Molecular Neurobiology Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
* Author for correspondence (e-mail: doleary{at}salk.edu)
Accepted 3 March 2003
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SUMMARY |
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Key words: Axon attraction, Axon branching, Axon guidance, Axon repellents, Chick visual system, Electroporation, Optic tectum, Eph receptors, Gradients, Recombinant retrovirus, Topographic maps
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INTRODUCTION |
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The developmental mechanism by which the DV retinal axis maps along the LM
tectal axis imposes unique requirements on the molecular activities that
control this process (Fig. 1A).
Initially, RGC axons from the same location in the retina enter the tectum at
its anterior border over a broad LM extent. In rats and mice, RGC axons
arising from the same DV retinal location are distributed across 80% of the LM
axis of the SC (Simon and O'Leary,
1992a; Simon and O'Leary,
1992b
; Hindges et al.,
2002
). In chick, RGC axons arising from the same DV retinal
location are found millimeters medial and lateral to the correct location of
their future termination zone (TZ) along the LM axis
(Nakamura and O'Leary, 1989
).
Even along the AP tectal axis, the guidance of the primary axons is
topographically inaccurate since they substantially overshoot the AP location
of their TZ (Yates et al.,
2001
). RGC axons establish their appropriately ordered connections
through interstitial branches that form perpendicular to the shaft of the
primary RGC axon, with a topographic bias for the AP location of the TZ
(Simon and O'Leary, 1992c
;
Yates et al., 2001
).
Subsequently, the interstitial branches extend medially or laterally across
the tectum to reach and arborize at the topographically correct position along
the LM axis (Nakamura and O'Leary,
1989
; Simon and O'Leary,
1992b
; Hindges et al.,
2002
) (present study). Thus, guidance of the primary RGC axon
mainly serves to put the axon in the vicinity of the target, whereas the
topographic formation and guidance of interstitial branches is the critical
determinant in setting up ordered connections.
|
Recent studies have shown that the EphB subfamily of receptor tyrosine
kinases and their ephrin-B ligands act as attractants to control, in part,
mapping of the DV retinal axis along the LM tectal axis
(Hindges et al., 2002;
Mann et al., 2002
;
McLaughlin et al., 2003
).
During map development in chicks and mice, EphB2, EphB3 and EphB4 are
expressed in a low to high DV gradient by RGCs, EphB1 is expressed uniformly
(Holash and Pasquale, 1995
;
Henkemeyer et al., 1996
;
Connor et al., 1998
;
Birgbauer et al., 2000
;
Hindges et al., 2002
) and one
of their ligands, ephrin-B1, is expressed in a low to high LM gradient across
the tectum (Braisted et al.,
1997
; Hindges et al.,
2002
). RGC axons arising from the same DV retinal location, and
therefore having similar levels of EphB receptors, extend interstitial
branches either medially up the ephrin-B1 gradient or laterally down it to
reach the LM location of their TZ (Fig.
1A). At least two distinct molecular activities are required to
control this mapping behavior: one activity, accounted for at least in part by
ephrin-B1-mediated attraction of branches medially up the gradient, shown by
analyses of EphB2; EphB3 mutant mice
(Hindges et al., 2002
), and a
second activity directs branches laterally. Modeling indicates that the
additional activity is a branch repellent expressed in a gradient similar to
ephrin-B1 (Hindges et al.,
2002
).
We suggest that ephrin-B1 is not only an attractant for interstitial
branches, but is also a repellent for them, and that a branch's response is
context-dependent and depends upon the DV origin of its primary RGC axon,
which determines its level of EphB receptors, and the origin of the branch
relative to the LM location of its future TZ, and therefore its position along
the ephrin-B1 gradient. Together, these parameters determine the level of EphB
signaling experienced by a given branch, and whether it is less or more than
that at the appropriate location of its TZ. The plausibility of a bifunctional
action of ephrin-B1 is suggested by evidence that ephrin-Bs can have either a
repellent or attractant affect on distinct populations of early and late
migrating neural crest cells (Santiago and
Erickson, 2002), and also findings that other guidance molecules,
such as netrin 1, act as a repellent or an attractant for different types of
axons (Colamarino et al., 1995) or for the same spinal axons depending upon
the intracellular level of cyclic nucleotides
(Ming et al., 1997
;
Song et al., 1997
). To test
this hypothesis, we have used RCAS vectors to ectopically express ephrin-B1 in
the developing chick tectum and analyzed the effect of ectopic domains of
ephrin-B1 on the trajectories and mapping of RGC axons, and the directional
extension and arborization of their interstitial branches.
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MATERIALS AND METHODS |
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Retroviral construction
We made three retroviral constructs based on the avian retroviral RCAS
vector (Fekete and Cepko,
1993). Total RNA was harvested from E8 chick tectum using a
QIAquick kit (Qiagen) and then cDNA was prepared by reverse transcription with
random primers. PCR amplification of full-length ephrin-B1 was performed with
nested primers specific for ephrin-B1. Four clones were sequenced; one of the
four clones with the correct predicted protein sequence was used. For the
RCAS-ephrin-B1-eGFP fusion construct, the stop codon of ephrin-B1 was replaced
with the start codon of eGFP and inserted into the ClaI site of the
RCAS vector. For the RCAS-ephrin-B1-IRES-eGFP construct, eGFP (Clontech) was
inserted into the SLIRES11 shuttle vector (a gift from C. Cepko). Then the
IRES-eGFP was amplified by PCR with primers containing a 5'
ClaI site and a 3' ClaI site protected by a 5'
guanine, thus preventing ClaI cleavage when methylated. The PCR
product was directly cloned into the ClaI site of RCAS, making
RCAS-IRES-eGFP. Full-length ephrin-B1 was inserted into the RCAS-IRES-eGFP
vector upstream of the IRES at the functional ClaI site. The third
clone, RCAS-eGFP was made by cloning eGFP into the SLAX shuttle vector, then
into the ClaI site of RCAS. All constructs were sequenced for
orientation and fidelity.
Retroviral vector electroporation
Eggs of the white Leghorn strain of chicken were obtained from a local
supplier (McIntyre Farms, Lakeside, CA) and incubated at 38°C for 40 hours
prior to electroporation. At stages 10-12
(Hamburger and Hamilton,
1951), eggs were windowed, 2.5 ml of albumin removed, and they
were injected in the mesencephalic ventricular space with a small amount of
RCAS-eGFP, RCAS-ephrin-B1-eGFP, or RCAS-ephrin-B1-IRES-eGFP plasmid DNA at 1-4
µg/µl mixed with Fast Green (to visualize the solution) as described by
Erkman et al. (Erkman et al.,
2000
). Parallel platinum-coated electrodes spaced at 4 mm were
placed along the embryo such that the embryo was centered and its anterior
posterior axis was parallel to the electrodes. A small drop of L15
(Gibco) was placed on the embryo and five square pulses of 50 milliseconds at
25 volts were applied by a T820 Electrosquare porator (BTX). Nine to 16 days
later embryos were perfused, dissected and examined with a confocal microscope
(Zeiss). Typically, only one tectal lobe was transfected and only animals in
which the entire infection was limited to one tectal lobe were analyzed
further.
Anterograde axon labeling
Anterograde labeling of RGC axons was done as described by Yates et al.
(Yates et al., 2001). Chicks
were injected with a small amount of a 10% solution of DiI (Molecular Probes)
in DMF into the retina and perfused 1 day later. Whole mounts of the optic
tectum contralateral to the injected eye were examined on a Zeiss confocal
microscope. Two-channel confocal microscopy was performed to record all DiI
and eGFP present in the optic tectum.
Quantification of branch orientation
Confocal images were taken with a 10x objective from which montages
were made and aligned with Adobe Photoshop software to create a full view of
each tectum. The medial and lateral borders of the emerging termination zone
(TZ) were marked using Adobe Photoshop software. The image was expanded so
individual DiI-labeled axons and their branches could be easily visualized and
unambiguously identified. All branches and their orientation were marked on
the image prior to knowledge of infection domain and blind to their precise
location within the tectum. All domains of eGFP in chicks electroporated with
RCAS-eGFP or RCAS-ephrin-B1-IRES-eGFP were marked, blind to location in
tectum, on each whole-mount montage. All eGFP marks were made blind to all DiI
labeling. The interstitial branch and eGFP tracings were overlayed and
branches in contact with eGFP markings were considered to be in a domain of
infection. The tectum was separated into three sections based on the exact
location of the nascent TZ. The orientation, length, relation to eGFP
expression (whether eGFP alone or eGFP coexpressed with ephrin-B1), and
position of the primary axon for all branches was recorded. RGC axons were
labeled with DiI and analyzed between E11 and E14 for the branch
directionality data presented in Fig.
3. E10-E13 chicks were used to collect data that relates branch
directionality to branch length.
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RESULTS |
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Each vector was transfected into the mesencephalic vesicle at E1.5 using in ovo electroporation (Fig. 1B). Transfection domains were limited to a single tectal lobe (not shown). We were able to obtain persistent expression of eGFP alone, or eGFP and ephrin-B1, to at least E17 with no detectable reduction in expression. Because the RCAS vector encodes a replication competent avian retrovirus, in addition to the viral vector being integrated into the genome of the host progenitor cells and being passed on to its progeny, the transfected/infected cells produce viable viral particles, which likely infect nearby progenitors, leading to larger domains of vector-expressing cells. Electroporated tecta were characterized by transfection/infection domains in columnar patterns extending from the ventricular zone to the pial surface (Fig. 1C), ensuring the presence of exogenous ephrin-B1 in the stratum opticum, the intratectal path of RGC axons and their branches, and the superficial layers of the tectum where the interstitial branches arborize, throughout the development of retinotectal topography. For simplicity, we will refer to the ectopic expression domains as transfections. To analyze the effects of the expression vectors on retinotectal mapping, a small number of RGC axons were anterogradely labeled by the fluorescent axon tracer DiI, injected into the eye contralateral to the transfected tectum, 16 hours before fixation.
Transfection domains of control RCAS vectors have no effect on
retinotectal development
As expected, the RCAS-eGFP vector had no effect on RGC axon trajectory, the
directional extension of interstitial branches, or location of the TZ
(n=3 tecta) (Fig. 1D).
Even within domains of eGFP, interstitial branches were evident and were
preferentially directed along the LM axis towards their TZ
(Fig. 1D inset). In contrast,
as described below, ectopic domains of ephrin-B1 created by transfection with
either the RCAS-ephrin-B1-eGFP vector or the RCAS-ephrin-B1-IRES-eGFP vector
had substantial effects on the arborization of RGC axons and the directional
extension of interstitial branches.
Ectopic domains of ephrin-B1 have no influence on the trajectories of
primary RGC axons
Surprisingly, the ectopic domains of ephrin-B1 in the tectum had no
influence on the growth or trajectories of primary RGC axons regardless of
whether they arose from ventral retina with high levels of EphB expression, or
from more dorsal retina with lower levels of EphB expression
(Fig. 2A-A'';
n=11 tecta). The topographic positioning of the TZ formed by the
DiI-labeled RGC axons in the ephrin-B1 transfected cases appears to be at the
topographically correct site, regardless of whether the TZ formed in a
location within the tectum heavily transfected with ectopic domains of
ephrin-B1, or lacking them entirely. This lack of effect differs from the
strong repellent effect that ectopic domains of ephrin-A2 have on temporal RGC
axons and on the topographic positioning of their TZs
(Nakamoto et al., 1996).
|
Perhaps the most dramatic examples of the repellent action of the ectopic domains of ephrin-B1 are their apparent ability to shape and distribute an arbor. Two such examples are illustrated in Fig. 2. In one example, a comet-like TZ had a bulbous head abutting an ectopic domain of ephrin-B1, and a narrow tail confined to a space hemmed by ectopic domains of ephrin-B1 (Fig. 2C-C''). In another example, an ectopic domain of ephrin-B1 splits what would likely have been a single high density arbor into two distinct high density components with a domain of substantially reduced density between them that is coincident with an elongated ectopic domain of ephrin-B1 (Fig. 2D-D'').
In the remaining four of the 10 `co-localized' cases, the DiI-labeled TZ was found at a tectal location coincident with small ectopic domains of ephrin-B1. These TZs were not consistently dense as in controls, but contained areas devoid of significant branching or arborization. In most instances, these areas of diminished branching and arborization corresponded precisely to small ectopic domains of ephrin-B1 (Fig. 2E-E''). Taken together, these findings indicate that high levels of ephrin-B1 act as a repellent or inhibitor of the branching process that leads to the formation of dense arborizations of RGC axons within the tectum.
Bi-functional effects of ephrin-B1 on directional extension of
interstitial branches
In chicks, as in rodents, arbors are formed by interstitial branches that
extend directionally from the shafts of primary RGC axons either medially up
the ephrin-B1 gradient, or laterally down it, to reach the LM location of
their TZ (Fig. 1A). Recent work
has shown that EphB forward signaling acts as an attractant to guide
interstitial branches medially up the gradient of ephrin-B1, which is
consistent with ventral RGCs, expressing high levels of EphBs, mapping to
medial tectum with high levels of ephrin-B1
(Hindges et al., 2002).
Although our findings above show that ectopic domains of ephrin-B1 do not
affect the trajectories of primary RGC axons, they do have a repellent or
inhibitory effect on their arborizations. Thus, in addition to ephrin-B1 being
an attractant for interstitial branches, it may also be a repellent for them,
and therefore have a bifunctional role in guiding interstitial branches along
the LM axis.
To address this issue, we analyzed the directional extension of all visible interstitial branches in control and ephrin-B1-transfected tecta of E11-E14 chicks 1 day after focal DiI injections were made into the retina. High-resolution confocal images were analyzed, often in three-dimensional projections to confirm the points of origin of branches and their directionality. All branches were digitally traced, blind to infection domains, from confocal images and their length, location, and orientation recorded. We also digitally marked all areas of eGFP, blind to RGC axon labeling, in all RCAS-ephrin-B1-IRES-eGFP animals. The quantification scheme is outlined in Fig. 3A.
Directional extension of branches in control tecta
In control tecta, temporal axons (n=14 tecta;
Fig. 3B) and nasal-ventral
axons (n=11 tecta; Fig.
3C) exhibited similar preferences in the directional extension of
interstitial branches towards the LM location of their future TZ. For both
populations, RGC axons located lateral to their future TZ preferentially
extended interstitial branches medially toward their future TZ, whereas axons
located medial to the TZ preferentially extended branches laterally
(Fig. 3C). At the LM location
of the TZ, we observed no bias in branch orientation. As expected, outside of
the TZ, directional branch extension is related to branch length. Branches
shorter than 25 µm in length are randomly oriented (53% extend towards
their future TZ; n=21 cases, 146 branches; 2 test
P=0.51). Branches greater than 100 µm in length are preferentially
directed towards their future TZ (69% extend towards the TZ; n=200
branches; P<<0.001). These data indicate that initial branch
formation along the primary axon occurs without a directional response to
molecular information that encodes position along the LM axis, but that
branches initially directed toward their TZ preferentially elongate toward
it.
Essentially no difference was observed in the directional extension of
branches toward the TZ from axons displaced lateral or medial to their TZ
(Fig. 3B,C), indicating that
equally effective mechanisms account for their guidance. The similarities in
these data from different retinal sites, and to published data from peripheral
temporal retina (Nakamura and O'Leary,
1989) suggest that RGC axons throughout the retina use the same
mechanism. Therefore, for the analysis of the effects of ephrin-B1, we focused
on ventral RGCs, which express high levels of EphBs and normally arborize in
medial tectum, which expresses high levels of endogenous ephrin-B1.
Directional branch extension is biased laterally within ectopic
domains of ephrin-B1
Within ectopic domains of ephrin-B1, regardless of the location within the
tectum and their relationship to the TZ, branches tended to extend laterally,
down the gradient of endogenous ephrin-B1. Even in cases where the primary
axon is adjacent to the forming TZ, but within an ectopic domain of ephrin-B1,
branches were directed away from the TZ
(Fig. 4). In addition, branches
extended from the same primary axon located lateral to the future TZ can
exhibit different directionalities that relate to whether they extend within
or outside of an ectopic domain of ephrin-B1: branches outside an ectopic
domain of ephrin-B1 preferentially extended medially towards the TZ up the
gradient of endogenous ephrin-B1, whereas branches within an ectopic domain of
ephrin-B1 preferentially extended laterally away from the TZ down the gradient
of endogenous ephrin-B1 (Fig.
4B-D, arrowheads). These findings suggest that high levels of
ephrin-B1 act as a repellent for interstitial branches.
|
To examine directly the effect of high levels of ephrin-B1 on directional branch extension, we divided the pool of branches into those that were in ectopic domains of ephrin-B1 and those that were not. Within ectopic domains of ephrin-B1, branches exhibited a significant bias to be directed laterally, down the endogenous ephrin-B1 gradient, regardless of their location along the LM axis (Fig. 3E). In contrast, in the same tecta, branches found outside the ectopic domains of ephrin-B1 exhibited the same directionality as in control tecta: those lateral to the TZ preferentially extended medially towards it and up the ephrin-B1 gradient, whereas those medial to the TZ preferentially extended laterally towards it and down the ephrin-B1 gradient (Fig. 3F, compare with 3B,C). Thus, the effect of the ectopic domains of ephrin-B1 on branch directionality is limited to the transfection domains.
The directional extension of branches is controlled, at least in part, by
the graded expression of ephrin-B1
(Hindges et al., 2002)
(present study). Because branch directionality is not random within the
ectopic domains of ephrin-B1, but is shifted toward a lateral bias, we
suspected that within the ectopic domains of ephrin-B1 protein, the overall
level of endogenous and transgene ephrin-B1 parallels the normal ephrin-B1
gradient, albeit at higher levels than the graded distribution in positions
adjacent to the transfection domains. To address this issue, we stained chick
tecta with an EphB2-Fc receptor affinity probe to reveal the distribution of
ephrin-B1 (Fig. 5). We found
that ephrin-B1 protein is concentrated in the stratum opticum, as previously
reported (Braisted et al.,
1997
), and exhibited a low to high LM gradient similar to
ephrin-B1 transcripts (Braisted et al.,
1997
). In addition, EphB2-Fc staining in tecta transfected with
RCAS-ephrin-B1-IRES-eGFP revealed the combined distribution of endogenous and
transgene ephrin-B1 protein. Qualitatively, the level of EphB2-Fc labeling
within the ectopic domains of ephrin-B1 appeared to equal or exceed the level
of staining in medial tectum, which has the highest level of endogenous
ephrin-B1. When an ectopic domain of ephrin-B1 is sufficiently large to detect
a gradient, a low to high LM gradient is evident, but at a higher overall
level of ephrin-B1 (Fig. 5D-F).
Taken together these findings suggest that at high levels, ephrin-B1 still
acts as a directional cue for interstitial branches of RGC axons, but rather
than being an attractant, it acts as a repellent.
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DISCUSSION |
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Our gain-of-function studies reported here were designed to test this hypothesis. If correct, branches that form in ectopic domains of ephrin-B1, created by transfection, would experience higher levels of ephrin-B1 than those found at their appropriate TZ, and therefore would be repelled laterally whether or not this would direct them towards their appropriate TZ. We find that within ectopic domains of ephrin-B1, interstitial branches of RGC axons show an aberrant bias to extend laterally down the gradient of ephrin-B1 irrespective of the DV origin of the primary RGC axon or the LM origin of the branch; even branches extended by RGC axons positioned lateral to their appropriate TZ are preferentially directed laterally away from it (Fig. 6A). Consistent with this aberrant lateral bias in directional branch extension, we show that within the ectopic domains the overall ephrin-B1 protein (endogenous and transgene) is distributed in a gradient that parallels the low to high LM gradient of endogenous ephrin-B1, but at a higher level of protein. Branches outside the ectopic domains of ephrin-B1 exhibit their normal directional extension such that they are preferentially directed medially or laterally toward the TZ depending on whether they originate lateral or medial to it.
|
In contrast to their interstitial branches, the primary RGC axons
themselves do not respond to either the endogenous ephrin-B1 gradient or the
ectopic domains of ephrin-B1 by altering their trajectories or stopping their
growth. These findings suggest that during normal development, ephrin-B1 acts
as a bifunctional guidance molecule to control selectively the directional
extension of interstitial branches extended by RGC axons arising from the same
DV position and presumably expressing the same subtypes and levels of EphB
receptors (Fig. 6C). In
addition, the ectopic domains of ephrin-B1 inhibit the arborization of RGC
axons and shape the distribution of arbors
(Fig. 6B). These findings are
consistent with a mechanism in which a high level of ephrin-B1 signaling
repels RGC axon branches. Therefore, ephrin-B1 may cooperate with ephrin-As to
restrict the size and shape of arbors formed by interstitial branches
(Fig. 6D). Our findings
indicate that ephrin-B1 may help limit arbors along the LM axis, whereas the
findings from other studies suggest that ephrin-As may serve to limit the
posterior extent of an arbor (Nakamoto et
al., 1996; Yates et al.,
2001
).
Although the bifunctional action of ephrin-B1 on the directional extension
of interstitial branches can in principle explain LM mapping, our findings do
not rule out that other, as yet unidentified, guidance molecules may
contribute to topographic mapping along this axis (see
Mui et al., 2002). Reverse
signaling of ephrin-Bs, which mediates RGC axon attraction in the frog
retinotectal system (Mann et al.,
2002
), may potentially contribute to this mapping; a caveat
however is that although ephrin-Bs are expressed in a countergradient to EphBs
in the RGC layer of chick retina, in contrast to EphBs, ephrin-Bs are not
detected along the length of RGC axons in vivo
(Braisted et al., 1997
). In
addition, other activities may be required to initiate branching along the RGC
axon shaft (Yates et al.,
2001
), and promote the arborization and laminar patterning of
interstitial branches (Cohen-Cory and
Fraser, 1995
; Inoue and Sanes,
1997
).
ephrin-B1 selectively affects directional branch extension and
arborization
During normal development, the primary RGC axons do not respond to
ephrin-B1, or any LM guidance information, by changing their trajectories; nor
are their trajectories affected by the ectopic domains of ephrin-B1. However,
the extension of interstitial branches, which extend either up or down the
ephrin-B1 gradient, are affected by ephrin-B1. Since short branches extend
randomly, whereas the orientation of long branches is biased towards their TZ,
we conclude that ephrin-B1 does not promote the formation of interstitial
branches, but directs their extension along the LM axis.
These findings are in strong contrast to the demonstration that primary RGC
axons, as well as their branches and arbors, are repelled or inhibited by
ephrin-As that control, in part, TN retinal mapping along the AP tectal axis
(Nakamoto et al., 1996;
Yates et al., 2001
). The
selective influence of ephrin-B1 on branches rather than primary RGC axons,
and the bifunctional action of ephrin-B1 on directional branch extension, may
underlie the inability to show differential DV retinal responses to LM tectal
cells or membranes using in vitro axon guidance assays and chick tissues
(Bonhoeffer and Huf, 1982
;
Walter et al., 1987
) or
differential DV retinal responses of chick retina to membranes of heterologous
cells transfected with ephrin-B1 or substrates of artificially clustered
ephrin-B1-Fc (T.McL., J. E. Braisted, D.D.M.O'L. unpublished observation). The
same types of in vitro assays effectively reveal the repellent or inhibitory
effect of posterior tectal membranes and ephrin-As on chick or rodent temporal
retinal axons (Walter et al.,
1987
; Simon and O'Leary,
1992a
; Drescher et al.,
1995
; Nakamoto et al.,
1996
; Monschau et al.,
1997
; Feldheim et al.,
1998
) and their interstitial branches
(Roskies and O'Leary, 1994
;
Yates et al., 2001
).
Why primary RGC axons and their branches respond to ephrin-A2 whereas only
the branches respond to ephrin-B1 is unclear. One possible explanation for
this is that EphB receptors may be differentially distributed on axons and
branches, and preferentially found on branches. This possibility is suggested
by the finding that EphA2 receptors are predominantly distributed to the
distal part of spinal commissural axons by a mechanism of RNA translation
within the axon and insertion of the locally synthesized EphA2 into the distal
part of the growing axon (Brittis et al.,
2002). This mechanism appears to account for the change in
commissural axon responsiveness to guidance cues at different points in their
pathway. By analogy, RNAs encoding EphBs may be preferentially translated at
branch points and exported to the membrane of newly formed branches which
could account for the selective effect of ephrin-B1 on directional branch
extension and arborization. Interestingly, in several systems, interstitial
branches have been found to extend from varicosities on the primary axon
(Bastmeyer et al., 1998
), and
that these varicosities form de novo as a prelude to branch formation
(Bastmeyer and O'Leary, 1996
).
The varicosities may act as pools enriched with RNAs and the machinery for RNA
translation into protein. Alternatively, EphBs may be transported
intra-axonally from the cell body, for example in association with vesicles,
and preferentially exported to the membrane of developing branches.
Potential mechanisms for context-dependent ephrin-B1-mediated branch
attraction and repulsion
The context in which a branch extends is a critical determinant in the
choice between attraction and repulsion. Our findings indicate that this
differential response is a locally controlled phenomenon since interstitial
branches extending from the same primary axon exhibit different responses
depending on whether they extend within or outside of ectopic domains of
ephrin-B1. Local changes in the intracellular environment of the axon and its
branches may be a critical parameter in determining these differential
responses to ephrin-B1, since in vitro studies have shown that changes in
cyclic nucleotides can change the response of axonal growth cones from
attraction to repulsion, or vice versa
(Song and Poo, 1999).
Furthermore, at least in some instances, these responses require local protein
synthesis (Campbell and Holt,
2001
). The substrate upon which an axon grows can also be an
important factor in determining its response to a guidance cue
(Hopker et al., 1999
), which
in principle may be affected by changes in concentrations of ephrin-B1.
Our findings indicate that for RGC axons originating from the same DV
position, and therefore expressing the same levels of EphB receptors, whether
an interstitial branch is repelled or attracted by ephrin-B1 depends upon
where along the LM tectal axis (and therefore the gradient of ephrin-B1) the
branch originates from the primary axon. It is possible that this is
controlled by a single guidance molecule with an attractant and repellent
function, dependent on distinct receptors. Dual activities (attraction and
repulsion) of one guidance molecule depending on the receptor complexes with
which it interacts have been shown for netrin
(Hong et al., 1999) and
semaphorins (Liu and Strittmatter,
2001
; Castellani and Rougon,
2002
). The ephrin-B ligands present in the dorsolateral migratory
path of melanoblasts to the skin act as an attractant for these
EphB-expressing cells, but act as a repellent for an earlier migrating
population of EphB-expressing neural crest cells that take a ventral path
(Santiago and Erickson, 2002
).
In addition, the repellent activity of ephrin-A5 mediated by EphA7 can be
suppressed and even changed to adhesion by co-expression of splice variants of
EphA7 that lack the kinase domain
(Holmberg et al., 2000
).
During the postnatal period of branch extension, RGCs express EphB2, EphB3 and
EphB4 in a DV gradient, and EphB1 uniformly. EphB2 and EphB3 have been shown
to transduce an attractant signal upon binding ephrin-B1
(Hindges et al., 2002
),
whereas EphB1 may transduce a repellent signal upon binding ephrin-B1.
Consistent with this possibility is the finding that ephrin-B1 interaction
with EphB1 or EphB2 results in different signaling complexes
(Stein et al., 1998
),
suggesting different cellular responses.
Alternatively, activation of each of the EphB receptors may result in a
similar response, but the response switches from attraction to repulsion at a
threshold level of EphB/ephrin-B1 signaling. A single axon guidance molecule
can act in vitro as an attractant or a repellent depending on the
intracellular cyclic nucleotide levels in the axon
(Ming et al., 1997;
Song et al., 1997
). Also the
degree of response (e.g. repulsion/growth cone collapse) can be modulated by
other signaling pathways such as neurotrophin/trk pathways
(Tuttle and O'Leary, 1998
).
The threshold at which the switch from branch attraction to repulsion occurs
could be determined by the proportion of EphB receptors occupied by ephrin-B1,
by absolute levels of EphB signaling, or possibly by differences in ephrin-B1
concentration that may affect its oligomer state. Support for this latter
suggestion comes from the work of Stein et al.
(Stein et al., 1998
) who show
that the oligomer state of ephrin-B1 (dimers, tetramers, and higher order
multimers) results in the formation of markedly different EphB1 and EphB2
signaling complexes, as well as differences in receptor phosphorylation and
cell attachment. Consistent with this mechanism, the concentration of
ephrin-B1 dimers or tetramers in a substrate of extracellular matrix molecules
has been shown to be a critical factor in EphB1-induced, integrin-mediated
attachment of various cell lines (Huynh-Do
et al., 1999
). Within a critical concentration range, cells attach
to their substrate in an integrin dependent fashion at a much higher density;
if the concentration of ephrin-B1 is either above or below this optimal level,
cell attachment is decreased. The ephrin-B1 concentration at which maximal
attachment is observed is oligomer dependent, with tetramers being most
effective at a lower concentration than dimers
(Huynh-Do et al., 1999
).
It is intriguing to speculate that an analogous mechanism of EphB receptor signaling acts as a `ligand-density sensor' to control DV retinotectal mapping. In such a model, the DV gradient of EphB receptors in the retina and the LM gradient of ephrin-B1 in the tectum would set the critical range of ephrin-B1 concentration at the appropriate LM position for DV retinotopic mapping. An interstitial branch would sense ephrin-B1 concentration through EphB receptors, which would titrate signaling pathways that promote branch extension toward the optimal ephrin-B1 concentration, for example by controlling the density of receptors (e.g. integrins) on its surface that mediate attachment to ECM components and cells in the tectum and cytoskeletal changes required for branch extension. The level of ephrin-B1 at the TZ would be the optimal concentration for maximal attachment; therefore a branch located either medial or lateral to the TZ would encounter a gradient that increasingly favored attachment in the direction of the TZ. If in principle this model is correct, it may warrant a reconsideration of the mechanisms of axon guidance by graded molecules and the terminology used to describe axonal responses to them.
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ACKNOWLEDGMENTS |
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