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The Journal of Neuroscience, June 1, 1999, 19(11):4428-4436
Chemoattraction and Chemorepulsion of Olfactory Bulb Axons by
Different Secreted Semaphorins
Fernando de
Castro1,
Lingjia
Hu2,
Harry
Drabkin2,
Constantino
Sotelo1, and
Alain
Chédotal1
1 Institut National de la Santé et de la
Recherche Médicale U106, Hôpital de la
Salpêtrière, 75013 Paris, France, and
2 University of Colorado Health Sciences Center, Denver,
Colorado 80262
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ABSTRACT |
During development, growth cones can be guided at a distance by
diffusible factors, which are attractants and/or repellents. The
semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel
sensory, sympathetic, motor, and forebrain axons. We found that in rat
embryos, the olfactory epithelium releases a diffusible factor that
repels olfactory bulb axons. In addition, Sema A and Sema IV, but not
Sema III, Sema E, or Sema H, are able to orient in vitro
the growth of olfactory bulb axons; Sema IV has a strong repulsive
action, whereas Sema A appears to attract those axons. The expression
patterns of sema A and sema IV in the
developing olfactory system confirm that they may play a cooperative
role in the formation of the lateral olfactory tract. This also
represents a further evidence for a chemoattractive function of
secreted semaphorins.
Key words:
semaphorin; olfactory system; chemorepulsion; chemoattraction; development; neuropilin
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INTRODUCTION |
The organization of axonal
projections in the rodent olfactory system has been extensively
characterized. Axons from olfactory receptor neurons in the olfactory
epithelium project ipsilaterally to glomeruli in the main olfactory
bulb, where they synapse on the dendrites of the mitral and tufted
cells. These neurons project ipsilaterally to the anterior olfactory
nucleus and to higher olfactory centers, including the piriform and
entorhinal cortex, and some amygdaloid nuclei, collectively referred to
as the primary olfactory cortex (Shipley and Ennis, 1996 ). The axons of
the mitral and tufted cells are located immediately under the pial
surface (Derer et al., 1977; Schwob and Price, 1984; Marchand and
Bélanger, 1991) and form the lateral olfactory tract (LOT).
The molecular mechanisms governing the establishment of axonal
projections from the olfactory receptor neurons to the olfactory bulb
have started to be unraveled (Wang et al., 1998). In contrast, the
development of bulbofugal projections is still poorly understood, although putative guidepost cells for LOT axons have been described (Sato et al., 1998). In the rat embryo, isolated fibers start to leave
the olfactory bulb by embryonic day 14 (E14), and by E15 a LOT has
clearly formed (Marchand and Bélanger, 1991; Pini, 1993;
López-Mascaraque et al., 1996). At this stage of development, the
great majority of postmitotic neurons in the olfactory bulb are mitral
cells (Bayer, 1983). Therefore, the early LOT is almost solely composed
of mitral cell axons. Organotypic co-cultures of olfactory bulb and
telencephalic vesicles or membranes has shown that the telencephalon
contains precisely localized, short-range positional cues that guide
LOT axons (Sugisaki et al., 1995; Hirata and Fujisawa, 1997). The
results of other in vitro assays (Pini, 1993; Hu and
Rutishauser, 1996) suggest that developing rat LOT axons can also be
guided from a distance by some unidentified diffusible chemorepulsive
factors produced by the septum or the neocortex.
Chemotropic factors that can attract and/or repel axons have been
described in a variety of systems (Tessier-Lavigne and Goodman, 1996).
To date, there are two characterized chemoattractants, netrin-1
(Kennedy et al., 1994) and the hepatocyte growth factor/scatter factor
(Ebens et al., 1996). Potential chemorepellents have been identified in
two gene families, the netrins and the semaphorins (Tessier-Lavigne and
Goodman, 1996; Mark et al., 1997). Five secreted semaphorins are known
in rodents, and they have been shown to bind receptors of the
neuropilin family (Chen et al., 1997; Feiner et al., 1997; He and
Tessier-Lavigne, 1997; Kolodkin et al., 1997; Takahashi et al.,
1998).
Several pieces of evidence suggest that secreted semaphorins could play
a role in the development of bulbofugal projections. Neuropilin-1 is
highly expressed on the axons of embryonic mitral cells, and
neuropilin-2 mRNA is present in all components of the olfactory
circuitry (Kawakami et al., 1995; Chen et al., 1997). Moreover
sema III/collapsin-1 mRNAs are found in the
olfactory bulb and in the LOT pathway (Giger et al., 1996;
Shepherd et al., 1996; Kobayashi et al., 1997). This has led us to
investigate whether secreted semaphorins can influence the growth of
LOT axons.
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MATERIALS AND METHODS |
Animals. Wistar rats (IFFA Credo, Lyon, France) were
used for the culture experiments and for in situ
hybridization studies. The day on which a vaginal plug was detected was
considered E0, and the day of birth was considered postnatal day 0. Pregnant rats were anesthetized with chloral hydrate (350 mg/kg,
i.p.).
Cloning of mouse semaphorin IV sequences. Mouse
semaphorin IV sequences were amplified by RT-PCR on a
random-primed template from mouse A9 cells using the following primers:
59H8, 5'-TTC AAC TTC CTG CTC AAC; and 39G5, 5'-GAA GAC CAT GCG AAT ATC,
which were obtained from the human H-sema IV sequence (Roche
et al., 1996). PCR conditions included 35 cycles with an annealing
temperature of 55°C with Taq DNA polymerase and buffer
(Promega, Madison, WI). The final products cloned into a T vector
prepared from pBluescript II KS and sequenced on an ABI377 through the
University of Colorado Cancer Center DNA Sequencing Core. The 0.9-kb
product is identical to base pairs 143-1056 of the recently published
mouse H-Sema IV homolog (Eckhardt and Meyerhans, 1998).
In situ hybridization. E14-E15 embryos (five to seven
animals each) were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PFA). Brains were post-fixed in
4% PFA, cryoprotected with 10% sucrose, and sectioned at 20 µm with
a cryostat. Antisense riboprobes were labeled with digoxigenin-dUTP
(Boehringer Mannheim, Mannheim, Germany) or 35S-UTP
(Amersham, Buckinghamshire, UK) as described elsewhere (Chédotal et al., 1998). Controls including hybridization with sense riboprobes prevented hybridization signals.
Explant cultures and co-cultures. The olfactory bulbs from
E14-E15 embryos were dissected out as a single piece, and 250-350 µm explants were obtained using fine tungsten needles (Pini, 1993). Only the rostral two-thirds of the bulb were used to try to eliminate most of the accessory olfactory bulb. Explants were co-cultured with
aggregates (~750 µm in diameter) of COS cells transfected with
secreted alkaline phosphatase (AP), using the AP-Tag-4 vector (a gift
from Dr. J. Flanagan, Harvard University, Boston, MA), or human
sema III-myc (Messersmith et al., 1995), human sema
IV-AP (Chen et al., 1997), mouse sema E-AP (Chen et
al., 1997), mouse sema A-myc, or mouse sema H-myc
(Chédotal et al., 1998). COS cell aggregates were prepared using
the hanging drop method (Kennedy et al., 1994). Expression was
controlled on Western blots using the monoclonal 9E10 anti-myc antibody
or a polyclonal anti-AP antibody (Dako, High Wycombe, UK). Explants
were embedded in rat tail collagen gel as previously described (Lumsden
and Davies, 1986), and cultured for 36-60 hr in DMEM (Seromed, Berlin,
Germany) supplemented with L-glutamine,
D-glucose, and 10% horse serum (all from Life
Technologies, Gaithersburg, MD). Co-cultures were incubated in a 5%
CO2, 37°C, 95% humidity incubator.
Explants were fixed for 1 hr in ice-cold 4% PFA. For the visualization
of neuronal processes, cultures were rinsed several times in 0.1 M PBS, blocked with 1% normal goat serum, incubated with a
neuron-specific anti-class III  -tubulin monoclonal antibody (1:3000,
clone TUJ-1; Babco, Richmond, CA; Moody et al., 1989), followed by an
HRP-conjugated donkey anti-mouse antibody (1:2000; Jackson
ImmunoResearch, West Grove, PA), and developed with a diaminobenzidine reaction. Other co-cultures were kept in 4% PFA, and
the olfactory bulb explant was injected with a small crystal of
lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'
tetramethylindocarbocyannine (DiI; Molecular Probes, Eugene, OR). After
7-14 d at 37°C in the dark, to allow the diffusion of the tracer,
explants were recorded and photographed under rhodamine fluorescent optics.
Quantification. After -tubulin immunostaining or DiI
labeling, the surface covered by the neurites growing from the explant was measured in the proximal and distal quadrants (Wang et al., 1996)
using Imstar (Paris, France) software. Microphotographs of each
individual explant were digitally scanned with a Nikon CP-9003 camera
and transfer to a computer (Imstar). The contour of the area covered by
the neuritic processes was acquired by hand with computer-aided filling
using a specially devised package from Imstar (see Fig. 4C).
This measure takes into account both neurite lengths and numbers. In
addition, the average length of the neuritic bundles (three to four
were used in each quadrant) was measured together with the distance
separating the explants from the COS aggregates. Moreover, the mean
number of neurites in both the proximal and distal quadrants was also
determined (see Table 2). Finally, individual cultures were
additionally classified as described in Table 1. Data were
statistically analyzed using the Student's t test or paired
t test and their corresponding nonparametric tests
(Mann-Whitney and Wilcoxon tests, respectively).
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RESULTS |
Early development of the rat lateral olfactory tract
At E14-E15 the olfactory bulb can be clearly distinguished at the
rostral end of the forebrain, allowing us to inject tiny DiI crystals
in the bulb primordium (Fig.
1E). Such injections lead to the anterograde tracing of a compact axonal bundle that runs
rostrocaudally just under the pial surface (Fig. 1) and corresponds to
the presumptive LOT. Dorsoventrally, the LOT is at equal distance from
the cortex and the septum (Fig. 1A-C). Laterally,
the LOT is near a relatively thin leaflet of mesenchymal cells that
represents the precursors of the frontal bone (Fig.
1B,D,F, arrowheads). They are so closed that the
lipophilic dye can, in some cases, diffuse from the LOT to the
mesenchyme (Fig. 1C). The schematic representation in Figure
7 summarizes the embryonic position of olfactory bulb projecting
neurons (in red) and possible interactions with closely
related cells.
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Figure 1.
Illustration of the localization of the LOT in rat
E15 embryos. Embryos have been injected in the OB with DiI or
4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide (DiA)
crystals. A-C, Coronal vibratome sections at the level
of the septum (S), viewed under bright-field
(A, B) or rhodamine filter. The LOT
(outlined in B, C) grows immediately
under the pial surface, in the vicinity of the mesenchyme precursor of
the frontal bone (B, arrowheads). D-F,
Horizontal sections corresponding to the area framed in
E. They show, at E15, the rostrocaudal extent of the LOT
(labeled axon fascicle in F) and the location of
the frontal bone (D, F, arrowheads). The dashed
line delimits the caudal extent of the olfactory bulb. The
injection site is shown in E (arrowhead).
Cx, Cortex; GE, ganglionic eminence;
OE, olfactory epithelium. Scale bars: A,
200 µm; B, C, 100 µm; D, F, 50 µm.
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LOT axons are repelled by Sema IV and attracted by Sema A
To test directly whether secreted semaphorins influence the growth
of bulbofugal axons, we cultured olfactory bulb explants (from E14-E15
rat embryos) with COS cells that had been transiently transfected with
expression constructs for all five known mammalian secreted semaphorins
(A, III/D, E, IV, and H). Explants were cultured for 36-60 hr, fixed,
and stained with an anti-class III -tubulin monoclonal antibody that
labels the entire population of axons growing from the explant (Moody
et al., 1989). The expression of the diverse epitope-tagged semaphorins
was verified by Western blotting (Fig.
2). Under nonreducing conditions, all
secreted semaphorins, with the exception of Sema IV (see below), run as a single band (at the expected molecular weight of ~95 kDa for myc-tagged Sema A, Sema III, and Sema H, and ~160 kDa for AP-tagged Sema E). In the case of Sema IV two bands were observed, one at ~160
kDa and another one at ~130 kDa, which could represent a partially
cleaved fragment. This suggests that in COS cells, most of the secreted
semaphorins are not cleaved before secretion, contrary to what has been
observed using 293T cells (Adams et al., 1997). Observations similar to
ours were made using an Fc-tagged collapsin-1 (Eickholt et al.,
1997).
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Figure 2.
Western blot analysis of epitope-tagged secreted
semaphorins. Recombinant proteins were collected in the supernatant of
transfected COS cells and analyzed with anti-myc (lanes
1-3) or anti-AP (lanes 4-6) antibodies.
The myc tag is at the C terminus, whereas the AP tag is at the N
terminus. In lane 6, a single band of ~70 kDa is
detected and corresponds to COS cells expressing only secreted AP. For
Sema H (lane 1), Sema A (lane 2), Sema
III (lane 3), and Sema E-AP (lane
4) a single band is observed at their expected molecular
weight. In the case of Sema IV-AP (lane 5) two bands are
detected, the top one at the expected molecular weight of 160 kDa and
the bottom one at ~130 kDa, probably corresponding to a partially
cleaved protein.
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We found that olfactory bulb axons grow symmetrically when confronted
with COS cells mock-transfected or transfected with an alkaline
phosphatase expression construct (Figs.
3A, 4; Table 1). We could not detect any effect of COS
cells secreting Sema III, Sema E, or Sema H (Fig. 3B-D,
Table 1). In all these cases the surface covered by neurites was
similar in the proximal and distal quadrants. In contrast, olfactory
bulb axons were strongly repelled when confronted with COS cells
expressing Sema IV (Figs. 3E, 4; Table 1). Repulsion could
be clearly detected after 36 hr in vitro but was stronger
after 60 hr; therefore, quantification was done at that stage. The area
covered by the neurites and the mean number of neurites in the distal
quadrant were more than two times larger than in the proximal quadrant
(Fig. 4B, Table 2). Neurites were also much longer in the
distal (372.4 ± 15.7 µm; n = 83) than in the
proximal (234.8 ± 9.8 µm; n = 83) quadrant, suggesting that Sema IV somewhat reduces the growth of olfactory bulb
(OB) axons. Moreover, a strong or moderate repulsion was observed in
54% of the cases (Fig. 4A, Table 1).
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Figure 3.
Illustration of the co-culture experiments.
E14-E15 rat olfactory bulb explants were co-cultured 60 hr next to
aggregates of control COS cells (A) or COS cells
transfected with H-sema III (B),
mouse sema E (C), mouse
sema H (D), H-sema IV
(E), or mouse sema A
(F). All explants were fixed and stained with
anti--tubulin antibodies. Olfactory bulb axons grow symmetrically,
in the case of control cells (A) and COS cells
expressing Sema III (B), Sema E
(C), and Sema H (D),
whereas they are strongly repelled by Sema IV-expressing COS cell
aggregates (E). With Sema A-expressing cells
(F), olfactory bulb axons grow in all quadrants,
but they are more numerous in the quadrant facing the COS cells
aggregate, indicative of an attraction. Scale bars:
A-D, 170 µm; E, F, 150 µm.
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View this table:
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Table 1.
Semiquantitative evaluation of the effect of secreted
Semaphorins and olfactory epithelium (OE) on axonal outgrowth of
olfactory bulb explants
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Figure 4.
A, Histogram summarizing the data
shown in Table 1 and illustrating the proportion (percent) of olfactory
bulb explants in which axons look attracted, repelled, or unaffected
when co-cultured next to control COS cells, Sema A- or Sema
IV-expressing cells, and olfactory epithelium. B,
Histogram showing the axonal surface (mean ± SEM) in the
different combinations of olfactory bulb explants and COS cells. All
explants were fixed and stained with anti--tubulin antibodies. The
axonal surface was measured in the distal (black bars)
and proximal (gray bars) quadrants, as indicated
in C. Olfactory bulb axons are strongly repelled by Sema
IV but attracted by Sema A. Control COS cells have no effect. The
asterisks denote differences between both quadrants
(proximal and distal) that are significant at the p = 0.0001 level (Mann-Whitney rank sum test and Wilcoxon signed rank
test). C, Schema of the method used to quantify neurite
outgrowth. An image analysis system was used to determine the overall
area covered by neurites in the proximal (P, gray area)
and distal (D, black area), as represented on the
right. This gives an estimate of neurite lengths and
numbers.
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Neurite outgrowth was also asymmetrical in the case of explants facing
Sema A-expressing COS cell aggregates. Surprisingly, Sema A appeared to
exert a moderately attractive effect on olfactory bulb neurites (Figs.
3F, 4; Table 1). The neurite surface in the
proximal quadrant was significantly higher of 33% when compared with
the distal one (Fig. 4B), and the mean number of
neurites in the proximal quadrant was very significantly increased
(Table 2). In addition, chemoattraction was observed in 40% of the
explants in comparison with 11% of control COS cells or 0% of Sema
IV-expressing cells (Fig. 4B, Table 1). Repulsion
with Sema A was observed in merely 9% of the explants (Fig.
4A, Table 1). This attractive effect is comparable
with the one that has been recently shown for Sema E on neocortical
axons (Bagnard et al., 1998). In addition, the average length of
neurite bundles was comparable in the proximal (273 ± 8 µm;
n = 92) and distal (231 ± 10 µm;
n = 92) quadrants and similar to the average distance
(266 ± 10 µm; n = 92) between the explants and
the COS aggregates. This shows that the Sema A has essentially a
directional effect and does not stimulate the growth. Repulsive
secreted semaphorins also have mostly a directional action (Messersmith
et al., 1995; Song et al., 1998).
Sema A and Sema IV expression in the rat olfactory system
We tried to determine the possible function of Sema A and Sema IV
in the development of the LOT in vivo by studying the
expression pattern of their mRNAs in E14-E15 rat embryos (Fig.
5). At E15, sema A mRNA was
not detected in the forebrain (Fig. 5A,B), as previously
mentioned (Püschel et al., 1996). Nevertheless, we could observe
a strong expression of sema A in the mesenchyme precursor of
the frontal bone extending from the base of the olfactory bulb
rostrally to the piriform cortex caudally (Fig. 5A,B).
Therefore, sema A expression is restricted to cells that are
juxtaposed to the pathway followed by LOT axons. sema IV was
expressed in the cortical plate of the neocortex, as previously
described (Chédotal et al., 1998), and in the ganglionic eminence
(Fig. 5C,D). High sema IV mRNA expression was
also observed in the olfactory epithelium (Fig. 5E) and was
absent from the septum (Fig. 5C,D). In addition, at E15,
sema IV mRNA was not expressed in the olfactory bulb
(results not shown).
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Figure 5.
Expression pattern of sema A and
sema IV in the developing head. Hybridizations were
performed with 35S-labeled (A-D) or
digoxigenin-labeled (E) riboprobes on coronal
(A-D) and horizontal (E)
sections of E15 rat brains. The prospective location of the LOT is
delimited by dashes in A-C. A,
B, sema A mRNA is found in the mesenchyme
precursor of the frontal bone (A, arrows), immediately
adjacent to the LOT. Expression starts at the base of the OB in
A and runs caudally to the level of the trigeminal
ganglion (V) that is also expressing
sema A (see B). C, D,
sema IV is expressed in the cortical plate
(arrowheads) and in the ganglionic eminence
(ge). Strong expression is also found in the
choroid plexuses (cp) and the skin. Note in
C and D that the septum
(S) is not expressing sema IV. In
E, sema IV expression is homogeneously
high in the olfactory epithelium (arrowheads). Scale
bars: A, 215 µm; B, D,
110 µm; C, 240 µm; E, and 300 µm.
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Chemorepulsion of olfactory bulb axons by the
olfactory epithelium
Because Sema IV repels LOT axons and is highly expressed in the
olfactory epithelium, we tried to determine whether we could show
long-range effects of the olfactory epithelium on olfactory bulb axons.
We therefore co-cultured in collagen gel E14-E15 olfactory bulb
explants next to olfactory epithelial explants. In 52% of the cases,
the growth of olfactory bulb axons, visualized with DiI, was opposed to
the epithelial explant (Figs. 4A,
6; Table 1), and neurites were
significantly more numerous in the distal quadrant than in the proximal
one (Table 2). This demonstrates that the embryonic olfactory
epithelium secretes a factor repulsive for olfactory bulb axons.
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Figure 6.
Olfactory bulb and olfactory epithelium
cocultures. OB explants were labeled with DiI and placed near an
unlabeled explant of olfactory epithelium (OE,
dotted line). Labeled axons grow almost exclusively on
the distal side of the explant opposite to the olfactory epithelium,
indicating that the OE releases a diffusible repulsive factor. Scale
bar, 200 µm.
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We also tried to co-culture olfactory bulb explants next to explants of
mesenchyme surrounding the LOT but could not observe clear attractive
or repulsive effects, because in 24 hr mesenchymal cells migrate in
the collagen gel and reach and partially cover OB explants (results not shown).
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DISCUSSION |
We found that olfactory bulb axons, which will form the LOT,
respond to Sema IV and Sema A but not to other secreted semaphorins. In
collagen gel assays, Sema IV repels olfactory bulb axons, whereas, more
surprisingly, Sema A acts as an attractant. In addition, in co-culture
experiments, the olfactory epithelium exercises a repulsive action on
the axons growing from olfactory bulb explants. Finally, the expression
patterns of sema A and sema IV in the embryonic
olfactory system and in structures next to the lateral olfactory tract
strongly suggest that these semaphorins might play a role in the
formation of the LOT.
Developing axons are known to be guided from a distance by diffusible
molecules that can be attractive, repulsive, or both (Tessier-Lavigne
and Goodman, 1996). Our results suggest that chemoattraction and
chemorepulsion exert a cooperative action during the development of the
LOT. First, we showed that the olfactory epithelium releases a
diffusible factor that repels olfactory bulb axons and could force them
to leave the olfactory bulb primordium and grow caudally. This
repellent effect is mimicked by Sema IV and not by any other secreted
semaphorins or by netrin-1, another stong chemorepellent (F. de Castro,
A. Chédotal, and C. Sotelo, unpublished observations). The
finding that sema IV is expressed in the olfactory
epithelium (also see Giger et al., 1998) at the time LOT axons grow
suggests that this molecule could be the epithelial-derived repellent
factor (Fig. 7; see below). In addition,
it is known that developing LOT axons never enter the embryonic
neocortex (Schwob and Price, 1984), which produces a repulsive factor
for LOT axons (Pini, 1993). sema IV, whose mRNA is highly
expressed in the cortical plate of the neocortex (Chédotal et
al., 1998; Giger et al., 1998 and present results), becomes an
excellent candidate to be the secreted factor preventing the invasion
of the neocortex by LOT axons (Fig. 7).
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Figure 7.
Schematic representation in horizontal
(A) and coronal (B) planes
of the chemorepulsive and chemoattractive factors that influence the
formation of the lateral olfactory tract. The olfactory epithelium
(OE) would released a repulsive factor (violet
circles), probably Sema IV, forcing LOT axons
(red) to leave the olfactory bulb. An unidentified
factor (green circles) produced by the septum
(S) would prevent LOT axons to cross the midline.
In addition, LOT axons would grow very superficially, being attracted
by Sema A (orange circles) secreted by the mesenchyme
precursor of the frontal bone (gray).
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Chemorepulsion has been observed in several neuronal systems
(Fitzgerald et al., 1993; Colomarino and Tessier-Lavigne, 1995; Tamada
et al., 1995; Varela-Echavarría et al., 1997; Chédotal et
al., 1998; Rochlin and Farbman, 1998; Tuttle et al., 1998), but the
first evidence came from the embryonic rat olfactory system in which
the septum produces a diffusible factor repelling olfactory bulb axons
(Pini, 1993; Fig. 7). This still uncharacterized activity was supposed
to explain why LOT axons grow away from the midline (Pini, 1993),
although recent in vitro results have shown that mitral cell
axons also elongate along their normal pathway without a septum
(Sugisaki et al., 1995). Because Sema IV and the other known secreted
semaphorins have not been found in the embryonic septum, the repulsive
factor, produced by the latter, might not belong to the semaphorin family.
In addition, olfactory bulb axons grow preferentially toward aggregates
of COS cells producing Sema A but not any other secreted semaphorins,
suggesting that chemoattraction could also be involved in the formation
of the LOT. In the embryonic rat head, sema A mRNA is almost
exclusively found in the frontal bone primordium that is apposed to the
forebrain parenchyma and therefore immediately adjacent to the LOT, at
a distance that could be smaller than 20 µm (Meller and Tetzlaff,
1975). Although direct evidence is still lacking, our results suggest
that Sema A released by the mesenchymal cells could attract in the CNS
axons of the lateral olfactory tract. At this early stage, the immature
meningeal covers are permeable, and the glia limitans is covered by a
basal membrane, which prevents axons to leave the CNS. This could
explain why LOT axons grow preferentially in the most superficial
zone of the forebrain (Fig. 7). Sema A-mediated attraction was weaker than Sema IV repulsion, a difference that could emerge from the heterogeneity of olfactory bulb axons together with a differential action on a large axonal population for Sema IV and on a smaller one
for Sema A (similarly, at E14 in the rat, Sema III repels only
NGF-sensitive dorsal root ganglion axons but not NT3-sensitive ones;
Messersmith et al., 1995). This assumption is based on the fact that
mitral and tufted cells are morphologically, neurochemically, and
functionally heterogeneous (Macrides et al., 1985; Shipley and Ennis,
1996).
It has recently been shown that Sema E can attract cortical axons
(Bagnard et al., 1998). The present results confirm that in the
vertebrate CNS, secreted semaphorins could promote the growth of
certain axonal populations in normal developmental conditions. In
addition, in cultured embryonic Xenopus spinal neurons, an experimental elevation of the cytosolic concentration of cGMP can turn
Sema III repulsive action into an attraction (Song et al., 1998).
Moreover, G-Sema I, a grasshopper transmembrane semaphorin that was
previously shown to inhibit the growth of other sensory axons (Kolodkin
et al., 1992), appears to promote the growth of axons in the subgenual
organ (Wong et al., 1997). Similarly, Sema A, attractive in this study,
is repellent for sympathetic axons (Adams et al., 1997; Takahashi et
al., 1998). Therefore, we have enough evidence to start thinking that
semaphorins might be bifunctional guiding cues, attractive and
repulsive, like the midline-derived netrin-1 (Serafini et al., 1994;
Colomarino and Tessier-Lavigne, 1995; Varela-Echavarría et al.,
1997). Non-neuronal tissues, such as the somites, are known to
influence the patterning of axonal projections in the peripheral
nervous system (Tannahill et al., 1998). In addition, diffusible
factors produced by the the notochord and other mesodermic tissues play
a role in neuronal differentiation in the CNS (LaMantia et al., 1993;
Rubenstein and Beachy, 1998). To our knowledge, the present results
represent the first evidence for a presumptive influence of
non-neuronal peripheral tissue on axon tract formation and orientation
in the vertebrate CNS.
The identity of the receptors involved in Sema A and Sema IV signaling
in olfactory bulb axons is still unknown. The transmembrane proteins
neuropilin-1 and -2 have been shown to be receptors, or components of
receptors, for vertebrate-secreted semaphorins (Chen et al., 1997;
Feiner et al., 1997, He and Tessier-Lavigne, 1997; Kolodkin et al.,
1997, Giger et al., 1998; Takahashi et al., 1998). Recently, it has
been shown that Sema IV-induced repulsion of sensory axons is mediated
by neuropilin-2 (Giger et al., 1998). Moreover, Sema A collapsing
activity also requires neuropilin-2, perhaps in combination with
neuropilin-1 (Takahashi et al., 1998). Both neuropilins are expressed
by mitral neurons and mitral cell axons at the time they leave the
olfactory bulb (Sugisaki et al., 1995; Kawakami et al., 1996; Chen et
al., 1997; F. de Castro and A. Chédotal, unpublished
observations), but in neuropilin-1 knock-out mice (Kitsukawa
et al., 1997) the LOT develops normally, suggesting that neuropilin-2
could be the receptor mediating Sema IV and Sema A responses. It would
be interesting to determine whether the repulsive and attractive
effects of Sema A are mediated by different receptors. In
Xenopus axons, it is clear that the same second messengers
may be used for both Sema III-mediated attraction and repulsion and
that neuropilin-1 is the receptor for both (Song et al., 1998).
Finally, the receptor might not be a neuropilin, because it has
recently been shown that virus-encoded semaphorin protein receptor, a
member of the plexin family of transmembrane proteins, is a receptor
for a viral semaphorin (Comeau et al., 1998), and that in
Drosophila, plexin A binds transmembrane semaphorins (Winberg et al., 1998). Whether plexins are receptors for other vertebrate-secreted or transmembrane semaphorins remains unknown (also
see Fujisawa and Kitsukawa, 1998).
Semaphorins have been involved in the development and regeneration of
other axonal connections of the olfactory system (Shepherd et al.,
1996; Pasterkamp et al., 1998). Collapsin-1/Sema III can induce the
collapse of olfactory axonal growth cones (Kobayashi et al., 1997) and
could serve as a stop signal for these axons, initially preventing them
from invading the olfactory bulb (Giger et al., 1996). Recently it has
been shown that Sema A and Sema E can antagonize Sema III, preventing
its binding to neuropilin-1 (Takahashi et al., 1998). Olfactory
receptor neurons express Sema IV, which also binds neuropilin-1 but
does not induce DRG growth cone collapse (A. Chédotal,
unpublished observations) and could also be an antagonist of
neuropilin-1. In agreement with Takahashi et al. (1998), Sema IV could
act as a repellent for olfactory bulb axons and an antirepellent for
olfactory axons, allowing them to enter the bulb even in the presence
of a high concentration of Sema III in their target territory. Finally,
several studies have shown that the formation of the olfactory bulb
depends on the olfactory receptor neurons, independently of synapse
formation (Graziadei et al., 1978; Stout and Graziadei, 1980; Gong and
Shipley, 1995). The factors responsible for these actions have not been identified but it would be interesting to determine whether it could be
a semaphorin.
In conclusion, during axonogenesis, LOT axons are probably influenced
by multiple diffusible factors, repulsive and attractive, in
combination with a variety of short-range cues (Sugisaki et al., 1995;
Sato et al., 1998).
| |
FOOTNOTES |
Received Nov. 23, 1998; revised March 8, 1999; accepted March 11, 1999.
A.C. and C.S. are supported by Institut National de la Santé et
de la Recherche Médicale and Grant BIO4-CT960-774 from the European Community (EC). F.d.C. is supported by EC Fellowship ERB-4001-GT-970077. H.D. is supported by Grants CA68383 and CA58187 from the National Institutes of Health. We thank Dr. D. J. Flanagan for the APtag4 vector and Drs. C. Christensen and A. W. Püschel for providing us with mouse sema H and
mouse sema A, respectively.
Correspondence should be addressed to Dr. Alain Chédotal,
Institut National de la Santé et de la Recherche Médicale
U106, Hôpital de la Salpêtrière, 47 Boulevard de
l'Hôpital, 75013 Paris, France.
| |
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K. T. N. Ba-Charvet, K. Brose, L. Ma, K. H. Wang, V. Marillat, C. Sotelo, M. Tessier-Lavigne, and A. Chedotal
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D. Bagnard, C. Vaillant, S.-T. Khuth, N. Dufay, M. Lohrum, A. W. Puschel, M.-F. Belin, J. Bolz, and N. Thomasset
Semaphorin 3A-Vascular Endothelial Growth Factor-165 Balance Mediates Migration and Apoptosis of Neural Progenitor Cells by the Recruitment of Shared Receptor
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Y Tashiro, M Miyahara, R Shirasaki, M Okabe, C. Heizmann, and F Murakami
Local nonpermissive and oriented permissive cues guide vestibular axons to the cerebellum
Development,
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E. Brambilla, B. Constantin, H. Drabkin, and J. Roche
Semaphorin SEMA3F Localization in Malignant Human Lung and Cell Lines : A Suggested Role in Cell Adhesion and Cell Migration
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ABSTRACT
INTRODUCTION
Substance via MeSH
