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First published online 1 March 2006
doi: 10.1242/dev.02312
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1 Department of Physiological Sciences, School of Life Science, The Graduate
University for Advanced Studies (Sokendai), 5-1 Higashiyama, Myodaiji, Okazaki
444-8787, Japan.
2 Division of Neurobiology and Bioinformatics, National Institute for
Physiological Sciences, Okazaki 444-8787, Japan.
3 Department of Morphological Neural Science, Graduate School of Medical
Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
4 Department of Morphological Brain Science, Graduate School of Medicine, Kyoto
University, Kyoto 606-8501, Japan.
5 Howard Hughes Medical Institute and The Jackson Laboratory, Bar Harbor, MN
04609, USA.
* Author for correspondence (e-mail: katsono{at}nips.ac.jp)
Accepted 3 February 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Network formation, Dcc, Unc5c, RCM, Dorsal funiculus, DRG, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The central projections of DRG axons in the spinal cord are
tightly regulated spatially and temporally, and different functional types of
sensory neurons send afferents to different target areas in the spinal cord
(Snider, 1994). During
development, DRG axons enter the spinal cord at the dorsal root entry zone
(DREZ) and then grow to the marginal zone of the spinal cord longitudinally to
form the dorsal funiculus without penetrating the dorsal mantle layer. After a
few days, proprioceptive afferents, which are involved in the muscle stretch
reflex, penetrate the mantle layer and project ventrally through the dorsal
layers. Subsequently, cutaneous sensory afferents start to send collaterals
into the dorsal mantle layer and terminate in the dorsal-most laminae of the
cord (Ozaki and Snider, 1997).
Therefore, the projection pattern of DRG axons shows a delay between the
formation of the dorsal funiculus and the extension of collaterals into the
dorsal mantle layer, called the `waiting period'. Based on this growth pattern
of DRG axons, it has been presumed that repellant and/or inhibitory cues
transiently prevent sensory afferents from penetrating the dorsal spinal cord
during the waiting period (Ozaki and
Snider, 1997). In other words, inhibitory cues are apparently
required for the correct patterning of sensory afferents. For example,
semaphorin (Sema) proteins are one of the candidates expressed in the spinal
cord, and repel DRG axons in vitro
(Messersmith et al., 1995;
Shepherd et al., 1997;
Masuda et al., 2003). However,
the trajectory of dorsal root afferents is apparently normal in
Sema3a and its receptor neuropilin 1 mutant embryos
(Kitsukawa et al., 1997;
Taniguchi et al., 1997). Thus,
the functions of Sema proteins cannot fully explain the waiting period in
vivo.
Netrin 1 was originally identified in the ventral midline of the neural
tube as a bifunctional guidance molecule during early embryogenesis
(Serafini et al., 1994;
Kennedy et al., 1994); it is a
long-range diffusible factor that exerts chemoattractive or chemorepulsive
effects for distinct developing neural cells depending on the specific
combination of Dcc and Unc5 receptors, thus regulating axon outgrowth and cell
migration (Serafini et al.,
1994; Colamarino and
Tessier-Lavigne, 1995a;
Keino-Masu et al., 1996;
Ackerman et al., 1997;
Leonardo et al., 1997;
Hong et al., 1999;
Yee et al., 1999). Netrin
secreted from cells in the floor plate directs many axons to the midline
(Colamarino and Tessier-Lavigne,
1995b; Tessier-Lavigne and
Goodman, 1996). Netrin 1 is also weakly expressed in the
developing dorsal spinal cord (Serafini et
al., 1996). However, the function of dorsally derived netrin 1 is
unknown.
In this study, we examined the mechanisms regulating the neural network formation of primary sensory axons in the spinal cord. Our results demonstrate that netrin 1 is transiently expressed near the DREZ during the waiting period, and that loss of netrin 1 results in aberrant invasion of cutaneous and proprioceptive afferents into the dorsal mantle layer without first growing along the marginal zone. Furthermore, netrin 1 suppresses axon outgrowth from DRG explants in vitro. Mutation of a netrin receptor Unc5c results in the aberrant projection of DRG axons. These findings clearly demonstrate that netrin 1 expressed in the dorsal spinal cord is necessary to prevent premature extension of primary sensory axons into the mantle layer and thus serves as a crucial signal for the proper formation of sensory neural networks.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Fazeli et al., 1997). Netrin 1
mutant mice were originally generated on a CD1 background but were mated onto
C57BL6 or ICR background. Mice homozygous for netrin 1, Dcc and
Unc5crcm mutations were obtained from heterozygote matings
and were identified as described previously
(Serafini et al., 1996;
Ackerman et al., 1997;
Fazeli et al., 1997).
Gli2 heterozygous mice were kindly provided by Dr C. C. Hui
(University of Toronto) (Mo et al.,
1997). These mice in a mixed background of 129/Sv and CD1 were
mated onto an ICR background. The genotyping of Gli2-deficient mice
was performed as described previously (Mo
et al., 1997). All procedures were approved by the Animal Research
Committee of the National Institute for Physiological Sciences. The plug date was considered to be embryonic day 0.5 (E0.5). Embryos at E11.5-18.5 were harvested from anesthetized pregnant mice. For histological analysis, the embryos were fixed with 4% paraformaldehyde (PFA) in PBS overnight at 4°C and then incubated in PBS containing 20% sucrose at 4°C, embedded in OCT compound (Sakura Finetechnical). Frozen sections (18 µm) were cut on a cryostat (CM3050; Leica) and mounted onto MAS-coated glass slides (Matsunami). All analyses in this study were performed on the spinal cord at cervical and thoracic levels.
In situ hybridization
The following cDNAs were generated by RT-PCR and used as probes: mouse
netrin 1 (GenBank Accession Number, NM_008744), Dcc, Unc5a, Unc5b,
Unc5c (Sugimoto et al.,
2001), lacZ, Ebf1 (gifts from Dr H. Takebayashi),
Pax3 (a gift from Dr O. Chisaka), Math1 (a gift from Dr R.
Kageyama), Lmx1b (a gift from Dr B. Lee) and Brn3a (a gift
from Dr E. E. Turner). Digoxigenin-labeled RNA probes were synthesized using
DIG RNA-labeling kit (Roche Diagnostics). In situ hybridization was performed
as described previously (Ding et al.,
2005a). DIG-labeled RNA hybrids were reacted with alkaline
phosphatase-conjugated anti-DIG antibody (Roche). Reaction product was
visualized by incubating the sections with nitrobluetetrazolium chloride and
5-bromo-4-chloro-3-indolylphosphate (Roche).
Immunohistochemistry and X-gal staining
Cryostat sections were immunohistochemically stained as previously
described (Ono et al., 2004).
Sections were incubated with primary antibodies overnight at 4°C, and were
then processed with the ABC method (Vector Lab.), following the manufacturer's
protocol. Detection with horseradish peroxidase was performed by incubation in
0.05% diaminobenzidine and 0.015% hydrogen peroxide in PBS. For
immunofluoresence, sections were labeled with species-specific secondary
antibodies conjugated to Alexa Fluor 488 or 594 (Molecular Probes) and with
Hoechst 33342 (Sigma) to visualize nuclei. The primary antibodies used were:
mouse anti-neurofilament M (1C8; culture supernatant; 1:5), rabbit anti-TrkA
(a gift from Dr L. F. Reichardt, UCSF; 1:2000) and rabbit anti-TrkC (Santa
Cruz Biotechnology; 1:200) antibodies. Immunostaining of whole embryos and DRG
explants was performed using the same method. For double labeling, in situ
hybridization with lacZ probe was carried out, followed by
immunostaining for neurofilament. X-gal staining was performed as previously
described (Ding et al.,
2005a).
BrdU labeling
Pregnant mice were given an intraperitoneal injection of BrdU (50 µg/g
body weight; Sigma) at E10.5 or E11.5. Embryos were isolated at E12.5. BrdU
was detected by immunostaining using mouse anti-BrdU antibody (BD Pharmingen;
1:500). For quantification, we used Photoshop (Adobe Systems) to divide the
dorsal spinal cord into two, dorsal and ventral, halves, and BrdU-labeled
neurons in the mantle layer were enumerated in two halves in six sections from
three wild-type and netrin 1 mutant mice.
DiI tracing of DRG axons
To label DRG axons, E12.5 and E13.5 embryos were fixed in 4% PFA, and small
crystals of
1,1'-dioctadecyl-3,3,3',3''-tetramethylindocarbocyanine
perchlorate (DiI; Molecular Probes) were put on the DRG. After incubation for
1-2 weeks at 37°C, 60 µm transverse sections were cut with a tissue
slicer (DSK Microslicer). Free-floating sections were counterstained by
Hoechst 33342 and mounted onto glass slides.
Collagen gel culture
An in vitro assay using collagen gel culture was performed as previously
described with a slight modification
(Kennedy et al., 1994;
Masuda et al., 2003).
Recombinant Sindbis virus for the expression of netrin 1 or EGFP was used, and
BHK cell aggregates were prepared as previously described
(Furuta et al., 2001;
Sugimoto et al., 2001). DRG
was dissected from the E13.5 ICR mouse embryos. DRG explants and BHK-cell
aggregates were embedded in rat-tail collagen gels separated by a distance of
200-1000 µm. The explants in gels were incubated for 24-48 hours in DMEM
containing 10% FBS, 50 ng/ml 7S nerve growth factor (NGF; Chemicon) and 50
ng/ml neurotrophin 3 (NT3; Sigma) at 37°C under 5% CO2.
Anti-netrin 1 rabbit polyclonal antibody (Oncogene Research Products) was
added at a concentration of 2 µg/ml to neutralize the effect of netrin 1
secreted from BHK cells. After incubation, these explants were fixed with 4%
PFA overnight at 4°C and placed in PBS for immunostaining. At least three
independent cultures were carried out for statistical analysis (see
below).
Quantification
For quantification of the disorganization of the dorsal funiculus, areas
occupied by the dorsal funiculus formed in the marginal zone and ectopic axon
bundles invading the mantle layer were measured by tracing the edges of
bundles, which were identified by staining with neurofilament antibody on
transverse sections of the spinal cord using ImageJ software (NIH). The
boundary between the marginal zone and the mantle layer of the dorsal spinal
cord was identified by NeuroTrace fluorescent Nissl stain (Molecular Probes).
Eighteen sections from three embryos were quantified.
To quantify the axon outgrowth from DRG explants, the total axon surface
area out of the explants stained by anti-neurofilament antibody was measured
using Photoshop (Adobe) and Matlab software (Media Cybernetics), and analyzed
using Student's t-test. In addition, axons from DRG explants
co-cultured with BHK cells were grouped into four quadrants: proximal (p),
distal (d) and two lateral quadrants. The data were expressed as the ratio
between the area of axons present in the proximal and distal quadrants (p/d
ratio) (Masuda et al.,
2003).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
At E11.5, when many DRG axons have reached the DREZ, netrin 1 mRNA was not
detected dorsally, although its signal was detected intensely in the floor
plate and the ventral ventricular zone
(Fig. 1A). At E12.5, when
sensory axons grow longitudinally along the dorsolateral margin of the cord,
and few collaterals enter the dorsal gray matter, netrin 1 expression was
observed in the dorsolateral region adjacent to the DREZ
(Fig. 1B,D). Signal intensity
seemed to be weaker in the dorsal spinal cord than in the floor plate or even
in the ventricular zone. In the E12.5 DRG, netrin receptors Unc5a and
Unc5c were expressed, whereas Dcc and Unc5b
transcripts were not detectable (Fig.
1F-I). At this stage, all receptors were expressed in the spinal
cord as reported by others (data not shown)
(Keino-Masu et al., 1996;
Ackerman et al., 1997;
Leonardo et al., 1997). In the
E13.5 spinal cord, when many collaterals have entered the mantle layer, netrin
1 mRNA intensity in the dorsal spinal cord was decreased when compared with
that at E12.5 (Fig. 1C,E).
Therefore, netrin 1 is transiently expressed or upregulated in the dorsal
spinal cord during the period of sensory axon outgrowth but prior to the entry
of these axons into the gray matter, suggesting that netrin 1 may have a role
in primary sensory axon patterning in the spinal cord.
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Next, the axonal pattern was visualized on transverse sections of the
spinal cord. In the E11.5 wild-type mice, the dorsal funiculus was formed in
the dorsolateral margin of the spinal cord (data not shown). Subsequently, the
dorsal funiculus developed well as a crescent-shape with a sharp inner border
facing the dorsal mantle layer, and very few collaterals invaded the mantle
layer at E12.5 (Fig. 2C). By
E13.5, collaterals had begun entering the mantle layer from the dorsomedial
region of the dorsal funiculus, projecting ventrally
(Fig. 2G). This projection
pattern was compatible with a previous observation by Ozaki and Snider
(Ozaki and Snider, 1997). In
netrin 1 mutant mice at E11.5, the formation of the dorsal funiculus was
almost identical to that observed in wild-type animals except for slight
dorsal extension of the funiculus (data not shown). At E12.5, the dorsal
funiculus was dramatically defasciculated with an unclear inner border, and
neurofilament-positive axons dispersed within the superficial part of the
dorsal mantle layer (Fig. 2D).
As the netrin 1 mutant allele harbors a lacZ insertion, lacZ
expression recapitulates the pattern of endogenous netrin 1
(Serafini et al., 1996;
Charron et al., 2003). We
performed immunostaining for neurofilament after in situ hybridization with a
lacZ probe in netrin 1 heterozygous and homozygous mice
(Fig. 2E,F). In netrin
1-deficient mice, many axon bundles directly invaded the
lacZ-expressing zone of the dorsal spinal cord
(Fig. 2F), whereas very few
axons entered the netrin 1-lacZ-expressing areas in heterozygous
animals (Fig. 2E). The
quantitative analysis demonstrated that the dorsal funiculus in the correct
superficial position was markedly decreased in netrin 1-deficient mice, less
than in heterozygous or wild-type animals at E12.5
(Table 1). By contrast,
aberrant projections in the dorsal gray matter were more common than normal
projections in the mutants. The dorsal funiculus in netrin 1 heterozygous mice
was normally formed with a sharp inner border, and was only slightly thinner
than that of wild-type mice (Table
1). At E13.5, the dorsal funiculus in the marginal zone of netrin
1 mutant mice is thinner than that of wild-type mice
(Fig. 2G,H). Moreover, thick
axon bundles were observed within the dorsal mantle layer in netrin 1 mutants
(Fig. 2H). The ectopic axon
bundles extended towards the ventricular zone and turned dorsally. In spite of
the disorganization of the dorsal funiculus, some neurofilament-positive
fibers extended ventrally from the ectopic dorsal funiculus as observed in the
normal spinal cord (Fig. 2G,H).
Disorganization of the dorsal funiculus was observed throughout the spinal
cord from E12.5 onwards, and the extent of disorganization became more severe
as the embryos matured (Fig. 2,
see Fig. S1 in the supplementary material).
|
). In
netrin 1 mutant mice, longitudinal tracts were established as observed in
wild-type mice (Fig. 3B). We next examined axonal trajectories on transverse sections. In the E12.5 netrin 1 mutants, DRG axons stacked near the DREZ, and some axons entered the lateral part of the dorsal gray matter (data not shown). The defects of axonal patterning were more severe at E13.5 than at E12.5 (Fig. 2). At E13.5, the dorsal funiculus had been formed in the dorsolateral margin of the wild-type spinal cord (Fig. 3C). By contrast, thick axon bundles directly invaded the dorsal mantle layer without elongating along the dorsolateral margin of the cord in netrin 1-deficient mice, as observed in neurofilament staining (Fig. 3D).
|
).
In wild-type mice, both TrkA- and TrkC-positive afferents grew into the
marginal zone of the spinal cord, and no axons entered the dorsal gray matter
at E12.5 (Fig. 3E,F). In netrin
1 mutant mice, TrkA-positive axons stayed near the DREZ, and several axons
aberrantly entered the dorsal gray matter
(Fig. 3G). TrkC-positive axon
bundles also penetrated the dorsal mantle layer
(Fig. 3H). These results
suggest that netrin 1 is required for the appropriate guidance of primary
sensory axons, including both cutaneous and proprioceptive afferents.
Suppression of axon outgrowth from DRG by netrin 1 in vitro
The above results strongly suggest that netrin 1 is necessary for the
accurate projection of DRG axons. To determine the function of netrin 1 in the
outgrowth of DRG axons, DRG explants were co-cultured in collagen gels with
aggregates of BHK cells expressing EGFP or netrin 1 via Sindbis virus
transfection. After 24-48 hours, total axonal outgrowth from DRG cultured
adjacent to netrin 1-expressing cells was dramatically inhibited when compared
with control cell aggregates that expressed EGFP
(Fig. 4A,B,D). Although less
axon outgrowth was observed in the presence of netrin 1-expressing cells,
there did not seem to be a specific effect on either axon attraction or
repulsion (p/d values: EGFP, 0.9; netrin 1, 1.0; not statistically
significant; see Materials and methods). Axon outgrowth was rescued by the
application of anti-netrin 1 antibody to the DRG co-cultures with netrin
1-expressing cells (Fig.
4B,C,D). These results support the hypothesis that netrin 1
provides an inhibitory cue for DRG axons, preventing these axons from
penetrating the dorsal gray matter directly.
Evidence that dorsally derived netrin 1 is crucial for the correct projection of sensory afferents
As netrin 1 expressed in the floor plate apparently influences the dorsal
spinal cord in the early stages
(Tessier-Lavigne et al.,
1988), we next examined whether netrin 1 secreted from the dorsal
spinal cord regulates the DRG axon pathfinding. To do this, we took advantage
of Gli2 mutant mice, which lack netrin 1 expression in the floor
plate because of a marked decrease in floor-plate cells
(Matise et al., 1999;
Charron et al., 2003). Netrin 1
expression was obviously weakened in the ventral region of the E12.5
Gli2-deficient spinal cord (Fig.
5A,B), while netrin 1 mRNA appeared to be normally expressed in
the dorsal spinal cord of mutants when compared with wild-type and
heterozygous mice (Fig. 5A,B).
Although the overall shape of the spinal cord was extremely abnormal in the
Gli2 knockout mice, the dorsal funiculus was correctly formed with a
sharp inner border, and lacked defasciculation or axonal invasion defects as
observed in netrin 1 mutant embryos (Fig.
5C,D). These results indicate that dorsally derived netrin 1
influences the axonal patterning of primary sensory afferents.
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;
Helms and Johnson, 2003;
Ding et al., 2005b). Moreover,
early-born neurons are important for the correct projection of DRG axons
(Ding et al., 2005b). To
examine netrin 1 expression in early-born neurons, we performed a BrdU
labeling experiment. We carried out immunostaining for BrdU after
lacZ in situ hybridization in netrin 1 heterozygous and homozygous
mice. Pregnant mice were given an injection of BrdU at E10.5 and allowed to
develop for another 48 hours. Many BrdU-positive cells in the dorsolateral
spinal cord were co-labeled with lacZ (see Fig. S2A,B in the
supplementary material). Moreover, we observed that lacZ-expressing
cells were correctly localized in the E12.5 netrin 1 heterozygous and
homozygous dorsolateral spinal cord (Fig.
2E,F), whereas many X-gal-positive cells were aberrantly
distributed in the dorsolateral spinal cord of the mutants at later stages
such as E14.5 (Fig. S2C,D). Therefore, early-born neurons express dorsally
derived netrin 1 and are correctly settled in the dorsolateral spinal cord
around E12.5 in netrin 1 mutants.
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;
Ninkina et al., 1993;
Akazawa et al., 1995;
Wang et al., 1997;
Chen et al., 1998). We were
unable to detect any obvious alternation in the expression pattern of these
markers between wild-type and netrin 1 mutant mice at the stages observed. For
example, Math1 and Pax3 expressions at E12.5 were observed
in the dorsal ventricular zone of the cord in both animals (Pax3,
Fig. 6C,D; Math1, data
not shown). Brn3a, Lmx1b and Ebf1 were also expressed in the
lateral margin of the dorsal ventricular zone and in the dorsal mantle layer
of both animals (Lmx1b, Fig.
6E,F; others, data not shown).
As mice lacking Dcc show abnormal ventral migration of early-born
neurons (Ding et al., 2005b),
we next performed a BrdU labeling experiment to examine the ventral migration
of dorsal neurons. We analyzed the distribution of E10.5 or E11.5 BrdU-labeled
neurons in the E12.5 dorsal spinal cord. As a result, we could not detect any
significant change in the ratio of BrdU-positive cells in the dorsal half and
ventral half of the dorsal spinal cord between wild-type and netrin 1 mutant
mice (Fig. 6G-K), indicating
that the ventral migration of dorsal neurons is nearly normal in netrin
1-deficient mice. Although the majority of early-born neurons migrated
ventrally (Fig. 6G,H), a few
BrdU-positive cells were found in the superficial part of the
wild/heterozygous dorsolateral mantle layer and in the ectopic cell island of
the mutant spinal cord at E12.5, following E10.5 BrdU injection (data not
shown). Therefore, local patterning of early-born neurons seems similar
between the two groups in this stage. These results suggest that defects of
axonal pathfinding in netrin 1 mutants are not secondary to abnormal neural
patterning in the dorsal spinal cord, especially in the early stage.
|
;
Fazeli et al., 1997). In the
E12.5 spinal cord of Dcc-deficient mice, the dorsal funiculus was
established in the correct position, and both TrkA- and TrkC-positive
afferents grew into the marginal zone as wild-type mice
(Fig. 7A,C,D; Figs
2,
3). The dorsal funiculus of
Dcc mutant mice seemed to be slightly defasiculated when compared
with that of wild-type mice, and some neurofilamant-positive fibers were
observed in the mantle layer (Fig.
7A; Fig. 2, data
not shown). However, the direct invasion of thick axon bundles into the dorsal
spinal cord, which was observed in netrin 1 mutant mice, was not found in
Dcc mutants at this stage, and thus the inner border of the dorsal
funiculus was relatively sharp (Fig.
7A,C,D; Figs 2,
3). As previously reported, in
E13.5 Dcc mutants, many fibers entered the dorsal mantle layer from
the lateral dorsal funiculus, whereas fibers also projected from the
dorsomedial region as seen in wild-type mice (see Fig. S3 in the supplementary
material) (Ding et al.,
2005b).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Furthermore, our in vitro studies elucidated the inhibitory effect of netrin 1
on axon outgrowth from DRG explants (see
Fig. 4). Taken together, our
results provide strong evidence that dorsally derived netrin 1 transiently
inhibits the direct invasion of DRG axons into the dorsal mantle layer of the
spinal cord and thus elaborates the waiting period.
Netrin 1 may act as a short-range guidance cue for primary sensory axons
In mouse DRG, Unc5 family receptors, but not Dcc, are
predominantly expressed during development (see
Fig. 1)
(Keino-Masu et al., 1996;
Ackerman et al., 1997;
Leonardo et al., 1997).
Unc5crcm mice show aberrant projections of DRG axons,
whereas the dorsal funiculus of Dcc mutant mice is normally
established at E12.5 (see Fig.
7). As netrin 1 mutant mice show more severe defects than
Unc5crcm mutants, it is probable that the netrin 1 signal
is mediated not only by Unc5c receptor but also by other receptors such as
Unc5a, which is also expressed in the DRG, and that moderate defects in the
Unc5crcm mutant dorsal spinal cord may be caused by
functional redundancy with such receptors. Thus, dorsal netrin 1 probably
functions in DRG axons through Unc5 family receptors around E12.5. In our
studies, netrin 1 expressed in the dorsal spinal cord is not as intense as
that in the floor plate and thus could be localized only close to the DREZ. We
observed slightly impaired projections of DRG axons in netrin
1+/– mice (see
Table 1), suggesting the dose
dependency of the defects in the dorsal spinal cord. Such a dose-dependent
response of DRG axons to netrin 1 was also examined in vitro; BHK cells
expressing netrin 1 were diluted with uninfected BHK cells, which were
co-cultured with DRG explants in the same experiment
(Liu et al., 2005). BHK cell
aggregates diluted at 1:10 (netrin 1-expressing cells: uninfected cells) had
an inhibitory effect, whereas those at 1:100 had no inhibitory effect (data
not shown). Therefore, netrin 1 may have an inhibitory effect at relatively
higher concentrations on DRG axons. Interestingly, it has been reported that
the ectopic expression of UNC5 elicits a short-range response in the absence
of the Drosophila Dcc homolog Frazzled, in which UNC5 causes the
axons to stop elongation, rather than directing them to repel away from the
source of netrin (Keleman and Dickson,
2001). Dorsal netrin 1 may act as a short-range inhibitory cue for
sensory afferents through Unc5 family receptors without cooperation of the
Dcc. The dorsal netrin 1 signal to DRG axons functions to form the dorsal
funiculus in the correct position: the marginal zone of the dorsolateral
spinal cord.
Multiple guidance molecules may elaborate the waiting period for sensory afferents in the developing spinal cord
Dorsal netrin 1 is expressed or upregulated in the restricted time window
of the waiting period at E12.5, while the arrest of axon collateral extension
into the dorsal spinal cord is observed from E10.5 to E14.5, depending on the
axon subclasses (Ozaki and Snider,
1997). Therefore, it is apparent that molecules other than netrin
1 also inhibit the invasion of axons into the dorsal spinal cord during early
and late phases of the waiting period. For example, Sema3a repels
NGF-responsive axons in vitro (Messersmith
et al., 1995). In addition, Wang et al.
(Wang et al., 1999) suggested
that slit controls the initial collateral branching of DRG axons in vitro,
whereas netrin 1 has no effect on this branching. Furthermore, transcription
factors and cell surface molecules, such as Drg11, Runx3, Er81 and F11, are
involved in the correct projection of DRG axons
(Arber et al., 2000;
Chen et al., 2001;
Inoue et al., 2002;
Perrin et al., 2001). These
observations suggest that multiple molecules orchestrate to elaborate the
total waiting period for sensory afferents. Further studies are required to
fully elucidate the mechanisms underlying the whole waiting period.
Dorsally derived netrin 1 is involved in dorsal spinal cord formation in both direct and indirect manners
During development, the waiting period has been demonstrated in many
regions (Schreyer and Jones,
1982; Ghosh and Shatz,
1992; Renzi et al.,
2000; Wang and Scott,
2000). The waiting period is thought to be important for the
formation of proper neural networks. In netrin 1 mutant mice, which lack the
middle phase of the waiting period, disorganization of the cytoarchitecture in
the dorsal spinal cord becomes more severe at E13.5 and later, although
defects in the early patterning of dorsal cells are subtle (see Figs
2,
6; Fig. S1 in the supplementary
material; data not shown). Furthermore, in netrin 1, Dcc and
Unc5crcm mutants, corticospinal tract abnormalities are
observed, and the dorsal funiculus is more disorganized in the perinatal stage
(Finger et al., 2002). It is
probable that the misrouting of pioneer DRG axons in the netrin 1-deficient
dorsal spinal cord directs the following DRG fibers and other axons to a more
abnormal course, which may prevent dorsal cells from localizing in the
appropriate positions in later stages. Recently, Ding et al.
(Ding et al., 2005b)
demonstrated that the abnormal migration of dorsal cells in
Dcc-deficient mice induces aberrant patterning of DRG axons through
Sema3a signaling. netrin 1 may control the axonal pathfinding of DRG axons by
affecting the migration of spinal cord neurons through Dcc and the outgrowth
of DRG axons through Unc5 during different developmental stages. Therefore,
the functional architecture of neural tissues is formed at least in part by
neuron-axon interactions. In this study, we elucidated that netrin 1 directly
regulates the timing and patterning of early DRG axons in the dorsal spinal
cord. In addition, netrin 1 may also influence dorsal cell patterning
indirectly at a later stage by regulating axonal pathfinding.
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/7/1379/DC1
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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