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The Journal of Neuroscience, December 15, 2001, 21(24):9690-9700
Neocortical Cell Migration: GABAergic Neurons and Cells in Layers
I and VI Move in a Cyclin-Dependent Kinase 5-Independent Manner
Edward C.
Gilmore1 and
Karl
Herrup1, 2
1 Department of Neurosciences, School of Medicine, Case
Western Reserve University, and 2 Alzheimer Research
Laboratory, University Hospitals of Cleveland, Cleveland, Ohio 44106
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ABSTRACT |
The adult mammalian cerebral cortex arises from a complex series of
neuronal migrations. The primitive layer known as the preplate is split
into an outer marginal zone and an inner subplate by invading cortical
plate neurons in an "inside-out" pattern of layering with respect
to time of neuronal origin. In cyclin-dependent kinase 5 (Cdk5)-deficient mice (cdk5 /),
the earliest born cortical neurons split the preplate, but later born
neurons arrest below the subplate, resulting in an ectopic
"outside-in" layer of neurons normally destined for layers II-V.
We have pursued this analysis in
cdk5/  wild-type chimeric mice
coupled with experiments in cell culture. In vitro
migration assays show no difference in migrational ability between
embryonic cdk5/ and wild-type
neurons. In cdk5/ chimeras,
layers I and VI are made up of both mutant and wild-type genotype
neurons, whereas layers II-V contain predominantly wild-type cells. In
addition, a thin layer of neurons is found below layer VI, made up of
cdk5/ cells; bromodeoxyuridine
labeling suggests that these neurons were destined for layers II-V.
Scattered cdk5/ cells are found
throughout layers II-V, but these neurons are always found to be
GABAergic. The findings suggest that Cdk5 is not required for migration
of either the deepest cortical plate neurons or the GABAergic neurons
from the ganglionic eminences. The migration of layer II-V pyramidal
neurons, however, is intrinsically blocked by Cdk5 deficiency, thus
suggesting that different neuronal cell types use distinct mechanisms
of migration.
Key words:
tangential migration; radial migration; chimeras; cyclin-dependent kinase 5; BrdU; cortical cell culture
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INTRODUCTION |
The cerebral cortex begins as a
single layer of pseudostratified neuroepithelial cells and develops
into a six-layered laminar structure (Boulder Committee, 1970 ). First
the preplate, a layer consisting primarily of transient populations,
forms. The preplate is split into two layers by radially migrating
neurons destined to form the true cortical plate (Marin-Padilla, 1978).
The first cohort of arriving neurons is followed by subsequent groups
of cells that migrate past the subplate, past earlier born cortical plate neurons, and stop just under the marginal zone. The end result is
a cerebral cortex that is born inside-out (Angevine and Sidman, 1961).
Tangential migration of GABAergic interneurons from their ventricular
origin in the ganglionic eminences adds additional complexity to the
process (Anderson et al., 1997; Tamamaki et al., 1997).
The range of gene products whose coordinate expression is required
during corticogenesis can be appreciated by considering the genetic
defects, in both humans and rodents, that are known to disrupt this
process. These mutations include the engineered null alleles of
cyclin-dependent kinase 5 (Cdk5; Ohshima et al., 1996; Gilmore et al.,
1998) and its activating subunit p35 (Chae et al., 1997; Kwon and Tsai,
1998). Cdk5 has close homology to other cyclin-dependent kinases, but
its highest expression and levels of detectable activity are found
within postmitotic neurons (Hellmich et al., 1992; Lew et al., 1992;
Tsai et al., 1993). Cdk5-deficient mice develop normally through the
point of early cortical plate neurons bisecting the preplate (Gilmore
et al., 1998). Later born neuronal cohorts, however, appear unable to migrate past the subplate and form an outside-in layer located beneath
the subplate; the mice subsequently die in the perinatal period
(Ohshima et al., 1996, 1998, 1999; Gilmore et al., 1998). The proper
migration of the earliest cortical plate neurons (layer VI) contrasts
with the failure of later born immigrants to layers II-V and suggests
that there are important differences between migratory programs of
these two populations. This bipartite effect on migratory ability is
not observed in reeler and related mutations, in which all
neocortical neurons reside below the subplate (Falconer, 1951; Caviness
and Sidman, 1973; Caviness and Rakic, 1978; Howell et al., 1997;
Sheldon et al., 1997; Sheppard and Pearlman, 1997; Ware et al., 1997).
Cdk5 may thus have a unique involvement in the migration of layer II-V neurons.
In this study, we demonstrate that
cdk5/ neurons are fully
competent to migrate in a simple culture model, in a manner
indistinguishable from wild-type neurons. In contrast, we demonstrate
that the in vivo radial migration of neurons in cerebral
cortical layers II-V is Cdk5-dependent even in the relatively normal
"terrain" of the Cdk5 chimera. Importantly, the tangential
migration of the GABAergic interneurons from the ganglionic eminence is
found to be Cdk5-independent. These findings imply differential
requirements for migration by early and late cerebral cortical
precursors, as well as by tangentially migrating GABAergic precursors.
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MATERIALS AND METHODS |
Migration assays. Cerebellar explant cultures were
established from embryonic day 18 (E18) fetuses (plug date being E0).
Pregnant cdk5+/ dams from
cdk5+/ matings were killed by
cervical dislocation, and their embryos were quickly removed into
ice-cold HEPES-buffered saline solution. Heads were removed, and bodies
were saved for PCR analysis (Ohshima et al., 1996; Gilmore et al.,
1998). Tissue was dissected into small pieces, ~50-150 µm in
diameter, in chilled HEPES-buffered saline solution. Five to eight
pieces of tissue were placed into 24 well plates coated with
poly-L-lysine (Sigma, St. Louis, MO) and laminin (Life
Technologies, Gaithersburg, MD) containing Neurobasal medium (Life
Technologies) supplemented with B-27 (Life Technologies), Penn-Strep at
100 U/ml (Life Technologies), and 2 mM
L-glutamine (Life Technologies). Cultures were
grown for 1 d before fixation. 4,6-Dimidino-2-phenylindole (DAPI)
staining was used to visualize cell nuclei. The location of cells
relative to the explant edge was determined with NIH Image 1.6, and
distances were calculated with Microsoft (Redmond, WA) Excel 98 software. Because cells do not uniformly migrate away from explants,
photographs were taken at varying angles of 0, 180, 270, or 90° (in
that order) from the center of the culture. The top of the plate was
considered 0°. If no cells migrated away from an explant, it was not
used for analysis.
Chimeric mice derived from cdk5/ embryonic
stem cells. cdk5/ embryonic
stem (ES) cell clones were obtained using the high G418 selection
method (Mortensen et al., 1992).
cdk5+/ ES cells, derived from
clone 57 (Ohshima et al., 1996), were cultured in 1 mg/ml G418 (Life
Technologies) for 6-7 d. Resistant colonies were picked and genotyped
individually as described previously (Ohshima et al., 1996, 1999). To
generate chimeric mice, cdk5/ ES
cells (57G23) were injected into blastocysts derived from C57BL/6
females mated with males from the ROSA26 transgenic strain. The cells
of these animals contain a  -galactosidase transgene driven by a
nearly ubiquitous cytomegalovirus promoter. As a result, histologically
detectable expression of -galactosidase is observed in nearly all
cells, including neurons (Magrassi and Graziadei, 1996; Zambrowicz et
al., 1997). Age-matched nonchimeric littermates, C57BL/6, and
C57BL/6 × ROSA26 mice were used as controls.
Histological and immunohistochemical analysis. Adult brains
from chimeras and controls were obtained after transcardial perfusion with 3% paraformaldehyde in 100 mM PIPES, pH 7.4, for 15 min. Brains were dissected and transferred overnight (or longer) to 20% sucrose and PBS at 4°C. For embryonic anti-GABA immunostaining, pregnant dams from cdk5+/ matings
were killed at E18.5. Embryos were transferred immediately to cold PBS.
Heads were transferred to 4% paraformaldehyde in 0.1 mM phosphate buffer for immersion fixation
overnight at 4°C followed by transfer to 18% sucrose and PBS
overnight (or longer) at 4°C. Bodies were retained for PCR genotyping
(Ohshima et al., 1996; Gilmore et al., 1998). Sections were cut on a
cryostat at 12 µm. All sections were kept at  70°C before use. For
5-bromo-4-chloro-3-indolyl--D-galactoside (X-gal) studies, coronal sections of forebrain were incubated for 4 hr
at 37°C with 1 mg/ml X-gal in 35 mM
K3Fe(CN)6, 25 mM Fe4(CN)6, 2 mM MgCl2, 0.01%
deoxycholic acid, and 0.02% Nonidet P-40 followed by several washes
with H2O. For bromodeoxyuridine (BrdU)
immunostaining, sections were acid-permeabilized with 2N HCl for 75 min
followed by neutralization with eight washes of PBS of 5 min each.
Immunocytochemistry was performed using primary antibody buffer
consisting of PBS with 0.5% Tween 20 and 5% goat serum. The primary
antibodies used and their dilutions were as follows: rat anti-BrdU
(Accurate Chemicals, Westbury, NY), 1:6; rabbit anti-Cdk5 (Santa Cruz
Biotechnology, Santa Cruz, CA), 1:100; rabbit anti-GABA (Sigma),
1:1000; mouse anti-NeuN (A60; a kind gift from Richard Mullen, Woods
Hole Oceanographic Institute, Woods Hole, MA), 1:50; and mouse
anti-calbindin (Sigma), 1:1000
Primary antibodies were visualized in PBS and 0.5% Tween 20 with 20%
goat serum. The following secondary antibodies were used: goat anti-rat
IgG conjugated to Cy3 (Chemicon, Temecula, CA), 1:400; goat anti-rabbit
IgG conjugated to Cy3 (Jackson ImmunoResearch, West Grove, PA), 1:400;
and goat anti-mouse IgG conjugated to FITC (Jackson ImmunoResearch),
1:200. Sections were counterstained with 1 µg/ml DAPI in PBS for 10 min, followed by two washes in PBS for 5 min.
Purkinje cell counts were performed on three nonadjacent midsagittal
sections. Cresyl violet staining was performed on coronal sections of
forebrain and sagittal sections of hindbrain according to standard
protocols. Cerebral cortical neurons were counted under 400×
magnification in a field ~600 µm in diameter for Cdk5 and NeuN
double staining. For GABAergic staining, cells were examined with both
light and fluorescent techniques and scored as -galactosidase (-gal)-positive if they contained an X-gal deposit within or immediately adjacent to the neuron (see examples in Fig. 6). The boundaries of the six cortical layers were determined with DAPI or NeuN.
All sections were examined by standard light and fluorescent techniques
on a Leitz (Wetzlar, Germany) Orthoplan microscope. Images were
captured with a Spot digital camera model 1.3.0 from Diagnostic
Instruments Inc.
Statistics. SD and Student's t tests were
performed using Microsoft Excel 98 software. The  value for the
Student's t test was always 0.05.
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RESULTS |
The use of chimeric mice has greatly aided the study of many
neurological mutations. Our laboratory has previously used this technique to analyze the developmental potential of the cerebellar cells in Cdk5-deficient mice. All
cdk5/ Purkinje cells are blocked
in their migration. This is true whether they are located in the mutant
or in association with wild-type neighbors as in
cdk5/ cdk5+/+ chimeric mice (Cdk5
chimeras). In contrast, all genetically wild-type Purkinje cells
migrate normally even in the chimera in the presence of mutant
neighbors (Ohshima et al., 1999). There are also cell-autonomous defects in cerebellar granule cell migration, but these defects are not
complete. Some cdk5/ granule
cells are capable of migrating from the external to the internal
granule cell layer despite their mutant genotype. The cells of the deep
cerebellar nuclei appear to migrate successfully regardless of their
cdk5 genotype (Ohshima et al., 1999).
Cell migration assays
We have shown that Cdk5 is required for proper migration of a
number of neuronal progenitors in vivo (Gilmore et al.,
1998; Ohshima et al., 1999). The cell biological nature of this
requirement, however, is not clear. To test for a potential defect in
the general locomotive ability of mutant neurons, explants from the
cerebella of E18 embryos derived from the mating of
cdk5+/ mice were grown in culture
for 1 d before fixation. During this time, extensive neurite
outgrowth and glial migration occurred along with significant neuronal
migration away from the explants. There was no apparent difference
between the number of granule cells that had migrated away from the
explants in wild-type (Fig. 1A) or
cdk5/ (Fig.
1B) cultures. To estimate the rates at which the
nerve cells migrate in vitro, their distance from the
nearest edge of the explant after 1 d in culture was calculated.
The results are shown in Figure 1C, and they illustrate that
there was no difference between the genotypes in the distance traveled
by individual neurons.
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Figure 1.
In vitro migration of cerebellar
neurons on poly-L-lysine and laminin after 1 d. Shown
here are the cell migration patterns from explants of wild-type
(A) and
cdk5/ (B)
embryonic cerebellum. Processes consisting of radial glia and neurons
are extended from the explants, and numerous migrating cells can be
seen. No qualitative differences are seen between the two genotypes.
C, Extent of migration represented as a frequency
histogram of the distance migrated by granule cells from explant. The
y-axis represents the relative percentage of neurons
that traveled a particular range of distances. The
x-axis represents the distance traveled from explant.
The data for wild-type mice are represented by stippled
bars; cdk5/ mice are
represented by cross-hatched bars. No difference is seen
between the genotypes. Scale bar, 100 µm.
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Cerebral cortex of chimeras
We have demonstrated previously that migrating cerebral cortical
precursors in cdk5/ mice migrate
and properly split the preplate, but later born cerebral cortical
precursors stall in their migration below the newly formed subplate
(Gilmore et al., 1998). The perinatal death of the mutant embryos
precludes the study of any late developmental event, including the
completion of cortical cell migration. To address this, we generated
cdk5/ cdk5+/+ chimeric mice. Cdk5 chimeras
survive into adulthood because of the presence of the wild-type cells,
yet because all cell populations of these animals are made up mixtures
of cdk5+/+ and
cdk5/ cells, the resulting
phenotype is both complex and highly informative. ES cells with both
copies of the cdk5 gene disrupted were injected into ROSA26
blastocysts. The neurons that descend from the cells of the host
blastocysts will be wild type at the cdk5 locus and, because
of their ROSA genotype, each will express bacterial -galactosidase (Magrassi and Graziadei, 1996; Zambrowicz et al., 1997). This genetic
combination allows mutant and wild-type neurons to be distinguished on
a cell-by-cell basis by two independent methods: wild-type neurons will
express both Cdk5 and -galactosidase, whereas mutant neurons will
express neither.
The interpretation of the findings in chimeras such as these depends on
the ability to determine the ratio of cells of the two genotypes.
Because both the host embryos and ES cells were agouti, coat color
could not be used. Instead we first examined the cerebellar parenchyma
for misplaced Purkinje cells using calbindin staining to help identify
the ectopic cells (results not shown) (Ohshima et al., 1999). To
quantitate the degree of chimerism, we performed midline Purkinje cell
counts of all animals. Within a chimera, the percentage of wild type in
one tissue (i.e., cerebellum) closely follows the ratio of the
genotypes in the rest of the animal (Soriano and Jaenisch, 1986). In
addition, in the Purkinje cell population, there is a fairly
homogeneous distribution of the cells of the two genotypes across the
two dimensions of the Purkinje cell layer (Mullen, 1977; Herrup and
Sunter, 1987). Four Cdk5 chimeric mice were generated with estimated
degrees of chimerism ranging from 60 to 76% wild type. Two potential
chimeras, 426B and 426C, had no ectopic
cdk5/ Purkinje cells, indicating
that they had essentially no
cdk5/ contribution, and we have
used them as wild-type, age-matched, littermate controls (Table
1).
Gross histological examination of Cdk5 chimeras indicated that the
cytoarchitecture of their cerebral cortices were not dramatically different from wild-type (Fig. 2). Such
variation as can be seen in Figure 2 cannot be distinguished from
differences that might be expected on the basis of differences in
either the anteroposterior or mediolateral location. Examination of
animals at young postnatal (Fig. 2A,B) and adult
(Fig. 2C,D) ages revealed similar cellular composition and
density in both wild-type (Fig. 2A,C) and chimeric (Fig. 2B,D) mice; both contain the typical six-layer
neocortical architecture. However, the density of cells in the
subcortical white matter appears greater in the chimeras. This results
in an apparent increase in the thickness of layer VI (Fig.
2B,D) compared with wild type (Fig.
2A,C).
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Figure 2.
Cresyl violet-stained coronal sections of cerebral
cortex. Both developing and mature cortex are illustrated here.
A, Postnatal day 12 wild type; B, Cdk5
chimera 326B; C, adult wild type; D,
chimera 423D. Cortical layers are numbered I-VI with
the white matter tract (WT) labeled along the
ventral surface. Although different regions of cerebral cortex
demonstrate variation in both composition and density of various layers
of cells, the basic pattern is present in both wild-type and chimeric
mice from early developmental times. Scale bar, 100 µm.
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Most of the chimeric cerebral cortex appears to be populated
extensively by wild-type neurons, whereas most of the
cdk5/ neurons are located below
cortical layer V. Cdk5 is an abundant antigen that is highly expressed
in all neurons, a fact that has been exploited previously in
examination of the cerebellum of Cdk5 chimeras (Ohshima et al., 1999).
In cerebral cortex of wild-type animals, all cells stained positive for
the neuron-specific marker NeuN (Mullen et al., 1992) (Fig.
3A,G, green) and
Cdk5 (Fig. 3B,H, red) and thus appear
yellow when the two images are combined (Fig. 3C,I). A much different pattern is seen in Cdk5
chimeras. Layers II and III contain nearly all wild-type neurons, as
indicated by the presence of both NeuN (Fig. 3D,
green) and Cdk5 (Fig. 3E, red,
F, yellow when photocombined), with a similar
composition found in layers IV and V (results not shown). Layer I, on
the other hand, contains a substantial number of
cdk5/ neurons (white
arrowhead) demonstrated by the presence of anti-NeuN (Fig.
3D, green) and the absence of Cdk5 (Fig.
3E, red) resulting in a green
appearance when photocombined (Fig. 3F). The
cdk5/ neurons in layer VI have a
similar staining pattern (Fig. 3J-L, white
arrowheads). In addition, a substantial number of Cdk5-deficient neurons are found in deep portions of cerebral cortex, primarily in the
white matter tract below layer VI (Fig. 3J-L,
bottom-most arrowheads). These observations suggest that
wild-type neurons are capable of proper migration within a chimeric
environment, whereas most of the
cdk5/ neurons are not. In every
chimera we examined, however, there were always a few
cdk5/ neurons in layers II-V
and many within layer I. To quantitate these observations, the genotype
ratios of the different neuronal layers were determined by profile
counts of NeuN-labeled cells. The raw counts are presented in Table
2, and the relative percentage of the
genotypes in each layer is presented graphically in Figure 4. The essentially wild-type chimeras
423B and C and other controls were not counted, because these animals
contained only cdk5+/+ neurons.
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Figure 3.
Immunostaining of cerebral cortex from
wild-type mice (A-C, superficial; G-I,
deep) and Cdk5 chimeric mice (D-F, superficial;
J-L, deep). Cdk5 immunostaining is shown in
red (Cy3 secondary; B, E, H, K);
the neuron-specific marker NeuN is shown in green (FITC
secondary; A, D, G, J). C, F, I,
L, digitally combined images. In layers II and III of both
wild-type animals (A-C) and chimeric animals,
e.g., 426D (D-F), most neurons are wild-type and
thus Cdk5-positive. Double staining with NeuN antibody
(green) demonstrates that nearly all of the
neurons are Cdk5-positive, resulting in yellow when
combined. There are exceptions to this situation, however. The
arrowhead in layer I of the Cdk5 chimera
(D-F) demonstrates a single
Cdk5/ neuron within layer I. In
deeper layers of cerebral cortex in wild-type animals
(G-I), all layer VI cells contain Cdk5. In
contrast, neurons in the deeper regions of Cdk5 chimeras are often
found to be cdk5/. The
arrowheads in J-L mark
cdk5/ neurons within the deep
portions of layer IV. The white matter tract contains occasional
cdk5+/+ neurons
(yellow) in both wild-type and Cdk5 chimeras.
Scale bar, 50 µm.
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Figure 4.
Graphic representation of data in Table 2. Results
from chimera 423A are shown in A, chimera 423D in
B, and chimera 326B in C. Stippled
bars represent the percentage of wild-type neurons within that
particular layer of cerebral cortex; cross-hatched bars
represent the per- centage of cdk5/
neurons. Sum is all of the neurons from the various
layers tallied together. Note the abundance of Cdk5-deficient neurons
in Layer I, Layer IV, and Sub
Layer VI compared with their near absence from the other
layers. Note also that the number of
cdk5/ neurons within a layer is
never zero.
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Previous studies of cerebral cortex of
cdk5/ embryos indicated that
only the earliest born cortical neurons are capable of entering the
cortical plate, whereas the rest remain trapped below the subplate in
the underplate (Gilmore et al., 1998). In Cdk5 chimeras, however, there
are infrequent but regular examples of cdk5/ neurons found in layers
II-V. We wished to determine the reason for this apparent discrepancy.
Cortical plate neurons derive primarily from the ventricular zone of
the dorsolateral ventricle, but this is not their only source. The
ganglionic eminences give rise to a large tangential migration of cells
that are the likely precursors to the cortical GABAergic interneurons
of cerebral cortex (de Carlos et al., 1996; Anderson et al., 1997;
Tamamaki et al., 1997; for review, see Parnavelas, 2000). To determine
whether cdk5/ cells in the upper
layers of the chimeric cerebral cortex were these GABAergic neurons,
immunostaining was performed with an anti-GABA antibody plus
histochemical staining with X-gal for the genotype marker
-galactosidase. The spatial location of the GABA-positive neurons is
the same in both wild-type mice (Fig. 5A) and all chimeras (Fig.
5B). An X-gal reaction product can be seen associated with
most GABAergic neurons of the primarily wild-type chimera 423C (Fig.
5C-E), identifying them as
cdk5+/+. Chimera 423D (30%
cdk5/; Table 1) has many more
GABAergic neurons without associated X-gal staining (Fig.
5F-H, arrowheads). The presence of these GABA+/X-gal
cells demonstrates that cdk5/
mutant GABAergic neurons survive and locate properly in the chimera. No
pattern was noted in the mediolateral or anteroposterior location of
-gal-positive and -negative neurons, indicating that both cdk5+/+ and
cdk5/ neurons are equally
capable of migrating throughout the cerebral cortex.
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Figure 5.
Cdk5-deficient GABAergic neurons are present
within the cerebral cortex of Cdk5 chimeras. An essentially wild-type
animal is shown in A, whereas a more balanced chimera is
illustrated in B. Note the similar distribution of
GABAergic cells in both animals. The remaining panels illustrate
GABAergic immunostaining combined with X-gal histochemistry in
wild-type (C-E) and Cdk5 chimeric
(F-H) brains. Sections were stained with X-gal
(D, G) and then were immunostained with anti-GABA
antibodies (red; C, F). The X-gal
staining (D, G) was digitally converted to
green on a black background to allow
overlay with GABA immunostaining for purposes of illustration
(E, H). A green dot is associated
with all GABAergic neurons in the wild-type chimera
(E). In contrast, in Cdk5 chimeric mice
(H), many GABAergic neurons are
X-gal-negative (arrowheads), indicating that they are
cdk5/. To further confirm the
presence of cdk5/ GABAergic
neurons, wild-type (I-K) and Cdk5
(L-N) chimeras were double-labeled with
antibodies to Cdk5 (I, L) and calbindin (J,
M), a marker for a subpopulation of GABAergic neurons
(Hendry et al., 1989; Van Brederode et al., 1990). The images were
digitally combined (K, N) for clarity. In normal
cortex (I-K), all GABAergic neurons are
Cdk5-positive (blue arrowhead). In the chimeric cortex
(L-N), although many of calbindin-positive
neurons are also Cdk5-positive (blue arrowhead), there
are substantial numbers of Cdk5-negative, calbindin-positive neurons
(white arrowheads). Scale bars: A, B, 100 µm; C-N, 50 µm.
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To quantitate this effect, GABAergic neurons were counted and scored
for the presence of X-gal precipitate. The results are shown in Table
3. The percentage of
GABA+/X-gal
neurons is 7% for 423B and 8% for 423C, 23% for 423A, and 40% for
423D. The essentially wild-type mice, 423B and C, having 7-8% GABA+/X-gal,
likely represent the false-negative rate, whereas the chimeric animals
423A and D have percentages that correspond approximately to the level
of chimerism in these animals (Table 1). To confirm that
cdk5/ GABAergic neurons exist
within the Cdk5 chimeric cerebral cortex, calbindin and Cdk5 double
immunostaining was performed (GABA and Cdk5 double immunostaining could
not be performed because of antibody incompatibility). Calbindin labels
a subset of GABAergic neurons within the cerebral cortex (Hendry et
al., 1989; Van Brederode et al., 1990; Gogelia and Hamori, 1992). In
the essentially wild-type mouse, 423C, all calbindin-positive neurons
were Cdk5-positive (Fig. 5I-K, blue arrowhead).
However, whereas the chimeric mouse, 423D, also contained Cdk5-positive
neurons (Fig. 5L-N, blue arrowhead), this animal
also had numerous Cdk5-negative, calbindin-positive neurons
(white arrowheads) in its cortex. This is additional
evidence for the presence of Cdk5-negative GABAergic neurons within the cerebral cortex of this animal.
To confirm the ability of cdk5/
GABAergic neurons to migrate properly, we examined both wild-type and
cdk5/ nonchimeric embryos. In
E18.5 cerebral cortex, anti-GABA immunostaining is qualitatively
distinct but quantitatively low compared with other brain regions
(results not shown). This situation is true in both wild-type (Fig.
6A) and
cdk5/ (Fig.
6B) animals. In E18.5 wild-type embryos, GABAergic
neurons are found within deeper portions of the developing cerebral
cortex [Fig. 6A, inset (magnified area of
D), D] (Del Rio et al., 1992). GABAergic neurons
are also found in the portion of the
cdk5/ cortex that contains the
early cortical plate (between the marginal zone and subplate, ~50
µM below the pial surface; Gilmore et al., 1998) [Fig. 6B, inset (magnified area of
D marked by arrowheads)]. These data validate
the evidence from the Cdk5 chimeras that GABAergic neurons do not
require Cdk5 activity for proper migration.
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Figure 6.
GABA staining is seen in both
cdk5+/+ (A) and
cdk5/ (B)
E18.5 embryos. Insets in A and
B are magnified in C and
D, respectively. Arrowheads indicate
locations of GABA-positive neurons in high-magnification images
(C, D). Scale bars: A, B, 100 mm;
C, D, 50 mm.
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Our findings demonstrate that most
cdk5/ neocortical neurons are
deficient in their migration, but the complete status of wild-type neurons remains unknown. We have already shown that the chimeric cerebral cortex appears normal in its overall appearance and layering pattern (Fig. 3) and that it is composed of primarily wild-type neurons
(Table 2, Fig. 4). It remains a possibility, however, that some
cdk5+/+ neurons are disrupted in
their migration by the presence mutant neurons during development. To
investigate the migration of a particular population, the nucleoside
analog BrdU was injected into pregnant dams at day 17 of gestation.
Cortical neurons born at this time normally migrate to layers II and
III in the mouse (Caviness, 1982; Nowakowski et al., 1989). Anti-BrdU
immunostaining of adult wild-type animals treated in this way during
their gestation reveals that most of the neurons labeled with BrdU are
indeed found within layers II and III (Fig.
7A). Interestingly, although many labeled neurons are also found in layers II and III in the chimera, many BrdU-labeled neurons remain in deep portions of cerebral
cortex below layer VI (Fig. 7B) where
cdk5/ neurons are found (Fig. 4,
Table 2). A similar result was found in young (postnatal day 12)
chimeras (Fig. 7C,D). Younger chimeras also display small,
faintly stained BrdU-positive nuclei throughout the cerebral cortex.
These nuclei are likely to belong to glial cells, because glial genesis
begins approximately at the time of BrdU injection. We presume that the
few large BrdU-labeled neurons not found within layers II and III or IV
are likely to be GABAergic neurons, which are also born at this time
(Miller, 1986).
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Figure 7.
Migration of
cdk5/ neurons is defective in the
Cdk5 chimera. Wild-type (A, C) and chimeric (B,
D) mice were labeled with BrdU at E17.5. Superimposed images of
DAPI (blue) and anti-BrdU (red) staining
are shown. BrdU-labeled neurons appear purple. In
wild-type adult (A) and juvenile (postnatal day
12; C) mice, most of the E17-labeled neurons settle in
layers II and III as expected (Caviness, 1982). In chimeras, many
E17-labeled neurons end up in layers II and III, but a number are found
deep to these layers as well. Similar results are found in both young
(D) and old (B) animals.
Note that small nuclei, faintly stained with BrdU, are present
throughout the cortex. Because of their small size, they are likely to
be glial cells. Scale bar, 100 µm.
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DISCUSSION |
The present analysis extends our previous studies of the defects
in neuronal migration of Cdk5-deficient neurons. We find that neurons
do not seem to require Cdk5 activity for simple migration activities
in vitro. This is in contrast to other mutants in which sometimes subtle defects in migration in vivo display
dramatic in vitro migration difficulties (Rezai and Yoon,
1972; Hatten et al., 1986; Reiner et al., 1993; Hirotsune et al.,
1998). If Cdk5 deficiency worked in this manner, we would expect to
find slower-migrating cells in our cultures from mutant cerebral
cortex. Although it is theoretically possible that only
Cdk5-independent neurons migrate away from the explants, we observed no
difference between cdk5/ and
wild-type explants in either the pattern or extent of cell movement. As
a whole, our results suggest that Cdk5 is not an essential part of the
basic movement machinery of a cell but instead plays a specific role in
the much more complicated process of directed neuronal migration
in vivo.
Chimeric animals are a powerful system in which to examine the adult
cellular phenotypes of mutants that die during development and to
determine how wild-type and mutant cells interact. Previous analysis of
cdk5/ embryos demonstrated that
early cortical plate precursors (layer VI) and preplate neurons do not
require Cdk5 to migrate properly, whereas all other cortical plate
neurons do (Gilmore et al., 1998). Analysis of the chimeras indicates
that the Cdk5 deficiencies are cell-autonomous defects in migration.
Most of the neurons in the cerebral cortex of the adult chimeras are
wild-type, whereas most of cdk5/
cells are located ectopically at the bottom of the cerebral cortex. Examination of the cerebral cortex of Cdk5 chimeras also reveals the
presence of mutant (cdk5/)
GABAergic neurons within the cerebral cortex. We conclude from this
that Cdk5 activity is not required for proper migration of GABAergic
neurons from ganglionic eminences into and throughout the cerebral
cortex (de Carlos et al., 1996; Anderson et al., 1997; Tamamaki et al.,
1997; for review, see Parnavelas, 2000).
Within cdk5/ mutant embryos,
only the earliest born cortical neurons are capable of migrating
through the subplate and separating it from the marginal zone (Gilmore
et al., 1998). In the chimeras, many
cdk5/ neurons are found mixed
with their wild-type counterparts within the deep strata of layer VI.
It is possible that some of these are true layer VI cells, because in
both the mutant and the chimera they migrate past the subplate and
separate it from the marginal zone, thus establishing a bona fide
cortical plate. It is also possible that, during normal development,
some neurons from the embryonic subplate survive into adulthood and
become located in the deep portions of layer VI, as suggested by Landry
et al. (1998). In addition,
cdk5/ GABAergic neurons deep
within the cortex can be confused with layer VI neurons. However, we
think that our evidence supports the conclusion that the
cdk5/
NeuN+ cells are true layer VI neurons for
two reasons. First, some cdk5/
neurons are located in more superficial portions of this layer, an
unlikely position for surviving subplate neurons. Second, in the mutant
Cdk5-deficient embryos, there is a distinct population of layer VI-like
neurons that separate the subplate from the marginal zone. Proper
migration of cdk/ layer VI
neurons within Cdk5-deficient mice is in contrast to the situation in
the reeler mouse, in which such cells are absent, and this
crucial event in corticogenesis never occurs (Gilmore et al.,
1998).
We have proposed previously, on the basis of the phenotype of the
cdk5/ embryo, that layer VI
neurons use a different mechanism of migration than do neurons in
layers II-V (Gilmore et al., 1998). We suggested that layer VI neurons
might use a Cdk5-independent mechanism to translocate their nucleus
through a cytoplasmic process that remains attached to the pial
surface, a mechanism that has been proposed previously (Morest, 1970;
Nowakowski and Rakic, 1979; Brittis et al., 1995). We also speculated
that radial glial-guided migration (Rakic, 1971a,b, 1972) might be used
by the subsequent neurons of layers II-V. Recent studies by Nadarajah
et al. (2001) using slice culture visualization of migration have found
new evidence in support of this model. Early precursors appear to move
their cell body in a manner consistent with nuclear translocation
(Nadarajah et al., 2001). GABAergic neurons not only migrate from the
ganglionic eminences and through the subventricular zone of the
cerebral cortex, they must at some point more deeply penetrate the
superficial cortical plate region of cerebral cortex. Because this
process appears unaffected by the absence of Cdk5, GABAergic neurons
must use different cues or different migrational mechanisms than layer II-V neurons to reach their final location.
The cell-autonomous requirement for Cdk5 shows some similarities to
other mouse mutants. Reeler mice are deficient in the large
extracellular protein reelin. This protein is thought to bind to
several different extracellular receptors, resulting in the
differential phosphorylation of the mouse disabled protein mDab1
(D'Arcangelo et al., 1995, 1997, 1999; Hirotsune et al., 1995; Ogawa
et al., 1995; Howell et al., 1997, 1999; Sheldon et al., 1997; Ware et
al., 1997; Rice et al., 1998; Hiesberger et al., 1999; Senzaki et al.,
1999; Trommsdorff et al., 1999) (for review, see Rice and Curran, 1999;
Gilmore and Herrup, 2000). Cerebellar Purkinje cells appear to require
intrinsic Dab1, because all
Dab1/ Purkinje cells in Dab1
chimeras are blocked in their migration (Goldowitz, 1998). Correct
migration would also appear to require a certain level of cell-cell
interaction, however, because some wild-type Purkinje cells remain
ectopically located in Dab1 chimeras that are made up primarily of
mutant cells (D. Goldowitz, personal communication). In addition,
recent evidence demonstrates a genetic association between Dab1 and the
Cdk5 activator p35 (Ohshima et al., 2001). The exact relationship
between reelin and Dab1 and p35 and Cdk5 for proper migration is
clearly a matter that requires further study.
The survival of substantial numbers of mutant cells in the mature
chimeras indicates that Cdk5 deficiency per se is not toxic to neurons.
GABAergic neurons of the cerebral cortex, Purkinje cells, deep
cerebellar nuclear neurons, granule cells, and nearly all neurons in
the embryo can survive without Cdk5, although precise quantitation has
not been performed for any population (Gilmore et al., 1998; Ohshima et
al., 1999). Nonetheless, in the chimera, cdk5/ neurons appear to be at a
significant long-term survival disadvantage compared with wild-type
neurons. Estimations of the percent chimerism from wild-type Purkinje
cell counts can be used to illustrate that the
percentage of cdk5/ neurons
present in the cerebral cortex is much lower than would be predicted
(Tables 1, 3). For 423A, the observed percentage is 15%, whereas the
expected percentage is 32%; for 423D, 19% is the observed percentage,
whereas 32% is expected. For chimera 326B, the numbers are even more
dramatic. On the basis of the Purkinje cell counts, 40% of the cells
of cerebral cortex should be mutant, yet we observed only 14%
cdk5/ cells. The three animals
combined have a lower than expected percentage of
cdk5/ neurons that is
statistically significant (p < 0.005). These calculations strongly suggest that
cdk5/ neurons are selected
against during development. It is noteworthy that the counts of
Cdk5-deficient neurons include the GABAergic neurons present in each
field; therefore, the deficiency of neurons derived from the
telencephalic ventricular zone is most likely even greater than these
approximate calculations would imply. The missing cells cannot be
explained by a deficit in the generation of
cdk5/ neurons, because no
detectable difference in the size of cerebral cortex is noted
between cdk5/ and wild-type
embryos at E18.5 (Ohshima et al., 1996; Gilmore et al., 1998).
Why cdk5/ neurons should be at
such a competitive developmental disadvantage is not clear. Ectopic
location alone does not prevent a neuron from finding a target.
Significant neuronal ectopia has been observed in other mouse mutants
such as reeler, scrambler (dab1/), and the null allele of
the Cdk5 activator p35; these displacements have no reported effect on
cell survival within the cerebral cortex. In addition, characterization
of the reeler mutant has shown repeatedly that mispositioned
neurons are fully capable of finding their correct target (Lemmon and
Pearlman, 1981; Simmons et al., 1982; Caviness and Frost, 1983; Frost
et al., 1986) even if the time course of connectivity is delayed
compared with wild type (Yuasa et al., 1994; Del Rio et al., 1997). If
ectopic cdk5/ neurons were
delayed in finding a target in chimeras, that could put them at a
competitive disadvantage compared with wild type. This situation may be
amplified further, because Cdk5 has been suggested to be important for
normal axonal outgrowth and synaptogenesis (Matsubara et al., 1996;
Nikolic et al., 1996, 1998; Xiong et al., 1997; Paglini et al., 1998;
Qi et al., 1998; Shuang et al., 1998; Fletcher et al., 1999). Thus,
lack of survival of cdk5/
neurons could be attributable to migrational, axonal, or synaptic defects, or to an interaction of all three.
| |
FOOTNOTES |
Received Dec. 1, 2000; revised Aug. 9, 2001; accepted Aug. 16, 2001.
This work was supported by National Institutes of Health Grant NS20591.
We thank Toshio Ohshima [Laboratory for Developmental Neurobiology,
Brain Science Institute, The Institute of Physical and Chemical
Research (RIKEN)] and Ashok Kulkarni (National Institute of Dental and
Craniofacial Research, National Institutes of Health) for use of
cdk5/ embryonic stem cells.
Correspondence should be addressed to Karl Herrup, Alzheimer Research
Laboratory, University Hospitals of Cleveland, 10900 Euclid Avenue,
Cleveland, OH 44106. E-mail kxh26{at}po.cwru.edu.
| |
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ABSTRACT
INTRODUCTION
