KIF2A regulates the development of dentate granule cells and postnatal hippocampal wiring
- The University of Tokyo, Japan
- King Abdulaziz University, Saudi Arabia
Figures
Figure 1
Hyperactivity, severe epilepsy, and eventual fatality in 3w-Kif2a-cKO mice.
(A–D) The generation of Kif2a-cKO mice. (A) Schematic diagram of the conditional Kif2a targeting strategy. The conventional targeting allele is also shown at the bottom. Tamoxifen-induced Cre …
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Figure 1—source data 1
The raw data of body weight, activity, and survival rate of WT and 3w-Kif2a-cKO mice.
- https://doi.org/10.7554/eLife.30935.004
Figure 2 with 2 supplements
3w-Kif2a-cKO mice develop epileptic hippocampi.
(A–D) Electroencephalography (EEG) recordings. (A) Representative baseline EEG recordings of WT and 3w-Kif2a-cKO hippocampi in postnatal week 6. Some spikes were observed in the cKO brain, even in …
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Figure 2—source data 1
EEG recordings of WT and 3w-Kif2a-cKO mice.
- https://doi.org/10.7554/eLife.30935.008
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Figure 2—source data 2
The latency and lasted time of PDS occurred in the 3w-Kif2a-cKO hippocampal slices.
- https://doi.org/10.7554/eLife.30935.009
Figure 2—figure supplement 1
The epileptic hippocampus in tamoxifen-injected Kif2a-cKO mice.
(A–D) The typical features of hippocampal sclerosis in 4w-Kif2a-cKO mice at postnatal week 12. The 4w-Kif2a-cKO mice clearly showed hippocampal atrophy (A), including CA1 loss (B), sprouting MFs in …
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Figure 2—figure supplement 1—source data 1
GFAP-positive area in WT and 3w-Kif2a-cKO dentate gyrus at P35.
- https://doi.org/10.7554/eLife.30935.007
Figure 2—Video 1
EEG recordings of 3w-Kif2a-cKO mice with hippocampal epilepsy.
This recording was obtained at postnatal week 5 in a mouse after an EEG operation at postnatal week 4. The epileptic convulsion continued throughout the video. The EEG wave for this convulsion is …
Figure 3
Defects in cell proliferation and cell migration were not significant in the 3w-Kif2a-cKO hippocampus.
(A) The injection scheme of thymidine analogs and tamoxifen. CldU and IdU were injected during postnatal weeks 2 and 3, respectively. The mice were fixed and double-stained with anti-CldU and …
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Figure 3—source data 1
The migrating distance, direction, and expression of birth-dating marker of WT and 3w-Kif2a-cKO DGCs at P35.
- https://doi.org/10.7554/eLife.30935.012
Figure 4 with 2 supplements
Zn-positive axon terminals of DGCs are aberrant and widespread throughout the whole ML in the 3w-Kif2a-cKO mice.
(A–B) The expression of postnatal KIF2A in the hippocampus at P21. Note that KIF2A is highly expressed in the dentate hilus (Hil) and stratum lucidum (SL) (A) where DGCs extend their axons, termed …
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Figure 4—source data 1
Timm grain intensity in WT and 3w-Kif2a-cKO dentate gyrus at P35.
- https://doi.org/10.7554/eLife.30935.019
Figure 4—figure supplement 1
The Zn-positive axons of DGCs abnormally extended into the SO and the entire ML in the CBZ-injected 3w-Kif2a-cKO mice.
(A–D) Representative images showing Timm staining of the hippocampus at P35. C and D are magnified images of the areas indicated by the red rectangles in A and B. Coronal cryosections in the …
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Figure 4—figure supplement 1—source data 1
Timm grain intensity in CBZ-injected-WT and CBZ-injected-3w-Kif2a-cKO dentate gyrus at P35.
- https://doi.org/10.7554/eLife.30935.015
Figure 4—figure supplement 2
More DGC axons were present throughout the ML of the 3w-Kif2a-cKO mice than in that of WT mice.
(A–B) Representative images of cryosections immunostained with anti-NFM and anti-MAP2 at P35. Images are 1-μm-thick confocal optical sections (OS). Compared with WT (A), 3w-Kif2a-cKO mice had more …
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Figure 4—figure supplement 2—source data 1
Intensity of NFM-staining in WT and 3w-Kif2a-cKO dentate gyrus at P35.
- https://doi.org/10.7554/eLife.30935.017
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Figure 4—figure supplement 2—source data 2
Intensity of synaptoporin-staining in WT and 3w-Kif2a-cKO dentate gyrus at P35.
- https://doi.org/10.7554/eLife.30935.018
Figure 5
DGCs developed aberrant morphological changes in axons, cell bodies, and dendritic spines in 3w-Kif2a-cKO mice.
(A) Schematics of unsolved questions in developing DGCs in the Kif2a-cKO dentate granule layer (DGL). In the postnatal hippocampus (left panel), DGCs continuously proliferated in the SGZ, migrated …
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Figure 5—source data 1
The population of the cells with apical axons in the outer ML, the cells with protrusion in the inner ML, and the ectopic cells in inner ML in WT and 3w-Kif2a-cKO dentate gyrus at P35.
- https://doi.org/10.7554/eLife.30935.021
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Figure 5—source data 2
The spine morphology of dendrites in IML and OML of WT and 3w-Kif2a-cKO dentate gyrus at P35.
- https://doi.org/10.7554/eLife.30935.022
Figure 6 with 4 supplements
Both axons and dendrites were aberrantly developed in cultured P3-Kif2a-cKO DGCs.
(A–L) Representative images of dissociated cultured DGCs immunostained with an axon marker (NFM or Tau1) and a dendrite marker (MAP2) at DIV1 (A–H) and DIV5 (I–L). At DIV1, both WT DGCs (A–D) and …
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Figure 6—source data 1
The character of neuronal processes from a cell body of WT and P3-Kif2a-cKO DGCs.
- https://doi.org/10.7554/eLife.30935.027
Figure 6—figure supplement 1
The loss of KIF2A induced MF sprouting in dissociated cultured DGCs.
(A–C) Characterization of dissociated cultured granule cells and confirmation of their loss of KIF2A. A-B. Representative images of cultured DGCs. Granule cells from the hippocampal dentate gyri of …
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Figure 6—figure supplement 1—source data 1
The number of Ankyrin G-positive processes from a cell body of of WT and P3-Kif2a-cKO DGCs at DIV3.
- https://doi.org/10.7554/eLife.30935.025
Figure 6—figure supplement 2
Transfection of KIF2A rescued MF sprouting in dissociated cultured DGCs.
(A–G) The rescue experiment. (A–D) Representative images of single cultured DGCs. DGCs were transfected with YFP or YFP-KIF2A at DIV3 and then observed at DIV4. Compared with the WT DGCs (A), cKO …
Figure 6—Video 1
Time lapse recordings of cultured WT DGC.
This recording was obtained at the end of DIV2 for 8 hr. The DGC controls the length of an axon and a dendrite. No protrusions from the cell body. An immunostained image of the recorded cell is …
Figure 6—Video 2
Time lapse recordings of cultured P3-Kif2a-cKO DGC.
This recording was obtained at the end of DIV2 for 8 hr. The DGC is rapidly elongating and branching an axon and dendrites, in addition to some aberrant protrusions from the cell body. An …
Figure 7
Schematic of the function of KIF2A in the postnatal hippocampus.
(A) KIF2A function in DGC development. At the early stages of development, both WT and cKO DGCs normally make axonal and dendritic processes. However, whereas WT DGCs regulate the length of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus muscu/us) | Kif2a | PMID:1447303 | MGl:108390 | |
| Strain, strain background (Mus muscu/us) | C57BU6NJcl, C57BU6JJcl | Clea Japan | ||
| Strain, strain background(Mus muscu/us) | CreERt(CAG-cre/Esr1) | The Jackson Laboratory | Tg(CAG-cre/Esr1 *)5Amc/J (Stock No: 004453) | |
| Strain, strain background(Mus muscu/us) | R26R (lacZ expression with the ROSA26 Cre reporter strain) | PMID:9916792 | ||
| Strain, strain background(Mus muscu/us) | Thy1-EGFP line M | The Jackson Laboratory | Tg(Thy1-EGFP)MJrs/J (Stock No: 007788) | |
| Cell line (Mus musculus) | CMTl-1 | Merck Millipore | 129/Sv-derived ES cell (ESC) line | |
| Antibody | anti-KIF2A (mouse monoclonal) | PMID: 7535303 | (1:200) | |
| Antibody | anti-KIF2A (rabbit polyclonal) | Thermo Fisher | Thermo Fisher: PA3-16833 | (1:1500) |
| Antibody | anti-GFAP(mouse monoclonal) | BO Transduction Laboratory | BO Bioscience: 610565 (Clone 52/GFAP) | (1:200) |
| Antibody | anti-BrdU (mouse monoclonal) for ldU | BO Pharmingen | BO Bioscience: 555627 (Clone 304) | (1:1000) |
| Antibody | anti-BrdU (rat monoclonal) for CldU | Accurate Chemicals | Accurate Chemicals (Clone: BU1/75) | (1:500) |
| Antibody | anti-Tau1 (mouse monoclonal) | Merck Millipore | Merck Millipore: MAB3410 (Clone PC1C6) | (1:1000) |
| Antibody | anti-Neurofilament M (mouse monoclonal) | Sigma | Sigma: N5264 (Clone NN18) | (1:600) |
| Antibody | anti-MAP2 (chicken monoclonal) | Abeam | Abeam: AB5392 (Clone 304) | (1:200) |
| Antibody | anti-Synaptoporin (rabbit polyclonal) | Synapitic Systems | SYSY:102002 | (1:300) |
| Antibody | anti-Prox1 (rabbit polyclonal) | Merck Millipore | Merck Millipore: AB5475 | (1:10000) |
| Antibody | anti-Ankyrin G (mouse monoclonal) | Thermo Fisher | Thermo Fisher: 338800 (Clone 4G318) | (1:600) |
| Antibody | Alexa 488-, 546-, 555-, 647, (Goat polyclonal) | Molecular Probes | (1: 200–1: 500) | |
| Recombinant DNA reagent | pEYFP-C1 (vector) | Clontech | ||
| Recombinant DNA reagent | YFP-Kif2a (full length) | PMID: 14980225 | Progenitors: pEYFP-C1 vector | |
| Commercial assay or kit | LA PCR Kit | Takara | TaKaRa:RR013A | |
| Chemical compound, drug | Tamoxifen | Sigma | Sigma:T5648 | (30 mg/kg body weight/day) |
| Chemical compound, drug | Pilocarpine | Sigma | Sigma: P6503 | (290 mg/kg body weight/day}, |
| Chemical compound, drug | Scopolamine methyl bromide | Sigma | sigma: 8502 | (1 mg/kg body weight/day) |
| Chemical compound, drug | ScaleView | Olympus | ||
| Chemical compound, drug | 5-iodo-2'-deoxyuridine | Sigma | Sigma: 17125 | 50 mg/kg |
| Chemical compound, drug | 5-chloro-2 '-deoxyuridene | Sigma | Sigma: C6891 | 50 mg/kg |
| Chemical compound, drug | poly-L-lysine | Sigma | Sigma: P-2636 | 100 mg/ml |
| Chemical compound, drug | Laminin | lnvitrogen | lnvitrogen: 23017–015 | 25 µg/ml |
| Software, algorithm | MicroMax 1. 3 | AccuScan Instrument | ||
| Software, algorithm | Clampex 9.2 software | Axon Instruments | ||
| Software, algorithm | IMARIS 7.2 | Bitplane:Zeiss | ||
| Software, algorithm | lmageJ 1.51 hr | NIH | ||
| Software, algorithm | SPSS V22 | IBM | ||
| Machine | MicroMax Monitor | AccuScan Instruments | ||
| Machine | Axopatch 1D amplifier | Axon Instruments |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.30935.031
