MRC Centre for Developmental Neurobiology, King's College London, New Hunt's House, Guy's Hospital Campus, London SE1 1UL, UK
* Author for correspondence (e-mail: uwe.drescher{at}kcl.ac.uk)
Accepted 14 July 2003
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SUMMARY |
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We provide evidence that the Slit family of axon guidance molecules and their Robo receptors contribute to the topographic targeting of basal vomeronasal axons. Robo receptor expression is confined largely to basal VNO axons, while Slits are differentially expressed in the AOB with a higher concentration in the anterior part, which basal axons do not invade.
Immunohistochemistry using a Robo-specific antibody reveals a zone-specific targeting of VNO axons in the AOB well before cell bodies of these neurones in the VNO acquire their final zonal position. In vitro assays show that Slit1-Slit3 chemorepel VNO axons, suggesting that basal axons are guided to the posterior AOB due to chemorepulsive activity of Slits in the anterior AOB.
These data in combination with recently obtained other data suggest a model for the topographic targeting in the vomeronasal projection where ephrin-As and neuropilins guide apical VNO axons, while Robo/Slit interactions are important components in the targeting of basal VNO axons.
Key words: Robo, Slit, Axon guidance, VNO, Vomeronasal, AOB, Chemorepulsion, Topographic, Olfactory, Pheromone, Mouse
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Introduction |
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The axonal projection between the VNO and the AOB is zonally organised.
Sensory neurones in the apical zone of the VNO project their axons to the
anterior half of the AOB, while the posterior AOB is innervated by axons of
the basal VNO (see Fig. 7).
This topography was revealed in expression studies on several molecular
markers, including vomeronasal receptors, the putative pheromone receptors and
G-proteins (Dulac and Axel,
1995
; Herrada and Dulac,
1997
; Jia and Halpern,
1996
; Matsunami and Buck,
1997
; Ryba and Tirindelli,
1997
). Co-expression of the marker proteins tau-lacZ and
tau-GFP with selected vomeronasal receptors led, for the first time, to the
visualisation of a vomeronasal map
(Belluscio et al., 1999
;
Del Punta et al., 2002b
;
Rodriguez et al., 1999
),
demonstrating that axons which express the same vomeronasal receptor terminate
within the AOB in a specific pattern of spatially conserved groups of
glomeruli. Genetic ablation of vomeronasal receptors disrupts this glomerular
convergence, and so vomeronasal receptors might act as bifunctional molecules,
working both to transduce signals from pheromones and to guide VNO axons. In
such mutants, however, vomeronasal axons still project mostly to the
appropriate half in the AOB suggesting that other guidance cues must be
involved in establishing the integrity of the zone-to-zone projection
(Belluscio et al., 1999
;
Rodriguez et al., 1999
).
Indeed, two families of axonal guidance molecules, the ephrins and the
semaphorins, were recently reported to be involved in the zonal projection of
apical vomeronasal axons (Cloutier et al.,
2000
; Knöll et al.,
2001
; Walz et al.,
2002
).
|
This raises the question of which guidance cues are involved in guiding
basal axons to the posterior AOB, as the zonal projection of basal axons (e.g.
in neuropilin 2 mutant mice) is normal
(Cloutier et al., 2000;
Walz et al., 2002
).
We present data that indicate that the Slit and Robo axon guidance
molecules are candidates to fulfil this function. Robo proteins were first
shown to regulate axon crossing at the Drosophila midline because of
repulsive interactions with Slits secreted from midline cells
(Brose and Tessier-Lavigne,
2000; Guthrie,
2001
; Nguyen-Ba-Charvet and
Chedotal, 2002
; Schimmelpfeng
et al., 2001
; Tear,
1999
). Although ipsilaterally projecting axons express Robo
constitutively and do not cross the midline, commissural axons are initially
Robo negative, which allows them to cross. Robo expression is only evident on
axons once they have crossed the midline and so prevents them from
re-crossing. In Drosophila, the differential expression of Robo is
controlled by commissureless (Georgiou and
Tear, 2002
; Keleman et al.,
2002
; Kidd et al.,
1998a
; Kidd et al.,
1998b
; Myat et al.,
2002
; Tear et al.,
1996
).
In vertebrates, three Robo proteins (Robo1, Robo2 and Rig1) and three Slits
(Slit1-Slit3) have been identified (Brose
et al., 1999; Fricke et al.,
2001
; Holmes et al.,
1998
; Itoh et al.,
1998
; Li et al.,
1999
; Marillat et al.,
2002
; Sundaresan et al.,
1998
; Yuan et al.,
1999a
; Yuan et al.,
1999b
). Besides an involvement in midline crossing
(Bagri et al., 2002
;
Hutson and Chien, 2002
;
Plump et al., 2002
), Robo and
Slit proteins have been implicated in the pathfinding - but not topographic
targeting - of olfactory, retinal, hippocampal and cranial motor axons by
exerting a repulsive action (Brose et al.,
1999
; Erskine et al.,
2000
; Hutson and Chien,
2002
; Li et al.,
1999
; Nguyen Ba-Charvet et
al., 1999
; Niclou et al.,
2000
; Ringstedt et al.,
2000
).
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Materials and methods |
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In situ hybridisation
All in situ hybridisation experiments were performed as previously
described (Knöll et al.,
2001). The cRNAs probes to rat Robo1, Robo2 and rat
Slit1-Slit3 (Brose et
al., 1999
) were kindly provided by Dr Marc Tessier-Lavigne
(Stanford University, Stanford, CA).
Expression analysis of Slits
For detection of Slits on sections of the accessory olfactory bulb, we used
the protocol of Conover et al. (Conover et
al., 2000). In brief, P1 embryos were fixed in ice-cold 4%
paraformaldehyde (PFA) for 3-5 hours, embedded in 5% low-melting agarose and
vibratome-sectioned at 100-150 µm. After blocking with 10% newborn goat
serum (NGS)/2% bovine serum albumin (BSA)/phosphate-buffered saline (PBS) for
1hour at 4°C, sections were incubated overnight at 4°C with 1 µg/ml
of the indicated Fc fusion proteins in 0.5x block. After extensive
washing, the sections were heat-treated for 30 minutes at 70°C and blocked
for 1 hour in 0.5x block with 0.1% Triton X-100. Subsequently, sections
were incubated with alkaline phosphatase conjugated anti-Fc antibody (1:1000;
Promega) overnight at 4°C. After extensive washing with PBS/0.1% Triton
X-100, binding of fusion proteins was revealed using BCIP/NBT as a substrate.
Sections were counterstained with DAPI and embedded in Moviol.
Immunohistochemistry
Animals at the stages indicated were fixed for 7 days in 4%
formaldehyde/PBS and then processed for immunohistochemistry using a series of
IMS solutions of ascending concentration (30%, 70%, 90% to absolute). Tissue
was then processed through a series of IMS/xylene (50:50), two solutions of
xylene and two solutions of paraffin wax before being embedded in wax.
Paraffin sections were cut (5 µm) and dewaxed overnight at 60°C. After
two changes in xylene (5 minutes each), three changes in absolute IMS (2
minutes each), 70% IMS (2 minutes), sections were blocked for 10 minutes in 3%
H2O2 followed by excess washes in tap water. Sections
were pressure-cooked in 10 mM citric acid/H2O (pH 6) for 5 minutes
to retrieve antigen sites. Slides were then washed in tap water and
equilibrated in 0.05 M TBS followed by blocking in 2% BSA/TBS prior to
overnight incubation with a rabbit polyclonal serum against Robo, S3, diluted
1:2000 at 37°C. This antiserum was raised against a 14 amino acid peptide
stretch in the Ig domain 1 of mouse Robo1, which is identical to that in mouse
Robo2 with the exception of one amino acid. Specific binding of the antiserum
can be blocked by preincubation with either the immunogen, Robo1-Fc or
Robo2-Fc indicating that the serum binds both Robo1 and Robo2 (L. Bannister
and V.S., unpublished). Neighbouring sections - 5 µm apart - were stained
with a rabbit anti ß-tubulin antibody (Cambridge Bioscience, Cambridge,
UK). After washing, the primary antibody was detected using the
tyramide-StreptABC/HRP method (Dako, Glostrup, Denmark) and DAB as a
substrate. Sections were counterstained in Harris' Haematoxylin and dehydrated
before mounting in DPX mounting medium (RA Lamb, East Sussex, UK).
Collagen assays
E14.5-E15.5 VNO explants were prepared as described by Cloutier et al.
(Cloutier et al., 2002) and co-cultured with COS cell-aggregates secreting
Slit1, Slit2 or Slit3 as previously described
(Patel et al., 2001).
Lipofection was used to transfect 6 cm2 dishes with a total amount
of 0.75 µg Slit2 DNA or 1.5 µg DNA for Slit1 and Slit3. Cultures were
incubated for 2-3 days at 37°C/5% CO2 in neurobasal medium
using B27 additives (both Invitrogen, Carlsbad, CA) and recorded with
phase-contrast microscopy using Lucia G software (Laboratory Imaging). Heparin
was not added to the culture medium. Quantification of neurite outgrowth was
carried out as described by Chen et al.
(Chen et al., 2000
).
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Results |
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The VNO also expressed Slit1 and Slit3, with Slit1 being expressed at higher levels (Fig. 1D,F). Both Slit1 and Slit3 RNAs were found in a patchy pattern, similar to that of Robos (Fig. 1G). Slit2 was not expressed in the sensory epithelium, but specifically labelled a region close to the vomeronasal vein (Fig. 1E). Expression of Slit2 in this region was undetectable by P21 (Fig. 4E).
Asymmetric distribution of Slits in the mouse AOB
VNO axons entering the AOB at its medial margin (see
Fig. 7) form a distinct nerve
layer (n) in the dorsal part of the AOB
(Fig. 2A). Mitral- and tufted
(m/t) cells located in the ventral part of the AOB elaborate dendrites to
establish synapses with VNO axons in glomeruli and send axons towards other
regions of the brain such as the amygdala
(Dulac, 2000;
Keverne, 2002
). The expression
of RNA coding for Slit1 and Slit3 in the AOB is confined to clusters of cells
at the anterior border of the AOB (arrows,
Fig. 2D,F). This asymmetric
distribution of Slit is preserved between E15.5 and P5, but is no longer
apparent at P21 (data not shown). Slit2 is not expressed in this anterior cell
group but is detected in cells scattered in the mitral/tufted (m/t) cell layer
throughout the AOB, with individual cells expressing different RNA levels
(Fig. 2E).
In addition, Robo2 RNA (Fig. 2B), and to a lesser extent Robo1 RNA (Fig. 2A), are uniformly expressed on m/t cells of the AOB. Robo2 is also strongly expressed on mitral and tufted cells of the main olfactory bulb (arrows, Fig. 2B). However, by using a Robo-specific antibody we observed staining of only the posterior part of the nerve layer at E15.5 (Fig. 5F), at P1, P4 (data not shown), and at P21 (Fig. 5G,H).
Given that Slits are secreted proteins, we reasoned that in particular the
high punctuate expression of Slits 1 and 3 at the anterior border of the AOB
might result in a differential (or graded) expression of Slit proteins within
the AOB. For this purpose we have generated and used a set of Robo-Fc fusion
proteins to specifically detect Slit expression. Instead of using a Robo1-Fc
fusion protein containing all five Ig domains, we have used sub-domains from
the extracellular part of Robo having different Slit binding capabilities
(Fig. 3A,B). Based on
structure-function analysis, only Ig domains 1 and 2, but not 3-5, are
involved in Slit binding (V.S., unpublished). Hence, a Fc fusion protein
containing Ig domains 1 and 2 (3, 4, 5 Robo1-Fc) was used to localise
Slit expression (Fig. 3C-E),
while (
1, 2) Robo2-Fc lacking Ig domains 1 and 2 served as a negative
control (Fig. 3F).
These analyses revealed a smooth gradient of Slit protein at the most medial part of the AOB being stronger in the anterior part and fading away towards the posterior part (Fig. 3C). More lateral to this position (Fig. 3D), Slit protein is found in the anterior but not the posterior AOB. In the central part of the AOB (Fig. 3E), there is a stronger Slit expression in the anterior part of the mitral/tufted cell layer than in its posterior part, which is evident in approximately half of the sections taken (n>10 mice, 2-3 sections/bulbus), while in the remaining sections an even expression of Slit was found. In summary, the asymmetric expression of Slit protein is present at the time of ingrowth of vomeronasal axons into the AOB and may function to steer ingrowing basal axons away from the topographically inappropriate anterior AOB (see below).
Thus, it appears that Slits are differentially expressed in the AOB, while Robo protein - similar to the situation in the VNO (see Fig. 5B) - might be transported into the axons leaving the cell body Robo protein negative. Consistent with this, we found Robo protein on axons of the lateral olfactory tract connecting the MOB with other olfactory processing centres of the brain (see below, Fig. 5G arrows).
Segregation of Robo- and Slit-expressing cells in the adult mouse
VNO
At birth, presumptive apical and basal cell bodies in the VNO sensory
epithelium are intermingled as revealed by a number of markers including
specific G subunits (Berghard and
Buck, 1996
; Berghard et al.,
1996
; Jia and Halpern,
1996
). This intermingling is resolved during the first postnatal
weeks, when apical and basal cell bodies become confined to their respective
zones in the VNO. To investigate a possible change in Robo and Slit RNA
expression that might coincide with this process, in situ hybridisation
experiments were performed at P21 (Fig.
4). Compared with earlier stages
(Fig. 1), Robo2 expression is
now confined to the basal zone of the VNO
(Fig. 4B), but its expression
level varies between individual cells (Fig.
4C). By contrast, Robo1 expression is downregulated as early as P5
(data not shown) and hardly detectable at P21
(Fig. 4A). The expression of
Slits is more restricted compared to earlier stages, with Slit3 mostly
confined to the apical VNO and Slit1 having a higher expression in the apical
than the basal VNO (Fig.
4D,F).
Expression of Robo protein on basal VNO axons
Given its function as an axon guidance receptor, we wished to determine the
localisation of Robo protein within the vomeronasal projection by
immunohistochemistry using an antibody, which detects both Robo1 and Robo2
(Hivert et al., 2002) (see
Materials and methods for details) on tissue sections.
At E15.5, VNO axons emanating from the VNO sensory epithelium and navigating dorsally along the nasal septum towards the AOB are strongly Robo positive (arrows, Fig. 5A,B). In the AOB, we observed stronger staining for Robo in the posterior than in the anterior part of the nerve layer (arrows, Fig. 5F). This is particularly interesting as this zone-specific localisation occurs prior to the segregation of the corresponding cell bodies in the VNO. In addition, we observed a differential expression of Robo across the mediolateral (ML) axis of the AOB at E15.5 (Fig. 5D) and P1 (data not shown). Here, the lateral aspect of the nerve layer shows higher Robo staining than its medial side, from which VNO axons enter the AOB (arrows, Fig. 5D; Fig. 7). However, this pattern is transient and by P21 there is no longer a differential distribution of Robo along the ML axis (data not shown).
Inspection of sagittal sections of the AOB at P21 shows that only the posterior half of the AOB nerve layer - which is formed by basal axons - is Robo positive (Fig. 5G,H; Fig. 7). These data are consistent with the expression of Robo2 RNA in the basal zone of the VNO and the downregulation of Robo1 RNA at P21 (Fig. 4).
Finally, axons of the main olfactory bulb, which terminate in glomeruli of the ventral part of the main olfactory bulb, are also Robo positive (Fig. 5G), suggesting that Robos and Slits may also be involved in the topographic mapping in the main olfactory system.
Robo-Slit interactions repel vomeronasal axons
The expression profile presented in this study suggests that basal VNO
axons are guided to the posterior AOB based on a chemorepulsive effect exerted
by a differential Slit expression in the AOB. To investigate this idea more
directly, we analysed the sensitivity of VNO axons to all three Slits in a
collagen gel co-culture system. Here, E14.5 VNO explants were arranged in
proximity to COS cell aggregates secreting Slit1, Slit2 or Slit 3 or
mock-transfected control cells (Fig.
6, see Materials and methods). Two to 3 days later, the response
of VNO axons to the cell clusters was monitored. In control experiments using
mock-transfected COS cell aggregates (Fig.
6A, n=33), we observed an almost radially symmetrical
outgrowth of VNO axons indicating an insignificant level of secretion of
repulsive molecules by COS cells per se
(Fig. 6A). By contrast, VNO
axons were robustly repelled from Slit-secreting COS cell aggregates, with
virtually no axons growing towards the Slit-producing cells
(Fig. 6B-D). This
chemorepulsion was observed even without the addition to the growth medium of
heparin, which is known to enhance Slit release into the medium
(Brose et al., 1999;
Nguyen Ba-Charvet et al.,
1999
; Hu,
2001
).
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Discussion |
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Robo and Slit expression patterns
We have shown that Robo receptors are expressed on vomeronasal axons
throughout the time at which this topographic projection is formed. During
early stages of development of this projection (P1) differential expression
patterns of Robos on apical versus basal axons are difficult to analyse,
because cell bodies from presumptive basal and apical axons are intermingled
within the VNO. Nevertheless, the patchy expression of Robo2 mRNA in
particular is in agreement with such a differential expression on apical
versus basal cell bodies. Robo1, however, appears to be rather uniformly
expressed in the VNO. At later stages of development, when apical and basal
cell bodies in the VNO have separated, Robo2 is obviously expressed only on
basal axons, while Robo1 expression is no longer detectable.
These data were confirmed by staining vomeronasal axons during their growth into the AOB, which uncovered a stronger Robo protein staining in the posterior AOB, the target area of basal axons, than in its anterior part. This indicates that - given that targeting of vomeronasal axons is topographically specific from the beginning - basal axons express Robo receptors more strongly than apical axons from early stages of development. Our data therefore suggest that basal axons are more sensitive to Slits than are apical ones and might react differently in a target area of differential Slit expression.
The VNO also expressed Slit1 and Slit3 mRNA, with the former being
expressed at higher levels than the latter. It is unknown whether the apparent
co-expression of Slits with Robos on sensory VNO neurones exerts any effects
on the sensitivity of these axons. Co-expression of Eph receptors and ephrins
on nasal retinal ganglion cell axons renders these axons less sensitive to
exogenously applied ephrin As (Hornberger
et al., 1999).
To uncover any differential Slit protein expression pattern in the AOB, we
have used a (3,4,5) Robo1-Fc fusion protein binding to all three Slits,
to probe sections from the AOB and demonstrated a differential protein
localisation of Slits in the AOB such that their concentration was higher in
the anterior than posterior part. This fits with the (stronger) Slit1 and
(weaker) Slit3 RNA expression at its anterior border, possibly in so-called
necklace glomeruli (Lipscomb et al.,
2002
; Shinoda et al.,
1989
). The actual contribution of Slit1 and Slit3 to this
differential expression can not be revealed by this staining method and might
require suitable antibodies (Rajagopalan
et al., 2000
; Simpson et al.,
2000
).
Vomeronasal axons are chemorepulsed by Slits
Functional investigation of the interaction of Slits with Robo-expressing
vomeronasal axons in collagen co-cultures showed a chemorepulsive activity of
all three Slits on vomeronasal axons from E14.5 explants. Given that at E15.5
Robo2 is restricted to basal axons only, while Robo1 is uniformly expressed on
both apical and basal axons, one might have expected a certain differential
sensitivity of basal versus apical axons, which we did not observe. This might
reflect technical limitations of the collagen gel assay and its
quantification, which result in a failure to detect subtle differences in
sensitivity.
Importantly, the collagen co-culture assays could be performed only at early embryonic stages (i.e. E14-E15.5), as at later (postnatal) stages there is little axon outgrowth from VNO explants. However, at early stages, the difference in Robo expression between apical and basal vomeronasal axons appears to be much smaller than at later stages. In particular, the expression level of the uniformly expressed Robo1 appears to be somewhat higher than that of the differentially expressed Robo2 (Fig. 1; and data not shown for E15.5). At later stages of development, i.e. at crucial times of topographic map formation, Robo1 expression is significantly downregulated, while the differential Robo2 expression is unchanged. Thus, we would expect a differential sensitivity of apical versus basal VNO axons in collagen co-culture assays for explants from older mice. However, as mentioned, the lack of axon outgrowth from postnatal VNO explants prevents us from demonstrating the anticipated difference in apical versus basal VNO axon sensitivity in vitro.
Segregation of basal and apical VNO axons in the AOB
The expression patterns of ephrin A/Ephs and Robos/Slits suggest a
mechanism by which basal and apical axons are guided to the posterior and
anterior AOB respectively. According to this model, guidance would result from
the sum of attractive forces exerted by the ephrin A/Eph system and the
repulsive forces acting via the Slit/Robo system, which appear to be different
for apical versus basal axons.
We propose that vomeronasal axons arrive at the AOB and diverge at the
medial margin between the anterior and posterior zones (see
Fig. 7). Here, apical axons,
which express higher concentrations of ephrin As than basal ones, turn into
the anterior AOB, because of an attraction towards higher concentrations of
EphA receptors in the anterior AOB
(Knöll et al., 2001).
Given that basal axons also express ephrin A proteins, they in principle would
also turn into the anterior AOB. However, because these basal axons express
higher concentrations of Robo receptors than apical axons, they would be
repelled from invading the anterior AOB, which contains higher concentrations
of Slit, and turn into the posterior AOB (for details see
Fig. 7).
The neuropilin 2/semaphorin class 3 axon guidance family contributes also
to the guidance of apical axons, possibly via a chemorepulsive mechanism
involving a semaphorin gradient through differential neuropilin/semaphorin
sequestering (Cloutier et al.,
2000; Walz et al.,
2002
).
Interplay between axon guidance molecules and vomeronasal
receptors
A subsequent step in the formation of the vomeronasal projection is the
establishment of vomeronasal receptor-specific, complex patterns of glomeruli
within the anterior and posterior zones, which requires the expression of the
vomeronasal receptors themselves (Belluscio
et al., 1999; Rodriguez et
al., 1999
). A role of `classical' axon guidance molecules in this
fine-tuning process appears possible and is consistent with the patchy
expression of Robo2 (shown here) and of ephrin A5
(Knöll et al., 2001
) in
the basal and apical VNO, respectively. This could lead to differential
sensitivities within the subpopulations of apical and basal axons, and might
lead to a differential targeting within the two zones of the AOB.
The development of the zonal topography
One of the special features of the vomeronasal system is the intermingling
of future apical and basal cell bodies in the VNO during early development,
and their subsequent separation during postnatal development. We show that at
the time when VNO cell bodies are still intermingled, the ingrowth of axons
into the AOB is already topographic or zone-specific. This is evident from
immunohistochemical analyses showing that Robo staining is largely restricted
to the posterior AOB already at E15.5 (Fig.
5) and P1 (data not shown). Thus, the segregation of apical and
basal cell bodies in the VNO is not a precondition for the zone-specific
targeting of their respective axons within the AOB. Robo is one of the
earliest markers that defines the zonal projection of vomeronasal axons, and
might therefore be particularly suited to investigate further details on the
formation of the zonal projection of vomeronasal axons.
Potential functions of Robos in the adult vomeronasal projection
It is known that the vomeronasal projection is continuously renewed
throughout life (Barber and Raisman,
1978; Gogos et al.,
2000
; Graziadei and Graziadei,
1979
; Graziadei et al.,
1978
; Martinez-Marcos et al.,
2000
; Moulton,
1974
). The expression of Robo2 and ephrin A5 on VNO axons of adult
mice might reflect an involvement of these molecules in the guidance of
later-forming axons. We observed a downregulation of Slit1, Slit3 and Epha6 in
the postnatal AOB. Thus, the zone-specific guidance might be maintained
through a mechanism different from the one used initially and could involve a
Robo2-mediated fasciculation of VNO axons along the axonal tracts established
earlier in development (Hivert et al.,
2002
).
Interactions between guidance families
Several different guidance molecules such as Robo, ephrin A and neuropilin
proteins are expressed in overlapping and complementary expression patterns on
VNO axons and were shown to be functionally involved in the zonal targeting of
VNO axons in the AOB. Thus the vomeronasal system provides - owing to its
clear-cut zonal bipartition - an excellent and simple model system with which
to study the integration of attractive and repulsive signalling pathways
controlled by these molecules.
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ACKNOWLEDGMENTS |
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