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The Journal of Neuroscience, May 1, 2002, 22(9):3568-3579
Deleted in Colorectal Carcinoma and Differentially Expressed
Integrins Mediate the Directional Migration of Neural Precursors in the
Rostral Migratory Stream
Shin-ichi
Murase and
Alan
F.
Horwitz
Department of Cell Biology, University of Virginia School of
Medicine, Charlottesville, Virginia 22908
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ABSTRACT |
Precursors of the olfactory interneurons migrate from the
subventricular zone via the rostral migratory stream (RMS). To
investigate the molecular mechanisms by which RMS cells migrate, we
used a slice preparation, which allows the migrating cells to be imaged at very high temporal and spatial resolution in the presence of added
inhibitors. Using immunohistochemistry, we first determined that the
 1-,  8-, and 1-integrin subunits and the 5- and  1-laminin subunits are expressed during embryonic day 16 to the early postnatal stage. During early postnatal days, v- and 6-integrins appeared, and their expression persisted throughout adulthood. The migrating cells also expressed the netrin receptors neogenin and Deleted in
Colorectal Carcinoma (DCC). Netrin-1 is expressed in olfactory mitral
cells. Anti-integrin antibodies inhibited the production of protrusions
as well as cellular translocation. In contrast, anti-DCC antibodies
primarily altered the direction of the protrusions; consequently, the
migration was no longer unidirectional, and the speed was reduced.
Thus, the interaction of DCC, possibly through an interaction with
netrin-1, contributes to the direction of migration by regulating the
formation of directed protrusions. In contrast, the integrins function
in production of protrusions and cellular translocation, with different
integrins participating at different developmental stages.
Key words:
chemoattraction; neural cell adhesion molecule; slice
culture; video microscopy; radial glia; time-lapse recording; polysialic acid
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INTRODUCTION |
In the rodent brain, the
subventricular zone (SVZ) is a mitotically active region that surrounds
the ependymal wall of the lateral ventricle and is characterized by a
high cell density (Allen 1912 ; Messier et al., 1958; Bryans, 1959;
Boulder Committee, 1970). Precursors of the olfactory interneurons
generated at the SVZ migrate tangentially, via the restricted route
called the rostral migratory stream (RMS), to the olfactory bulb, where
they then differentiate into periglomerular cells and granule cells (Altman, 1969; Luskin, 1993; Lois and Alvarez-Buylla, 1994). This migration does not end when the olfactory bulb reaches adult size; rather, it continues in the adult, and it is neither gliophilic nor
axonophilic, as reported in many other brain regions (Rakic, 1971;
Hynes et al., 1986; Lois and Alvarez-Buylla, 1994). The mechanism of
this migration is unclear. The polysialylated form of neural cell
adhesion molecule (PSA-N-CAM) appears to play an important role, on the
basis of studies using knock-out mice (Tomasiewicz et al., 1993; Cremer
et al., 1994), enzymatic removal of polysialic acid (PSA) (Ono et al.,
1994), and transplantation (Hu et al., 1996). One plausible explanation
for the influence of PSA-N-CAM on the migration is that the PSA moiety
reduces the adhesive properties of N-CAM (Hoffman and Edelman, 1983;
Sadoul et al., 1983; Rutishauser et al., 1985) and allows the cells to
translocate in the RMS. The absence of PSA, in contrast, causes the
cells to adhere more strongly and thus inhibits migration. Although the
above studies support a role for PSA-N-CAM, other molecules likely
contribute to migration along the RMS. For example, the alterations in
migration are only partial in mice with an inactive N-CAM gene (Chazal
et al., 2000), suggesting that additional mechanisms contribute to the
directed migration. Interestingly, the migrating neuronal precursors in
the adult have the appearance of chains, which coalesce to form the RMS
(Lois et al., 1996), whereas those in the neonates form thicker streams
more akin to intertwined ropes than to chains (Kishi, 1987; Kishi et
al., 1990). Electron microscopic analyses in the adults show that glial
cells and their processes cover the migrating cells (Jankovski and
Sotelo, 1996; Lois et al., 1996) and form the walls of longitudinally
arranged canals commonly referred to as "glial tubes" (Peretto et
al., 1997). In the neonatal RMS, however, the migrating precursors do
not appear to be surrounded by such glial processes and instead migrate
through a large extracellular space (Kishi et al., 1990). Thus, it
seems likely that the molecular mechanisms underlying migrations along
the RMS differ in adults and neonates.
In the present study, we used slice technology to visualize the nature
of RMS migrations at high spatial and temporal resolution (Wu et al.,
1999; Knight et al., 2000). We also identified additional adhesion
molecules, extracellular matrices, and chemoattractive factors that are
expressed in the RMS during development. We found that six integrin
subunits and the receptor for netrins were differentially expressed and
contributed to migration. The integrins provide traction for cellular
translocation, whereas the netrins appear to contribute to the
direction of the migrations.
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MATERIALS AND METHODS |
Immunohistochemistry. Wild-type mice (ICR or CD-1
strains) and rats (SD strain) were purchased from Hilltop Lab Animals,
Inc. (Scottdale, PA) and housed using a 12 hr light/dark cycle.
Heterozygotes of N-CAM knock-out mice (Cremer et al., 1994) were
purchased from The Jackson Laboratory (Bar Harbor, ME). Homozygotes
were obtained by breeding the heterozygotes, and brain tissues of the
homozygotes were used to confirm the specificity of anti-N-CAM and
anti-PSA monoclonal antibodies. Fifteen homozygous, 30 heterozygous,
and 15 wild-type mice were characterized. For immunostaining, the mice
or rats were anesthetized with halothane. To obtain the brains of
fetuses, deep anesthesia was used on the pregnant mice, and the
abdominal cavity was opened to remove the fetuses. The mother's diaphragm was cut to ensure that she had been killed. The
embryos still under the crossover anesthesia were perfused through the aorta with fixatives consisting of either 4% paraformaldehyde and 0.1 M phosphate buffer or acid-ethanol (5% acetic acid in ethanol). The postnatal mice were anesthetized with halothane and
perfused through the aorta with the above fixatives. The dissected brains were immersed in 20% sucrose and PBS, frozen in powdered dry
ice, and embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN).
Parasagittal or horizontal sections (20 µm) of brains were cut using
a cryostat and mounted on silane-coated slides. For immunoperoxidase
staining, sections were treated with primary antibodies for 42 hr at
4°C and then incubated with peroxidase-conjugated secondary
antibodies for 2 hr at room temperature. The immune complexes on the
sections were detected using a peroxidase substrate consisting of
diaminobenzidine-tetrahydrochloride as described previously
(Murase, 1995).
Antibodies. Anti-1-integrin [antibody (AB) 1934;
Chemicon, Temecula, CA], anti-v-integrin (AB1930; Chemicon), and
anti-1-integrin (AB1938; Chemicon) antibodies were assayed for
specificity by preabsorption using the peptides against which the
antibodies were raised and Western blotting as described previously
(Murase and Hayashi, 1996, 1998a,b). The function-blocking
anti-1-integrin rat monoclonal antibody (clone 9EG7) was obtained
from PharMingen (San Diego, CA); its specificity has been reported
previously (Lenter et al., 1993; Lenter and Vestweber, 1994). The
anti-v-integrin (T-20), anti-3-integrin (C-20),
anti-6-integrin (C-19 and N-20), and anti-8-integrin (C-19) and
anti-neogenin (C-20) antibodies were obtained from Santa Cruz
Biotechnology, (Santa Cruz, CA). The specificities of these antibodies
for immunohistochemistry were verified by absorbing each primary
antibody with 0.5 mM corresponding antigen-peptide solution
overnight at 4°C before application to brain sections, which was then
followed by the diaminobenzidine reaction. Other control experiments
included omission of primary antibodies. Anti-tenascin-C rat monoclonal
antibody (clone MTn-12) was obtained from Sigma (St. Louis, MO); its
specificity and immunoreactivity in the forebrain have been reported
previously (Aufderheide and Ekblom, 1988; Jankovski and Sotelo, 1996).
The function-blocking anti-Deleted in Colorectal Carcinoma (DCC; clone
AF5, which was raised against the extracellular domain of DCC),
anti-netrin-1 (PC364), and its control peptide were from Oncogene
Research Products (Cambridge, MA); their immunoreactivities and the
function-blocking activity of AF5 have been reported previously
(Keino-Masu et al., 1996; Madison et al., 2000). Clone Men-B for
PSA-N-CAM was a gift from G. Rougon (Centre National de la Recherche
Scientifique, Marseille, France) (Rougon et al., 1986), anti-4-
and 5-laminins were gifts from J. H. Miner (Washington
University School of Medicine, St. Louis, MO) (Miner et al., 1997), and
anti-2-laminin was a gift from K. Sekiguchi (Osaka University,
Osaka, Japan) (Fukushima et al., 1998). Clones 5A5 (PSA-N-CAM), AG1 and
5B8 (N-CAM), and 2E8 and D18 (1-laminin) were obtained from
the Developmental Studies Hybridoma Bank, (DSHB), University of
Iowa, Department of Biological Sciences (Iowa City, IA). Anti-collagen
I (AB765), anti-collagen IV (AB756), anti-fibronectin (AB2033),
anti-vitronectin (AB1903), anti-2-integrin (AB1936),
anti-3-integrin (AB1920), anti-4-integrin (AB1924),
anti-5-integrin (AB1928-P and AB1949), anti-6 integrin
[monoclonal AB (MAB) 1972 and clone GoH3], anti-4-integrin (AB1922), anti-5-integrin (AB1926), and anti-2-laminin (MAB1922) were all obtained from Chemicon. Anti-2-laminin clones C4, D5, and
D7 were from DSHB. Anti-adenosine A2B receptor (R-20),
anti-1-laminin (M-20), and anti-1-laminin (C-19) were obtained
from Santa Cruz Biotechnology. These antibodies did not react with any
cellular or extracellular components along the RMS (data not shown).
Preparation of brain slices. Slices were prepared from
embryonic day 18 (E18) and postnatal day 0 (P0)-P16 mice using
modifications of the methods of Stoppini et al. (1991) and
Knight et al. (2000). For each individual experimental determination,
two slices from each brain, at a minimum, were used, and the results
were confirmed by at least two additional determinations. Slices that
contained the entire migratory stream, which originated from the
anterior portion of the SVZ (SVZa) and ended at the center of the
olfactory bulb, were selected for further studies. The postnatal mice
were anesthetized with halothane and decapitated. For fetal brains, the
pregnant mice were deeply anesthetized, and the abdominal cavity was
opened to remove the fetuses. The mother's diaphragm was cut to ensure
that she had been killed. While still under crossover anesthesia, the
fetuses were decapitated, and their brains were removed. The brains
were placed into cell culture media 1 (CCM1) (Hyclone, Logan, UT)
medium at 4°C. The brains were then transferred into CCM1 or F-12
medium (Invitrogen, Rockville, MD) containing 5% heat-inactivated
horse serum (Sigma) and penicillin and streptomycin antibiotics
(Sigma). They were then embedded CCM1 medium containing 8% agarose
(Sigma type IX) and sliced into 200 µm parasagittal sections using a
vibratome. The slices were cultured on a Millicell-CM membrane
(Millipore, Bedford, MA) according to the method of Stoppini et al.
(1991).
DiI labeling and time-lapse videomicrography. Small crystals
of the lipophilic dye DiI (D-3911; Molecular Probes, Eugene, OR) were
placed onto the SVZa, the RMS, or the central portion of the olfactory
bulb of the slices using a microneedle (Fig. 1). Images were acquired using a Nikon
IX-70 inverted microscope fitted with a cooled CCD camera (Photometrics
CH250). The microscope was also equipped with a Ludl motorized XYZ
stage and heating insert (Medical Systems Corp.). Electronic shutters
regulated fluorescence illumination. Image acquisition and processing
used Inovision software. After 5 hr of incubation, a field in each slice was selected that retained good morphology and had fluorescently labeled (migrating) cells adjacent to the DiI crystals. In a typical experiment, nearly all of the DiI-treated slices displayed appropriate labeling. The slices were illuminated with either a mercury or halogen
lamp. A tetramethyl rhodamine isothiocyanate filter cube was used to
observe the DiI-labeled cells. Fluorescence images were recorded from
multiple fields every 5 min using a 10× objective. Images were
typically captured over 3-10 hr using 0.015-0.20 sec exposures. The
time-lapse movies (available at www.jneurosci.org) were analyzed for
cells that migrated to the olfactory bulb along the rostral migratory
stream.
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Figure 1.
Experimental design for assays of cell migration
in the RMS. Two hundred-micrometer-thick sagittal slices were cut from
brains dissected from embryonic day 18 or postnatal day 0-16 mice
using a vibratome. Slices containing the entire RMS were selected and
placed on Millicell inserts in CCM1 medium containing HEPES and 5%
horse serum. Crystals of DiI were placed on the RMS using a
microneedle. By selecting the position of the DiI crystals, migrating
cells in every part of the RMS can be visualized. Time-lapse images of
fluorescent cells were recorded over 3-10 hr using an inverted
microscope.
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Function-blocking assays. The tissue slices that
showed migrating cells labeled with DiI were selected and cultured in
CCM1 medium containing function-blocking antibodies against
1-integrin (Mendrick et al., 1995), 1-integrin (9EG7; Lenter et
al., 1993; Lenter and Vestweber, 1994), v-integrin (H9.2B8; Maxfield
et al., 1989), 3-integrin (2C9.G2; Frieser et al., 1996), or DCC (AF5; Keino-Masu et al., 1996). The function-blocking antibodies for
integrins were purchased from PharMingen (San Diego, CA), and the
specificities and function-blocking activities of these are reported in
the publications cited above. For negative controls, isotype-compatible
antibodies, from a matched species, directed against keyhole limpet
hemocyanin, and anti-trinitrophenol were used. MAB 5A5, an IgM directed
against PSA (DSHB) was used at 10 µg/ml to assay the effect of PSA on
migration in early postnatal mice, when PSA expression is very weak or
not found. MAB 8D9, an anti-L1 MAB (DSHB), was used as a negative
control for AF5. Anti-integrin and control antibodies were used at 20 µg/ml, and anti-DCC and its control were used at 5 µg/ml.
Migration parameters. The time-lapse movies were analyzed
for migration speed and direction. The center of the soma of a
migrating cell was traced at 5 min intervals, and the speed and
direction of migration during the time-lapse recording were determined
as described by Knight et al. (2000). Each value represents the
mean ± SD. Statistical analysis was performed by one-way ANOVA
with Scheffé's multiple comparison procedure (significance of
p < 0.01).
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RESULTS |
Expression of adhesion and guidance molecules in the RMS
To determine the repertoire of molecules that contribute to the
migration of neural precursors during different stages of development,
we investigated the expression of two classes of adhesion molecules in
the RMS of both embryos and adults. Figure 2A shows a
Nissl-stained sagittal section containing the entire route of the RMS
in a P4 rat. PSA-N-CAM was reported previously to contribute to the
migration of neural precursors in the RMS (Ono et al., 1994).
Surprisingly, the expression of N-CAM and PSA in the RMS, as assayed by
four independent monoclonal antibodies; e.g., the 5B8 and AG1 MABs,
directed against N-CAM, and the 5A5 and Men-B MABs, directed against
PSA epitopes, revealed only weak expression of N-CAM (Fig.
2B) and PSA (Fig. 2C) before P4-P5. In
contrast, neurons and axons in the cerebral cortex, corpus callosum,
olfactory tubercle, and caudate-putamen surrounding the RMS and SVZ
were positive for N-CAM and PSA. The PSA immunoreactivity in the RMS
peaked at ~P30 (Fig. 2D) at a level that persisted in the adult, whereas the region adjacent to the RMS showed very low
immunoreactivity. These four MABs did not react with RMS and olfactory
bulb sections from N-CAM knock-out homozygotes (data not shown).
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Figure 2.
N-CAM, PSA, and laminin expression in the rostral
migratory stream. A, Nissl staining of a sagittal
section from a postnatal day 4 rat forebrain. The RMS, which is
characterized by a high cellular density, begins at the anterior
portion of the SVZ and ends at the center of the olfactory bulb
(OB). B, C, N-CAM and PSA in the RMS of
early postnates. B and C are adjacent
sections from a P4 rat forebrain that are stained with anti-N-CAM
(clone 5B8) and anti-PSA (clone MenB) MABs, respectively. The SVZ and
the RMS stain very weakly, whereas the borders of the RMS show strong
staining. D, PSA immunostaining of a P30 mouse
forebrain. In contrast to weak expression of PSA in P4 RMS shown in
C, PSA is expressed strongly in the RMS
(arrows) and the olfactory bulb (OB).
E, 5 subunit of laminin in the RMS
(arrows) from an embryonic day 18 mouse shows punctate
staining. Relatively strong staining is also seen in the choroid plexus
(CPX) of the lateral ventricle
(LV) and blood vessels
(BV). F, 1-Laminin is expressed
in the RMS (arrows) from a P0 rat brain. Blood vessels
(BV) and the meninges
(M) are also stained. Scale bars:
A-D, F, 1 mm; E, 50 µm.
AOB, Accessory olfactory bulb; CC, corpus
callosum; CP, caudate-putamen; CX,
cerebral cortex; OT, olfactory tubercle.
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Laminin, which supports migration of many neurons, is also expressed in
the RMS and basement membranes of blood vessels. The 5 and 1
subunits were observed in a punctate pattern in the RMS during E16-E20
and E16-P4, respectively (Fig. 2E,F), whereas other laminin subunits, including 1, 2, 4, 1, 2, and
2, were not expressed detectably. Although most of the laminin
immunoreactivity in the RMS was highly punctate, in some cases the
staining tended to resemble cellular contours. However, we were unable
to discern at this resolution whether the staining was intracellular or
extracellular. Collagen type I, fibronectin, and vitronectin were not
prominently expressed along the RMS but were seen in blood vessels.
Tenascin-C was also observed along the RMS, as reported previously
(Jankovski and Sotelo, 1996; Thomas et al., 1996; data not shown).
These data suggest that other adhesion molecules, in addition to N-CAM,
might participate in RMS migrations, especially at relatively early
developmental stages. Therefore, we assayed for the expression pattern
of integrins, because they can mediate interactions of cells with
laminin. Six integrin subunits were expressed on putative migratory
cells in the RMS: 1, v, 1, 3, 6, and 8.
Interestingly, the relative expression of these integrins changed
during development. They were rarely found in cells migrating radially
from the center of the olfactory bulb, suggesting that the integrins
mainly function in the tangential migrations. The glial tube
surrounding the RMS also did not express these integrins. The 1 and
8 subunits appeared initially in the RMS (Figs.
3A,B); their expression was
followed by expression of the 1 subunit (Fig. 3C).
Expression of the 1 and 8 subunits diminished at P3-P4,
coinciding with the expression of the v subunit, which began to
appear at ~P2-P3 and continued into the adult (Fig. 3D).
The 1 subunit was expressed until P10, whereas the 6 subunit
appeared at ~P10 and continued to be expressed in the adult (Fig.
3E). The 3 subunit appeared in the adult RMS beginning at
P28 and then persisted (Fig. 3F). These dynamic
integrin expression patterns are summarized in Figure
4.
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Figure 3.
Six integrins are differentially expressed in the
rostral migratory stream. The 1 (A) and 8
(B) integrin subunits are expressed
(arrows) from the anterior horn of the subventricular
zone to the center of the olfactory bulb from P0 mice.
C, The 1-integrin subunit is expressed in the RMS
(arrows), blood vessels (BV), and
the choroid plexus (CPX) of the lateral ventricle
(LV) of a P2 mouse. D, The
v-integrin subunit is found in the RMS (arrows) from
a P30 mouse. E, The 6-integrin subunit is expressed
in the RMS (arrows) of a P15 mouse. F,
The 3-integrin subunit is observed in P30 rat RMS
(arrows). Scale bar, 1 mm. AOB, Accessory
olfactory bulb; CC, corpus callosum; CX,
cerebral cortex.
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Figure 4.
Summary of the stage-specific expression of
integrin and laminin subunits in the RMS. Tenascin-C is expressed along
the sides of the RMS but not in the RMS itself.
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Visualizing RMS migrations
The presence of the integrins on migrating cells prompted us to
use a preparation with which we could visualize migration at high
temporal and spatial resolution and assay the effects of antibody
reagents for their effects on migration in the RMS. For this, we
prepared 200-µm-thick slices from neonatal mouse forebrains, which
were embedded in agarose gels and sliced using a vibratome (Fig. 1).
The viability of the cells in the slices was assayed by trypan blue
exclusion. Dye-incorporating dead cells were rarely found in the RMS or
other brain regions (data not shown) up to P16, although they were
common in slices from older brains (>P18). Therefore, we only examined
slices from P0-P16 mice. The migrating cells were labeled by placing a
small crystal of DiI on the slice in the anterior region of the SVZ or RMS.
Actively migrating cells labeled by DiI were found in the RMS
during the younger neonatal stages that we studied. As shown in Figure
5A (collage and Movie 1, available at www.jneurosci.org), the overall direction of migration was
highly persistent and directed toward the olfactory bulb (Fig.
5B). No labeled cells were observed to migrate into regions
surrounding the RMS. The migrating cells were highly polarized, with
long leading processes and relatively small somata. This morphology is
similar to that observed by silver impregnation (Kishi, 1987). Once at
the olfactory bulb target, the cells tended to meander without clear
direction or substantial net movement (Movie 4, supplemental data,
available at www.jneurosci.org). Thus, the slices appear to be viable
and to reflect the migrations present in the native brain.
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Figure 5.
Integrins mediate migration of neural
precursors in the RMS. Brain slices from a P12 mouse were labeled with
DiI and cultured in CCM1 medium with 5% horse serum for 5 hr in the
presence of either control or anti-integrin antibodies. Three hours
after addition of DiI, the migration was confirmed by fluorescence time
lapse, and then a control or function-blocking anti-v-integrin
antibody was added for (Figure legend continued.) 5 hr, after which
migration was recorded over the next 3 hr. A, Time-lapse
sequence of three cells (a-c) in a slice migrating from
the SVZ (bottom) toward the olfactory bulb
(top) in the presence of a control antibody. The
arrow pointing to each cell shows the leading process,
and the line shows the cell body. The interval between
each image is 5 min. See Movie 1 (available at www.jneurosci.org).
B, Graphical representation of the migration of the
three cells (a-c) in A. Each
point represents the position of the cell body at 5 min
time points. Note the unidirectional pathway and the bursts of rapid
migration followed by slower meandering. C, Time-lapse
sequence of images of seven cells (d-j) in a slice
migrating from the SVZ (left) to the olfactory bulb
(right) in the presence of an anti-v-integrin
antibody. Seven cells (d-j) are marked for reference.
See Movie 2 (available at www.jneurosci.org). D,
Graphical representation of the migration of the seven cells as
described in C. The olfactory bulb is at the
right. Note the inhibited migration. The interval
between each image is 5 min. Scale bars, 50 µm.
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Some parameters characterizing the migration were quantified. The
average speeds of migration in P3, P5, and P12 control slices were
98 ± 26, 85 ± 19, and 94 ± 20 µm/hr, respectively.
These values did not show any statistically significant differences when compared with each other (Fig. 6).
The average values result from the heterogeneous cellular movements
that are characterized by rapid bursts of migration, which in turn are
followed by a period of meandering or rest. The heterogeneous nature of
the migration is apparent in the rose plots (Fig. 5B) and is
similar to that reported for somitic migrations (Knight et al., 2000). The rose plots also reveal the highly directed nature of the
migrations.
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Figure 6.
Inhibited migration of RMS cells by
function-blocking anti-integrin antibodies. Living brain slices were
prepared from P3, P5, and P12 mice, and the cells were labeled with a
small crystal of DiI placed on the center of the RMS. The slices were
cultured in CCM1 medium supplemented with HEPES and 5% horse serum.
The slices were preincubated with anti-integrin antibodies for 5 hr,
and the migrating cells were traced by time-lapse recording for 3 hr.
At P3, when 1- and 1-integrins are expressed, corresponding
blocking antibodies reduced the migration speed. Anti-3 antibody did
not inhibit the migration significantly. At P5, when v- and
1-integrins are expressed, antibodies against these integrins
inhibited the migration speed. At P12, when v-integrin is expressed,
anti-v-integrin antibody inhibited the speed as well; however,
anti-1 and -3 antibodies did not inhibit the migration speed
significantly, and they are not expressed at this stage. Each value
represents the mean ± SD. Statistical analysis was performed by
one-way ANOVA with Scheffé's multiple comparison procedure
(significance of p < 0.01). The groups with
asterisks do not differ from each other, nor do the
nonmarked groups, but in all other comparisons, the differences are
significant.
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Integrins mediate migration of neural precursors
Using this in situ system, we assayed the effect
of anti-integrin antibodies that inhibit integrin-mediated adhesion on
RMS migration. Because slices from P0-P4 mice express 1- and
1-integrins prominently, we assayed the effects of anti-1 and
anti-1 antibodies on migration during these stages. Similarly, we
used P8-P16 slices to assay for the role of v integrins, because
they are strongly expressed at these stages. Function-blocking
antibodies for 8 and 6 are not available, so we were unable to
determine the roles of these subunits, and the role of the 3 subunit
in the adult RMS was not accessible using our conditions. Although our
immunohistochemical data show only weak if any PSA expression in the
RMS during E14-P5, we also used an anti-PSA antibody (5A5 MAB) to
assay for the effect of PSA on RMS migration.
The migration rate of antibody-treated (20 µg/ml) groups is
summarized in Figure 6. Antibodies against the corresponding integrins inhibited the migration during the stages at which they are expressed. In P3 slices, for example, 1- and 1-integrins were expressed prominently, and addition of anti-1 or anti-1 antibodies
decreased the migration speed to ~20% of the controls, which were
treated with either anti-trinitrophenol or anti-keyhole limpet
hemocyanin antibodies. Anti-1 and anti-3 antibodies did not
affect the migration in the slices from P7 or P10 mice, stages at which
neither 1 nor 3 integrins are expressed (data not shown).
We applied anti-1- and anti-v-integrin antibodies to the slices
from P1 or P5 brains, which express both of these integrins. We also
added anti-1-integrin antibodies to P0 slices, which do not express
the v-integrin but do express 1. The migration speed in the
presence of these antibodies, added either alone or together (P1),
decreased, but the antibodies did not show an additive effect when
added together (Fig. 6). In P5 slices, v- and 1-integrins are
also expressed prominently. Addition of either anti-v or anti-1
decreased the migration to ~22% of the control levels. Finally, P12
slices were treated with anti-v antibodies, and the speed was
suppressed to 25% of the control level; however, control antibodies
and anti-3 function-blocking antibodies did not inhibit this
migration. The migration rates for all of the controls were similar to
those of slices not treated with control antibody (data not shown).
The inhibitory effect of anti-integrin antibodies did not show
synergistic effects when two different anti-integrin antibodies were
added simultaneously. The inhibitory effect of anti-integrin antibodies
was so pronounced that many of labeled cells did not move over the
time of measurement, and protrusions were only rarely observed (Fig.
5C,D, Movie 2, available at www.jneurosci.org). These
results show that the different integrins expressed in the different
stages mediate the migration of the neural precursors in the RMS. The
migration speed of RMS cells from a P4 brain slice was not changed by
incubation with the anti-PSA MAB (data not shown).
Expression of chemoattractive molecules in the RMS
In the above experiments, we showed that particular integrins are
required for cellular translocation in the RMS. We next asked what
molecules contribute to their guided migration toward the olfactory
bulb. Our time-lapse recordings showed highly directed migration in the
RMS toward the olfactory bulb (Fig. 5A,B). Relatively few
cells showed retrograde migration, and when observed, it was transient.
Thus, it seems likely that a chemoattractant produced in the olfactory
bulb might determine the direction in the RMS. Therefore, we assayed
for the presence of receptors for netrins, because they serve guidance
roles for growth cones and neuroblasts (Kennedy et al., 1994; Yee et
al., 1999).
Using antibodies specific for netrins and the netrin receptors neogenin
and DCC, we assayed for their presence in cells residing in the RMS. We
observed strong expression for both of them in migrating cells during
E15-P5, a time during which there is a massive migration from the SVZa
to the olfactory bulb (Fig.
7A,B). In contrast, the
adenosine A2b receptor, another putative netrin-1 receptor (Corset et
al., 2000; Stein et al., 2001) was not observed (data not shown). The
protein expression of these netrin receptors in this study complements
the pattern of mRNA expression reported earlier (Gad et al., 1997).
Netrin-1, in contrast to neogenin and DCC, was expressed in the mitral
cells (Fig. 7C,D). Netrin-3 mRNA was not expressed (data not
shown), and netrin-2 homologs have not been identified in the rodent
(Wang et al., 1999). Taken together, these localizations suggest that
netrin-1 produced by the mitral cells may function to produce a
chemogradient through the forebrain that attracts migrating cells to
the olfactory bulb.
View larger version (90K):
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Figure 7.
Expression of DCC, neogenin, and netrin-1 and
function of DCC in RMS migrations. A, Neogenin, a
netrin-1 receptor, immunoreactivity coincides with the contour of RMS
beginning at the anterior horn of the lateral ventricle
(LV) and ending at the center of the olfactory
bulb in a P0 mouse. B, The rostral part of the RMS
strongly expresses the DCC protein, which is also present in the
lateral olfactory tract (LOT) in P2 rats.
AOB, Accessory olfactory bulb. C,
Netrin-1 protein is expressed in the basal portion of olfactory mitral
cells in the mitral cell layer (MCL) from an embryonic
day 18 mouse. EPL, External plexiform layer;
GCL, granule cell layer; GL, glomerular
layer; ONL, olfactory nerve layer. (Figure legend continued.) D,
Preabsorbed netrin-1 antibody (control) was prepared by coincubation of
antibody and antigen peptide. The absorbed antibody did not show
immunoreactivity. E, Sequence of 21 time-lapse images of
a living slice from a P3 mouse treated with anti-DCC antibody as
described in the legend to Figure 5. The olfactory bulb is located at
the top. Three migrating cells (a-c) are
indicated. Arrows indicate retracting leading process,
and crossed arrows indicate processes pointing toward
the anterior region of the subventricular zone (bottom).
Note that the migration is no longer unidirectional, and that the
processes form and retract frequently, which also distinguishes these
migrations from those of normal cells shown in Figure 5. The interval
between each image is 10 min. See Movie 3 (available at
www.jneurosci.org). F, Graphical representation of three
migrating cells (a-c) shown in E. Cell
bodies are tracked as described in Figure 5, and the time interval is 5 min. S, Start point; E, end point for
cell b. See Movie 3 (available at www.jneurosci.org).
G, Inhibited migration of cells by anti-DCC
function-blocking antibodies. Living brain slices were prepared from P3
mice, and then the cells were labeled with DiI placed at the center or
end of the RMS. The slices were cultured in CCM1 medium with HEPES and
5% horse serum with or without anti-DCC antibody for 5 hr, and the
migration was observed by time-lapse recording. Each value represents
the mean ± 1 SD. Statistical analysis was performed by one-way
ANOVA with Scheffé's multiple comparison procedure (significance
of p < 0.01). The control groups without anti-DCC
antibody differ from every other group with anti-DCC. The groups with
asterisks do not differ from each other, nor do the
nonmarked groups, but in all other comparisons, the differences are
significant. In contrast to cell treated with anti-integrin antibodies
(Fig. 6), these cells show a larger net translocation. Scale bars:
A, B, 1 mm; C, D, 100 µm;
E, 50 µm.
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|
To determine whether netrin-1 and its receptors contribute to the
directed migration of the neural precursors in the RMS, we used the
slice system described above to assay the effects of a
function-blocking antibody directed against DCC. The addition of an
anti-DCC antibody (5 µg/ml) perturbed the migration of RMS cells in
the slices. In the presence of antibody, the RMS cells continued to
extend single, long processes; however, they were directed not only
toward the olfactory bulb, as seen in the controls, but also in a
variety of other directions, including toward the SVZa and at right
angles to these directions. Thus, the cells appeared to have lost their
sense of direction (Fig. 7E,F, Movie 3, available at
www.jneurosci.org). This bizarre, nondirected movement of the leading
processes was not observed when cells were treated with any of the
anti-integrin antibodies.
The nondirected extension of protrusions led to parallel alterations in
migration. The cells no longer showed bursts of rapid migration, but
instead, they tended to meander without a clear direction (Fig.
7F). A quantitative analysis of the antibody effects on migration speed showed that the apparent speed was suppressed to
~30% of the control values (p < 0.01) in
both the center and end portions of the RMS (Fig. 7G).
Together, these data implicate DCC and its related proteins in the
directed migration of RMS cells toward the olfactory bulb.
| |
DISCUSSION |
The RMS is a specific route by which neural precursors migrate
from the SVZa to the olfactory bulb. In this study, we used a slice
preparation to image the migrating cells in the RMS at high temporal
and spatial resolution. The overall objective was to reveal the
cellular and molecular mechanisms that underlie these migrations. Using
this preparation, we made a number of interesting observations. They
include the differential expression of integrins that selectively
mediate migration at different developmental stages, the apparent role
of DCC in polarizing protrusions and directing migration, the high
polarity of the cells, and the highly directed and restricted nature of
the migrations that this polarity produces.
How do they migrate?
Previous studies on the nature of migrations in the RMS have
relied on evaluations of static, morphologic observations (Kishi, 1987;
Kishi et al., 1990). Our observations extend these studies by
characterizing the nature of the cellular dynamics and pathways of the
migrating cells. The cells migrating in the RMS are highly polarized
with a single, prominent, and very long-lived protrusion that is
polarized along the direction of migration. The cell body moves in the
direction of this protrusion, thus resulting in a net translocation.
The migrations are also characterized by bursts of rapid migration,
which in turn are followed by a "resting" phase. At the end points
of migration, the protrusions are less directed, with little net
displacement, and the cells begin to differentiate into mature neurons
(Movie 4, available at www.jneurosci.org). These migration features are
similar to those reported recently for the migration of myogenic
precursors from the somite to the limb (Knight et al., 2000). In
contrast to this previous study, the RMS migrations are more highly
directed, with most cells migrating unidirectionally along a highly
restricted pathway toward the olfactory bulb. The migrations from the
somite to the limb, in contrast, occur over a broad and less well
defined pathway, with cells frequently migrating away from the target
followed by a reorientation toward it.
The migration speeds estimated here (~80-100 µm/hr) are comparable
with those measured for cells from SVZ explants migrating in Matrigel
(122 µm/hr; Wichterle et al., 1997) and glial progenitors migrating
in SVZ slices (85-89 µm/hr; Kakita and Goldman, 1999). However, they
are faster than those estimated in the adult RMS by tracing
[3H]thymidine-labeled migrating cells
(30 µm/hr; Lois and Alvarez-Buylla, 1994). This difference in
migration speeds for cells in the neonates and adults may be
attributable to the morphological difference between them or the
methods used to compute the speed. The neonatal RMS is thicker than
that in the adult and is not enclosed by glial tubes (Kishi, 1987;
Kishi et al., 1990). A more likely explanation, however, is that the
[3H]thymidine method is based on the two
time points at which the location of the cells is measured, i.e., the
injection site of [3H]thymidine and the
location of migrating cells at the time of killing. The start-stop
nature of migration in the RMS would result in significant differences
when estimates are made at higher time resolution.
Attempts to identify the molecules that produce guided migrations along
the RMS have focused on the role of PSA-N-CAM (Ono et al., 1994; Hu,
2000). The movement of migrating neural precursors is characterized by
a "chain migration," especially in the adult (Doetsch and
Alvarez-Buylla, 1996; Lois et al., 1996), collagen gel (Hu et al.,
1996), or Matrigel (Wichterle et al., 1997), in which the migrating
cells move in association with each other (Rousselot et al., 1995).
This type of migration is thought to enable cells to use neighboring
cells as their substrate for translocation and quick migration. The
enzymatic removal of PSA results in the dispersion of chains into
single cells both in subventricular zone explants in Matrigel and in
adult mice (Hu, 2000). This result is surprising, because PSA is
believed to have an anti-adhesive activity attributable to its size and
negative charge. Thus, although the current evidence points to a role
for PSA-N-CAM in chain migration of neural precursors, its function for
the RMS migration remains unclear. In this context, it is interesting
that migrating chains are not observed in newborns (Hu, 2000; Pencea et
al., 2001), a time at which we detected little PSA-N-CAM in the RMS.
Interestingly, PSA-deficient cells migrate when transplanted into the
RMS of wild-type mice, showing that PSA is not required for their
migration per se (Hu et al., 1996). The paucity of PSA expression in
the RMS during early developmental stages, a time during which robust migrations also occur, supports this interpretation.
Although PSA-N-CAM is implicated in migrations in the RMS, it is also
evident that other molecules contribute. We show here that six
integrins are differentially expressed in a developmental, stage-specific manner, and those assayed, e.g., 1, 1, and v, are necessary for the migration at the developmental stage at which
each is expressed. Unfortunately, we were unable to test the functional
role of 3, 6, and 8 subunits because of unavailability of
function-blocking antibodies or technical problems in culturing slices
from young adults. We also identified ECM proteins that are
expressed in RMS and may serve as potential ligands for these integrins. These include two laminin subunits in addition to
tenascin-C, whose expression was reported previously (Jankovski and
Sotelo, 1996). The concomitant expression of 5- and 1-laminins
during E16-E19 and E16-P6, respectively, and of 1- and
1-integrins, also at late gestational stages, suggests that RMS
cells use 11-integrin to migrate along laminin-10 or -11, or
both, because laminin-10 and -11 are composed of 511 and
521 subunits, respectively. It is also possible that the cells
use a novel laminin, because we were unable to identify either 1- or
2-laminins in the RMS. In any case, laminins are effective
substrates for neural migration (Liang and Crutcher, 1992). The
resolution of these studies does not reveal how or where the laminin is
organized with respect to the migrating cells; however, the punctate
deposition is similar to that seen for other ECM proteins. Tenascin-C
is reported to be rich in the adult RMS and is proposed to play a role
in the migration of RMS (Jankovski and Sotelo, 1996; Thomas et al.,
1996). We found that it is also expressed in the RMS of younger mice. Tenascin-C is a ligand for both v3- and v6-integrins
(Yokosaki et al., 1996) and, therefore, might be one of the ligands
recognized by migrating cells. However, mice deficient in tenascin-C do
not show any apparent phenotype (Saga et al., 1992), suggesting that other ECM proteins in the RMS might also serve as a functional substrate for migration.
What directs the migration toward the distant olfactory bulb?
We next asked what directs the migrations on the
restricted pathway to the distant olfactory bulb. Previous in
vitro studies suggested that chemorepulsive factors from the
caudal region of the septum (Hu and Rutishauser, 1996) or choroid
plexus (Hu, 1999), e.g., the Slit proteins, might repel migrating cells
(Hu, 1999; Wu et al., 1999). The presence of Slits from these tissues
is thought to produce a gradient of chemorepulsive activity that prevents the cells from migrating in the caudal direction and thus
contributes to directing them toward the olfactory bulb. However, the
presence of the Slits does not explain why migrating cells do not
migrate into the tissues that do not express them, such as the putamen
or corpus callosum that surround the RMS. The glial tubes (Peretto et
al., 1997) surrounding RMS in the adult may also help contain the
migrating cells so they do not enter regions along the RMS pathway;
however, this specific structure is not present in the neonates (Kishi
et al., 1990).
We hypothesized, therefore, that chemoattractive factors generated at
the olfactory bulb contribute to the directed migration of RMS cells.
Our data suggest that DCC and neogenin expressed in the migrating cells
appear to recognize a chemogradient of netrin-1 secreted from the
mitral cells. However, netrin-1 expression is greatly reduced by P4,
suggesting that another chemoattractant may be expressed at subsequent
stages. Yee et al. (1999) reported that netrins can act over relatively
long distances. Netrin-1, generated by the floor plate close to the
pons, guides the migration of cells from the dorsal rhombencephalic
neuroepithelium to basilar pons, a distance of at least 3 mm. Netrin-1,
produced by the olfactory mitral cells, might function similarly in
guiding perinatal RMS migrations over a distance of <1.5 mm. The
unidirectional migration after P6 might then be controlled by Slits or
unidentified molecules. Although our data suggest a role for netrin-1
in the directional migration, it remains possible that other
netrin-like molecules that are recognized by DCC mediate the migration.
In Figure 8, we present a diagram
depicting a working model for the roles of DCC and netrin and integrins
in the migration of neural precursors from the SVZ to the center of
olfactory bulb. Slit proteins from the septum inhibit migration out of
the RMS into the septum by their repulsive activity. Integrins and
laminins provide the traction for the motive force, and PSA-N-CAM
provides the cellular milieu where the cells can move easily and
develop chains. The function of tenascin-C, although present in the
RMS, remains unclear. On arrival at the center of the olfactory bulb, the neural precursors begin to migrate radially and differentiate into
mature neurons. The molecules responsible for this process are not
known; however PSA-N-CAM does not appear to be involved (Ono et al.,
1994; Hu et al., 1996). Our immunocytochemistry suggests that integrins
are also not involved. Thus the molecular mechanisms that mediate
radial migration and neuronal differentiation are important targets for
future study.
View larger version (29K):
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Figure 8.
Diagram depicting a working hypothesis for the
roles of DCC and netrin and integrin in the migration of neural
precursors from the SVZ to the center of the olfactory bulb. Netrin-1
secreted from mitral cells attracts DCC- or neogenin-expressing
migrating cells, or both, to the olfactory bulb. Slit proteins from the
septum inhibit migration out of the RMS into the septum or surrounding
tissues by their repulsive activity. Integrins and laminins provide the
traction for the motive force, and PSA-N-CAM provides the cellular
milieu where the cells can move easily and maintenance of chains. The
short arrow indicates the differentiation of neural
precursors to granule cells; the long arrow marks the
periglomerular cells. EPL, External plexiform layer;
GCL, granule cell layer; GL, glomerular
layer; MCL, mitral cell layer; ONL,
olfactory nerve layer.
|
|
| |
FOOTNOTES |
Received Sept. 5, 2001; revised Feb. 6, 2002; accepted Feb. 6, 2002.
This work was supported by National Institutes of Health Grants GM23244
and GM53905 (A.F.H.), by the Cell Migration Consortium (A.F.H.), and by
a Human Frontier Science Program Organization award (S.M.). We thank
Dr. J. H. Miner, Dr. G. Rougon, and Dr. K. Sekiguchi for providing antibodies.
Correspondence should be addressed to Shin-ichi Murase, Department of
Cell Biology, University of Virginia School of Medicine, P.O. Box
800732, Charlottesville, VA 22908. E-mail: sm4fh{at}virginia.edu.
| |
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