1 Department of Molecular Neurobiology, Institute of Development, Aging and
Cancer, Tohoku University, Aoba-ku, Sendai 980-8575, Japan
2 Graduate School of Life Sciences, Tohoku University, Aoba-ku, Sendai 980-8575,
Japan
* Author for correspondence (e-mail: yuji{at}idac.tohoku.ac.jp)
Accepted 29 October 2003
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SUMMARY |
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Key words: Chick, Trochlear nerve, Axon guidance, MHB, Neuropilin 2, Sema3F
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Introduction |
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In contrast to the segmental pattern of spinal nerves, each cranial nerve
shows a specific fiber organization and function. The trochlear nerve, which
is the fourth cranial nerve (IV), consists of a somatic motor component whose
nucleus is located in the rostral part of the rhombomere 1
(Lumsden and Keynes, 1989;
Mastick and Easter, 1996
). The
trochlear nerve is unique among cranial nerves in that its axons project
dorsally to decussate at the dorsal midline and exit the brain
contralaterally. During the dorsal projection the axons elongate along the
midbrain-hindbrain boundary (MHB). This trajectory suggests that the
interneuromeric boundary may affect the organization of the axonal tract
(Chédotal et al., 1995
;
Mastick and Easter, 1996
).
However, the molecular mechanism that regulates the establishment of the
trochlear axon trajectory along the MHB remains undefined.
It was reported that neuropilin 2 mutant mice exhibit
disorganization of several cranial nerves and spinal nerves. In homozygous
mutant mice, the trochlear motor axons showed aberrant trajectory, or were
missing, although their nucleus was present
(Giger et al., 2000;
Chen at al., 2000
). Trochlear
neurons express neuropilin 2, and trochlear motor axons were repelled
by Sema3F-transfected cells in vitro
(Giger et al., 2000
).
Moreover, recent paper reported that most of the trochlear nerve were absent
in Sema3F-null mutant mice (Sahay
et al., 2003
). It was thus suggested that neuropilin 2-mediated
Sema3F repulsion may guide trochlear axons.
In this study we examined the distribution of neuropilin 2 and Sema3F mRNA in chick embryos. Unexpectedly, neuropilin 2 expression was detected not only in the trochlear nucleus but also in the neuroepithelium of the dorsal isthmus. Sema3F expression was localized to the caudal part of the midbrain along the MHB, and in the rhombic lip. This expression pattern suggested that Sema3F repulsion may guide trochlear axons along the MHB. However, it was not clear if neuropilin 2 in dorsal isthmus is involved in trochlear axon guidance. To resolve this, we have used a gain-of-function approach in chick embryos, and further explored the roles of neuropilin 2 and Sema3F in trochlear axon guidance. In ovo electroporation revealed that Sema3F indeed repels trochlear axons in vivo, suggesting that Sema3F provides an inhibitory barrier to guide these axons along the MHB. By contrast, ectopic neuropilin 2 inactivated this repulsive activity, i. e. the invasion of trochlear axons into the tectum occurred after neuropilin 2 misexpression. Binding assay using a soluble neuropilin 2 has demonstrated that ectopic neuropilin 2 cancels neuropilin 2 binding activity distributed in the midbrain. We suggest that neuropilin 2 can neutralize the midbrain-evoked repulsive activity in the dorsal isthmus to navigate trochlear axons toward the decussation point. We finally propose a model in which the interaction of neuropilin 2 with its ligands plays decisive roles in trochlear axon guidance.
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Materials and methods |
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Flat-mounted embryos were observed under a fluorescence microscope (BX51, Olympus) and photographed with a cooled CCD digital camera (ORCA-ER, Hamamatsu). Fluorescence images were also captured on a confocal laser scanning unit (CSU10, Yokokawa). Three-dimensional images were reconstructed from serial z-axis images using volume rendering software (Aquacosmos, Hamamatsu). Rotated images were captured and processed by a confocal laser scanning system (FLUOVIEW, Olympus). Red fluorescent color of Alexa594 or Texas Red was converted to magenta in figures. For immunohistochemistry, embryos were fixed in 4% paraformaldehyde and embedded in paraffin wax. Sections (5 µm) were reacted with primary (3A10) and secondary (HRP-conjugated anti-mouse IgG) antibodies. The specimen was colored with 3,3'-diaminoobenzidine (DAB) substrate and counter-stained with hematoxylin.
Cloning of neuropilin 2 and Sema3F
A cDNA fragment for chick neuropilin 2 was amplified from E3 chick brain
mRNA by RT-PCR using the degenerate primers
(5'-MRMGACTGCAAGTATGAYT-3' and
5'-GTTCTCCAGTGTGGTGCAGCT-3'). A PCR product of 2.1 kb was
identified as a chick homologue of neuropilin 2 covering the b1
domain to cytoplasmic domain, and was subsequently used to synthesize a
DIG-labeled anti-sense RNA probe. This fragment was used to clone the
full-length coding region by screening an E2 chick cDNA library. The amino
acid sequence and coding sequence of neuropilin 2 were registered in the DNA
data bank (Accession Number AB090235).
A cDNA fragment for chick Sema3F was amplified from E3 chick brain mRNA
using degenerate primers (5'-ANTCTAYTTCTTCTTCCGAGAR-3' and
5'-CCAGGCACAGTANGGATC-3'). The PCR product corresponding to base
pairs 729-1611 of the coding region was subcloned into pBluescript and was
identified as the chick homologue of Sema3F after sequencing. This
0.9 kb fragment was used to synthesize a DIG-labeled anti-sense RNA probe for
Sema3F. The same fragment was used to isolate the full-length coding
region by screening an E2 chick cDNA library. The amino acid sequence and its
corresponding coding sequence were registered in the DNA data bank (Accession
Number, AB072930). The gene trees were constructed by CLUSTALW alignment
(Thompson et al., 1994).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as described previously
(Watanabe and Nakamura, 2000).
Digoxigenin- or FITC-labeled RNA probes were synthesized according to the
manufacturer's protocols (Promega; Ambion). Some specimens were subsequently
reacted with 3A10 antibody to detect axonal fibers.
Construction of expression vectors and in ovo electroporation
Chick Sema3F-coding region was inserted into the epitope tagging
vector pCMV-Tag5B (Stratagene). The coding region with a C-terminal Myc tag
was inserted into a pMiwII expression vector derived from pMiwSV, which
carries the chick ß-actin promoter and RSV enhancer
(Wakamatsu et al., 1997). The
coding region of neuropilin 2 was inserted into the pMiwIII expression vector,
modified from pMiwII at the multiple cloning sites.
Fertile chick eggs were incubated at 38°C in a humidified atmosphere.
Embryos were staged according to Hamburger and Hamilton
(Hamburger and Hamilton,
1951). In ovo electroporation was performed using stick electrodes
as described previously (Funahashi et al.,
1999
; Nakamura et al.,
2000
). The Sema3F construct (pMiwII-Sema3F-c-myc) or the
neuropilin 2 construct (pMiwIII-NP2) was mixed with the GFP expression vector
(pEGFP-N1; Clonetech) in a final concentration at 0.5 µg/µl or 1
µg/µl. DNA solution of 0.1-0.2 µl in Tris-EDTA buffer was injected
into the central canal with a micropipette. For transfection at stage 14, a
pair of electrodes (0.5 mm diameter, 1.0 mm length and 4 mm distance between
the electrodes; Unique Medical Imada, Natori, Japan) was placed on the dorsal
(anode) and ventral (cathode) sides of the embryos to achieve dorsal-specific
transfection. Unilateral transfection at stage 11 was performed as previously
described (Watanabe and Nakamura,
2000
). A rectangular pulse of 25 V for 50 ms was emitted four
times at 1 second intervals by the electroporator (CUY21, Tokiwa Science,
Fukuoka, Japan). GFP fluorescence coincides with the expression sites of the
transgene (Momose et al.,
1999
).
DiI-labeling of trochlear axons
Normal or neuropilin 2-transfected embryos were fixed with 4%
paraformaldehyde and brain tissues were dissected out. Trochlear nucleus were
immunostained using an anti-ISL1 primary antibody (40.3A4; DSHB) and the
secondary antibody (HRP-conjugated anti-mouse IgG; Jackson) without detergents
throughout the process. Incubation in DAB substrate in the presence of 0.03%
cobalt chloride faintly colors the trochlear nucleus, where small crystals of
DiI were placed. Then the specimens were incubated at 37°C until trochlear
axons were labeled anterogradely.
AP fusion protein-binding assay
A DNA fragment for neuropilin 2 ectodomain without signal sequence,
transmembrane domain and cytoplasmic domain was amplified from chick
neuropilin 2-coding region by PCR using the primers
(5'-AAGCTTTGGCGGCGGAGACAGCTCAGCCG-3' and
5'-GGATCCTGGGAAAGCTGTGATGGGTTC-3'). A
HindIII-BamHI fragment of amplified DNA was subcloned into
HindIII-BglII site of pAPtag5 (GenHunter) to produce
neuropilin 2 ectodomain fused with alkaline phosphatase at its C terminus.
This construct was transfected into 293T cells with TransFast (Promega)
according to manufacture's protocol. Cell culture supernatant containing AP
fusion protein (NP2dC-AP) was used as a probe for binding reaction as
described below.
Brain tissues were dissected out in PBS, blocked with 10% fetal bovine
serum in PBS for 5 minutes, and then incubated with 50% dilution of NP2dC-AP
for 1 hour at room temperature. After washing three times with PBS, tissues
were fixed in 4% paraformaldehyde for 1 hour, and washed three times again in
PBS. Then endogenous AP activity was heat-inactivated at 65°C for 3 hours
and AP activity was detected by NBT/BCIP staining for several hours. The
staining reaction was stopped within 1 hour for detection of abundant Sema3F
misexpressed by electroporation. The procedure was modified from Flanagan et
al. (Flanagan et al., 2000).
Some embryos were subsequently reacted with 3A10 antibody to detect axonal
fibers.
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Results |
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Expression of neuropilin 2 in trochlear neurons and neuroepithelium of the dorsal isthmus
It has been suggested that neuropilin 2 is implicated in trochlear axon
guidance. In mutant mice homozygous for neuropilin 2, the projection
of trochlear motor axons was aberrant or missing
(Giger at al., 2000;
Chen et al., 2000
). Sema3F is
the instructive agent for the neurons that express neuropilin 2, a
functional receptor for Sema3F
(Chédotal et al., 1998
;
Chen et al., 1998
;
Giger et al., 1998
). Actually,
Sema3F can repel trochlear axons in vitro
(Giger at al., 2000
). We have
isolated a chick neuropilin 2 cDNA, whose deduced amino acid sequence
is highly homologous to human and mouse neuropilin 2. Molecular phylogenetic
analysis showed the amino acid sequence clustering with human and mouse
counterparts, indicating that it corresponds to chick neuropilin 2
(Fig. 2A). We have also
isolated a chick Sema3F cDNA, whose deduced amino acid sequence is clustered
with human and mouse Sema3F (81% identity for both;
Fig. 2B).
|
|
Expression of Sema3F along the midbrain-hindbrain boundary
Subsequently, we examined the expression of Sema3F in chick
embryos focusing on the midbrain-hindbrain region. Faint expression of
Sema3F was observed throughout the ventral midbrain from stage 10-11
(Fig. 4A). The expression
became intense after stage 12 (Fig.
4B), having extended dorsally to the caudal part of the midbrain
by stage 14 (Fig. 4C). At stage
16, which is just before the emergence of the first trochlear axons
(Fig. 1B), the expression was
confined to the most caudal part of the midbrain
(Fig. 4D). During the dorsal
projection of the trochlear axons, the caudal limit of Sema3F
expression was clearly demarcated, as if it provided a linear boundary along
the MHB (Fig. 4D-F).
|
In the hindbrain, Sema3F expression was prominent in rhombomeres 3 and 5 until stage 14 (Fig. 4A-C). In the rhombic lip, Sema3F expression was detected during stages 16-20 (Fig. 4D-F). Outside of the brain, Sema3F was expressed in the otic vesicle and the optic vesicle (Fig. 4A,B).
The spatial relationship between Sema3F expression and the projection of trochlear axons was then examined. At stage 19, two lines of Sema3F expression were identified; along the MHB and in the rhombic lip (Fig. 4K). Weak expression was also detected in the most ventral part of the isthmus, medial to the trochlear neurons. During the dorsal projection, the axons elongated in the most rostral hindbrain, caudal to a line of Sema3F expression domain along the MHB (Fig. 4K-M). In the ventral isthmus, trochlear axons proceeded dorsally with some distance from the Sema3F expression domain along the MHB (Fig. 4M). In the dorsal isthmus, although Sema3F expression was very weak or missing, we could not detect any trochlear axons that cross the MHB.
In vivo repulsive action of Sema3F on trochlear axons
Assuming that Sema3F protein is an inhibitory guidance cue that provides
repulsive activity, it is likely that Sema3F excludes trochlear axons to guide
them along the MHB. The question remains, however, as to whether Sema3F
protein is repulsive in vivo for trochlear axons. To address this, Sema3F was
transfected by in ovo electroporation into dorsal isthmus during projection of
trochlear axons in the hindbrain.
First, correct translation of Sema3F protein was ascertained in vivo by
translation of Myc tag at the C terminus of the Sema3F construct.
Co-electroporation of the Sema3F plasmid (pMiwII-Sema3F-c-myc) and a GFP
vector (pEGFP-N1) allowed us to monitor the transfection by GFP fluorescence
(Momose et al., 1999).
Electroporation was carried out at stage 14 to misexpress Sema3F in the MHB
region prior to the projection of trochlear axons. In the normal brain,
trochlear axons had crossed the dorsal midline by stage 23
(Fig. 5A).
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In summary, the results show that Sema3F protein provides functional guidance activity in vivo by repelling trochlear axons during the dorsal projection.
Neuropilin 2 misexpression causes invasion of trochlear axons into tectum
As neuropilin 2 is expressed in the neuroepithelium in the dorsal
isthmus, but not in the mantle layer, it is unlikely that neuropilin 2 serves
as the ligand sensor for axons at this site. Nevertheless, its spatial
expression pattern strongly suggests that it may be involved in the guidance
of trochlear axons in the dorsal isthmus. To explore its role in the
neuroepithelium, we misexpressed neuropilin 2 in the MHB and observed the
effects on axon pathfinding.
Full-length neuropilin 2 cDNA inserted in the expression vector, pMiwIII-NP2 was electroporated into the brain at stage 11 together with a GFP expression vector (pEGFP-N1). In the transfected side at stage 22, it was notable that so many axons were deflecting and invading the tectum (Fig. 6A-C). On flat-mount specimens, it was obvious that deflecting axons originated from the ipsilateral trochlear nucleus (Fig. 6E,F), indicating that dorsally projecting trochlear axons had deflected towards the midbrain and crossed the MHB to invade the tectum (Fig. 6I). By contrast, in the untransfected side of the same embryo, trochlear axons never crossed the MHB, but extended toward the dorsal decussation point (Fig. 6D). Confocal images show that the aberrant axons were trochlear axons, and that they were distinct from the tectobulbar axons and the tract of the mesencephalic nucleus of the trigeminal nerve which extended caudally from the tectum and fasciculate to form the caudal longitudinal pathway (Fig. 6G,H). The abnormal projection of these trochlear axons to tectum was observed in most of the transfected embryos (18/20 GFP-positive embryos; Table 1). We have confirmed that neuropilin 2 misexpression did not affect Sema3F and Fgf8 expression (data not shown).
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Neuropilin 2 masks its ligand to navigate trochlear axons
The result of neuropilin 2 misexpression indicates that the inhibitory
barrier at the MHB was weakened or dismissed by the transfection of neuropilin
2 so that trochlear axons could invade the tectum. It is plausible that
ectopic neuropilin 2 has bound its ligand and canceled its repulsive activity
against the trochlear axons. To verify the possibility, we have examined the
distribution of neuropilin 2 ligand in normal and neuropilin 2-transfected
embryos.
Distribution of the ligand can be visualized by receptor-ligand interaction
with a soluble AP fusion protein (Flanagan
et al., 2000). For this purpose, neuropilin 2 ectodomain fused to
the N terminus of AP (NP2dC-AP) was prepared, and reacted with brain tissues.
First, we checked if NP2dC-AP actually detect the ligand distribution of
neuropilin 2 in vivo. After co-electroporation of GFP and Sema3F, the
GFP-positive region, i. e. the Sema3F-misexpressing region, coincided with
NP2dC-AP staining, indicating that this probe can detect the ligand
distribution in the brain (Fig.
7G,H). Then, potential neuropilin 2 ligand distribution was
examined on normal embryos with NP2dC-AP. At stage 17, when trochlear axons
began to elongate, strong AP staining was found in the whole midbrain and the
rhombic lip, but the isthmic region was devoid of staining
(Fig. 7A-C). This staining
pattern was kept until stage 19 when trochlear axons approach the decussation
point (Fig. 7D-F). Thus,
trochlear axons could proceed dorsally in the isthmic region which was devoid
of the neuropilin 2 ligand. Treatment with intact AP protein gave no apparent
staining as observed after NP2dC-AP treatment (data not shown).
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Discussion |
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Neuropilin 2 conveys chemorepulsion to trochlear axons
Neuropilin 1 and neuropilin 2 are the receptors that directly bind to class
3 semaphorins. As only neuropilin 2 is expressed in the trochlear nucleus
during the dorsal projection (Y.W., unpublished), neuropilin 2 is the only
known receptor for class 3 semaphorins in trochlear axons. However, caudal
populations of the trochlear motoneurons do not express neuropilin 2
(see Fig. 3H-L). It is thus
inferred that trochlear axons from neuropilin 2-negative caudal nucleus should
be insensitive to the repulsive activity. Accordingly, foregoing axons from
the rostral trochlear nucleus may act as pioneer axons and the axons from the
caudal nucleus may follow them. This idea is supported by the axonal phenotype
of Sema3F-transfected embryos, because both rostral and caudal trochlear axons
turned back when they met ectopically transfected Sema3F. Sema3F-insensitive
axons may have followed the preceding axons that had been repelled by the
ectopic Sema3F.
Existence of neuropilin 2 ligands as the barrier for trochlear axons
Sema3F repelled trochlear axons in vivo at a distance from the
Sema3F-transfected domain, indicating that diffusible Sema3F molecules serve
as the inhibitory barrier for trochlear axons in the midbrain by preventing
them from crossing the MHB. However, expression of Sema3F mRNA in
caudal midbrain was not obvious along the dorsal MHB. Therefore, it is not
known Sema3F is sufficient to provide the barrier all along the MHB. To
resolve this issue, we have examined the distribution of neuropilin 2 ligand,
using a soluble neuropilin 2 ectodomain-AP fusion protein as a probe. In the
hindbrain, strong AP staining was found in the rhombic lip, which coincided
with Sema3F mRNA expression. However, the staining was seen in the
whole midbrain, which was different from the line shape expression of
Sema3F mRNA in the caudal margin of the midbrain. This indicates that
another ligand(s) for neuropilin 2 should be involved in trochlear axon
guidance as the repulsive cue. The most plausible candidate is Sema3C
(formerly SemE or Collapsin 3), which is the nearest family member of Sema3F
(see Fig. 2B) and it can bind
to neuropilin 2 (Chen et al.,
1997). We have examined expression of Sema3C mRNA in
mid-hindbrain region, but we have recognized it only in the oculomotor
nucleus, which is not comparable with the broad distribution of neuropilin 2
ligand in the midbrain (Y.W., unpublished). Therefore, in chick embryo,
unidentified ligand(s) for neuropilin 2 may participate in trochlear axon
guidance together with Sema3F.
It is noteworthy that Sema3F transfection caused typical effects on
trochlear trajectory. When trochlear axons encountered ectopic Sema3F en
route, the axons ceased elongation and the distal axons turned to
neuroepithelium. It seemed as if axons avoided surrounding repulsive agents.
In fact, they were caught between the ectopic Sema3F dorsally, and the
floorplate which provides repulsive activity to trochlear axons (see
Fig. 5G-K)
(Colamarino and Tessier-Lavigne,
1995; Serafini et al.,
1996
). In some cases, these axons subsequently changed the course
to reverse direction and went out of the brain. Interestingly, in normal
embryos, distal regions of trochlear axons including growth cones similarly
converge in neuroepithelium, where neuropilin 2 is expressed. As neuropilin 2
can neutralize the repulsive activity of its ligands as discussed below,
trochlear axons may have converged in the neuroepithelium to avoid the
repellants in normal embryos. However, it remains elusive how trochlear axons
subsequently decussate and exit the brain.
Neuropilin 2 neutralize the repulsive activity of its ligands
In the neuropilin 2-transfected brain, the trochlear axons acted as if
there was no inhibitory barrier and crossed the MHB, while Sema3F
mRNA localization at the MHB was unchanged. Therefore, the aberrant axonal
projection should be due to functional loss of the inhibitory barrier at the
MHB after neuropilin 2 misexpression. It is known that neuropilin 2 can form a
homodimer or multimer and bind to Sema3F in vitro in the absence of other
components, such as plexins (Chen et al.,
1998; Feiner et al.,
1997
; Nakamura et al.,
1998
; Takahashi et al.,
1999
; Tamagnone, 1999). As exogenous neuropilin 2 binds to Sema3F
in vivo, it is plausible that the ectopic neuropilin 2 bound to endogenous
ligands in the midbrain, and masked them from the neuropilin 2-positive
trochlear axons. Consequently, the repulsive activity of the neuropilin 2
ligands might be cancelled. Without the inhibitory barrier, trochlear axons
could penetrate the MHB and invade the ipsilateral tectum region. This
interpretation is strongly supported by the result that the binding affinity
of the neuropilin 2 ligands with neuropilin 2 ectodomain was lost in
neuropilin 2-transfected midbrain. These results suggest that neuropilin 2
masked its ligands to neutralize their repulsive activity against trochlear
axons.
There is another possibility that ectopic neuropilin 2 may homophilically
interact with neuropilin 2 positive trochlear axons to navigate them into the
tectum. This idea comes from the fact that neuropilin 2 in cultured cell line
can homophilically interact with exogenous neuropilin 2 in trans
(Chen et al., 1998). If it is
true in vivo, misexpressed neuropilin 2 must interact with exogenous
neuropilin 2. However, in our experiment, misexpressed neuropilin 2 did not
bind with a soluble neuropilin 2 ectodomain and instead bound with its ligands
(see Fig. 7J). Thus, the
aberrant trochlear trajectory should be caused by the ligand-receptor binding
rather than receptor-receptor interaction.
In this context, it is particularly interesting that in normal development neuropilin 2 is expressed in the neuroepithelium of dorsal isthmus. As a membrane-bound receptor, neuropilin 2 must be immobilized on the neuroepithelial cell surface in the dorsal isthmus and be able to bind its ligands. Importantly, the ligands including Sema3F are distributed in the midbrain and the rhombic lip, adjacent to the neuropilin 2 expression domain. Based on the results of neuropilin 2 misexpression, we propose that neuropilin 2 traps repulsive cues in the dorsal isthmus and neutralizes its activity. This may provide the ligand-free space for trochlear axons to pass through the dorsal isthmus despite of being surrounded by repulsive sources.
Our results suggest that neuropilin 2 has a novel biological activity to
function in axon guidance, albeit indirectly by masking its ligands. A similar
mechanism is suggested in the modulation of ligand distribution by receptor
binding for axonal guidance in Drosophila. The Netrin receptor,
Frazzled can capture and accumulate secreted Netrin protein to navigate
photoreceptor axons (Hiramoto et al.,
2000). A common feature between the two guidance mechanisms of
Frazzled and neuropilin 2 is that the membrane-bound receptor traps diffusible
ligand on the regions where axons extend, and this process does not
necessarily induce intracellular signaling events. This may provide a general
mechanism to modify the distribution of ligand to achieve elaborate axon
guidance.
Other guidance cues for trochlear axon guidance along the MHB
It was observed that trochlear axons elongate dorsally and simultaneously
deflect in a rostral direction throughout the dorsal projection. When the
inhibitory barrier of the midbrain was blocked by neuropilin 2 misexpression,
both rostral and caudal trochlear axons deflected toward the rostral
direction. These results indicate that some attractant or repellant cue exists
for the general rostral direction of trochlear axons. In rodent embryos, broad
expression of Sema3F mRNA in the hindbrain suggest that Sema3F could
be a hindbrain-derived repulsive agent
(Giger et al., 2000). In chick
embryos, neuropilin 2 ligands seem to be diffused in the hindbrain (see
Fig. 7A,J). However, Sema3F may
be inadequate to explain the hindbrain-derived repulsion because chick
neuropilin 2 expression is absent in caudal trochlear nucleus. In
addition, this rostral guidance activity was not cancelled after neuropilin 2
misexpression.
It was reported recently that Fgf8 protein had acted as a chemoattractant
for trochlear axons in vitro and in vivo
(Irving et al., 2002). Fgf8
could be a candidate for the general rostral guidance cue for trochlear axons,
because its mRNA is localized at the rostral margin of the hindbrain along the
MHB, just caudal to Sema3F expression domain. In this context,
trochlear axons may be attracted rostrally towards the
Fgf8-expressing domain, the source of Fgf8 protein. However, after
neuropilin 2 misexpression, distribution of Fgf8 mRNA was similar to
that in the normal embryo, indicating that aberrant trochlear axons initially
extended toward the Fgf8-expressing domain, but subsequently crossed
it; i.e. the axons moved away from the Fgf8 source. This is not explained
solely by the model of Fgf8-evoked chemoattraction, as trochlear axons finally
project away from the source of Fgf8 protein. Therefore, we suspect that other
guidance cue(s) distinct from Fgf8 is also involved in the guidance of chick
trochlear axons toward the rostral direction.
Roles for neuropilin 2 and its ligands in trochlear axon guidance
Based on the results presented here, we propose a model for the trochlear
axon guidance as schematically summarized in
Fig. 8A. Trochlear axons
project dorsally in response to the repulsive signal from the floor plate
(Colamarino and Tessier-Lavigne,
1995; Serafini et al.,
1996
). During the dorsal projection, trochlear axons incline to
the rostral direction in response to unidentified rostral guidance cue(s). As
repulsive cues including Sema3F exist in the midbrain, they do not cross the
MHB, but instead elongate along the MHB. In the dorsal isthmus, trochlear
axons are surrounded by the sources of repellants in the midbrain and the
rhombic lip. Nevertheless, they can proceed to reach the decussation point, as
neuropilin 2 in the dorsal isthmus may bind to its ligands to neutralize their
repulsive activity. The caudal trochlear axons that do not express neuropilin
2 also incline to the rostral direction, and join the foregoing axons in the
dorsal isthmus to reach decussation point. Thus, when the neuropilin 2 is
misexpressed in the midbrain, the midbrain-evoked repulsive activity is
cancelled and trochlear axons deflected to the tectum by crossing the MHB
(Fig. 8B). The caudal trochlear
axons cannot join the rostral foregoing axons, because deflection seems to
have occurred in the ventral isthmus before joining, so that the caudal axons
independently invade the tectum. In this regard, neuropilin 2 has dual
functions in axon guidance: neuropilin 2 in trochlear neurons plays a direct
role in midbrain-evoked repulsive guidance along the MHB; and neuropilin 2 in
the neuroepithelium indirectly provides an axonal pathway toward the dorsal
decussation point by canceling the repulsion.
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ACKNOWLEDGMENTS |
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