1 Department of Neurosciences, Lerner Research Institute, The Cleveland Clinic
Foundation, Cleveland, OH 44195, USA
2 Department of Cell Biology and Program in Neuroscience, Harvard Medical
School, Boston, MA 02115, USA
* Author for correspondence (e-mail: nakamom{at}ccf.org)
Accepted 11 September 2002
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SUMMARY |
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Key words: Eph receptors, Ephrins, Olivocerebellar projection, CNS
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INTRODUCTION |
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In contrast, neural areas that are involved in higher-order information
processing, such as multisensory integration and sensorimotor integration,
require more complex projection patterns. One of the common ways used to build
up the central neural maps is to arrange the incoming afferent fibers into
distinct domains in the target region, as seen in the mosaic of two
compartments (the patches and matrix) in the striatum, and in the modular
columnar organization in the cerebral cortex. However, little is known about
the molecular mechanisms that establish such discontinuous maps.
Interestingly, although it has long been thought that neuronal activity plays
the main role in development of the cortical columnar organization, recent
studies have suggested that the formation of ocular dominance column in the
visual cortex may also be dependent on molecular cues
(Crowley and Katz, 2000).
The neuronal projection from the inferior olive (IO) in the myelencephalon
to the cerebellum has been used as a favorite model system to study
domain-specific neural maps (the olivocerebellar projection)
(Altman and Bayer, 1997;
Brodal and Kawamura, 1980
).
Previous studies have revealed that the axons of IO neurons project to
Purkinje cells in the contralateral side of the cerebellum, with at least two
overlapping patterns. First, IO axons are organized along the rostrocaudal
axis of the cerebellum (Furber,
1983
; Furber and Watson,
1983
). In chicken, neurons in the rostromedial IO project to the
caudal cerebellum, while neurons in the caudolateral IO project to the rostral
cerebellum (Chédotal et al.,
1997
; Furber,
1983
). Second, discrete areas of the IO project to different
domains in the cerebellar cortex that are aligned mediolaterally (parasagittal
domains) (Buisseret-Delmas and Angaut,
1993
). Whereas this mediolateral arrangement is ordered with
respect to the local origin of IO neurons, neighboring domains of the IO do
not always project to adjacent cerebellar domains, thus forming a
discontinuous mapping with sharp boundaries. Although it has been predicted
that the matching of domain-specific labels between the incoming axons and
target cells would be the mechanism for the formation of the olivocerebellar
projection (Sotelo and Chédotal,
1997
; Wassef et al.,
1992
), the molecular nature of such labels remains to be
elucidated.
In recent years, Eph receptor tyrosine kinases and their membrane-bound
ligands, ephrins, have been implicated in neuronal network formation. Both the
Eph and ephrin families can be divided into two groups based on structural
features and binding affinities; i.e. EphA1-A9, EphB1-B6, ephrin-A1-A6
(glycosyl phosphatidylinositol (GPI)-anchored), and ephrinB1-B3
(transmembrane) (Eph Nomenclature
Committee, 1997; Menzel et
al., 2001
) [see Eph nomenclature web site
(http://cbweb.med.harvard.edu/eph-nomenclature/)
for update]. With a few exceptions, the groupings of the Eph receptors and
ephrins correspond to the receptor-ligand interaction (ephrin-A ligands
preferentially bind to EphA receptors, and ephrin-B ligands to EphB
receptors), although there are wide variations in affinity within each group
(Flanagan and Vanderhaeghen,
1998
). Their functions in neuronal projection are most typically
shown in development of the retinotectal (retinocollicular, in mammals)
system. In this system, EphA receptors and ephrin-A ligands are expressed in
complementary gradients along the anteroposterior axes of the projecting and
target fields, respectively (Cheng et al.,
1995
; Drescher et al.,
1995
). The receptor-ligand interaction mediates topographically
specific repulsive signals, and thus acts as a major determinant of this
continuously graded neuronal projection
(Feldheim et al., 2000
;
Monschau et al., 1997
;
Nakamoto et al., 1996
). While
many central neural maps show discontinuously segregated patterns, however,
whether or how the Eph-ephrin system acts as the molecular mechanism to
establish such domain-specific projections is still unknown. In the present
study, we performed expression analyses and functional characterization of Eph
receptors and ephrins in the chicken olivocerebellar system. Our results
suggest that in the olivocerebellar projection, EphA receptors and ephrin-A
ligands provide domain-specific positional information that guides axons to
their correct target domains.
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MATERIALS AND METHODS |
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Organotypic hindbrain cultures
Organotypic cultures of the chicken hindbrain were performed basically as
previously described (Chédotal et
al., 1997), except that we prepared explants at E9 or 10, by which
time the domain patterns in the cerebellum can be clearly identified.
Axon tracing experiments
A tiny crystal of DiI and DiA (Molecular Probes) was injected into
different domains of either the IO or the cerebellum in the organotypic
hindbrain culture on the first to forth day in vitro (corresponding to E9-12
in ovo). After incubation for 64-72 hours (anterograde tracing) or for 40-48
hours (retrograde tracing), projection patterns were analyzed under a
fluorescence microscope. Using the same explants, affinity probe in situ (with
EphA3-AP or ephrin-A2-AP) or RNA in situ hybridization was performed to
determine exact insertion sites of axon tracers and labeled neurons. In
retrograde labeling, 30 µm-frozen sections through the IO were cut, and
subjected to RNA in situ hybridization with EphA probes.
Retrovirus preparation and injection
Retrovirus stocks of RCAS-ephrin-A2, RCAS-EphA3C, and RCAS-AP were
prepared as described previously (Fekete
and Cepko, 1993a
; Nakamoto et
al., 1996
). RCAS-EphA3
C contains chicken EphA3 sequences
from nucleotide 173-1756 fused to short 5' coding region derived from
mouse EphA3 cDNA (nucleotide 89-235)
(Sajjadi et al., 1991
) in a
retroviral vector RCASBP(B) (Fekete and
Cepko, 1993b
). The virus solution with dye tracer was injected
into the rostral hindbrain region of E2 [Hamburger and Hamilton stage (HH)
10-12] chick embryos in windowed eggs. Virus-mediated expression was evaluated
by RNA in situ hybridization, affinity probe in situ, or alkaline phosphatase
(AP) staining. Only the explants in which the virus-derived gene expression
was restricted to the cerebellum were used for the axon tracing
experiments.
In vitro membrane substratum assay
The membrane substratum assay was performed as previously described
(Frisén et al., 1998).
Briefly, 293T cell membranes were prepared 68-72 hours after transfection with
pcDNAI-ephrin-A2 or pcDNAI control plasmid (Invitrogen) using the calcium
phosphate method, and were mixed with membranes from the anterior third of E8
tecta (2:1) to create a more permissive growth substratum. Medial or lateral
IO explants (175 µm in width, 500 µm in length) were prepared from E8
embryos, and were placed onto the homogeneous membrane carpets. After 60
hours, neurite outgrowth was scored on a graded 0-4 scale, in which 0 was no
or very sparse outgrowth from a living explant and 4 was very robust growth.
Statistical significance of the data was determined using Student's unpaired
t-test.
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RESULTS |
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Based on the expression patterns of EphA3, EphA5 and EphA6, at least four classes of IO areas could be distinguished (Fig. 1P,Q): (i) EphA3-positive, EphA5-positive, EphA6-positive (EphA3(+), EphA5(+), EphA6(+)) area in the medial part of the IO (Area M1); (ii) EphA3-negative, EphA5-strong positive, EphA6-positive (EphA3(-), EphA5(++), EphA6(+)) areas in the caudal part of the intermediate region (Area Ic) and in the most medial part of the IO (Area Mm); (iii) EphA3-negative, EphA5-positive, EphA6-negative (EphA3(-), EphA5(+), EphA6(-)) area in the rostral-intermediate region (Area Ir); and (iv) EphA3-negative, EphA5-negative, EphA6-negative (EphA3(-), EphA5(-), EphA6(-)) region in the lateral part of the IO (Area L).
Ligand-AP detects receptor activity that corresponds to the
area-specific mRNA expression patterns
Since the interaction between Eph receptors and ephrins is promiscuous,
with a single ligand binding multiple receptors with different affinities, we
were next interested in evaluating how the expression of EphA receptors was
reflected on the total receptor activity in each IO area. To test this, we
performed affinity probe in situ (Cheng et
al., 1995), in which the extracellular domain of ligands fused to
an AP-tag was used as a probe to detect the distribution of receptor activity
(Fig. 1G,K,O).
As expected, an ephrin-A2-AP probe detected a high receptor activity in Area M1 (EphA3(+), EphA5(+), EphA6(+)). Interestingly, Areas Ic and Mm (EphA3(-), EphA5(++), EphA6(+)) also showed strong receptor activity that was comparable to that in Area M1, presumably reflecting the strong expression of EphA5. Weak receptor activity was observed in Area Ir (EphA3(-), EphA5(+), EphA6(-)), whereas no activity could be seen in Area L (EphA3(-), EphA5(-), EphA6(-)). No binding activity was detectable for ephrin-B2-AP (K. N. and M. N., unpublished), suggesting that EphB receptors are not expressed at significant levels in the IO. The results showed that ligand-AP could indeed detect receptor activity in the IO that correlated with the expression patterns revealed by RNA in situ hybridization.
Domains of cerebellar Purkinje cells detected by expression and
activity of ephrin-A ligands
It has recently been shown immunohistochemically that the ephrin-A2 and
ephrin-A5 proteins are distributed in parasagittal domains of Purkinje cells
in the developing chicken cerebellum
(Karam et al., 2000). In the
present study, to directly relate their expression patterns to the
olivocerebellar map, we re-examined expression of ephrin-A2 and
ephrin-A5 in the chicken cerebellum by RNA in situ hybridization.
Based on the expression patterns, we named the parasagittal domains
alphabetically from the midline to the lateral sides (domains A to F;
Fig. 2H). At E10,
ephrin-A2 was most strongly expressed in domain C (lobules I-IXab),
and moderately in domains A (lobules I-VIII) and E (lobules II-VII)
(Fig. 2A,D). ephrin-A5
was expressed in the rostral part of domains A and C (lobules I-V), and in
domains D (lobules I-VIII) and F (lobules III-VIII)
(Fig. 2B,E). No
ephrin-A2 or ephrin-A5 expression was detected in domain B,
which consists of the rostral oval region and the caudal polygonal region that
are connected by a very narrow band. Consistent with the previous report
(Karam et al., 2000), the
parasagittal domains of ephrin-A2 and ephrin-A5 represent
their expression in the Purkinje cell layer
(Fig. 2G and data not
shown).
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Next, we tested how the ephrin-A ligand activity in the cerebellum is recognized by cognate EphA receptors by affinity probe in situ (Fig. 2C,F). Consistent with the RNA expression patterns, the rostral part of domain C (ephrin-A2(++), ephrin-A5(+)) showed strong ligand activity, followed by the rostral domains A (ephrin-A2(+), ephrin-A5(+)) and E (ephrin-A2(+), ephrin-A5(-)). Significant ligand activity was also observed in the rostral domain D (ephrin-A2(-), ephrin-A5(+)). Weak ligand activity was detected in the middle regions of domains A (ephrin-A2(+), ephrin-A5(-)) and C (ephrin-A2(++), ephrin-A5(-)) after longer incubation. No ligand activity was detected in domain B (ephrin-A2(-), ephrin-A5(-)).
It should be noted that since not all members of the Eph and ephrin families have been cloned in chicken, it is possible that other members are expressed in the IO or the cerebellum with distinct patterns. However, our results from affinity probe in situ indicate that such additional molecules, if any, are not expressed in a way that overwhelms the domain-pattern described above.
In the experiments described below, we focused on the parasagittal domains A-C in the cerebellum, where three regions could be distinguished based on the ephrin-A expression (Fig. 2H); (i) the rostral region (lobules I-V) of domains A and C, where both ephrin-A2 and ephrin-A5 are expressed (ephrin-A2(+), ephrin-A5(+) in domain A; ephrin-A2(++), ephrin-A5(+) in domain C); (ii) the middle region of domains A (lobules VI-VIII) and C (lobules VI-IXab), where only ephrin-A2 is expressed (ephrin-A2(+), ephrin-A5(-) in domain A; ephrin-A2(++), ephrin-A5(-) in domain C); and (iii) domain B, where no ephrin-A2 or ephrin-A5 expression is detected (ephrin-A2(-), ephrin-A5(-)).
Domains defined by Eph receptors and ephrins correspond to
olivocerebellar mapping domains
If the ephrin-A ligands in the cerebellum and EphA receptors in the IO act
as domain-specific labels, it is likely that their expression patterns
correlate with the olivocerebellar projection pattern. To study this, we
utilized an organotypic culture system of the whole hindbrain, which was
originally developed by Sotelo's group
(Chédotal et al., 1996)
(Fig. 3). It has previously
been shown that in this system, the topography of the olivocerebellar
projection is preserved (Chédotal et
al., 1997
). We have also confirmed that the domain patterns of
EphA receptors and ephrins are preserved in the explants (data not shown and
Figs
4,5,6).
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In the organotypic hindbrain culture, we first performed anterograde tracing experiments by inserting a tiny crystal of DiI into different areas of the IO. When Area L (EphA3(-), EphA5(-), EphA6(-)) was marked with DiI, two different patterns were observed depending on the exact dye-insertion sites. In most cases, dense axonal plexures were detected in the rostral part of domain C (ephrin-A2(++), ephrin-A5(+)) (Fig. 4A-C). In contrast, if the most caudal part of Area L was marked, labeled axons projected to the rostral part of domain A (ephrin-A2(+), ephrin-A5(+)) (Fig. 4D-F). Axons from Area Ml (EphA3(+), EphA5(+), EphA6(+)) projected to the caudal part of domain B (ephrin-A2(-), ephrin-A5(-)) (Fig. 4M-O). Interestingly, when Area Ic (EphA3(-), EphA5(++), EphA6(+)) was marked, labeled axons were found to project to the rostral oval region of domain B (ephrin-A2(-), ephrin-A5(-)) (Fig. 4G-I). Finally, axons from Area Ir (EphA3(-), EphA5(+), EphA6(-)) were found to project to the middle region of domain C (ephrin-A2(++), ephrin-A5(-)) (Fig. 4J-L). No clear projection pattern was reproducibly obtained by marking Area Mm.
To confirm the results of the anterograde labeling, we next performed retrograde axon tracing. In each explant, we inserted a crystal of two different dyes (DiI and DiA) into different cerebellar domains, so that the positional relationship between the labeled IO neurons would be clear.
The results were consistent with the anterograde labeling. When a dye crystal was inserted into the caudal part of domain B, labeled cells were detected in the medial part of the IO as a narrow band that extended rostrocaudally (Fig. 5B,D). To determine the identity of the labeled area, we cut sections through the IO of the labeled explant and performed RNA in situ hybridization with EphA probes. Both EphA3 and EphA5 RNAs were detected in the area, indicating that labeled cells were in Area Ml (Fig. 6A-D). In contrast, when the rostral part of domain C was marked, the labeled cells localized in the caudolateral part of the IO (Fig. 5C,D,K,L,O,P,S,T). In section RNA in situ hybridization, this part was identified to be in Area L, as neither EphA3 nor EphA5 was expressed there (Fig. 6M-P). The most caudal part of Area L was labeled when the dye was inserted into the rostral domain A (Fig. 5R,T, Fig. 6Q-T). Similarly, the middle region of domain C received axons from Area Ir (Fig. 5N,P, Fig. 6I-L). Finally, when the rostral oval region of domain B was marked, labeled cells were identified in Area Ic of the IO (Fig. 5F,H,J,L, Fig. 6E-H).
Taken together, the axon tracing experiments revealed that in the olivocerebellar projection, the projecting areas in the IO defined by EphA receptor expression correlate well with the target domains in the cerebellum defined by ephrin-A ligand expression (summarized in Fig. 7). First, the receptor and ligand expressions were in a reciprocal or inverse correlation; areas in the IO with high EphA receptor activity project to cerebellar domains with no or low ephrin-A ligand activity, and vice versa, suggesting that the receptor-ligand interaction mediates repulsive signals. Second, IO areas expressing different combinations of Eph receptors mapped to distinct cerebellar domains. It is also important to note that in all different retrograde labeling combinations, DiI- and DiA-labeled areas in the IO do not overlap, but are complementary, further supporting the idea that Eph receptors and ephrins act as domain-specific molecular labels.
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Retroviral misexpression of ephrin-A2 disrupts the olivocerebellar
projection pattern
If the Eph-ephrin interaction functions as the molecular mechanism that
guides IO axons to their correct target domains in the cerebellar cortex, it
would be expected that by modifying their expression patterns, the
olivocerebellar projection pattern would be altered correspondingly. To test
this possibility, we overexpressed ephrin-A2 in the developing
cerebellum, using a retrovirus-mediated gene expression system, and examined
the effects on the projection pattern.
The RCAS-ephrin-A2 retrovirus (Nakamoto
et al., 1996) was injected into the hindbrain region of the neural
tube at E2 in ovo (HH 10-12). We took care to localize the virus infection to
the rostral part of the hindbrain, from which the cerebellum develops.
Efficiency and patterns of ectopic expression were examined later by affinity
probe in situ using an EphA3-AP probe. As shown in
Fig. 8B, the vast majority of
cerebella infected with the virus showed massive ephrin-A2 expression
that covered most of the cerebellar surface.
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We next tested for effects of ectopic ephrin-A2 expression on the projection pattern, by retrograde labeling in explants prepared from virus-infected embryos. In each explant, we double-labeled the rostral domain C (ephrin-A2(++), ephrin-A5(+)) and the caudal domain B (ephrin-A2(-), ephrin-A5(-)). After infection with a negative control virus RCAS-AP (n=28), the labeling patterns in the IO were indistinguishable from those observed in wild type (uninfected) explants (Fig. 8G-I). In contrast, 85% (22/26) of the explants infected with the RCAS-ephrin-A2 virus showed aberrant projection patterns. In most of the aberrant cases (18/26), the caudal part of domain B was invaded by axons from more lateral parts of the IO, and only a small number of Area Ml neurons projected there (Fig. 8J-L). Only in some cases (4/26), significant numbers of Area Ml cells were labeled together with cells in the lateral parts (data not shown). These results indicate that when ephrin-A2 is overexpressed in the developing cerebellum, most Area Ml axons fail to project to the normal target domain (the caudal domain B), and lateral IO axons project to not only their normal target domain, but also the caudal domain B. Consistent with these results, when axons of Area Ml were anterogradely-labeled, the labeled axons stalled on the ventral border of the cerebellum (Fig. 8Q,R), suggesting that ectopic ephrin-A2 expression prevents Area Ml axons from entering the cerebellum.
Ephrin-A2 inhibits outgrowth of IO axons in vitro in a
region-specific manner
Our in vivo results suggest that ephrin-A2 prevents IO axons with high
receptor activity from invading cerebellar domains with high ligand activity,
through repulsion or growth inhibition of axons. To directly test this, we
used an in vitro membrane substratum assay
(Frisén et al., 1998;
Walter et al., 1987
). Since it
is not readily feasible to separate individual areas of the IO with reasonable
reproducibility, we instead compared axonal behavior between the medial (high
EphA receptor activity) and lateral (low to no EphA receptor activity) parts
of the IO. The medial and lateral parts of the IO were separately dissected
out, and were cultured on homogeneous membrane carpets prepared from
ephrin-A2- or mock-transfected 293T cells
(Fig. 9).
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Axon outgrowth from lateral IO explants showed no significant difference between on control [n=17, score 3.6±0.1(s.e.m.)] and ephrin-A2 [n=18, score 3.6±0.1(s.e.m.)] membranes. In contrast, axon outgrowth from the medial IO was significantly inhibited on the ephrin-A2 membrane [n=17, score 1.3±0.3(s.e.m.)], compared to the control membrane [n=13, score 2.6±0.3, P<0.001) These results indicate that ephrin-A2 inhibits growth of IO axons in a region-specific manner.
Retroviral overexpression of a truncated EphA3 receptor significantly
reduces the ephrin-A ligand activity in the cerebellum and causes aberrant
olivocerebellar projection
In the developing chicken cerebellar cortex, several EphA receptors (EphA3,
EphA4, EphA5, and EphA7) are expressed in distinct and overlapping domains of
Purkinje cells (Karam et al.,
2000; Lin and Cepko,
1998
) (K. N. and M. N., unpublished). The expression patterns
suggest that the receptor-ligand interaction could occur in developing
cerebellum, raising the possibility that Eph receptors in the cerebellum could
modulate the function of co-expressed ligands and thus affect the projection
pattern. To test this, we used a retrovirus vector encoding the EphA3
receptor, which has high affinity both to ephrin-A2 and ephrin-A5. To reduce
the risk that the misexpressed receptor itself transduces signals and exerts
secondary effects, we used a truncated form of the EphA3 receptor in which
most of the cytoplasmic domain is deleted (EphA3
C). Infection of the
RCAS-EphA3
C virus caused a widespread expression of the receptor, which
could be detected by affinity probe in situ with an ephrin-A2-AP probe
(Fig. 8E). We then tested how
the endogenous ephrin-A ligand activity was recognized after the retrovirus
infection, using an EphA3-AP probe. Surprisingly, no ligand activity could be
detected in cerebella infected with the EphA3
C virus
(Fig. 8C), suggesting that the
binding sites of endogenous ligands were occupied by the retrovirus-derived
receptor.
If the binding sites of the endogenous ephrin-A ligands are occupied by the
virus-derived EphA3C receptor, one can expect that medial IO axons with
high EphA receptors could now invade the high-ligand domains in the
cerebellum. We tested this in axon tracing experiments using hindbrain
explants infected with the RCAS-EphA3
C virus
(Fig. 8M-O). As predicted, when
the rostral part of domain C was marked with a tracer dye, retrogradely
labeled cells were observed in a very broad area that covered most of the IO
(22/25, 88%), indicating that this cerebellar domain was now invaded not only
by axons from Area L, but also those from the other (medial and intermediate)
areas of the IO. In anterograde tracing, labeled axons from Area Ml were found
to project to the most of the cerebellum, without showing any
domain-specificity (Fig. 8S,T).
These results suggest that medial IO axons do not recognize the repulsive cues
that are normally provided by ephrin-A ligands, and therefore can project to
high-ligand domains in the cerebellum. Interestingly, the caudal region of
domain B still receives axons mainly from Area Ml, indicating that some
mapping order is still preserved.
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DISCUSSION |
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Modulation of the ligand function and olivocerebellar map by
co-expressed receptors
In the developing chicken cerebellum, not only ephrin-A ligands, but also
EphA receptors are expressed in parasagittal domains of Purkinje cells. Since
Purkinje cells send their axons to the deep cerebellar nuclei or the
vestibular nuclei, one may expect that one of the major functions of EphA
receptors in Purkinje cells is to act as axon guidance receptors to establish
the olivonuclear and olivovestibular projections. While the present study does
not directly address this issue, consistent with the prediction,
region-specific expression of ephrin-A2 and ephrin-A5 were
detected in the deep cerebellar nuclei by RNA in situ hybridization (K. N. and
M. N., unpublished).
Results of the RCAS-EphA3C retrovirus experiments suggest the
possibility that EphA receptors in the cerebellum also play roles in the
olivocerebellar mapping. First, the endogenous ligand activity in the
cerebellum, which can normally be detected with EphA3-AP, becomes undetectable
after overexpression of the EphA3
C receptor. Second, in those
cerebella, medial IO axons with high receptor activity invade high ligand
domains, which is consistent with the loss of detectable ligand activity in
the field. Although the present study did not directly test the function of
endogenous Eph receptors, these findings suggest the possibility that
endogenous receptors in the target field could function as a part of the
positional information, by modulating the function of co-expressed ligands. In
this regard, it is intriguing that in the middle part of domain C of the
wild-type cerebellum, where several EphA receptors are co-expressed with
ephrin-A2, the ligand activity detected with EphA3-AP was much weaker
than expected from ephrin-A2 RNA expression
(Fig. 2D,E). Previous studies
have shown that in the retinotectal system, the function of Eph receptors in
the projecting field (retina) can be modulated by co-expressed ligands
(Hornberger et al., 1999
).
Together with the present study, these findings suggest that in the Eph-ephrin
system, the value of positional information in the projecting and target
fields is determined through receptor-ligand interaction.
Whereas it has recently been shown that an ephrin-A ligand can transduce
signals upon interaction with cognate EphA receptors
(Davy et al., 1999), it is
unlikely that overexpression of EphA3
C disrupted the projection pattern
by mediating attractive or repulsive signals through ephrin-A ligands
expressed on IO axons. First, in the wild-type embryo, expression and activity
of ephrin-A ligands are localized in the lateral IO (K. N. and M. N.,
unpublished), which maps to cerebellar domains with low or no EphA receptor
expression, and therefore does not fit the concept that the ligands on axons
mediate attractive signals. Second, because axons from the lateral IO show
massive innervation to the cerebella that overexpress EphA3
C, it is
also unlikely that the ligands on axons mediate repulsive signals.
Mechanisms by which the Eph-ephrin system guides IO axons to the
target domains
A variety of models could account for the mechanisms by which the
Eph-ephrin system establishes the olivocerebellar target selection pattern.
Some possible models are illustrated in
Fig. 10. Because it is
unlikely from our results that ephrin-A ligands act as axon attractants in
this map, only models that involve repulsive or inhibitory signals are
considered. In addition, when we discuss receptors and ligands in these
models, they represent `active' or `unoccupied' portions.
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In the first model (Fig.
10A), the mapping pattern is determined by the total amount of
receptor activity on the growing axons and of ligand activity in the target
domains, regardless of which or how many receptors contribute to the activity.
Projecting areas with high receptor activity would be repelled from domains
with high ligand activity, and would then map to no or low ligand domains. The
activity crucial for target selection could either be absolute or relative
amounts; the latter has been shown to be the case for Eph receptors in the
retinocollicular projection (Brown et al.,
2000). This model is compatible with our results that high areas
of the receptor activity in the IO map to low domains of the ligand activity
in the cerebellum, and vice versa.
In the second model (Fig.
10B), the projecting and target domains are characterized by
combinations of receptors and ligands, respectively. This model is based on
the assumption that in vivo, individual receptors could interact with a
limited and distinct set of ligands. Then, axons would be arranged into
distinct target domains through differential repulsion according to the
receptor sets they express. In this regard, it is noteworthy that although the
Eph-ephrin interaction shows promiscuity in in vitro binding assays, a recent
study suggested that EphA receptors may interact with more selective set of
ligands in vivo (Janis et al.,
1999). This model seems to match some of our axon tracing results
that cannot be clearly explained by the first model. For example, Areas M1 and
Ic project to different cerebellar domains, although both areas contain
comparable level of total receptor activity detected by ephrin-A2-AP.
Different combinations of receptors are expressed in these areas, and they may
be responsible for the differential projections.
The third model is a combination of the concepts of the first and second
models (Fig. 10C). In this
model, the axons project to the target domain where they receive the minimal
repulsive signal. The amount of repulsive signal may be determined by both
expression levels of receptors/ligands and in vivo affinity of individual
receptor-ligand pairs. As postulated in the mass action model
(Nakamoto et al., 1996), the
amount of repulsive signals could be most simply given as
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The second and third models involve combinations of receptors and ligands
acting as `molecular codes' in target selection. This `Eph code' model is also
attractive in that it might explain why so many Eph receptors and ephrins
exist in vertebrates. The large number of receptors and ligands, together with
their complex expression patterns, might be required to make combinational
molecular codes for the formation of the complex neuronal network.
Intriguingly, it has been shown that combinations of the Robo receptor protein
(the Robo code) expressed on axons are the major determinant of the position
of longitudinal axon tracts in Drosophila
(Rajagopalan et al., 2000;
Simpson et al., 2000
). Further
studies, using gain- and loss-of-function approaches for each receptor and
ligand, will be required to determine whether such `Eph codes' exist and
function, and which models can best explain the underlying mechanism for this
complex neuronal projection.
Possible cooperation with other molecular mechanisms
Considering the complexity of the olivocerebellar map, it seems likely that
in addition to the Eph-ephrin system, other molecular systems are also
involved in the selection of target domains. For example, the models in
Fig. 10 do not explain how the
incoming axons detect the correct direction to grow within the cerebellum. In
addition, model 1 has the problem of explaining why axons with no receptors
map to the high-ligand target domains. These issues could be explained, for
example, by considering other classes of molecules, such as domain-specific
attractants or adhesion molecules that interact with specific subsets of IO
axons, and other domain-specific repellent system. Interestingly, in the
cerebellum infected with the RCAS-EphA3C virus, the caudal domain B is
still innervated by Area M1 axons (Fig.
8M,O), which might be suggesting involvement of other molecular
systems. A number of molecules, including semaphorins
(Rabacchi et al., 1999
),
cadherins (Arndt et al., 1998
;
Hirano et al., 1999
), and an
immunoglobulin family member BEN/SC-1
(Chédotal et al., 1996
),
have been shown to be expressed in specific mapping domains of the
olivocerebellar system. While it is not clear yet whether these molecules
could be involved in the olivocerebellar mapping, their domain-specific
expression does suggest possible functions in patterning this complex
projection.
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ACKNOWLEDGMENTS |
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