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First published online 16 May 2007
doi: 10.1242/dev.02854

Development 134, 2273-2282 (2007)
Published by The Company of Biologists 2007
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Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex

Toshio Ohshima1,*, Motoyuki Hirasawa2, Hidenori Tabata3, Tetsuji Mutoh4, Tomoko Adachi1, Hiromi Suzuki1, Keiko Saruta1, Takuji Iwasato5, Shigeyoshi Itohara5, Mistuhiro Hashimoto6, Kazunori Nakajima3,7, Masaharu Ogawa4, Ashok B. Kulkarni2 and Katsuhiko Mikoshiba1,8

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.


Figure 1
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  		Fig. 1. Visualisation of migrating neurons in Cdk5+/+ and Cdk5-/- mice by introduction of T{alpha}1-EGFP plasmid into VZ cells by in utero electroporation. EGFP was introduced into (A) Cdk5+/+ and (B) Cdk5-/- mouse embryos at E14.5, and brains were fixed at E17.5. Sections were stained with anti-GFP antibody. Insets show areas indicated by arrows (a,b) at higher magnification. (A) Over the period of 3 days, neurons migrated into the CP with the typical bipolar shape (a) in Cdk5+/+ mice. Migrating neurons in the CP extend their leading processes toward the pia and their axons (arrowhead) toward the VZ. In the SVZ-IZ, GFP-positive neurons of multipolar shape (b) are observed at this stage. (B) By contrast, in Cdk5-/- mice neurons arrested in the SVZ-IZ in a multipolar shape (a), and their axons (arrowhead) run oblique within the SVZ-IZ. CP, cortical plate; SVZ, subventricular zone; IZ, intermediate zone; VZ, ventricular zone. Scale bar: 25 µm.

 

Figure 2
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  		Fig. 2. Cell-autonomous impairment of the multipolar-to-bipolar transformation within the SVZ-IZ in neurons expressing Cdk5-DN. Mixtures of CRC-Cdk5-DN and CAG-RFP were used in different ratios for in utero electroporation of E14.5 mouse embryos. (A,A') The distribution and morphology of RFP-positive cells (red) were analysed in coronal sections at E18.5. Higher magnifications of the indicated areas of 100% (a) and 50% Cdk5-DN (b) are shown in A'. Dose-dependent disturbance of neuronal migration by CRC-Cdk5-DN was also cell-autonomous. When only Cdk5-DN was used (100%), RFP-positive neurons arrested within the SVZ-IZ in the multipolar shape (arrows in A'). This result matched the phenotype of Cdk5-/- neurons, indicating that defective Cdk5 activity in migrating neurons caused migration arrest in a cell-autonomous manner. When Cdk5-DN was used at 50%, migration delay was observed. RFP-positive neurons have bipolar shape in CP but some of the RFP-positive neurons have branched leading processes (arrows in A'b). MZ, marginal zone; CP, cortical plate; SVZ-IZ, subventricular zone-intermediate zone; VZ, ventricular zone. Scale bar: 100 µm. (B) Bin distribution of RFP-positive neurons at each ratio of Cdk5-DN. The cortical mantle was divided into ten equally spaced bins and the percentage of cells in each bin calculated.

 

Figure 3
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  		Fig. 3. Time-lapse imaging of migrating neurons expressing either CAG-EGFP or CGC-Cdk5-DN. (A) Representative results are shown for mouse brain slices electroporated with either CAG-EGFP (GFP) or CGC-Cdk5-DN (Cdk5-DN) at E14.5. (B) Based on their morphology and behaviour, GFP-positive neurons were divided into two categories: bipolar shape (bipolar) or multipolar shape (multipolar) when observed at 4 hours (4 h) and 24 hours (24 h) post-electroporation, and the percentage of each is shown for CAG-EGFP and CGC-Cdk5-DN brain slices. (C) The percentage of GFP-positive neurons that transformed from multipolar to bipolar during a 20-hour peroid was calculated for each slice and subjected to statistical comparison. Multipolar-to-bipolar transformation was impaired in neurons expressing Cdk5-DN (GFP, 40.15±14.25%; Cdk5-DN, 7.75±3.77%, n=4); *, P<0.001.

 

Figure 4
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  		Fig. 4. Time-lapse analysis of migratory behaviour of Cdk5-/- neurons. (A) Cdk5+/+ and Cdk5-/- mouse embryos were infected with Adex-CAG-Lyn-Venus at E12.5, and brain slice cultures were started at E14.5. Representative images are shown of migrating neurons after 24 hours (24 h) and 48 hours (48 h) of culture. Scale bar 10 µm. (B) GFP-positive neurons were classified as multipolar or bipolar by their morphology and behaviour at 24 and 48 hours, and the percentage is shown for each genotype. *, P<0.01.

 

Figure 5
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  		Fig. 5. In situ hybridisation in the coronal sections from Cdk5+/+ and Cdk5-/- mouse embryos at E16.5 using Neurod1 and doublecortin probes. The expression of these genes in the premigratory zone of Cdk5-/- embryos was comparable to that in Cdk5+/+ embryos, indicating that initial neuronal differentiation is not disturbed in Cdk5-/- mice. P, pia; V, ventricle. Scale bars: 100 µm.

 

Figure 6
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  		Fig. 6. Cortex-specific Cdk5 knockout mice. (A) Loss of Cdk5 in the cerebral cortex and hippocampus was confirmed by western blot analysis. Samples from each brain region were dissected from embryonic or postnatal mouse brains at the indicated stages and subjected to western blot analysis for Cdk5 proteins. Cdk5 protein levels in the cerebral cortex (cc) were low compared with those in other brain regions (ex) at E15.5 and P2. At P10, brains were separated into the cerebral cortex (CC), hippocampus (Hipp), thalamus (Th), and brain stem (BS) and analysed for Cdk5 and actin by western blot. (B) Reduction in the Cdk5 protein level (Cdk5/actin) was confirmed in cerebral cortex at P10. Mean±s.d.; n=6; *, P<0.01. (C) Immunostaining of the coronal section of the cerebral cortex from control and CxCdk5KO mice at P10 with anti-Cdk5 antibody. Cdk5 staining was observed in all regions of the control mouse brain at P10. This staining pattern was missing in the cerebral cortex and hippocampus from CxCdk5KO mice. Higher magnification of the area indicated by the arrow in the right-hand panel reveals the presence of Cdk5-positive interneurons, which derive from the ganglionic eminence where Cre recombinase is not expressed; these are also GABA positive (data not shown). Scale bar: 200 µm; inset, 100 µm. (D) Comparison of cerebral cortex in Nissl-stained sagittal sections revealed abnormal laminar structure in CxCdk5KO mice. In the cerebral cortex, cell-sparse structures of layer I (I) and white matter (W.M.) observed in the control were not evident in the cerebral cortex in CxCdk5KO mice. In the hippocampus, pyramidal neurons failed to form a confined layer in CxCdk5KO mice. Scale bar: 100 µm.

 

Figure 7
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  		Fig. 7. Inverted cortex in CxCdk5KO mice. (A) Bin distributions of BrdU-positive cells shown as mean percentage of three mice for control (Cont.) and CxCdk5KO mice (CxCdk5KO) at indicated time points. The cortical mantle was divided into ten equally spaced bins, and the percentage of BrdU-positive cells in each bin is shown. Birth-dating BrdU labelling from E12.5 to E16.5 revealed a typical inside-out pattern of generation of the laminar structure in the cerebral cortex of control mice at P10. This pattern was inverted, resulting in an outside-in pattern in the motor cortex (a) and somatosensory cortex (b) of CxCdk5KO mice. (B) In situ hybridisation study with layer markers Cux2, Er81 and Foxp2 at P10. In the control, Cux2-positive, Er81-positive and Foxp2-positive neurons accumulated in layer II/III, layer V and layer VI, respectively. This pattern was inverted in the CxCdk5KO cortex. Scale bar: 100 µm.

 

Figure 8
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  		Fig. 8. Morphologies of GFP-labelled neurons at E14.5 in the cerebral cortex of control and CxCdk5KO mice. GFP immunostaining of coronal sections from CxCdk5KO mice and their littermate controls which were electroporated at E14.5 with CAG-EGFP plasmid. Brains were fixed at E17.5, P1 or P3 and stained with anti-GFP antibody (green). GFP-positive neurons in CxCdk5KO mice were positioned deep down and had multipolar morphology. Scale bars: 100 µm. The bottom-right panel is a higher magnification image of GFP-positive cells in the cerebral cortex of CxCdk5KO mice at P3 obtained with a confocal microscope. Scale bar: 20 µm.

 

Figure 9
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  		Fig. 9. Defective dendritic development in CxCdk5KO mice. (A) Position and (B) detailed morphologies of layer V neurons in CxCdk5KO;YFP-H double-transgenic mice. (A) Typical examples of the cerebral cortex in coronal sections from control and CxCdk5KO mice at P14. Bin distributions of YFP-positive layer V neurons are shown (control, black; CxCdk5KO, grey). (B) Confocal images of the cerebral cortex of control (Cont.;YFP-H) and CxCdk5KO (CxCdk5KO;YFP-H) mice at P14. Abnormal dendritic structures (arrows) and axonal trajectories (arrowheads) are present in CxCdk5KO mice. Scale bar: 50 µm.

 

Figure 10
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  		Fig. 10. DiI labelling of commissural neurons in P10 CxCdk5KO and Cdk5-/- mice. DiI crystals were placed in the cerebral cortex of fixed brains of (A) P10 CxCdk5KO and (B) E18.5 Cdk5-/- mice and littermate controls. Vibratome sections (100 µm) were imaged using a fluorescent (A,B) or confocal (inset in B) microscope. (A) Abnormal dendrite development of commissural neurons in CxCdk5KO mice. Scale bar: 25 µm. (B) In Cdk5+/+ mice, cortical pyramidal neurons extended their apical dendrites toward the pia. By contrast, Cdk5-/- neurons extended their dendrites in multiple directions. Inset, confocal image of abnormal dendrite structures of Cdk5-/- commissural neurons.

 

Figure 11
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  		Fig. 11. Reduced Map2 expression in the CxCdk5KO cortex, and unaltered pyramidal morphology of layer VI neurons in Cdk5-deficient brains. (A) Map2 immunostaining revealed defective dendrite structures of pyramidal neurons in CxCdk5KO mice. Apical dendrites of pyramidal cortical neurons were typically Map2-positive in the control (inset, arrowheads). This pattern was missing, with only a few Map2-positive radial dendrites observed in the superficial layer of the CxCdk5KO cortex (inset, arrowheads). Insets are higher magnifications of the areas indicated by the arrows. Overall, staining of Map2 immunoreactivity was reduced in the CxCdk5KO cortex. (B) Protein levels for Map2, NF-M and actin were examined in the dissected cerebral cortices from control and CxCdk5KO mice at P10. The reduction in Map2/actin in CxCdk5KO mice was also confirmed by western blotting. *, P<0.01. The level of NF-M/actin was not significantly altered in CxCdk5KO mice. (C,D) Cortico-thalamic neurons, corresponding to layer VI neurons, extended their apical dendrite toward the pia in Cdk5-/- (C) and CxCdk5KO (D) mice. Arrows indicate apical dendrites and arrowheads indicate their axons. Scale bars: 100 µm.

 

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© The Company of Biologists Ltd 2007