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 Previous Article | Next Article 
The Journal of Neuroscience, May 15, 2002, 22(10):4036-4044
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
Toshio
Ohshima1,
Masaharu
Ogawa2,
Kyoko
Takeuchi2,
Satoru
Takahashi3,
Ashok B.
Kulkarni3, and
Katsuhiko
Mikoshiba1
1 Laboratory for Developmental Neurobiology and
2 Cell Culture Development, Brain Science Institute, The
Institute of Physical and Chemical Research (RIKEN), Wako, Saitama
351-0198, Japan, and 3 Functional Genomics Unit, National
Institute of Dental and Craniofacial Research, National Institutes of
Health, Bethesda, Maryland 20892
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ABSTRACT |
Cyclin-dependent kinase 5 (Cdk5)/p35 is a serine/threonine kinase,
and its activity is detected primarily in postmitotic neurons. Mice
lacking Cdk5/p35 display migration defects of the cortical neurons in
the cerebrum and cerebellum. In this study, we demonstrate that
although most brainstem nuclei are found in their proper positions, the
motor nucleus of the facial nerve is ectopically located and neurons of
the inferior olive fail to position correctly, resulting in the lack of
their characteristic structures in the hindbrain of Cdk5 / mice.
Despite the defective migration of these neurons, axonal exits of the
facial nerve from brainstem and projections of the inferior cerebellar
axons appear unchanged in Cdk5/ mice. Defective neuronal migration
in Cdk5/ hindbrain was rescued by the neuron-specific expression of
Cdk5 transgene. Because developmental defects of these structures have
been reported in reeler and Dab1 mutant mice, we
analyzed the double-null mutants of p35 and Dab1 and found more
extensive ectopia of VII motor nuclei in these mice. These results
indicate that Cdk5/p35 and Reelin signaling regulates the selective
mode of neuronal migration in the developing mouse hindbrain.
Key words:
Cdk5; p35; reelin; disabled-1; facial branchiomotor
neuron; inferior olive
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INTRODUCTION |
Cyclin-dependent kinase 5 (Cdk5) is
a member of the family of Cdks. Unlike other Cdks, Cdk5 activity is
detected mainly in postmitotic neurons (Tsai et al., 1993 ). Association
of Cdk5 with a neuron-specific regulatory subunit, either p35 or its
isoform p39, is critical for its kinase activity (Lew et al., 1994;
Tsai et al., 1994; Tang et al., 1995). Cdk5/ mice exhibit perinatal death associated with disruption of the cortical laminar structures, apparently because of defective neuronal migration (Ohshima et al., 1996; Gilmore et al., 1998), whereas p35/ mice display milder
phenotypes than Cdk5/ mice because of the redundancy of Cdk5
regulatory subunits (Chae et al., 1997; Kwon and Tsai, 1998; Ohshima et
al., 2001). Moreover, p35/p39/ mice display a phenotype
identical to that of Cdk5/ mice, confirming redundancy in these
subunits (Ko et al., 2001). Neuronal birth-date labeling by
bromodeoxyuridine revealed an inverted layer structure in the cerebral
cortex of Cdk5/ mice (Gilmore et al., 1998). An inverted pattern of
layer structure in the cerebral cortex is also a well known
characteristic of reeler and scrambler/yotari
mice. These mutant mice exhibit nearly identical phenotypes, suggesting
that the gene products mutated in these mice, Reelin and Dab1,
respectively, act in a common signaling pathway during cortical
development (D'Arcangelo et al., 1995; Ogawa et al., 1995; Sheldon et
al., 1997; Yoneshima et al., 1997; Rice et al., 1998; Howell et al., 1999). In wild-type mice, successive waves of migrating neurons split
the preplate into the marginal zone and subplate and form the cortical
plate in an inside-out manner. In reeler and
scrambler/yotari mutants, the migrating cortical neurons
appear incapable of splitting the preplate, and the cortical-plate
neurons stack up in inverted order beneath the preplate. In Cdk5/
and p35/ mice, the earlier-born neurons successfully split the
preplate; however, the late-born neurons stack up in an inverted layer
under the subplate. Two general modes of neuronal migration have been
described in the developing nervous system: nuclear translocation (also
called nucleokinesis) and "locomotion" (Book and Morest, 1990), and
both the modes have been observed in the slice cultures of the cerebral cortex (Miyata et al., 2001; Nadarajah et al., 2001). We have proposed
that earlier-born neurons might use a Cdk5-independent nuclear-translocation mode, whereas the migration mode of late-born neurons is Cdk5-dependent in the cerebral cortex (Gilmore et al., 1998).
Recently, developmental defects of brainstem structures, including the
lack of the inferior olive (IO), have been reported in Cdk5/ and
p35/p39/ mice (Ko et al., 2001). However, detailed characterization of neuronal migration defects in these abnormalities remains to be elucidated. To characterize Cdk5-dependent and
-independent modes of neuronal migrations, we have analyzed neuronal
migrations in detail in the hindbrain of Cdk5/ mice. Selective
defects in neuronal migration were identified in the facial nucleus and IO; however, the rest of brainstem nuclei, including the pontine nucleus, formed normally. Because neuronal migration defects in the
facial nucleus and IO have been reported previously in
reeler mice, we have analyzed any correlation of Cdk5/p35
with Reelin signaling and its effect on neuronal migration using
double-mutant mice for p35 and Dab1.
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MATERIALS AND METHODS |
Animals. Cdk5 and p35 mutant mice were generated as
described previously (Ohshima et al., 1996, 2001) and maintained in
C57BL/6J and in 129/Sv × C57BL/6J backgrounds, respectively.
Yotari mutants were maintained in C57BL/6 × 129/SvJ
hybrid background (Yoneshima et al., 1997). Double-mutant mice were
obtained after crossing each mouse line, and genotypes for Cdk5, p35,
and Dab1 alleles were determined by PCR as described previously
(Ohshima et al., 2001). p35-Cdk5 × Cdk5 null (Cdk5TgKO) mice were
generated as described previously (Tanaka et al., 2001). All
mice were housed in the standard mouse facility and were fed an
autoclaved diet and water.
Immunohistochemical study and in situ
hybridization. Anesthetized mice were perfused
transcardially with 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4. Ten micrometer cryostat sections were stained
with 0.9% toluidine blue solution for Nissl stain. For
immunohistochemistry, antibodies were diluted in PBS/0.01% Triton
X-100 and 5% bovine serum albumin. Primary antibodies were polyclonal
anti-p75 NGF receptor (Chemicon, Temecula, CA; diluted 1:200),
polyclonal anti-neurofilament 150 kDa (Chemicon; 1:400), polyclonal
anti-choline acetyltransferase (ChAT) (Chemicon; 1:2000), 2H3 (obtained
from the Developmental Studies Hybridoma Bank, University of Iowa, Iowa
City, IA; monoclonal supernatant; 1:200), polyclonal anti-peripherin
(Chemicon; 1:400), and anti-Dab1 (gift from Dr. J. Cooper, Seattle,
WA; 1:400). For fluorescent staining, fluorescein isothiocyanate-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG (Chemicon) were used at a 1:400 dilution. For paraffin sections, the heads were removed from embryos and immediately fixed by immersion in a fixative consisting of 95% ethanol and 5% acetic acid. Tissue blocks were embedded in paraffin after dehydration using absolute ethanol and xylene and sequentially sectioned at 10 µm thickness on a
rotary microtome. After mounting on glass slides, the sections were
air-dried on a hot plate at 44°C and then stained with 0.5% cresyl
violet for Nissl staining. Whole-mount immunohistochemistry with 2H3
antibody was performed as described previously (Taniguchi et al.,
1997). In situ hybridizations were performed using
digoxigenin-labeled probe as described previously (Ohshima et al.,
2001), and the following probes were used: reelin (D'Arcangelo et al.,
1995), Dab1 (Howell et al., 1997), peripherin (Escurat et al., 1990), cadherin-8 (Korematsu and Redies, 1997), Phox2b (Pattyn et al., 2000),
ER81 (Arber et al., 2000), and Brn3b (Wyatt et al., 1998).
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate tracing: retrograde labeling of IO. After intracardiac perfusion with 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4, a small occipital craniotomy was performed to
expose the cerebellum of embryonic day 18.5 (E18.5) Cdk5+/+ and
Cdk5/ embryos. Several small crystals of
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI) were inserted medially and laterally into the cerebellar tissue
of the right hemicerebellum to label most of the IO neurons. The brains
along with their skulls were kept in the same fixative for 4-6 weeks
in the dark at 37°C. The brains were then dissected from their
skulls, embedded in 2% agarose, and cut into sections at a thickness
of 100 µm with a vibratome. The sections were photographed with a
rhodamine filter.
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RESULTS |
Selective migration defects in the facial branchiomotor and IO
neurons of Cdk5/ mice
We first compared the overall structure of the E18.5 hindbrains of
Cdk5+/+ mice with that of the Cdk5/ mice using Nissl-stained coronal and sagittal sections (Fig.
1A-L). This comparison
revealed that several neuronal nuclei in Cdk5/ mice, the IVth, Vth,
Xth and XIIth cranial motor nerve nuclei, were comparable with those in
the Cdk5+/+ mice. Among the derivatives of the rhombic lip, pontine
nuclei of Cdk5/ mice formed normally compared with the Cdk5+/+ mice
(Fig. 1K,L); however, the typical
structure of IO was found to be absent in Cdk5/ mice (Fig.
1F,J). We also found a specific loss of facial
branchiomotor (FBM) nucleus (Fig. 1B,H) and an
appearance of an ectopic mass consisting of relatively large cells in
the dorsal pons in the Cdk5/ hindbrain (Fig. 1D,L,M). Immunostaining of this ectopic mass
with neurofilament antibodies confirmed that it consisted of
postmitotic neurons (Fig. 1N,O). This ectopic
neuronal mass was located at the site at which FBM neurons arise;
therefore, we assume that this structure is caused by the arrested
migration of FBM neurons. To further confirm that the ectopic neurons
are indeed FBM neurons, we used available antibodies and RNA probes to
detect the markers that are expressed either in the FBM neurons or in
cranial nuclei. Both FBM neurons in the wild-type mice and ectopic
neurons in the Cdk5/ mice stained positive for p75 nerve growth
factor receptor and ChAT (Fig.
2A-F). For
in situ hybridization, we used the following probes as
markers for FBM neurons: peripherin (Escurat et al., 1990), Phox2b
(Pattyn et al., 2000), and cadherin-8 (Korematsu and Redies, 1997).
In situ hybridization studies with these probes revealed
that ectopic neurons were positive for Phox2b and cadherin-8 as well as
peripherin as early as E14.5, when migration defects of FBM neurons
became detectable in Cdk5/ mice (Fig.
3A-L). The normal appearance
of the VIIth ganglia in Cdk5/ mice was confirmed by whole-mount
immunostaining with the 2H3 antibody at E11.5 (data not shown). At
E12.5, using islet-1 immunostaining, we found that comparative numbers
of FBM neurons were generated in Cdk5/ mice (data not shown). These
data strongly indicated that the ectopic neuronal mass consists of the
migration-defective FBM neurons. Although neuronal cell bodies of FBM
neurons in Cdk5/ mice were ectopically located, their axons (facial
nerve) extended normally and exited at the sites identical to those of
the wild-type mice (Fig. 2G-I). Thus, despite their
defective migration, the specification and axonal trajectory of FBM
neurons are not disturbed by Cdk5 deficiency. To study the correlation
between the defective migration of FBM neurons and Cdk5-kinase
deficiency, we subsequently analyzed the localization of FBM neurons in
p35/ and p35/Cdk5+/ mice at E18.5. We previously reported a
10% residual activity of Cdk5 kinase in p35/ hindbrain at
postnatal day 5 (P5) (Ohshima et al., 2001). Additional reduction of
this residual activity is expected in p35/Cdk5+/ mice. In
p35/ mice, the location of FBM neurons is comparable with that in
p35+/+ mice. However, defective migration of FBM neurons, which
appeared as elongated shapes of VIIth nuclei, was apparent in
p35/Cdk5+/ mice at E18.5 (Fig. 3P,Q), as well as in
the adult p35/Cdk5+/ mice (data not shown). These results
indicate that migration of FBM neurons is also dependent on Cdk5-kinase
activity.
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Figure 1.
Selective loss of the VIIth motor nucleus and IO
in Cdk5/ mice. Nissl-stained coronal (A-J,
M) and sagittal (K, L) sections from
Cdk5+/+ (A, C, E, G, I, K) and Cdk5/
(B, D, F, H, J, M) mice at E18.5. Genotypes are
indicated as wild type (WT) (Cdk5+/+) and
knock-out (KO) (Cdk5/). A-J and
M are paraffin sections stained with cresyl violet.
K and L are frozen sections stained with
toluidine blue. G-J and M are higher
magnifications of a specific part of the sections in A, B, E,
F, and D, respectively; the magnified area in
each section is indicated by a letter on the
bottom right corner in A, B, and
D-F. A loss of typical structures of the facial
nerve nucleus (B, H) and IO (F,
J) is observed in the null mice. As seen in the sagittal
sections, the appearance and position of the pontine nucleus
(arrowheads) are similar in Cdk5+/+ and Cdk5/ mice
(K, L). In Cdk5/ hindbrain, an abnormal cell mass is
seen in L and M. This ectopic mass
(M and red arrow in
L) consists of postmitotic neurons positively stained
with the anti-neurofilament antibodies NF-150 (N)
and 2H3 (O). Black arrows in
K and L indicate the external granule
cell layer of the cerebellum. The red arrow in
K indicates the location of VIIth nucleus in Cdk5+/+
mice. Arrowheads in K and L
indicate pontine nucleus. A-F, G-J, and
M-O are at the same magnifications, respectively. Scale
bars: G, N, 120 µm; L, 700 µm.
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Figure 2.
Immunohistochemical
characterization of a neuronal mass of facial branchiomotor neurons.
Serial coronal sections from Cdk5+/+ (A-C) and
Cdk5/ (D-F) mice at E18.5 were stained with
anti-p75 (A, D), anti-NF150 (B, E), and
anti-ChAT (C, F) antibodies. Both the facial
nucleus in Cdk5+/+ mice and the neuronal mass in Cdk5/ mice are
positive for these antibodies. G-I, Coronal sections
from Cdk5+/+ (G, H) and Cdk5/
(I) mice at E14.5 stained with
anti-peripherin antibody. The facial nerves (fn),
axonal bundles of FBM neurons, are formed early after the neurons are
generated and extend dorsally toward the exit point at the r4 level.
The facial nerve bends in parallel to the migration of the cell
bodies, forming the "genu" of the facial nerve. Thus, the facial
nerve exits at the rostral level (G) from the
facial nucleus (VII in H) in Cdk5+/+ mice. In
Cdk5/ mice, the facial nerve extends from the neuronal mass (VII)
and exits at the same level (I). These
results indicate that the neuronal mass in Cdk5/ mice consists of
migratory-arrested FBM neurons at the site at which they were born.
Scale bars: F, 160 µm; I, 400 µm.
Vg, Vth ganglia; VII, VIIth ganglia;
VII+VIIIg, VIIth and VIIIth ganglia; WT,
wild type; KO, knock-out.
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Figure 3.
In situ hybridization studies
for the Cdk5 mutant hindbrain. Sections were probed with either
peripherin (A-D, G-J) or Phox2b (E, F,
K, L); these sections were from either Cdk5+/+ (A, C, E,
G, I, K) or Cdk5/ (B, D, F, H, J, L)
mouse embryos of age E18.5 (A, B), E14.5
(C-F), and E16.5 (G-L).
The sections shown are either coronal (A-F) or
sagittal (G-L) sections. The ectopic neuronal
mass was identified as VIIth motor nuclei because it stained positive
for Phox2b as well as peripherin (arrows in
B and H). I and
J are higher magnifications of G and
H, as indicated with arrows,
respectively. The arrow in A indicates the VIIth
motor nucleus in Cdk5+/+ mice. E, F, and K,
L are serial sections of C, D and I,
J, respectively, hybridized with Phox2b probe. In Cdk5/
mice (N, O) at E18.5, the motor nuclei of the Vth
(arrows in O), the Xth
(arrowheads in M and
N), and the XIIth (arrow in
M and N) nuclei are comparable with
Cdk5+/+ mice (M), as shown by the
expression pattern of peripherin. However, segregation of the Xth
(arrowheads) and XIIth (arrow) nuclei was
less clear in Cdk5/ mice (N). Although the
VIIth nucleus (arrows) appeared normal in p35/ mice
(P), an elongated shape of the VIIth nucleus
(arrows) was observed in p35/Cdk5+/ mice
(Q) at E18.5, as visualized by peripherin
expression. The arrowhead in P indicates motor
nuclei of the Vth nerve in p35/ mice. Scale bars: B,
400 µm; C, 80 µm; N, 100 µm.
WT, Wild type; KO,
knock-out.
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We subsequently attempted to identify the IO neurons in Cdk5/ mice
by two methods: (1) in situ hybridization with the
IO-specific riboprobes ER81 (Chotteau-Lelievre et al., 1997; Arber et
al., 2000) and Brn3b (Wyatt et al., 1998) and (2) a retrograde labeling of IO by DiI placement in the cerebellum. Neurons destined for IO are
generated in the rhombic lip at E11 and migrate through the submarginal
(intraparenchymal) stream. The IO appears at E14 as a cell crescent
located at the ventral and medial aspects of the rhombencephalon (Fig.
4A), and then a
progressive modeling leads to its typical foliated shape at E16 in the
wild type (Fig. 4C). By in situ hybridization
with ER81 (Fig. 4A-F) and Brn3b (data not
shown) probes, we found that IO neurons in Cdk5/ mice were
diffusely distributed without forming a typical crescent shape in the
medulla at E14.5 (Fig. 4B). This diffuse pattern of
IO neurons observed at E14.5 in the mutant mice persisted through E18.5, whereas typical foliated subdomains were identified in the
wild-type mice (Fig. 4C,D). The contralateral IO neurons
were labeled by placing DiI in the cerebellum in the wild-type mice (Fig. 4G). In the Cdk5/ mice, DiI-positive neurons were
diffused without segregating into a typical foliated shape at the
contralateral side, as revealed by in situ
hybridization (Fig. 4G,H). Thus, the lack of typical
structure of the IO complex in Cdk5/ mice is apparently a result of
the positioning disturbance of IO neurons through intramural streams
from the lower rhombic lip. Interestingly, dislocated IO neurons were
found at the parenchyma of the medulla. A comparison of both types of
labeling revealed that more neurons stained positive for the IO marker
than the DiI-labeled ones, indicating that only a subpopulation of IO
neurons have correct axonal projection to the cerebellum. The
extramural migratory stream, which forms the lateral reticular nucleus
and external cuneate nucleus, was found to be comparable in appearance
in Cdk5/ and Cdk5+/+ mice at E14.5 (Fig. 4I,J;
also see higher magnifications in the insets of I
and J).
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Figure 4.
IO neurons are positioned
abnormally in Cdk5/ mice. Using the IO neuronal marker ER81
(A-F) and retrograde labeling with DiI
(G, H), IO neurons were identified in Cdk5/
mice (B, D, F, H). These neurons distribute
diffusely in the parenchyma of the medulla at E14.5
(B) and at E18.5 (D, F, H),
whereas they form a typical structure in the wild-type neurons at E14.5
(A) and E18.5 (C, E, G).
Arrows in E and F indicate
ER81-positive neurons in the sagittal sections of Cdk5+/+
(E) and Cdk5/ (F) mice
at E18.5. Crossing fibers of the olivary commissure are clearly seen
(arrows in G, H;
insets, higher magnifications). In Cdk5/ mice, the
number of crossing fibers is decreased, but commissure fibers run
correctly beyond the midline
(H). The posterior extramural
stream in Cdk5/ mice (J) is comparable with
that in Cdk5+/+ mice (I) at E14.5, as
shown in the Nissl-stained sagittal sections
(arrows in I and J;
insets, higher magnifications). Scale bars:
A, 400 µm; C, F, 800µm;
inset in I, 160 µm. WT,
Wild-type; KO, knock-out.
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Synergistic contribution of Reelin signaling with Cdk5/p35 in
the migration of FBM and IO neurons
Because the specific defects in the migration of FBM and IO
neurons seen in Cdk5/ mice have also been reported in
reeler mice (Goffinet, 1983, 1984), we have analyzed reelin
and Dab1 expression in Cdk5 mutant mice. We first analyzed the
expression pattern of Reelin and Dab1 in the Cdk5+/+ and Cdk5/ mice
by in situ hybridization at E14.5 and by
immunohistochemistry at E18.5. Dab1 expression was clearly seen in the
FBM neurons by in situ hybridization (Fig.
5B,E), whereas Reelin
expression was observed in the surrounding areas of FBM neurons in the
wild-type mice (Fig. 5A,D). Expression of Dab1 transcripts
and protein was clearly seen in the migration-defective FBM neurons of
the Cdk5/ mice (Fig. 5H,K,O).
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Figure 5.
Reelin signaling in the FBM neuronal migration.
A-L, In situ hybridization study in
Cdk5+/+ (A-F) and Cdk5/
(G-L) mice at E14.5. D-F and
J-L are higher magnifications of A-C
and G-I at indicated areas
(arrows) in each panel,
respectively. The FBM neurons express Dab1 mRNA (B, E, H,
K), and Reelin mRNA is expressed in the surrounding area
at the migratory termination of FBM neurons in the wild-type mice
(A, D). Cadherin-8 (Cad8) expression
confirms the identification as FBM neurons in the wild-type (C,
F) and Cdk5/ mice (I, L).
Migration-arrested FBM neurons also express Dab1
(O) as well as peripherin
(M) and neurofilament
(N), as shown by the immunostaining of the
coronal serial sections of E18.5 Cdk5/ brain with anti-Dab1,
anti-peripherin, and anti-NF-150 antibodies, respectively. Scale bars:
A, 300 µm; D, 80 µm; M, 400 µm. WT, Wild type; KO, knock-out.
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To study the combined effects of the inactivation of p35 and Dab1, we
also studied the migration patterns of FBM and IO neurons in Dab1
mutant yotari mice (Yoneshima et al., 1997) and
p35/Dab1yot/yot mice (Ohshima et al.,
2001). In yotari (Dab1yot/yot)
mice, the elongated shapes of the VIIth nuclei were clearly observed at
E18.5 (Fig. 6A,C,G).
Interestingly, additional defects in the migration patterns of FBM
neurons were observed in
p35/Dab1yot/yot mice at E18.5 (Fig.
6B,D,H) as well as at postnatal day 10 (data not shown). Because an abnormal shape of subdivisions of the IO complex
has been reported previously in reeler mice (Goffinet, 1983), we subsequently analyzed the IO complex in these mutant mice
(Fig. 6I-K). Although we found a similar
abnormality in yotari mice (data not shown), interestingly,
in p35/Dab1yot/yot mice, the dorsal
accessory olive was found to be shifted laterally compared with those
seen in Dab1yot/yot or p35/ mice (Fig.
6M, arrows). These data indicate the
synergistic contributions of Cdk5/p35 and Reelin signaling in the
positioning of FBM and IO neurons as well as cortical neurons (Ohshima
et al., 2001).
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Figure 6.
Abnormal FBM neuronal migration in
Dab1yot/yot and
p35/Dab1yot/yot mice. Disturbance of radial
migration of FBM neurons in yotari,
Dab1yot/yot, mice is shown in Nissl-stained
(A) and peripherin-stained
(C) coronal serial sections at E18.5.
Deterioration of FBM neuronal migration is advanced in
p35/Dab1yot/yot mice as seen in Nissl-stained
(B) and peripherin-stained
(D) coronal sections at E18.5. These
abnormalities are confirmed by in situ hybridization
with a peripherin probe in the sagittal sections from p35+/+
(E), p35/ (F),
Dab1yot/yot (G), and
p35/Dab1yot/yot (H)
mice at P0. Dashed lines indicate the margin of
sections. I-K, Serial sections in coronal sections are
either Nissl-stained (I) or analyzed by
in situ hybridization with peripherin probe
(J) and cadherin-8 (Cad8) probe
(K). Abnormally located FBM neurons in
p35/Dab1yot/yot mice are identified by their
cadherin-8 mRNA expression (K). L,
M, Migration abnormality of the IO neurons is also detected in
rostral (L) and caudal IO
(M) sections of
p35/Dab1yot/yot mice with Nissl stain at P0.
Arrows in L indicate ectopically located
FBM neurons; arrows in M indicate the
dorsal accessory olive, which is located laterally in
p35/Dab1yot/yot mice. Scale bars:
A, 300 µm; E, 240 µm; I, 60 µm. V, Vth nucleus; VII, Vth
nucleus.
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Radial fiber morphology as determined by immunostaining for nestin was
not found to be altered in the Cdk5/ hindbrain (data not shown).
Therefore, it is unlikely that defective migration of FBM and IO
neurons is caused by a defect in the radial fibers. We also confirmed
that these migration defects observed in Cdk5/ mice occur primarily
because of neuronal Cdk5 deficiency in a double-transgenic mouse model
(Cdk5TgKO) in which expression of neuron-specific Cdk5-transgene
"rescued" the Cdk5-null phenotype (Tanaka et al., 2001). We
examined the location of FBM and IO neurons in Cdk5TgKO mice and
wild-type mice. The locations of both FBM and IO neurons were found to
be normal in the adult Cdk5TgKO brain (Fig.
7). Although Cdk5 is expressed in both
neurons and glial cells, a complete rescue of the migration defects of
FBM and IO neurons in Cdk5TgKO also indicates that neuronal migration defects in the hindbrain of Cdk5/ mice are cell autonomous, as
shown previously in Cdk5+/+ × Cdk5/ chimeric mice (Ohshima et al.,
1999).
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Figure 7.
Abnormalities of FBM and IO neurons are rescued by
neuron-specific expression of Cdk5 transgene. No abnormality is found
in the VIIth motor nucleus and IO complex in Nissl-stained coronal
sections from adult Cdk5TgKO mice (B, D) compared with
age-matched wild-type mice (A, C). A-D
are at the same magnification. Scale bar, 1 mm.
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DISCUSSION |
Our previous studies revealed that abnormal neuronal migration in
Cdk5/ mice was associated with defective layer structures in the
cerebrum and cerebellum of these mice (Ohshima et al., 1996; Gilmore et
al., 1998). In the present study, we have performed an
additional detailed analysis of neuronal positioning in the hindbrain
of Cdk5/ mice and identified novel migration defects in FBM and IO
neurons. Despite these defects in specific neurons, the neurons in
other nuclei migrate normally in the hindbrain, indicating that the
migration in a selected neuronal population is Cdk5-dependent.
Interestingly, defective neuronal migration in Cdk5/ hindbrain
overlaps with the abnormal neuronal migration seen in the hindbrain of
the mice with defective Reelin signaling. Most importantly, our study
demonstrates a synergistic role of Cdk5/p35 and Reelin signaling in the
migration of a specific neuronal population of the hindbrain.
Many cell populations migrate over long distances and follow complex
trajectories from their site of origin to their final destination
during the formation of the vertebrate CNS. There are two general modes
of neuronal migration in the CNS: nuclear translocation and locomotion
(Book and Morest, 1990; Nadarajah et al., 2001). In nuclear (or somal)
translocation, the neuron first extends a leading process in the
direction of migration, then moves the nucleus (or soma) through this
process to its destination. This type of migration has been described
in pontine neurons (Yee et al., 1999), in the cerebellum (Hager et al.,
1995), and in many other brain areas (Morest, 1970; Gray and Sanes,
1991; Snow and Robson, 1995). Conversely, in the locomotion type of
neuronal migration, movement of the entire neuron is involved,
including its leading and tailing processes. The radial movement of
neurons along the radial fiber is a well defined example of
locomotion migration in the cerebral cortex (Rakic, 1972;
O'Rourke et al., 1992). The locomotion type of migration of late-born
neurons in the cerebral cortex is believed to be Cdk5-dependent because
of the defective migration observed in Cdk5/ and p35/ mice
(Gilmore et al., 1998; Kwon and Tsai, 1998).
Among the derivatives from the rhombic lip, migrating granule
precursors and pontine neurons exhibit characteristic unipolar morphologies, and a single leading process appears to guide their migration (Ono and Kawamura, 1990; Wingate and Hatten, 1999; Yee et
al., 1999), indicating that this type of migration is of a nuclear
translocation mode. In the Cdk5/ mice, the external granule cell
layer of the cerebellum (Ohshima et al., 1999) and the pontine nucleus
form normally (Fig. 1L), indicating that this type of
migration of granule precursors and pontine neurons is Cdk5-independent. IO neurons are also derivatives of the rhombic lip,
but their migration is quite different from that of pontine neurons and
other neurons of precerebellar nuclei, which migrate through the
anterior extramural stream and posterior extramural stream (Altman and
Bayer, 1987, 1997). IO neurons use a distinct migratory pathway called
the intramural migration (submarginal) stream (Altman and Bayer, 1997).
In Netrin-1-deficient mice, the number of IO neurons is remarkably
decreased, and some of them are located ectopically along the
intramural migration stream (Bloch-Gallego et al., 1999).
Because these ectopic IO neurons lack retrograde DiI labeling from the
cerebellum, these neurons are believed to be unable to reach their
axons to their targets in the cerebellum (Bloch-Gallego et al., 1999).
In Cdk5/ mice, ectopically located IO neurons are not positioned
along their migration stream. They do not make clusters but rather are
distributed diffusely in the parenchyma of medulla. Despite their
abnormal location, IO neurons in Cdk5/ mice seem to have made
correct projections to the cerebellum, as shown in our DiI study,
indicating that the climbing fiber is apparently established
independently of the ectopic nature of IO neuron position.
The FBM neurons are known to take a complex migration pattern (Auclair
et al., 1996; Garel et al., 2000). They are born in the basal plate of
rhombomere 4 (r4) between E9 and E14 and migrate caudally through r5
and then dorsally and radially in r6. The migration of the cell bodies
of FBM neurons is completed by E14. In Cdk5/ mice, FBM neurons are
settled in the location where they are born; therefore, initial
tangential migration along the ventral midline is impaired. Despite the
defective migration of FBM neurons, the facial nerves formed comparably
to those in wild-type littermates and exited correctly at the r4 level.
Thus, axonal path-findings of FBM neurons might not be disturbed in
Cdk5/ mice. ChAT staining also indicated that differentiation of
ectopic FBM neurons to cholinergic neurons occurs normally in Cdk5/ mice.
Normal migration of FBM neurons in p35/ mice is apparently a result
of redundancy of the Cdk5 activators p35 and p39, particularly because
of their overlapping expression in the developing brainstem (Ohshima et
al., 2001). However, p35/Cdk5+/ mice display misplaced FBM
neurons because of their disturbed radial migration within r6 along the
radial fibers in the medulla. This prompts us to believe that the
radial migration of FBM neurons is of the locomotive type, a radial
fiber-guided migration, and that it may be Cdk5-dependent. Interestingly, Reelin signaling is also involved in this radial migration of FBM neurons, because these neurons express Dab1 (Carroll et al., 2001), and Reelin expression is seen in the area surrounding the facial nucleus at the termination of the migration of these neurons. Furthermore, retarded radial migration of FBM neurons and
abnormalities in the architectonic organization of the facial nucleus
were described in reeler mice (Goffinet, 1984; Terashima et
al., 1993), and our present findings indicating profound disturbances in the last phase of radial migration of FBM neurons in Dab1 mutant yotari mice confirm the important role of Reelin signaling
in the migration of FBM neurons.
In the compound mutant mice of p35 and Dab1 or Reelin, an enhanced
deterioration was observed in the migration defects of the Purkinje
cells in the cerebellum as well as in the pyramidal neurons in the
hippocampus, indicating synergistic effects of inactivation of Cdk5/p35
and Reelin signaling on the migration of these cortical neurons
(Ohshima et al., 2001). In the present study, we have observed similar
effects in the radial migration of FBM neurons. In
p35/Dab1yot/yot mice, radial migration
of FBM neurons is much more defective than in either the p35/ or
Dab1 mutant. Most notably, p35/ mice have no obvious abnormality in
the location of the facial nucleus, whereas the compound mutants do
have clear ectopia. This strengthens our belief that Reelin signaling
plays a crucial role in the radial migration of FBM neurons. We have
not found any abnormality in the radial fiber morphology in Cdk5/
mice; moreover, neuron-specific expression of the Cdk5 transgene
completely rescues the defective migration of FBM and IO neurons in
Cdk5/ mice, indicating that these neuronal migration defects are
caused primarily by Cdk5 kinase deficiency and are cell-autonomous.
Our analysis of neuronal migration in the hindbrain of Cdk5/ mice
revealed that the migration of selective populations of neurons is
Cdk5-dependent. The impact of Cdk5 deficiency on the neuronal
positioning in these neurons is quite distinct. The migration of FBM
neurons was completely arrested at the site where they were born,
whereas IO neurons migrate close to their final destinations but fail
to form the cellular alignment into distinct structures of IO. These
observations prompt us to group the defects of neuronal positioning
caused by Cdk5 deficiency into three different categories. The first
category contains the FBM neurons and Purkinje cells whose migrations
are completely arrested; these neurons are located at the site where
they were born. In the second category, the defects disturb the
cellular alignment to form a distinct and novel layer or structure in
the brain. This includes IO neurons and mitral cells in the olfactory
bulb (Ko et al., 2001; T. Ohshima, M. Ogawa, and K. Mikoshiba,
unpublished observations). These neurons distribute diffusely near
their final destination. The third category contains neurons with a
type of defect intermediate between these two patterns. Late-born
neurons of the cerebral cortex and pyramidal neurons of the
hippocampus can be placed into this group. Several studies indicate
that Cdk5/p35 kinase may regulate actin and/or microtubule dynamics
(Paudel et al., 1993; Nikolic et al., 1998; Niethammer et al., 2000;
Sasaki et al., 2000) and cell adhesion mediated by N-cadherin (Kwon et
al., 2000) during cortical development. Therefore, it is reasonable to
hypothesize that an imbalance in Cdk5-mediated regulation of the
actin/microtubule dynamics may cause migration defects similar to those
seen in the first category. Conversely, defective regulation of cell
adhesion may affect the cell-to-cell interactions important for the
cellular alignment needed to form a distinct layer and structure, such
as the IO and mitral cell layer of the olfactory bulb. At the same
time, it should be pointed out that some of the migration modes of
certain hindbrain neurons are Cdk5-independent. Thus, the precise
delineation of Cdk5-dependent migration and its relationship with
Reelin signaling will certainly provide valuable insights into the
molecular and cellular mechanisms of neuronal migration underlying the
precise positioning of the neurons in normal and abnormal brain development.
| |
FOOTNOTES |
Received Oct. 4, 2001; revised Feb. 5, 2002; accepted Feb. 20, 2002.
This work was partially supported by grants-in-aid from the Ministry of
Education, Science, and Culture of Japan to T.O. and by National
Institute of Dental and Craniofacial Research, National Institutes of
Health Grant Z01DE00694-010DIR to A.B.K. We thank Drs. B. W. Howell and J. A. Cooper for the gift of Dab1 antibody and Dab1
cDNA. The 2H3 and islet-1 antibodies were obtained from the
Developmental Studies Hybridoma Bank (University of Iowa, Iowa City,
IA). We thank Drs. T. Saito, M.-M. Portier, C. Redies, J.-F. Brunet,
T. M. Jessell, and D. S. Latchman for in situ
probes. We also thank Drs. Y. Sugitani and T. Terashima for technical advice and Dr. May Jo Danton for critical reading of this manuscript.
Correspondence should be addressed to Toshio Ohshima, Laboratory for
Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. E-mail:
ohshima{at}brain.riken.go.jp.
| |
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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.,
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[Abstract]
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S. Takahashi, T. Saito, S.-i. Hisanaga, H. C. Pant, and A. B. Kulkarni
Tau Phosphorylation by Cyclin-dependent Kinase 5/p39 during Brain Development Reduces Its Affinity for Microtubules
J. Biol. Chem.,
March 14, 2003;
278(12):
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION
