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The Journal of Neuroscience, January 15, 2001, 21(2):527-540
Mode and Tempo of Tangential Cell Migration in the Cerebellar
External Granular Layer
Hitoshi
Komuro1, 2,
Ellada
Yacubova1,
Elina
Yacubova1, and
Pasko
Rakic2
1 Department of Neurosciences, Lerner Research
Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, and
2 Section of Neurobiology, Yale University School of
Medicine, New Haven, Connecticut 06510
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ABSTRACT |
After their final mitosis, cerebellar granule cells remain in the
external granular layer (EGL) for 20-48 hr before initiating their
radial migration across the molecular layer (ML), but the significance
of this latent period is not well understood. In the present study, we
used a confocal microscope to examine morphogenetic changes and
behavior of postmitotic granule cells restricted to the EGL in slice
preparations of the postnatal mouse cerebellum. We found that,
coincident with the extension of two uneven horizontal processes
oriented parallel to the longitudinal axis of the folium, postmitotic
granule cells start to migrate tangentially in the direction of the
larger process. Interestingly, their morphology and the speed of cell
movement change systematically with their position within the EGL. The
rate of tangential cell movement is fastest (~14.8 µm/hr) in the
middle of the EGL, when cells have two short horizontal processes. As
granule cells elongate their somata and extend longer horizontal
processes at the bottom of the EGL, they move at a reduced rate
(~12.6 µm/hr). At the interface of the EGL and ML where cells
migrate tangentially at the slowest rate (~4.1 µm/hr), their somata
round and then begin to extend couples of the descending processes into
the ML. After the stationary period, granule cells abruptly extend a
single vertical process and initiate the transition from tangential to radial migration, reshaping their rounded somata into a vertically elongated spindle. These observations suggest that tangential migration
of granule cells within the EGL may provide the developmental mechanisms for their appropriate allocation across parasagittal compartments of the expanding cerebellar cortex.
Key words:
cerebellar development; granule cell; neuronal cell
migration; confocal microscopy; brain slice preparation; fluorescent
carbocyanine dye; rate of cell movement
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INTRODUCTION |
Morphological transformation and
translocation of cerebellar granule cells within the external granular
layer (EGL) offer an opportunity to study the basic mechanisms of
neuronal migration and differentiation. This secondary proliferative
layer originates at the lateral aspect of the rhombencephalon (rhombic
lip by His, 1890 ) and spreads quickly over the entire surface of the
cerebellar hemispheres during the prenatal development (for review, see
Sidman and Rakic, 1982; Rakic, 1985). Early studies using
3H-thymidine labeling have confirmed the
general concept, proposed by Ramon y Cajal (1911), that cerebellar
granule cells originate in the EGL and, after their final cell
division, migrate radially across the molecular layer (ML) to the
internal granular layer (IGL) where they reside in the adult cerebellum
(Miale and Sidman, 1961; Fujita et al., 1966; Fujita, 1967). Combined
application of electron microscopy, Golgi-impregnation method, and
3H-thymidine autoradiography indicated
that postmitotic granule cells remain in the EGL for >20 hr as they
form, in succession, three cytoplasmic processes that change the shape
of the cell from round to bipolar and then to the three-polar
form (Rakic, 1971, 1972, 1973). More recently, the use of
replication-incompetent retrovirus suggests that granule cells may
migrate tangentially in both rostrocaudal and mediolateral planes
before onset of their radial migration (Ryder and Cepko, 1994).
Although radial migration of granule cells along the Bergmann glial
processes in the ML have been extensively analyzed (for review, see
Rakic, 1981, 1990; Hatten and Mason, 1990), little is known about the
early behavior of postmitotic granule cells within the EGL before they
start their descent to the ML. Yet, the tangential movement within the
EGL is critical to understanding how cerebellar compartments form, as
well as how clonally related cells become allocated to the IGL in the
expanded cerebellar hemispheres. This is an important issue because,
after radial migration begins, the granule cells cannot change any more
than their mediolateral or anteroposterior position (Komuro and Rakic,
1998a). During early development, the cerebellum expresses distinct,
stripe-like compartments as revealed by the expression of specific
genes and molecules, containing candidate molecules that may generate
attractive or repulsive signals to migrating cells (Hawkes and Leclerc,
1987; Herrup and Kuemerle, 1997; Lin and Cepko, 1998; Ozol et
al., 1999; Alcantara et al., 2000). The differential expression of
various spatial cues raises the possibility that postmitotic granule
cells may migrate within the EGL to find their assigned position in appropriate compartments before establishing their contact with Bergmann glial fibers that will carry them to the IGL.
A major impediment in the analysis of granule cell behavior in the EGL
was the availability of a reliable assay system. For example, the use
of 3H-thymidine labeling alone could not
reveal the extent, direction, and rate of cell migration within a
histologically homogeneous cell layer such as the EGL. Moreover,
dissociated cells and microexplant culture systems do not retain the
ambient cellular environment and cytoarchitecture of the living
cerebellum. However, the development of acute slice preparation (for a
description of the method, see Komuro and Rakic, 1999) allows direct
observation of the granule cell movement within its natural cellular
milieu (Komuro and Rakic, 1992, 1993, 1995, 1998a,b). The study of
granule cell behavior in real time demonstrates that, at the various
levels of the EGL, postmitotic granule cells exhibit a different
morphology and rate of tangential movement before the onset of radial
migration that sets the site of its journey to the final destination.
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MATERIALS AND METHODS |
Cerebellar slice preparations. Postnatal day 10 (P10) mice (CD-1) were killed by decapitation, in
accordance with institutional guidelines. Cerebella were quickly
removed from the skull and placed in cold (5°C) HBSS. Before
cutting, cerebella were embedded in 20% gelatin and sectioned
transversely or sagittally into 300- to 400-µm-thick slices on a
vibrating blade microtome (VT1000S; Leica, Nussloch, Germany).
After sectioning, pia mater and gelatin were carefully removed under a
dissecting microscope.
1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate labeling. To label granule cells in the EGL,
cerebellar slices were incubated for 3 min at room temperature in a
fluorescent lipophilic carbocyanine dye
[1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI)] (7.2 µg/ml) (Molecular Probes, Eugene, OR), which was added
to the cell culture medium (Honig and Hume, 1986). The incubation
medium consisted of minimum essential medium (Life Technologies,
Rockville, MD) supplemented with 40 mM
glucose, 1.8 mM glutamine, 24 mM NaHCO3, penicillin (90 U/ml), and streptomycin (90 µg/ml). After rinsing in the incubation
medium, brain slices were maintained in an incubator (37°C, 95%
O2-5% CO2) for an
additional 2 hr to allow diffusion of DiI molecules in the plasma
membrane of the granule cells. Slices were then transferred into the
chamber of a microincubator (PDMI-2; Medical Systems Corp., Greenvale, NY) attached to the stage of an inverted microscope (DM IRBE; Leica).
The rate of cell movement is closely related to the temperature of the
medium; lowering the medium temperature slows cell movement (Rakic and
Komuro, 1995). Therefore, the chamber temperature was kept at 37.0 ± 0.5°C using a temperature controller (TC-202; Medical Systems
Corp.), and the slices were provided with constant gas flow (95%
O2-5% CO2). To prevent
movement of the slice preparation during observation, a nylon net glued
to a small silver wire ring was placed over the preparations.
Observation of granule cell migration. A laser scanning
confocal microscope (TCS SP; Leica) was used to visualize migrating granule cells labeled with DiI in the slices (Komuro and Rakic, 1995,
1998a; Rakic and Komuro, 1995). The use of this microscope permitted
high-resolution imaging of migrating neurons up to 120 µm deep within
the tissue slices. The tissue was illuminated with a 488 nm wavelength
light from an argon laser through an epifluorescence inverted
microscope equipped with a 40× oil-immersion objective (numerical
aperture 1.25; Leica), and fluorescence emission was detected at
530 ± 15 nm. To clearly resolve movement of migrating cells,
image data typically were collected at an additional electronical zoom
factor of 1.5-3. Time-lapse imaging of live, fluorescently labeled
cells can produce phototoxic effects in the imaged cells. Indeed, when
well stained cells were imaged with very high incident illumination
intensity or were imaged too frequently, we invariably saw changes in
the structure or dynamics of the migrating granule cells. However, when
the incident illumination was sufficiently attenuated, the labeled
specimens could be imaged (5 min intervals) for many hours without
signs of photodynamic damage (Komuro and Rakic, 1995, 1998a). To
protect the migrating granule cells labeled with DiI from any cytotoxic
effect of the laser beam, the excitation light level was reduced by
99%. To avoid injured granule cells located near the sectioning
surfaces, we examined the shape and behavior of migrating granule cells
located 15-50 µm below the surface of the slices. If granule cells
showed no evidence of changes in cell shape or motility for >300 min,
the brain slice was discarded. Shock to the tissue during sectioning of
slices can disrupt cell movement and prevent cells from migrating.
Accordingly, the present study is based on the analysis of
approximately one-half of the healthy slices that had displayed active
cell migration. This sampling procedure favored slices in which cells
displayed visible and robust movement and alteration of morphology
shortly after sectioning. Images of the postmitotic granule cells in a single focal plane or up to 40 different focal planes along the z-axis were collected with laser scans every 5-10 min for
up to 10 hr and recorded on an external drive (Jaz 2GB; Iomega, Roy, UT). At the beginning and end of each recording session for each preparation, frame images were recorded with 40× (electronical zoom
factor of 1) or 20× (electronical zoom factor of 1) magnification to
determine the orientation of the slice preparations, the borders between the EGL and the ML, and the position of granule cells within
the EGL by optical sectioning of several different focal planes along
the z-axis. Statistical significance between experimental groups was tested by Student's t test.
The length/width ratio of the soma and leading process length of each
migrating granule cell were determined manually with a mouse-driven
imaging software package. The leading process length was defined as the
linear distance between its tip and its base. The distance traveled by
a migrating granule cell was defined as the absolute value of the
changes in its position during the entire time-lapse session.
5-Bromo-2'-deoxy-uridine labeling and
immunohistochemistry. To determine the time course of granule cell
migration, postnatal 10-d-old mice were injected intraperitoneally with
5-bromo-2'-deoxy-uridine (BrdU) (50 mg/kg body weight), which was
incorporated into the DNA of dividing cells during the S-phase of the
cell cycle, and were killed 2 hr, 1 d, and 2 d later (Kuhn et
al., 1996). After an anesthetic overdose, all animals were
transcardially perfused with 4% paraformaldehyde. Brains were removed,
post-fixed in 4% paraformaldehyde for 24 hr, and stored in a 30%
sucrose solution. Brains were embedded in Tissue-Tek (Sakura
Finetechnical Corp., Torrance, CA) and sectioned horizontally into
30-µm-thick slices on a cryostat. For detection of BrdU-labeled
nuclei in tissue sections, the following DNA denaturation steps
proceeded the incubation with anti-BrdU antibody: 2 hr incubation in
50% formamide-2× SSC (0.3 M NaCl and 0.03 M sodium citrate) at 65°C, 5 min rinse in 2×
SSC, 30 min incubation in 2N HCl at 37°C, and 10 min rinse in 0.1 M boric acid, pH 8.5. After completion of DNA
denaturation, cells that had incorporated BrdU into DNA were detected
by an anti-BrdU monoclonal antibody (BrdU Labeling and Detection Kit I;
Boehringer Mannheim, Indianapolis, IN) and fluorescein-conjugated secondary antibody. Fluorescent signals were detected and processed using a confocal microscope.
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RESULTS |
Itinerary of postmitotic granule cells within the EGL
To determine the time course of translocation of granule cells
after their final mitosis within the EGL, we injected BrdU into
postnatal 10-d-old mice and killed them 2, 24, and 48 hr later. Two
hours after injection, BrdU-labeled cells were localized at the
outermost two rows of the EGL, showing directly that granule cell
precursors proliferate within the top level of the EGL, whereas the
middle and bottom levels of the EGL contain unlabeled postmitotic granule cells (Fig.
1Aa). Within the next
24 hr, the BrdU-labeled cells spread across the entire EGL, and some
(Fig. 1Ab, white arrowheads) begin their
descent to the ML. Two days after injection, ~50% of BrdU-labeled
cells have left the EGL and translocated their soma into the ML,
Purkinje cell layer (PCL), or IGL (Fig. 1Ac).
Previous studies indicated that the postsynthetic
(G2) phase and the mitotic (M) phase of the cycle
of the granule cell precursor in 10-d-old mouse cerebellum last 2 and 0.5 hr, respectively (Fujita, 1967). Together, these results
demonstrate that, after final cell division, postmitotic granule cells
remain in the EGL between 20 and 48 hr before the initiation of their
radial migration across the ML.
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Figure 1.
A, Time course of translocation of
postmitotic granule cells in the EGL. P10 mice were injected with BrdU
intraperitoneally and killed 2 hr (a), 1 d
(b), and 2 d (c)
later. Two hours after injection, BrdU-labeled cells were localized at
the top level of the EGL. One day after injection, the BrdU-labeled
cells occupied the entire EGL. Two days after injection, approximately
half of BrdU-labeled cells left the EGL and translocated to the ML,
PCL, and IGL. B, Morphology of granule cell precursors
and postmitotic granule cells in the EGL of P10 mouse cerebellum. The
granule cells in the EGL at the pyramis were visualized 2 hr after
DiI staining. The granule cell precursor
(a) had a round soma without any long processes.
Postmitotic granule cells (b, c) had
spindle-shaped cell bodies oriented parallel to the longitudinal axis
of the folium with two horizontal processes. On the EGL-ML border, the
postmitotic granule cell (d) had a rounded cell
body with vertically oriented short processes penetrating the ML. Scale
bars, 10 µm. PC, Purkinje cell.
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We next examined the morphological characteristics of granule cells
located at different levels of the EGL. Transverse sections were
obtained from postnatal 10-d-old mouse cerebellum, and the cells were
visualized 2 hr after staining with DiI (Fig. 1). The cell located near
the top level of the EGL (Fig. 1B, a) has
a round shape, lacking the long processes as described in previous Golgi (Ramon y Cajal, 1911) and electron microscopic (Rakic, 1971, 1972, 1985) studies. In the middle and bottom levels of the EGL, the
cells assume a spindle shape, oriented parallel to the longitudinal axis of the folium with two horizontal processes emanating from the
opposite poles of their soma (Fig. 1B,
b, c). The two processes of the cell in the
middle level of the EGL are always different in size; one process is
thicker and ~50 µm long, similar to a leading process with a large
filopodium at its tip (white arrow), whereas the other
process is thin and short (25 µm), reminiscent of an axon. In
contrast, both horizontal processes of the cell in the bottom level of
the EGL are usually longer than 100 µm (white arrowheads)
and uniform in diameter. At the border between the EGL and the ML, the
granule cell (Fig. 1B, d) again acquires a
rounded shape of its soma as it emanates the third, vertically oriented
process as it penetrates the ML (open arrow). These results confirm that postmitotic granule cells undergo dynamic changes in their
morphology while remaining in the EGL (Rakic, 1971, 1985).
Tangential cell migration in the middle level of the EGL
We used confocal microscopy to examine dynamic changes in the
shape and behavior of the postmitotic granule cells at different levels
of the EGL in real time. For example, the cell illustrated in Figure
2A was visualized 2 hr
after staining, and its movement was recorded in optical sections at 10 min intervals for up to 150 min. At the beginning of the recording, it
had a horizontally oriented, spindle-shaped cell body, a thin trailing
process, and a voluminous leading process as described in
Golgi-impregnated sections (Ramon y Cajal, 1911; Rakic, 1971) and
electron microscopic preparations (Rakic, 1985). The distal portion of
the leading process had large motile lamellipodia (white small
arrowheads). During the observation period, the granule cell soma
(white asterisks) with an elongated bipolar shape (length vs
width ratio of 2.0-3.5) (Fig.
2B,D) was initially located ~17
µm away from the pial surface, near the midline of the pyramis, and
was gradually displaced toward the left side of the cerebellar
hemisphere at a rate of 18.8 µm/hr. Although the distance traversed
by the granule cell gradually increased, the cell body did not move
linearly (Fig. 2C). Rather, the migration speed of the cell
body fluctuated between phases of rapid advancement to a complete
curtailment of movement, similar to the saltatory movement of the glial
cell-associated migration observed in the ML (Komuro and Rakic, 1995,
1998a).
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Figure 2.
Tangential migration of a granule cell in the
middle level of the EGL. A, During the observation
period, the granule cell soma (white asterisks)
gradually moved toward the left side of the cerebellar hemisphere at a
rate of 18.8 µm/hr. The distal portion of the leading process had
large motile lamellipodia (small white arrowheads). Time
interval (in minutes) is indicated on the top right of
each photograph. Scale bar, 10 µm. The total distance traversed by
the granule cell soma (B), the direction and
distance traveled by the soma during each 10 min of the testing period
(C), the length/width ratio of the soma
(D), and the leading process length
(E) were plotted as a function of elapsed time.
In C, positive values represent forward cell movement,
and negative values represent backward cell movement.
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At the beginning of the observation, a voluminous leading
process extended laterally, and its length increased from 35 to 71 µm
(Fig. 2E). However, 60 min later, the tip of the
leading process (white arrows) stopped growing. The
shortening of the leading process from 71 to 40 µm (Fig.
2E) is attributable to the translocation of
the granule cell soma within the leading process rather than its active
retraction. The present results demonstrate that, in the middle level
of the EGL, the postmitotic granule cell, which has a short leading
process and a thin trailing process, migrates laterally (parallel to
the folial folds) with a speed similar to that observed in the ML for
cell migrating radially (Komuro and Rakic, 1995, 1998a). We also found
that, in the middle level of the EGL, the spindle-shaped cell body of a
granule cell (white asterisks) initially located ~18 µm
away from the pial surface near the midline of the pyramis remained stationary for >3 hr, while its voluminous leading process
(white arrows) continued to grow (Figs.
3A-C). During a 220 min
observation period, the length of the leading process doubled (from 46 to 95 µm) (Fig. 3D,E). The
leading process often developed actively motile filopodia (open
arrows) near the tip and lamellipodia at the center (white
small arrowheads), although their soma do not migrate during
extension of the horizontal processes.
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Figure 3.
Postmitotic granule cell extending its voluminous
leading process without active cell movement. A, In the
middle level of the EGL, the cell body of a granule cell (white
asterisks) remained stationary for >3 hr, while its voluminous
leading process (white arrows) extended continuously.
Time interval (in minutes) is indicated on the top left
of each photograph. Scale bar, 10 µm. The total distance traversed by
the granule cell soma (B), the direction and
distance traveled by the soma during each 5 min of the testing period
(C), the leading process length
(D), and changes in the leading process length
during each 5 min of the testing period (E) were
plotted as a function of elapsed time.
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Among 124 granule cells examined in the middle level of the EGL in
transverse or sagittal slices, 92 cells (83% of total cells) migrated
in the transverse plane (parallel to the folium) at a speed of >10
µm/hr, 17 cells (9%) migrated at the speed between 5 and 10 µm/hr,
and 15 cells (7%) moved at the rate <5 µm/hr or remained
stationary. Moreover, we did not find any postmitotic granule cells
showing the anteroposterior movement (perpendicular to the folium) in
the middle level of the EGL. Thus, the majority of the postmitotic
granule cells located in the middle level of the EGL actively migrate
in the transverse plane.
Direction and rate of cell movement at the bottom of the EGL
Next we examined granule cell behavior and change in their
morphology at the bottom of the EGL. A typical example of a
spindle-shaped granule cell located in the bottom level of the EGL (29 µm away from the pial surface) with two long (over 100 µm)
horizontal processes oriented parallel to the longitudinal axis of
folia is provided in Figure
4A. During the
observation period, its cell body (white asterisks)
exhibited saltatory movement and migrated toward the lateral side of
the hemisphere at the rate of 15.7 µm/hr (Figs.
4B,C), which is comparable with
that observed in the ML and IGL (Komuro and Rakic, 1995, 1998a). The
cell changed its shape during its movement, possibly as a result of
squeezing past various obstructions in its environment (Fig.
4D). Interestingly, 260 min after the beginning of
the recording, the horizontally extended leading process bent slightly
toward the ML. However, after 340 min of recording, the tip of the
leading process again changed the direction of extension toward the
middle of the EGL (Fig. 4A, arrowheads).
Although the tips of horizontally extending leading processes often
exhibit upward and downward movement within the bottom of the EGL, the
leading processes did not penetrate into the ML and did not transform
into vertical processes. These results demonstrate that postmitotic
granule cells located in the bottom level of the EGL do not stop their
movement but continue to migrate in the mediolateral direction.
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Figure 4.
Tangential movement of a granule cell in the
bottom level of the EGL. A, During the observation
period, the spindle-shaped soma of the granule cell having two long
horizontal processes (white arrowheads) migrated toward
the left side of the hemisphere. Time interval (in minutes) is
indicated on the top right of each photograph. Scale
bar, 10 µm. The total distance traversed by the granule cell soma
(B), the direction and distance traveled by the
soma during each 10 min of the testing period
(C), and the length/width ratio of the soma
(D) were plotted as a function of elapsed
time.
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A small number of cells reverse the direction of cell body movement
during the tangential migration at the bottom level of the EGL. For
example, we found that the horizontally oriented granule cell body
(white asterisks) located ~30 µm away from the pial
surface initially moved toward the left aspect of the hemisphere (Fig.
5A). However, 80 min after the
beginning of the recording, the granule cell changed the direction of
cell movement and started to migrate toward the right hemisphere (Fig.
5B,C). It is likely that, during
the transition period, while reversing the direction of cell movement,
the granule cell altered its polarity. For example, during the first
140 min of recording, one horizontal process (white arrows)
extending from the left side of the cell body was thicker, resembling
the leading process, whereas the other horizontal process (open
arrows) extending from the right side of the cell body was
thinner, resembling the trailing process. However, 160 min after
recording, the left side process became thinner, resembling the
trailing process, whereas the right side process became thicker, resembling the leading process. The granule cell also changed the shape
of its soma during the transition periods of cell polarity (Fig.
5D). Moreover, the cell slightly increased the rate of
movement after changing the direction of migration from 14.6 µm/hr
(toward the left side) to 17.9 µm/hr (toward the right side) (Fig.
5B). These results suggest that postmitotic granule cells
located in the bottom level of the EGL may be able to alter the
direction of cell movement, indicating the possibility that their cell
polarity may not be rigid, but reversible, possibly in response to
changes in the local cues.
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Figure 5.
A reverse in the direction of tangential movement
of a granule cell in the bottom level of the EGL. A, The
horizontally oriented cell body (white asterisks) of the
granule cell initially moved toward the left hemisphere at a rate of
14.6 µm/hr, and 80 min later, changed its direction of movement and
started to migrate toward the right hemisphere at a rate of 17.9 µm/hr. Black circles represent a reference point. Time
interval (in minutes) is indicated on the bottom right
of each photograph. Scale bar, 10 µm. The total distance traversed by
the granule cell soma (B), the direction and
distance traveled by the soma during each 10 min of the testing period
(C), and the length/width ratio of the soma
(D) were plotted as a function of elapsed time.
In B and C, positive values represent
cell movement toward the left hemisphere, and negative values represent
cell movement toward the right hemisphere.
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Among the 120 granule cells examined in the bottom level of the EGL,
the longest process extending from one side of the horizontally oriented granule cells was 190 µm in the length. The two horizontal processes were usually not of even length. For example, Figure 6, A and B,
illustrates that the granule cell located at the bottom level of the
EGL (36 µm away from the pial surface) had one long (157 µm) and
one short (54 µm) horizontal process. During the period of our
observation, the cell moved toward the right side of the hemisphere at
a reduced rate of 9.1 µm/hr while altering its cell body shape
(Fig. 6C-E). During this period, the cell did not form
filopodia and lamellipodia, except for the single small extension at
the tip of the process. These results suggest that, at the bottom level
of the EGL, granule cells with the longer horizontal processes move at
a reduced rate.
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Figure 6.
Slowdown of tangential movement of a granule cell
with longer horizontal processes. A, During the period
of observation, the cell with longer horizontal processes moved toward
the right side of the hemisphere at a reduced rate of 9.1 µm/hr.
Interestingly, the cell did not protrude filopodia and lamellipodia
from its cell body and horizontal processes. Time interval (in minutes)
is indicated on the top right of each photograph. Scale
bars, 10 µm. B, A photograph representing the long
horizontal process of tangentially migrating granule cell shown in
A. The total distance traversed by the granule cell soma
(C), the direction and distance traveled by the
soma during each 10 min of the testing period
(D), and the length/width ratio of the soma
(E) were plotted as a function of elapsed
time.
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Cessation of tangential migration at the interface of the EGL
and ML
How do granule cells change the direction of migration from
tangential to radial? How do granule cells initiate their radial migration through the ML? Are there specific signs indicating the time
and place for the initiation of radial migration? These questions are
critical for understanding the cellular and molecular mechanisms
underlying the transition between the two modes of granule cell
migration. To address these issues, we examined the changes in
morphology and behavior of horizontally oriented granule cells situated
near the border between the EGL-ML. Figure
7 illustrates a granule cell located near
the border between the EGL and ML that extrude large third processes
from the center of its soma. During the period of recording, this
highly motile process with lamellipodia (open arrows) and
filopodia (white arrows) at the tip extended from the
spindle-shaped cell body into the ML (Fig. 7A). The length
versus width ratio of the cell body during this period was consistently
low, with a range of 3.2-2.1 (Fig. 7D). During extension of
the vertical process, a horizontally oriented cell body
(asterisks) exhibited the tangential movement toward the
right hemisphere at a significantly reduced speed of 3.8 µm/hr (Fig.
7B,C). These results suggest that
significant reduction in tangential cell body movement occurs during
extrusion of vertical processes into the ML as a sign for ending
tangential migration at the interface of the EGL and ML. Furthermore,
the newly extended vertical processes may actively search for potential
guidance cues responsible for changing the direction of cell movement
from tangential to radial.
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Figure 7.
Slowly migrating granule cell with descending
processes near the EGL-ML border. A, The highly motile
processes having lamellipodia-like structure (open
arrows) and filopodia (white arrows), extended
from the cell body. During the process extension, the horizontally
oriented cell body (asterisks) exhibited the tangential
movement toward the right hemisphere of the cerebellum at a
significantly reduced rate of 3.8 µm/hr. Time interval (in minutes)
is indicated on the bottom left of each photograph.
Scale bar, 10 µm. The total distance traversed by the granule cell
soma (B), the direction and distance traveled by
the soma during each 5 min of the testing period
(C), and the length/width ratio of the soma
(D) were plotted as a function of elapsed
time.
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Initiation of radial migration
How and at which site do granule cells alter their mode and
direction of migration from glia-independent, tangential movement in
the EGL to the Bergmann glia-dependent radial movement in the ML? How
do granule cells generate their axon, known as parallel fibers, during
the transition phase from one form of migration to another? To address
these questions, we have examined the onset of radial migration of
granule cells at the interface between the EGL and the ML. At the
beginning of recording, a granule cell (asterisks) located
at the interface between the EGL and ML (dotted line)
retained two elongated horizontal processes, whereas its nucleus and
surrounding cytoplasm started to enter into the short vertical process
descending into the ML (Fig.
8A). It took ~30 min
for the completion of the translocation of its nucleus and surrounding
cytoplasm from the horizontally extended process to the vertical
process. After the completion of change in nucleus orientation, the
soma quickly moved toward the bottom of the ML (Fig.
8B,C). During the translocation,
the shape of its soma transformed from a sphere (a length vs width
ratio of 1.5-1.8) to a vertically elongated spindle (a length vs width
ratio of 2.5-4.0) (Fig. 8D). As a result of the
translocation of the soma within the leading process, the granule cell
develops a thin trailing process connected with two horizontal
processes (immature parallel fiber) (Fig. 8A). These
results demonstrate in real time that two horizontal processes emitted
from each side of granule cell soma at the bottom level of the EGL
transform into future parallel fibers, as deduced from the light and
electron microscopic observations (Rakic, 1971).
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Figure 8.
Initiation of vertical migration and formation of
a T-shaped axon. A, At the beginning, a granule cell
(asterisks) located near the EGL-ML border
(dotted line), had vertically oriented soma with two
horizontal and one vertical process. The cell remained stationary for
the first 30 min before its soma quickly moved radially toward the
bottom of the ML. As a result, the granule cell developed a trailing
process (small arrow) and a T-shaped axon. Time interval
(in minutes) is indicated on the bottom right of each
photograph. Open arrows represent rapid extension of the
horizontal process of other granule cells. Scale bar, 10 µm. The
total distance traversed by the granule cell soma
(B), the direction and distance traveled by the
soma during each 10 min of the testing period
(C), and the length/width ratio of the soma
(D) were plotted as a function of elapsed
time.
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Although the majority of parallel fibers develop from the two
pre-existing horizontal processes of a tangentially migrating granule
cell, we observed here another mechanism for the formation of parallel
fibers. Figure 9A illustrates
that, during the initiation of vertical migration near the EGL and ML
border (dotted line), a vertically oriented granule cell may
initiate a new process, which becomes one side of the parallel fibers.
During the first 30 min of recording, the granule cell
(asterisks) had a single horizontal process extending toward
the left side of the cerebellum. After 40 min of recording, the cell
developed a new small process (open arrow) at the rear part
of the vertically elongated soma directed toward the right side (Fig.
9A). Thus, the T-shaped axon consisting of a trailing
process and the two arms of the future parallel fiber is formed during
the course of downward movement of the soma (Fig.
9B,C). Interestingly, during the
process of the initiation of radial migration, the soma of the granule
cell became transiently round (Fig. 9D) and withdrew its
vertical process. However, 40 min after rounding, the soma assumed its
spindle shape and re-extended its vertical process toward the ML (Fig.
9A). These results demonstrate that, in some cases, one side
of a parallel fiber originates from a single horizontal process of
laterally migrating granule cells in the EGL, whereas the other side
develops later in a separate manner.
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Figure 9.
Extrusion of one side of the parallel fibers
during initiation of vertical migration of a granule cell.
A, During the first 30 min, the granule cell
(asterisks) located near the EGL-ML border
(dotted line), had a single horizontal process extending
toward the left side. Forty minutes later, the cell developed a small
horizontal process (open arrow) at the rear part of its
soma toward the right side. As a result of downward movement of the
soma, the cell developed a T-shaped axon branch. Time interval (in
minutes) is indicated on the bottom right of each
photograph. Scale bar, 10 µm. The total distance traversed by the
granule cell soma (B), the direction and distance
traveled by the soma during each 5 min of the testing period
(C), and the length/width ratio of the soma
(D) were plotted as a function of elapsed
time.
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Speed and direction of granule cell migration depend on their
position in the EGL
To further elucidate the behavior of postmitotic granule cells in
the EGL, we examined the relationship between the vertical levels of
granule cells within the EGL, their morphology (Fig. 10A-C), and the rate
of their migration (Fig. 10D). In the postnatal 10-d-old mouse cerebellum, the EGL in the pyramis is ~30 µm wide and consists of approximately five to six rows of tightly packed granule cells (proliferating cells at the top two rows, and postmitotic cells at the bottom three to four rows). To categorize the morphology and behavior of the postmitotic granule cells within the EGL, we
divided the thickness of the EGL into five levels: the first (I) and
second (II) levels (proliferating cell layer), within 12 µm of the
pial surface; the third (III) level (postmitotic cell layer), within 18 µm of the pial surface; the fourth (IV) level (postmitotic cell
layer), within 24 µm of the pial surface; and the fifth (V) level
(postmitotic cell layer), over 24 µm from the pial surface. As
presented quantitatively in the histograms (Fig.
10A-D), the morphology and behavior of the
postmitotic granule cells depends on their vertical position. For
example, the rate of granule cell movement is the fastest (14.8 ± 1.2 µm/hr) in the third (III) level when cells have a short, leading
process-like fiber (32.9 ± 1.3 µm) and horizontally oriented
spindle-shaped soma (a length vs wide ratio of 3.0). In the fourth (IV)
level, the granule cells slow down their movement to 12.6 ± 0.6 µm/hr while extending their soma (a length vs wide ratio of 3.4) and leading-like process (94.5 ± 2.0 µm). Cell movement of granule cells is the slowest (4.1 ± 0.4 µm/hr) in the fifth (V) level after their somata become round (a length vs wide ratio of 2.2) and the
leading-like processes further extend horizontally (130.0 ± 7.3 µm). These data suggest that postmitotic granule cells alter their
shape and rate of movement in different levels of the EGL.
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Figure 10.
A-E, Histograms of soma length
(A), soma width (B),
leading process length (C), movement
rate(D), and movement direction
(E) of granule cells in the EGL. To categorize
the morphology and behavior of the postmitotic granule cells within the
EGL, we divided the width of the EGL of the P10 mouse into five levels:
levels I, II (proliferating cell layer;
within 12 µm of the pial surface); level III
(postmitotic cell layer; within 18 µm of the pial surface);
level IV (postmitotic cell layer; within 24 µm of the
pial surface); and level V (postmitotic cell layer; over
24 µm from the pial surface). Each column represents
the average values obtained from 80 migrating granule cells in level
III, 75 cells in level IV, and 64 cells in level V. *p < 0.05 and **p < 0.01 indicate statistical significance.
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The real-time observation of identified granule cells also revealed a
difference in direction of tangential cell migration between the
midline and lateral regions of the cerebellum (Fig. 11E). Among 105 granule cells examined at the midline region of the cerebellum, 50 cells (48%) migrated toward the right side of the hemisphere, 53 cells
(50%) migrated toward the left side, and only three cells (2%)
reversed the direction of their cell movement either from right side to
left side or from left side to right side. Among 92 granule cells
examined in the left hemisphere, 53 cells (58%) migrated toward the
right side of the hemisphere (toward the midline regions), 37 cells
(40%) migrated toward the left side, and only two cells (2%) reversed
their direction of cell movement. Among 96 granule cells examined in
the right hemisphere, 36 cells (38%) migrated toward the right side of
hemisphere, 59 cells (61%) migrated toward the left side (toward the
midline regions), and only two cells (2%) reversed the direction of
movement. These observations indicate that postmitotic granule cells
located in the left or right side of the hemisphere tend to migrate
toward the midline region, whereas the cells located in the midline
region do not exhibit a preference in the movement direction between the left and right side. Furthermore, postmitotic granule cells located
in the left or right side of the hemispheres may move in response to
localized guidance cues. At present, the source or nature of either
attractive or repulsive signals has not been identified.
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Figure 11.
A, Shift of the
geography of developing cerebellar cortex over time. At P10, the
granule cell precursors proliferate within ~19-21 hr of the cell
cycle within levels I and II of the EGL. Approximately 21 hr after
their final cell division, tangentially migrating granule cells change
the orientation of their somata from horizontal to vertical and start
to leave the EGL. As a result, their horizontal processes become
parallel fibers, and the EGL-ML border moves up toward the pial
surface (as seen at P11). Therefore, the granule cell, which initially
migrates tangentially at level III of the EGL at P10, will be located
at the level V after 24 hr (at P11), without undergoing downward
movement. B, Schematic drawing illustrating the
hypothesis that granule cells may be specified to settle in a
particular A-P compartment and have to travel tangentially over
relatively long distances in the middle and the bottom of the EGL to
reach a target compartment. Granule cells that underwent final cell division in
the same area at the top level of the EGL may settle in different
compartments in the IGL. For example, green granule
cells may be instructed to enter a green compartment,
whereas red granule cells may be instructed to enter a
red compartment. Furthermore, postmitotic granule cells
located in the left or right side of the hemisphere tend to migrate
toward the midline region, whereas the cells located in the midline
region do not exhibit a preference in the movement direction between
the left and right side.
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DISCUSSION |
The real-time observation in living slice preparations reveals
that, after their last mitotic division, prospective granule cells
remain in the EGL from 1 to 2 d. During that period, the majority
of postmitotic cells become oriented in the transverse plane, and their
two horizontal processes transform into parallel fibers, as has been
correctly inferred from the static Golgi preparations (Ramon y Cajal,
1911). In addition, we have observed substantial tangential migration
during the period between the last cell division and the onset of their
descent to the ML. The slice preparations expose vividly that cell
shape, mode, and tempo of migration changes systematically according to
their position within the EGL. In the middle of the EGL (Fig.
11A, level III), which is situated below the proliferative cell layers (levels I,
II), the initially round, postmitotic cells elaborate
two short but uneven horizontal processes, oriented parallel to the
axis of the future folium and begin to migrate tangentially at the
fastest rate. At the bottom level of the EGL (level
IV), granule cells acquire spindle-shaped somata and
further extend two horizontal processes while moving at a slightly
reduced rate. At the interface of the EGL and ML (level
V), granule cells round their somata and reduce
significantly their movement before forming the third vertical process
that enters the radially developing ML. If we assume that postmitotic granule cells spend equal time at each level and that they do not
reverse significantly their direction of movement, the average distances traveled by granule cells during the tangential phase of
their migration will be over 220 µm, although individual cells may
have a considerable longer trajectory. Furthermore, studies with
3H-thymidine indicate that the transit
time of the postmitotic granule cells in the EGL increases during early
postnatal development (Altman, 1972; Rakic, 1973, 1985), suggesting
that late-generated granule cells move farther from the site of their
birthplace during the tangential migration in the EGL than the
early-generated granule cells.
Change in cell position, from the middle to the bottom level and then
to the EGL-ML border, appears to be attributable to passive
displacement as suggested from the electron microscopic reconstructions
(Rakic, 1971). Because granule cell precursors divide approximately
every 20 hr within the top two levels of the EGL (Fujita et al., 1966;
Fujita, 1967), the surface of the EGL expands rapidly outward.
Moreover, as the cell soma leaves the territory of the EGL and their
original horizontal processes transform into parallel fibers, the ML
increases in thickness, and the EGL-ML border shifts toward the pial
surface. Therefore, tangentially migrating granule cells situated at
the middle level of the EGL relocate to the EGL-ML border within 24 hr
without undergoing an active downward movement, as illustrated in
Figure 11A.
Although the cellular substrate responsible for the guidance of
tangential cell migration in the EGL has not been experimentally established, it was suggested that the basic cellular mechanism may be
similar to those involved in tangential movement of immature neurons in
the medulla and the pons that are mediated by homotypic interactions
between migrating neurons and neurites of the previously generated
neurons (Rakic, 1985, 1990; Ono and Kawamura, 1989, 1990). In support
of this hypothesis, it was reported that, in the microexplant cultures,
granule cells move along the bundles of parallel fibers formed by
previously generated granule cells (Nagata and Nakatsuji, 1990; Komuro
and Rakic, 1996). This mode of granule cell movement may be similar to
tangential migration of neuronal precursors from the telencephalic
subventricular zone to the olfactory bulb, which occurs via chain
migration and without the guidance of radial glia or axonal processes
(Wichterle et al., 1997). However, an alternative possibility that
cannot be excluded is that the tangential movement of granule cells may be guided by heterotypic gliophilic interactions at multiple glancing contacts with the vertically oriented Bergmann glial processes. This
has been suggested for the tangentially migrating cortical neurons in
the embryonic cerebrum (Rakic et al., 1974; O'Rourke et al., 1997).
Furthermore, it might be possible that near the EGL-ML border the
descending processes of tangentially migrating granule cells interact
repulsively with the Purkinje cell dendrites because it was reported
that repulsive interactions between ephrin-B1 on migrating granule
cells and EphA4 on dendrites of Purkinje cells may determine the
migratory pathway of granule cells in the ML of embryonic chick
cerebellum (Karam et al., 2000).
Molecular mechanisms orchestrating the behavior of postmitotic granule
cells in the EGL are also not well understood. It has been suggested
that directionality of migration may depend on the extracellular matrix
molecules localized on the ambient substrates (Fishell
and Hatten, 1991; Rivas and Hatten, 1995), as it does in other systems
(Rakic et al., 1994; Bronner-Fraser, 1995; Anton et al., 1999). Indeed,
a variety of extracellular matrix molecules are expressed in the EGL
(Chuong, 1990), and dissociated granule cells have been shown to
migrate predominantly on the extracellular matrix molecules in the
absence of either glial or axonal processes (Liesi, 1992; Fishman and
Hatten, 1993). The molecules from the Netrin family have been shown to
be involved in directing tangential migration in the EGL (Alcantara et
al., 2000). In contrast, initiation of radial migration may depend
critically on neuron-glia cell adhesion molecules (Ng-CAM) because the
application of Ng-CAM antibody inhibits the relocation of
3H-thymidine-labeled granule cells from
the EGL to the ML (Chuong et al., 1987) but does not affect the granule
cell migration along the glial processes in dissociating cultures
(Edmondson et al., 1988).
The results reviewed above suggest that tangential and radial migration
of the granule cells may depend on different signals emanating from the
local substrates. However, intrinsic programs may also be involved in
the determination of cell fate and in control of its migratory behavior
in the EGL (Jankovski et al., 1996). In the microexplant cultures, for
example, granule cells first migrate radially along the bundles of
their neurites, and then, the cells abruptly change their orientation
by 90° and begin to translocate their nucleus within the newly
developed neurite without any contact with glial cells (Nakatsuji and
Nagata, 1989; Nagata and Nakatsuji, 1990). This suggests that changes
in the direction of cell migration may be governed by intrinsic signals and the arrangement of the cytoskeleton (Rakic et al., 1996). Finally,
it was reported that migrating granule cells express several genes at
specific points along their pathway and that some of these genes encode
for the specific receptors activated by the cell adhesion molecules
(Kuhar et al., 1993; Hatten and Heintz, 1995; Hatten et al., 1997).
Therefore, systematically alternating gene expression during the
tangential migration of granule cells, combined with signals
originating from the cellular elements of the EGL, may function as
modulators of cell shape and their migratory behavior.
Does the tangential migration of postmitotic granule cells contribute
to the allocation of granule cells across the various functional
compartments of the cerebellar hemispheres? The developing cerebellum
expresses distinct parasagittal, stripe-like compartments oriented in
the anteroposterior direction (A-P compartments) that are dramatically
revealed by the expression of specific genes and molecules (Hawkes and
Leclerc 1987) (for review, see Herrup and Kuemerle, 1997; Ozol et al.,
1999). Cellular organization is different in each parasagittal
compartment, which contains a unique set of Purkinje cell phenotypes
that can generate molecules that attract or repel migrating granule
cells. The fact that most granule cells travel tangentially,
perpendicular to the orientation of these parasagittal compartments,
indicates that neurons may be instructed to enter a given compartment
before initiation of their radial migration, as schematically shown in
Figure 11B. It was suggested that appropriate
allocation of the species-specific number of granule cells per Purkinje
cell is orchestrated by signals emanating from their dendrites (Zecevic
and Rakic, 1978), which may have attracting and/or repulsive properties
(Alcantara et al., 2000). In the developing forebrain, immature neurons
destined for the radial columns or layers of the cerebral cortex seem
to be specified in the proliferative ventricular zone, before the initiation of their radial migration (Rakic, 1988; McConnell and Kaznowski, 1991, 1995). It is possible that the granule cells of the
cerebellum are also specified to settle in a given A-P compartment
similar to the formation of the protomap in the developing forebrain
(for review, see Rubenstein and Rakic, 1999). However, it is unclear
whether postmitotic granule cells cross A-P compartment boundaries
during their tangential migration in the EGL before settling in a given
A-P compartment, because each A-P compartment varies in position, as
well as width, and are overlapping each other (Herrup and Kuemerle,
1997). It may be significant that radial migration of granule cells
occur predominantly in the restricted pathways (named granule cell
raphes) that are connecting the EGL and IGL (Lin and Cepko, 1998). The
existence of granule cell raphes suggests that postmitotic granule
cells may migrate tangentially until they reach the position of granule
cell raphes in the EGL. Although granule cell raphes have not been
identified in mice, tangentially migrating granule cells may start
their radial migration in response to localized environmental cues. The
combined use of acute slice preparations and confocal microscopy allows
study of the role of various molecules and receptors in tangential cell movement in the natural cellular milieu.
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FOOTNOTES |
Received Aug. 14, 2000; revised Oct. 6, 2000; accepted Oct. 19, 2000.
This work was supported by the United States Public Health Service
(P.R) and the Cleveland Clinic Foundation (H.K). We thank Dr. K. Wikler
for his useful suggestions and comments on this manuscript.
Correspondence should be addressed to Dr. Hitoshi Komuro, Department of
Neurosciences/NC30, Lerner Research Institute, The Cleveland Clinic
Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail:
komuroh{at}ccf.org.
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ABSTRACT
INTRODUCTION
