Department of Anatomy, University of Cambridge, Downing Street, Cambridge
CB2 3DY, UK
* Present address: Centre for Cell and Molecular Dynamics, Department of Anatomy
and Developmental Biology, University College London, 5th Floor Rockefeller
Building, Gower Street, London WC1E 6BT, UK
Author for correspondence (e-mail:
dt10003{at}mole.bio.cam.ac.uk)
Accepted 29 November 2002
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SUMMARY |
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Key words: Axon guidance, Neural development, Chick embryo, Notochord, Spinal nerves, Dorsal root ganglia, Semaphorin
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INTRODUCTION |
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The initial trajectory of DRG and motor axons within the somites is further
restricted within the dorsoventral plane, because growing axons avoid the
notochord lying medially to the DRG and the dermamyotome which lies laterally
(Fig. 1A). Insights into the
mechanisms that influence DRG axon trajectory in the periphery have come from
experiments involving tissue explants cultured in three-dimensional collagen
gels (Keynes et al., 1997;
Nakamoto and Shiga, 1998
). It
was found that the notochord and dermamyotome, and to a lesser extent ventral
neural tube, secrete repellents acting on DRG axons over considerable
distances. It is probable, therefore, that tissues lying either side of the
DRG channel axon trajectories by repulsion in vivo. It has also been found
that the notochord has axon-repulsive activity at the appropriate
developmental stages for early outgrowing DRG axons
(Keynes et al., 1997
;
Nakamoto and Shiga, 1998
).
Notochord removal experiments in ovo have lent further support for such a role
of the notochord, together with its surrounding matrix, in axon repulsion
(Stern et al., 1991
;
Tosney and Oakley, 1990
). In
contrast, surgical removal of dermamyotome has proved less informative as
residual ectoderm, which is also a source of repulsive cues, regenerates in
situ to maintain a source of repulsion
(Keynes et al., 1997
;
Tosney, 1987
).
|
Although the molecules mediating axon repulsion by the notochord and
dermamyotome remain to be identified, dermamyotome-mediated repulsion may be
explained, in part, by SEMA3A (collapsin 1), a secreted member of the
semaphorin family (Castellani and Rougon,
2002; He et al.,
2002
; Raper,
2000
). SEMA3A activity is mediated through homodimers of
neuropilin 1, forming part of the semaphorin receptor complex
(He and Tessier-Lavigne, 1997
;
Kolodkin et al., 1997
;
Miyazaki et al., 1999b
;
Nakamura et al., 1998
;
Takahashi et al., 1998
).
SEMA3A protein has the appropriate repulsive activity on DRG and motor axons,
as their growth cones collapse in response to it
(Luo et al., 1993
;
Varela-Echavarria et al.,
1997
). SEMA3A mRNA is also present at high levels in the
dermamyotome at the appropriate stages
(Adams et al., 1996
;
Shepherd et al., 1996
;
Wright et al., 1995
).
Furthermore, in mice lacking Sema3a or neuropilin 1, some
axons are seen to project aberrantly towards the dermamyotome
(Kitsukawa et al., 1997
;
Taniguchi et al., 1997
;
White and Behar, 2000
). A role
for SEMA3A in contributing to repulsion from the notochord is less obvious.
Although SEMA3A is expressed in the notochord, the level of expression is much
lower than that in the dermamyotome
(Shepherd et al., 1996
). Also,
in Sema3a and neuropilin 1 knockout mice, no defects
consistent with semaphorin-mediated repulsion by the notochord have been
described in the initial trajectories of DRG or motor axons
(Kitsukawa et al., 1997
;
Taniguchi et al., 1997
;
White and Behar, 2000
).
Two other semaphorin family members are also expressed at low levels in the
chick embryo notochord, namely SEMA3C (collapsin 3) and SEMA3D (collapsin 2)
(Luo et al., 1995). SEMA3C,
whose activity is thought to be mediated through heterodimers of neuropilin 1
and 2 (Chen et al., 1998
;
Raper, 2000
;
Renzi et al., 1999
;
Takahashi et al., 1998
), has
been reported to induce the collapse of sympathetic chain ganglia (SCG) but
not DRG axons (Adams et al.,
1997
; Chen et al.,
1998
; Koppel et al.,
1997
; Takahashi et al.,
1998
). Moreover, SEMA3D does not induce collapse of either DRG or
sympathetic growth cones (Koppel et al.,
1997
; Raper,
2000
). It seems unlikely, therefore, that SEMA3C or SEMA3D have a
direct influence on DRG axon trajectory. In mice, expression of the neuropilin
1-dependent semaphorin, Sema3e, has been found in the perinotochordal
mesenchyme (Miyazaki et al.,
1999a
; Miyazaki et al.,
1999b
). Although the biological activity of chick SEMA3E protein
has yet to be reported, mouse Sema3E protein appears to repel DRG axons
(Miyazaki et al., 1999a
;
Miyazaki et al., 1999b
). As
described above, the phenotype of neuropilin 1 knockout mice, which
has brought into question the role of neuropilin 1-dependent semaphorins, also
raises questions concerning the role of other semaphorins, such as SEMA3C,
which are thought to act through neuropilin 1 together with neuropilin 2. The
role of secreted semaphorins acting through neuropilin 2, such as SEMA3F
(Chen et al., 1998
;
Raper, 2000
;
Renzi et al., 1999
), also
remains unclear. Although defasciculation of peripheral spinal nerves has been
reported in neuropilin 2 knockout mice, defects in initial axon
trajectory consistent with neuropilin 2-dependent repulsion by the notochord
and dermamyotome have not been described
(Giger et al., 2000
).
Recently, it has been shown that slit genes are expressed in the
notochord, raising the possibility that they could contribute to repulsion of
peripheral spinal axons. Consistent with this is the observation that
slit2 expression precedes, and continues beyond, the initial
outgrowth of DRG axons (Holmes and
Niswander, 2001; Li et al.,
1999
). Expression of slit3 has also been reported in the
notochord at similar stages (Holmes and
Niswander, 2001
). Slit2 protein has been shown to repel many
different axon types, including olfactory bulb, hippocampal, spinal motor
column and retinal axons (Brose et al.,
1999
; Erskine et al.,
2000
; Li et al.,
1999
; Nguyen Ba-Charvet et
al., 1999
; Niclou et al.,
2000
; Ringstedt et al.,
2000
; Yuan et al.,
1999
). DRG axons, however, are not repelled by Slit2 in collagen
gel assays, and DRG growth cones do not collapse in response to Slit2
(Nguyen Ba-Charvet et al.,
1999
; Niclou et al.,
2000
). Further evidence against a role for Slit2 in motor axon
repulsion has come from an examination of the repulsive properties of the
floor plate, which expresses high levels of Slit2
(Holmes and Niswander, 2001
;
Holmes et al., 1998
;
Yuan et al., 1999
). When
soluble robo/Fc receptor fusion proteins are used to inhibit Slit2, motor axon
repulsion by the floor plate is unaffected
(Patel et al., 2001
). At first
sight, this argues against a role for Slit2 in peripheral spinal axon
guidance, but recent studies have suggested that a membrane-bound N-terminal
Slit2 fragment is not a preferred substratum for DRG axon growth
(Nguyen-Ba-Charvet et al.,
2001
). Thus, the role of Slit proteins in peripheral spinal axon
guidance remains unclear.
In order to investigate the molecular nature of peripheral spinal axon repulsion by the notochord, we undertook a screen for axon repellents secreted by the notochord and identified SEMA3A as a candidate repellent. Furthermore, experiments in which notochord-derived SEMA3A repulsion is inhibited indicate that, although SEMA3A contributes to the notochord activity, other unidentified factors are also critical in determining spinal sensory axon trajectories in the periphery.
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MATERIALS AND METHODS |
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Collagen gel cultures
Tissue explants were co-cultured in collagen gels essentially as described
previously (Keynes et al.,
1997; Ohta et al.,
1999
). Tissues were cultured in 15 mm four-well dishes (Nunc) and
rat-tail type VII collagen was obtained from Sigma or Roche. Unless otherwise
stated, DRG and SCG explants were positioned 250-500 µm from notochord or
COS cell explants, whereas retinal and olfactory bulb explants were positioned
200-400 µm away. DRG, SCG and olfactory bulb cultures were incubated for
24-30 hours and retinal explants for 48-72 hours. All tissues were cultured in
DMEM (Sigma) containing gentamycin (50 µg/ml, Sigma) with either 50 ng/ml
NGF (Alomone) or 50 ng/ml NT3 (a gift from Regeneron Pharmaceuticals) for DRG
and SCG cultures; ITS (Sigma) for olfactory bulb cultures, or 3% chick embryo
extract (Ohta et al., 1999
)
for retinal explants. Neuropilin 1/Fc and neuropilin 2/Fc fusion proteins were
obtained from R&D Systems. After incubation, cultures were fixed in 4%
paraformaldehyde, 15% sucrose, phosphate-buffered saline pH 7.4.
Evaluation of axon growth
Axon outgrowth in the quadrant proximal to the notochord or COS cell
co-explant was compared with the distal quadrant and scored as described
previously (Keynes et al.,
1997). A score of zero represents no outgrowth towards the
co-explant; two, a weak and/or patchy outgrowth; four, more consistent
outgrowth which does not extend closer to the co-explant than
50 µm;
six, growth approaches the co-explant closer than
50 µm, but does not
contact it; eight, axons contact the co-explant, but some asymmetry between
proximal and distal remains; ten, no difference in outgrowth in the proximal
versus distal quadrants, with axons growing into and/or past the co-explant.
At least three independent experiments were performed for each assay.
Photography was performed using an Axiovert microscope with dark-field or
phase-contrast optics (Zeiss). Statistics were performed using a
Kruskal-Wallis non-parametric test followed by a Bonferroni/Dunn post-hoc
ANOVA test at the 1% significance level.
COS cell transfection
COS-7 cells were transfected with a pCAGGS
(Niwa et al., 1991) vector
containing SEMA3A, SEMA3C or vector control using LipofectAMINE (Invitrogen)
as described previously (Ohta et al.,
1999
). Full-length chick SEMA3C was cloned from a chick notochord
cDNA expression library based on published sequences (accession number
AF022946). COS cell aggregates were made by the hanging drop method
(Kennedy et al., 1994
).
SEMA3A-expressing COS cells were mixed with vector-transfected cells to
moderate the amount of SEMA3A secreted by the aggregate. Aggregates were
sub-divided before placing in collagen gels. To control for SEMA3A expression
levels, the same COS cell aggregate was used in comparing the effects of
neuropilin 1/Fc with controls.
Whole-mount in situ hybridisation
Whole-mount in situ hybridisation was performed as described previously
with minor variations (Nieto et al.,
1996). Probes were generated by PCR from a notochord cDNA library
and sub-cloned into pCR2.1 (Invitrogen). Hybridisation was performed at
65°C. Post-in situ hybridisation, embryos were embedded in TissueTek OCT
compound (Sakura) and 30 µm sections cut using a cryostat. The following
chicken sequences were used: SEMA3A, nucleotides 2221-2869 (Accession number,
GGU02528); SEMA3C, nucleotides 1786-2197 (Accession number, AF022946); and
neuropilin 2 nucleotides 1170-1785 (Accession number, AF417236).
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RESULTS |
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To explore the potential contribution of SEMA3A to notochord-mediated
repulsion, we exploited the sensitivity of different DRG axon populations,
selected by growth medium containing different neurotrophins
(Farinas et al., 1998;
Martin-Zanca et al., 1990
;
Phillips and Armanini, 1996
),
to repulsion by SEMA3A. Older DRG axons grown in NGF, but not NT3, are
repelled by SEMA3A in vitro (Messersmith
et al., 1995
; Pond et al.,
2002
; Puschel et al.,
1996
; Shepherd et al.,
1997
). This is thought to limit the extension of NGF-, but not
NT3-, responsive axons in vivo to the dorsal spinal cord, as they are repelled
by SEMA3A expressed in the ventral spinal cord
(Messersmith et al., 1995
;
Pond et al., 2002
;
Puschel et al., 1996
;
Shepherd et al., 1997
). The
differential SEMA3A sensitivity also correlates with the expression of
neuropilin 1, which is required for SEMA3A-mediated repulsion. NGF-responsive
axons maintain neuropilin 1 expression, whereas NT3-responsive axons lose
neuropilin 1 expression around the time that they project into the spinal cord
(Fu et al., 2000
;
Pond et al., 2002
).
We therefore employed collagen gel co-cultures to assay repulsion by the
notochord of different populations of stage 36 (day 10) DRG axons. Our results
indicate that stage 36 DRG axons cultured in the presence of NGF, but not NT3,
are repelled by stage 17-21 (day 3) notochords
(Fig. 2A,B,F; mean score
± standard error of the mean is 1.2±0.1 versus 7.9±0.2).
Although this is consistent with the action of SEMA3A, it does not exclude the
action of other semaphorins acting through neuropilin 1, or of other
unidentified repellents. In contrast to the differential responsiveness of
stage 36 DRG axons to SEMA3A when cultured in NGF versus NT3, it has been
noted that earlier DRG axons are responsive to SEMA3A when cultured in either
neurotrophin (Pond et al.,
2002; Puschel et al.,
1996
; Shepherd et al.,
1997
). We therefore examined repulsion by the notochord of earlier
DRG axons, grown from stages closer to the times of initial axon outgrowth in
vivo. Stage 26-27 (day 5) DRGs were used, being the earliest stages at which
DRGs can be isolated and cultured reproducibly
(Masuda et al., 2000
). Similar
to the response of stage 29-31 DRG axons to SEMA3A
(Pond et al., 2002
;
Shepherd et al., 1997
),
earlier DRG axons (stage 26-27) cultured in either neurotrophin are strongly
repelled by the notochord, consistent with a role for SEMA3A
(Fig. 2C,D,F; mean scores are
0.8±0.2 and 1.6±0.2 for NGF and NT3 cultures, respectively).
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Similar to peripheral DRG axons, axons from SCGs, lying ventral to the
DRGs, avoid the notochord and do not cross into the contralateral side. SCG
axons are known to be sensitive to repulsion by several semaphorins, including
SEMA3A, SEMA3C and SEMA3F (Adams et al.,
1997; Chen et al.,
1998
; Koppel et al.,
1997
; Takahashi et al.,
1998
). We therefore tested whether SCG axons are sensitive to
notochord repulsion. Stage 36 SCG axons were strongly repelled by the
notochord (mean score 0.3±0.1), and were significantly more sensitive
to notochord repulsion than similar stage DRG axons cultured in NGF
(Fig. 2E,F;
P<0.001). To examine whether the differential sensitivity of SCG
and DRG axons to notochord repulsion could be accounted for by differences in
SEMA3A responsiveness, we compared the repulsion of SCG and DRG axons to
SEMA3A-expressing cells within the same collagen gel. Qualitatively, SCG and
DRG axons of both stages showed a similar extent of repulsion to SEMA3A
secreted from COS cell aggregates (Fig.
3). The finding that SCG axons are more repelled by the notochord
than stage 36 DRG axons (Fig.
2F) is consistent with the secretion of a repellent additional to
SEMA3A (see below). Taken together, these results implicate a neuropilin
1-binding semaphorin, probably SEMA3A, as a candidate molecule that
contributes to notochord-derived axon repulsion.
|
Inhibition of notochord repulsion by soluble neuropilins
To test whether neuropilin 1-binding semaphorins, including SEMA3A, are
secreted by the notochord, we employed soluble neuropilin receptor proteins,
comprising the extracellular domains of rat neuropilin 1 or neuropilin 2 fused
to the human IgG complement-binding fragment (Fc), to titrate out semaphorin
activity in collagen gel assays. To control for the effectiveness of the
neuropilin 1/Fc receptor reagent, we first used neuropilin 1/Fc to inhibit
SEMA3A repulsion derived from COS cells expressing SEMA3A. In these
experiments we used SEMA3A-expressing cell aggregates that matched the extent
of repulsion obtained with notochord explants. Using vector-transfected cells,
with or without neuropilin 1/Fc, no significant change in the growth of stage
36 DRG axons was observed (Fig.
4A,E). However, in co-cultures of stage 36 DRGs and
SEMA3A-expressing cells, axon repulsion was significantly reduced in the
presence of the neuropilin 1/Fc (P<0.0001), and many more axons
approached and occasionally contacted the cell aggregate (from a mean score of
1.4±0.2 to 5.0±0.8; compare
Fig. 4B,C,E). As a control, the
addition of 10 µg/ml neuropilin 2/Fc did not significantly reduce repulsion
(Fig. 4D,E), consistent with
the finding that SEMA3A binds neuropilin 2 only with low affinity
(Chen et al., 1997).
|
When stage 36 DRGs were cultured in NGF with stage 17-21 notochords in the presence of neuropilin 1/Fc, repulsion of DRG axons was significantly reduced in a dose-dependent manner (P<0.0001). In the presence of neuropilin 1/Fc, the mean score increased from 1.6±0.2 in control co-cultures to 3.2±0.2 and 5.7±0.4 for 3 µg/ml and 10 µg/ml neuropilin 1/Fc, respectively (Fig. 4F,G,J). In these experiments, the reduction of repulsion was equivalent to that seen using neuropilin 1/Fc to block SEMA3-mediated repulsion (see above). The observation that neuropilin 2/Fc does not cause a similar reduction in repulsion (Fig. 4H-J; mean score 1.6±0.2 and 2.1±0.2 for 3 µg/ml and 10 µg/ml neuropilin 2/Fc, respectively) indicates that the Fc domain is not responsible for reducing notochord-induced repulsion, and also suggests that neuropilin 2-binding semaphorins are unlikely to be involved. Taken together, these results are in keeping with the proposition that the repulsive activity secreted by the notochord has a major component based on SEMA3A.
We next tested whether the neuropilin/Fc receptor reagents could reduce the repulsion by the notochord of earlier (stage 26-27) DRG axons as well as SCG axons. Overall, in these experiments a reduction in repulsion was evident, but this was significantly less than for stage 36 DRG axons (P<0.0001) and was more variable (Fig. 5). At 10 µg/ml neuropilin 1, the mean score for DRG and SCG axon repulsion increased from 0.6±0.2 to 2.3±0.3, and from 0.2±0.1 to 2.2±0.2, respectively (Fig. 5A,D,G). At 10 µg/ml neuropilin 2, an apparent reduction in repulsion was observed for DRG and SCG axons, but this was not statistically significant at the 1% level (Fig. 5B,E,G; the mean score increased from 0.6±0.2 to 1.0±0.3, and from 0.2±0.1 to 1.2±0.4, respectively). When combinations of neuropilin 1/Fc and neuropilin 2/Fc were used, the reduction in repulsion was not significantly different from that for neuropilin 1/Fc alone (Fig. 5C,F,G; at 5 µg/ml of each neuropilin/Fc the mean score increased from 0.6±0.2 to 2.4±0.5, and from 0.2±0.1 to 2.0±0.3, for DRG and SCG axons, respectively; at 10 µg/ml of each neuropilin/Fc the respective values were 2.7±0.5 and 1.7±0.3).
|
Because neuropilin 1/Fc receptor reagents were significantly less effective
in inhibiting notochord repulsion of early DRG and SCG axons compared with
later DRG axons, even though they appear equally sensitive to SEMA3A (see
above), it is probable that multiple repellents are secreted by the notochord.
Although much of the repulsive activity on early DRG axons appears not to be
mediated by neuropilin-binding proteins, our results support a role for SEMA3A
nonetheless. Neuropilin 1-binding molecules are secreted by the notochord, as
there is a significant reduction in repulsion of DRG and SCG axons with
neuropilin 1/Fc. A role for neuropilin 2 is not revealed by these experiments,
but the expression of neuropilin 2 in mouse
(Chen et al., 1997) and chick
DRGs (Fig. 5) could indicate a
potential contribution of neuropilin 2.
Expression of SEMA3C and neuropilin 2 in the chick embryo trunk
SCG neurons express both neuropilin 1 and neuropilin 2, and respond to
semaphorins that bind either receptor (Chen
et al., 1998; Giger et al.,
1998
). The finding that SCG axons are more repelled by the
notochord than stage 36 DRG axons raises the possibility that SCG axons also
respond to a neuropilin 2-binding semaphorin, such as SEMA3C. We therefore
confirmed the expression of SEMA3C in the notochord by wholemount in situ
hybridisation (Fig. 6A,B), in
keeping with previous data (Luo et al.,
1995
). SEMA3C expression was also noted in the trunk at sites
similar to SEMA3A, such as the posterior half-sclerotome of more anterior
somites (Fig. 6B), consistent
with expression of the mouse homologue
(Puschel et al., 1995
).
|
Although it has been reported that DRG axons are insensitive to SEMA3C
(Adams et al., 1997;
Chen et al., 1998
;
Koppel et al., 1997
;
Takahashi et al., 1998
), very
early DRG axons have not been tested. If these axons are SEMA3C-sensitive,
they should express neuropilin 2. We examined the expression of neuropilin 2
in the trunk and found strong expression in the anterior but not posterior
half-sclerotome of the earliest forming somites, with expression being
maintained in more anterior somites in regions where neural crest cells
coalesce to form DRGs (Fig.
6C-E). By stage 26, neuropilin 2 expression had decreased in the
DRGs, but was detectable in both DRGs and their axons
(Fig. 6F). The spinal motor
columns of the ventral neural tube were also seen to express neuropilin 2
(Fig. 6D). This expression
pattern is reminiscent of mouse neuropilin 2
(Chen et al., 1997
).
These results are consistent with a possible role for a neuropilin 2-binding semaphorin in notochord-mediated repulsion, but the inability of neuropilin 2/Fc to prevent repulsion (Fig. 5G) argues against this. As a further test we examined whether DRG axons taken from early stages are able to respond to a source of SEMA3C (Fig. 7). As a control, we found that SCG axons were strongly repelled by cells expressing SEMA3C (Fig. 7B,E; mean score 1.7±0.3); however, DRG axons taken from stage 26-27 were not repelled (Fig. 7D,E; mean score 8.8±0.2). Therefore, although the notochord may secrete SEMA3C, it is probable that SEMA3C does not account for the repulsion of these DRG axons, as they do not respond to SEMA3C and express neuropilin 2 only weakly.
|
The notochord repels retinal axons
Several other axon populations have well-characterised responses to known
axon repellents. These can therefore be employed in assays of notochord
repulsion to explore the nature of the repellents secreted by the notochord.
For example, retinal axons are known to be insensitive to SEMA3A
(Luo et al., 1993;
Vermeren et al., 2000
). When
stage 26-29 retinal explants were cultured together with notochord, retinal
axons were repelled (Fig. 8;
mean score 2.4±0.3). Furthermore, when retinal and notochord explants
were cultured together in the presence of soluble neuropilins, there was no
significant reduction in repulsion (Fig.
8; mean scores 2.1±0.5 and 0.9±0.3 in the presence
of 10 µg/ml neuropilin 1 or neuropilin 2, respectively), again suggesting
that the notochord secretes axon repellents that act independently of
neuropilins.
|
slit2 and slit3 are expressed by the notochord in vivo
(Holmes and Niswander, 2001;
Li et al., 1999
), and Slit2
can act as a repellent for retinal axons
(Erskine et al., 2000
;
Niclou et al., 2000
;
Ringstedt et al., 2000
),
raising the possibility that Slit2 is secreted by the notochord to repel
retinal axons. We examined this possibility by exploiting the observation that
olfactory bulb axons are repelled by Slit2
(Li et al., 1999
;
Nguyen Ba-Charvet et al.,
1999
; Yuan et al.,
1999
). When stage 36 olfactory bulb explants were cultured
together with notochord, we found that axons were not repelled
(Fig. 8; mean score
7.4±0.4), suggesting that Slit2 does not mediate notochord repulsion of
retinal axons. As olfactory bulb axons can be repelled by SEMA3F and not
SEMA3A (de Castro et al.,
1999
), this further suggests that neuropilin 2-binding semaphorins
do not contribute to the repulsive activity of the notochord. Overall, our
results strongly suggest that unidentified axon repellents, additional to
semaphorins, mediate peripheral spinal axon repulsion by the notochord.
![]() |
DISCUSSION |
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Several factors may explain why blocking repulsion from SEMA3A-expressing cells did not reduce the score to that of control cells. There was individual variability in the scores; some cultures were completely disinhibited whereas others were only partially so. This may reflect the difficulty in standardising the amount of SEMA3A being secreted by the cell aggregates. Also, the semi-quantitative scoring system that we use is particularly sensitive to low levels of repulsion and scores of five and above (which represents half the numerical scale) are in the realms of weak to no repulsion. Finally, as the neuropilin receptor reagents are from rat sequences, they may have less efficacy. Despite these concerns, we conclude that neuropilin 1/Fc can be used as an effective tool in collagen gel assays to detect the presence of repulsive molecules such as SEMA3A.
Taken together, our results strongly suggest that SEMA3A is secreted by the notochord and repels DRG axons. However, although it is probable that a major repellent for stage 36 axons is SEMA3A, our results indicate that other repellents contribute to the activity of the notochord on stage 26-27 DRG and stage 36 SCG axons. In particular, we noted that neuropilin 1/Fc treatment reduces notochord repulsion of stage 26-27 DRG axons to a much lesser extent than that of stage 36 DRG axons.
Other semaphorins as notochord-derived axon repellents
Although our results are consistent with SEMA3A being secreted by the
notochord, they do not exclude the possibility that other semaphorins act in
concert, because in our inhibition experiments neuropilin 1/Fc could bind to
semaphorins other than SEMA3A. Several other semaphorins, including SEMA3C and
SEMA3D have been shown to be expressed by the notochord in chick embryos
(Luo et al., 1995;
Shepherd et al., 1996
), and it
remains to be determined whether any further semaphorins are also expressed
here. It is unlikely that SEMA3D directly repels DRG axons because it does not
induce collapse of their growth cones
(Koppel et al., 1997
). SEMA3E
is also expressed at low levels in the perinotochordal region, and it may
contribute to repulsion by the notochord as it has the appropriate repulsive
activity for DRG axons (Miyazaki et al.,
1999a
; Miyazaki et al.,
1999b
).
Our results raise the possibility of a role for SEMA3C, because it is
expressed at moderate levels by the notochord. SEMA3C is thought to operate
through a combination of neuropilin 1 and 2
(Chen et al., 1998). Although
we find that DRGs express neuropilin 2, our inhibition experiments suggest
that signalling through this receptor is not functionally significant. This is
also supported by our direct tests of early DRG axon repulsion by SEMA3C, in
which we find no evidence to support a role for this semaphorin. As we find
higher expression of neuropilin 2 in stage 17-20 DRGs, it remains possible
that SEMA3C plays a role in the earliest stages of axon outgrowth.
SEMA3C, in addition to SEMA3A, may also contribute to repulsion of SCG axons by the notochord, as it has the appropriate activity on SCG axons and is expressed in the notochord. We also found a diminution of repulsion when notochord-SCG co-cultures were treated with neuropilin 2/Fc (repulsion score increased from 0.2±0.1 to 1.2±0.4), although this was not statistically significant at the 1% level (P<0.012). This may reflect the difficulty in scoring small differences in repulsion in cultures showing strong repulsion.
It has also been reported that SEMA3F, a neuropilin 2-binding semaphorin,
is expressed in the perinotochordal region
(Eckhardt and Meyerhans,
1998). However, olfactory bulb axons are not repelled by the
notochord but are repelled by SEMA3F (de
Castro et al., 1999
), further suggesting that neuropilin 2 does
not mediate notochord repulsion. Again, this is consistent with the absence of
defects in the early guidance of peripheral spinal nerves in mice lacking
neuropilin 2 (Giger et al.,
2000
). The role of SEMA3F in vivo remains somewhat unclear as it
has been recently reported that mouse DRG axons are repelled by Sema3F in
vitro (Dionne et al., 2002
).
It is also of particular note that both SEMA3A and SEMA3C are expressed in
posterior half-somites (Eickholt et al.,
1999
; Shepherd et al.,
1996
), a well-characterised repulsive barrier to peripheral spinal
nerves (Tannahill et al.,
2000
). As it is thought that peripheral nerve segmentation is
based on contact repellents, rather than secreted repellents, any contribution
of semaphorins to this repulsive mechanism will probably be short- rather than
long-range.
The role of other molecules in axon repulsion by the notochord
The apparently normal pathfinding of peripheral spinal axons with respect
to the notochord in mice lacking Sema3a, neuropilin 1 or
neuropilin 2 would also suggest that factors other than semaphorins
mediate axon repulsion by the notochord. Consistent with this, our results
show that neuropilin 1/Fc is unable to inhibit a large proportion of the
repulsion of stage 26-27 DRG axons by the notochord. In these experiments, it
is probable that the neuropilin 1/Fc reagent is not saturated as it can almost
completely inhibit repulsion by the notochord of stage 36 DRG axons.
Furthermore, retinal axons, which do not express neuropilins
(Takagi et al., 1995;
Takahashi et al., 1998
), are
still repelled by the notochord. This latter result might be explained by
secretion of molecules from the notochord that do not normally function as DRG
axon repellents. For example, although we have previously shown that sonic
hedgehog, which is strongly expressed by the notochord, is unable to repel DRG
axons in collagen gel experiments (Keynes
et al., 1997
), in some situations it can interfere with the growth
of retinal axons (Trousse et al.,
2001
). Similarly, Slit proteins can repel retinal but not DRG
axons (Erskine et al., 2000
;
Niclou et al., 2000
;
Ringstedt et al., 2000
), and
they are also expressed by the notochord
(Holmes and Niswander, 2001
;
Li et al., 1999
). Our finding
that retinal but not olfactory bulb axons are repelled by the notochord
suggests, however, that notochord repulsion is not mediated by Slit2.
Clearly, the simplicity of axon repulsion by the notochord at the
anatomical level is underlain by a considerable complexity at the molecular
level. Although our results implicate SEMA3A, the precise identity and
relative contributions of the relevant molecules remain to be elucidated. If
many different molecules are involved, the use of multiple knockouts may be
required to assess the individual contribution of each molecule in the absence
of others. In addition to long-range axon repellents described in this paper,
a full characterisation of midline repulsion by the notochord will also need
to consider molecules that act at short-range, in the immediate vicinity of
the notochord. For example, the perinotochordal matrix is rich in
extracellular matrix components, such as chondroitin sulphate proteoglycans
(Landolt et al., 1995;
Newgreen et al., 1986
;
Pettway et al., 1990
;
Ring et al., 1996
), which are
known to inhibit axon growth in other systems
(Anderson et al., 1998
;
Becker and Becker, 2002
;
Chung et al., 2000
;
Dou and Levine, 1994
;
Fichard et al., 1991
;
Silver, 1994
).
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ACKNOWLEDGMENTS |
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