Figure 1.
lag mutation preferentially affects the brain.
(A) The lag mutant phenotype. The littermate normal control mouse and the lag mutant mouse faced each other. Upper panel: general appearance. Lower panel: enlarged head image. Asterisk indicates the mutant mouse and red arrow indicates the flat head of the mutant mouse. (B) Behavior test for ataxia. Quantitative analysis of the duration the littermate normal control mice (n = 16) and the lag mutant mice (n = 16) stood on a narrow platform. Error bars represent SD. (C) Survival curves. Survival rate of the littermate normal control mice (open circles, n = 41) and the lag mutant mice (closed circles, n = 18) from postnatal day 1 (P1) to P25. (D) Whole brain images of the littermate normal control mouse and the lag mutant mouse at P12. Arrowheads indicate the translucent olfactory bulb and spinal cord of the lag mutant brain. Bar, 10 mm.
Figure 2.
Positional cloning of Kif14 in the lag mutant mouse.
(A) LOD score plot of the lag locus on chromosome 1 for the F2 intercrosses. The microsatellite markers are shown on the lower side. (B) Fine mapping analysis of the lag locus. Black boxes represent DBA/2N allele homozygotes; white boxes represent C57BL/6 allele homozygotes and heterozygotes. The identification number of each mouse is listed at the top (# number). (C) Candidate genes mapped to the crucial region. Kif14 exon-intron structure, positions of the 3′ UTR, and start site of translation are indicated according to the NCBI reference assembly build 37. (D) G/A substitution at the 3′ splice acceptor site of Kif14 exon 5. The genomic sequence analysis of exon5 splice junction of Kif14 gene was performed. The red asterisk denotes the mutation site for lag.
Figure 3.
Identification of exon skipping in mRNA from lag/lag mouse.
(A) Northern blot analysis of Kif14 mRNA. PolyA RNA from whole brains of +/+, lag/+, lag/lag mice was subjected to agarose gel-electrophoresis and then transferred to a nylon membrane. The membrane was hybridized with a Kif14 cDNA probe. (B) Transcripts analysis of Kif14 splice acceptor site mutation. Agarose gel-electrophoresis of Kif14 PCR products from first-strand cDNA prepared from whole brains of +/+, lag/+, lag/lag mice. (C) Sequence analysis of the three Kif14 transcripts identified in the lag/lag mouse. The Kif14 transcript in the lag/lag mouse exhibited skipping of an 11-bp segment of exon 5, entire exon 5, and exons 5 and 6 as a result of a G to A substitution at the acceptor site of exon 5. (D) Western blot analysis of Kif14 protein at E14.5 and P12. Whole brain extracts (40 µg of proteins) from +/+, lag/+, lag/lag mice at E14.5 and P12 were subjected to SDS-PAGE, followed by immunoblotting with the anti-Kif14 rabbit polyclonal antibody. Arrow indicates full-length Kif14. Asterisk indicates the non-specific band.
Figure 4.
(A) The transgenic rescue construct. pCAGGS-Kif14 carries a strong constitutive promoter (CAG pro, CAG promoter), mouse Kif14 cDNA, SV40 early splice region/polyadenylation signal (pA), human cytomegalovirus immediate early enhancer (HCMVIEE), ampicillin resistance gene (Amp r) and E. coli replicate origin (ColE1 ori). (B) Transgenic mice were crossed with lag/+ mice to yield Tg:lag/+ mice. Tg:lag/+ mice were crossed with lag/lag mice to obtain Tg mice in a lag/lag background. (C) Expression levels of Kif14 in three lines of Tg:lag/lag mice. Whole brain extracts (20µg of proteins) from E14.5 wild-type mouse, P12 wild-type mouse, P12 lag/lag mouse, and P12 Tg:lag/lag mice lines (Tg26l:lag/lag, Tg28L:lag/lag, and Tg29L:lag/lag) were subjected to SDS-PAGE, followed by immunoblotting with the anti-Kif14 rabbit polyclonal antibody. Arrow indicates full-length Kif14. Asterisk indicates the non-specific band. (D) Behavior test for ataxia. Quantitative analysis of the duration the littermate wild type (+/+) mice (n = 16), the lag mutant (lag/lag) mice (n = 16), and the Tg:lag/lag mice (Tg#26l:lag/lag, n = 16; Tg#28L:lag/lag, n = 16; Tg#29L:lag/lag, n = 16) stood on a narrow platform. Error bars represent SD. (E) Dorsal views of P16 whole-brain from Kif14 transgenic rescue (Tg29L:lag/lag) and non-transgenic littermate normal control mouse (lag/+) mice. Bar, 5 mm. (F) Sagittal brain sections from Kif14 transgenic rescue (Tg29L:lag/lag) mouse (Fa), non-transgenic littermate normal control mouse (lag/+) (Fb), and lag mutant mouse (lag/lag) (Fc), at P16 were immunostained with the anti-MBP antibody. Bar, 2 mm. (G) Sagittal brain sections from Kif14 transgenic rescue (Tg29L:lag/lag) mouse (Ga), non-transgenic littermate normal control mouse (lag/+) (Gb), and lag mutant mouse (lag/lag) (Gc), at P16 were counterstained with hematoxylin. ac, anterior commissure; CX, cortex; DC, deep cerebellar nuclei; DG, dentate gyrus; fi, fimbria; Hi, hippocampus; Ic, inferior colliculus; OB, olfactory bulb; ot, optic tract; Pn, pontine nuclei; RMS, rostral migratory stream; Sc, superior colliculus; Spc, spinal cord. Bars, 2 mm.
Figure 5.
(A) Schematic of the KO allele, in which exon 5 was deleted by homologous recombination in ES cells. (B) Behavior test for ataxia. Quantitative analysis of the duration the littermate wild-type mice (n = 8) and the Kif14 KO mice (n = 8) stood on a narrow platform. Error bars represent SD. (C) The Kif14 KO mouse phenotype. The littermate wild-type control mouse and the Kif14 KO mouse faced each other. Red arrow indicates the flat head of the Kif14 KO mouse. (D) Whole brain images of the littermate wild-type control mouse and the Kif14 KO mouse at P12. Arrowheads indicate the translucent olfactory bulb and spinal cord of the Kif14 KO brain. Bar, 5 mm. (E) Sagittal brain sections from the littermate wild-type control mouse (Ea) or Kif14 KO mouse (Eb) at P12 were immunostained with the anti-MBP antibody. Bars, 2 mm. (F) Sagittal brain sections from the littermate wild-type control mouse (Fa) or Kif14 KO mouse (Fb) at P12 were counterstained with hematoxylin. ac, anterior commissure; CX, cortex; DC, deep cerebellar nuclei; DG, dentate gyrus; fi, fimbria; Hi, hippocampus; Ic, inferior colliculus; OB, olfactory bulb; Pn, pontine nuclei; RMS, rostral migratory stream; Sc, superior colliculus; Spc, spinal cord. Bars, 2 mm.
Figure 6.
Hypomyelination in lag/lag mice.
(A) Littermate wild type (Aa) and lag/lag mutant (Ab) whole brain sagittal sections at P14 were immunostained with the anti-MBP antibody. ac, anterior commissure; cc, corpus callosum; fi, fimbria; Hi, hippocampus; OB, olfactory bulb; Pn, pontine nuclei. Bars, 1 mm. (B) Littermate wild type (Ba) and lag mutant (Bb) spinal cord coronal sections at P14 were immunostained with the anti-MBP antibody. (Bc) is an enlarged image of the boxed area in (Bb). AH, anterior funiculus; df, dorsal funiculus; PH, posterior funiculus; py, pyramidal tract. Bars in (Ba) and (Bb), 1 mm. Bar in (Bc), 200 µm. (C) Protein levels of various myelin components in the brains of both the littermate wild type and the lag mutant mice. Myelin-related proteins from whole brain extracts from +/+, lag/+ and lag/lag mice were analyzed by quantitative immunoblotting with various antibodies against the indicated proteins. 18 µg of protein for Kif14 detection. 12 µg of protein for MAG, CNPase, MBP, and β-tubulin detection. Arrow indicates full-length Kif14. Asterisk indicates the non-specific band. (D) Oligodendrocyte morphology in the littermate wild type and the lag mutant mice. Upper panels: semi-thin sections of optic nerves were stained with toluidine blue. Star indicates enlargement of subarachnoid space. Bars, 1 mm. Lower panels: higher magnification of the optic nerves using transmission electron microscopy. OL, oligodendrocyte. Bars, 5 µm. (E) The number of oligodendrocytes in cross section of optic nerve (n = 6). Error bars represent SD.
Table 1.
Data of microarray analysis on RNA samples isolated from brains of P14 wild-type and mutant laggard mice.
Figure 7.
No noticeable difference between OPCs of lag/lag mice and those of wild type mice.
(A) Coronal sections of wild-type and lag/lag mice cerebral cortex at E17 were counterstained with the fluorescent Nissl stain Neurotrace (red) and immunostained with anti-Olig2 antibody (green). CP, cortical plate; IZ, intermediate zone; VZ/SVZ, ventricular zone/subventricular zone. Bars, 100 µm. The number of Olig2 immuno-positive cells are shown in the right panel (n = 6). Error bars represent SD. (B) Coronal sections of wild-type and lag/lag mice cortex at E17 were counterstained with the fluorescent Nissl stain Neurotrace (red) and immunostained with anti-PDGFRα antibody (green). Error bars represent SD. CP, cortical plate; IZ, intermediate zone; VZ/SVZ, ventricular zone/subventricular zone. Bars, 100 µm. The number of PDGFRα immuno-positive cells are shown in the right panel (n = 4). (C) Quantitative real time-PCR analysis of Kif14, Olig2, Pdgfra and Mbp in +/+, lag/+, lag/lag mice at E14.5 and P6. Each gene was amplified from whole brain polyA RNA. β-actin was used as a positive control for each real time-PCR. Error bars represent SD.
Figure 8.
Disrupted cytoarchitecture of the cerebellar cortex of lag/lag mice.
(A) Littermate wild type cerebellar cortex sagittal sections and the adjoining sections at P14 were counterstained with hematoxylin (Aa and Ab) and immunostained with the anti-calbindin antibody (Ac and Ad). (Ab) and (Ad) are enlarged images of the boxed areas in (Aa) and (Ac), respectively. DC, deep cerebellar nuclei; IC, inferior colliculus; IGrL, internal granule cell layer; ML, molecular layer; OGrL, outer granule cell layer; PCL, Purkinje cell layer; SC, superior colliculus. Bars in (Aa) and (Ac), 1 mm. Bars in (Ab) and (Ad), 100 µm. (B) lag mutant cerebellar cortex sagittal sections and the adjoining sections at P14 were counterstained with hematoxylin (Ba and Bb) and immunostained with the anti-calbindin antibody (Bc and Bd). (Bb) and (Bd) are enlarged images of the boxed areas in (Ba) and (Bc), respectively. DC, deep cerebellar nuclei; IC, inferior colliculus; IGrL, internal granule cell layer; ML, molecular layer; OGrL, outer granule cell layer; PCL, Purkinje cell layer; SC, superior colliculus. Bars in (Ba) and (Bc), 1 mm. Bars in (Bb) and (Bd), 100 µm. (C) Laminar structure of the cerebellum. Littermate wild type and lag mutant cerebellar cortex sagittal sections and the adjoining sections at P14 were counterstained with hematoxylin and immunostained with the anti-calbindin antibody. Bars, 100 µm. (D) Littermate wild type (Da) and lag mutant (Db) cerebellar cortex sagittal sections at P12 were immunostained with the anti-cleaved caspase-3 antibody. Littermate wild type (Dc) and lag mutant (Dd) cerebellar cortex sagittal sections at P12 were subjected to TUNEL (terminal deoxynucleotide transferase mediated dUTP nick end labeling) analysis. The insets are enlarged images of the boxed areas. Bars, 100µm.
Figure 9.
Disrupted cytoarchitecture in the neocortex and hippocampus.
(A) Littermate wild type (Aa) and lag mutant (Ab) whole brain coronal sections at P14 were counterstained with hematoxylin. (Ac) and (Ad) are enlarged from boxed areas in (Aa) and (Ab), respectively. (Ae) and (Af) are enlarged form (Ac) and (Ad), respectively. The arrows in (Af) indicate ectopic large pyramidal cells in layer 1. Bars in (Aa) and (Ab), 1 mm. Bars in (Ac) and (Ad), 200 µm. Bars in (Ae) and (Af), 100 µm. (B) Littermate wild type (Ba) and lag mutant (Bb) whole brain coronal sections at P14 were immunostained with the anti-Cux1. Littermate wild type (Be) and lag mutant (Bf) were immunostained with the anti-Foxp2 antibody. (Bc), (Bd), (Bg) and (Bh) are enlarged from boxed areas in (Ba), (Bb), (Be) and (Bf), respectively. Bars in (Ba), (Bb), (Be) and (Bf), 1 mm. Bars in (Bc), (Bd), (Bg) and (Bh), 200 µm. (C) The number of Cux1- and Foxp2-immunopositive cells in the cerebral cortex of the wild type and lag mutant mice. Cell counts were performed at 100 µm intervals using a counting grid. (D) Littermate wild type (Da) and lag mutant (Db) hippocampal sagittal sections at P14 were counterstained with hematoxylin. CA, Cornu Ammonis; S, subiculum; DG, dentate gyrus. Bars, 1 mm.
Figure 10.
The increased apoptosis and the decreased cell proliferation during development of the cerebral cortex in lag/lag mice.
(A) Pregnant mice were intraperitoneally injected with BrdU 2 h before sacrifice. Littermate wild type (+/+) (Aa, Ab, Ac, Ad, Ae, Af) and mutant (lag/lag) (Aa’, Ab’, Ac’, Ad’, Ae’, Af’) cerebral cortex coronal sections at E12.5 and 15.5 were stained with hematoxylin, anti-BrdU antibody and TUNEL. CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; VZ/SVZ, ventricular zone/subventricular zone. Bars, 100 µm. (B) The number of BrdU immuno-positive cells (n = 4). Error bars represent SD. Asterisk indicates statistical significance (t test; *, p<0.05). (C) The number of TUNEL immuno-positive cells in (n = 4). Cell counts were performed in the sensory-motor cortex. Error bars represent SD. Asterisks indicate statistical significance (t test; *, p<0.01).