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The Journal of Neuroscience, July 15, 1999, 19(14):6017-6026
Migration Defects of cdk5 / Neurons in
the Developing Cerebellum is Cell Autonomous
Toshio
Ohshima1, 2,
Edward C.
Gilmore4,
Glenn
Longenecker1,
David M.
Jacobowitz3,
Roscoe O.
Brady2,
Karl
Herrup5, and
Ashok B.
Kulkarni1
1 Functional Genomics Unit, Gene Targeting Facility,
National Institute of Dental and Craniofacial Research,
2 Developmental and Metabolic Neurology Branch, National
Institute of Neurological Disorders and Stroke, and
3 Laboratory of Clinical Science, National Institute of
Mental Health, National Institutes of Health, Bethesda, Maryland 20892, 4 Department of Neuroscience and 5 Alzheimer
Research Laboratory, Case Western Reserve Medical School, Cleveland,
Ohio 44106
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ABSTRACT |
Cyclin-dependent kinase 5 (Cdk5) is a member of the family of cell
cycle-related kinases. Previous neuropathological analysis of
cdk5 / mice showed significant
changes in CNS development in regions from cerebral cortex to
brainstem. Among the defects in these animals, a disruption of the
normal pattern of cell migrations in cerebellum was particularly
apparent, including a pronounced abnormality in the location of
cerebellar Purkinje cells. Complete analysis of this brain region is
hampered in the mutant because most of cerebellar morphogenesis occurs
after birth and the cdk5/ mice
die in the perinatal period. To overcome this disadvantage, we have
generated chimeric mice by injection of
cdk5/ embryonic stem cells into
host blastocysts. Analysis of the cerebellum from the resulting
cdk5/  cdk5+/+ chimeric mice shows that the
abnormal location of the mutant Purkinje cells is a cell-autonomous
defect. In addition, significant numbers of granule cells remain
located in the molecular layer, suggesting a failure to complete
migration from the external to the internal granule cell layer. In
contrast to the Purkinje and granule cell populations, all three of the
deep cerebellar nuclear cell groupings form correctly and are composed
of cells of both mutant and wild-type genotypes. Despite similarities
of the cdk5/ phenotype to that
reported in reeler and
mdab-1/
(scrambler/yotari) mutant brains, reelin
and disabled-1 mRNA were found to be normal in
cdk5/ brain. Together, the data
further support the hypothesis that Cdk5 activity is required for
specific components of neuronal migration that are differentially
required by different neuronal cell types and by even a single neuronal
cell type at different developmental stages.
Key words:
neuronal migration; cdk5; cerebellar
development; Purkinje cell; granule cell; cell autonomous
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INTRODUCTION |
The cell cycle in all eukaryotes is
controlled by a large family of serine/threonine protein kinases, the
cyclin-dependent kinases (Cdks), that are activated by binding to
regulatory subunits known as cyclins (Meyerson et al., 1992 ; Sherr,
1993). One unique member of the Cdk family is Cdk5. Unlike other Cdks,
Cdk5 expression and kinase activity are not high during cell division.
Furthermore, despite the wide distribution of its expression in the
organism (Hellmich et al., 1992; Meyerson et al., 1992; Tsai et al.,
1993), Cdk5 kinase activity is detected only in the nervous system
(Tsai et al., 1993). These unique characteristics are because of
the fact that, although Cdk5 has the ability to bind certain cyclins, no kinase activity results from this interaction (Xiong et al., 1992;
Kanaoka et al., 1997). Two regulatory subunits that do activate Cdk5
are known as p35 and p39, and their expression is found only in
neuronal tissue (Lew et al., 1994; Tsai et al., 1994; Tang et al.,
1995). Curiously, neither p35 nor p39 has significant homology with the
traditional cyclins (Lew et al., 1994; Tsai et al., 1994; Tang et al.,
1995; Ohshima et al., 1996a). The appearance of active Cdk5 is
correlated with the cessation of neurogenesis and the beginning of
differentiation of neuronal cells in the developing brain (Tsai et al.,
1993). Its endogenous substrates are unknown, but Cdk5 purified from
nervous tissue is capable of phosphorylating neuronal cytoskeletal
components, including neurofilament proteins (Lew et al., 1992;
Shetty et al., 1993) and the microtubule-associated protein tau
(Kobayashi et al., 1993; Paudel et al., 1993) in vitro.
These findings indicate that Cdk5 may have unique functions in the
development and differentiation of the brain, possibly through
regulating the phosphorylation of neuronal cytoskeletal molecules.
The cerebellum is a powerful system for studying neuronal development.
In the adult, it is a relatively simple structure consisting of a cell
sparse molecular layer, a single layer of Purkinje cells, the internal
granule cell layer (IGL), and deep white matter tracts that include the
axons of Purkinje cells en route to their target, the deep cerebellar
nuclei (DCN). Both Purkinje cells and neurons of the DCN are born in
the germinal layer of the fourth ventricle before migrating to their
final location. The granule cell precursors, originally derived from
the rhombic lip, migrate as a dividing population to cover the external
surface of the developing cerebellum forming the external granule cell
layer (EGL). The neurons of EGL cease division postnatally and then
migrate radially along the Bergmann glial fibers to the internal
granule cell layer. As granule cells migrate into IGL, they form
parallel fiber axons that synapse with the Purkinje cell dendrites in
the molecular layer.
We have reported previously on the generation and initial
characterization of cdk5/ mice
(Ohshima et al., 1996b). Mutant cdk5/
mice exhibit perinatal mortality that is associated with a unique CNS pathology (Ohshima et al., 1996b). Disruptions of lamination are observed in the olfactory bulb, as well as the cerebral and cerebellar cortices (Ohshima et al., 1996b, 1997). Neuronal
birthdate labeling by bromodeoxyuridine injections reveals an
abnormal neuronal migration pattern in the cerebral cortex of
cdk5/ mice (Gilmore et al., 1998).
Unfortunately, cdk5/ mice die around
birth. Thus, the abnormal brain organization cannot be characterized in
brain areas, such as cerebellum in which much of development occurs postnatally.
To delineate the precise role of Cdk5 during postnatal cerebellar
development, we have generated a series of
cdk5/ cdk5+/+ chimeric mice (cdk5
chimeras). The chimeras have an abnormal localization of a
subpopulation of Purkinje cells, indicating a cell-autonomous defect in
neuronal migration in these large cdk5/ neurons. A substantial granule
cell migration deficit is also apparent, a defect that would have been
undetectable in the nonchimeric mutant. Finally, comparison of the
cerebellar phenotype of the chimera with the previously described
reeler and scrambler/yotari mutants (Ogawa et
al., 1995; Sweet et al., 1996; Gilmore and Herrup, 1997; Goldowitz et
al., 1997; Gonzalez et al., 1997; Howell et al., 1997b; Sheldon
et al., 1997; Ware et al., 1997; Yoneshima et al., 1997) reveals
similarities but also significant differences. The levels and
distributions of the mRNAs for reelin (D'Arcangelo et al.,
1995) and disabled-1 (Howell et al., 1997a) are normal in
cdk5/ mice, consistent with the
suggestion that the Cdk5 deficiency disrupts a distinct migration process.
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MATERIALS AND METHODS |
Cdk5 mutant mice. The
cdk5+/ mouse line is maintained on a
C57BL/6 × 129/SvJ hybrid background. Genotyping of the offspring was performed by Southern blot analysis as described previously (Ohshima et al., 1996b). All mice were housed in the standard mouse
facility (Association for Assessment and Accreditation of Laboratory
Animal Care accredited) and were fed autoclaved diet and water.
Chimeric mice derived from cdk5/
embryonic stem cells.
cdk5/ embryonic stem (ES) cell
clones were obtained using high G418 culture method (Mortensen et al.,
1992). cdk5+/ ES cells, derived from
clone-57 (Ohshima et al., 1996b), were cultured in 1 mg/ml G418 (Life
Technologies, Bethesda, MD) for 6-7 d, and the resistant
colonies were picked and genotyped individually as described previously
(Ohshima et al., 1996b). To generate chimeric mice, two independent
clones of cdk5/ ES cells (57G23 and
57G25) were injected into C57BL/6 blastocysts. The cerebella from three
chimeric mice of each line were examined. Each had a high percentage of
agouti (cdk5/) coat color.
Age-matched C57BL/6 (cdk5+/+) mice were
used as controls.
Histopathological and immunohistochemical analysis. For
in situ hybridization, embryonic brains [embryonic day 16.5 (E16.5)] were immersed in 4% paraformaldehyde overnight,
dehydrated, and embedded in paraffin. The tissue sections were cut at
6-8 µm and stained with hematoxylin. For immunocytochemistry of
embryos, E18.5 brains were immersed in 4% paraformaldehyde overnight,
equilibrated in PBS-20% sucrose-0.8% paraformaldehyde at 4° C,
embedded in OCT (Tissue-Tek), frozen in dry ice, and cryostat sectioned
at 10 µm. Adult brains from 8- to 12-week-old chimeras and controls were obtained after transcardial perfusion with PBS, followed by 10%
buffered formalin (Fisher Scientific, Pittsburgh, PA). After a 1 hr
post-fix in the same fixative, brains were kept in 20% sucrose-PBS
for 2 d, and sections were cut on a cryostat at 20 µm. All
cryostat sections were kept at  70°C before use. For immunohistochemistry, the following antibodies were used at the indicated dilutions: mouse anti-calbindin IgG D-28K, monoclonal (Sigma)
at 1:1000 for double staining; rabbit anti-calbindin D-28K, polyclonal
(gift from Sylvia Christakos) at 1:1000 for single staining;
rabbit anti-Cdk5, polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA)
at 1:100; and mouse anti- 2/3 GABAA receptor bd17
(Boehringer Mannheim, Mannheim, Germany) at 1:100. For bright-field immunohistochemistry, HRP-conjugated secondary antibody was visualized using diaminobenzidene (DAB) reaction product as specified by Vectastain Elite protocol (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin. For immunofluorescence, mouse IgG primary antibodies were visualized with
fluorescein-conjugated secondary (Jackson ImmunoResearch, West Grove,
PA) at 1:200. Rabbit primary antibodies were detected with
Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1:400 for
rabbit primary antibodies. For confocal microscopy of study granule
cells, the Cdk5 antibody was detected with Oregon Green-conjugated
anti-rabbit IgG (Molecular Probes, Eugene, OR), and the bd17 monoclonal
was detected with Cy3-conjugated anti-mouse IgG (Jackson
ImmunoResearch) at 1:400. All sections were examined by standard light,
fluorescent, or confocal microscopic techniques.
reelin and disabled-1 cDNA fragments. Two cDNA
fragments of reelin, rl-1, corresponding to nucleotides
10,228-10,665, and rl-2, corresponding to nucleotides 10,860-11,459
(GenBank accession number U24703), were amplified by PCR methods using
a mouse brain cDNA pool (Marathon-Ready cDNA, catalog #7450-1;
Clontech, Palo Alto, CA). The mouse disabled-1 cDNA
fragment, corresponding to nucleotides 1626-2116 (GenBank accession
number Y08379), was amplified by the same PCR method. After subcloning
these fragments into T-vector (Promega, Madison, WI), nucleotide
sequencing was performed to confirm the identity of the insert with the
reported sequences.
RNA isolation, Northern blot analysis, and in situ
hybridization. RNA was isolated from individual brains at E16.5 by
the acid guanidinium thiocyanate-phenol-chloroform method
(Chomczynski and Sacchi, 1978). Twenty micrograms of total brain
RNA was heat-denatured size-fractionated by electrophoresis through a
1.0% formaldehyde-agarose gel and transferred to a nylon membrane
(Nytran; Schleicher & Schuell, Keene, NH) by capillary blotting as
described previously (Sambrook et al., 1989). cDNA fragments were
32P-radiolabeled by random priming. Hybridization was
performed using the reelin (rl-1) and disabled-1
cDNA probes in 6× SSC-50% formamide at 42°C overnight. The filter
was washed twice in 0.1× SSC-0.1% SDS at 50°C for 30 min. After
striping the probe, the same filter was used for hybridization with the
mouse GAPDH probe.
For in situ hybridization, E16.5 brains were immersed fixed
in 4% paraformaldehyde, embedded in paraffin, and sectioned at 6-8
µm. 35S-Labeled riboprobes for reelin (rl-1
and rl-2) were generated by in vitro synthesis.
Hybridization was performed as described previously (Fox and
Cottler-Fox, 1993; Yoshida et al., 1996). Briefly, after
deparaffinization and rehydration, the sections were incubated with 0.2 N HCl, rinsed with DEPC-treated water, and then digested with
proteinase K solution at 37°C. The sections were incubated
successively in 0.1 M triethanolamine buffer, pH 8.0, succinic anhydride solution (1% solution in 0.1 triethanolamine buffer), and 0.1 M triethanolamine buffer. The
prehybridization and hybridization were performed in a solution
containing 50% formamide and 10% dextran sulfate at 45°C. After
overnight hybridization, the slides were washed sequentially with 2×
SSC, formamide wash solution, Triton X-100 wash solution, and
0.1× SSC. Then slides were exposed to RNase A at 37°C, washed in 2×
SSC, and dehydrated in 0.3 M ammonium acetate and graded
alcohols. The slides were exposed in the dark at 4°C for 3 weeks
after being coated with autoradiography emulsion (NTB-3; Eastman Kodak,
Rochester, NY).
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RESULTS |
Cerebellar formation in cdk5/ mice
At birth, the cortex of the normal mouse cerebellum consists of
two layers: a layer of Purkinje cells, several cells thick, and a
superficial external granule cell layer. In the E18.5 cerebellum of the
cdk5/ mouse, this layered arrangement
is disrupted. Although the EGL forms normally and is normal in
appearance (Ohshima et al., 1997), the Purkinje cell layer
cannot be readily distinguished. To identify the location of the
different cerebellar cell types, immunocytochemical studies were
performed on E18.5 brains. In cdk5+/
mice at E18.5, calbindin-positive Purkinje cells are located, as
expected, as a broad layer of medium- to large-sized cell bodies in the
developing cerebellar plate (Fig.
1A). In contrast, in cdk5/ mice, virtually no Purkinje
cells are found in this cortical position. Instead, nearly all
calbindin-positive cells are located deep in the cerebellar parenchyma
(Fig. 1B). These observations imply that there is a
disturbance of the prenatal migration pattern of Purkinje cells in the
cdk5/ cerebellum with little or no
change in the initial migration of the granule cells to form the
EGL.
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Figure 1.
Calbindin staining of sagittal sections from E18.5
cerebellum reveals migration deficiencies of Purkinje cells in
cdk5/ mutants. Anti-calbindin
antibody is visualized through the red-brown DAB reaction product, seen
beneath the external granule cell layer (arrowheads) in
normal (cdk5+/) cerebellum
(A). Note that the Purkinje cells in this mouse
are found in their proper cortical location. In
cdk5/ mice
(B), calbindin-positive Purkinje cells are
ectopically located deep in the cerebellum, near the ventricle at the
bottom the cerebellum. Note the presence of a normal appearing external
granule cell layer (arrowheads) in the
cdk5/ cerebellum. Scale bar, 100 µm.
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The cdk5/ cdk5+/+ chimeric cerebellum
Because much of the process of cerebellar maturation occurs in the
postnatal period, ES cell chimeric mice were generated with both
cdk5/ and
cdk5+/+ cells. Two clones of
cdk5/ ES cells were obtained by
selection in high G418 medium (see Materials and Methods). Injection of
the homozygous mutant ES cells into wild-type blastocyst was used to
generate six cdk5 chimeric mice with highly variegated
coats. All chimeras were analyzed at 2 to 3 months of age. Chimeric
mice appeared to have normal viability without noticeable ataxia. The
cerebella of all six chimeras examined are either equal to or smaller
in size than a typical wild-type mouse (Fig.
2A,B).
Although smaller in size, the typical trilaminar pattern of the
cerebellar cortex is always readily apparent, and the cerebellar
lobules are well formed. The IGL is normal in position and cell
composition, and the Purkinje cell layer is well formed, consisting of
a row of calbindin-positive cells (Figs. 2B,
3B).
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Figure 2.
Sagittal cresyl violet-stained sections of adult
wild-type (A, C) and
cdk5/ chimeric (B,
D, E) cerebellum. The chimeric cerebellum
(B) is normal in foliation and cellular
distribution but is reduced in size compared with wild-type cerebellum
(A). The molecular layer
(ml) of normal mice is cell-sparse
(C, higher magnification of the boxed
area in A). A monolayer of Purkinje cells is
found between the molecular layer and the cell dense internal granule
cell layer (igl). Beneath the internal granule
cell layer are the white matter tracts, which contain myelinated axons
of Purkinje cells that will synapse on neurons of the deep cerebellar
nuclei. Cell density in the molecular layer of chimeras varies from
region to region (D, E, higher
magnification of the boxed areas in B).
Despite this, the Purkinje cell layer and internal granule cell layer
are normal in appearance. Large cells are seen in aberrant locations
(arrowheads in the white matter of E).
Scale bars: (in A) A, B,
200 µm; (in C) C-E, 50 µm.
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Figure 3.
Horizontal sections through deep nuclear region of
adult wild-type (A, C, D)
and chimeric (B, E-H) cerebella.
Sections were double immunostained for calbindin (A-C,
E, G) and Cdk5 (D,
F, H). Wild-type cerebellum
(A) with the ventricle (v)
to the left shows prominent calbindin staining in the
Purkinje cell layer of the cerebellar cortex. Only rarely are
calbindin-positive neurons found near the ventricles. Calbindin
immunostaining of chimeric cerebellum (B)
indicates that, although correctly located Purkinje cells are
calbindin-positive, there are many ectopic Purkinje cells
(ep) found within the parenchyma of the cerebellum near
the regions of the deep cerebellar nuclei (dcn). No
calbindin-positive neurons are apparent within the internal granule
cell layer. In wild-type mice, there is complete congruence between
calbindin-positive (C) and Cdk5-positive
(D) neurons. Within the region of the Purkinje
cell layer, there are some large neurons in both wild-type and chimeric
mice that are Cdk5-positive but not calbindin-positive. These are
probably other large neurons of the cerebellar cortex, such as basket,
Lugaro, or Golgi cells. All calbindin-positive neurons
(E) within the Purkinje cell layer of chimeras
are wild type, i.e., Cdk5-positive (F). However,
calbindin-positive Purkinje cells in the chimera (ectopically located
near the ventricle in the top left corner of
G) are mutant, i.e., Cdk5-negative
(H). Insets in
G and H are higher magnifications of the
areas indicated by the white boxes. Scale bars: (in
A) A, B, 150 µm; (in
C) C-H, 100 µm.
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In wild-type animals, the molecular layer contains only a few well
separated basket and stellate cells. The bulk of this layer consists
mainly of parallel fibers (granule cell axons) and their targets, the
dendrites of Purkinje cells. The molecular layer of the cdk5
chimeras, however, has an abundance of granule-like cells (Fig.
2D,E). In all chimeras examined,
the density of these granule-like cells varies from almost complete
saturation of the molecular layer (Fig. 2D) to almost
wild-type sparseness (Fig. 2E). Within the same
animal, the density varies from region to region in both the
mediolateral and anteroposterior dimension. The anatomical location of
the cell-dense and cell-sparse regions between different chimeric
cerebella does not appear to follow a reproducible pattern. Because
granule cells should have completed migration to the IGL by the time
these animals were examined, it appears that some aspect of the granule
cell migratory process must be deficient. The migratory defect of the
granule cells is not entirely unexpected because, in mice lacking p35
(one of the Cdk5 activators), an excess of granule cells is found in
the molecular layer (Chae et al., 1997). Interestingly, the defect in
cdk5 chimeras appears to be much more severe.
In addition to the normally positioned Purkinje cells of the Purkinje
cell layer of the chimeras, an unusually large number of neuronal-like
cells are located near the white matter (Fig. 2E) and
in regions occupied by the DCN. These neurons are found in positions
ranging from near the fourth ventricle to just below the IGL at the
bottom of a sulcus (Fig. 2E). The identification of
these cells as mutant Purkinje cells was confirmed by
immunohistochemical techniques, using antibodies against calbindin. In
wild-type mice, staining with the Purkinje cell-specific calbindin
antibody stains large cells exclusively in the Purkinje cell layer
(Fig. 3A). In the chimeric brains, all of the Purkinje cells
in the cortical Purkinje cell layer are immunopositive for calbindin,
as expected. However, many of the unidentified large neurons in the
deep cerebellar core region are also calbindin-positive. This finding
demonstrates that a subset of the Purkinje cells in the chimera are in
an abnormal location (Fig. 3B).
The presence or absence of Cdk5 protein can be used to determine the
genotypes of the Purkinje cells in the chimera. As expected, all
calbindin-positive neurons in wild-type brains are Cdk5-positive (Fig.
3C,D). In the chimera, all of the large
calbindin-positive neurons in the Purkinje cell layer are also
Cdk5-positive, confirming their cdk5+/+
genotype (i.e., they were Purkinje cells derived from the wild-type C57BL/6 blastocyst) (Fig.
3E,F). It is highly
significant that none of the calbindin-positive cells seen within the
Purkinje cell layer are cdk5/. This
means that no mutant Purkinje cell is able to achieve its correct
location in cerebellar cortex. In contrast, nearly all ectopic Purkinje
cells are found to be cdk5/ (Fig.
3G,H). Because no
cdk5/ Purkinje cells are found within
the cerebellar cortex and virtually all Cdk5-positive Purkinje cells
are located in the cerebellar cortex, it appears that the migration
defect of Cdk5-deficient Purkinje cells is cell autonomous and fully penetrant.
The status of the DCN neurons was also investigated. There are no
specific immunohistochemical markers for DCN neurons comparable with
calbindin for Purkinje cells. Therefore, neurons of the DCN were
identified by their size, their location within the cerebellum, and by
the presence of a "halo" of calbindin staining surrounding their
cell bodies (resulting from presynapic terminals of calbindin-positive Purkinje cells). In wild-type mice, all neurons in the DCN are Cdk5-positive, as expected (Fig.
4A,B).
In the chimera, many of the identifiable DCN neurons are also
Cdk5-positive (Fig. 4C,D). However there are also
many clear examples of DCN neurons that are
cdk5/ (Fig.
4C,D, arrowheads). No attempt was made
to quantitate the number of
cdk5/ neurons because of the
difficulties inherent in their identification within the DCN. It is of
interest to note that there is a nearly complete segregation of DCN
neurons and ectopic Purkinje cells (Fig.
3B,G,H). This
phenomena has been observed before in both reeler and
scrambler mice (Goffinet, 1984; Goldowitz et al.,
1997). Although ectopic Purkinje cells can be found adjacent to
DCN neurons (Fig. 3G,H,
insets), they do not exist as a mixture, indicating a
developmental mechanism that keeps the populations separate.
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Figure 4.
High magnification of deep cerebellar nuclei from
wild-type (A, B) and chimeric
(C, D) cerebella. Sections were double
immunostained with calbindin (A, C) and
Cdk5 (B, D). Wild-type deep cerebellar
nuclei neurons can be identified by location and the presence of a
calbindin-positive ring surrounding their cell body. The chimeric deep
cerebellar nuclei contain both Cdk5-positive and Cdk5-deficient
neurons. In the chimera, many deep cerebellar nuclei neurons contain
Cdk5; however, some do not (arrowheads). Scale bar, 50 µm.
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Abundant granule cells can be identified in the molecular layer of
chimeras, although they are rarely found there in wild-type animals.
Antibodies to 2/3 GABAA receptor (bd17) were used
identify the plasma membrane of granule cells (Ewert et al., 1992;
Laurie et al., 1992; Fritschy and Mohler, 1995). Whereas other cell
types within the cerebellum may be recognized by bd17, granule cells can be distinguished from all other cell types by their small diameter,
5-8 µm. Because of the very small size of granule cells, confocal
microscopy was needed to define their genotype (based on the presence
or absence of Cdk5). Wild-type animals show a low but detectable level
of Cdk5 protein in all granule cells of adult animals (Fig.
5A,B).
In contrast, the granule cells of the chimeric IGL are a mixture of
cdk5+/+ and
cdk5/ cells (Fig.
5C,D). Furthermore, the granule cells found
within the molecular layer are nearly all Cdk5-deficient (Fig.
5E,F). This indicates that
the block in cell migration that leaves many granule cells stranded
within the molecular layer is intrinsic to
cdk5/ neurons. However, the presence
of cdk5/ cells correctly located in
the IGL indicates that this block is not absolute.
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Figure 5.
Cdk5 staining of granule cells in wild-type and
chimeras visualized with confocal microscopy. Double immunostaining
with antibodies 2/3 GABAA receptor (bd17) (Ewert et
al., 1992) and Cdk5. The cell surface localization of bd17
(red) surrounds the Cdk5-positive
(green) granule cell (Laurie et al., 1992;
Fritschy and Mohler, 1995). Higher magnifications of boxed
areas of A, C, and
E are shown in B, D, and
F, respectively. Granule cells are identified by
membrane staining with bd17 and a diameter of 5-8 µm. The relative
Cdk5 staining in granule cells is much lower than in Purkinjecells (A,
top left corner, and C, bottom
right corner). Granule cells of wild-type mice all contain Cdk5
(A, B). A few capillaries are stained by
Cy3-conjugated anti-mouse IgG antibodies (arrowheads in
A) and should not be confused with Cdk5-negative granule
cells. The granule cell layer of the chimeric IGL contains both
cdk5+/+ and
cdk5/ neurons (C,
D). These can be seen at high magnification in
D, with cdk5+/+
granule cells indicated by the arrowheads and
cdk5/ granule cells indicated by
asterisks. The ectopic granule cell neurons of the
chimeric molecular layer are Cdk5-deficient (E,
F). Scale bar: A,
C, E, 20 µm; B,
D, F, 5 µm.
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reelin and disabled-1 mRNA expression
is normal
Although it appears that there are distinctions between the
cerebellar phenotypes of the cdk5/
and reeler/scrambler mutants (Goffinet, 1984; Sweet et al.,
1996; Goldowitz et al., 1997), they do have many facets in common. The similarities between mutants prompted us to explore the expression of
reelin and disabled-1 mRNA (the respective
products of these two genes) in the embryonic brains of
cdk5/ mice to determine whether
alteration of expression of either of them could account for some of
the defects found in cdk5/ mice. cDNA
fragments were obtained by PCR amplification using primer sets designed
from the published GenBank sequence data for reelin and
disabled-1. Correct amplifications were confirmed by
sequencing (see Materials and Methods). The labeled fragments were used
for Northern blot analysis and for in situ hybridization studies. The results illustrate that the mRNA expression levels of
reelin and disabled-1 are not changed in the
cdk5/ brain (Fig.
6). The distributions of
reelin mRNA in the brains from
cdk5+/+ and
cdk5/ mice were also examined by
in situ hybridization using 35S-labeled
riboprobes. As reported in earlier studies (D'Arcangelo et al., 1997;
Schiffmann et al., 1997), reelin mRNA is distributed in the
epithelium of the olfactory bulb, the marginal zone of the cerebral
cortex, and the EGL of the cerebellum in the wild-type mice at E16.5
(Fig.
7A,B).
In cdk5/ mice, the distribution and
level of reelin mRNA remain unchanged (Fig.
7C,D).
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Figure 6.
Northern blot analysis of mRNA expression for
reelin, disabled-1
(mdab-1), and GAPDH in E16.5 brains of
cdk5+/+ and
cdk5/ mice reveals no difference
between wild-type and mutant mice.
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Figure 7.
Expression pattern of reelin mRNA
in sagittal section of cerebrum (A, C)
and cerebellum (B, D) by in
situ hybridization. Antisense probes (synthesized from rl-1)
hybridized to E16.5 Cdk5+/+
(A, B) and
Cdk5/ (C,
D) show normal levels and distribution of
reelin mRNA in the olfactory bulb (left,
facing arrowheads), along with the marginal zone of the
cerebral cortex (arrowheads along top).
Note the reelin expression within the external granule cell layer of
the cerebellum (arrowheads) of wild-type and
Cdk5/ mice (B,
D, respectively). No significant hybridization is
observed when sense probe is used (data not shown). Two different cDNA
fragments (rl-1 and rl-2) were used as probes, and the same
hybridization patterns were obtained (rl-2, data not shown). Scale
bars: A, C, 500 µm; B,
D, 800 µm.
|
|
| |
DISCUSSION |
The results presented here demonstrate that the cerebellar defects
of the cdk5/ mutation are partially
rescued by the presence of wild-type cells in
cdk5/ cdk5+/+ chimeras. Although the cause of
perinatal death of cdk5/ embryos
remains unknown, it is clear from the long-term survival of the
chimeric mice in this study that it is not a result of a toxic effect
of cdk5/ cells during development. In
most chimeras, the cerebellum is slightly smaller than in wild-type,
but the typical trilaminar configuration of the cerebellar cortex is
readily apparent, and DCN are present in their proper location. This
phenotypic rescue, however, occurs only at the level of cerebellar
structure; at the level of the individual cells, there are a number of
defects that distinguish the chimeric and wild-type brains. In the
chimera, many Purkinje cells are normal in position, but many others
are ectopically located deep in the cerebellar parenchyma. Likewise, many granule cells participate in the formation of a well defined IGL,
but the molecular layer is dense with ectopic granule cells. The close
correlation between abnormal phenotype and mutant genotype leads us to
interpret these findings to mean that the defect in migration caused by
the absence of the Cdk5 protein is cell autonomous in all affected cells.
The data for the cerebellar Purkinje cells are definitive evidence in
support of the conclusion that the migrational defects of the
cdk5/ neurons are cell autonomous. At
E18.5 in cdk5/ mice, most of the
calbindin-positive cells are located deep in the central mass of the
cerebellum. The failure of these Purkinje cells to achieve a normal
adult position is reminiscent of the defect in cell migration described
previously in cdk5/ cerebral cortex
(Ohshima et al., 1996b; Gilmore et al., 1998). The tight coupling
between Purkinje cell genotype and the migration phenotype in the
chimera adds significantly to the overall picture of the migration
defect. All Purkinje cells in the normal Purkinje cell layer of the
chimera are cdk5+/+, as revealed by their
staining with anti-Cdk5 antibody. In addition, the Purkinje cells that
are ectopically positioned in the chimera are all
cdk5/. These data show that both the
presence of wild-type cells in the chimeric environment does not rescue
the migration defect of the cdk5/
Purkinje cells and the presence of
cdk5/ mutant cells does not interfere
with the normal migration of the neighboring wild-type Purkinje cells.
The normal configuration of the DCN and their mixed genotype
composition in the chimera indicate that the cell movements required to
form the three nuclear groups (lateralis, interpositus, and medialis)
probably occur by a Cdk5-independent process. The DCN neurons migrate
along a complex pathway before settling within the three nuclei (Altman
and Bayer, 1985). However, it is unclear whether the migration
mechanism of the DCN neurons involves glial guidance. Although we
cannot exclude the possibility that
cdk5/ DCN neurons do not follow the
same pathway to reach their final destination as the wild-type neurons,
we find this improbable because we have observed many
cdk5/ DCN neurons in the correct
location, correctly surrounded by Purkinje cell innervation. Our
interpretation is that the migration of the DCN neurons is not
dependent on Cdk5. This is important because the DCN are generated at a
similar time and in the same location as Purkinje cells (Pierce, 1975;
Altman and Bayer, 1978). We have shown previously that some of the
neurons of the deeper layers of cerebral cortex achieve their normal
position even in the nonchimeric
cdk5/ mutant (Gilmore et al., 1998).
Although there is nothing in the migratory path taken by DCN cells
(Altman and Bayer, 1985) that would suggest that the mechanics
of migration are different from those used by the Purkinje cells, the
cdk5/ mutant and the cdk5
chimera plainly identify this difference. This reinforces the
conclusion of our earlier work in cerebral cortex (Gilmore et al.,
1998) that important yet unidentified differences exist in the
mechanics of neuronal migration across different cell types.
The behavior of the granule cells in the chimera is also consistent
with this idea. Although the IGL is made up of both
cdk5/ and
cdk5+/+ granule cells, nearly all granule
cells in the molecular layer are Cdk5-deficient. Thus, although many
mutant granule cells are incapable of completing migration, others are
seemingly unaffected. One possible explanation for this finding is that
there are distinct granule cell populations, each with a different
requirement for Cdk5 activity. Another possibility is that wild-type
granule cells are capable of rescuing some mutant granule cells. Such a
rescue has been reported when granule cells from in the cerebellar
mutant weaver are transplanted to a wild-type EGL or
cultured in vitro with wild-type cells (Gao et al., 1992;
Gao and Hatten, 1993). We favor an alternative explanation, however,
one that assumes the existence of compensatory mechanisms in the
granule cell population that reduces the impact of the Cdk5 deficiency.
The nature of these mechanisms is unclear, but the milder phenotype of
the p35/ mouse would fit well with
this explanation (Chae et al., 1997). The implication of this
hypothesis is that the ability of granule cells to migrate is near some
threshold level in the cdk5/ mutant.
It is worth noting that the long tangential migration of the granule
cell precursors from the rhombic lip to the anterior-most and
medial-most regions of the cerebellar surface appears undisturbed by
the absence of the Cdk5 kinase and is likely, therefore, to be
Cdk5-independent. Thus, once again, the Cdk5 mutation
partitions neuronal migration into different types: an early
Cdk5-independent form and a later Cdk5-dependent form. The granule cell
data suggest that a single cell type can use both types sequentially
during development.
The close chromosomal proximity of loci for cdk5 (Ohshima et
al., 1995) and reeler on chromosome 5 (Dernoncourt et al.,
1991; Goffinet and Dernoncourt, 1991) raised concerns that the
reelin gene could be downregulated because of an
insertion-induced alteration in a long-distance enhancer located in the
vicinity of cdk5. This might reduce the levels of
Reelin, thus possibly contributing to the observed phenotype. To
address this, we analyzed the expression levels and pattern of
reelin mRNA in wild-type and
cdk5/ mouse brains and found no
difference. Furthermore, the expression levels of disabled-1
were also found to be similar to wild type, suggesting overall that the
migration defect of cdk5/ neurons is
not mediated by changes in the expression of either of these two
phenotypically related genes.
The characterization of the cdk5/
cellular phenotype in the adult cerebellum allows the first direct
comparison of the effect of the mutation with those of null alleles of
the Cdk5 activator p35. In p35/
cerebella, there is a normal Purkinje cell layer and a slight increase
in the density of granule-like cells in the molecular layer (Chae et
al., 1997). Our results contrast with this picture. The
cdk5/ Purkinje cells are totally
blocked in their migratory ability, and the granule cell block is much
more severe. These observations, as well as those reported previously
in cerebral cortex, suggest that nearly all of the differences between
cdk5/ and
p35/ mice, including the final
location of subplate (Ohshima et al., 1996b; Chae et al., 1997; Gilmore
et al., 1998; Kwon and Tsai, 1998), are caused by compensatory
actions of multiple activators of Cdk5, such as p39. Therefore, the
differences found in the cerebellar phenotypes, along with previously
characterized differences of cerebral cortical phenotypes, lead to the
hypothesis that alternative Cdk5 activators can substitute when p35 is
not present to induce Cdk5 activity. This hypothesis predicts that the
p35/ phenotypes are a subset of the
cdk5/ phenotypes.
It is of interest to compare the cell migration phenotypes of the
cdk5/ mouse with those found in other
cerebellar mutants. In both reeler and the mdab-1
mutant scrambler, a small but significant number of mutant
Purkinje cells successfully complete migration to the Purkinje cell
layer (Mariani et al., 1977; Goldowitz et al., 1997). In contrast, none
of the cdk5/ Purkinje cells in the
chimeras successfully migrate to the cerebellar cortex. This suggests
that the cdk5/ mutation causes a more
severe arrest of Purkinje cell migration than either the
reeler or scrambler mutation. A far more dramatic difference is that no granule cell migration defect is reported in
reeler/scrambler mice, yet Cdk5-deficient mice have
significant cell-autonomous defects in granule cell migration. The
cdk5/ granule cell migration
deficiency is most reminiscent of weaver heterozygous mice,
wv/+. Mice homozygous for the weaver gene
wv/wv have extensive granule cell pathology (Rakic and
Sidman, 1973a,b; Herrup and Trenkner, 1987; Smeyne and Goldowitz,
1989). In heterozygous wv/+ mice, the cerebellar structure
is primarily normal, yet a subset of granule cells remain trapped in
the molecular layer, even while most granule cells successfully migrate
into the IGL (Rakic and Sidman, 1973a). Studies of chimeric mice have
indicated that this migration block is a cell-autonomous defect
(Goldowitz and Mullen, 1982). However, other studies have indicated
that the migration ability of the wv/wv granule cells,
although normally deficient, can be rescued through interaction with
wild-type neurons (Gao et al., 1992; Gao and Hatten, 1993). It is
unclear how the genetic defect in weaver mice, a point
mutation in the inwardly rectifying potassium channel girk2,
leads the granule cell migration defect (Patil et al., 1995; Rossi et
al., 1998). It would be of interest to determine, however, if the block
in weaver granule cells is in any way related to Cdk5
activity in the deficient granule cells.
| |
FOOTNOTES |
Received Dec. 23, 1998; revised May 3, 1999; accepted May 5, 1999.
This work was supported by National Institutes of Health Grant
NS20591 to K.H., National Institute of Dental and Craniofacial Research, and National Institute of Neurological Disorders and Strokes,
Division of Intramural Research, to A.K. It was also supported
by Japan Society for the Promotion of Science Research Fellowship for Japanese Biomedical and Behavioral Researchers at
National Institutes of Health to T.O.
Drs. Ohshima and Gilmore contributed equally to this work.
Correspondence should be addressed to Dr. Toshio Ohshima, Laboratory
for Developmental Neurobiology, Brain Science Institute, The Institute
of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama
351-0198, Japan.
| |
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Y.-T. Chen, L. L. Collins, H. Uno, and C. Chang
Deficits in Motor Coordination with Aberrant Cerebellar Development in Mice Lacking Testicular Orphan Nuclear Receptor 4
Mol. Cell. Biol.,
April 1, 2005;
25(7):
2722 - 2732.
[Abstract]
[Full Text]
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V. Andres
Control of vascular cell proliferation and migration by cyclin-dependent kinase signalling: new perspectives and therapeutic potential
Cardiovasc Res,
July 1, 2004;
63(1):
11 - 21.
[Abstract]
[Full Text]
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S. Kesavapany, N. Amin, Y.-L. Zheng, R. Nijhara, H. Jaffe, R. Sihag, J. S. Gutkind, S. Takahashi, A. Kulkarni, P. Grant, et al.
p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons
J. Neurosci.,
May 5, 2004;
24(18):
4421 - 4431.
[Abstract]
[Full Text]
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M. Hirasawa, T. Ohshima, S. Takahashi, G. Longenecker, Y. Honjo, Veeranna, H. C. Pant, K. Mikoshiba, R. O. Brady, and A. B. Kulkarni
Perinatal abrogation of Cdk5 expression in brain results in neuronal migration defects
PNAS,
April 20, 2004;
101(16):
6249 - 6254.
[Abstract]
[Full Text]
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P. M. Rodier
Environmental Causes of Central Nervous System Maldevelopment
Pediatrics,
April 1, 2004;
113(4/S1):
1076 - 1083.
[Abstract]
[Full Text]
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V. Hammond, L.-H. Tsai, and S.-S. Tan
Control of Cortical Neuron Migration and Layering: Cell and Non Cell-Autonomous Effects of p35
J. Neurosci.,
January 14, 2004;
24(2):
576 - 587.
[Abstract]
[Full Text]
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J. L. Rosales, B.-C. Lee, M. Modarressi, K. P. Sarker, K.-Y. Lee, Y.-G. Jeong, R. Oko, and K.-Y. Lee
Outer Dense Fibers Serve as a Functional Target for Cdk5{middle dot}p35 in the Developing Sperm Tail
J. Biol. Chem.,
January 9, 2004;
279(2):
1224 - 1232.
[Abstract]
[Full Text]
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J. L. Hallows, K. Chen, R. A. DePinho, and I. Vincent
Decreased Cyclin-Dependent Kinase 5 (cdk5) Activity Is Accompanied by Redistribution of cdk5 and Cytoskeletal Proteins and Increased Cytoskeletal Protein Phosphorylation in p35 Null Mice
J. Neurosci.,
November 19, 2003;
23(33):
10633 - 10644.
[Abstract]
[Full Text]
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S. S. Kholmanskikh, J. S. Dobrin, A. Wynshaw-Boris, P. C. Letourneau, and M. E. Ross
Disregulated RhoGTPases and Actin Cytoskeleton Contribute to the Migration Defect in Lis1-Deficient Neurons
J. Neurosci.,
September 24, 2003;
23(25):
8673 - 8681.
[Abstract]
[Full Text]
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B.-S. Li, W. Ma, H. Jaffe, Y. Zheng, S. Takahashi, L. Zhang, A. B. Kulkarni, and H. C. Pant
Cyclin-dependent Kinase-5 Is Involved in Neuregulin-dependent Activation of Phosphatidylinositol 3-Kinase and Akt Activity Mediating Neuronal Survival
J. Biol. Chem.,
September 12, 2003;
278(37):
35702 - 35709.
[Abstract]
[Full Text]
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S. Kesavapany, K.-F. Lau, S. Ackerley, S. J. Banner, S. J. A. Shemilt, J. D. Cooper, P. N. Leigh, C. E. Shaw, D. M. McLoughlin, and C. C. J. Miller
Identification of a Novel, Membrane-Associated Neuronal Kinase, Cyclin-Dependent Kinase 5/p35-Regulated Kinase
J. Neurosci.,
June 15, 2003;
23(12):
4975 - 4983.
[Abstract]
[Full Text]
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H. Patzke, U. Maddineni, R. Ayala, M. Morabito, J. Volker, P. Dikkes, M. K. Ahlijanian, and L.-H. Tsai
Partial Rescue of the p35-/- Brain Phenotype by Low Expression of a Neuronal-Specific Enolase p25 Transgene
J. Neurosci.,
April 1, 2003;
23(7):
2769 - 2778.
[Abstract]
[Full Text]
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A. Diez-Juan and V. Andres
Coordinate Control of Proliferation and Migration by the p27Kip1/Cyclin-Dependent Kinase/Retinoblastoma Pathway in Vascular Smooth Muscle Cells and Fibroblasts
Circ. Res.,
March 7, 2003;
92(4):
402 - 410.
[Abstract]
[Full Text]
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C. Gao, S. Negash, H. T. Guo, D. Ledee, H.-S. Wang, and P. Zelenka
CDK5 Regulates Cell Adhesion and Migration in Corneal Epithelial Cells
Mol. Cancer Res.,
November 1, 2002;
1(1):
12 - 24.
[Abstract]
[Full Text]
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L. Keshvara, S. Magdaleno, D. Benhayon, and T. Curran
Cyclin-Dependent Kinase 5 Phosphorylates Disabled 1 Independently of Reelin Signaling
J. Neurosci.,
June 15, 2002;
22(12):
4869 - 4877.
[Abstract]
[Full Text]
[PDF]
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T. Ohshima, M. Ogawa, K. Takeuchi, S. Takahashi, A. B. Kulkarni, and K. Mikoshiba
Cyclin-Dependent Kinase 5/p35 Contributes Synergistically with Reelin/Dab1 to the Positioning of Facial Branchiomotor and Inferior Olive Neurons in the Developing Mouse Hindbrain
J. Neurosci.,
May 15, 2002;
22(10):
4036 - 4044.
[Abstract]
[Full Text]
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P. Sharma, Veeranna, M. Sharma, N. D. Amin, R. K. Sihag, P. Grant, N. Ahn, A. B. Kulkarni, and H. C. Pant
Phosphorylation of MEK1 by cdk5/p35 Down-regulates the Mitogen-activated Protein Kinase Pathway
J. Biol. Chem.,
January 4, 2002;
277(1):
528 - 534.
[Abstract]
[Full Text]
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E. C. Gilmore and K. Herrup
Neocortical Cell Migration: GABAergic Neurons and Cells in Layers I and VI Move in a Cyclin-Dependent Kinase 5-Independent Manner
J. Neurosci.,
December 15, 2001;
21(24):
9690 - 9700.
[Abstract]
[Full Text]
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B.-S. Li, M.-K. Sun, L. Zhang, S. Takahashi, W. Ma, L. Vinade, A. B. Kulkarni, R. O. Brady, and H. C. Pant
Regulation of NMDA receptors by cyclin-dependent kinase-5
PNAS,
September 26, 2001;
(2001)
211428098.
[Abstract]
[Full Text]
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J. Ko, S. Humbert, R. T. Bronson, S. Takahashi, A. B. Kulkarni, E. Li, and L.-H. Tsai
p35 and p39 Are Essential for Cyclin-Dependent Kinase 5 Function during Neurodevelopment
J. Neurosci.,
September 1, 2001;
21(17):
6758 - 6771.
[Abstract]
[Full Text]
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D. S. Smith, P. L. Greer, and L.-H. Tsai
Cdk5 on the Brain
Cell Growth Differ.,
June 1, 2001;
12(6):
277 - 283.
[Abstract]
[Full Text]
[PDF]
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T. Ohshima, M. Ogawa, Veeranna, M. Hirasawa, G. Longenecker, K. Ishiguro, H. C. Pant, R. O. Brady, A. B. Kulkarni, and K. Mikoshiba
Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain
PNAS,
February 8, 2001;
(2001)
51628498.
[Abstract]
[Full Text]
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T. Rashid, M. Banerjee, and M. Nikolic
Phosphorylation of Pak1 by the p35/Cdk5 Kinase Affects Neuronal Morphology
J. Biol. Chem.,
December 21, 2001;
276(52):
49043 - 49052.
[Abstract]
[Full Text]
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T. Ohshima, M. Ogawa, Veeranna, M. Hirasawa, G. Longenecker, K. Ishiguro, H. C. Pant, R. O. Brady, A. B. Kulkarni, and K. Mikoshiba
Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain
PNAS,
February 27, 2001;
98(5):
2764 - 2769.
[Abstract]
[Full Text]
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B.-S. Li, M.-K. Sun, L. Zhang, S. Takahashi, W. Ma, L. Vinade, A. B. Kulkarni, R. O. Brady, and H. C. Pant
From the Cover: Regulation of NMDA receptors by cyclin-dependent kinase-5
PNAS,
October 23, 2001;
98(22):
12742 - 12747.
[Abstract]
[Full Text]
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TOP
ABSTRACT
INTRODUCTION
