Download Eph Receptors: Two Ways to Sharpen Boundaries

Current Biology Vol 15 No 6
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growing primordium in the tissues
which form the vasculature and by
becoming basally localised within
these cells, creating an entirely
new direction of flow [7,8].
These often rapid changes in PIN
polarity within cells would appear
to be greatly facilitated by the fact
that PIN proteins are continuously
cycled between endosomal
compartments and the plasma
membrane [10,18]. This trafficking
of PINs is dependent on the
Arabidopsis ARF guanine nuclotide
exchange protein GNOM [18], but
what is far from clear is how the
changes in subcellular targeting
are signalled. One protein that is
almost certainly involved is the
protein kinase PINOID (PID) [19].
Overexpression and loss-offunction of PID lead to opposite
effects on the apical-basal
localisation of certain PIN proteins
[20]. Interestingly, PID transcription
is also auxin-inducible, raising the
exciting possibility that auxin could
regulate the dynamic changes in its
own flux [19,20].
Plants differ from animals in that
a large part of their development
occurs post-embryonically and
with continuous reference to
environmental conditions. This
developmental plasticity is a
reflection of the way in which auxin
gradients can be erected,
dismantled, moved around, and
turned upside down. Thus the
French flag can be replaced with
that of the European Union when
it’s politically expedient.
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1Department
of Biology, University of
York, Box 373, York YO10 3DH, UK.
2Umeå Plant Science Centre, SLU, S90183, Umeå, Sweden.
E-mail: [email protected]
DOI: 10.1016/j.cub.2005.03.012
Eph Receptors: Two Ways to
Sharpen Boundaries
Eph receptors and ephrins can sharpen domains within developing
tissues by mediating repulsion at interfaces. An Eph receptor has
now been shown also to regulate cell adhesion within tissue
subdivisions.
Dalit Sela-Donenfeld and
David G. Wilkinson
An important problem in
developmental biology is to
understand how precise spatial
patterns of cell types are
maintained despite the potential
for rearrangement by cell division
and intercalation. This question
applies to a key phase in the
patterning of many tissues, in
which they are subdivided into
domains, each with a distinct
regional identity that specifies a
particular set of cell types.
One way in which the precision
of regional patterning can be
maintained is by the inhibition of
cell mixing between domains [1,2].
Two types of mechanism have
been found to restrict cell
intermingling. First, cells can be
confined within a regional domain
because they preferentially adhere
to each other, for example by
homophilic adhesion via
cadherins. A classical
demonstration of this is the sorting
that occurs when cells with
different affinities are mixed,
driven by cells minimising contact
Dispatch
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with others that have different
adhesive properties [3].
A second mechanism involves
the mutual inhibition of cell
invasion via bidirectional
activation of Eph receptors and
ephrins at the interface of domains
[4,5]. In this issue, Cooke et al. [6]
now report the important finding
that an Eph receptor regulates cell
affinities within tissue
subdivisions, and so may sharpen
boundaries by modulating cell
adhesion both at interfaces and
within regional domains.
Repulsion at Interfaces
Interactions between Eph receptor
tyrosine kinases and ephrins
mediate cell-contact-dependent
signalling in which both
components can transduce signals
that trigger cell responses [4].
Binding between members of the
vertebrate Eph receptor and ephrin
families falls largely into two
classes: GPI-anchored ephrinAs
bind to EphA receptors, and
transmembrane ephrinBs bind to
EphB receptors and to EphA4.
Collectively, Eph and ephrin
proteins are expressed in many,
perhaps all, tissues during
development, and have key roles in
the repulsive guidance of migrating
cells and axonal growth cones at
boundaries, or along gradients, of
complementary expression [4,5].
Similarly, Eph–ephrin signalling can
restrict the intermingling of cells
across interfaces within tissues,
which studies in the hindbrain have
suggested may involve
bi-directional cell responses.
The hindbrain is transiently
subdivided during development
into repeated segments called
rhombomeres, each expressing a
distinct set of transcription factors
that underlie regional specification
[1]. The precision of this segmental
organisation involves the formation
of sharp interfaces by restriction of
cell intermingling between
adjacent rhombomeres [7]. Several
ephrinBs and interacting EphA4
and EphB receptors are
segmentally expressed in the
hindbrain, largely (but not entirely)
in a complementary manner, such
that Eph–ephrinB interactions and
bi-directional signalling could
occur at the interface of segments.
The results of dominant-negative
blocking [8], mosaic
overexpression [9] and cell
transplantation [10] experiments
suggest that Eph–ephrinB
signalling at interfaces underlies
the restriction of mixing across
hindbrain boundaries.
Furthermore, bi-directional
Eph–ephrinB signalling at the
interface of in vitro cell aggregates
was found to bi-directionally
restrict cell invasion [11]. These
findings suggest that localised
activation of Eph receptors and
ephrinBs at the interface of
complementary expression
domains underlies a mutual
repulsion that prevents
intermingling.
Cell Affinity within Segments
Cooke et al. [6] further examined
the role of Eph–ephrin interactions
in the hindbrain in loss-of-function
‘knockdown’ experiments by
injecting antisense morpholino
oligonucleotides into zebrafish
embryos. As anticipated,
knockdown of EphA4 — which is
expressed in rhombomeres r3 and
r5 — resulted in fuzzy interfaces
between r3/r5 and adjacent
segments. However, mosaic
knockdown experiments in which
EphA4-knockdown cells were
transplanted into an uninjected
embryo, or vice versa, led to an
unexpected result: that cell
sorting occurred within r3 and r5,
with EphA4-knockdown cells
segregating to the edges and
uninjected cells to the centre of
these segments (Figure 1A,B).
Detection of r3/r5-specific gene
expression revealed that the
segregated EphA4-knockdown
cells remained within r3/r5, rather
than mixing into adjacent territory,
which can most easily be
explained by continued
expression of other Eph receptors
that mediate repulsion by ephrin
at segment interfaces. Indeed,
there is a stronger disruption of
the interfaces between r3/r5 and
r4 when both EphA4 and
ephrinB2a — expressed in r1/r4/r7
— are knocked down. The sorting
of EphA4-knockdown cells shows
that EphA4 regulates cell–cell
affinity within r3/r5, which the
results of time-lapse experiments
suggest contributes to the normal
restriction of cell mixing between
A
ephrinB2
EphA4
B
C
Current Biology
Figure 1. EphA4 and boundary
sharpening.
(A) Mosaic knockdown of EphA4 protein
in r3/r5 is achieved by transplantation of
cells between uninjected (strong red)
and EphA4 morpholino-injected (light
red) embryos. (B) EphA4-knockdown
cells are found to segregate to the
borders of r3/r5. This sorting suggests
that EphA4 regulates cell affinity
(indicated by lines connecting cells) that
is decreased (dotted lines) following
EphA4 knockdown. (C) Taken together
with previous findings, EphA4 sharpens
the borders of hindbrain segments via
interactions with complementary ephrins
that mediate repulsion across interfaces
and by regulating cell affinity within
segments.
segments. Taken together with
previous findings, Eph–ephrin
interactions thus contribute to the
sharpening of segments by
regulating both repulsion at
interfaces and cell affinity within
rhombomeres (Figure 1C).
Regulation of Adhesion versus
Repulsion
The results of the mosaic EphA4
knockdown experiments [6] imply
Current Biology Vol 15 No 6
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that EphA4 regulates cell affinity,
and thus is being activated,
throughout r3/r5. Indeed, although
most functional studies have
focussed on complementary
Eph–ephrin expression, overlaps
in expression occur in many
tissues, including the hindbrain.
Furthermore, there has been
growing evidence that Eph
receptor activation can trigger
alternative adhesion or repulsion
responses that might be regulated
by overlapping expression with
ephrins [5].
An intriguing feature of the
Eph–ephrin system is that the
nature of the cell response may
depend upon the degree of
clustering and activation [5]. Low
level Eph receptor clustering or
activation promotes cytoskeletal
assembly, invasion and adhesion,
whereas increased clustering or
activation triggers cytoskeletal
disassembly and repulsion (for
example [12]). Overlapping
expression of Eph receptors and
ephrins suppresses cell repulsion
responses [13] and promotes
adhesion [14]. A biochemical basis
for this suppression is suggested
by the finding that co-expressed
ephrin interacts in cis with Eph
receptor to form inactive
complexes, and inhibits trans
Eph–ephrin interactions between
cells [15]. Consequently,
overlapping Eph–ephrin expression
may cause low-level activation that
promotes an adhesive response.
These findings suggest a model
in which EphA4 has overlapping
expression with some ephrins
within r3/r5 that underlies low
level activation and cell adhesion,
and complementary expression
with other ephrins that promote
high-level activation and
repulsion across boundaries. It
will be interesting to uncover
whether ephrins regulate cell
affinity within even-numbered
segments, or if their only role is to
participate in bi-directional
repulsion
Interfaces and Boundary Cells
The findings of Cooke et al. [6]
also raise the question of whether
Eph receptors and ephrins have
additional roles at segment
interfaces. An important
mechanism for patterning of some
tissues is the formation of a
signalling centre at the interface of
regional domains. Distinct
boundary cells form at the
interface of hindbrain segments
[16,17], and recent work suggests
that expression of Wnt signals and
Notch activation at hindbrain
boundaries in zebrafish regulates
neurogenesis and the localisation
of boundary cells [18,19]. As
blocking or knockdown of EphA4
leads to a depletion of boundary
cells [6,8], Eph–ephrin signalling
could have a direct role in
boundary cell specification. This is
an attractive possibility since
boundary cells are induced by
interactions between adjacent
segments [20], at the site of bidirectional Eph–ephrinB activation.
On the other hand, there may be
an indirect relationship in which
boundary cell formation requires a
stable interface between segments
that is disrupted by blocking of
Eph–ephrin signalling. In view of
evidence that local interactions
switch the segmental identity of
any isolated cells that move into an
adjacent segment (reviewed in [2]),
there may be more intermingling
occurring than suggested by the
fuzzy interfaces of r3/r5 gene
expression following loss of EphA4
function [6,8]. Further studies will
thus be required to address the
relative contribution of the
regulation of cell movement and
identity to the formation of sharp
interfaces, and the potential role of
Eph-ephrin signalling in boundary
cell specification.
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Divisional of Developmental
Neurobiology, National Institute for
Medical Research, The Ridgeway, Mill
Hill, London NW7 1AA, UK.
E-mail: [email protected]
DOI: 10.1016/j.cub.2005.03.013