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Review articles
Control of retinal growth and
axon divergence at the chiasm:
lessons from Xenopus
Fanny Mann and Christine E. Holt*
Summary
Metamorphosis in frogs is a critical developmental
process through which a tadpole changes into an adult
froglet. Metamorphic changes include external morphological transformations as well as important changes in
the wiring of sensory organs and central nervous system.
This review aims to provide an overview on the events
that occur in the visual system of metamorphosing
amphibians and to discuss recent studies that provide
new insight into the molecular mechanisms that control
changes in the retinal growth pattern as well as the
formation of new axonal pathways in the central nervous
system. BioEssays 23:319±326, 2001.
ß 2001 John Wiley & Sons, Inc.
Introduction
The visual system of amphibians undergoes profound
remodeling during metamorphosis. In tadpoles, the two eyes
are placed laterally with no binocular overlap. As the skull
changes shape during metamorphosis, laterally positioned
eyes migrate dorsofrontally, leading to a substantial degree of
binocular overlap.(1) During embryonic development, retinal
ganglion cell axons from the two eyes project to the contralateral side of the brain.(2) Concomitant with the metamorphic
change in eye position, however, a new pattern of retinal
projections begins to develop, connecting the retina to several
nuclei in the ipsilateral thalamus.(3±5) These uncrossed
retinothalamic projections subserve the new acquisition of
binocular vision appropriate to the predatory lifestyle of the
adult frogs (Fig. 1). Another marked change occurring in the
metamorphosing visual system is found in the pattern of cell
production in the retina. In amphibians, retinal growth results
from the proliferation of stem cells located at the periphery of
Department of Anatomy, University of Cambridge, Cambridge, UK.
*Correspondence to: Christine E. Holt, University of Cambridge,
Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK.
E-mail: [email protected]
Abbreviations: CMZ, ciliary marginal zone; D3, type III deiodinase;
IOPA, iopanoic acid; PTU, goitrogen propylthiouracil; RXR, retinoid X
receptor; T2, diiodothyronine; T3, triiodothyronine; TH, thyroid
hormone; TR, thyroid hormone receptor.
BioEssays 23:319±326, ß 2001 John Wiley & Sons, Inc.
the retina, in the ciliary marginal zone (CMZ).Whereas in
tadpoles the retina grows by proliferating symmetrically, a
sudden shift to an asymmetrical growth pattern is observed
when metamorphosis begins, with more cells being added at
the ventral and temporal margin than at the dorsal and nasal
ciliary margin.(6±8) At metamorphic climax, the ratio of dividing
cells in dorsal and ventral CMZ reaches 1:10.(8) This is
attributed to an increased number of progenitor cells in the
ventral compared to the dorsal margin.(9) It has been proposed
that the extensive proliferation in the ventral margin serves
the production of neurons in the portion of the retina that
views the new binocular visual field.(8) The asymmetric retinal
growth may also compensate for the changes in eye position
throughout metamorphosis.(1,8) Because the retinal position
relative to the body is changing, visuomotor readjustments
are required, but these are minimized by the fact that the
asymmetrical growth pattern allows the retina produced before
metamorphosis to view constant regions of the body-centered
visual space during ocular migration.
Thyroid hormone and the control of
retinal growth
Metamorphosis in amphibians is under the control of thyroid
hormone (TH), which acts directly on tissue to induce
metamorphic transformations.(10) In the amphibian Xenopus
laevis, the shift in histogenetic pattern from symmetric to
asymmetric growth in the developing retina occurs in late
tadpoles beginning at stage 54,(6±8) about 30 days postfertilization, when circulating levels of TH begin to rise.(11)
Asymmetric growth persists through metamorphic climax
(stages 58±64) and declines thereafter. There are several
lines of evidence that TH can trigger the change in cell
production by a direct action on the eye. Kaltenbach and
Hobbs first showed in Rana pipiens that implanting an
intraocular TH-releasing pellet in tadpoles causes an increase
in retinal cell proliferation.(12) Similarly, in Xenopus laevis, slow
release of TH into the eye of premetamorphic tadpoles
stimulates cell proliferation restricted to the treated eye.(9) In
the latter case, although cells in both dorsal and ventral retina
are capable of responding to exogenous TH, a greater
proliferation response occurs in the ventral retina, giving rise
prematurely to an asymmetric pattern of retinal proliferation.(9)
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periphery of the retina.(14,15) Taken together, the results
confirm that changing levels of endogenous thyroxine at the
onset of metamorphosis initiates the change in retinal growth
pattern by inducing an asymmetric ventral but not dorsal
increase in retinal cell production (Fig. 2A).
Figure 1. Eye position and pattern of retinal projections in
premetamorphic and postmetamorphic amphibians. A: In
tadpole, the two eyes are placed laterally and most or all of
the retinal ganglion cells (orange) project contralaterally to the
optic tectum. B: During metamorphosis, the two eyes migrate
dorsofrontally leading to the emergence of a binocular field,
and a novel pattern of retinal projections to the ipsilateral
thalamus develops. These ipsilateral projecting fibers arise
from newly generated retinal ganglion cells (blue) that sit in
the ventrotemporal portion of the retina viewing the binocular
visual space.
Reciprocally, the metamorphic increase in ventral retina
proliferation can be inhibited by treatment with the goitrogen
propylthiouracil (PTU), a chemical that blocks the production
of thyroxine in the thyroid gland by preventing the iodination of
tyrosine.(13) Xenopus tadpoles reared in the presence of PTU
stay permanently premetamorphic as they stop their morphological development at about stage 54. PTU-treated animals
remain viable, increase in size and their eyes continue to grow
by addition of neurons symmetrically at the dorsal and ventral
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Figure 2. The type III deiodinase (D3) enzyme controls the
asymmetric responsiveness of retinal progenitors to thyroid
hormone (TH) during metamorphosis. A: During normal
metamorphosis, as levels of circulating TH increase, retinal
growth changes from a symmetric to an asymmetric pattern of
proliferation, with more cells being generated in ventral retina
compared to dorsal retina. Increased proliferation in ventral
retina is directly stimulated by TH and produces a dorsal
displacement of the optic nerve head (onh) within the retina.
B: D3 enzyme, which degrades TH, is expressed in dorsal
(black) but not ventral (white) retinal progenitor cells. In
tadpoles treated with iopanoic acid (IOPA), an inhibitor of D3
activity, dorsal progenitor cells are able to respond to
exogenous TH by showing an increased rate of proliferation
as ventral progenitors normally do. C: In tadpoles carrying the
D3 transgene, the ventral retina appears resistant to the
proliferating changes that can be induced by exogenous TH.
Review articles
TH is known to act on target tissues through two thyroid
hormone receptors (TR alpha and TR beta) and their heteromeric partner, the retinoid X receptor (RXR).(16,17) TRs can
enter the cell nuclei and function as hormone-dependent
transcription factors to regulate gene expression.(17) In
Xenopus laevis, about 35 genes that are transcriptionally
upregulated during tadpole tail resorption initiated by TH have
been isolated and sequenced.(18,19) The known TH response
genes encode proteins that range from thyroid hormone
receptors themselves to proteolytic enzymes, transcription
factors and extracellular matrix components. Interestingly,
TH-inducible genes encoding the transcription factors xBTEB,
TH/bZip and FRA-2 are most actively upregulated in tissues
that grow at metamorphosis, including cartilage, muscles,
brain and spinal cord, than in structures that undergo cell death
and resorption.(20) This suggests a general role for these TH
response genes in controlling the programmed metamorphic
growth and cell proliferation throughout the entire body of the
tadpole, and possibly in the ventral retina. However, the
mechanism by which TH preferentially influences cell proliferation in the ventral retina versus dorsal retina has long
remained unsolved.
After rotating the eye of embryos between stages 26 and
35/36, Beach and Jacobson found that dorsal and ventral
retina are intrinsically specified to grow differentially at
metamorphosis and that these differences are determined
early during embryogenesis.(21) Theoretically, an asymmetrical distribution of thyroid hormone receptors in the ventral
and dorsal retina could explain the greater growth of the ventral
retina as the result of TH stimulation. This is unlikely to be the
case because a study of TR transcripts did not detect an
asymmetrical distribution in the retina.(22) The recent understanding of the mechanism controlling asymmetric growth in
the retina comes from the study of a TH-inducible gene
product, the type III deiodinase (D3), an enzyme that degrades
TH by converting the active triiodothyronine (T3) hormone into
an inactive diiodothyronine (T2) molecule.(23) First isolated
from the Xenopus tadpole tail,(18,23) D3 is also expressed in
other tissues of the metamorphosing animal, predominantly in
those structures that present a reduced response to TH,(24)
and is upregulated in tissues after completing metamorphic
changes.(20,25) Transgenic tadpoles that overexpress D3
usually undergo spontaneous metamorphosis, but often arrest
their development at the climax of metamorphosis and only
rarely complete the late morphologic transformations, such as
tail resorption, that require high levels of circulating TH.(26)
Moreover, transgenic tadpoles fail to undergo the gross
metamorphic changes that can be prematurely induced by
treatment with exogenous TH.(26) The results indicate a
function of D3 in protecting the tissues from the effect of TH,
and suggest a role for D3 in controlling the differential
proliferation response of dorsal and ventral retina to TH
stimulation. In a recent study, Marsh-Armstrong, Brown and
colleagues examined the pattern of D3 expression in the retina
during metamorphosis and found an asymmetric distribution,
with D3 mRNA expression predominant in the dorsal CMZ, the
area of least growth at metamorphosis.(27) D3 mRNA colocalizes with X-Notch-1 mRNA, a marker for retinal progenitor
cells.(28) This asymmetric pattern was established as early as
stage 35/36 well before the development of the thyroid gland,
suggesting that the differential D3 expression in the metamorphosing retina is not a secondary effect of TH induction.(27)
Treatment of premetamorphic tadpoles with iopanoic acid
(IOPA), an inhibitor of D3 activity,(29) caused exogenous TH to
stimulate proliferation in the dorsal retinal margin to the same
level as the proliferation induced by TH in the ventral retina
(Fig. 2B) .(27) In contrast, tadpoles expressing the D3 transgene are resistant to the increased cell proliferation normally
observed in the ventral retina as the result of exogenous TH
treatment (Fig. 2C) .(27) Moreover, as spontaneous metamorphosis proceeds in D3 transgenic animal, the ventral retina
shows reduced proliferation that parallels a decrease in the
number of X-Notch-1 mRNA-positive progenitors in the ventral
CMZ.(27) These results provide evidence for a novel mechanism regarding how different cells respond differentially to the
same level of a circulating hormone; asymmetric D3 expression in the eye controls the retinal growth pattern at metamorphosis by inactivating TH in the dorsal retina and thus
preventing dorsal CMZ progenitors from responding to TH.
Development of ipsilateral retinothalamic
projections
During embryonic development, virtually all retinal ganglion
cell axons from the two optic nerves cross each other at the
ventral midline of the diencephalon, forming the optic chiasm,
and project into the contralateral side of the brain. As metamorphosis proceeds, a novel pattern of retinal projections
develops that connect the retina to the ipsilateral thalamus.(3)
These projections first appear at the beginning of metamorphosis at stage 54 in Xenopus laevis,(5) about three weeks
after crossed retinal projections started to develop in the
embryo.(2) Cells giving rise to the ipsilateral retinothalamic
projections lie in the ventral and temporal periphery of the
adult retina,(4,30) in the regions produced by the differential
growth induced by TH.(5) Little is known, however, about the
regulatory events that control the delayed appearance of a
new retinal pathway in metamorphosing amphibians.
Because PTU-reared tadpoles fail to develop ipsilateral
retinothalamic projections,(15) it has been proposed that TH
controls the production of ipsilateral retinal connections.
Hoskins and Grobstein showed that injecting TH into the eye
of PTU-reared tadpoles induces the formation of projections to
the ipsilateral thalamus, without influencing the pattern of
projection from the uninjected eye.(14,15) The authors concluded that TH-induced changes restricted to the retina are
sufficient to cause the development of a new pattern of retinal
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projections, whereas additional changes along the central
retinal pathway seem unnecessary for normal axon guidance.
Hoskins proposed that TH triggers the production of a new
population of retinal ganglion cells, which differs from the other
populations by expressing a cytochemical tag that allows them
to follow a preexisting ``ipsilateral route blueprint''.(31) According to this view, the ipsilateral labeled path would already be
present in premetamorphic tadpoles but, in the absence of TH
induction, retinal ganglion cell axons would ignore it.(31)
This hypothesis does not fit, however, with the results from
other experiments reporting that tadpoles fail to develop
precociously uncrossed retinothalamic projections after intraocular implantation of a TH pellet(4) or when reared in the
presence of exogenous TH.(32) One possible explanation is
that the retina is not ``competent'' to respond to a TH signal and
to produce ipsilaterally projecting ganglion cells until tadpoles
reach metamorphosis. To address this issue, Nakagawa, Holt
and colleagues used a transplantation approach in which
marginal zone tissue harvested from the ventrotemporal retina
of metamorphosing frog, supposedly competent to the TH
signal, was transplanted into the eye of young tadpoles.(32)
Axons from the transplant projected to the tectum in the
contralateral side of the brain, either in the presence or the
absence of exogenous TH, indicating that retinal ganglion
cells, which normally form ipsilateral projections during
metamorphosis, do not project ipsilaterally when placed into
a younger environment.(32) It is unlikely that the host environment has respecified the transplant as it retained a topographic
specificity that coincides with its ventral origin even when
placed into dorsal host retina.(32) The heterochronic graft
experiments strongly suggest that molecular cues to guide
axons ipsilaterally are not expressed in the young embryo.
Finally, the finding that D3 transgenic Xenopus do form small
uncrossed projections at metamorphosis(27) provides additional support for the idea that changes other than those
induced by TH in the retina are needed for the formation of a
new uncrossed pathway.
The role of Eph family molecules in guiding
ipsilateral retinothalamic projections
The optic chiasm is the choice point where contralateral and
ipsilateral fibers diverge during development of visual projections. In rodents, in vitro experiments suggest that inhibitory
cues expressed in the developing optic chiasm differentially
affect crossed and uncrossed axons and may play an important role in selectively guiding uncrossed retinal axons away
from the ventral midline into the ipsilateral optic tract.(33±35)
The molecular nature of these guiding cues is unknown,
however. The search for candidate molecules recently led to
focus on the Eph family of molecules. The Eph receptors form
the largest family of receptor tyrosine kinases with the 14
known members falling into two subclasses: EphA and EphB.
With few exceptions, these interact respectively with A-type
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(GPI-anchored) and B-type (transmembrane) ephrin ligands.
The inhibitory interactions between Eph receptors and ephrins
have been found to play important roles in axon guidance and
formation of topographic projections.(36) There is also an
increasing body of evidence that suggests a role for Eph family
molecules in regulating axon growth at the midline of the
central nervous system. For example, genetic analysis of mice
lacking Eph receptors revealed defects in the formation of
commissures in the forebrain,(37,38) guidance of contralaterally
projecting tectal axons,(39) and decussation of the corticospinal tract.(40) Recent investigations that examined the molecular nature of axon guidance at the optic chiasm in rodents
have involved ephrin-A ligands. Using receptor affinity probe in
situ, Marcus, Mason and colleagues found that A-type ephrins
are expressed along the ventral aspect of the optic chiasm.(41)
In vitro, the addition of EphA-Fc chimeric protein, a competitive
inhibitor of Eph/ephrin interactions, reduced the inhibition of
axon growth observed when retinal neurites contact aggregates of ventral diencephalic cells.(41) EphA-Fc equally
influenced the behavior of ipsilaterally and contralaterally
projecting axons, however.(41) These results suggest that
ephrin-A ligands act to repel retinal axon growth from inappropriate regions of the ventral diencephalon during the formation
of the optic chiasm, but a role in regulating the divergence of
crossed and uncrossed axons seems unlikely.
Nakagawa et al. studied the expression pattern of Eph
receptors in the eye of metamorphosing Xenopus laevis using
probes consisting of the ephrin-binding domains fused to
alkaline phosphatase, a technique introduced by Cheng and
Flanagan.(42) As in other species previously examined,(43±48)
Eph receptors in Xenopus were found more highly concentrated in the ventral and temporal regions of the developing
retina than in the rest of the retina, with EphB receptors
distributed in the ventralmost region of the retina and EphA
receptors abundant in the temporal retina.(32) The ventral
expression of EphB receptors is established well before
metamorphosis begins and could be detected early during
embryonic development.(32) This striking coincidence between
the expression patterns of Eph tyrosine kinase receptors and
the region of the retina giving rise to uncrossed projections
suggests a function for the Eph family of molecules in guiding
ipsilaterally projecting axons in Xenopus laevis. According to
this idea, the delayed appearance of a new ipsilateral pathway
at metamorphosis would result from concomitant changes in
the expression pattern of Eph receptor ligands, presumably at
the chiasmatic region. Thus Nakagawa and collaborators
studied the expression of ephrin ligands in the tadpole and frog
optic chiasm. Whereas A-type ephrin ligands could not be
detected in the chiasm of tadpoles, ephrin-B ligand protein
expression was found to be upregulated in the optic chiasm
at stage 55 when metamorphosis begins, and expression
persists throughout metamorphosis(32) (Fig. 3A,B). To address
whether B-type ephrins can guide axons ipsilaterally, the
Review articles
placing a crystal of the lipophilic agent DiI into the ventral but
not the dorsal retina, thus indicating that ectopic ephrin-B2
specifically influenced the behavior of retinal cells carrying
EphB receptors.(32) Taken together, these results provided
direct evidence for a role of ephrin-Bs in directing the divergence of ipsilaterally and contralaterally projecting axons at
the optic chiasm of metamorphosing frogs (Fig. 4). In normal
animals, however, ipsilateral projections arise from regions of
the retina that express EphB receptors as well as EphA
Figure 3. Distribution of transmembrane ephrin-B ligands in
the developing optic chiasm revealed by in situ binding of
EphB4 ectodomain fused to alkaline phosphatase. A: Frontal
section through the chiasm of stage 54 Xenopus laevis
tadpole. B: Frontal section through the chiasm of stage 60
Xenopus laevis tadpole. C: Horizontal section through the
ventral diencephalon of an E16.5 mouse embryo. The
arrowheads indicate the position of the chiasm. On, optic
nerve. Scale bar, 100 mm (A,B) and 200 mm (C). Adapted from
Nakagawa et al. 2000.(32)
zebrafish ephrin-B2 gene was introduced into the chiasm of
stage 19±20 tadpoles, long before the onset of normal ephrinB ligand expression here.(32) Precocious expression of ephrinB2 induced an increased number of retinal projections to the
ipsilateral side of the brain at stage 41, when retinal projections
are normally almost completely crossed. Anterograde labeling
of precocious uncrossed projections could be performed by
Figure 4. Model for a role of EphB receptors and ephrin-B in
regulating retinal fibers crossing at the optic chiasm. A: In
premetamorphic tadpole, cells in a small region of the ventral
retina express EphB receptors (yellow), but corresponding
ephrin-B ligands are not detected along the retinal pathway in
the central nervous system. Retinal fibers arising from the
entire retina enter the brain, cross the optic chiasm and project to the contralateral side of the brain. B: The asymmetric
pattern of retinal growth induced by thyroid hormone at metamorphosis gives rise to an increased number of new retinal
ganglion cells in the ventral retina, most of them expressing
EphB receptors (yellow). Simultaneously, ephrin-B ligands
are upregulated in the chiasmatic region (red). Repulsive
interactions between Eph receptors and their ligands may
guide growing axons from ventral retina (green) away from
the optic chiasm into the ipsilateral optic tract.
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receptors. A role of ephrin-B ligands in guiding EphA receptor
expressing axons ipsilaterally is made possible by the fact
that one of the EphA receptors, EphA4, has been found to
interact significantly with some of the transmembrane ephrinB ligands.(49)
These results indicate a new role for ephrin-Bs at the
ventral midline of the frog diencephalon. Interestingly, ephrinB ligands have been found to be also expressed in the ventral
midline of the developing hindbrain(50) and spinal cord(51±55) in
rodents. In the spinal cord, cognate receptors are expressed
on ipsilaterally projecting axons and are upregulated on
commissural axons after crossing the midline.(55) Moreover,
in vitro experiments indicated that B-type ephrins are able to
induce the collapse of commissural growth cones.(55) These
observations suggest a conserved role of Eph/ephrin interactions in sorting out ipsilaterally and contralaterally projecting
axons at the ventral midline of the nervous system.
How can these results be reconciled with those of Hoskins
and Grobstein, which showed a prominent role of TH on the
retina itself for the induction of ipsilateral retinothalamic
projections?(14,15) One possibility is that, in Hoskins and
Grobstein's experiments, TH injected into the retina of PTUreared tadpole leaked out of the eye and also affected the
chiasmatic region, inducing metamorphic-like changes such
as ephrin-B upregulation. Although TH alone does not appear
to be able to stimulate ephrin-B protein expression in early
stage tadpoles, it does so in tadpoles near metamorphosis.(32)
Alternatively, in Hoskins and Grobstein's experiments, signals
to guide ipsilateral projections could have been upregulated in
a TH-independent manner in the chiasm of PTU-reared
tadpoles during the 3 weeks delay between the ocular injection
of TH and the analysis of the retinal projection patterns. The
neotenic Ambystoma mexicanum, which does not undergo
metamorphosis due to the absence of endogenous TH,
develops delayed ipsilateral retinal projections,(4) suggesting
that the molecules that normally guide axons ipsilaterally are
regulated in the absence of TH. This alone, however, can not
explain why the Hoskins and Grobstein's studies found that
projections from the uninjected eye were not affected and
continue to project contralaterally even in the possible
presence of cues to project ipsilaterally. This could be
accounted for by the mitogenic activity that TH has on the
retinal margins.(9,12,32) In the injected eye, TH induced the
proliferation and the production of Eph-expressing retinal
ganglion cells that are guided along the ipsilateral retinal
pathway, whereas in the non-injected eye of PTU-reared
animal, the reduced proliferation rate was not sufficient to give
rise to a significant number of ipsilaterally projecting ganglion
cells.
Future perspectives
Several points remain to be clarified to fully understand how
ipsilateral retinothalamic projections form at metamorphosis.
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First, it is known that ipsilaterally projecting cells represent only
a subpopulation (about 10%) of the ganglion cells generated
during TH-induced growth of the retina.(30) A higher proportion
of cells in the ventrotemporal region of the retina appear to
express Eph receptors, however. Why do only few of them
respond to the ephrin-B signal at the chiasmatic region?
Co-expression of ephrin ligands and Eph receptors on retinal
ganglion cells has been found to regulate axon sensitivity to
ephrin ligands in vitro.(56) Such a mechanism could explain the
differential response of ganglion cells to ephrin-Bs at the optic
chiasm. It is also possible that different levels of Eph receptor
expression or a specific combination of Eph receptors on
individual retinal ganglion cells could modulate how axons
respond to ephrin-B signal.
Second, in the experiments by Nakagawa, Holt and
colleagues, ipsilateral retinal projections, prematurely induced
by ephrin-B2 expression in the chiasm of tadpoles, target the
optic tectum whereas ipsilaterally projections formed normally
during spontaneous metamorphosis terminate in the thalamus. This difference may be due to the absence of a labeled
pathway to the thalamus in early embryos, as crossed retinothalamic projections normally develop later around stage 49.(5)
The signals that selectively guide retinal ganglion cell axons
towards the tectum or the thalamus are so far unknown,
however.
Finally, a long-standing question remaining to be answered
is how retinal axon divergence at the chiasm is regulated in
mammals. Do ephrin-Bs play a role during development of the
mammalian visual pathway similar to that found in metamorphosing Xenopus laevis? Several lines of evidences argue in
favor of this idea. In the mouse, for example, ipsilaterally
projecting axons arise from the ventrotemporal portion of the
retina,(57,58) the region of highest expression of both A-type
and B-type Eph receptors.(59) Furthermore, ephrin-B expression has been recently reported in the optic chiasm of
embryonic mice at the time of major retinal axon outgrowth(32,41) (Fig. 3C). Although there is no direct evidence
implicating Eph family of molecules in directing crossed and
uncrossed projections in rodents, it is tempting to speculate
that ephrin-Bs are at play. It will certainly be interesting to
investigate whether aberrant crossing occurs at the chiasm of
EphB/ephrin-B mutant mice.
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