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First published online 16 May 2007
doi: 10.1242/dev.02854
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1 Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute,
Wako, Saitama 351-0198, Japan.
2 Functional Genomics Section, CDBRB, NIDCR, NIH, Bethesda, MD 20892, USA.
3 Department of Anatomy, Keio University School of Medicine, Shinjuku-ku, Tokyo
160-8582, Japan.
4 Laboratory for Cell Culture Development, RIKEN Brain Science Institute, Wako,
Saitama 351-0198, Japan.
5 Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Wako,
Saitama 351-0198, Japan.
6 Hashimoto Research Unit, RIKEN Brain Science Institute, Wako, Saitama
351-0198, Japan.
7 Department of Molecular Neurobiology, Institute of DNA Medicine, Jikei
University School of Medicine, Tokyo 105-8461, Japan.
8 Department of Molecular Neurobiology, Institute of Medical Science, University
of Tokyo, Minato-ku, Tokyo, Japan.
* Author for correspondence (e-mail: ohshima{at}brain.riken.go.jp)
Accepted 21 March 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neuronal migration, Cerebral cortex, Mouse, Cdk5, Map2 (Mtap2)
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This unique layering is achieved via coordinated radial
migration of neurons from the ventricular zone (VZ) to their final
destination. Proper positioning of neurons is particularly crucial for the
formation of laminated cortical structures, such as the cerebral cortex,
hippocampus and cerebellum. Recently, time-lapse imaging of migrating cerebral
cortex neurons identified specific phases in radial neuronal migration
(Nadarajah et al., 2001;
Tabata and Nakajima, 2003;
Noctor et al., 2004). After
the final division, immature neurons transiently become multipolar, with
multiple neurites within the subventricular zone (SVZ) and lower intermediate
zone (IZ), which is also known as the premigratory zone. Then, neurons change
their shape from multipolar to bipolar just before migrating from the
premigratory zone to the cortical plate (CP). The molecular and cellular
mechanisms that regulate this transition remain to be elucidated.
Cdk5 is a unique serine/threonine kinase with close homology to other Cdks
but its kinase activity is mainly detected in postmitotic neurons
(Dhavan and Tsai, 2001).
Association of Cdk5 with a neuron-specific regulatory subunit, either p35
(CdK5r1 - Mouse Genome Informatics) or its isoform p39, is crucial for kinase
activity. Previous studies by us and other groups revealed that Cdk5 and its
activating subunit p35 have crucial roles in the formation of cortical
structures of the developing mouse brain, and this is brought about mainly by
the regulation of neuronal migration
(Ohshima et al., 1996;
Gilmore et al., 1998;
Chae et al., 1997;
Ohshima et al., 2001).
Cdk5-/- mice exhibit substantial migratory deficits
throughout the brain (Ohshima et al.,
1996; Gilmore et al.,
1998). In the cerebral cortex, neocortical
Cdk5-/- neurons destined for layers II to V stall below
the subplate (Gilmore et al.,
1998). A birth-date labelling study indicated that there are
migration defects in Cdk5-/- neurons
(Gilmore et al., 1998), but
these abnormalities need to be elucidated in detail.
Here we report our analysis of the migratory behaviour of
Cdk5-/- neurons in the developing cerebral cortex. We
found that Cdk5-/- neurons have severe impairment of the
multipolar-to-bipolar transition during their radial migration.
Cdk5-/- mice die in the perinatal period before cerebral
cortex layer formation is completed
(Ohshima et al., 1996). To
overcome this early lethality, we generated cortex-specific Cdk5-deficient
mice by mating Emx1Cre mice
(Iwasato et al., 2000) and
fCdk5/fCdk5 mice (Hirasawa et
al., 2004). Using this mutant mouse line, we demonstrate that the
inability of cortical neurons to transition from the multipolar phase of
radial migration results in abnormal dendritic development and defective
axonal trajectories of pyramidal neurons in the postnatal cerebral cortex.
Further analysis of mutant mice indicated that Cdk5-dependent
multipolar-bipolar transition of migrating neurons is a priori for proper
corticogenesis in the developing mouse brain.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cdk5 loxP-flanked mice were generated as described
(Hirasawa et al., 2004). The
YFP-H mouse line (Feng et al.,
2000) was obtained from Jackson Laboratory, MA. The
Emx1Cre mouse line was generated by the knock-in strategy; Cre
recombinase activity was restricted to the Emx1 expression region
(Iwasato et al., 2000) and
detected as early as E10 as described
(Cappello et al., 2006).
Emx1Cre conditional Cdk5 KO mice
(fCdk5/-;Emx1Cre/+, CxCdk5KO) were analysed along
with littermate controls. Reeler mice (B6C3Fe-a/a-rl, Jackson
Laboratory) were mated with YFP-H mice to obtain reeler;YFP-H mutant
mice. Mice were fed ad libitum and provided with care as per RIKEN BSI
guidelines.
In utero electroporation and time-lapse imaging
Female Cdk5 heterozygotes were mated with Cdk5
heterozygote males as well as ICR males (Japan SLC, Shizuoka, Japan) and,
after becoming pregnant, were subjected to in utero electroporation as
described previously (Tabata and Nakajima,
2001; Tabata and Nakajima,
2003). T
1-EGFP was generated as described
(Tabata and Nakajima, 2001).
To generate CAG-EGFP and CAG-RFP, the cDNA of EGFP and DsRed2 from pEGFP-N1
and pDsRed2-N1 (Clontech, Palo Alto, CA) were inserted, respectively, into
pCAGGS, which was kindly provided by Dr J. Miyazaki
(Niwa et al., 1991).
T1-EGFP efficiently labelled migrating neurons, and CAG-EGFP labelled
migrating neurons as well as their progenitors (i.e. radial glial cells), as
reported previously (Tabata and Nakajima,
2001). In order to express both Cdk5-DN and EGFP in the same
cells, bi-expression vector CAG-GFP-CAG-Cdk5-DN (CGC-Cdk5-DN) was generated as
follows. To generate CAG-EGFP-CAG (CGC), a 2.3 kb fragment containing the CAG
promoter and the EcoRI site was amplified with pCAGGS by PCR, and
inserted into the HindIII site downstream of the EGFP gene in
CAG-EGFP. CAG-RFP-CAG was generated in the same way using CAG-RFP. Cdk5-DN
(N144) was then inserted into CAG-EGFP-CAG and CAG-RFP-CAG to generate
CGC-Cdk5-DN and CRC-Cdk5-DN, respectively. Time-lapse imaging of brain slices
was conducted as described previously
(Tabata and Nakajima, 2003).
Briefly, coronal brain slices (200 µm) from the anterior one-third of the
cerebral cortex were embedded in collagen gel on a permeable filter
(Millicell-CM, 0.4 µm pore size, Millipore). The filter was placed on a
glass-bottom dish with Neurobasal medium containing B27 (Invitrogen, San
Diego, CA) and was incubated at 37°C in 5% CO2 on a laser
microscope (FV300, Olympus). Ten to twenty optical z-sections at 5
µm intervals were captured every 30 minutes and all focal planes were
merged.
Dissociated neuronal culture and imaging analysis
E14.5 cortical neurons of Cdk5+/+ and
Cdk5-/- were cultured as described
(Hayashi et al., 2002) with
slight modification. Briefly, the cerebral cortex was excised, incubated with
0.25% trypsin in PBS for 10 minutes at 37°C, dissociated, and washed in
culture medium which consisted of DMEM/Ham's F-12 mixture (Sigma, St Louis,
MO) supplemented with N-2 supplement (Invitrogen), 5% fetal bovine serum, 5%
calf serum, 4 ng/µl EGE, 8 ng/µl basic FGF, penicillin and streptomycin.
The dissociated cells were cultured on fibronectin-coated dishes, on which an
astrocyte feeder was cultured outside, at 3x104
cells/cm2 in culture medium for 3 days. After washing with PBS,
cultured neurons were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate
buffer for 10 minutes at room temperature. After washing with PBS three times,
cells were incubated with blocking buffer (1% bovine serum albumin and 0.2%
Triton X-100 in PBS) at room temperature for 30 minutes. The cells were then
incubated with anti-ßIII-tubulin antibody (1:500 Tuj1, Covance, Denver,
PA) at room temperature for 1 hour followed by Alexa-Fluor 488 (Molecular
Probes, Eugene, OR). Cell images were acquired with a fluorescent microscope.
The percentage of pyramidal neurons among Tuj1-positive cells was calculated
in randomly selected fields. Pyramidal neurons were defined as cells that had
a pyramidal cell body with one major dendrite that gradually tapered off from
the cell body (Threadgill et al.,
1997). Data are shown as means±s.d.
Bromodeoxyuridine injection, immunohistochemical staining and in situ hybridisation
For bromodeoxyuridine (BrdU) experiments, pregnant dams were injected with
BrdU (100 µg/g, intraperitoneal) at the indicated age and analysed at P10.
Paraffin sections (8 µm) of fixed brain (4% PFA in 0.1 M phosphate buffer)
were cut and stained with monoclonal anti-BrdU antibody as described
(Gilmore et al., 1998). For
the immunohistochemical analysis, frozen sections (15 µm) were cut and
stained as described (Gilmore et al.,
1998; Ohshima et al.,
2001) using the following primary antibodies: polyclonal
antibodies for GFP (MBL, Tokyo, Japan) and Cdk5 (C-8, Santa Cruz, CA) and
monoclonal antibody for Map2 (Chemicon). In situ hybridisation was performed
as described (Ohshima et al.,
2002) in the coronal sections from CxCdk5KO and control mice at
P10 using Foxp2 (Ferland et al.,
2003), Er81 (Arber et
al., 2000) and Cux2
(Nieto et al., 2004) as
probes. For in situ hybridisation in the coronal sections from
Cdk5+/+ and Cdk5-/-, embryos at E16.5
were stained using Neurod1 and Dcx as probes. Mouse cDNAs
for Neurod1 and Dcx were the FANTOM3
(The FANTOM Consortium, 2005)
and IMAGE clones, respectively.
1,1'-dioctadecyl-3-3-3'-3'-tetramethylindiocarbocyanine perchlorate (DiI) tracing
DiI tracing was performed as described previously
(Ohshima et al., 2002).
Briefly, after intracardiac perfusion with 4% PFA in 0.1 M phosphate buffer
(pH 7.4), brains were removed from the skulls. Several small crystals of DiI
were inserted into the dorsal thalamus or cerebral cortex of
Cdk5+/+ and Cdk5-/- embryos at E18.5
or CxCdk5KO mice and their littermate controls at P10. The brains were kept in
the same fixative for 3-4 weeks in the dark at 37°C. The brains were
embedded in 2% agarose, cut into 100 µm sections with a vibratome and
photographed with a rhodamine filter.
Western blot analysis
Protein extraction and western blotting were carried out as described
(Ohshima et al., 2005) with
the following primary antibodies: polyclonal antibody for Cdk5 (C-8), Map2
(Chemicon), NF-M (NF-150, Chemicon) and actin (-actin, Sigma). Data are
shown as mean±s.d. and analysed by Student's t-test.
P<0.05 was considered statistically significant.
Adenovirus preparation, infection and time-lapse imaging
The adenoviral vector construction was carried out basically as previously
described (Hashimoto and Mikoshiba,
2004). The adenoviral vector Adex-CAG-Lyn-Venus expresses
membrane-targeted improved YFP called Venus
(Nagai et al., 2002) under
control of the CAG promoter. Lyn-Venus was generated by fusing the sequence of
the palmitoylation site of tyrosine kinase Lyn (amino acids 246-307) to the
N-terminus of Venus. The detailed feature of Adex-CAG-Lyn-Venus will be
described elsewhere (T.M., M.H. and M.O., unpublished). Pregnant mice were
deeply anesthetised with sodium pentobarbitone at 50 µg/g body weight.
Adenoviral vectors (2.0x106 pfu total) were injected into the
ventricle of the embryos at E12.5. The embryos were removed later (E14.5).
Preparation and culture of brain slices were carried out as described above. A
Leica ASMDW workstation was used for multidimensional live tissue imaging
(Leica, Heidelberg, Germany). The microscope setup consisted of an inverted
microscope (DMIRE2, Leica) and multipoint time-lapse stage controlled by ASMDW
software (Leica). The deconvolution Deblur software (Leica) was used for
deblurring the z-stacked images. Then, the deconvoluted images were
processed to AVI movies by ASMDW and Premiere software (Adobe, San Jose,
CA).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
first analysed the morphology of migrating later-born
Cdk5-/- neurons. To visualise the morphology of migrating
cortical neurons in the cerebral cortex, we introduced an enhanced GFP (EGFP)
by in utero electroporation (Tabata and
Nakajima, 2001; Tabata and
Nakajima, 2003) at embryonic day 14.5 (E14.5). Embryos were fixed
2 (E16.5), 3 (E17.5) or 4 days (E18.5) after in utero electroporation, and the
morphologies of GFP-labelled neurons examined by immunostaining with anti-GFP
antibody. At E16.5, most GFP-positive cells were localised in the premigratory
zone. GFP-labelled radial glial cells exhibited a bipolar morphology, and
their fibres extended toward the pia in the cerebral cortex of
Cdk5+/+ as well as Cdk5-/- embryos
(see Fig. S1 in the supplementary material). When we examined E17.5 embryos,
GFP-labelled neurons exhibited either a multipolar morphology at the
premigratory zone or a bipolar morphology at the upper IZ and CP in
Cdk5+/+ embryos (Fig.
1A), indicating multipolar-to-bipolar transition during radial
migration from the premigratory zone (SVZ and lower IZ) to the CP as
previously reported (Tabata and Nakajima,
2003; Noctor et al.,
2004). In the Cdk5+/+ brain, GFP-labelled
neurons extended their axons towards the IZ and then turned sharply towards
the midline (Fig. 1A). By
contrast, GFP-labelled neurons in the Cdk5-/- brain
migrated upward, keeping their multipolar morphology; bipolar neurons were not
observed in the premigratory zone or CP
(Fig. 1B). Even 4 days after
labelling (E18.5), no GFP-positive cells in a bipolar shape were detected in
the cerebral cortex of Cdk5-/- embryos (data not shown).
Axonal trajectories of GFP-labelled Cdk5-/- neurons ran
obliquely within the cortex (arrowhead in
Fig. 1B). These results
indicate a specific deficit of multipolar-to-bipolar transition during the
migration from the premigratory zone to the CP in Cdk5-/-
neurons.
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|
). To
examine the morphology and migratory behaviour of the neurons expressing
Cdk5-DN, we utilised a dual CAG promoter vector, CAG-RFP-CAG-Cdk5-DN
(CRC-Cdk5-DN), in which RFP and Cdk5-DN were transcribed via their own CAG
promoter within a single plasmid. When we introduced only the CRC-Cdk5-DN
plasmid (100% Cdk5-DN), the most RFP-positive neurons were positioned within
the premigratory zone and had the multipolar shape
(Fig. 2). Few neurons were
bipolar and the intensity of RFP in these neurons was relatively low
(Fig. 2), indicating a lower
level of Cdk5-DN expression. When we applied CRC-Cdk5-DN in different
concentrations by diluting with CAG-RFP, different degrees of migration
defects were observed (Fig. 2).
In the brains with a CAG-RFP dilution of 20% (1:4) or 50% (1:1), a delayed
migration of neurons expressing Cdk5-DN was observed in the CP, but the
majority of RFP-positive neurons transformed into the bipolar shape within the
upper IZ. However, careful observation at higher magnification revealed that
some RFP-positive neurons in the CP had branched leading processes
(Fig. 2A'b, arrows).
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).
Based on the morphology and behaviour of GFP-positive neurons at 4 and 24
hours after onset of observation, each neuron was classified as either
`multipolar', in which a neuron has extended multiple (more than three)
neurites, or `bipolar', in which a neuron has one or two neurites (also
identified as leading and tailing processes), as described
(Tabata and Nakajima, 2003;
Noctor et al., 2004). The
morphology and behaviour of GFP-positive neurons were already different at 4
hours between brain slices with CAG-EGFP and those with CGC-Cdk5-DN. These
differences were enhanced at 24 hours (Fig.
3A,B; see Movie 1 in the supplementary material). Within 20 hours,
40.15% (s.d. 14.25) of multipolar GFP-positive neurons became bipolar in the
brain slices with CAG-EGFP, whereas only 7.75% (s.d. 3.77) of multipolar
neurons became bipolar in those with CGC-Cdk5-DN
(Fig. 3C). These results
clearly indicate that neurons specifically require Cdk5 for
multipolar-to-bipolar transition in the premigratory zone.
Time-lapse imaging of migratory behaviour of Cdk5-/- neurons
We carried out time-lapse recordings of migratory behaviour in
Cdk5-/- brain slices from different stages of
corticogenesis. Cdk5-/- brain slices were particularly
sensitive to mechanical stress in the advanced stages of corticogenesis.
Therefore, we focused on the analysis of migratory behaviours of
Cdk5-/- neurons at an early stage. We introduced
CAG-Lyn-Venus at E12.5 using the adenoviral infection system and recorded
migratory behaviour for 48 hours from 2 days after adenoviral infection. On
this schedule, adenoviral infection occurred at the dorsal telencephalon and
labelled mostly layer V neurons as described previously
(Hashimoto and Mikoshiba,
2004). In the control slices, we observed both multipolar and
bipolar shapes in the premigratory zones and in the CP, which just starts to
form at this stage. However, in Cdk5-/- slices, a higher
percentage of GFP-positive cells remained in multipolar shapes at 48 hours
(Fig. 4; see Movie 2 in the
supplementary material). Time-lapse imaging of early-stage
Cdk5-/- embryos revealed impaired transition of neurons
from multipolar to bipolar shapes even at the early stage of neuronal
migration.
|
) defined a pyramidal neuron as a neuron with a single
dendrite that gradually tapers off from the cell body; with this definition,
they found that about 45% of cultured cortical neurons from rat cerebral
cortex exhibit the morphology of pyramidal neurons at 3 DIV. Using the same
definition, we conducted dissociated cultures of cortical cells isolated from
the cerebral cortex of E14.5 brains and observed no difference in the
percentage of pyramidal neurons generated from Cdk5-/-
embryos (45.8%, s.d. 5.7) compared with those from Cdk5+/+
embryos (46.2%, s.d. 5.2) at 3 DIV (see Fig. S2 in the supplementary
material). These results indicate that defective morphology is a unique
phenomenon observed in migrating Cdk5-/- neurons in
vivo.
Neuronal differentiation of Cdk5-/- neurons in vivo
A recent study suggested the coupling of neuronal migration with
neurogenesis and neuronal differentiation is induced by proneural bHLH genes
that upregulate the expression of doublecortin (Dcx) and p35
mRNAs in migrating neurons (Ge et al.,
2006). Because Cdk5 is involved in neuronal differentiation
(Dahvan and Tsai, 2001), it is possible that the defects in radial migration
and cellular polarities of cerebral cortical neurons described here are
secondary consequences of defective neuronal differentiation. To evaluate
neuronal differentiation in migrating Cdk5-/- neurons, we
performed in situ hybridisation for the expression of Neurod1, Dcx
and p35 mRNAs in Cdk5+/+ and
Cdk5-/- embryos at E16.5. Neurod1 and
Dcx mRNA expression levels in Cdk5-/- neurons in
the SVZ and IZ were comparable to those in Cdk5+/+ neurons
(Fig. 5). Therefore, initial
differentiation of migrating Cdk5-/- neurons occurs
normally, and migration defects in Cdk5-deficient cortical neurons are not
caused by defective neuronal differentiation.
Cdk5-deficient cortex in the postnatal stage
Loss of Cdk5 in the developing cerebral cortex of cortex-specific Cdk5 conditional knockout mice
We found that multipolar-to-bipolar transition of cortical neurons in
Cdk5-/- mice was impaired and this might affect the
morphology of cortical pyramidal neurons in the postnatal cerebral cortex.
However, we cannot examine the postnatal morphology of pyramidal neurons in
Cdk5-/- mice because they die perinatally
(Ohshima et al., 1996). In
order to investigate the Cdk5 loss-of-function phenotype in the cortex of the
postnatal mouse brain, we used the Cre/loxP conditional knockout system. To
delete the Cdk5 gene in a cortex-specific manner, we utilised the
Emx1Cre mouse, in which Cre recombinase is expressed in the cerebral
and hippocampal cortices as early as E10
(Cappello et al., 2006).
Cortex-specific Cdk5 conditional KO (CxCdk5KO) mice were generated as
Emx1Cre/+;fCdk5/- by mating
Emx1Cre/+;Cdk5+/- with
fCdk5/fCdk5 mice.
To confirm the loss of Cdk5 expression in the cortex of CxCdk5KO mice, we determined the levels of Cdk5 protein at E15.5, postnatal day 2 (P2) and P10. The cerebral cortex was dissected out from the brain, and homogenised brain samples were subjected to western blot analysis (Fig. 6). A substantial decrease in Cdk5 protein was observed in the cerebral cortex from CxCdk5KO mice at E15.5 and P2 (Fig. 6A). We also detected low levels of Cdk5 protein in the hippocampus as well as in the cerebral cortex of CxCdk5KO mice at P10 [Fig. 6B; Cdk5/actin control with mean of 1 (s.d. 0.14); CxCdk5KO with a mean of 0.189 (s.d. 0.08), n=4, P<0.01]. Immunohistochemical analysis of the CxCdk5KO brain revealed a loss of Cdk5 immunoreactivity in the specific brain areas expressing Cre recombinase, including the cerebral and hippocampal cortices. Throughout these areas, we detected Cdk5-positive interneurons that derived from the ganglionic eminence, from which Cdk5 was not removed in CxCdk5KO mice (Fig. 6C, inset). Most CxCdk5KO mice survived their perinatal period and no difference in body weight was evident between CxCdk5KO mice and littermate controls at P0 (i.e. at day of birth; control, 1.32±0.84; CxCdk5KO, 1.38±0.04, n=5). However, CxCdk5KO mice had retarded growth, with their body weight being significantly lower in the early postnatal days (see Fig. S3A in the supplementary material), and died between two and three weeks of age.
Inverted cortex in CxCdk5KO mice
Nissl staining revealed a defective layer structure of the cerebral cortex
in CxCdk5KO mice, although the cerebral wall thickness was not changed
(Fig. 6C,D) and no significant
loss of neocortical neurons was observed (data not shown). Limited posterior
extension of the cerebral cortex was also observed in the CxCdk5KO mice (see
Fig. S3B,C in the supplementary material). In the hippocampus, neurons that
normally form pyramidal cell layers of CA1 to CA3 were scattered throughout
the hippocampus without forming layer structures
(Fig. 6D). To analyse the
positioning of cortical neurons according to their birth date, we performed
serial birth-dating by BrdU (Fig.
7A). BrdU was injected into pregnant females carrying embryos of
E12.5 to E16.5 and analysed at P10. In the control, neurons at each birth date
segregated and formed a distribution peak in an `inside-out' manner
(Fig. 7A). However, the
distribution of neurons with the same birth date was completely inverted
(`outside-in') in the cerebral cortex of CxCdk5KO mice
(Fig. 7A). To confirm the
inverted nature of the cortex of CxCdk5KO mice, we conducted in situ
hybridisation with layer markers Foxp2, Er81 (Etv1 - Mouse Genome
Informatics) and Cux2 (Cutl2 - Mouse Genome Informatics). In
agreement with previous reports (Ferland
et al., 2003; Arber et al.,
2000; Nieto et al.,
2004), Foxp2-positive neurons were distributed in layer
VI, the deepest layer, whereas Er81-positive neurons were positioned
in layer V, and Cux2-positive neurons were in superficial layers II
and III in the cerebral cortex of control mice
(Fig. 7B). However, in the
CxCdk5KO cerebral cortex, Foxp2-positive neurons occupied a
superficial position and Cux2 occupied the deeper position. These
results confirmed an inversion of the cerebral cortex layer structure in
CxCdk5KO mice.
|
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|
). Because the pyramidal neurons of a subset of layer V
neurons in the cerebral cortex, as well as those in the hippocampus, are
YFP-positive in the YFP-H line after P10
(Feng et al., 2000), we could
observe dendrite, soma and axon morphologies of these neurons in detail. We
applied this strategy to analyse the morphological features of Cdk5-deficient
pyramidal neurons in the postnatal cortex of CxCdk5KO mice. In the control
mice, a typical pyramidal morphology of layer V neurons was observed: a single
apical dendrite extending toward the pia with secondary branches, basal
dendrites and a single axon exiting from the ventricular side and running
straight down (Fig. 9A).
However, in CxCdk5KO mice, the dendrite morphology of layer V neurons was
largely abnormal, in addition to their inverted position in the cortex
(Fig. 9B). In the majority of
layer V neurons, the apical dendrite was difficult to identify; instead,
multiple dendrites extended directly from the soma
(Fig. 9B). Furthermore, an
abnormal axonal trajectory was also observed in the cortex of CxCdk5KO mice;
each neuron had a single axon, but the exit site of the axon was not
consistent and axons ran obliquely within the cortex
(Fig. 9B). To elucidate the
relationship between dendritic abnormalities and the ectopic position of layer
V pyramidal neurons, we analysed the morphologies of layer V neurons in
reeler;YFP-H mice. We observed an abnormal orientation of apical
dendrites of layer V neurons, similar to previous reports
(Terashima et al., 1983).
Importantly, layer V pyramidal neurons preserved pyramidal morphology in the
cerebral cortex of reeler mice, despite their ectopic localisation
(see Fig. S4 in the supplementary material). This result indicates that
abnormal dendritic structures in the CxCdk5KO mice are not a secondary
consequence of ectopic localisation of layer V neurons. We further analysed the morphology of callosal commissural (CC) neurons in CxCdk5KO mice at P10 using retrograde DiI labelling. In the control, mainly layer II/III pyramidal neurons were labelled by DiI, and these neurons had typical pyramidal morphology as described above (data not shown). However, labelled CC neurons had an abnormal morphology in CxCdk5KO mice, similar to layer V neurons in CxCdk5KO;YFP-H mice (Fig. 10A). These results indicate that the abnormal morphology of pyramidal neurons occurred throughout the cortex in CxCdk5KO mice. By DiI labelling, we also found an abnormal dendritic morphology and axonal trajectory in the contralateral cerebral cortex in Cdk5-/- mice at E18.5 (Fig. 10B).
|
|
).
Overall, staining of Map2 decreased in the cerebral cortex of CxCdk5KO mice at
P10 (Fig. 11A), consistent
with the decreased protein level of Map2
[Fig. 11B; Map2/actin control
with a mean of 1 (s.d. 0.10) versus CxCdk5KO with a mean of 0.04 (s.d. 0.16),
n=4, P<0.01]. Because the protein level of NF-M
(neurofilament, medium polypeptide; also known as Nefm - Mouse Genome
Informatics) was not changed [NF-M/actin control with mean of 1 (s.d. 0.12)
versus CxCdk5KO with mean of 0.78 (s.d. 0.06), n=4,
P=0.055], the specific decrease in Map2 might reflect the
developmental deficit of the dendritic structure of pyramidal neurons, i.e.
that defects in the dendrite structures of pyramidal neurons are associated
with a lower level of Map2 in the cerebral cortex of CxCdk5KO mice.
Map2-positive apical dendrites were evident in the cerebral cortex of the
controls (Fig. 11A). Such
radial staining of Map2 was missing in the cerebral cortex of CxCdk5KO mice.
Instead, Map2-positive staining was detected in the superficial layer, where
layer VI neurons existed in CxCdk5KO mice
(Fig. 11A). The morphology of
layer VI neurons, which extend their axons toward the dorsal thalamus, was
examined by retrograde labelling with DiI. As in our previous report
(Gilmore et al., 1998), we
found that layer VI neurons, although placed in the superficial layer,
preserved the pyramidal morphology in the Cdk5-/-
embryonic brain (Fig. 11C). We
also found that layer VI neurons had typical pyramidal morphology in the
cerebral cortex in CxCdk5KO mice at P10
(Fig. 11D). |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Gilmore et al., 1998). In the
present study, we report that Cdk5 is mainly involved in the transitional
phase of neurons from the multipolar to bipolar during radial neuronal
migration. When we suppressed Cdk5 using Cdk5-DN, we observed a severe
impairment of multipolar-to-bipolar transition in neurons expressing Cdk5-DN
in the time-lapse imaging study (Fig.
3), as well as in vivo (Fig.
2). This result is consistent with our observation of the
behaviour and morphology of migrating Cdk5-/- neurons
(Figs 1,
4). Our observation of neurons
expressing Cdk5-DN differs from a previous study that found a substantial
delay in the migration of neurons expressing Cdk5-DN after they became bipolar
(Hatanaka et al., 2004).
However, Hatanaka et al. used an equal ratio of EGFP and Cdk5-DN plasmid. When
we used a 50% (1:1) dilution of Cdk5-DN, some neurons became bipolar, but
their migration was delayed within the CP
(Fig. 2). Interestingly, some
of these neurons had branched leading processes
(Fig. 2A'b, and milder
morphological abnormalities that have been observed in
p35-/- neurons (Gupta
et al., 2003) (T.O., T.A. and K.M., unpublished). These results
indicate that strong suppression of Cdk5 activity causes impaired
multipolar-to-bipolar transition, just like that seen in the
Cdk5-/- mice, and moderate suppression of Cdk5 delays
radial migration within the CP after acquisition of the bipolar shape,
although these bipolar neurons sometimes have branched leading processes.
These observations also suggest that the multiple morphologies we observed in
Cdk5-/- migrating neurons were not simply the result of
migration arrest at the multipolar stage of radial migration. When we cultured
the Cdk5-/- neurons from the cerebral cortex, however,
Cdk5-/- neurons were able to acquire the pyramidal
morphology (see Fig. S2 in the supplementary material), indicating that loss
of Cdk5 does not severely affect the ability of cortical neurons to acquire
the pyramidal morphology. Morphological change during transition from
multipolar to bipolar might require dynamic rearrangement of the cytoskeleton
of migrating neurons and might require Cdk5 activity. Recent studies indicate
that this transitional process is considered a stage of neocortical
development vulnerable to disruption, as RNAi for migration-related proteins
including Lis1 (Pafah1b1 - Mouse Genome Informatics) and Dcx disrupts this
transition as well (reviewed by Bai et al.,
2003; Tsai et al.,
2005; LoTurco and Bai,
2006).
The most interesting finding is the abnormal morphology of pyramidal
neurons in the cerebral cortex of CxCdk5KO mice. These neurons failed to
develop an apical dendrite structure and extended multiple dendrites. Because
this defect was observed in Cdk5-/- mice at E18.5
(Fig. 10B), as well as in
CxCdk5KO mice postnatally (Figs
8,
9), it is likely that this
abnormality occurs as a consequence of the inability of neurons to acquire a
bipolar morphology during their radial migration. Our analysis with DiI
labelling indicated that abnormal pyramidal morphology occurred throughout the
cortical neurons corresponding to layer II/III and layer V
(Fig. 10A). Our preliminary
results from Golgi staining of the CxCdk5KO mouse brain are consistent with
these results (T.O., T.A. and K.M., unpublished). It is known that intrinsic
afferent input plays an important role in the sculpting of the mature neuronal
dendritic tree. Therefore, we cannot exclude the possibility that the ectopic
position of Cdk5-deficient pyramidal neurons might be at least partially
responsible for the abnormalities of dendrite structures described above.
However, our analysis of reeler mice indicates that the typical
pyramidal morphology, with a single apical dendrite, is preserved in layer V
neurons even in their ectopic positions in this mutant (see Fig. S4 in the
supplementary material). Another possible explanation for abnormal dendrite
structure in CxCdk5KO mice is that the absence of Cdk5 causes neuronal
apoptosis (Li et al., 2002),
and the remaining neurons exhibit dendrite abnormalities. However, we did not
detect any significant loss of cortical neurons in postnatal-stage CxCdk5KO
mice (data not shown). Therefore, we consider that the morphological
abnormality in CxCdk5KO mice is cell-autonomous and tightly related to the
transitional defect from multipolar to bipolar during neuronal migration.
Why does Cdk5 deficiency cause substantial defects in the radial migration
of cortical neurons? Recent studies identified Cdk5 substrates that are
related to neuronal migration, including Dcx, Nudel (Ndel1 - Mouse Genome
Informatics) and Fak1 (Ptk2 - Mouse Genome Informatics)
(Tanaka et al., 2004b;
Sasaki et al., 2000; Niethmmer
et al., 2000; Xie et al.,
2003). Phosphorylation of Nudel, Dcx and Fak1 at
Cdk5-phosphorylation sites is considered important to organise the link
between centrosome and nucleus through their association with microtubules
(Tanaka et al., 2004a). siRNA
of Dcx causes premature arrest of migrating multipolar neurons in the
premigratory zone (Bai et al.,
2003). Inhibition of Rac1 arrests the migration of cerebral
cortical neurons in the multipolar morphology
(Kawauchi et al., 2003);
therefore, improper regulation of RhoGTPase in migrating neurons might impair
their transformation from multipolar to bipolar. Other studies suggest that
RhoA activity is inhibited within the IZ for proper radial neuronal migration
(Hand et al., 2005;
Ge et al., 2006).
Interestingly, Cdk5 phosphorylates p27 (CdKn1b - Mouse Genome Informatics) at
Ser10 and stabilises p27 in the cytoplasm of migrating neurons
(Kawauchi et al., 2006).
Because p27 inhibits RhoA activity (Besson
et al., 2004), Cdk5-mediated phosphorylation of p27 regulates RhoA
activity during radial neuronal migration
(Kawauchi et al., 2006). Cdk5
also phosphorylates the actin-binding protein filamin A, and a decrease in the
expression of filamin A results in the accumulation of multipolar cells in the
premigratory zone (Nagano et al.,
2004). Therefore, it is clear that Cdk5 phosphorylates multiple
substrates and regulates the dynamics of microtubules and of the cytoskeletal
protein actin in the transitional phase from multipolar to bipolar. Cdk5 also
regulates nucleokinesis, which maintains a proper distance between the nucleus
and centrosome (Tanaka et al.,
2004a). Recent studies indicate involvement of non-muscle type
myosin II in the nucleokinesis at the rear of migrating neurons
(Schaar and McConnell, 2005;
Bellion et al., 2005) and the
possible involvement of Cdk5 in this process remains to be elucidated.
A previous study demonstrated that early-born neurons, which correspond to
layer VI, migrate by somal translocation and that this migration is
Cdk5-independent (Nadarajah et al.,
2001). Our results from in situ hybridisation
(Fig. 7B) and DiI labelling
experiments (Fig. 11C,D)
suggest that layer VI neurons migrate properly and obtain a pyramidal
morphology. Our explanation for why Cdk5 deficiency has little impact on layer
VI neurons is that layer VI neurons use somal translocation as their migration
mode and skip the transitional process from the multipolar to bipolar
morphology (Noctor et al.,
2004). Thus, they can migrate properly and obtain a pyramidal
morphology in the absence of Cdk5. In this sense, our current study supports
the proposed idea that early- and later-born neurons use distinct molecular
and cellular mechanisms for their radial migration
(Nadarajah et al., 2001;
Hatanaka et al., 2004). An
alternate explanation is that neurons need to migrate a relatively short
distance in the early stages of cortical development; therefore,
Cdk5-/- neurons would be able to reach the pia even in the
multipolar morphology or in the incompletely transformed bipolar shape, such
as the branched shape, previously reported in p35-/-
neurons (Gupta et al.,
2003).
Although there is as yet no direct evidence in migrating neurons for a
transition from the leading process to apical dendrite, the bipolar shape in
migrating neurons is likely to be a fundamental requirement for the pyramidal
neurons to obtain their correct morphology
(Hatanaka and Murakami, 2002).
Our results provide evidence to support this view, because the inability to
acquire a bipolar morphology during migration resulted in abnormal dendrite
development in Cdk5-deficient pyramidal neurons. In addition, recent
observations suggest that migrating neurons already extend their axons
(Noctor et al., 2004;
Hatanaka and Murakami, 2002).
In the present study, we also observed that extensions of axons from migrating
neurons (Fig. 1A) and axonal
trajectories are impaired in Cdk5-deficient neurons
(Fig. 1B;
Fig. 9), probably because of
their defective migration. Based on our findings in the Cdk5-deficient mouse
models, we propose that the transition of migrating neurons into a bipolar
shape is essential to form the pyramidal morphology in which neurons extend
their single apical dendrite toward the pia, and extend single axons towards
the bottom of the cortex, and that this radial axis is brought about by the
active radial migration of neurons in a Cdk5-dependent manner.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/12/2273/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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