The optic chiasm as a midline choice point
Scott E Williams, Carol A Mason1 and Eloı́sa Herrera
The mouse optic chiasm is a model for axon guidance at the
midline and for analyzing how binocular vision is patterned.
Recent work has identified several molecular players that
influence the binary decision that retinal ganglion cells make at
the optic chiasm, to either cross or avoid the midline. An ephrin-B
localized to the midline, together with an EphB receptor and a
zinc-finger transcription factor expressed exclusively in the
ventrotemporal retina where ipsilaterally projecting retinal
ganglion cells are located, comprise a molecular program for
the uncrossed pathway. In addition, the mechanisms for axon
divergence in the optic chiasm are discussed in the context
of other popular models for midline axon guidance.
Addresses
Department of Pathology, Center for Neurobiology and Behavior,
Columbia University, College of Physicians and Surgeons,
630 W. 168th Street, New York, NY 10032, USA
1
e-mail: cam4@columbia.edu
Current Opinion in Neurobiology 2004, 14:51–60
This review comes from a themed issue on
Development
Edited by Barry Dickson and Christopher A Walsh
0959-4388/$ – see front matter
ß 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.conb.2004.01.010
Abbreviations
CAM
cell adhesion molecule
comm
commissureless
CS-PGs chondroitin sulfate proteoglycans
DCC
deleted in colorectal cancer
HS-PGs heparan sulfate proteoglycans
LMC
lateral motor column
RGC
retinal ganglion cell
robo
roundabout
Sema
semaphorin
Shh
sonic hedgehog
VT
ventrotemporal
Introduction: the biological function of the
optic chiasm
Most animals with frontally located eyes have binocular
vision, such that the central (binocular) portion of the
visual field is perceived by both eyes and bilaterally
processed in higher visual centers. The major functional
consequence of this arrangement is depth perception,
which in most mammals is established at the level of the
optic chiasm. This X-shaped structure is actually a partial
decussation, where axons from regions of retina that
perceive each visual hemifield segregate together and
project to the same side of the brain. Thus, axons in
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the nasal retina project to contralateral visual centers,
whereas those in temporal retina project ipsilaterally. In
general, as the eyes are displaced more laterally, the
region of binocular overlap in the visual field decreases,
and the number of ipsilaterally projecting retinal ganglion cells (RGCs) in the temporal retina decreases
accordingly. In mice, which have poor binocular vision,
only around 3% of the RGCs in the adult project ipsilaterally, and these are confined to the ventrotemporal (VT)
crescent [1,2].
In the past few years, much progress has been made in
understanding the molecular cues, both guidance receptors and transcription factors, which contribute to retinal
axon divergence at the chiasm midline. In addition,
significant advances have been made toward identifying
molecules that shape the chiasm, by channeling RGC
axons along their proper course through the ventral diencephalon. Here, we review recent progress in identifying
the guidance systems that contribute to these developmental processes.
Midline structures: are they all the same?
Although much has been learned about the factors that
regulate crossing behavior at the midline through genetic
analysis of the ventral nerve cord in Drosophila, it has
become increasingly clear that these mechanisms are not
necessarily conserved in the vertebrate spinal cord, nor
indeed in other midline structures in higher vertebrates
[3–5]. Anatomically, the chiasm differs from both the
ventral nerve cord and spinal cord in that there is but a
single population of commissural neurons, the RGCs,
which diverge at the midline (Figure 1). In the retina,
the cell bodies of crossing and non-crossing axons are
spatially segregated. By contrast, commissural and ipsilaterally projecting spinal neurons have cell bodies
located at different positions along the dorsoventral axis,
and few reliable markers are available to distinguish
among them [3].
The optic chiasm also differs developmentally from the
floor plate, the ventral midline of the spinal cord. The
ventral diencephalon, on which the chiasm forms, arises
from the rostral end of the neural tube, and is patterned by
prechordal mesoderm rather than by the notochord. The
proper specification of ventral diencephalic cells requires
the combined activities of Sonic hedgehog (Shh) and
bone morphogenetic protein 7 (BMP7), whereas Shh
activity appears to be sufficient for patterning the ventral
spinal cord [6]. Perhaps as a consequence of these embryological differences, the guidance molecules expressed by
each of these midline structures vary (Figure 1). The
Current Opinion in Neurobiology 2004, 14:51–60
52 Development
Figure 1
(a)
Attractants
Netrin
Repellents
Slit1
Semaphorins
Slit2
Ephrin-Bs
Slit3
(1)
Ipsilateral axons
fra +, comm -, robo1/2/3 +
(2)
Commissural axons
fra +, comm +, robo1/2/3 +
(3)
(b)
Ipsilateral axons
???
Commissural axons
Expression:
Robo1, DCC, Npn-2, EphB1
(1)
Sensitivity:
pre-crossing (1): Netrin-1
post-crossing (2): Slits,
Sema3s, ephrin-Bs
(2)
fp
(c)
vt
Ipsilateral axons
Robo2 +
DCC +
EphB1 +
Npn / Plexins?
Contralateral axons
Robo2 +
DCC +
EphB1 Npn / Plexins?
Current Opinion in Neurobiology
Guidance molecules and midline models. Schematic diagrams comparing function of representative families of guidance cues in three different
midline models: (a) Drosophila ventral nerve cord (ventral view), (b) vertebrate ventral spinal cord (‘open-book’ preparation), and (c) mouse optic
chiasm (horizontal plane). Dotted line indicates the midline. (a) In order for commissural axons (light green) to cross the midline only once, they
must first be attracted to the midline (1), acquire a repulsiveness that drives them out (2), then be prevented from recrossing (3). Netrin (cyan) is
responsible for this initial attraction, while the latter two behaviors are mediated by the repulsive activity of slit (yellow). The differential expression
of the robo receptor and its regulation by comm determine whether an axon crosses the midline [12,14]. (b) The vertebrate spinal cord and
Drosophila ventral nerve cord are similarly arranged, in that both netrins and slits are secreted by the midline glia. Consistent with genetic evidence
Current Opinion in Neurobiology 2004, 14:51–60
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The optic chiasm as a midline choice point Williams, Mason and Herrera 53
vertebrate floor plate and midline of the fly ventral nerve
cord both express netrin, which attracts commissural
axons to the midline, and slits, which repel them once
fibers have crossed (or in the case of ipsilateral axons,
prevents them from approaching the midline in the first
place) [7]. Commissural axons express the attractive
netrin receptor deleted in colorectal cancer (DCC)/
frazzled (fra), and repulsive slit receptors of the roundabout (robo) family [8–11]. However, the similarities
between these two ventral midline choice points end
here. Drosophila robo and slit mutants exhibit severe
midline crossing defects [9,12], whereas this does not
appear to be the case in vertebrate Slit1; Slit2 mutants,
perhaps because of redundant function of either Slit3 or
Semaphorins (Sema) present at the spinal cord midline
[10,13]. Moreover, in Drosophila, pre-crossing commissural axons express low levels of robo because of the
commissureless (comm) gene product, which encodes a
transmembrane protein that binds robo and traffics it into
late endosomes, thus preventing premature repulsion by
midline slit [14,15]. In vertebrates, there appears to be
no comm homolog, so Robo levels must be regulated by
some other mechanism.
Interestingly, these same guidance systems are also utilized by RGC axons, but are applied to entirely different
ends (Figure 1). Retinal axons express DCC (as well as
the repulsive receptor UNC-5), but netrin is absent from
the chiasm region [16–18]. Instead, netrin is expressed at
the optic nerve head, and in netrin or DCC mutants RGC
axons fail to exit this region [19]. Additionally, RGCs
express Robo2, and both Slit1 and Slit2 are found in the
ventral diencephalon [20–23]. However, loss-of-function
analyses in mouse suggest that, rather than acting as
midline gatekeepers, Slits appear to establish a repulsion-free corridor that prevents axons from straying from
their path. In Slit1; Slit2 double mutants, RGC axons
often wander into regions where Slits are normally found,
forming an ectopic chiasm anterior to where axons normally cross [24].
Unfortunately, this ‘surround repulsion’ model is somewhat simplistic, and is confounded by other experimental
observations. For instance, in Slit1; Slit2 mutants, axons
do not stray in all areas where Slits are expressed, and no
guidance defects are detected in either of the single
mutants, even in regions where the expression of the
two Slit genes does not overlap. These findings could be
explained if other guidance systems function redundantly
in RGC repulsion. In support of this, Sema5A functions to
channel RGC axons within the optic nerve [25], and Shh
seems to restrict RGCs to the proper dorsoventral position
around the chiasm midline [26]. A second issue is that the
Robo2 loss-of-function phenotype appears to be stronger
than that of the Slit1; Slit2 double mutants. In the zebrafish robo2 mutant, astray, retinal axons make guidance
errors into anterior regions where slit2 and slit3 are
expressed [27], similar to those observed in Slit1; Slit2
mouse mutants. However, in severe astray alleles, retinal
axons also make ipsilateral and retino–retinal guidance
errors, and often wander throughout the diencephalon
[23]. As these behaviors are not observed in Slit1; Slit2
mutants, it is possible that there may be other ligands for
Robo2 other than Slits, or even modifiers of robo–slit
interactions [11]. Analysis of mouse Robo2 mutants should
clarify this issue.
The chiasm midline: cues for divergence
One of the first classes of molecules found to influence
the uncrossed population in vivo are the chondroitin
sulfate proteoglycans (CS-PGs), extracellular matrix proteins that are generally thought to be unfavorable for axon
growth [28,29]. In mouse, CS-PGs are expressed by the
early population of neurons in the ventral diencephalon,
and enzymatic removal of the chondroitin moieties has an
age-dependent effect on RGC guidance [30,31]. If treated at an early age (E13) when few retinal axons have
reached the chiasm, most axons stall or are misrouted, and
many fail to cross the midline. At later ages, however, the
same treatment reduces the ipsilateral projection, having
no apparent effect on crossing axons. As the permanent
uncrossed projection develops later than the crossed
projection, this could indicate that CS-PGs are important
for the guidance of early pioneers. However, the mechanism of CS-PG action remains unclear, as exogenous
application of CS-PGs can have a similar disruptive
influence on axon growth as enzymatic removal [32,33].
Moreover, CS-PGs are expressed in the retina as well
[31], so chondroitinase treatment may interfere with
interactions among retinal axons as well as with chiasm
(Figure 1 Legend Continued) in Drosophila, commissural axons in Netrin-1 mouse mutants often fail to reach the midline [68]. However, no
obvious defects are observed in Slit1; Slit2 mutants. This may be partially explained by the fact that Slit3 (dark green) is expressed in the floor plate
(fp) in addition to Slit1 (yellow) and Slit2 (light green). Moreover, Semaphorins (red) are also found there, and both Slit2 and Sema3B appear to function
redundantly in repelling post-crossing commissural axons [10]. In addition to their presence at the midline, ephrin-Bs (brown) are found in the
intermediate spinal cord, functioning to constrain axons (which express EphB1 only after crossing) to their proper longitudinal tracts [69,70]. Although
much is known about what regulates the process of crossing the midline, little is known about what prevents ipsilateral axons from crossing in the first
place. (c) Unlike either of the other model systems, netrin is not present at the chiasm midline. Instead, the decision of whether or not to cross
is decided in large part by the differential expression of EphB1 in ventrotemporal retina (vt), which confers responsiveness to the repellent
ephrin-B2 (brown) present on midline radial glia [40]. Slit1 (yellow) and Slit2 (light green) are also present in the ventral diencephalon, as is the
transmembrane Semaphorin5A (red), and each appears to play a general role in repelling all retinal axons from inappropriate regions [24,25]. Netrin-1
(cyan) is required to attract retinal axons to the optic nerve head [19], but it is unclear what drives them out, although there is evidence that
retinal axons alter their responsiveness to Netrin-1 at different points along their trajectory [18].
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Current Opinion in Neurobiology 2004, 14:51–60
54 Development
cells. Along these lines, it has recently been shown that
CS-PGs are also important for maintaining fiber order as
axons approach the chiasm [34], which suggests that
perhaps the observed guidance defects may be a secondary result of disorganization in the optic nerve. Future
work will be required to determine whether the mechanism of CS-PG action is dependent on the structural
moieties they contain and/or the context of other guidance cues with which they may interact, as has been
shown for the related heparan sulfate proteoglycans
(HS-PGs) [35–37].
One set of guidance molecules whose role in retinal axon
divergence appears to be clearer are the B-subclass Ephs
and their ephrin ligands. The pioneering work demonstrating a role for ephrin-Bs in directing the uncrossed
retinal projection was conducted in the Holt laboratory,
using Xenopus laevis as a model system [38]. The development of the Xenopus visual system differs from that of
the mouse in that the formation of the ipsilateral projection is dependent upon thyroid hormone and occurs at a
relatively late stage [39]. In the tadpole, the retinal projection is initially completely crossed but at metamorphosis,
an ipsilateral visual projection develops as a result of a new
wave of RGC neurogenesis in VT retina (Figure 2). The
Holt laboratory demonstrated that an ephrin-B is present
at the chiasm coincident with the formation of this
uncrossed component at metamorphosis, and premature
misexpression of ephrin-B2 in the ventral diencephalon
induced an ectopic ipsilateral projection [38].
A recent study in mouse has expanded upon these findings and demonstrated that ephrin-B2 is not only sufficient but also required for the formation of the ipsilateral
projection [40]. Using EphB4-Fc, a soluble receptor
ectodomain highly specific for ephrin-B2, the authors
were able to block the development of the ipsilateral
projection in semi-intact preparations of the visual system. The repulsive effect of ephrin-B2 appears to be
mediated by EphB1 (Figure 2), as this receptor’s expression is restricted to ipsilateral RGCs, and EphB1 mutants
exhibit a severely reduced ipsilateral projection.
EphB1, receptor for the uncrossed retinal
projection, and its regulation
Given the number of EphB receptors present in the retina
[40,41–44], it seems likely that many of them function
redundantly. Indeed, this is the case for intraretinal
guidance, as dorsal axons in EphB2; EphB3 double
mutants overshoot the optic nerve head, but neither
EphB2 nor EphB3 single mutants display a phenotype
[42]. However, EphB1 appears to act alone with respect to
midline guidance at the chiasm. Although its mRNA is
expressed highly in ventral retina, EphB2 mutants have
no apparent chiasm defects, and the phenotype of EphB1;
EphB2; EphB3 triple mutants is no more severe than that
of EphB1 single mutants [40]. This raises an interesting
Current Opinion in Neurobiology 2004, 14:51–60
question: how does ephrin-B2 signal preferentially
through EphB1?
The simplest explanation is that ephrin-B2 could have a
higher affinity for EphB1 than for either EphB2 or
EphB3, but this does not seem to be supported by
biochemical data, as the dissociation constant of binding
with ephrin-B2 is higher for EphB1 than for EphB2 or
EphB3 [45]. Alternatively, it is possible that the specificity of downstream effectors accounts for this selective
signaling through EphB1. For instance, the adaptor protein Grb7 appears to interact specifically with EphB1, but
not EphB3 [46], and could therefore transduce EphB1
signals more effectively. Finally, each EphB may be
subject to differential spatial control at the protein level.
EphB1 protein — alone or in combination with other
EphBs — may be expressed at much higher levels on
ipsilateral axons, thus endowing VT axons with a ‘suprathreshold’ level of signaling compared to dorsal or ventronasal axons. Unfortunately, although the expression
patterns of their mRNAs are known, the absence of good
antibodies has made it difficult to ascertain the localization of EphB proteins. Recent findings [47,48] have also
raised the possibility that guidance cues may be locally
synthesized in growth cones from anterogradely transported mRNAs. Thus, an intriguing possibility is that
EphB1 may be locally translated as axons approach the
midline, endowing VT growth cones with special
response properties distant from the retina.
A final lingering question is why a residual ipsilateral
projection remains in EphB1 mutants. One possibility is
that a second repulsive mechanism, not involving Bsubclass Ephs and ephrins, functions redundantly.
Another scenario, supported by findings from monocular
enucleation studies, is that adhesive interactions between
axons from opposing eyes play an auxiliary role in the
proper formation of the ipsilateral optic tract. If one eye is
removed at the beginning of retinal axon outgrowth (E12E13), before many retinal axons have reached the chiasm,
the uncrossed component is diminished [49,50]. Thus,
the presence of axons from the opposite eye assists in the
proper formation of the ipsilateral optic tract, as in this
paradigm, fiber–fiber interactions from opposing eyes are
essentially eliminated. Cell adhesion molecules (CAMs)
are attractive candidates to mediate these fiber–fiber
interactions, and in support of this, it has recently been
shown that mice lacking L1, an immunoglobulin superfamily CAM, have a diminished uncrossed projection at
E14 and E15 [51]. Perhaps, then, the proper formation of
the ipsilateral projection requires the coordinated efforts
of both repulsive and adhesive guidance cues.
Regulatory genes controlling the retinal axon
projection at the chiasm midline
As we have seen, recent studies have shed light on the
guidance molecules involved in the navigation of retinal
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The optic chiasm as a midline choice point Williams, Mason and Herrera 55
Figure 2
(a)
Pre-metamorphic Xenopus
(stage 40)
(b)
Early mouse
(E12.5)
D
D
T
N
T
N
V
V
Chiasm midline
Chiasm midline
Post-metamorphic Xenopus
(stage 60)
Late mouse
(E15.5)
D
T
D
T
N
V
EphB1
EphB2
EphB1 + EphB2
N
V
Ipsilateral RGC
Contralateral RGC
ephrin-B2
Current Opinion in Neurobiology
Comparative model of RGC guidance at the optic chiasm of Xenopus and mouse. Depicted on the left in each panel are the gradients of EphB
receptors in the ganglion cell layer relative to the positions of the cell bodies of RGCs that project ipsilaterally (red dots) and contralaterally
(light green dots). On the right are schematics of frontal views of the chiasm region illustrating the paths of ipsilateral (red) and contralateral (light green)
axons. (a) In pre-metamorphic Xenopus, all RGC axons project contralaterally, regardless of their origin in the retina, as no ephrin-B is present at
the chiasm. However, at this stage, a ventral gradient of EphB2 (yellow) is already present in the retina. EphB1, although expressed in the inner
nuclear layer, appears to be absent from the ganglion cell layer [44]. At metamorphosis, the ventral gradient of EphB2 broadens, and EphB1 (cyan)
expression is initiated exclusively in the ventrotemporal crescent. Simultaneously, ephrin-B (brown) is upregulated in the chiasm, serving to
selectively repel VT axons, which express the highest levels of EphB receptors (green). (b) In mouse, the early projection from the retina includes
both ipsilaterally and contralaterally projecting RGCs, and ephrin-B2 is expressed in the chiasm during this period. The pattern of receptors in the
mouse retina differs from Xenopus in that EphB2 is initially broadly expressed, whereas EphB1 is found in a population of RGCs in dorsocentral retina,
a region that gives rise to a transient population of uncrossed axons [50]. Later in development, EphB2 forms a ventral gradient, whereas EphB1
becomes confined to ventrotemporal retina, a pattern that resembles that of the post-metamorphic frog. As in Xenopus, VT axons express the
highest levels of EphB receptors, are most sensitive to ephrin-Bs, and are therefore repelled from the chiasm midline.
axons at the optic chiasm. Less is known, however, about
what controls the ability of individual retinal axons to
express these specific guidance molecules. The emerging
view in other models used to study neural identity and
axon trajectory is that each neuronal subtype possesses an
intrinsic capacity to detect its own unique path soon after
it becomes postmitotic [52]. This ability is conferred by
the expression of specific sets of transcription factors. The
LIM homeodomain proteins exercise this function by
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specifying different subsets of motoneurons in the spinal
cord, and directing their response to guidance cues in
muscle targets in the limb.
In the visual system, several regulatory genes expressed
in the developing retina have been reported to play a role
in retinal axon guidance at the optic chiasm. Mice lacking
Vax1, Vax2, Pax2 or Brn3b exhibit different defects in
retinal axon trajectory at the chiasm: first, a reduction in
Current Opinion in Neurobiology 2004, 14:51–60
56 Development
the ipsilateral projection, as in Vax2 KO mice [53,54],
second, a complete absence of contralateral projection, as
in Pax2 KO mice [55], third, a larger proportion of ipsilateral axons, as is the case for Brn3b KO mice [56], or
fourth, a failure of RGC axons to penetrate the brain,
instead terminating in large whorls at the base of the
hypothalamus, as occurs in Vax1 mutants [57]. Vax1,
Vax2 and Pax2 are expressed early in eye development,
before RGCs even differentiate. The possibility that these
early genes, Vax1, Vax2 and Pax2, play a direct role in axon
divergence at the chiasm cannot be ruled out. However,
from their spatio-temporal expression pattern, it seems
more likely that these genes are involved in morphogenesis
and regional specification of the eye and/or the chiasm [58],
therefore affecting events upstream of mechanisms
directly controlling axon divergence at the optic chiasm.
Brn3 genes, however, are different in this respect, as they
are expressed postmitotically. The number of axons
projecting ipsilaterally is increased in Brn3b/ mice
and this misrouting is partially prevented when Brn3c
is also removed [56]. It is possible that Brn3b/Brn3c may
control the production of ipsilaterally projecting RGCs,
but the fact that Brn3b and Brn3c are not expressed
exclusively in ipsilateral or contralateral RGCs, along
with the pathfinding defects at several decision points
along the visual pathway in Brn3b KO mice [59], argues
for a more general function of these proteins in axon
pathfinding, rather than directly controlling divergence at
the optic chiasm.
Very recently, Zic2, a zinc finger transcription factor
involved in early neural patterning, has been identified
Figure 3
(a)
(b)
Transcription factors
D
X
N
T
Zic2
Axon guidance factors
EphB1
V
Y
EphrinB2
Crossed axons
Uncrossed axons
(c)
E12
E14
E16
E18
Age
Crossed RGC axon passage
through optic chiasm
Uncrossed RGC axon passage
though optic chiasm
DC
VT
Zic2 expression in retina
EphB1 expression in retina
VT
DC
VT
Current Opinion in Neurobiology
Molecular players mediating the establishment of the uncrossed projection at the optic chiasm. (a) Expression pattern of the transcription factor
Zic2 in the RGCs of VT retina (red), which give rise to the uncrossed retinal projection. Different transcription factor(s) may be expressed only in
crossed RGCs (green) (X). It is also probable that a set of transcription factors are localized at the ventral diencephalon (Y), that are important for
specification of the cell/molecular cues of the chiasm midline and crucial to retinal axon divergence. (b) Summary diagram of the distribution of
EphB1 (red) and ephrin-B2 (brown) proteins in RGCs and the chiasmatic midline. Uncrossed RGC axons from VT retina turn away from
ephrin-B2-expressing midline glia near the midline, whereas crossing RGCs (green) traverse the ephrin-B2 zone. Whether the crossing axons actively
overcome the inhibitory cues or use an entirely different molecular mechanism to cross the midline, is not known. (c) The expression of the
transcription factor Zic2 and guidance receptor EphB1 closely overlap in time, during the outgrowth of the permanent uncrossed projection
through the optic chiasm, and space, as both Zic2 and EphB1 are expressed in postmitotic cells in VT retina.
Current Opinion in Neurobiology 2004, 14:51–60
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The optic chiasm as a midline choice point Williams, Mason and Herrera 57
as the first regulatory gene directly involved in axon
divergence at the optic chiasm [60]. Zic2 is expressed
postmitotically in VT retina only in RGCs that project
ipsilaterally, during their extension through the optic
chiasm. Zic2 seems to be crucial to directing the ipsilateral retinal projection, as mice expressing low levels of
this protein show a great reduction in the number of
uncrossed axons. Interestingly, Zic2 expression in VT
retina matches the spatiotemporal expression of EphB1
(Figure 3).
of Zic2 in vivo in the retina causes the same phenotype as
total removal of Zic2.
Interestingly, Zic2 expression in VT retina appears to be
conserved in both mammals and amphibians, precisely
mirroring the extent of binocularity. One lingering question, however, is whether or not this Zic2-based mechanism for axon divergence and binocular patterning is
conserved in humans as well.
Conclusions
A comparable situation to retinal axon-chiasm patterning
is the motor neuron projection from the lateral motor
column (LMC) to the dorsoventral axis of the developing
limb. Medial (LMCm) and lateral (LMCl) axons innervate
the dorsal and ventral limb mesenchyme, respectively.
Thus, although motoneuron axons do not project to a
midline structure as in the visual system, in both models
axons project in a binary manner. The topographic projections from the LMC are established, in part, through
LIM homeodomain protein control of EphA receptors
and ephrin-A ligands in motor neurons and limb mesenchymal cells [61]. In the visual system, the Zic2/EphB1/
ephrin-B2 pathway could function in a similar manner,
with Zic2 regulating EphB1 expression temporally, to
mediate recognition of the inhibitory ligand ephrin-B2 on
the radial glial cells at the midline (Figure 3). Despite the
close spatio-temporal relationship between Zic2 and
EphB1, whether Zic2 regulates EphB1 in VT retina or
is simply expressed in a parallel program, remains to be
tested. Although Zic2 and EphB1 are coexpressed in VT
retina, EphB1 — but not Zic2 — is present in dorsocentral
retina at the time when the early transient ipsilateral
projection forms, which suggests that EphB1 expression
at this time must be controlled by transcription factors
other than Zic2 (Figure 3).
Progress has been made on understanding how the
uncrossed pathways diverge from the crossed pathway,
in Xenopus and in the mouse optic chiasm. Zic2 and
EphB1 are both expressed concomitantly in the ventrotemporal retina, site of origin of the uncrossed RGCs. The
results from gain- and loss-of-function experiments
in vitro and in vivo strongly argue that this transcription
factor and guidance receptor comprise major determinants of the uncrossed pathway, with ephrin-B2 as a key
inhibitory ligand at the midline. Future experiments will
reveal whether or not there is a link between Zic2 and
EphB1, and how EphB1 receptors are handled at the
midline ([63,64,65]; Also see Holt and co-workers, this
issue). Despite all of the advances in unveiling mechanisms of the uncrossed pathway, little is understood about
mechanisms for crossing the midline. A crucial question is
whether crossing axons actively overcome inhibitory cues
or use a different type of mechanism — such as CAMmediated cell adhesion [49,51,66,67] — to traverse the
midline. Now that molecular factors and genes have been
ascribed to the uncrossed path, investigations of crossing
mechanisms should be facilitated. Soon, we should no
longer be puzzled by the proposition ‘to cross, or not to
cross’.
Update
Zic2 may also regulate genes other than EphB1. In
support of this view, Zic2kd/kd mice (genetically modified
mice with low levels of Zic2 expression) exhibit a phenotype that does not perfectly match that of mice lacking
EphB1. In both Zic2kd/kd and EphB1 mutants, there is a
strong reduction in the number of fibers that project
ipsilaterally, but in Zic2kd/kd mice, there is an additional
defasciculation phenotype in which retinal axons wander
from the distal optic nerve. This phenotype suggests that
Zic2 may control axon patterning in the retina by coordinating the regulation of multiple guidance genes. Zic2
may function, too, at the midline, as Zic2 is also expressed
in the chiasm [62], in a rather different pattern than the
other regulatory genes expressed in this region ([58], E
Herrera, CA Mason, unpublished). On the other hand,
alteration of Zic2 expression in retinal explants in vitro is
sufficient to change the behavior of RGC neurites in
response to cues provided by chiasmatic cells [60],
which argues that Zic2 acts primarily in the retina. It will
be important to test whether or not restricted perturbation
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Recently, a report has described the generation of a
neural-specific conditional knockout of the heparan sulfate (HS) polymerizing enzyme EXT1 (Nes-EXT1-null),
which is essential for HS synthesis [71]. These mutants
display multiple neural patterning defects in cell proliferation and commissure formation. In the optic chiasm of
Nes-EXT1-null mice, many retinal axons aberrantly project into the contralateral optic nerve. This phenotype
shows a genetic interaction with Slit2, which has been
shown to bind to HS-PGs [35,36]. As HS is expressed in
both the retina and along the pathway their axons take in
the chiasm [72], it remains to be seen how HS functions
mechanistically in optic chiasm development.
Acknowledgements
We wish to sincerely apologize to those colleagues whose work has
contributed greatly to our thinking, and was not cited due to space
limitations. We are grateful to the National Institutes of Health National
Eye Institute (NEI-EY12736, T32 EY13933 and NINDs P030532), and
the Human Frontiers Science Program for supporting the research conducted
in our laboratory.
Current Opinion in Neurobiology 2004, 14:51–60
58 Development
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