MRC Centre for Developmental Neurobiology, 4th Floor New Hunt's House, King's College, Guy's Campus, London SE1 1UL, UK
* Author for correspondence (e-mail: sarah.guthrie{at}kcl.ac.uk)
Accepted 11 August 2005
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SUMMARY |
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Key words: Motoneurons, Hindbrain, Slit, Robo, Repulsion, Rat, Chick
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Introduction |
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The floor plate produces repulsive signals
(Guthrie and Pini, 1995),
ensuring that motor axons and cell bodies do not cross the midline, and that
BM/VM axons grow dorsally. Two molecules thought to mediate this effect are
netrin 1 and semaphorin 3A (Sema3a), both of which repel BM/VM axons in
collagen gel co-cultures, while only Sema3a repels the SM population
(Colamarino and Tessier-Lavigne,
1995
; Varela-Echavarría
et al., 1997
). However, there is no definitive evidence to suggest
that netrin 1 or Sema3a operate in vivo to shape cranial motor axon pathways.
Netrin 1 is expressed by the floor plate
(Kennedy et al., 1994
), but in
netrin 1 mutants, no motor axon pathfinding defects have been reported
(Serafini et al., 1996
). No
central defects in motoneuron projections have been reported in mice mutant
for Sema3a or its receptor neuropilin 1
(Taniguchi et al., 1997
;
Kitsukawa et al., 1997
).
Moreover, Sema3a is not expressed by the hindbrain floor plate and therefore
cannot account for the repulsive effects of this tissue in vitro
(Varela-Echavarría et al.,
1997
; Chilton and Guthrie,
2003
), although it is expressed by the mesenchyme underlying the
hindbrain in the chick (Anderson et al.,
2003
) and in the mouse (V.V., M. Studer and S.G., unpublished). It
therefore remains unknown whether additional chemorepellents are involved in
motor axon repulsion.
We have therefore investigated the roles of the Slit guidance molecules and
their Robo receptors in cranial motor axon guidance. In Drosophila,
the Slit axon guidance molecules are important regulators of midline crossing
(Kidd et al., 1999;
Rajagopalan et al., 2000
;
Simpson et al., 2000
), and in
vertebrates Slit proteins are involved in the guidance of several groups of
axons, including post-crossing commissural axons, cortical axons and retinal
axons (Zou et al., 2000
;
Nguyen Ba-Charvet et al.,
1999
; Shu et al.,
2002
; Plump et al.,
2002
). Evidence that Slit2 repels spinal motor axons (SM) was
obtained in rodents (Brose et al.,
1999
), but the effects of Slit proteins on cranial motoneurons
have not been tested.
In this paper, we show that expression patterns of Slit and Robo genes are
consistent with their playing a role in cranial motor axon pathfinding. Using
a well-established culture system for rat cranial motoneurons (see
Caton et al., 2000), we find
that Slit1 and Slit2 inhibit and repel the axons of dorsally projecting
(BM/VM), but not ventrally projecting (SM), axons in vitro. Mice deficient in
Robo or Slit gene function show axon navigation defects, with motor axons
projecting aberrantly into or across the midline. In order to test the effects
of focal overexpression of Slit proteins or of dominant-negative Robo
receptors, we performed electroporation experiments in chick embryo
hindbrains. Overexpression of Slit in chick hindbrains caused axon
navigation errors, while BM/VM motor axons, which expressed dominant-negative
Robo proteins, did not project away from the floor plate, and failed to exit
the hindbrain correctly.
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Materials and methods |
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Collagen gel co-cultures
E12 rat embryo hindbrains were dissected into bilateral explants of the
ventral third of the neuroepithelium, which contains motoneuron somata, and
the floor-plate region, at the levels of rhombomere (r) 1, r2/3, r4/5, r6 and
r7/8 (Naeem et al., 2002).
Spinal cord explants were unilateral, and from thoracic levels. In some
experiments motoneuron subpopulations were labelled before dissection using
fluorescent axon tracers (Caton et al.,
2000
).
HEK293T cells were transfected with full-length human myc-tagged Slit
expression constructs (hSlit1, hSlit2 or hSlit3; kind gift of Dr S. Sakano,
Asahi Kasei Corporation) in pcDNA3.1 (Invitrogen)
(Itoh et al., 1998) and made
into clusters as described previously
(Varela-Echavarría et al.,
1997
). Transfection was confirmed using antibody staining against
Myc epitopes on the fusion proteins, while mock-transfected cells served as
controls. Cell clusters were made in hanging drops and co-cultured with
explants in collagen gels for 48 hours as previously described
(Guthrie and Lumsden, 1994
;
Caton et al., 2000
), in medium
supplemented with heparin sulphate (50 ng/ml; Sigma, UK)
(Brose et al., 1999
;
Hu, 2001
).
Immunohistochemistry on collagen co-cultures was performed using
anti-neurofilament antibody for levels r2-8 (2H3, 1:100, DSHB, USA), or
anti-SC1 antibody for trochlear r1 explants (F84.1, 1:20, kind gift of W.
Stallcup) as described previously (Caton et
al., 2000
).
Assessment and quantitation of axon outgrowth in collagen gel co-cultures
All assessments of axon outgrowth were carried out blind. The response of
F84.1-stained trochlear axons to the cell clusters was determined by measuring
the angle between the lateral edge of the explant, and a line drawn down the
centre of the fan of projecting trochlear axons (Scion Image programme) on
both sides of the explant. In cases where the lateral explant edge was not
straight, a line of best fit parallel to the floor plate was drawn. The `angle
difference' was calculated as the angle for the opposing side minus that for
the facing side. Differences between data sets were evaluated using Student's
t-test (two-tailed).
R2-8 explants were observed under phase contrast after 48 hours and
semi-quantitative analysis was performed using a 0-5 index as described
previously (Caton et al., 2000)
where 0 indicates no axonal outgrowth and 5 indicates maximal axonal
outgrowth. A net score (facing score opposing score) was determined
for each explant and statistical analysis of the data sets was performed using
the Mann-Whitney U-test. Quantitative analysis (Scion Image programme) was
carried out on neurofilament-stained explants, and involved counting the
number of pixels surrounding each half of the explant. The number of pixels on
the facing side was expressed as a percentage of the total number of pixels
and results were tested statistically using the two-tailed t-test.
For dextran-labelled explants, the number of axons facing towards the cell
cluster was expressed as a percentage of the total number of labelled axons
and group comparisons were performed using Student's t-test
(two-tailed).
Analysis of Robo and Slit mutant mice
A Cre-flox strategy was used to generate a frame-shift mutation in the
Robo1 or Robo2 gene, which induced a stop codon, and
consequently led to mRNA decay and a `null' phenotype. Exon 5 (an Ig domain)
of Robo1 or Robo2 on a mouse BAC was floxed and used to
generate a targeting vector (Southern or Western blot analyses were performed
using standard techniques). ES cell cultures and generation of mice was
carried out as previously described
(Mombaerts et al., 1996).
Founders were then mated with mice expressing Cre (under the actin promoter),
yielding mice lacking the exon 5 cassette. Genotypes were assessed using PCR
analysis primers and conditions are available on request. Sequence
analysis from E14 tissue samples confirmed the frame shift, and in situ
hybridisation showed an absence of Robo1 or Robo2 mRNA in
the spinal cord (data not shown). Absence of Robo1 or Robo2 protein was
confirmed by western blot (data not shown). Slit1 and Slit2
mutant mice were a gift of Dr M. Tessier-Lavigne and have been described
previously (see Plump et al.,
2002
; Bagri et al.,
2002
). In E11.5 embryos, dorsally projecting cranial motoneurons
were labelled by injecting the lipophilic dye DiI into the cranial sensory
ganglia [trigeminal, geniculate, petrosal and nodose to label respectively the
trigeminal, (r2/r3) facial (r4/r5), glossopharyngeal (r6) and vagus (r7/8)
motoneurons] as described previously
(Guthrie and Lumsden, 1992
).
Briefly, hindbrain motor axons extend through, or in association with, these
ganglia and therefore DiI injected into these regions labels the entire
motoneuron via the membrane. As motoneurons are the only neurons with cell
bodies within the hindbrain that extend axons via the ganglia, one can
unequivocally say that motoneurons are labelled by this process (see
Fig. 7E). Neurons and their
entire axons are nicely shown by this labelling process, and therefore even
when labelling multiple exit points simultaneously it is possible to clearly
see whether motor axons from a particular axial level target to their correct
exit point (e.g. whether r3 axons exit at r2; see
Fig. 7D).
Electroporation of chick embryos in ovo
Hens' eggs were incubated to stage 10-11 and processed according to Momose
et al. (Momose et al., 1999).
The fourth ventricle was microinjected with the appropriate DNA construct:
hSlit1-myc or GFP, truncated
Robo1
-GFP or truncated
Robo2
-GFP or myristylated GFP, each
regulated by a ß-actin promoter with a CMV enhancer. The truncated
Robo1
-GFP and Robo2
-GFP
transcripts consisted of the extracellular and transmembrane domain, but with
the cytoplasmic domain deleted and a GFP tag substituted. Embryos were
incubated to stages 17-19 and immunohistochemistry was performed as described
previously (Guidato et al.,
2003
) using anti-SC1 (DSHB, USA; 1:10), anti-Myc (Autogen
Bioclear, UK; 1:100), anti-islet1/2 (4D5, DSHB, USA; at 1:100) and
anti-neurofilament H (1:600, Chemicon, UK).
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Results |
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Slit proteins are expressed by the floor plate and the rhombic lip
In E11 and E12 rat embryos, we observed Slit1 and Slit2
expression within the floor plate at all axial levels
(Fig. 1B,C; data not shown).
Retrograde labelling of BM/VM or SM neurons in combination with in situ
hybridisation for Slit1 showed no expression by motoneurons
(Fig. 1D,E). Slit2 was
also expressed throughout the floor plate, and in the caudal hindbrain (r5-8)
in the regions of the differentiating SM neurons, as has been observed in the
spinal cord (Fig. 1C)
(Zou et al., 2000).
Slit2 (and to a lesser extent Slit1) was also expressed in the
rhombic lip, which lies lateral to the motor exit points
(Fig. 1B,C). In situ
hybridisation for Slit2 following retrograde dextran labelling of
BM/VM cranial motoneurons clarified that there was no overlap
(Fig. 1F), but retrograde
labelling of SM neurons confirmed that they express Slit2
(Fig. 1G). Slit3
expression at E11 and E12 was similar to that of Slit2, but with no
detectable expression in the rhombic lip, or in SM neurons (data not shown).
In the chick embryo (stage 18), Slit1 and Slit2 were
expressed in the floor plate, and Slit2 was expressed in the rhombic
lip, consistent with previous data (data not shown)
(Gilthorpe et al., 2002
).
Thus, Slit genes are expressed within the floor plate, consistent with a role in motor axon chemorepulsion from the midline, while Slit2 expression in the rhombic lip might `hem in' motor axon projections dorsally.
Robo proteins are expressed broadly in the neuroepithelium and by motoneurons
At E11, Robo2 but not Robo1 was expressed in columns
corresponding with differentiating motoneurons
(Fig. 1I; data not shown). At
E12, Robo1 and Robo2 expression domains were similar,
including the ventral half of the neuroepithelium and differentiating
motoneurons (Fig. 1H,J). Axon
tracing followed by in situ hybridisation and vibratome sectioning
demonstrated that dorsally projecting BM/VM neurons and ventrally projecting
SM neurons express Robo1 and Robo2
(Fig. 1K,L,M,N). In the chick
hindbrain (stage 18), expression of Robo1 and Robo2 also
encompassed the region of differentiating cranial motoneurons (data not
shown).
Immunostaining using the S3 anti-Robo1/2 antibody
(Sundaresan et al., 2004) and
anti-neurofilament staining showed that both dorsally projecting motor axons
(e.g. trigeminal) and ventrally projecting somatic motor axons (e.g.
hypoglossal) express Robo proteins (Fig.
1O-R). Our expression data therefore demonstrate that at the mRNA
level nascent motoneurons express Robo2 at E11 and then both
Robo1 and 2 at E12, while Robo1 and/or Robo2 proteins are
expressed by SM and BM/VM populations at E12. These expression patterns are
consistent with the idea that Robo proteins (and in particular Robo2 at early
stages) might transduce a Slit signal that drives motor axons away from the
floor plate and later limits their trajectories dorsally.
Slit1 and Slit2 repel cranial motor axons in vitro
Collagen gel co-cultures were used to investigate the responses of cranial
motoneurons (in bilateral E12 rat hindbrain explants) to cell clusters
secreting Slit1-Slit3 (Fig.
2A). Previous studies have shown that these explants contain a
high proportion of motoneurons, which project from the lateral sides of the
explant (Guthrie and Pini,
1995; Varela-Echavarría
et al., 1997
; Caton et al.,
2000
). Explants were dissected from axial levels containing the
trochlear nucleus (r1), trigeminal nucleus (r2/3), facial and abducens nuclei
(r4/5), glossopharyngeal nucleus (r6), vagus, and cranial accessory and
hypoglossal nuclei (r7/8) (Fig.
2A, compare with Fig.
1A). These were grown in collagen gels with their lateral sides
400-500 µm away from clustered cells that had been transiently transfected
with Slit1, Slit2 or Slit3, or mock-transfected as controls.
Following 48 hours incubation, r2-8 explants were immunostained using
anti-neurofilament antibodies, while those from r1 (trochlear) levels were
immunostained using the anti-F84.1 antibody.
Semi-quantitative assessment of outgrowth from r2-8 explants was made under phase contrast, scoring each explant quadrant on a 0-5 index (see Materials and methods). In the presence of Slit1 and Slit2-secreting cell clusters, explants showed a consistent tendency to extend more axons from the side facing away from the cluster, indicating inhibition of growth by Slit proteins (Fig. 2B,C). Explants cultured in apposition to mock-transfected cells showed no inhibition of outgrowth or slight inhibition (Fig. 2E). The degree of asymmetry was analysed by subtracting the outgrowth score away from the cluster from that towards the cluster and deriving a percentage of explants showing each net score (see Fig. 2F-M). Values for explants cultured with Slit1/2 cells were consistently shifted to the left for all axial levels (i.e. showing more inhibition) compared with the corresponding control group. This inhibition was statistically significant for all axial levels, and appeared to be more pronounced for cultures with Slit2 (Fig. 2; see legend for P values).
Further quantitation of axon outgrowth on separate batches of r2-8 immunostained explants was performed by counting pixels on the side facing towards and away from the cell cluster (see Materials and methods; Fig. 2D), and showed a significant level of outgrowth inhibition (Fig. 2D) by Slit1/2 for all axial levels. At the trigeminal (r2 and 3) level, this inhibition was particularly striking, as Slit1- and Slit-2-exposed explants had a mean of 31% and 27%, respectively, of outgrowth present on the facing side, both of which resulted in a P value of less than 8x108 when compared with the control group (Fig. 2B,C,E). For explants from other axial levels, Slit1 and Slit2 inhibited outgrowth with a mean of 33-36% of pixels on the facing side, thus showing a significant difference compared with controls.
Slit3-secreting cells did not cause any inhibition of outgrowth at any
axial level (data not shown). The expression constructs for all three Slit
proteins differed only in their inserts, while transfection levels were
similar for all three constructs (data not shown), and previous work using an
identical transfection system and the same constructs showed that the protein
yield from cells transfected with Slit3 was similar to that seen with Slit1
and Slit2 (Patel et al.,
2001). Therefore, technical differences are unlikely to explain
this result, pointing to a bona fide lack of response to Slit3.
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Individual motoneuron subpopulations were also labelled via their dorsal (BM/VM) or ventral (SM) exit points using fluorescent dextran tracers, and the mean percentages of axons facing the cell cluster were quantitated. We found that for cultures with mock-transfected cells, BM/VM axons displayed symmetrical outgrowth, while Slit proteins inhibited BM/VM axon outgrowth from all axial levels (Fig. 3E,K,L,N,O). This represents significant inhibition, which was greatest for trigeminal levels (see Fig. 3 legend), consistent with the results obtained by quantitating total axon outgrowth from r2/3 explants. However, abducens (r5) and hypoglossal (r8) ventrally projecting SM neurons extended axons towards Slit-secreting cell clusters without impediment, showing equal outgrowth from towards and away-facing explant borders (Fig. 3H,J,M). Thus, ventrally projecting SM cranial motoneurons failed to respond to the Slit proteins.
We also performed co-cultures of SM spinal cord explants with
Slit-secreting cells and observed radial outgrowth that was indistinguishable
from that in the presence of mock-transfected cell clusters
(Fig. 3C,F,H,I). This result is
different from that obtained by (Brose et
al., 1999), who showed inhibition of spinal SM axon outgrowth by
Slit. Taken together, our results show that Slit1 and Slit2 inhibit and repel
cranial BM/VM axons, but not cranial or spinal SM axons. Trochlear axons
project dorsally, despite being classified as SM subclass, and in our assays
behave more like BM/VM axons (i.e. show repulsion). Of course, this result
does not preclude a role for Slit/Robo proteins in other aspects of SM
neuronal development, while a possible role in axon pathfinding of the
oculomotor nerve (not tested here) remains possible.
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Disruption of this pattern of axon projections was observed in mice
deficient in Slit or Robo gene function. DiI labelling showed that there was
no overall reduction in numbers of motoneurons relative to wild-type embryos,
and that axons targeted normally to their exit points. Cranial motor axon
pathways in Slit1/ mutants resembled those
in wild-type animals, as previously reported for other projections
(Plump et al., 2002;
Long et al., 2004
). However,
defects were detected in
Slit1/;Slit2/
animals, in which axons within r4/5 failed to cross the floor plate directly,
and instead projected for short distances longitudinally between fascicles
(Fig. 4C), or entered the floor
plate and failed to exit (Fig.
4D). Similar defects were observed in Robo1/2 double
heterozygotes (data not shown). In Robo1/ or
Robo2/ mutants, more severe defects were
observed, and while the projection pattern viewed at low power was grossly
normal (Fig. 4G), at r4/5
levels axons entered the midline and projected rostrally and caudally within
the floor plate, forming long fascicles
(Fig. 4E,F). Defects of motor
axon pathfinding also occurred at other axial levels. Within r2/3, some
trigeminal axons projected longitudinally between fascicles, rather than
growing laterally towards their exit point
(Fig. 4H), and at r2/3 and r6
levels, axons crossed or looped back across the midline
(Fig. 4I,J). Ectopic
projections were quantitated in a subset of embryos of each genotype
(Table 1). For Slit1/2
mutants, Robo1/2 double heterozygotes and Robo1 mutants, the
numbers of axons crossing the floor plate at r4/5 level were similar to those
in wild-type embryos. However, for Robo2 mutants these numbers were strongly
increased. As IEE axons normally cross the floor plate at r4/5, excessive
crossing is likely to represent facial BM axons. In addition, axons were seen
projecting longitudinally within the floor plate in all genotypes, with the
greatest numbers seen in Robo2 mutants. Embryos in which BM or IEE
neurons only were labelled showed longitudinal axon fascicles, suggesting that
both populations exhibit this behaviour.
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Electroporation of Slit or dominant-negative Robo proteins in vivo causes motor axon pathfinding errors
We next tested the effects of overexpression of Slit1, or of
dominant-negative Robo1 or Robo2 expression plasmids in
chick hindbrains in ovo. These plasmids were electroporated into chick embryos
at stages 10-11, and embryos were harvested at HH stage17-19 to examine motor
axon pathways. A Slit1-Myc construct was used, or GFP as a
control, and anti-SC1 antibodies were used to detect early motor axon
projections (Simon et al.,
1994).
In control GFP electroporations, axons projected normally at all axial levels on both the electroporated and non-electroporated sides of the embryo; at r3 and r5 levels axons curved rostrally towards their exit points in r2 and r4, respectively (Fig. 5A). By contrast, motor axons showed misprojections within the Slit1-Myc-expressing region. At r3 and r5 levels, some axons projected caudally to the r4 or r6 exit point, respectively, rather to their correct, rostral exit point (Fig. 5B-D; n=12/12 for r3; n=12/16 for r5). At r6 level, glossopharyngeal motor axons displayed distinct misprojections within the region of ectopic Slit1-Myc expression, overshooting their dorsal exit point, and forming tangles (Fig. 5E,F; n=14/14). These phenomena was also observed at r7/8 levels (n=4/4; data not shown). At all axial levels, axon stalling was also frequently observed within the Slit-Myc expressing domain (Fig. 5D; n=14/20), with axons failing to reach their exit points; this suggests that motor axon growth is inhibited in response to ectopic Slit protein in vivo.
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By contrast, chick hindbrains electroporated with either the
Robo1-GFP or the Robo2
-GFP
constructs showed dramatic axon guidance defects in the BM/VM axon pathways.
Anti-neurofilament immunostaining confirmed that these defects were not due to
global effects on hindbrain axon pathways
(Fig. 6F). The majority of
Robo1
-GFP-expressing motor axons failed to project away from
the midline, and instead stalled or projected parallel to the floor plate
(Fig. 6B,C). Axons that did
project dorsally had wandering or looping trajectories, and very few
Robo1
-GFP-expressing motor axons reached their dorsal exit
points (<20 in n=5 embryos at r2/3 level; <15 in n=7
embryos at r4/5 level; 0 in n=7 embryos at r6 level). Embryos
containing Robo2
-GFP-electroporated motor axons showed very
similar defects to those described above
(Fig. 6D-F; n=19/19
cases).
When electroporated hindbrains were viewed at higher magnification, myr-GFP
labelled axons extended away from the floor plate
(Fig. 6J), except at r4/5 axial
level, where floor plate-crossing axons of IEE neurons were found. Analysis of
embryos expressing dominant-negative Robo proteins showed that electroporated
motoneurons resided in the floor plate at many levels other than r4/5, and
their GFP-labelled axons often behaved abnormally. Many axons extended into
the floor plate and either grew longitudinally, crossed to the other side, or
looped back to the side of origin (Robo1-GFP 31 axons
in 9 embryos; Robo2
-GFP 14 axons in 8 embryos;
Fig. 6G-J). These data suggest
that Robo1
-GFP-expressing neurons/axons do not
respond to repellent guidance cues found at the midline.
To determine whether electroporated BM/VM axons were able to traverse their
exit points into the periphery, we immunostained transverse cryosections of
electroporated hindbrains with anti-Islet1/2 and anti-neurofilament
antibodies. In these sections, axons expressing myr-GFP could be
observed projecting from both ventral (SM) and dorsal (BM/VM) exit points into
the periphery (Fig. 6L). By
contrast, Robo1-GFP and
Robo2
-GFP-expressing axons projected via ventral and not
dorsal exit points (Fig.
6K,M,N; insets). A small proportion of
Robo1
-GFP and
Robo2
-GFP-expressing motor axons were seen to project
dorsally (as in the flat-mounted preparations) but did not traverse the exit
point. The most likely interpretation of these results is that a lack of
Slit-Robo signalling not only hampers the ability of motor axons to pursue an
initial trajectory away from the midline, but also impairs their ability to
locate and traverse the dorsal exit points.
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Discussion |
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Expression, in vitro and in vivo studies are consistent with a repellent role for Slit
This study has demonstrated that Slit1 and Slit2 are
expressed by the floor plate and the rhombic lip, consistent with previous
studies (E9.5-E11.5) (Brose et al.,
1999; Holmes et al.,
1998
; Yuan et al.,
1999
; Zou et al.,
2000
; Gilthorpe et al.,
2002
). Our study is the first to show that cranial motor
populations express Robo1 and Robo2 at early stages in the
rat (Fig. 7A).
|
|
BM/VM cranial motor axons fail to exit the midline or the hindbrain when Slit-Robo signalling is attenuated
Experiments in which Slit-Robo signalling was attenuated either in mouse
mutants or using dominant-negative approaches in the chick showed strikingly
similar phenotypes. In Slit1/2 double mutants, signalling via Slit3
would be expected to occur (although our in vitro experiments in the rat
suggest that Slit3 is not repulsive), whereas we were unable to generate
Robo1/2 double mutants. Thus, we did not assess motor axon
projections in mice in the complete absence of Slit-Robo signalling. However,
attenuating this signalling might be expected to result in a randomisation of
axon projections with a loss of projections away from the midline. The most
striking phenotypes were seen in Robo2 mutants, suggesting that
Robo2 may be the crucial receptor for cranial motor axon guidance in
rodents, consistent with our observation in rat embryos of an earlier onset of
expression of Robo2 in hindbrain motoneurons
(Fig. 7D). It is interesting
that motor axon phenotypes observed at r4/5 levels were similar to those seen
in Ephb2/ mice and
Gata3+/ mice; in both cases, aberrant longitudinal
motor axons in the floor plate were attributed to IEE projections
(Cowan et al., 2000;
Karis et al., 2001
). This
raises the possibility that Gata3 might regulate levels of Robo
proteins and Eph receptors required for midline exit by IEE axons. Our
favoured interpretation is that both facial BM and IEE axons projected
longitudinally in Robo2 mutants, while aberrant midline crossing by
BM/VM axons occurred at all axial levels, but particularly r4/5.
The most striking effect of reducing Slit-Robo signalling in chicks was to
disrupt BM/VM axon projections away from the midline and prevent them from
exiting the neural tube (Fig.
7C). Therefore, Slit signalling seems to polarise motoneurons,
allowing them to extend only one laterally orientated axon. The failure of
BM/VM axons expressing the dominant-negative receptor to reach the periphery
might imply that cranial motoneurons require early and transient Slit-Robo
signalling in order to later manifest sensitivity to other cues, such as exit
point-derived signals or HGF (Guthrie and
Lumsden, 1992; Caton et al.,
2000
; Naeem et al.,
2002
). Transient Slit exposure might constitute part of a switch
that changes motor axon growth from a repulsive mode (away from the midline)
to an attractant one (towards the periphery).
Which are the chemorepellents for BM/VM and SM axons in vivo?
In vitro assays revealed a differential effect of Slit proteins on cranial
motoneuron subpopulations, with strong repulsion by Slit1 and Slit2 of BM/VM
axons, but not SM axons. Another study has reported repulsion of spinal motor
axons (though not of cranial SM axons) by Slit2
(Brose et al., 1999), and the
reasons for the discrepancy is unclear, but may be technical. Interestingly,
Patel et al. (Patel et al.,
2001
) showed that addition of Robo1-Fc to co-cultures of spinal
motor explants and floor-plate tissue did not block floor-plate-mediated
repulsion, thereby suggesting that Slit proteins were not involved in this
process. As SM neurons of the caudal hindbrain themselves express Slit2 at
early stages of development, it is possible that the endogenous Slit2
expression desensitises SM axons to exogenous ligands, as has been shown for
ephrin A proteins (Hornberger et al.,
1999
). The expression of Slit2 by this motoneuron population might
also affect its ability to respond to netrin 1, as Slit2 has been shown to
bind netrin 1 (Brose et al.,
1999
) and SM neurons do not respond to netrin 1 in vitro
(Varela-Echavarría et al.,
1997
), despite expressing the Unc5h1 receptor
(Barrett and Guthrie,
2001
).
Thus, cranial SM neurons do not respond to either netrin 1 or Slit
proteins; as SM axons exit the hindbrain ventrally and then project rostrally
or caudally in mesenchyme on either side of the notochord and close to the
midline, a lack of responsiveness to midline repellents might be a
prerequisite of the pathway. Only Sema3a has thus far been identified as a
chemorepellent for cranial SM neurons, and this ligand is not expressed by the
hindbrain floor plate, leaving undetermined which floor-plate-secreted
repellent prevents SM axons from crossing the midline
(Guthrie and Pini, 1995).
Sema3a expression by the notochord
(Anderson et al., 2003
) might
dictate the position of the longitudinal tracts within the mesenchyme.
Here, we have provided in vivo evidence that Slit-Robo signalling is involved in cranial motor axons repulsion from the midline; currently evidence of an in vivo role for netrin 1 and Sema3a in this process is lacking. The relative contribution of different repellent mechanisms remains to be evaluated.
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ACKNOWLEDGMENTS |
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