REVIEWS

Mechanisms of ephrinEph
signalling in development, physiology
anddisease
Artur Kania13 and Rdiger Klein4,5

Abstract | Eph receptor Tyr kinases and their membrane-tethered ligands, the ephrins, elicit
short-distance cellcell signalling and thus regulate many developmental processes at the
interface between pattern formation and morphogenesis, including cell sorting and positioning,
and the formation of segmented structures and ordered neural maps. Their roles extend into
adulthood, when ephrinEph signalling regulates neuronal plasticity, homeostatic events and
disease processes. Recently, new insights have been gained into the mechanisms of ephrinEph
signalling in different cell types, and into the physiological importance of ephrinEph in different
organs and in disease, raising questions for future research directions.

Institut de Recherches
Cliniques de Montral (IRCM),
Montral, Quebec H2W 1R7,
Canada.
2
Dpartement de Mdecine,
Universit de Montral,
Montral, Quebec H3T 1J4,
Canada.
3
Division of Experimental
Medicine, Departments of
Biology, Anatomy and Cell
Biology, and Integrated
Program in Neurosciences,
McGill University, Montral,
Quebec H3A 1A3, Canada.
4
Department of Molecules
Signaling Development,
Max Planck Institute of
Neurobiology,
AmKlopferspitz 18,
82152Martinsried,
Germany.
5
Munich Cluster for Systems
Neurology (SyNergy),
81377Munich, Germany.
artur.kania@ircm.qc.ca;
rklein@neuro.mpg.de
1

doi:10.1038/nrm.2015.16
Published online 21 Jan 2016

Eph proteins belong to the superfamily of transmembrane Tyr kinase receptors. They were initially
identified in human carcinomas, in which they are
significantly overexpressed 1. This triggered a vast
number of studies, which contributed to describing
their functions and importance in many biological
processes. Now, it is well established that, by binding
their membrane-tethered ephrin ligands, Eph proteins allow short-distance cellcell communication
(FIG.1a). This activates direct signalling pathways that
affect the cellular cytoskeleton, leading primarily to
cell repulsion but also in some instances to cell adhesion. Consequently, many processes that involve fast
changes in cellular motility and/or morphology depend
on ephrinEphsignalling.
One example of a process regulated by ephrinEph
signalling is developmental cell sorting at tissue compartment boundaries, through which two separate cellular populations sharing a stable border can emerge
from initially intermingled populations. Another
important ephrinEph signalling-mediated process
discussed here is axon guidance in neurons, which leads
to the collapse or immobilization of axonal growth cones.
Cancer, synaptic plasticity and many physiological processes such as vasculogenesis involve short-distance
cellcell communication and also rely on ephrinEph
signalling. In addition, there is increasing evidence that
this pathway functions in the regulation of cell differentiation, proliferation and apoptosis. What makes
ephrinEph signalling so versatile? One possible reason
is the variety of its signalling modes, which include Eph
proteins behaving as classical receptorsand ephrins

as ligands; Eph proteins behaving as ligands signalling to ephrins; and Ephs and ephrins simultaneously
eliciting intracellular signals in the cells that express
them(FIG.1).
This Review briefly discusses the classical experi
ments that uncovered the principles of molecular
mechanisms of ephrinEph signalling, including an
overview of more recent experiments on structure
function analysis. Arguably, the most insights into
ephrinEph function came from studies of the
developing nervous system, a subject extensively
discussed here. In addition, we provide an overview
of ephrinEph function that includes the development ofnon-neural tissues. We also consider the role
of ephrinEph in adult tissues, including stem cell
compartments, as well as its involvement in human
diseases such as cancer and neurodegeneration, two
important fields in which its functional principles are
still emerging. Because of their detailed review elsewhere, we do not discuss the roles of ephrins as viral
receptors2. Many aspects of Eph signalling have reached
scientific maturity as distinct disciplines, such that a
number of its core principles are well understood at
a molecular level, allowing us to propose some overarching themes of Eph function. Thus, we consider
the various ephrinEph signalling modes from the
perspective of different cell types and across evolution
(BOX1). Finally, we raise questions that might be considered as new research directions, such as how different
functionalities might be encoded by Ephephrin subtype differences and how ephrinEph signalling might
intersect with other signalling pathways.

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a

Cell 1
Ephrin

Cell repulsion
Cellcell adhesion
Cell proliferation
Tissue boundary formation
Cell migration
Axon guidance

Extracellular
space
Eph
Cell 2

Ephrin-B
PDZ

P
TM

Intracellular

Ephrin-A

RBD

GPI

Cell membrane

LBD

RBD

RBD

LBD

Sushi

Extracellular
space

Sushi
EGF

Eph

Cys-rich domain
EGF

FN1
FN2

Cell membrane
Intracellular

FN1

TM

P TK
P

SAM
PDZ

Ephrin:Eph
forward

FN2
Eph:ephrin
reverse

EphrinEph
bidirectional

Parallel

Anti-parallel

Intracellular
Ephrin
Extracellular
space
EPH
Axon guidance
A process during which
neurons extend axonal
processes (see definition of
neurons, below) that are
directed towards their
innervation targets such as
other neurons or muscles.

Neurons
Specialized cells transmitting
nerve impulses. Neurons
comprise a cell body
containing the nucleus;
dendrites, which are short
processes extending from the
cell body that receive nerve
impulses; and an axon, which is
a long process that transmits
nerve impulses to the neurons
postsynaptic target.

Axonal growth cones

Highly motile, fan-like
structures at the tip of the
extending axons. They often
contain receptors for axon
guidance cues, signalling from
which reorganizes the
underlying actin cytoskeleton,
resulting in changes in the
direction of axon extension.

Intracellular

Figure 1 | Domain composition and signalling modes of Ephs and ephrins. a|Eph Tyr kinase receptors and ephrin ligands
are located at the cell membrane and are implicated in short-distance signalling between
cells. They
Nature neighbouring
Reviews | Molecular
Celldirect
Biology
cellular processes such as cell repulsion, cellcell adhesion, cell proliferation, tissue boundary formation, cell migration and
axon guidance, among others. b|Ephephrin signalling relies on specific functional domains present in Ephs and ephrins
(shown schematically on the left). On the extracellular side, Eph receptors are composed of a ligand-binding domain (LBD),
which binds to the receptor-binding domain (RBD) of ephrins, followed by a Cys-rich domain (encompassing the sushi and
epidermal growth factor (EGF)like domain) and two fibronectin (FN) domains. The intracellular side of Eph receptors is
composed of the transmembrane region (TM), the Tyr kinase domain (TK), the sterile alpha motif (SAM) and the PDZ domain.
Ephrins are divided into A and B classes: the A class is linked to the membrane via a glycosylphosphatidylinositol (GPI)
linkage, whereas the B class has a transmembrane domain and an intracellular PDZ domain. Ephephrin interaction results
in phosphorylation of Tyr residues (P) found in the juxtamembrane domain between the TK and TM domains, as well as in
the TK and SAM domains. The juxtamembrane phosphorylation is required for TK function, which is crucial for many Eph
signalling-induced biological responses. Tyr phosphorylation also occurs on intracellular domains of ephrin-B molecules
and is implicated in reverse signalling (see FIG.1c). The ribbon diagram on the right represents the crystal structure of
EphA2 complexed with ephrinA5, showing that the major site of ephrinEph interactions are contacts between the Eph
LBD (dark blue) and the ephrin RBD (light blue). c|Ephephrin signalling can occur in various modes, depending on the
direction of signal flow. Here, ephrin and Eph are depicted using the domain-colour scheme as in parts a and b. Arrow
indicates a signalling event, and an X indicates the absence of signalling. Forward signalling, defined here as ephrin:Eph,
involves signal transduction from ephrins to Ephs; reverse signalling (Eph:ephrin) involves signalling from Ephs to ephrins;
and bidirectional signalling involves the simultaneous activation of pathways downstream of ephrins and Ephs (ephrin
Eph; also used here when the signalling direction is unknown). Parallel signalling occurs when ephrins and Ephs on the same
cell signal in response to Ephs and ephrins, respectively, present on a neighbouring cell. These different signalling modes
can be elicited by either A or B class Ephs and ephrins. Anti-parallel signalling is a special case of simultaneously occurring
forward signalling, whereby ephrinEph signals are propagated in both directions. The ribbon diagram in part b is reprinted
from REF.24, Nature Publishing Group.

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Box 1 | Evolutionary perspective on ephrinEph signalling
Considering the many signalling modes of ephrinEph complexes, it would be
interesting to examine the ephrinEph system from an evolutionary perspective.
Isthere a default or evolutionarily conserved mode of ephrinEph signalling? Did
bidirectional, forward or reverse signalling arise first? In evolutionarily ancient animals,
how many Eph- and ephrin-encoding genes are there? What do their sequences tell us
about their signalling modes?
Some answers to these questions are emerging. Ephrin-B ligands probably evolved
earlier than ephrin-A ligands, as cnidarians (Hydra vulgaris) have three class B but no
class A ephrins. In this case, the expression patterns of the class B ephrins suggest roles
in tissue boundary formation163, and the signalling probably occurs largely in the
forward direction. The Drosophila melanogaster ephrin belongs to class B, although,
unusually, it contains three predicted transmembrane domains164, as opposed to the
one found in other organisms. Caenorhabditis elegans has four class A ephrins and a
single Eph (VAB1). In this species, reverse signalling is predominant, but
kinase-independent forward signalling via phosphoinositide 3-kinase or Abl kinase has
been documented33.
Altogether, studying the evolutionarily intermediate species does not provide us with
further clues about the origins of signalling modes. However, even without considering
the evolution of Eph and ephrin sequences, one might still predict the hierarchy of
separate signalling modes and obtain insights into how they might have evolved. The
forward signalling mode could be considered as the ancestral mode, allowing signalling
in one direction with the Eph-expressing cell as the receiver. Such signalling could be
easily adapted for bidirectional signalling, whereby the coexpression of ephrin and
Eph on two apposed cells would result in ephrinEph signalling in both directions,
initially in an anti-parallel orientation (FIG.1c). The next signalling mode in this
hierarchy might be parallel signalling, whereby Eph and ephrin coexpressed in the
same cell can signal in parallel, through different signalling pathways. An additional
way to increase signalling diversity could be the introduction of different intracellular
effectors or splice variants that convert a normally repulsive forward signalling mode to
an adhesive one, such as when alternative splicing of the EphA7 mRNA creates EphA7
receptors lacking the intracellular domain and promoting adhesion21.

Mechanisms of ephrinEph signalling

All Eph receptors are composed of the following
domains: an extracellular globular ligand-binding
domain; a Cys domain (comprising sushi and epidermal
growth factor (EGF)-like motifs); fibronectin domains;
a transmembrane domain; an intracellular Tyr kinase
domain adjacent to a sterile alpha motif (SAM); and a
PDZ domain (FIG.1b). Eph receptors have been classified
into EphA or EphB subfamilies3 (there are nine EphA
and five EphB rodent members in current genomic databases4) depending on whether they preferentially bind to
membrane-anchored (glycophosphatidylinositol (GPI)linked) or transmembrane ephrin ligands, referred to as
ephrin-As and ephrin-Bs, respectively. Some notable
exceptions to this are EphA4, which binds to ephrin-B
ligands3, and EphB2, which binds to ephrinA5 (REF.5).
Ephrins, by contrast, are composed of a receptor-binding
globular domain and a GPI link in the case of ephrin-A
ligands, or a transmembrane domain and PDZ domains
in the case of ephrin-B ligands. Currently, there are
five ephrin-A ligands and three ephrin-B ligands in the
rodent and human genomes4.

Sterile-alpha motif
(SAM). A protein domain with a
predicted helical structure that
can induce proteinprotein
interactions.

Signalling modes. The classic model of Eph and ephrin

function in neighbouring cells involves ephrins acting as in trans ligands of Eph receptors, resulting in
cell repulsion (ephrin:Eph, or forward signalling).
However, Eph receptors can also act as in trans ligands
for ephrins (Eph:ephrin or reverse signalling), eliciting

either cell repulsion or adhesion. In addition, both Eph

proteins and ephrins can simultaneously act as receptors and ligands, leading to bidirectional or parallel and
antiparallel signalling, depending on the distribution of
Ephs and ephrins between interacting cells, as well as
the direction of signalling in single ephrinEph pairs
(FIG.1c). Ephrins can also induce signalling cascades
independently of Eph proteins, a mode of signalling
that seems to be evolutionarily conserved6. Owing to
this complexity of signalling modes, in this Review we
use the term ephrinEph signalling when the direction
of signalling is unclear, including simultaneous reverse
and forward signalling.
Activation of signalling. A number of detailed discussions of the multitude of Eph signalling mechanisms
exist at present7,8. In brief, in most cases, to elicit robust
Eph receptor signalling, ephrins must be presented as
multimers9. This results in the formation of signalling
clusters, in which Eph receptors form arrays intercalated
with ephrins10, the size of which correlates with the
strength of the signal11 (FIG.2a), and which might partly
explain the diverse cellular responses that are elicited by
Eph activation. Some studies argue that Ephs and ephrins
can interact on the surface of the same cell (in cis), and
that this attenuates Eph signalling, possibly by inhibiting the formation of Eph clusters12,13. One of the first
forward ephrin:Eph signalling events is the activation of
the kinase activity of the Eph receptor 14, which results
in theautophosphorylation of Eph juxtamembrane
Tyr residues (FIG.2a), an event that is crucial15 for Ephdirected cellular responses. In reverse Eph:ephrin signalling, the phosphorylation of the intracellular ephrin-B
domain is also an important early event 16,17, and is
mediated by Src family kinases18.
Crucial insights into the cellular consequences of
ephrinEph signalling came from pioneering experiments in the context of cellular boundary formation. In
these experiments, the forced expression of full-length
EphBs and ephrin-B ligands, or EphBs and ephrin-B
ligands lacking the intracellular domains, resulted in
different intermixing behaviour of neighbouring cells,
reflecting the importance of Eph and ephrin phosphorylation events for signalling and the regulation
of boundary formation19,20. EphA7 receptor splicing
variants that lack a complete cytoplasmic domain and
promote adhesion also highlight the importance of Eph
receptor-mediated intracellular signalling events for
Eph-mediated repulsion: these truncated receptors presumably bind to their cognate ligand without initiating
the normal intracellular repulsive cascades21.
Mechanistic insights from structural studies. Structure
function experiments and crystal structure analyses
have provided considerable insight into Eph signalling mechanisms. The first ephrinEph structure to be
solved showed the existence of tetrameric lattices with an
EphB molecule contacting two ephrin-B molecules, and
an ephrin-B ligand contacting two EphB molecules22.
Similar conclusions were drawn from ephrin-AEphA
crystals. However, because different residues were found

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to mediate ephrin-AEphA interactions compared with
ephrin-BEphB interactions, and because ephrin-A class
interactions displayed a more open conformation than
ephrin-B class interactions, subsequent work began to
explore the structural basis for class specificity 23. Further
characterization of ephrinA1EphA2 and ephrinA5
EphA2 crystals has shown that ephrin-AEphA have
functional interactions beyond their ligand-binding
domains, which are likely to facilitate the formation
of ephrinEph multimeric clusters10. Comparing the
crystal structure ofEphA2 in the absence of ligand with
that of ephrinA5EphA2 complexes suggests a seeding
mechanism through which small ephrin-AEphA clusters can recruit unbound ephrin-A and EphA molecules
to form stable ephrin-AEphA arrays at sites of cellcell
contact 24 (FIG.2b).
Further experiments on EphA family members
revealed that EphA2 clusters are large multimeric lattices, whereas EphA4 clusters are oligomeric, implying
that even same-class Eph receptors can have distinct
ectodomain and clustering properties25. In this study,
the introduction of mutations in the domains that mediate clustering resulted in EphA2 acquiring EphA4like
clustering properties, leading to decreased cell adhesion
and an EphA4like propensity to induce cytoskeleton
collapse. Similar rules seem to apply to EphB signalling, as experiments using artificial EphB dimerizers
suggest that the EphB2mediated cytoskeleton collapse
responses are a consequence of the small-sized clusters
induced by EphB2 and are proportional to the abundance of EphB2 multimers versus dimers26. In summary,
these studies suggest that differential clustering properties encoded by subtle protein sequence differences
enhance the relatively limited diversity of Ephs and
ephrins to elicit a diverse array of cellular responses.

Rho GTPases
Molecular switch proteins that
belong to the Ras, Rho, Rab,
Arf and Ran families, which are
known for their control of actin
polymer stability and for
generally coupling cell
membrane receptor function
tocytoskeleton dynamics.

Synapse
A narrow contact point across
which a neuron sends chemical
and electrical signals to its
postsynaptic target, frequently
another neuron.

Intracellular ephrinEph signalling effectors. Signals

generated by ephrin binding to Eph receptors involve
their interaction with specific intracellular proteins,
including non-catalytic region of Tyr kinase adaptor
protein 1 (Nck1) and Nck2, phosphoinositide 3kinase
(PI3K), Src family kinases, Vav2, Vav3 and eph
exin (FIG.2c). In turn, these effectors are coupled to
RhoGTPases such as Rac1 and RhoA, which can modulate
the cytoskeleton.
For reverse signalling, Src family kinases seem to be
crucial for signalling mediated by both ephrin classes.
In addition, Ret and p75 are transmembrane effectors
of class A ephrin signalling 2729, and the Grb4Pak1
Dock180 complex specifically interacts with the carboxyl
terminus of B class ephrins30,31 (FIG.2d). The importance
of many of these signalling effectors is well documented
biochemically, and a growing number of studies are
providing supporting invivo evidence, forexample, in
the case of Src, Nck1, Nck2 and PI3K3234. Furthermore,
proteomic experiments and protein network-mapping
studies examining signalling induced in cells expressing either only ephrin-B ligands or only EphB, which
is a scenario typically observed at tissue boundaries,
have shown that different phosphorylated protein-
binding modules are activated in each of the two cell

populations. This might drive the activation of distinct

signalling cascades, leading to differential outcomes
incellsreceiving forward signals and their n
eighbouring
cells receivingreverse signals35.
Many Eph-triggered cellular responses eventually
lead to cytoskeletal rearrangements, such as the collapse
of the cytoskeleton, by controlling the balance between
small GTPase activation and inactivation. To list a
few examples, EphA4 regulates the guanosine nucleo
tide exchange factors (GEFs) ephexin 1 (REFS36,37)
andVav2 (REF.38), linking EphA4 activity to RhoA and
endocytosis, respectively. EphB also signals to RhoA,
but here ephexin 5 functions as a mediator 39. In addition, EphA4 can modulate Rac1, but this function is
mediated by the GTPase-activating protein (GAP) 2
chimerin4043. Once intracellular signalling is initiated,
the repulsive cellular responses seem to rely on the
dissociation ofephrins from Ephs through proteolytic
cleavage of ephrins44,45 and/or Eph receptors46, an event
that has been proposed to terminate ephrinEph signalling. Intriguingly, a mutation in EphA4 that prevents
it from being proteolytically cleaved does not have any
obvious effects on cellular responses activated by forward ephrin-A:EphA signalling. Notably however, this
mutation results in higher EphA4 protein levels; thus,
ephrin-As could be more efficiently bound in cis in this
scenario, thereby lowering the number of ephrin-A molecules that are free to bind EphA receptors in trans and
balancing out forward signalling 47.
The endocytosis of ephrinEph ligandreceptor
complexes is also thought to terminate ephrinEph
signalling, and, at least in some contexts, it is bidirectional: when EphB-only and ephrin-B-only expressing
cells are mixed, EphB is endocytosed by the ephrinBexpressing cell, and ephrin-B is endocytosed by the
EphB-expressing cell4850. Interestingly, endocytosis of
the ephrinEph complex included the intact proteins
together with surrounding plasma membrane, suggesting that the cells can engulf parts of the neighbouring
cells49. The direction of endocytosis largely depends
on the cell type, for example, glial cells are effective at
engulfing EphB2, but not ephrinBs from neurons51.

Neural development
Arguably, the most advances in the study of ephrinEph
signalling have been made in the context of the developing nervous system, perhaps owing to the variety
of developmental processes that can be studied in this
context and the array of molecular tools that allow the
manipulation of these processes to be interpreted precisely. Generally, in the nervous system, ephrinEph signalling contributes to events relying on short-distance
cellcell communication, such as neuronal progenitor
proliferation, the guidance of axonal growth cones
towards their synaptic targets, and synapse formation.
Neurogenesis and neuronal migration. Neurons arise
from progenitors, the multipotent and proliferative nature of which is tightly controlled to generate
the appropriate proportions of postmitotic neuronal
subtypes; these often then migrate great distances to

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Neural tube closure
An early embryonic
development event during
which the ectodermal layer
that contains the precursors of
the nervous system forms an
elongated trough, the walls of
which eventually rise up and
fuse at their dorsal edge,
forming the neural tube.

CajalRetzius cells
Cells with complex
morphologies in the most
superficial layer of the cortex.
They initially disperse from
discrete developing brain
regions in a dimension
tangential to the surface of the
cortex to produce an
even-spaced tiling. They
secrete Reelin, a large protein
that is important for the radial
migration of cortical neurons.

produce highly ordered neuronal circuits. EphrinEph

signalling has a role in all of these processes, regulating
neural tubeclosure21,52, which is one of the first neuro
developmental events, and the self-renewal, differentiation53, proliferation54, adhesion55 and apoptosis56 of
neural progenitors. The influence of ephrins on these
processes extends into the adult nervous system57, as
outlinedbelow.
Paralleling its contribution to the establishment of
tissue boundaries, ephrinEph signalling is also crucial
for the dispersion of CajalRetzius cells: mathematical
modelling, time-lapse microscopy and genetic evidence
suggest that stochastic ephrinEph repulsive contacts

Cerebral cortex
The outer layer of the brain,
which is more prominent in
higher vertebrates and is
composed of grey and
whitematter.

between these cells eventually result in their even distribution in the plane tangential to the surface of the
developing cerebral cortex58. Once this dispersion occurs,
CajalRetzius cells secrete Reelin, which promotes the
migration of differentiated neurons in the plane perpendicular to the surface of the cortex (radial migration), allowing the newly born neurons to migrate past
older ones59. This then results in the establishment of
a stereotypical inside-out neuronal arrangement, which
is characteristic of the mammalian brain. Surprisingly,
Reelin also binds ephrin-B ligands, which activates
Reelin signalling, possibly by ephrin-Bs forming a complex with Reelin receptors (FIG.2d). The significance of
b

a
Intracellular

Intracellular

Cell membrane

Ephrin

Ephrin
Extracellular
space

Eph

Eph
P

Intracellular

P
P

P
P

P
Intracellular

Intracellular
Ephrin-A

Ephrin-B3

Ephrin-B
EphA

Extracellular
space
EphA4
Intracellular

Ephrin-A,B

EphB

EphA or
EphB

P
P
P
Vav2 or 3 Ephexin 1
Endocytosis
P
Vav2 or 3
RhoA

P
Ephexin 5

Actin cytoskeleton

Actin cytoskeleton

Actin cytoskeleton

Actin cytoskeleton

Axon repulsion

Axon repulsion

Synapse formation

Axon and cell repulsion

P
Nck1 or 2
2-chimerin
Rac1

P
Src

RhoA

RhoA

d
EphB

EphB2 or EphB3

EphB3

EphA
Reelin

Ephrin-B
Src

Ephrin-B1

Ephrin-B3 ApoER2

P
PDZ

P
Pak1

Ephrin-B1,
Ephrin-B2,
Ephrin-B3
P

p75
Fyn

Ret
Ephrin-A

Grb4

Dock180

Dab1

Rac1
Actin cytoskeleton

Eector recruitment
Cellular response

Axon tract
formation

Axon pruning

Migration

Repulsion

Attraction

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Cortical interneurons
Diverse set of GABAergic
inhibitory neurons with
relatively short axons that
regulate the activity of
excitatory pyramidal cells.

Growth cone turning

A response arising from the
stabilization or collapse of the
actin cytoskeleton in growth
cones. Turning is induced by
axonal guidance cues, which
trigger attraction or repulsion
of growth cones.

this Reelinephrin interaction is supported by the fact

that mice deficient in ephrin-B ligands display defects
in the radial migration of neurons that are similar to
those observed in mice lacking Reelin. Moreover, these
defects caused by the lack of Reelin can be rescued by
overexpressing ephrin-B60. EphB can also associate with
Reelin61, indicating the existence of a complex ephrin
EphReelin interplay that seems to be important for the
proper organization of the mammalian brain61.
Radial columns are the basic organizational units
of the cerebral cortex and are established through the
radial migration of neurons originating from a common proliferative site. These columns contain neurons
that are mostly closely related by lineage, but also some
cells from neighbouring columns, owing to lateral dispersion of neurons. Ephrin-A:EphA forward signalling
and ephrinB1 reverse signalling are important for
these short-distance, lateral migratory events, but the

Figure 2 | Intracellular signalling pathways of Ephs and ephrins. a|The binding of

an ephrin monomer to an Eph receptor initiates signalling through the formation of an

ephrinEph heterotetramer, and this leads to the formation of higher-order clusters
through the recruitment of other ligand-bound Eph receptors. EphrinEph signal
strength in both forward and reverse directions, depicted by the arrows, increases with
the recruitment of additional ligand-bound receptors. Tyr phosphorylation events are
also shown. b|A higher-order cluster elucidated from the ephrinA5EphA2 complex
crystal structure. Molecules are coloured as in FIG.1b. Intracellular domains and cell
membrane links are not shown. This structure reflects the interaction of EphA and
ephrinA in trans in a situation in which these proteins might be present on apposed cells,
the cell membranes of which are represented by the horizontal lines. c|Schematic
depicting the molecular pathways employed following forward signalling that couple
Eph receptors to the actin cytoskeleton, leading to changes in the morphology of axonal
growth cones, synapses and cellcell boundaries. Initially, the intracellular domains of
Ephs are phosphorylated. This regulates the recruitment of various effector proteins,
including the non-catalytic regions of Tyr kinase adaptor protein 1 (Nck1) and Nck2, Vav2
and Vav3, Src, 2chimerin and ephexins, which interact with the carboxyl terminus of
Eph receptors. These effector proteins then influence actin cytoskeleton organization,
either more directly or by modulating the activity of the small GTPases RhoA and Rac1.
Inthe context of axon repulsion, 2chimerin inhibits Rac1 and activates RhoA, which
leads to cytoskeleton collapse. The activation of EphA receptors by ephrin-A leads to
Vav2 (or Vav3)dependent endocytosis of the ligandreceptor complex, resulting in
growth cone collapse and axon repulsion. EphA activation triggers the phosphorylation
of ephexin 1, enhancing its guanine nucleotide exchange (GEF) activity towards RhoA,
which results in cytoskeleton destabilization and axon repulsion. Ephrin-B binding to
EphB causes the phosphorylation and degradation of the GEF ephexin 5, lowering its
activation of RhoA that normally inhibits excitatory synapse formation. Src family
kinases, including Src, interact with ephrin-activated Eph receptors and lead to axon and
cell repulsion. d|The cellular response elicited by Eph:ephrin (reverse) signalling
depends on the intracellular context. Generally, ephrin-B binding of EphB receptors
leads to Src family kinase recruitment and the phosphorylation of the intracellular
domain of ephrin-B. This, in turn, can result in coupling the signalling to proteins
containing the PDZ domain, which shapes axon tract formation. Phosphorylation might
also result in the recruitment of other effectors, which then elicit cellular responses, for
example, recruitment of the Grb4Pak1Dock180 (growth factor receptor bound protein
4Ser/Thr p21activated kinase 1dedicator of cytokinesis 180) complex induces axon
pruning. The outcome of Eph:ephrin signalling can also be modified by other
components, such as Reelin. Reelin binds to its receptor (very low-density lipoprotein
receptor (VLDLR) or apolipoprotein E receptor 2 (ApoER2)) and to ephrinB1 and
ephrinB3, and following phosphorylation of the intracellular domain of the ephrin leads
to the activation of effector proteins such as disabled 1 (Dab1), which in turn affects
neuronal cell migration. By contrast, ephrin-A ligands are not phosphorylated and need
to be coupled to transmembrane molecules to elicit cellular responses. As an example,
p75 coupling leads to cell repulsion involving the Src family kinase Fyn, whereas coupling
to Ret triggers cell attraction. Part b is adapted from REF.24, Nature Publishing Group.
Parts c and d are adapted with permission from REF.165, Company of Biologists.

mechanism behind their involvement remains unclear,

as ephrin-A and ephrin-B1 loss-of-function experiments
decrease and increase lateral dispersion, respectively 62,63.
In addition to the radially migrating neurons generated
in the cortical proliferative zones, many cortical inter
neurons originate at a distance from the cerebral cortex itself and migrate there in a direction tangential to
the radial columns. This process has been found to be
mediated by simultaneous forward and reverse EphA4
and ephrin-A and ephrin-B signalling 64. Thus, these
recent experiments position ephrinEph signalling
as an important mediator of short- and long-distance
neuronal migratory events, but the mechanism of their
integration with more classical neuronal migration
signals such as Reelin remains unclear.
Axon guidance. EphrinEph signalling is particularly
well-suited for guiding axonal growth cones. In general,
long-distance growth cone guidance comprises a series
of decision-making steps involving specific growth cone
guidance cues. Some of these steps depend on ephrin
Eph signals, which drive regional differences in actin
dynamics within a growth cone, resulting in localized
growth cone turning and thereby a choice of a particular axon trajectory. Once axons reach their target, they
become topographically ordered, reflecting the relative
position of the cell bodies of neurons that give rise to
them, as demonstrated by the branching patterns of
muscle-innervating nerves and the stereotypically
localized synaptic termini of sensory system neurons
that innervate different brain structures. The nature of
the cellular responses of a growth cone may vary along
its trajectory, where, for example, intermediate guidance
decisions might be mostly repulsion-based, whereas synapse localization might depend more on adhesion. The
precision and versatility of ephrinEph signalling has a
role in all of these events, as outlined in detailbelow.
During early brain development, distant neuronal
structures become interconnected. For example, thalamic neurons form connections with cortical neurons,
and this is followed by the establishment of anti-parallel
projections from the cortex to the thalamus. In mice,
the ubiquitous replacement of the intracellular domain
of EphB1 and EphB2 by galactosidase mirrors the
phenotype of loss of function of the gene encoding
ephrinB1 in the intervening tissues such as the ventral
telencephalon65. This indicates that the establishment of
neural connections between the thalamus and the cortex
depends on forward ephrin-B:EphB signalling. Similarly,
spinal axons ascending towards the brain and the later-
developing descending corticospinal axons form two
closely apposed axonal tracks in the dorsal spinal cord,
which are important for communication between the
brain and the spinal cord. Formation of these tracks
depends on ephrinB3 at the spinal cord midline and
EphA4 expressed in ascending and descending axons66.
A canonical axon guidance event mediated by
ephrinEph signalling involves the decision of whether to
cross the midline of the nervous system. The axonsthat
do cross the midline are called contralateral axons
(orcommissural if they cross a structure called the

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Thalamus
A structure located at the base
of the brain, functioning as a
relay station for sensory
information on its way to the
cerebral cortex.Optic chiasm
A structure at the midline of
the vertebrate nervous system,
where nerves originating in
both retinas converge and
cross to innervate brain
structures (mostly) on the side
of the nervous system opposite
to the eye.

Locomotor behaviour
phenotypes
Behavioural phenotypes that
are characterized by abnormal
animal movement. Here, it
mainly refers to phenotypes
caused by defects in spinal
cord neuronal circuits
concerned with coordinated
activation of muscles involved
in locomotion.

commissure), whereas axons that remain repelled from

the midline are referred to as ipsilateral. The classic example of this is provided by the retinal ganglion axons that
innervate the visual areas of the brain. In more primitive animals, most such projections are contralateral by
default, meaning that most retinal ganglion axons cross
the midline, becoming commissural axons. When ipsilateral axons are present, their numbers correlate with
the extent of binocular vision. The proportion of ipsilateral versus contralateral trajectory selection is regulated
by Zic2, a transcription factor expressed in retinal neurons67, which promotes the expression of axonal EphB
receptors and thus the sensitivity of axons to ephrin-B
ligands at the optic chiasm midline. Consequently, more
Zic2expressing neurons result in a greater number of
axons repelled from the midline, thus enlarging the field
a

no EphB EphB

of binocular vision68,69 (FIG.3a,b). EphB1 expression is

sufficient for axon repulsion from the midline and thus
ipsilateral projection specification, whereas expression of
the highly similar EphB2 is not, and EphB1 and EphB2
domain-swapping experiments revealed that the extracellular and the juxtamembrane domains of EphB1 are
crucial for the ipsilateral trajectory of retinal axons70.
Although it is unclear whether it is direct, the activation of
EphB transcription by Zic2 in ipsilateral retinal neurons
seems to occur in other neuronal populations, including
those originating in the spinal cord and the thalamus71.
The importance of ephrinEph signalling in midline
crossing has also been confirmed by the locomotor behaviour phenotypes that are seen upon the loss of ephrinEph
signalling. For example, EphA4knockout mice display a
kangaroo-like hopping gait72, a behavioural defect caused
c

Zic2

Retina

EphA low,
Ephrin-A high
N

EphA high,
Ephrin-A low in cis
T

b
Ephrin-B
in optic
chiasm cells

Ephrin-B
Optic
chiasm cell

Repulsion
EphB

Ipsilateral
axon
EphA

Tectum

Ephrin-A

Figure 3 | Visual system development controlled by ephrinEph signalling. a|Retinal

ganglion neurons innervate
Nature Reviews | Molecular Cell Biology
avariety of brain regions involved in vision, including the tectum (or the superior colliculus in mammals). Following their
growth in the optic nerve, the axons arrive at the optic chiasm, where most cross the nervous system midline and
innervate the contralateral tectum (so called contralateral axons, shown in grey). Some axons are, however, repelled from
crossing the midline (so called ipsilateral axons, shown in blue) and these express the transcription factor Zic2, which
promotes the expression of EphB receptors6769. b|In response to ephrin-Bs expressed by the cells at the midline of the
optic chiasm, EphB-expressing axons are repelled through ephrin-B:EphB (forward) signalling and prevented from
entering the midline, thus staying on the ipsilateral side of the nervous system68,69. c|Ephrin signalling specifies the
positioning of retinal axon termini within the optic tectum. In the retinal neurons, EphA receptor expression follows a
medio-lateral gradient whereby neurons in the temporal region of the retina (T) express high levels of EphAs (red), and
neurons in the nasal region of the retina (N) express low concentrations of EphA (orange). Complementarily, ephrin-As
(orange) are present in an anterioposterior gradient in the tectum, such that anterior tectum neurons express low levels of
ephrin-A and posterior tectum neurons express high levels of ephrin-A. Because EphA receptor expression levels in retinal
neurons determine their sensitivity to ephrin-A in the tectum, temporal axons with high level of EphA (red) are prevented
from growing to the posterior of the tectum and terminate in more anterior regions, whereas retinal neurons from the
nasal regions, which contain low levels of EphA (orange) can grow and terminate in the posterior tectum. Altogether, this
results in a topographically ordered termination map77. In addition, a counter-gradient of ephrin-As exists in the retinal
neurons, such that those that express low levels of EphAs express high levels of ephrin-As. This may attenuate the
responsiveness of EphA-expressing axons by blocking the function of EphA receptors in cis and fine-tuning their
responses to ephrin-As in the tectum80. There is also a counter-gradient of EphA in the tectum, which could be involved in
repulsive EphA:ephrin-A (reverse) signalling to retinal axons, such that temporal retinal axons that express high levels of
ephrin-A are repelled down the EphA gradient in the tectum to innervate posterior targets77. Axonaxon interactions also
play a part in these processes80,84. For the sake of simplicity, we have omitted EphB gradients in the retina and ephrin-B
gradients in the tectum, which are important for guiding retinotectal axons in the dorsoventral axis of the tectum84.
Parts a and c are adapted from Garcia-Frigola,C. & Herrera,E. Zic2 regulates the expression of Sert to modulate
eye-specific refinement at the visual targets. EMBO J. (2010) 29, 31703183. Reproduced with permission from EMBO.

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Topographic maps
In reference to the distribution
of parts or features on the
surface of animals. Here, this
term refers to the orderly
distribution of nerves
originating in sensory organs,
such as the retina, where the
relative position of nerves at
the origin (retina) is maintained
at their target (for example, the
superior colliculus), thus
providing the nervous system
with an internal representation
of the external world.

Projection neurons
Neurons, the axons of which
extend a long distance within
the nervous system relative to
local interneurons that
innervate nearby targets.

Motor neurons
Neurons, the synaptic targets
of which lie outside the nervous
system, including those that
innervate muscles and ganglia
of the autonomic nervous
system.

Dendritic branching
Complex branches, similar to
those of trees, that form when
dendritic processes extend
from the neuronal cell body.

Filopodia
Thin cytoplasmic extensions
found at the forefront of
migrating cells or growth cones.

by ipsilaterally fated axons inappropriately crossing the

midline of the spinal cord. More recent work indicates
that leftright limb alternation requires that axons from
EphA4expressing excitatory spinal neurons are repelled
by ephrinB3 at the midline of the ventral spinal cord73,74.
Many axonal connections form topographic maps
where the position of projection neurons relative to one
another is maintained in the relative positions of the
innervated targets. The original experiments providing
evidence for the role of forward ephrin:Eph signalling
in the establishment of such ordered neuronal arrangements were carried out in the context of the retinotopic
maps formed by ganglion neurons innervating the
tectum or the superior colliculus, two brain regions
involved in vision. Sperrys theory that the development
of such connections is controlled by chemical labels75
was functionally proven by the demonstration that
ephrin-As and EphAs specify these connections along
the anterioposterior dimension of the tectum76. The
work on ephrinEph signalling in topographic map
formation that followed is reviewed elsewhere77, but,
briefly, key points include that gradients of Eph receptors in retinal neurons respond to graded expression of
ephrins on their targets78,79, that ephrin expression in
retinal neurons attenuates Eph receptor function in cis 80,
and that, depending on their concentration, ephrins can
promote or inhibit axonal growth81 (FIG.3c). Similarly,
ephrin-B:EphB signalling is important for the formation
of the retinotopic maps in the orthogonal dimension82,
with evidence for both forward and reverse signalling
playing an important part 83. A recent study expanded
these findings even further by showing that ephrinEphdependent topographic map formation is also driven by
ephrinA5dependent axonaxon interactions84, similar
to those controlling peripheral nerve patterning 85.
Many ephrinEph signalling ideas were elaborated
on in the context of spinal motor axon innervation of
their target muscles in the limb. Here, two major divisions of motor neurons send their axons through either of
two major limb nerves: the dorsal nerves or the ventral
nerves, depending on the expression of Eph proteins on
the motor axons. The ventral limb expresses ephrin-A
ligands, which repel EphA-expressing motor axons into
the dorsal nerve86,87, whereas the dorsal limb expresses
ephrin-Bs, which repel EphB-expressing axons into
the ventral nerve88 (FIG.4a,b). Eph expression in motor
neurons is restricted by the LIM homeodomain transcription factors Isl1 and Lhx1, and ephrin expression
in the limb is restricted by the LIM homeodomain
transcription factor Lmx1b89. In some motor axon
populations, the repulsive forward ephrin-A:EphA signalling can also be complemented by attractive reverse
EphA:ephrin-A signalling, working together 90 with the
response to GDNF (glial cell line-derived neurotrophic
factor) signalling through Ret and GFR (GDNF family
receptor)28. This suggests the presence of simultaneous
forward and reverse ephrinEph signalling in these neurons (FIGS1c,4c). This configuration is facilitated by the
coexistence of EphAs and ephrin-A ligands in separate
cell membrane domains91, and is supported by genetic
evidence92. Similarly to what has been observed in the

visual system80, ephrin and Eph coexpression in some

motor axons seems to result in ephrin ligands binding
to Eph receptors to attenuate their signalling 13 (FIG.4d).
Synaptogenesis. Once the axonal growth cone is in the
vicinity of its synaptic target, it is converted into a synapse, an often stereotypically localized structure but
one whose morphology remains paradoxically plastic.
EphrinEph signalling can specify dendritic branching
patterns and the general location of synapses in, for
example, the neuromuscular system93. Synapse formation involves highly motile postsynaptic dendritic
filopodia interacting with arriving axons in a cellular
handshake. This process depends on an EphB receptor-initiated increase in filopodial motility 94, but is independent of EphB kinase activity 95. A more recent study
suggests that EphA7 functions through the Src kinase
and mTOR (mammalian target of rapamycin) regulator
Tsc1 (tuberous sclerosis 1) to control the complexity of
dendritic shafts and spines, which serve as a precursors
to synapses. In line with this, EphA7 overexpression
results in decreased dendritic branching and, consistent
with this, its loss results in increased dendritic branching
and enhanced synaptic function96.

Development of non-neuronal structures

One of the roles of ephrinEph signalling is to prevent cell mixing to establish stable tissue boundaries.
Many embryonic boundaries are initially unstable
to allow cell movement, but proper tissue separation
requires them to become immobile. This process relies
on balanced adhesive and repulsive forces: there must be
sufficient adhesion to allow cell movement and strong
enough repulsion to prevent cells from invading the
opposing tissue. The formation of embryonic boundar
ies is required for many processes, including the separ
ation of the cardinal embryonic germ layers, division of
a large tissue into smaller segments that will eventually
acquire different identities, and shaping the morphology of branched tubular networks such as those formed
by blood and lymphatic vessels. The short-distance
attractive and repulsive signals elicited by ephrinEph
interactions are highly suitable for all instances of
boundaryformation.
Tissue separation. A classic example of ephrinEph
function in tissue separation is its contribution to hindbrain segmentation20,97. This process depends on a set of
ephrinEph pairs, most prominently EphA4 together
with ephrinB2 and ephrinB3 ligands98, transducing
anti-parallel bidirectional signals across the tissue
boundary (FIG.1c). In Xenopus laevis, early in embryonic
development, during gastrulation, mesodermal tissues
separate from dorsal ectoderm. Migration of meso
dermal cells over the ectoderm involves alternating cycles
of attachment mediated by unknown adhesion proteins
and detachment triggered by ephrin-B:EphB signalling99.
In X.laevis, ephrinEph signalling is also responsible for
generating the necessary anti-adhesive forces during
convergent extension movements, enabling the partitioning of the dorsal mesoderm into notochord and

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Lateral LMC

Medial LMC

Spinal
cord

Spinal
cord

in cis
EphA (low) + ephrin-A + EphB

EphA + ephrin-A + Ret +GFR

EphA + GDNF

Ephrin-B

Dorsal

Dorsal
Ephrin-A

Ephrin-A

Ventral
Limb

Ventral limb
Intracellular limb
mesenchyme

Dorsal limb

Ephrin-A

Ventral limb

Ephrin-A

Extracellular

EphA4

Ephrin-A

Dorsal limb

Ephrin-Bs

GDNF
GDNF
GFR

EphA
Intracellular
axon

Ventral
Limb

EphA

EphBs

Ret
+

Figure 4 | Spinal motor neuron axon guidance in the limb controlled by multiple modes of ephrinEph signalling.
Nature Reviews | Molecular Cell Biology
a,b|Schematics depicting the spinal cord, the limb divided into the dorsal and ventral halves, and spinal motor axons
originating in the lateral motor column (LMC) exiting through the spinal root and selecting one of the two limb
nerves.Motor neurons in the lateral division of the LMC (purple) express EphA and some ephrin-A on their axons and,
upon entering the limb, turn away from ephrin-A located in the ventral limb and towards EphA and GDNF (glial cell
line-derived neurotrophic factor) expressed in the dorsal limb (a).Motor neurons in the medial division of the LMC
(burgundy) express EphB, ephrin-A and some EphA on their axons and, upon entering the limb, turn away from ephrin-B
inthe dorsal limb and into the ventral limb that contains ephrin-A (b). c,d|Signalling pathways underlying spinal motor
neuron axon guidance in the limb. Modes of ephrinEph signalling between the limb and lateral LMC axons include
ephrin-A:EphA forward signalling repelling () lateral LMC axons from the ventral limb and EphA:ephrinA reverse
signalling mediating attraction (+) of these axons towards the dorsal limb. This axon trajectory choice is further enhanced
by the attraction of these axons towards GDNF, mediated through Ret and GFR (GDNF family receptor). Furthermore,
coincident activation of the reverse EphA:ephrin-A and the GDNFRetGFR signalling pathways results in synergistic
attraction of lateral LMC axons into the dorsal limb (c). Medial LMC axons are repelled from the dorsal limb through
repulsive signalling from ephrin-Bs in the dorsal limb to axonal EphBs. At the same time, ephrin-A expression in these
axons attenuates residual EphA function by cis interaction, enabling them to enter ephrin-Aexpressing ventral limb
mesenchyme (d). Diagrams in parts c and d are adapted with permission from REF.165, Company of Biologists.
Angiogenesis
A physiological process that
generates new blood vessels
from pre-existing ones.
Sprouting is a form of
angiogenesis in which the
vessel-forming cells, the
endothelial cells, proliferate
into the surrounding tissue and
form sprouts that eventually
form loops to become a new
vessel. Pruning describes the
cropping and removal of
existing vessels.

Lymphangiogenesis
A physiological process that
generates new lymphatic
vessels from pre-existing ones.

paraxial mesoderm100. In this case, ephrinEph signalling

contracts the cell cortex, thereby destabilizing adhesive
structures. Under these conditions, cadherin clustering,
which can serve as a readout for adhesion, seems to be
inhibited through an unknown mechanism, allowing the
cells to slide against eachother 101,102.
Blood and lymphatic vessel development. The development of blood vessels (angiogenesis) and of the lymphatic
vasculature (lymphangiogenesis) depends on a single
ligandreceptor pair: ephrinB2 andEphB4.
The expression of ephrinB2 in arterial territories and
of EphB4 in venous territories may help to establish borders between these two compartments, and their absence
in mice causes severe defects in blood vessel growth and

remodelling 103106. Formation of the two earliest major

vessels, the dorsal aorta and the cardinal vein, requires
ephrinB2:EphB4 repulsive signalling in endothelial cells.
As shown in zebrafish and more recently in mouse, the
dorsal aorta assembles first, containing a mixed population of precursor cells with arterial (ephrinB2+) and
venous (EphB4+) signatures. EphrinB2:EphB4 repulsive
signalling promotes vessel sprouting by repelling venous
endothelial cells away from those with an arterial fate.
EphB4+ endothelial cells then assemble the cardinal
vein107,108 (FIG.5a).
EphrinB2:EphB4 signalling also promotes sprouting
angiogenesis, which has been best studied in the postnatal mouse retina. In this system, vascular endothelial growth factors (VEGFs) activate their receptors on

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endothelial cells and promote cell sprouting, motility
and proliferation. Their activity depends on the endocytosis and turnover of VEGF receptors in sprouting cells. EphrinB2 and its intracellular interactors,
the clathrin-associated protein disabled 2 (Dab2) and

the cell polarity regulator PAR-3, promote clathrin-

dependent VEGF receptor internalization and thereby
downstream signalling, inducing sprouting 109111. This
function of ephrinB2 depends on its PDZ-binding site,
but not on its ability to induce Tyr phosphorylation, and

a
Denitive dorsal aorta
Primitive dorsal aorta

Intracellular
Ephrin-B2
Extracellular
EphB4
Intracellular
Repulsion

Primitive cardinal vein

Denitive cardinal vein

b
Ependymal cell
Neuroblast

Progenitor
cell

Proliferation and
dierentiation
Soluble
cues

Cyclin D
MAPK

NSC
GFR

GFR

NSC
Notch

Progenitor cell
Notch

Soluble
cues

Cyclin D
MAPK

Eph

Jagged

Eph

Capillary

Ephrin-B2

Capillary

Figure 5 | Blood vessel development and adult neurogenesis are regulated by ephrinEph signalling. a|Dorsal aorta
Nature Reviews | Molecular Cell Biology
and cardinal vein development. In zebrafish (19.5hours post-fertilization) and mouse
(612-somite stage), the primitive
dorsal aorta forms first and consists of arterial-fated (pink) and venous-fated (blue) endothelial cells expressing ephrinB2
and EphB4, respectively. At the border of these heterogeneous cell populations (boxed region) ephrinB2 and EphB4
interact, thereby driving repulsive behaviour in the venous-fated cells. As a result, venous-fated endothelial cells segregate
from the primitive dorsal aorta and participate in the formation of the cardinal vein (24hours post-fertilization in fish; after
the 12-somite stage in mice). This process will eventually lead to definitive dorsal aorta and cardinal vein that uniformly
display arterial and venous specification, respectively. b|Regulation of adult neurogenesis by vascular endothelial cells in
the subventricular zone of the adult brain. Quiescent neural stem cells physically contact vascular endothelial cells.
Inseton the right: at the contact site, the neural stem cells receive chronic JaggedNotch and ephrinB2:Eph (forward)
signalling, which antagonizes the signalling induced by growth and differentiation factors (soluble cues). This maintains
stemness and prevents depletion of the stem cell pool. Inset on the left: progenitor cells that arise from neural stem cells
instead lose stable vascular contact and turn off Notch and Eph signalling, allowing the cells to respond to the soluble cues
and to differentiate into neuroblasts. Multi-ciliated ependymal cells line the walls of the lateral ventricle. GFR, growth
factor receptor; NSC, neural stem cell. Part a is adapted with permission from Pitulescu,M.E. & Adams,R.H. Eph/ephrin
molecules a hub for signaling and endocytosis. Genes Dev. 24, 24802492 (2010), CSH Press.

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Frontonasal neural crest

Subpopulation of neural crest
cells that gives rise to certain
midfacial bones.

Neural crest
A multipotent migratory stem
cell population in vertebrates
that generates a diverse cell
lineage, including peripheral
neurons and glia, melanocytes,
smooth muscle, craniofacial
cartilage and bone.

Stem cell niche

A local tissue
microenvironment that
interacts with stem cells to
maintain them and to regulate
their cell fate. Stem cells divide
relatively infrequently to
generate transit-amplifying or
progenitor cells, which divide
rapidly and expand in numbers
before generating more
matureprogeny.

Subgranular zone
(SGZ). A specific region of the
hippocampus, defined as the
thin layer at the border
between the hippocampal
granule cell layer and hilus.

Hippocampus
A brain structure that is
important for the consolidation
of short-term to long-term
memory and spatial
navigation.

Subventricular zone
(SVZ). Specialized layer in the
developing or adult brain. In
the adult SVZ, astrocytes are
the stem cells which generate
transit-amplifying or progenitor
cells, which then differentiate
into neuroblasts. Multi-ciliated
ependymal cells line the walls
of the lateral ventricle.

might be mediated by Src family kinases110,111. Whether

VEGF activity is also controlled by EphB4 signalling is
currentlyunknown.
Recently, a novel pathway controlled by ephrinB2 Tyr
phosphorylation was shown to control vessel p
runing in
the eye, independently of VEGF receptor signalling. At
sites of normal vessel pruning, signals inducing ephrin-B
dephosphorylation were found to promote endothelial
cell death and vessel degeneration112.
The lymphatic vasculature is an independent vascular network that unidirectionally transports interstitial liquid and contents from the periphery into the
venous branch of the circulation. Unidirectional fluid
flow through the vessels is controlled by intraluminal
valves113. During embryonic development, lymphatic
endothelial cells differentiate from the cardinal vein
and other venous sources. EphrinB2 and EphB4 are
expressed by lymphatic vessels and are required for
lymphangiogenesis111,114. EphrinB2 reverse signalling
(presumably induced by EphB4), via the Cterminal
PDZ-binding site, but not through interactions mediated
via the phosphorylated Tyr residue, is required for lymphatic sprouting in the skin and for valve formation and
maintenance114. EphrinB2 is also required for venous
valve formation and maintenance, indicating that lymphatic and venous valves share common molecular regu
lators113,115, but the mechanism of action of ephrinB2
remains to be clarified.
Finally, ephrinB2 is also necessary for the normal
function of mural cells of the mesenchymal lineage,
such as pericytes and vascular smooth muscle cells,
which, among other functions, wrap developing blood
vessels and stabilize them. Conditional inactivation
of the mouse gene encoding ephrinB2 in mural cells
changes their morphology and behaviour, ultimately
altering vessel architecture and causing haemorrhaging 116. Mechanistically, the function of ephrinB2 in
mural cells is coupled to the membrane distribution,
endocytosis and signalling output of platelet-derived
growth factor receptor (PDGFR) 117, which suppresses the differentiation and promotes the prolifer
ation of vascular smooth muscle cells. Studies in
cultured vascular smooth muscle cells suggested that
the function of ephrinB2 is complex and is required
for the balanced activation of multiple signalling
cascades in the PDGFpathway.
Skeletal patterning. EphrinEph signalling across tissue boundaries is also essential in bone development,
and aberrant signalling has been directly linked to
craniosynostosis, a human developmental bone dis
order that involves a premature fusion of the sutures
of certain cranial bones. The gene encoding ephrinB1
is located on the Xchromosome in mice and humans,
and mouse ephrinB1 is expressed in frontonasal neural crest cells. EphrinB1null/O male and ephrinB1null/null
female mice display cleft palate, shortening of the skull
and sternal abnormalities118,119. Importantly, the expression of ephrinB1 in heterozygous females is mosaic,
owing to random Xinactivation, and occurs in the
opposite pattern to the expression of EphB receptors118:

in a population of cells, some are devoid of the ligand

and overexpress the receptors, whereas others do not
express the receptors and instead overexpress the ligand.
Consequently, it was proposed that, in heterozygous
females, new ectopic ligandreceptor interfaces can be
created that might lead to aberrant skeletal patterning.
Accordingly, females heterozygous for ephrinB1 display
additional skeletal phenotypes such as polydactyly and
craniofrontonasal syndrome, the hallmarks of which
include craniosynostosis120,121.
Craniosynostosis may result from the aberrant establishment of the neural crestmesoderm boundary at the
future cranial suture, caused by the patchy expression
and sorting of ephrinB1positive and ephrinB1-
negative cells. Such ectopic ephrinEph boundary formation is thought to affect cell proliferation through the
MAPKERK signal transduction pathway 122.

Adult physiology
The molecular-level understanding of adult physiological processes has been limited by the complexity of adult
tissues and their interactions at the organismal level, perhaps explaining why our understanding of ephrinEph
signalling in adults lags behind what we know happens
in embryos. One emerging principle is that embryonic
ephrinEph signalling cascades are often redeployed in
adults. The relatively direct relay of ephrinEph signalling to proteins controlling the cytoskeleton, and thus
cellular morphology, and its ability to affect cellcell
signalling over very short distances, makes it particularly well-suited to function in controlling the adult stem
cell niche, the stability of neuronal synapses and the balance between bone resorption and deposition, as well as
energy metabolism.
Adult stem cells and regeneration. The stem cell niche
has two apparently opposing functions: it maintains
stem cells in a quiescent and undifferentiated state and
simultaneously regulates their differentiation into proliferating progenitor cells. Several adult stem cell niches
express Ephs and ephrins, and their functions in these
compartments are beginning to be understood. The role
of ephrinEph signalling in adult neural stem cell niches
is complex, owing to the fact that many diverse cell types,
including the vasculature, interact at these sites. Here,
we highlight the general trends and discuss the most
recent work that has offered new insights into the role of
ephrinEph signalling in adult stem cells and its impact
on the regenerative capabilities of human tissues.
EphrinEph signalling in neural stem cells has
been already extensively reviewed123, so here we provide only a brief overview, focusing on adult stem cells.
Neurogenesis in the adult rodent brain is restricted to
the subgranular zone (SGZ) of the hippocampus and the
subventricular zone (SVZ) lining the lateral ventricles.
In the SGZ, EphB1 is expressed by cycling stem cells
and transient amplifying cells. EphB1 forward signalling activated by ephrinB3 in neurons of the granular layer inhibits stem cell proliferation124. Similarly,
ephrinA2:EphA7 signalling has an anti-proliferative
effect on the SVZ stem cellniche.

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a

Villus
Ephrin-B

EphB2
Ephrin-B

Crypt

Abl
Cyclin D1
Proliferation

PI3K
Migration

EphB2

b
?

Ephrin-A

Osteoclast
Osteoblast

EphA2
Dierentiation

Ephrin-A2

Ephrin-B1 or B2

EphA2

Dierentiation

EphBs

Bone

Figure 6 | Maintenance of the intestinal stem cell

nicheReviews
and bone
homeostasis
Nature
| Molecular
Cellare
Biology
regulated by ephrinEph signalling. a|Ephrin-BEphB signalling is required to maintain
the adult intestinal stem cell niche. The small intestine contains many protrusions, villi, to
increase the intestinal absorptive surface. These have crypts at their bases that contain
stem cells and Paneth cells (both in green). Stem cells and Paneth cells express EphB
receptors, and this expression is essential for their correct positioning at the base of the
crypt. As stem cells differentiate into proliferating progenitor cells (blue cells along
thecrypt), they gradually lose EphB2 expression. Consequently, a gradient of EphB2
(bluetriangle) is formed along the crypt, with the highest EphB2 expression at the base.
Bycontrast, ephrinB expression is strongest at the villuscrypt boundary, forming an
opposing gradient to the EphB2 gradient (red triangle). The inset shows ephrin-B:EphB2-
activated signalling pathways in progenitor cells as they proliferate and migrate up the
crypt axis. Loss of ephrin-B:EphB (forward) signalling disrupts cell positioning and
proliferation. b|EphrinEph signalling has opposing effects on bone homeostasis. Ephrin-B
reverse signalling reduces bone resorption by inhibiting osteoclast differentiation,
whereas EphB forward signalling in osteoblasts promotes their differentiation and,
consequently, bone formation. In contrast to the inhibitory effects of the ephrinB reverse
signalling in osteoclasts, ephrin-A:EphA signalling seems to stimulate their differentiation.
Which cells interact to elicit these responses is a currently unsolved question.

Paneth cells
One of the principal cell types
of the intestinal epithelium, an
important player in its defence
against pathogenic
microorganisms.

Hence, in contrast to the intestinal stem cell niche

(see below), most studies on adult neurogenesis point
to an anti-proliferative effect of Ephephrin signalling.
Recent work also implicates Ephs and ephrins in the differentiation of neural stem cells. EphrinA2 seems to act
cell-autonomously in progenitor cells to inhibit neurogenesis, and it is under the control of EphA7, which is
expressed by stem cells, astrocytes and ependymal cells125.
EphrinB2 presented by hippocampal astrocytes activates
EphB4 forward signalling in neural stem cells and, via

activation of catenin, promotes neuronal differentiation without affecting proliferation126. In the SVZ, quiescent stem cells contact blood vessels, and this interaction
enforces quiescence and promotes stemness. Specifically,
ephrinB2 and Jagged1, a Notch ligand, presented by vascular endothelial cells, activate their respective receptors
on stem cells, which suppress proliferation and differentiation127 (FIG.5b). When stem cells detach from the blood
vessels, Eph and Notch signalling is suspended and cells
divide and differentiate.
The stem cell compartment of the intestinal epithelium is perhaps the best-understood system with respect
to ephrinEph signalling (FIG.6a). Intestinal stem cells
reside in the base of the crypts, where they divide and
give rise to progenitor cells that continue to divide as
they migrate up the crypt axis towards the villus. Upon
leaving the crypt and approaching the villus, cells stop
dividing and differentiate. As detailed below, ephrinEph
signalling controls cell positioning along the cryptvillus
axis and the proliferation of progenitor cells. Canonical
Wnt signalling promotes mitogenesis and the transcription of EphB2 and EphB3 in intestinal stem cells through
cateninTcf (the transcriptional effector complex of the
Wnt pathway). The same pathway also negatively regulates the transcription of ephrinB1 and ephrinB2, which
are expressed by differentiated cells outside the crypt. The
opposing expression of receptors and ligands maintains
the organization of proliferating progenitor cells in the
crypt and differentiated cells in the villus128. The signalling pathways regulating proliferation are distinct from
signalling pathways involved in cell positioning: cell positioning requires PI3K but not EphB2 Tyr kinase activity,
whereas cell proliferation is mediated by EphB2 kinase
activity (an effect that is not a secondary consequence of
cell positioning 129) by Abl kinase to increase cyclin D1
levels33 (FIG.6a).
Pathways regulating adult stem cells are important in
defining the regenerative capacities of tissues. Atissue
displaying highly limited capabilities to regenerate is the
adult mammalian central nervous system (CNS). Here,
regeneration after the injury is prevented by cell-intrinsic
suppressors of growth signalling, andbycell-extrinsic
mechanisms such as an inhibitory growth environment
consisting of glial- and myelin-derived proteins. Work
over the past decade has shown that ephrin:Eph signalling contributes to the unfavourable environment that
limits anatomical regeneration and functional recovery
after CNS injury. EphrinA3 and ephrinB3 are myelin-
associated proteins that activate EphA4 signalling in
injured axons to contribute to their limited regener
ative growth130134. This potent axon repulsion activity of
ephrinEph signalling is emerging as a major obstacle
to nerve regeneration following injury. Interestingly,
although myelin-producing oligodendrocytes of the
CNS seem to inhibit the growth of injured axons, myelin-
producing glial cells of the peripheral nervous system,
namely Schwann cells, support axonal growth after injury.
Here, ephrin-B:EphB signalling promotes rather than
inhibits regeneration. Schwann cells that migrate into
the nerve wound express EphBs (mostly EphB2), which
interact with ephrin-Bexpressing fibroblasts, activating a

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signalling cascade in the Schwann cell that leads to accumulation of the transcription factor Sox2 in the nucleus.
Sox2mediated transcription, by an unknown mechanism, causes the recruitment of Ncadherin to Schwann
cell contacts and the formation of Schwann cell cords,
which serve as a substrate to s upport the regrowth of
sensory axons135.
Synaptic plasticity. One of the foundations of nervous
system function is electrical impulse transmission from
one neuron to another via synapses, the strength of which
is modulated by experience-dependent plasticity. The
control of calcium dynamics by glutamate neurotransmitter receptors is an essential part of this process and is
modulated by ephrinB2:EphB signalling 136. Additional
observations in hippocampal slices led to the idea that
EphB2 acts postsynaptically to promote different forms
of synaptic plasticity 137139. Despite a significant body of
work, the exact mode of ephrin-B:EphB signalling in synaptic plasticity remains unclear, in part because ephrin-B
ligands may function either pre- or postsynaptically at
different synapses140.
EphrinEph signalling also modulates the morphology of the dendritic synapse, which is a crucial determinant of the strength of synaptic transmission. The use
of knockout mutant mice has highlighted a functionally redundant requirement of three EphB receptors in
endritic spine morphogenesis141. During the formation
d
of dendritic spines, EphB signalling modulates the activity of small GTPases, which in turn regulate the actin
cytoskeleton140. However, there is also evidence for a role
for ephrin-B reverse signalling in dendritic spine formation142, so a unifying model is still missing. More recently
ephrinA3 present on hippocampal synapse-associated
glia was shown to signal to EphA4expressing neurons,
leading to dendritic spine retraction, thereby effectively
controlling dendritic spine morphology and consequently the strength of synaptic transmission143. This idea
has been extended by more precise genetic manipulations
and a link to neurotransmitter transporter levels144,145.
According to these studies, astrocytes receive a signal
provided by EphA4 localized to neuronal dendrites via
ephrinA3, which then controls the abundance of astrocytic glutamate transporters and regulates glutamate
concentrations near synapses and synaptic plasticity.

Dendritic spines
Dendrites are structures
originating at the neuronal
soma, which in some neurons
can be highly complex, similar
to a tree branch network.
Dendrites receive impulses
from other neurons through
synapses, which are mainly
located on spine-like
protrusions of dendrites.

Bone homeostasis. EphrinEph signalling controls bone

homeostasis in adult mice, a process that requires a fine
balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts (FIG.6b). Initially
it was shown that ephrinB2 reverse signalling (possibly
initiated by EphB4) inhibits osteoclast differentiation;
however, conditional deletion of ephrinB2 resulted
in more efficient osteoclast formation invitro, but not
invivo 146. Subsequent work found that ephrinB1 is
actually the predominant player in this system. Targeted
deletion of the gene encoding ephrinB1 in osteoclast
precursors results in reduced bone volume caused by an
increase in osteoclast numbers and bone resorption147.
Because ephrinB1 has low affinity for EphB4, other EphB
receptors, such as EphB2 or EphB3, might interact with

ephrinB1 to regulate bone homeostasis146. More recently,

ephrin-AEphA signalling was shown to enhance osteoclastogenesis and to suppress osteoblastogenesis in ways
that raised the possibility that osteoclasts and osteoblasts
can be regulated separately by ephrinEph but also may
signal to each other via ephrinEph interactions148,149.
Insulin secretion. A relatively newly identified function
for ephrinEph signalling is the mediation of cellcell
communication between pancreatic islet -cells and
the regulation of insulin secretion. Ephrin-A:EphA forward signalling between -cells inhibits insulin secretion, whereas reverse signalling stimulates it. Thus,
bidirectional ephrin-AEphA signalling between -cells
seems to control a fundamental process underlying
energy metabolism. This process can be further linked to
the effect of ephrinEph signalling on the activation of a
small GTPase protein, Rac1: EphA:ephrin-A reverse signalling enhances Rac1 activity, whereas ephrinA:EphA
forward signalling reduces Rac1 activity, and these
changes in turn regulate the events leading to insulin
secretory granule fusion with the cell membrane. This
suggests that ephrinEph signalling regulation could
be potentially exploited to find new treatments for
type2diabetes150.

EphrinEph signalling in disease

First identified in the erythropoietin-producing
hepatoma cell line, the role of ephrinEph signals in
cancer and other human diseases has been substantiated
by a robust line of research over the past 10years.
In addition to angiogenesis, ephrinEph signalling
has a role in the control of tissue architecture and cell
motility, as described above, which both are involved in
cancer development151. For example, expression of wildtype EphB2 blocks the uncontrolled proliferation of cells
carrying human EPHB2 mutations that are associated
with prostate cancer 152, indicating that EphB2 signalling
suppresses tumour growth. EphB signalling has a similar
role in intestinal adenoma (a type of colorectal cancer
caused by constitutive activation of the Wnt pathway),
in which it restricts cell migration and inhibits invasive
growth. In these cancers, high EphB expression promotes
the growth of epithelial evaginations, but these are prevented from spreading by ephrin-B-expressing cells
surrounding the adenoma153. When tumours progress
to carcinomas, EphB expression is downregulated and
ligand-imposed repulsion is lost, allowing tumour cells to
invade the surrounding territory 154. EphB2 kinase activity is dispensable for the specific localization of tumour
cells within intestinal crypts or villi, which on the basis
of pharmacological evidence seems to be mediated
through PI3K. However, EphB2 Tyr kinase activity is
important to promote Abl- and cyclin D1driven cell
proliferation33, indicating that Eph receptors display both
anti- and pro-oncogenic properties. This has been further substantiated by studies on EphA2 signalling, which
has been shown to restrict the migration of glioma and
prostate cancer cells when activated by ephrinA1, but to
enhance cell motility in the absence of its ligand by acting
as a phosphorylation substrate of the Akt kinase155.

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Tip cell
A specialized cell found at the
leading edge of growing blood
vessels, often displaying
filopodia.

The effects of ephrinEph signalling on the motility of

metastatic cells can simultaneously depend on multiple
Eph receptors and the expression of the various ephrin
ligands on neighbouring cells, including other cancer
cells156, raising interesting parallels with the ephrin
Eph-mediated random dispersion of cortical neurons58. Some progress into how EphA signalling might
modulate cancer cell invasiveness came from apposing
EphA2expressing cells with membrane-bound ephrin-A
ligands that were restricted from moving in the plane of
the cell membrane. It has been suggested that EphA activation is regulated by the ephrin-AEphA cluster size,
which is supported by the observation that the invasive potential of cells in this setup correlates with the
extent of receptor mobility within the cell membrane157.
Furthermore, EphA7 is a tumour suppressor for follicular lymphomas, and its extracellular domain displays
anti-tumour effects against human lymphomas invivo,
possibly through the attenuation of EphA2 signalling 158.
Finally, Ephephrin signalling is important in pathological angiogenesis and thus crucial for tumour growth.
One important piece of evidence for this points to the
PDZ domain of ephrinB2 as required for VEGFR2 (vascular endothelial growth factor receptor 2) localisation
and signalling in tip cell filopodia110.
In the context of neurological disorders, mutations in
the human EPHA4 gene correlate with increased survival
of patients with amyotrophic lateral sclerosis (ALS) and
in ALS mouse models159. Moreover, the hippocampal
synaptic dysfunction observed in Alzheimer disease
mouse models is attenuated by small molecule blockers
of EphA4 function160, possibly reflecting EphA4 function
in dendritic spine morphology 143. Because the increased
EphB2 function in the memory centres of Alzheimer
disease animal models also has beneficial effects on
cognitive tasks161, the imbalance of ephrinEph signalling in synaptic plasticity and spine morphology might
be an important molecular defect in Alzheimer disease.
Finally, common variants of the human EPHA2 gene have
been linked to cortical cataracts, which are essentially an
age-related increased opacity of the crystalline lens of the
eye162, but the mechanism of this remains elusive.
These results collectively implicate ephrinEph signalling as an important target in therapies against c ancer
and neurological disorders in humans. The efforts
to develop therapeutics against Ephs and ephrins are
reviewedelsewhere7.

Open questions and future perspectives

It is now clear that Ephs and ephrins can elicit very different physiological responses, even in the same cell
Hirai,H., Maru,Y., Hagiwara,K., Nishida,J. & Takaku,F.
A novel putative tyrosine kinase receptor encoded by
the eph gene. Science 238, 17171720 (1987).
2. Barquilla,A. & Pasquale,E.B. Eph receptors and
ephrins: therapeutic opportunities. Annu. Rev.
Pharmacol. Toxicol. 55, 465487 (2015).
3. Gale,N.W. etal. Eph receptors and ligands comprise
two major specificity subclasses and are reciprocally
compartmentalized during embryogenesis. Neuron 17,
919 (1996).
4. Cunningham,F. etal. Ensembl 2015. Nucleic Acids
Res. 43, D662D669 (2015).
1.

type. In addition, there are multiple Ephs and ephrins

within the same class, with similar binding affinities,
but their interactions may lead to largely different cellular outcomes. The prototypical observation in this
case is the disparate cellular response to EphA2- versus
EphA4mediated activation by ephrinA5, leading to
the promotion of cell adhesion or cell collapse, respectively 25. An important question now is how these different functions are mechanistically achieved. Clearly,
as suggested by EphA2 and EphA4, this is partly owing
to the different three-dimensional structures formed by
ephrinA5EphA2 and ephrinA5EphA4. How this
translates into the recruitment of different intracellular
effectors might be elucidated by proteomic analyses of
intracellular signalling pathways, and such analyses have
already been initiated in the context of cellcell inter
actions elicited by ephrin-BEphB signalling. Moreover,
the molecular mechanisms that allow the Eph-expressing
and ephrin-expressing cells to respond differently to
ephrinEph signalling activated upon cellcell contact
are unknown, although some clues are emerging with
extensive proteomics analysis. For example, it seems that
SH2- and phosphorylated Tyr-binding proteins are used
more extensively to signal in EphB-expressing cells than
in ephrin-Bexpressing cells35.
Currently, it is also not known how ephrinEph
signalling is linked to the cytoskeleton and gene transcription. Clearly, in some instances, ephrinEph can
influence cell proliferation and cell identity, but the signalling pathways mediating these events remain unclear.
Proteomic and signalomic analyses are likely to provide
important data in this direction.
Yet another research avenue for the future will be
to investigate the intersection of ephrinEph signalling
with other signalling pathways, as normal cell function
is clearly the consequence of the integrative function of
multiple signalling pathways. Some insights have been
gathered in that area, by showing that reverse ephrinEph
signalling intersects with VEGFR and p75 or Ret pathways28,111. Many other such intersections are likely to exist
and await exploration. For instance, there is now evidence
of synergistic integration between ephrin-B and netrin
signals in spinal motor neuron growth cones168.
Finally, the latest discoveries on the role of ephrin
Eph signals in diseases are exciting but currently lack
detailed insights. Therefore, further research focused
on gathering mechanistic understanding of the involvement of ephrin signalling in pathological conditions
is needed. This will open up the possibility of introducing novel therapies for cancer, spinal cord injury
andneurodegeneration.

5. Himanen,J.P. etal. Repelling class discrimination:

ephrinA5 binds to and activates EphB2 receptor
signaling. Nat. Neurosci. 7, 501509 (2004).
6. Chin-Sang,I.D. etal. The divergent C.elegans ephrin
EFN4 functions in embryonic morphogenesis in a
pathway independent of the VAB1 Eph receptor.
Development 129, 54995510 (2002).
7. Boyd,A.W., Bartlett,P.F. & Lackmann,M.
Therapeutic targeting of EPH receptors and their
ligands. Nat. Rev. Drug Discov. 13, 3962 (2014).
An interesting review, along with reference2,
froma therapeutics development perspective.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

Kullander,K. & Klein,R. Mechanisms and functions of

Eph and ephrin signalling. Nat. Rev. Mol. Cell Biol. 3,
475486 (2002).
9. Davis,S. etal. Ligands for EPH-related receptor
tyrosine kinases that require membrane attachment
or clustering for activity. Science 266, 816819
(1994).
10. Himanen,J.P. etal. Architecture of Eph receptor
clusters. Proc. Natl Acad. Sci. USA 107,
1086010865 (2010).
11. Egea,J. etal. Regulation of EphA4 kinase activity is
required for a subset of axon guidance decisions
8.

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suggesting a key role for receptor clustering in Eph
function. Neuron 47, 515528 (2005).
12. Carvalho,R.F. etal. Silencing of EphA3 through a cis
interaction with ephrinA5. Nat. Neurosci. 9, 322330
(2006).
13. Kao,T.J. & Kania,A. Ephrin-mediated cis-attenuation
of Eph receptor signaling is essential for spinal motor
axon guidance. Neuron 71, 7691 (2011).
14. Kullander,K. etal. Kinase-dependent and kinaseindependent functions of EphA4 receptors in major
axon tract formation invivo. Neuron 29, 7384
(2001).
15. Zisch,A.H., Kalo,M.S., Chong,L.D. & Pasquale,E.B.
Complex formation between EphB2 andSrc requires
phosphorylation of tyrosine 611 in the EphB2
juxtamembrane region. Oncogene 16, 26572670
(1998).
16. Holland,S.J. etal. Bidirectional signalling through
theEPH-family receptor Nuk and its transmembrane
ligands. Nature 383, 722725 (1996).
17. Brckner,K., Pasquale,E.B. & Klein,R. Tyrosine
phosphorylation of transmembrane ligands for Eph
receptors. Science 275, 16401643 (1997).
18. Palmer,A. etal. EphrinB phosphorylation and reverse
signaling: regulation bySrc kinases and PTPBL
phosphatase. Mol. Cell 9, 725737 (2002).
19. Mellitzer,G., Xu,Q. & Wilkinson,D.G. Eph receptors
and ephrins restrict cell intermingling and
communication. Nature 400, 7781 (1999).
20. Xu,Q., Mellitzer,G., Robinson,V. & Wilkinson,D.G.
Invivo cell sorting in complementary segmental
domains mediated by Eph receptors and ephrins.
Nature 399, 267271 (1999).
21. Holmberg,J., Clarke,D.L. & Frisn,J. Regulation of
repulsion versus adhesion by different splice forms
ofan Eph receptor. Nature 408, 203206 (2000).
22. Himanen,J.P. etal. Crystal structure of an Eph
receptorephrin complex. Nature 414, 933938
(2001).
23. Himanen,J.P. etal. Ligand recognition by Aclass Eph
receptors: crystal structures of the EphA2 ligandbinding domain and the EphA2/ephrinA1 complex.
EMBO Rep. 10, 722728 (2009).
24. Seiradake,E., Harlos,K., Sutton,G., Aricescu,A.R.
&Jones,E.Y. An extracellular steric seeding
mechanism for Ephephrin signaling platform
assembly. Nat. Struct. Mol. Biol. 17, 398402 (2010).
25. Seiradake,E. etal. Structurally encoded intraclass
differences in EphA clusters drive distinct cell
responses. Nat. Struct. Mol. Biol. 20, 958964
(2013).
Important insights into differential intra-class
signalling properties of Eph receptors.
26. Schaupp,A. etal. The composition of EphB2 clusters
determines the strength in the cellular repulsion
response. J.Cell Biol. 204, 409422 (2014).
A functional link between Eph signalling cluster size
and the quality of cellular responses.
27. Marler,K.J. etal. A TrkB/EphrinA interaction controls
retinal axon branching and synaptogenesis.
J.Neurosci. 28, 1270012712 (2008).
28. Bonanomi,D. etal. Ret is a multifunctional coreceptor
that integrates diffusible- and contact-axon guidance
signals. Cell 148, 568582 (2012).
Synergistic interaction between Ephephrin and
GDFNRetGFR signalling.
29. Lim,Y.S. etal. p75NTR mediates ephrinA reverse
signaling required for axon repulsion and mapping.
Neuron 59, 746758 (2008).
30. Cowan,C.A. & Henkemeyer,M. The SH2/SH3 adaptor
Grb4 transduces Bephrin reverse signals. Nature
413, 174179 (2001).
31. Xu,N.J. & Henkemeyer,M. EphrinB3 reverse
signaling through Grb4 and cytoskeletal regulators
mediates axon pruning. Nat. Neurosci. 12, 268276
(2009).
A genetic analysis of ephrinB3 point mutants that
revealed new reverse signalling effectors.
32. Fawcett,J.P. etal. Nck adaptor proteins control
theorganization of neuronal circuits important
forwalking. Proc. Natl Acad. Sci. USA 104,
2097320978 (2007).
33. Genander,M. etal. Dissociation of EphB2 signaling
pathways mediating progenitor cell proliferation and
tumor suppression. Cell 139, 679692 (2009).
Insight into the divergence of mitogenic and
cytoskeletal signals evoked by EphB activation.
34. Kao,T.J., Palmesino,E. & Kania,A. SRC family
kinases are required for limb trajectory selection by
spinal motor axons. J.Neurosci. 29, 56905700
(2009).

35. Jrgensen,C. etal. Cell-specific information

processing in segregating populations of Eph receptor
ephrin-expressing cells. Science 326, 15021509
(2009).
Modelling of protein network signalling reveals
unexpected differences in Ephephrin and ephrin
Eph signalling when Eph-expressing cells contact
ephrin-expressing cells.
36. Sahin,M. etal. Eph-dependent tyrosine
phosphorylation of ephexin1 modulates growth cone
collapse. Neuron 46, 191204 (2005).
37. Shamah,S.M. etal. EphA receptors regulate growth
cone dynamics through the novel guanine nucleotide
exchange factor ephexin. Cell 105, 233244 (2001).
38. Cowan,C.W. etal. Vav family GEFs link activated Ephs
to endocytosis and axon guidance. Neuron 46,
205217 (2005).
39. Margolis,S.S. etal. EphB-mediated degradation of
the RhoA GEF Ephexin5 relieves a developmental
brake on excitatory synapse formation. Cell 143,
442455 (2010).
Degradation of ephexin5 promotes
EphB-dependent excitatory synapse development
and is mediated by Ube3A, a ubiquitin ligase linked
to human cognitive disorders.
40. Beg,A.A., Sommer,J.E. & Martin,J. H.
&Scheiffele,P. 2chimaerin is an essential EphA4
effector in the assembly of neuronal locomotor
circuits. Neuron 55, 768778 (2007).
41. Iwasato,T. etal. Rac-GAP -chimerin regulates motorcircuit formation as a key mediator of EphrinB3/
EphA4 forward signaling. Cell 130, 742753 (2007).
42. Wegmeyer,H. etal. EphA4dependent axon guidance
is mediated by the RacGAP 2chimaerin. Neuron 55,
756767 (2007).
43. Kao,T.J., Nicholl,G.C., Johansen,J.A., Kania,A.
&Beg,A. A. 2chimaerin is required for Eph
receptor-class-specific spinal motor axon guidance
andcoordinate activation of antagonistic muscles.
J.Neurosci. 35, 23442357 (2015).
Functional analysis of developmental motor axon
guidance errors caused by 2chimerin mutation.
44. Hattori,M., Osterfield,M. & Flanagan,J.G.
Regulated cleavage of a contact-mediated axon
repellent. Science 289, 13601365 (2000).
45. Janes,P.W. etal. Adam meets Eph: an ADAM substrate
recognition module acts as a molecular switch for
ephrin cleavage in trans. Cell 123, 291304 (2005).
46. Lin,K.T., Sloniowski,S., Ethell,D.W. & Ethell,I.M.
EphrinB2induced cleavage of EphB2 receptor is
mediated by matrix metalloproteinases to trigger cell
repulsion. J.Biol. Chem. 283, 2896928979 (2008).
47. Gatto,G., Morales,D., Kania,A. & Klein,R. EphA4
receptor shedding regulates spinal motor axon
guidance. Curr. Biol. 24, 23552365 (2014).
A mutation that abolishes EphA4 cleavage causes
axon errors in unexpected ways.
48. Mann,F., Miranda,E., Weinl,C., Harmer,E. &
Holt,C.E. Btype Eph receptors and ephrins induce
growth cone collapse through distinct intracellular
pathways. J.Neurobiol. 57, 323336 (2003).
49. Marston,D.J., Dickinson,S. & Nobes,C.D. Racdependent trans-endocytosis of ephrinBs regulates
Ephephrin contact repulsion. Nat. Cell Biol. 5,
879888 (2003).
Localized phagocytosis of an ephrin-Bexpressing
cell by an EphB-expressing cell.
50. Zimmer,M., Palmer,A., Khler,J. & Klein,R.
EphBephrinB bidirectional endocytosis terminates
adhesion allowing contact mediated repulsion.
Nat.Cell Biol. 5, 869878 (2003).
Evidence that ephrin-BEphB signalling complex
endocytosis is bidirectional and required for
efficient repulsion.
51. Lauterbach,J. & Klein,R. Release of full-length EphB2
receptors from hippocampal neurons to cocultured
glial cells. J.Neurosci. 26, 1157511581 (2006).
52. Ji,Y.J. etal. EphrinB2 affects apical constriction
inXenopus embryos and is regulated by ADAM10
andflotillin1. Nat. Commun. 5, 3516 (2014).
53. Qiu,R. etal. Regulation of neural progenitor cell state
by ephrinB. J.Cell Biol. 181, 973983 (2008).
54. North,H.A. etal. Promotion of proliferation in the
developing cerebral cortex by EphA4 forward
signaling. Development 136, 24672476 (2009).
55. Arvanitis,D.N. etal. Ephrin B1 maintains apical
adhesion of neural progenitors. Development 140,
20822092 (2013).
56. Depaepe,V. etal. Ephrin signalling controls brain size
by regulating apoptosis of neural progenitors. Nature
435, 12441250 (2005).

254 | APRIL 2016 | VOLUME 17

57. Jiao,J.W., Feldheim,D.A. & Chen,D.F. Ephrins as

negative regulators of adult neurogenesis in diverse
regions of the central nervous system. Proc. Natl
Acad. Sci. USA 105, 87788783 (2008).
58. Villar-Cervio,V. etal. Contact repulsion controls the
dispersion and final distribution of CajalRetzius cells.
Neuron 77, 457471 (2013).
Dispersion of migrating neurons depends on
ephrinEph signalling.
59. Rakic,P. Specification of cerebral cortical areas.
Science 241, 170176 (1988).
60. Sentrk,A., Pfennig,S., Weiss,A., Burk,K. &
AckerPalmer,A. Ephrin Bs are essential components
of the Reelin pathway to regulate neuronal migration.
Nature 472, 356360 (2011).
61. Bouch,E. etal. Reelin induces EphB activation. Cell
Res. 23, 473490 (2013).
62. Dimidschstein,J. etal. EphrinB1 controls the
columnar distribution of cortical pyramidal neurons
byrestricting their tangential migration. Neuron 79,
11231135 (2013).
63. Torii,M., Hashimoto-Torii,K., Levitt,P. & Rakic,P.
Integration of neuronal clones in the radial cortical
columns by EphA and ephrinA signalling. Nature 461,
524528 (2009).
64. Steinecke,A., Gampe,C., Zimmer,G., Rudolph,J.
&Bolz,J. EphA/ephrin A reverse signaling promotes
the migration of cortical interneurons from the medial
ganglionic eminence. Development 141, 460471
(2014).
65. Robichaux,M.A. etal. EphB receptor forward
signaling regulates area-specific reciprocal thalamic
and cortical axon pathfinding. Proc. Natl Acad.
Sci.USA 111, 21882193 (2014).
66. Paixo,S. etal. EphrinB3/EphA4mediated guidance
of ascending and descending spinal tracts. Neuron 80,
14071420 (2013).
Ephrin-B:EphA4 forward signalling is required for
two major axonal tracts linking the spinal cord and
the brain.
67. Herrera,E. etal. Zic2 patterns binocular vision by
specifying the uncrossed retinal projection. Cell 114,
545557 (2003).
68. Garcia-Frigola,C., Carreres,M.I., Vegar,C., Mason,C.
& Herrera,E. Zic2 promotes axonal divergence at the
optic chiasm midline by EphB1dependent and
-independent mechanisms. Development 135,
18331841 (2008).
69. Williams,S.E. etal. EphrinB2 and EphB1 mediate
retinal axon divergence at the optic chiasm. Neuron
39, 919935 (2003).
70. Petros,T.J., Shrestha,B.R. & Mason,C. Specificity
and sufficiency of EphB1 in driving the ipsilateral
retinal projection. J.Neurosci. 29, 34633474
(2009).
71. Escalante,A., Murillo,B., Morenilla-Palao,C., Klar,A.
& Herrera,E. Zic2dependent axon midline avoidance
controls the formation of major ipsilateral tracts in the
CNS. Neuron 80, 13921406 (2013).
72. Dottori,M. etal. EphA4 (Sek1) receptor tyrosine
kinase is required for the development of the
corticospinal tract. Proc. Natl Acad. Sci. USA 95,
1324813253 (1998).
73. Kullander,K. etal. Role of EphA4 and EphrinB3 in
local neuronal circuits that control walking. Science
299, 18891892 (2003).
74. Borgius,L. etal. Spinal glutamatergic neurons defined
by EphA4 signaling are essential components of
normal locomotor circuits. J.Neurosci. 34,
38413853 (2014).
75. Sperry,R.W. Chemoaffinity in the orderly growth of
nerve fiber patterns and connections. Proc. Natl Acad.
Sci. USA 50, 703710 (1963).
76. Drescher,U. etal. Invitro guidance of retinal ganglion
cell axons by RAGS, a 25 kDa tectal protein related to
ligands for Eph receptor tyrosine kinases. Cell 82,
359370 (1995).
77. Triplett,J.W. Molecular guidance of retinotopic map
development in the midbrain. Curr. Opin. Neurobiol.
24, 712 (2014).
78. McLaughlin,T., Lim,Y.S., Santiago,A. & OLeary,D.D.
Multiple EphB receptors mediate dorsal-ventral
retinotopic mapping via similar bifunctional responses
to ephrinB1. Mol. Cell. Neurosci. 63, 2430 (2014).
79. Frisen,J. etal. EphrinA5 (AL1/RAGS) is essential for
proper retinal axon guidance and topographic
mapping in the mammalian visual system. Neuron 20,
235243 (1998).
80. Hornberger,M.R. etal. Modulation of EphA receptor
function by coexpressed ephrinA ligands on retinal
ganglion cell axons. Neuron 22, 731742 (1999).

www.nature.com/nrm
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
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.
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e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
6
1
0
2

REVIEWS
81. Hansen,M.J., Dallal,G.E. & Flanagan,J.G. Retinal
axon response to ephrinAs shows a graded,
concentration-dependent transition from growth
promotion to inhibition. Neuron 42, 717730
(2004).
82. Hindges,R., McLaughlin,T., Genoud,N.,
Henkemeyer,M. & OLeary,D. EphB forward signaling
controls directional branch extension and arborization
required for dorsal-ventral retinotopic mapping.
Neuron 35, 475487 (2002).
83. Thakar,S., Chenaux,G. & Henkemeyer,M. Critical
roles for EphB and ephrinB bidirectional signalling in
retinocollicular mapping. Nat. Commun. 2, 431
(2011).
84. Suetterlin,P. & Drescher,U. Target-independent
EphrinA/EphA-mediated axonaxon repulsion as a
novel element in retinocollicular mapping. Neuron 84,
740752 (2014).
Ephrin-Adriven axonaxon interactions pattern
visual system topographic maps.
85. Wang,L., Klein,R., Zheng,B. & Marquardt,T.
Anatomical coupling of sensory and motor nerve
trajectory via axon tracking. Neuron 71, 263277
(2011).
Coupling of sensory and motor nerves via
ephrinEph signalling.
86. Eberhart,J., Swartz,M.E., Koblar,S.A.,
Pasquale,E.B. & Krull,C.E. EphA4 constitutes a
population-specific guidance cue for motor neurons.
Dev. Biol. 247, 89101 (2002).
87. Helmbacher,F., Schneider-Maunoury,S., Topilko,P.,
Tiret,L. & Charnay,P. Targeting of the EphA4 tyrosine
kinase receptor affects dorsal/ventral pathfinding of
limb motor axons. Development 127, 33133324
(2000).
88. Luria,V., Krawchuk,D., Jessell,T.M., Laufer,E.
&Kania,A. Specification of motor axon trajectory by
ephrinB:EphB signaling: symmetrical control of
axonal patterning in the developing limb. Neuron 60,
10391053 (2008).
Ephrin-B:EphB forward signalling guides motor
axons in the limb.
89. Kania,A. & Jessell,T.M. Topographic motor
projections in the limb imposed by LIM homeodomain
protein regulation of ephrinA:EphA interactions.
Neuron 38, 581596 (2003).
90. Kramer,E.R. etal. Cooperation between GDNF/Ret
and ephrinA/EphA4 signals for motor-axon pathway
selection in the limb. Neuron 50, 3547 (2006).
91. Marquardt,T. etal. Coexpressed EphA receptors and
EphrinA ligands mediate opposing actions on growth
cone navigation from distinct membrane domains. Cell
121, 127139 (2005).
92. Dudanova,I. etal. Genetic evidence for a contribution
of EphA:EphrinA reverse signaling to motor axon
guidance. J.Neurosci. 32, 52095215 (2012).
93. Feng,G. etal. Roles for ephrins in positionally
selective synaptogenesis between motor neurons
andmuscle fibers. Neuron 25, 295306 (2000).
94. Kayser,M.S., Nolt,M.J. & Dalva,M.B. EphB
receptors couple dendritic filopodia motility to
synapse formation. Neuron 59, 5669 (2008).
Dendrite motility requires ephrinEph signalling,
linking it to synapse formation.
95. Soskis,M.J. etal. A chemical genetic approach
reveals distinct EphB signaling mechanisms during
brain development. Nat. Neurosci. 15, 16451654
(2012).
96. Clifford,M.A. etal. EphA7 signaling guides cortical
dendritic development and spine maturation.
Proc.Natl Acad. Sci. USA 111, 49944999 (2014).
97. Cooke,J.E., Kemp,H.A. & Moens,C.B. EphA4 is
required for cell adhesion and rhombomere-boundary
formation in the zebrafish. Curr. Biol. 15, 536542
(2005).
98. Cayuso,J., Xu,Q. & Wilkinson,D.G. Mechanisms
ofboundary formation by Eph receptor and ephrin
signaling. Dev. Biol. 401, 122131 (2015).
99. Rohani,N., Parmeggiani,A., Winklbauer,R.
&Fagotto,F. Variable combinations of specific ephrin
ligand/eph receptor pairs control embryonic tissue
separation. PLoS Biol. 12, e1001955 (2014).
100. Wallingford,J.B., Fraser,S.E. & Harland,R.M.
Convergent extension: the molecular control of
polarized cell movement during embryonic
development. Dev. Cell 2, 695706 (2002).
101. Fagotto,F., Rohani,N., Touret,A.S. & Li,R.
Amolecular base for cell sorting at embryonic
boundaries: contact inhibition of cadherin adhesion
byephrin/ Eph-dependent contractility. Dev. Cell 27,
7287 (2013).

102. Fagotto,F. Regulation of cell adhesion and cell sorting

at embryonic boundaries. Curr. Top. Dev. Biol. 112,
1964 (2015).
103. Adams,R.H. etal. Roles of ephrinB ligands and EphB
receptors in cardiovascular development: demarcation
of arterial/venous domains, vascular morphogenesis,
and sprouting angiogenesis. Genes Dev. 13, 295306
(1999).
104. Wang,H.U., Chen,Z.F. & Anderson,D.J. Molecular
distinction and angiogenic interaction between
embryonic arteries and veins revealed by ephrinB2
and its receptor EphB4. Cell 93, 741753 (1998).
105. Gerety,S.S. & Anderson,D.J. Cardiovascular
ephrinB2 function is essential for embryonic
angiogenesis. Development 129, 13971410
(2002).
106. Gerety,S.S., Wang,H.U., Chen,Z.F. &
Anderson,D.J. Symmetrical mutant phenotypes of
the receptor EphB4 and its specific transmembrane
ligand ephrinB2 in cardiovascular development.
Mol.Cell 4, 403414 (1999).
107. Herbert,S.P. etal. Arterial-venous segregation by
selective cell sprouting: an alternative mode of blood
vessel formation. Science 326, 294298 (2009).
108. Lindskog,H. etal. Molecular identification of venous
progenitors in the dorsal aorta reveals an aortic origin
for the cardinal vein in mammals. Development 141,
11201128 (2014).
The molecular handle on cardinal vein development
is ephrinB2:EphB4 repulsive signalling.
109. Nakayama,M. etal. Spatial regulation of VEGF
receptor endocytosis in angiogenesis. Nat. Cell Biol.
15, 249260 (2013).
110. Sawamiphak,S. etal. EphrinB2 regulates VEGFR2
function in developmental and tumour angiogenesis.
Nature 465, 487491 (2010).
111. Wang,Y. etal. EphrinB2 controls VEGF-induced
angiogenesis and lymphangiogenesis. Nature 465,
483486 (2010).
112. Salvucci,O. etal. EphrinB2 controls vessel pruning
through STAT1JNK3 signalling. Nat. Commun. 6,
6576 (2015).
113. Bazigou,E. & Makinen,T. Flow control in our vessels:
vascular valves make sure there is no way back.
Cell.Mol. Life Sci. 70, 10551066 (2013).
114. Mkinen,T. etal. PDZ interaction site in ephrinB2 is
required for the remodeling of lymphatic vasculature.
Genes Dev. 19, 397410 (2005).
115. Bazigou,E. etal. Genes regulating lymphangiogenesis
control venous valve formation and maintenance in
mice. J.Clin. Invest. 121, 29842992 (2011).
116. Foo,S.S. etal. EphrinB2 controls cell motility and
adhesion during blood-vessel-wall assembly. Cell 124,
161173 (2006).
117. Nakayama,A. etal. EphrinB2 controls
PDGFRinternalization and signaling. Genes Dev. 27,
25762589 (2013).
118. Compagni,A., Logan,M., Klein,R. & Adams,R.H.
Control of skeletal patterning by ephrinB1EphB
interactions. Dev. Cell 5, 217230 (2003).
119. Davy,A., Aubin,J. & Soriano,P. EphrinB1 forward
and reverse signaling are required during mouse
development. Genes Dev. 18, 572583 (2004).
120. Twigg,S.R. etal. Mutations of ephrinB1 (EFNB1),
amarker of tissue boundary formation, cause
craniofrontonasal syndrome. Proc. Natl Acad. Sci. USA
101, 86528657 (2004).
121. Wieland,I. etal. Mutations of the ephrinB1 gene
cause craniofrontonasal syndrome. Am. J.Hum. Genet.
74, 12091215 (2004).
122. Bush,J.O. & Soriano,P. EphrinB1 forward signaling
regulates craniofacial morphogenesis by controlling
cell proliferation across Ephephrin boundaries.
Genes Dev. 24, 20682080 (2010).
123. Laussu,J., Khuong,A., Gautrais,J. & Davy,A. Beyond
boundaries Eph:ephrin signaling in neurogenesis.
Cell Adh. Migr. 8, 349359 (2014).
124. Chumley,M.J., Catchpole,T., Silvany,R.E.,
Kernie,S.G. & Henkemeyer,M. EphB receptors
regulate stem/progenitor cell proliferation, migration,
and polarity during hippocampal neurogenesis.
J.Neurosci. 27, 1348113490 (2007).
125. Holmberg,J. etal. EphrinA2 reverse signaling
negatively regulates neural progenitor proliferation
and neurogenesis. Genes Dev. 19, 462471 (2005).
126. Ashton,R.S. etal. Astrocytes regulate adult
hippocampal neurogenesis through ephrinB signaling.
Nat. Neurosci. 15, 13991406 (2012).
127. Ottone,C. etal. Direct cellcell contact with the
vascular niche maintains quiescent neural stem cells.
Nat. Cell Biol. 16, 10451056 (2014).

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

128. Batlle,E. etal. -catenin and TCF mediate cell

positioning in the intestinal epithelium by controlling the
expression of EphB/ephrinB. Cell 111, 251263 (2002).
129. Holmberg,J. etal. EphB receptors coordinate
migration and proliferation in the intestinal stem cell
niche. Cell 125, 11511163 (2006).
130. Duffy,P. etal. Myelin-derived ephrinB3 restricts axonal
regeneration and recovery after adult CNS injury.
Proc.Natl Acad. Sci. USA 109, 50635068 (2012).
131. Goldshmit,Y., Galea,M.P., Wise,G., Bartlett,P.F.
&Turnley,A.M. Axonal regeneration and lack of
astrocytic gliosis in EphA4deficient mice. J.Neurosci.
24, 1006410073 (2004).
132. Benson,M.D. etal. EphrinB3 is a myelin-based
inhibitor of neurite outgrowth. Proc. Natl Acad.
Sci.USA 102, 1069410699 (2005).
133. Goldshmit,Y. etal. EphA4 blockers promote axonal
regeneration and functional recovery following spinal
cord injury in mice. PLoS ONE 6, e24636 (2011).
134. Kempf,A. etal. Upregulation of axon guidance
molecules in the adult central nervous system of
NogoA knockout mice restricts neuronal growth
andregeneration. Eur. J.Neurosci. 38, 35673579
(2013).
135. Parrinello,S. etal. EphB signaling directs peripheral
nerve regeneration through Sox2dependent Schwann
cell sorting. Cell 143, 145155 (2010).
136. Dalva,M.B. etal. EphB receptors interact with NMDA
receptors and regulate excitatory synapse formation.
Cell 103, 945956 (2000).
137. Grunwald,I.C. etal. Kinase-independent requirement
of EphB2 receptors in hippocampal synaptic plasticity.
Neuron 32, 10271040 (2001).
138. Henderson,J.T. etal. The receptor tyrosine kinase
EphB2 regulates NMDA-dependent synaptic function.
Neuron 32, 10411056 (2001).
139. Contractor,A. etal. Trans-synaptic Eph receptor
ephrin signaling in hippocampal mossy fiber LTP.
Science 296, 18641869 (2002).
140. Klein,R. Bidirectional modulation of synaptic functions
by Eph/ephrin signaling. Nat. Neurosci. 12, 1520
(2009).
141. Henkemeyer,M., Itkis,O.S., Ngo,M., Hickmott,P.W.
& Ethell,I.M. Multiple EphB receptor tyrosine kinases
shape dendritic spines in the hippocampus. J.Cell
Biol. 163, 13131326 (2003).
142. Segura,I., Essmann,C.L., Weinges,S. &
AckerPalmer,A. Grb4 and GIT1 transduce ephrinB
reverse signals modulating spine morphogenesis and
synapse formation. Nat. Neurosci. 10, 301310
(2007).
143. Murai,K.K., Nguyen,L.N., Irie,F., Yamaguchi,Y.
&Pasquale,E.B. Control of hippocampal dendritic
spine morphology through ephrinA3/EphA4 signaling.
Nat. Neurosci. 6, 153160 (2003).
144. Filosa,A. etal. Neuronglia communication via EphA4/
ephrinA3 modulates LTP through glial glutamate
transport. Nat. Neurosci. 12, 12851292 (2009).
145. Carmona,M.A., Murai,K.K., Wang,L., Roberts,A.J.
& Pasquale,E.B. Glial ephrinA3 regulates hippocampal
dendritic spine morphology and glutamate transport.
Proc. Natl Acad. Sci. USA 106, 1252412529 (2009).
146. Zhao,C. etal. Bidirectional ephrinB2EphB4
signaling controls bone homeostasis. Cell Metab. 4,
111121 (2006).
147. Cheng,S. etal. Targeted disruption of ephrin B1 in
cells of myeloid lineage increases osteoclast
differentiation and bone resorption in mice. PLoS ONE
7, e32887 (2012).
148. Irie,N. etal. Bidirectional signaling through
ephrinA2EphA2 enhances osteoclastogenesis and
suppresses osteoblastogenesis. J.Biol. Chem. 284,
1463714644 (2009).
149. Wilkinson,D.G. Regulation of cell differentiation by
Eph receptor and ephrin signaling. Cell Adh. Migr. 8,
339348 (2014).
150. Konstantinova,I. etal. EphAEphrin-Amediated cell
communication regulates insulin secretion from
pancreatic islets. Cell 129, 359370 (2007).
151. Pasquale,E.B. Eph receptors and ephrins in cancer:
bidirectional signalling and beyond. Nat. Rev. Cancer
10, 165180 (2010).
152. Huusko,P. etal. Nonsense-mediated decay microarray
analysis identifies mutations of EPHB2 in human
prostate cancer. Nat. Genet. 36, 979983 (2004).
153. Cortina,C. etal. EphBephrinB interactions suppress
colorectal cancer progression by compartmentalizing
tumor cells. Nat. Genet. 39, 13761383 (2007).
154. Batlle,E. etal. EphB receptor activity suppresses
colorectal cancer progression. Nature 435,
11261130 (2005).

VOLUME 17 | APRIL 2016 | 255

.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
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l
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.
d
e
t
i
m
i
L
s
r
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h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
6
1
0
2

REVIEWS
155. Miao,H. etal. EphA2 mediates ligand-dependent
inhibition and ligand-independent promotion of cell
migration and invasion via a reciprocal regulatory loop
with Akt. Cancer Cell 16, 920 (2009).
156. Astin,J.W. etal. Competition amongst Eph receptors
regulates contact inhibition of locomotion and
invasiveness in prostate cancer cells. Nat. Cell Biol.
12, 11941204 (2010).
A combinatorial code of Eph receptors switches
migration properties of cancer cells.
157. Salaita,K. etal. Restriction of receptor movement
alters cellular response: physical force sensing by
EphA2. Science 327, 13801385 (2010).
158. Oricchio,E. etal. The Eph-receptor A7 is a soluble
tumor suppressor for follicular lymphoma. Cell 147,
554564 (2011).
159. Van Hoecke,A. etal. EPHA4 is a disease modifier of
amyotrophic lateral sclerosis in animal models and in
humans. Nat. Med. 18, 14181422 (2012).
EphA4 expression levels might determine the
susceptibility to motor neuron disease.
160. Fu,A.K.Y. etal. Blockade of EphA4 signaling
ameliorates hippocampal synaptic dysfunctions in

mouse models of Alzheimers disease. Proc. Natl

Acad. Sci. USA 111, 99599964 (2014).
161. Ciss,M. etal. Reversing EphB2 depletion rescues
cognitive functions in Alzheimer model. Nature 469,
4752 (2010).
EphB2 degradation as a consequence of amyloid-
binding as a potential Alzheimer disease
pathogenic mechanism.
162. Jun,G. etal. EPHA2 is associated with age-related
cortical cataract in mice and humans. PLoS Genet. 5,
e1000584 (2009).
163. Tischer,S., Reineck,M., Soding,J., Munder,S. &
Bottger,A. Eph receptors and ephrin class B ligands
are expressed at tissue boundaries in Hydra vulgaris.
Int. J.Dev. Biol. 57, 759765 (2013).
164. Bossing,T. & Brand,A.H. Dephrin, a transmembrane
ephrin with a unique structure, prevents interneuronal
axons from exiting the Drosophila embryonic CNS.
Development 129, 42054218 (2002).
165. Klein,R. Eph/ephrin signalling during development.
Development 139, 41054109 (2012).
166. Garcia-Frigola,C. & Herrera,E. Zic2 regulates the
expression of Sert to modulate eye-specific refinement

256 | APRIL 2016 | VOLUME 17

at the visual targets. EMBO J. 29, 31703183

(2010).
167. Pitulescu,M.E. & Adams,R.H. Eph/ephrin molecules
a hub for signaling and endocytosis. Genes Dev. 24,
24802492 (2010).
168. Poliak,S. etal. Synergistic integration of Netrin and
ephrin axon guidance signals by spinal motor
neurons. eLife http://dx.doi.org/10.7554/eLife.10841
(2015).

Acknowledgements

R.K. is supported by the Max Planck Society and by grants

from the European Research Council (ERC) and the Deutsche
Forschungsgemeinschaft (Munich Cluster for Systems
Neurology, SyNergy). A.K. is supported by the Canadian
Institutes of Health Research, National Sciences and
Engineering Research Council of Canada, Canada Foundation
for Innovation, Brain Canada and the W. Garfield Weston
Foundation. The authors thank Daniel Morales for critical
comments on the manuscript.

Competing interests statement

The authors declare no competing interests.

www.nature.com/nrm
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