulsion, can participate in cell adhesion. During neurulation, ephrin-A5 is co-expressed with its cognate receptor EphA7 in cells at the edges of the dorsal neural folds. Three different EphA7 splice variants, a full-length form and two truncated versions lacking kinase domains, are expressed in the neural folds. Co-expression of an endogenously expressed truncated form of EphA7 suppresses tyrosine phosphorylation of the full-length EphA7 receptor and shifts the cellular response from repulsion to adhesion in vitro. It is concluded that alternative usage of different splice forms of a tyrosine kinase receptor can mediate cellular adhesion or repulsion during embryonic development (Holmberg, 2000).

Because both ephrins and Eph receptors are membrane anchored, suppression of repulsive signal transduction could hypothetically turn these proteins into cell-adhesion molecules. Alternatively, the adhesive effect may be indirect where the ligand-receptor interaction may result in kinase-independent signal transduction that could affect other molecules with adhesive properties. Notably, mutations in the gene encoding the C. elegans Eph receptor, VAB-1, result in defective ventral enclosure, a process resembling neurulation. Analysis of different vab-1 mutants reveals that this process is independent of VAB-1 kinase activity, suggesting that the kinase-independent adhesive functions of Eph family receptors may be evolutionarily conserved. Over the past few years it has become clear that several molecules involved in axon guidance and cell migration, including netrins, semaphorins and slits, have both attractive and repulsive actions. The dual function of these molecules is regulated by interaction with different receptors or by modulation of cyclic-nucleotide-dependent signal transduction pathways. The differential response to ephrin-A5 is quite possibly the first example of how the response to a ligand can be modulated between repulsion or adhesion by alternative use of different splice forms of a receptor (Holmberg, 2000 and references therein).

Ephrins are developmentally regulated molecules that may contribute to axonal pathfinding through their binding to Eph receptor tyrosine kinases. In many cases, ephrins act as negative molecules that stimulate growth cone collapse, although some forms may promote axonal growth. The role played by ephrin-B1 during rat postnatal cerebellar development has been addressed. Ephrin-B1 is expressed by both granule and Purkinje neurons whereas EphB is present in granule neurons in early postnatal cerebellum at a time coincident with axonal and dendrite outgrowth. Stably transfected 3T3 cells overexpressing ephrin-B1 enhance survival and neurite growth from cultured cerebellar granule neurons, an effect that is inhibited by the presence of an excess of a soluble EphB protein. Ephrin-B1-induced neuritogenesis is correlated with an increased expression of certain neuronal-specific microtubule-associated proteins (MAPs). Cerebellar granule neurons plated on stably transfected 3T3 cells overexpressing ephrin-B1 show an up-regulation of the expression of axonal MAPs such as Tau and phosphorylated MAP2C compared with neurons cultured on control 3T3 cells. The level of expression of these axonal MAPs is similar to that found in neurons plated on poly-L-lysine. Interestingly, there is a noteworthy up-regulation of somatodendritic MAPs such as high-molecular-weight MAP2 and mode II-phosphorylated MAP1B in neurons cultured on stably transfected 3T3 cells overexpressing ephrin-B1 compared with neurons plated on either control 3T3 cells or poly-L-lysine. In view of these data, it is suggested that ephrin-B1 favors dendritogenesis of granule neurons during cerebellum development (Moreno-Flores, 2002).

Eph receptors are widely expressed during cerebral cortical development, yet a role for Eph signaling in the generation of cells during corticogenesis has not been shown. Cortical progenitor cells selectively express one receptor, EphA4, and reducing EphA4 signaling in cultured progenitors suppresses proliferation, decreasing cell number. In vivo, EphA4^-/- cortex has a reduced area, fewer cells and less cell division compared with control cortex. To understand the effects of EphA4 signaling in corticogenesis, EphA4-mediated signaling was selectively depressed or elevated in cortical progenitors in vivo. Compared with control cells, cells with reduced EphA4 signaling are rare and mitotically inactive. Conversely, overexpression of EphA4 maintains cells in their progenitor states at the expense of subsequent maturation, enlarging the progenitor pool. These results support a role for EphA4 in the autonomous promotion of cell proliferation during corticogenesis. Although most ephrins are undetectable in cortical progenitors, ephrin B1 is highly expressed. These analyses demonstrate that EphA4 and ephrin B1 bind to each other, thereby initiating signaling. Furthermore, overexpression of ephrin B1 stimulates cell division of neighboring cells, supporting the hypothesis that ephrin B1-initiated forward signaling of EphA4 promotes cortical cell division (North, 2009).

The cerebral cortex is a laminated sheet of neurons composed of the arrays of intersecting radial columns. During development, excitatory projection neurons originating from the proliferative units at the ventricular surface of the embryonic cerebral vesicles migrate along elongated radial glial fibres to form a cellular infrastructure of radial (vertical) ontogenetic columns in the overlaying cortical plate. However, a subpopulation of these clonally related neurons also undergoes a short lateral shift and transfers from their parental to the neighbouring radial glial fibres, and intermixes with neurons originating from neighbouring proliferative units. This columnar organization acts as the primary information processing unit in the cortex. The molecular mechanisms, role and significance of this lateral dispersion for cortical development are not understood. This study shows that an Eph receptor A (EphA) and ephrin A (Efna) signalling-dependent shift in the allocation of clonally related neurons is essential for the proper assembly of cortical columns. In contrast to the relatively uniform labelling of the developing cortical plate by various molecular markers and retrograde tracers in wild-type mice, alternating labelling of columnar compartments was found in Efna knockout mice that are caused by impaired lateral dispersion of migrating neurons rather than by altered cell production or death. Furthermore, in utero electroporation showed that lateral dispersion depends on the expression levels of EphAs and ephrin-As during neuronal migration. This so far unrecognized mechanism for lateral neuronal dispersion seems to be essential for the proper intermixing of neuronal types in the cortical columns, which, when disrupted, might contribute to neuropsychiatric disorders associated with abnormal columnar organization (Torii, 2009).

During forebrain morphogenesis, there is extensive reorganisation of the cells destined to form the eyes, telencephalon and diencephalon. Little is known about the molecular mechanisms that regulate region-specific behaviours and that maintain the coherence of cell populations undergoing specific morphogenetic processes. his study shows that the activity of the Eph/Ephrin signalling pathway maintains segregation between the prospective eyes and adjacent regions of the anterior neural plate during the early stages of forebrain morphogenesis in zebrafish. Several Ephrins and Ephs are expressed in complementary domains in the prospective forebrain and combinatorial abrogation of their activity results in incomplete segregation of the eyes and telencephalon and in defective evagination of the optic vesicles. Conversely, expression of exogenous Ephs or Ephrins in regions of the prospective forebrain where they are not usually expressed changes the adhesion properties of the cells, resulting in segregation to the wrong domain without changing their regional fate. The failure of eye morphogenesis in rx³ mutants is accompanied by a loss of complementary expression of Ephs and Ephrins, suggesting that this pathway is activated downstream of the regional fate specification machinery to establish boundaries between domains undergoing different programmes of morphogenesis (Cavodeassi, 2013).

Ephrins and limb innervation and patterning

The distribution of members of the Eph family were examined during muscle precursor cell development. The EphA4 receptor tyrosine kinase and its ligand, ephrin-A5, are expressed by muscle precursor cells and forelimb mesoderm in unique spatiotemporal patterns during the period when muscle precursors delaminate from the dermomyotome and migrate into the limb. To test the function of EphA4/ephrin-A5 interactions in muscle precursor migration, targeted, in ovo electroporation was used to express ephrin-A5 ectopically, specifically in the presumptive limb mesoderm. In the presence of ectopic ephrin-A5, Pax7-positive muscle precursor cells are significantly reduced in number in the proximal limb, compared with controls, and congregate abnormally near the lateral dermomyotome. In stripe assays, isolated muscle precursor cells avoid substrate-bound ephrin-A5 and this avoidance is abolished by addition of soluble ephrin-A5. These data suggest that ephrin-A5 normally restricts migrating, EphA4-positive muscle precursor cells to their appropriate territories in the forelimb, disallowing entry into abnormal embryonic regions (Swartz, 2001).

The genetic mechanisms that regulate the complex morphogenesis of generating cartilage elements in correct positions with precise shapes during organogenesis, fundamental issues in developmental biology, are still not well understood. By focusing on the developing mouse limb, importance was confirmed of transcription factors encoded by the Sall gene family in proper limb morphogenesis, and it was further shown that they have overlapping activities in regulating regional morphogenesis in the autopod (the distal elements of a limb that will give rise to the wrist and the fingers in the forelimb, and the ankle and toes in the hindlimb). Sall1/Sall3 double null mutants exhibit a loss of digit1 as well as a loss or fusion of digit2 and digit3, metacarpals and carpals in the autopod. Sall activity affects different pathways, including the Shh signaling pathway, as well as the Hox network. Shh signaling in the mesenchyme is partially impaired in the Sall mutant limbs. Additionally, the data suggest an antagonism between Sall1-Sall3 and Hoxa13-Hoxd13. Expression of Epha3 and Epha4 is downregulated in the Sall1/Sall3 double null mutants, and, conversely, is upregulated in Hoxa13 and Hoxd13 mutants. Moreover, the expression of Sall1 and Sall3 is upregulated in Hoxa13 and Hoxd13 mutants. Furthermore, by using DNA-binding assays, it was shown that Sall and Hox compete for a target sequence in the Epha4 upstream region. In conjunction with the Shh pathway, the antagonistic interaction between Hoxa13-Hoxd13 and Sall1-Sall3 in the developing limb may contribute to the fine-tuning of local Hox activity that leads to proper morphogenesis of each cartilage element of the vertebrate autopod (Kawakami, 2009).

Ephrins and heart and vascular system development

The vertebrate circulatory system is composed of arteries and veins. The functional and pathological differences between these vessels have been assumed to reflect physiological differences such as oxygenation and blood pressure. Ephrin-B2, an Eph family transmembrane ligand, marks arterial but not venous endothelial cells from the onset of angiogenesis. Conversely, Eph-B4, a receptor for ephrin-B2, marks veins but not arteries. ephrin-B2 knockout mice display defects in angiogenesis by both arteries and veins in the capillary networks of the head and yolk sac as well as in myocardial trabeculation. These results provide evidence that differences between arteries and veins are in part genetically determined and suggest that reciprocal signaling between these two types of vessels is crucial for morphogenesis of the capillary beds (Wang, 1998).

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of vascular morphogenesis. EphrinB2 may have an active signaling role, resulting in bi-directional signal transduction downstream of both ephrinB2 and Eph receptors. To separate the ligand and receptor-like functions of ephrinB2 in mice, the endogenous gene was replaced by cDNAs encoding either carboxyterminally truncated (ephrinB2^DeltaC) or, as a control, full-length ligand (ephrinB2^WT). While homozygous ephrinB2^WT/WT animals are viable and fertile, loss of the ephrinB2 cytoplasmic domain results in midgestation lethality similar to ephrinB2 null mutants (ephrinB2^KO). The truncated ligand is sufficient to restore guidance of migrating cranial neural crest cells, but ephrinB2^{DeltaC/DeltaC} embryos show defects in vasculogenesis and angiogenesis very similar to those observed in ephrinB2^KO/KO animals. These results indicate distinct requirements of functions mediated by the ephrinB carboxyterminus for developmental processes in the vertebrate embryo (Adams, 2001).

The cues and signaling systems that guide the formation of embryonic blood vessels in tissues and organs are poorly understood. Members of the Eph family of receptor tyrosine kinases and their cell membrane-anchored ligands, the ephrins, have been assigned important roles in the control of cell migration during embryogenesis, particularly in axon guidance and neural crest migration. The role of EphB receptors and their ligands during embryonic blood vessel development has been investigated in Xenopus laevis. In a survey of tadpole-stage Xenopus embryos for EphB receptor expression, expression of EphB4 receptors was detected in the posterior cardinal veins and their derivatives, the intersomitic veins. However, vascular expression of other EphB receptors, including EphB1, EphB2 or EphB3, could not be observed, suggesting that EphB4 is the principal EphB receptor of the early embryonic vasculature of Xenopus. Ephrin-B ligands are expressed complementary to EphB4 in the somites adjacent to the migratory pathways taken by intersomitic veins during angiogenic growth. RNA injection experiments were performed to study the function of EphB4 and its ligands in intersomitic vein development. Disruption of EphB4 signaling by dominant negative EphB4 receptors or misexpression of ephrin-B ligands in Xenopus embryos results in intersomitic veins growing abnormally into the adjacent somitic tissue. These findings demonstrate that EphB4 and B-class ephrins act as regulators of angiogenesis, possibly by mediating repulsive guidance cues to migrating endothelial cells (Helbling, 2000).

Angiogenesis can be divided into three phases: initiation (induction of sprouting); invasion (cell proliferation, migration and matrix degradation), and maturation (remodeling, lumen formation and differentiation of endothelial cells). To date, three growth factor systems (VEGF, angiopoietins and ephrins) have been identified as critical players of angiogenesis. As revealed by the vascular phenotypes obtained from gene inactivation and gain-of-function experiments, each signaling system appears to have an essential role during at least one particular phase of angiogenic growth of intersomitic veins. A tentative hierarchy of the signaling systems essential for intersomitic vein development may therefore be deduced. Analysis of heterozygous VEGF mutant embryos, which are less affected than those homozygously deficient for VEGF, has revealed a strong decrease in intersomitic vein sprouts. VEGF is therefore involved in the initiation of intersomitic vein sprouting. Angiopoietin-1 and its receptor Tie-2 appear to be critical later during maturation and stabilization of the intersomitic veins. Indeed, mutant mice initially form intersomitic veins, which in case of Ang-1 mutants undergo subsequent regression in older embryos. Finally, analysis of ephrin-B2- and double EphB2/EphB3-deficient mice as well Xenopus embryos disrupted in EphB4 signaling indicate a requirement for EphB receptors and their ligands during the invasion phase of intersomitic vein development. Therefore, EphB signaling appears to act downstream of VEGF and its receptors, but upstream of the angiopoietin signaling system (Helbling, 2000 and references therein).

EphrinB2, a transmembrane ligand of EphB receptor tyrosine kinases, is specifically expressed in arteries. In ephrinB2 mutant embryos, there is a complete arrest of angiogenesis. However, ephrinB2 expression is not restricted to vascular endothelial cells, and it has been proposed that its essential function may be exerted in adjacent mesenchymal cells. Mice have been generated in which ephrinB2 is specifically deleted in the endothelium and endocardium of the developing vasculature and heart. Such a vascular-specific deletion of ephrinB2 results in angiogenic remodeling defects identical to those seen in the conventional ephrinB2 mutants. These data indicate that ephrinB2 is required specifically in endothelial and endocardial cells for angiogenesis, and that ephrinB2 expression in perivascular mesenchyme is not sufficient to compensate for the loss of ephrinB2 in these vascular cells (Gerety, 2002).

Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between the endocardium and myocardium. Ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1, and BMP10 expression and signaling, and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression, and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro-cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. It is suggested that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease (Grego-Bessa, 2007).

A mutual coordination of size between developing arteries and veins is essential for establishing proper connections between these vessels and, ultimately, a functional vasculature; however, the cellular and molecular regulation of this parity is not understood. This study demonstrates that the size of the developing dorsal aorta and cardinal vein is reciprocally balanced. Mouse embryos carrying gain-of-function Notch alleles show enlarged aortae and underdeveloped cardinal veins, whereas those with loss-of-function mutations show small aortae and large cardinal veins. Notch does not affect the overall number of endothelial cells but balances the proportion of arterial to venous endothelial cells, thereby modulating the relative sizes of both vessel types. Loss of ephrin B2 or its receptor EphB4 also leads to enlarged aortae and underdeveloped cardinal veins; however, endothelial cells with venous identity are mislocalized in the aorta, suggesting that ephrin B2/EphB4 signaling functions distinctly from Notch by sorting arterial and venous endothelial cells into their respective vessels. These findings provide mechanistic insight into the processes underlying artery and vein size equilibration during angiogenesis (Kim, 2008).

Other roles of Ephrins in development

The epidermis of the nematode C. elegans is an epithelium that undergoes epiboly during embryogenesis, where it encloses the embryo ventrally. Ventral enclosure is the result of epidermal cell shape changes and involves two steps: (1) the extension of four anterior leading cells to the ventral midline, and (2) enclosure of the posterior epidermis. A catenin/cadherin system is required for normal movement of the anterior epidermal cells, and likely functions within epidermal cells to modulate cytoskeletal behavior. The Eph receptor VAB-1 is required in neurons for epidermal morphogenesis during C. elegans embryogenesis. Two models have been proposed for the nonautonomous role of VAB-1: neuronal VAB-1 might signal directly to epidermis, or VAB-1 signaling between neurons might be required for epidermal development. The ephrin VAB-2 (also known as EFN-1) is a ligand for VAB-1 and can function in neurons to regulate epidermal morphogenesis. In the absence of VAB-1 signaling, ephrin-expressing neurons are disorganized. vab-2/efn-1 mutations synergize with vab-1 kinase alleles, suggesting that VAB-2/EFN-1 may partly function in a kinase-independent VAB-1 pathway. These data indicate that ephrin signaling between neurons is required nonautonomously for epidermal morphogenesis in C. elegans (Chin-Sang, 1999).

How might lack of VAB-1 in neurons cause defects in the epidermis? Two models have been proposed for this nonautonomy of VAB-1: the 'steric hindrance' model and the 'reverse signaling' model; these models are not mutually exclusive. In the steric hindrance model, VAB-1 signaling operates between neuroblasts and neurons, and lack of VAB-1 causes neurons to be disorganized; enclosure is defective because the epidermal cells cannot move over the abnormal neuronal substrate. In the reverse signal model, VAB-1 signals directly from neurons to the epidermal cells; in vab-1 mutants such cues are absent and epidermal cells fail to migrate normally. Analysis of vab-2/efn-1 supports a steric hindrance model for the nonautonomous role of ephrin signaling. VAB-2/EFN-1 is mostly expressed in neuroblasts or neurons, and neuron-specific expression of VAB-2/EFN-1 can partly rescue epidermal defects of vab-2/efn-1 mutants, showing that VAB-2/EFN-1 can function in neurons to regulate epidermal development. Furthermore, mutations in the VAB-1 receptor cause disorganization of the VAB-2/EFN-1-expressing neurons. The spreading of VAB-2/EFN-1-expressing cells in vab-1 mutants likely reflects abnormal cell migration or adhesion, since vab-1 mutants do not display cell fate transformations (Chin-Sang, 1999).

This role of VAB-1 in preventing cell spreading is strikingly reminiscent of the cell sorting functions of Eph signaling in vertebrate neurogenesis. It has been concluded that VAB-2/EFN-1 signaling to VAB-1 occurs between neuronal precursors, and regulates cell adhesion or movement. In the absence of VAB-1 or VAB-2/EFN-1, neuroblasts fail to close up the ventral gastrulation cleft and as a result, descendant neurons are disorganized. Disorganized neurons might block epidermal movements directly (true 'steric hindrance') or indirectly: for example, the boundary between VAB-1 and VAB-2/EFN-1-expressing cells might attract migrating epidermal cells; if this boundary is disorganized, epidermal migrations would be disrupted. An analogous situation might occur in vertebrate angiogenesis, where ephrin signaling between endocardial cells is required for the development of overlying myocardial trabeculae. Such models do not rule out signaling from neurons to epidermis, possibly via other ephrins; however, of the three other C. elegans ephrins, at least two are expressed mainly in neurons. The small number of Eph receptors and ephrins in C. elegans suggests that it will be feasible to dissect the complete network of Eph/ephrin signaling in a simple animal (Chin-Sang, 1999).

The C. elegans genome encodes a single Eph receptor tyrosine kinase, VAB-1, which functions in neurons to control epidermal morphogenesis. Four members of the ephrin family of ligands for Eph receptors have been identified in C. elegans. Three ephrins (EFN-1/VAB-2, EFN-2 and EFN-3) have been previously shown to function in VAB-1 signaling. Mutations in the gene mab-26 affect the fourth C. elegans ephrin, EFN-4. efn-4 also functions in embryonic morphogenesis, and it is expressed in the developing nervous system. Interestingly, efn-4 mutations display synergistic interactions with mutations in the VAB-1 receptor and in the EFN-1 ephrin, indicating that EFN-4 may function independently of the VAB-1 Eph receptor in morphogenesis. Mutations in the LAR-like receptor tyrosine phosphatase PTP-3 and in the Semaphorin-2A homolog MAB-20 disrupt embryonic neural morphogenesis. efn-4 mutations synergize with ptp-3 mutations, but not with mab-20 mutations, suggesting that EFN-4 and Semaphorin signaling could function in a common pathway or in opposing pathways in C. elegans embryogenesis (Chin-Sang, 2002).

Ephrins and semaphorins regulate a wide variety of developmental processes, including axon guidance and cell migration. The roles of the ephrin EFN-4 and the semaphorin MAB-20 have been studied in patterning cell-cell contacts among the cells that give rise to the ray sensory organs of Caenorhabditis elegans. In wild-type, contacts at adherens junctions form only between cells belonging to the same ray. In efn-4 and mab-20 mutants, ectopic contacts form between cells belonging to different rays. Ectopic contacts also occur in mutants in regulatory genes that specify ray morphological identity. efn-4 and mab-20 reporters were used to investigate whether these ray identity genes function through activating expression of efn-4 or mab-20 in ray cells. mab-20 reporter expression in ray cells is unaffected by mutants in the Pax6 homolog mab-18 and the Hox genes egl-5 and mab-5, suggesting that these genes do not regulate mab-20 expression. mab-18 is found to be necessary for activating efn-4 reporter expression, but this activity alone is not sufficient to account for mab-18 function in controlling cell-cell contact formation. In egl-5 mutants, efn-4 reporter expression in certain ray cells is increased, inconsistent with a simple repulsion model for efn-4 action. The evidence indicates that ray identity genes primarily regulate ray morphogenesis by pathways other than through regulation of expression of semaphorin and ephrin (Hahn, 2003).

Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migration processes during development. The molecular cloning and tissue distribution are reported of zebrafish transmembrane ephrins that represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrin B sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously to several Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands are confined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins are expressed as early as 30% epiboly and during gastrula stages: in the germ ring, shield, prechordal plate, and notochord. Ectopic overexpression of dominant-negative soluble ephrin B constructs yields reproducible defects in the morphology of the notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does not completely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesis of the prechordal plate and the notochord is likely a consequence of a cell movement defect. These observations suggest Eph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation (Chan, 2001).

Eph receptor tyrosine kinases (RTK) and their ephrin ligands are involved in the transmission of signals that regulate cytoskeletal organization and cell migration, and are expressed in spatially restricted patterns at discrete phases during embryogenesis. Loss of function mutants of Eph RTK or ephrin genes result in defects in neuronal pathfinding or cell migration. Soluble forms of human EphA3 and ephrin-A5, acting as dominant negative inhibitors, interfere with early events in zebrafish embryogenesis. Exogenous expression of both proteins results in dose-dependent defects in somite development and organization of the midbrain-hindbrain boundary and hindbrain. The nature of the defects as well as the distribution and timing of expression of endogenous ligands/receptors for both proteins suggest that Eph-ephrin interaction is required for the organization of embryonic structures by coordinating the cellular movements of convergence during gastrulation (Oates, 1999).

Somitogenesis involves the segmentation of the paraxial mesoderm into units along the anteroposterior axis. A role for Eph and ephrin signaling in the patterning of presomitic mesoderm and formation of the somites is shown. Ephrin-A-L1 and ephrin-B2 are expressed in an iterative manner in the developing somites and presomitic mesoderm, as is the Eph receptor EphA4. The role of these proteins was examined by injection of RNA, encoding dominant negative forms of Eph receptors and ephrins. Interruption of Eph signaling leads to abnormal somite boundary formation and reduced or disturbed myoD expression in the myotome. Disruption of Eph family signaling delays the normal down-regulation of her1 and Delta D expression in the anterior presomitic mesoderm and disrupts myogenic differentiation. It is suggested that Eph signaling has a key role in the translation of the patterning of presomitic mesoderm into somites (Durbin, 1998).

Targeted inactivation of the Eph receptor ligand ephrinB1 in mouse causes perinatal lethality, edema, defective body wall closure, and skeletal abnormalities. In the thorax, sternocostal connections are arranged asymmetrically and sternebrae are fused -- defects that are phenocopied in EphB2/EphB3 receptor mutants. In the wrist, loss of ephrinB1 leads to abnormal cartilage segmentation and the formation of additional skeletal elements. It is concluded that ephrinB1 and B class Eph receptors provide positional cues required for the normal morphogenesis of skeletal elements. Another malformation, preaxial polydactyly, is exclusively seen in heterozygous females in which expression of the X-linked ephrinB1 gene is mosaic, so that ectopic EphB-ephrinB1 interactions led to restricted cell movements and the bifurcation of digital rays. These findings suggest that differential cell adhesion and sorting might be relevant for an unusual class of X-linked human genetic disorders, in which heterozygous females show more severe phenotypes than hemizygous males (Compagni, 2003).

How can ephrinB1-EphB interactions regulate segmentation and patterning processes? One possibility might be that enhanced proliferation of chondrogenic cells leads to a size expansion of cartilaginous condensations and eventually triggers the bifurcation of digits. Arguing against such a passive growth-controlled mechanism, ephrinB1 mutant limbs do not contain enlarged skeletal elements, and numbers of proliferating cells are comparable between mosaic areas. The A class receptor EphA7 facilitates chondrogenic condensation, but ephrinB1 KO, KO/+, and control mesenchyme show very similar chondrogenic capacities. Furthermore, the polydactyly in ephrinB1 KO/+ limbs is independent from changes in the Shh pathway (Compagni, 2003).

Interdigital zones forming between bifurcated digits in ephrinB1 KO/+ mutants are largely devoid of programmed cell death. BMPs and their receptors regulate apoptosis of interdigital cells, and, indeed, transcripts for bmp2, bmpr-1a, and msx-1 were initially absent in ectopic IDZs, which instead showed residual expression of the chondrogenic marker sox9. This indicates that morphological alterations induced by ectopic EphB-ephrinB1 signaling precede and are, to a certain extent, independent from changes in the genetic programs controlling the development of digits and IDZs (Compagni, 2003).

Previous work has shown that the Eph/ephrin system plays important roles in the guidance of neuronal growth cones and migrating cells by providing repulsive cues. Moreover, complementary patterns of Eph and ephrin expression in adjacent hindbrain rhombomeres are critically involved in restricting cell movement and intermingling, as confirmed by studies in zebrafish embryos. In mosaic KO/+ limbs, evidence was found for reduced cell mixing between mosaic areas of mesenchyme expressing EphB receptors and ephrinB1, respectively. It is hypothesized that EphB-ephrinB1 signaling interfaces provide interacting cells with repulsive cues, which, when properly located, lead to diverging cell movements and the splitting of growing digits (Compagni, 2003).

During somitogenesis, segmental patterns of gene activity provide the instructions by which mesenchymal cells epithelialize and form somites. Various members of the Eph family of transmembrane receptor tyrosine kinases and their Ephrin ligands are expressed in a segmental pattern in the rostral presomitic mesoderm. This pattern establishes a receptor/ligand interface at each site of somite furrow formation. This study uses the fused somites (fss) mutant as an in vivo system to study the role of Eph/Ephrin signaling during somite morphogenesis. fss encodes Tbx24, a T box transcription factor involved in maturation of the presomitic mesoderm. fss mutants lack anterior-posterior polarity within presumptive segments of the rostral PSM and fail to form somites. In the fused somites mutant, lack of intersomitic boundaries and epithelial somites is accompanied by a lack of Eph receptor/Ephrin signaling interfaces. These observations suggest a role for Eph/Ephrin signaling in the regulation of somite epithelialization. Restoration of Eph/Ephrin signaling in the paraxial mesoderm of fss mutants rescues most aspects of somite morphogenesis. (1) Restoration of bidirectional or unidirectional EphA4/Ephrin signaling results in the formation and maintenance of morphologically distinct boundaries. (2) Activation of EphA4 leads to the cell-autonomous acquisition of a columnar morphology and apical redistribution of ß-catenin, aspects of epithelialization characteristic of cells at somite boundaries. (3) Activation of EphA4 leads to nonautonomous acquisition of columnar morphology and polarized relocalization of the centrosome and nucleus in cells on the opposite side of the forming boundary. These nonautonomous aspects of epithelialization may involve interplay of EphA4 with other intercellular signaling molecules. These results demonstrate that Eph/Ephrin signaling is an important component of the molecular mechanisms driving somite morphogenesis. A new role is proposed for Eph receptors and Ephrins as intercellular signaling molecules that establish cell polarity during mesenchymal-to-epithelial transition of the paraxial mesoderm (Barrios, 2003).

The definitive retinal progenitors of the eye field are specified by transcription factors that both promote a retinal fate and control cell movements that are critical for eye field formation. However, the molecular signaling pathways that regulate these movements are largely undefined. Both the FGF and ephrin pathways impact eye field formation. Activating the FGF pathway before gastrulation represses cellular movements in the presumptive anterior neural plate and prevents cells from expressing a retinal fate, independent of mesoderm induction or anterior-posterior patterning. Inhibiting the FGF pathway promotes cell dispersal and significantly increases eye field contribution. EphrinB1 reverse signaling is required to promote cellular movements into the eye field, and can rescue the FGF receptor-induced repression of retinal fate. These results indicate that FGF modulation of ephrin signaling regulates the positioning of retinal progenitor cells within the definitive eye field (Moore, 2004).

Retinal development consists of a series of steps that progressively restrict the available cell fates. First, a subset of embryonic cells are prevented from expressing a retinal fate by inherited maternal factors, whereas others become biased toward retinal fates due to their position within the neural inductive field of the animal hemisphere. As the CNS is regionalized, part of the anterior neural plate is specified as the eye field. Potential retinal progenitors need to be positioned within the eye field to receive the local environmental signals that will direct their ultimate fates. Only after these steps are accomplished do the steps of eye organogenesis, cellular lamination, and phenotype specification occur. Although there has been great progress in understanding how retinal cell type specification occurs, the molecular mechanisms that control which embryonic cells become specified as the definitive retinal progenitors in the eye field remain largely undefined (Moore, 2004).

An accepted hypothesis of how the eye field forms is that signals from surrounding anterior structures regionalize the anterior neural plate. The presumptive eye field then expresses several transcription factors that initiate the retina developmental program, e.g., rx1, pax6, and six3. However, cellular movements during gastrulation and neurulation, directed in part by eye field transcription factors, also are critical, and the signaling factors involved in these early steps of eye field formation have not been identified (Moore, 2004).

Several FGF family members have been implicated in affecting cell movements during gastrulation, and the anterior expression patterns of some FGFs and their receptors are consistent with a role in the morphogenetic movements of eye field cells. Therefore, whether FGF signaling prior to gastrulation plays a role in determining which embryonic cells form the eye field was investigated. Using a constitutively active FGF receptor, enhanced FGF signaling was demonstrated to prevent the normal retinal progenitors from populating the presumptive eye field, suggesting that low levels of FGF signaling are normally required for cells to adopt a retinal fate. This was confirmed by demonstrating that reduced FGF signaling, accomplished by expression of a dominant-negative receptor, enhances the number of cells that become retinal progenitors. It is further reported that ephrinB1 signaling during gastrulation is required for retinal progenitors to move into the eye field, and that this movement can be modified by activating the FGF pathway. These results demonstrate that FGF modulation of ephrin signaling is important for establishing the bona fide retinal progenitors in the anterior neural plate (Moore, 2004).

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of embryonic blood vascular morphogenesis. However, the molecular mechanisms required for ephrinB2 transduced cellular signaling in vivo have not been characterized. To address this question, two sets of knock-in mice have been generated: ephrinB2^DeltaV mice expressed ephrinB2 lacking the C-terminal PDZ interaction site, and ephrinB2^5F mice expressed ephrinB2 in which the five conserved tyrosine residues were replaced by phenylalanine to disrupt phosphotyrosine-dependent signaling events. This analysis revealed that the homozygous mutant mice survive the requirement of ephrinB2 in embryonic blood vascular remodeling. However, ephrinB2^{DeltaV/DeltaV} mice exhibit major lymphatic defects, including a failure to remodel their primary lymphatic capillary plexus into a hierarchical vessel network, hyperplasia, and lack of luminal valve formation. Unexpectedly, ephrinB2^5F/5F mice display only a mild lymphatic phenotype. These studies define ephrinB2 as an essential regulator of lymphatic development and indicate that interactions with PDZ domain effectors are required to mediate its functions (Makinen, 2005).

Eph/ephrin interactions modulate muscle satellite cell motility and patterning.

During development and regeneration, directed migration of cells, including neural crest cells, endothelial cells, axonal growth cones and many types of adult stem cells, to specific areas distant from their origin is necessary for their function. It has recently been shown that adult skeletal muscle stem cells (satellite cells), once activated by isolation or injury, are a highly motile population with the potential to respond to multiple guidance cues, based on their expression of classical guidance receptors. In vivo, differentiated and regenerating myofibers dynamically express a subset of ephrin guidance ligands, as well as Eph receptors. This expression has previously only been examined in the context of muscle-nerve interactions; however, it is proposed that this expression might also play a role in satellite cell-mediated muscle repair. Therefore, whether Eph-ephrin signaling would produce changes in satellite cell directional motility was investigated. Using a classical ephrin 'stripe' assay, it was found that satellite cells respond to a subset of ephrins with repulsive behavior in vitro; patterning of differentiating myotubes is also parallel to ephrin stripes. This behavior can be replicated in a heterologous in vivo system, the hindbrain of the developing quail, in which neural crest cells are directed in streams to the branchial arches and to the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite. It is hypothesized that guidance signaling might impact multiple steps in muscle regeneration, including escape from the niche, directed migration to sites of injury, cell-cell interactions among satellite cell progeny, and differentiation and patterning of regenerated muscle (Stark, 2011).

EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity

Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. This study identified the transmembrane ligand EphrinB2 (see Drosophila Ephrin) and its receptor EphB4 (see Drosophila Eph) as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function (Frye, 2020).

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Search PubMed for articles about Drosophila Ephrin

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Biological Overview

date revised: 5 November 2020