Article Figures & Data
Figures
- Figure 1.
gfp knock-in mice for the Apc2 gene. A, Schematic representation of the structure of the endogenous allele (genomic), targeting vector (vector), and targeted allele (targeted). The genomic sequence of the head region of exon 2 in the targeted allele is shown below together with the encoded amino acid sequence. The protein coding exons of Apc2 are indicated by black boxes. For construction of the targeting vector, an egfp-Neo cassette was inserted in frame in the signal sequence of APC2 after the first two amino acids, Met-Ala, to yield fusion to the N terminus of GFP through a linker sequence. The diphtheria toxin A (DTA) gene cassette was placed at the 3′ terminus of the homologous region in the targeting vector for negative selection. The region used as a probe for Southern blotting is indicated by a bold bar. B, Southern blot analysis of SalI and BamHI double-digested genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. C, RT-PCR analysis of total RNA from the brain of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. D, Western blot analysis of brain extracts from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice using an anti-APC2, GFP, or GAPDH antibody. E, Wild-type and homozygous Apc2-deficient E14.5 embryos. F, Wild-type and homozygous Apc2-deficient P180 mice. Scale bars: E, 1 mm; F, 2 mm.
- Figure 2.
GFP expression in heterozygous Apc2-deficient mice. A, Live fluorescent image of GFP in an E14.5 heterozygous Apc2-deficient embryo. White dotted line outlines the embryo. B, A sagittal section immunostaining of an E14.5 heterozygous Apc2-deficient embryo with an anti-GFP antibody. GFP expression was observed in the telencephalon (tc), mesencephalon (mc), spinal cord (sc), dorsal root ganglion (drg), and liver (lv). C, Immunostaining of a coronal section through the middle head of an E14 heterozygous Apc2-deficient embryo. The telencephalon (tc), retina (rt), and olfactory epithelium (oe) were positive. D, Immunostaining of a coronal section through the anterior head of an E14.5 heterozygous Apc2-deficient embryo. The olfactory epithelium (oe) was positive. E, Immunostaining of a coronal section through the middle trunk of an E14.5 heterozygous Apc2-deficient embryo. The spinal cord (sc) and dorsal root ganglion (drg) were positive. F, Immunostaining of a coronal section through the posterior trunk of an E14.5 heterozygous Apc2-deficient embryo. Neurons in the intestine (int) were positive. G, Expression of GFP in neurons but not in glial cells. Immunostaining of dissociated cultures from P0 heterozygous Apc2-deficient mice with anti-GFP (green) and anti-Tuj1 (top, red) antibodies or anti-GFAP (bottom, red) antibodies. All GFP-positive cells coexpress neuron-specific marker, Tuj1, but not glia-specific marker, GFAP. Scale bars: A, B, 1 mm; C–F, 500 μm; G, 25 μm.
- Figure 3.
Preferential expression of Apc2 in postmitotic neurons. A, Sagittal section in situ hybridization with antisense probe for Apc2 of E16.5 forebrain or P5 cerebellum of wild-type mice. Apc2 expression was observed in the region where postmitotic neurons are distributed. B, A sagittal section immunostaining of the cortex of E13.5 heterozygous Apc2-deficient mouse with anti-GFP (green) and anti-Ki67 (red), a proliferation marker. MZ, marginal zone; CP, cortical plate; IMZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. C, A sagittal section of cerebellum of P5 heterozygous Apc2-deficient mouse was stained with anti-GFP (green) and anti-Ki67 (red). D, A sagittal section immunostaining of the cortex of E13.5 heterozygous Apc2-deficient mouse with anti-GFP (green) and anti-Tuj1 (red), a postmitotic neuronal marker. E, A sagittal section immunostaining of the cortex of E13.5 heterozygous Apc2-deficient mouse with anti-GFP (green) and anti-NeuN (red), a postmitotic neuronal marker. F, A sagittal section immunostaining of the cortex of E13.5 heterozygous Apc2-deficient mouse with anti-GFP (green) and anti-doublecortin (DCX, red). G, A sagittal section immunostaining of the cortex of E13.5 heterozygous Apc2-deficient mouse with anti-GFP (green) and anti-doublecortin-like kinase (DCLK, red). H, A sagittal section immunostaining of the cortex of E13.5 heterozygous Apc2-deficient mouse was stained with anti-GFP (green) and anti-Reelin, a marginal zone marker (red). Scale bars: A, 500 μm; B–H, 50 μm.
- Figure 4.
Morphological abnormalities in the brain of Apc2-deficient mice. A, Sagittal sections of P30 cerebral cortex stained with anti-NeuN (green, neuron-specific nuclear protein). Quantification was performed by measuring the distribution of NeuN-positive cells in each bin where the cortex was divided into 10 equal areas. The cell density of NeuN-positive cells in each area was plotted as histograms for wild-type (gray bars) or Apc2-deficient (dark bars) mice. Values are shown as the mean ± SEM. The asterisk indicates a significant difference between the two values in the same area by Student's t test (*p < 0.05, **p < 0.01). B, Sagittal sections of P20 cerebellum stained with anti-NeuN (green) and anti-calbindin D-28K (red). Arrows and arrowheads indicate ectopically distributed Purkinje and granule cells, respectively. Quantification was performed by measuring the distribution of NeuN-positive cells in the molecular layer (ML) and granule cell layer (GCL). The density of NeuN-positive cells in each area was plotted as histograms of wild-type (gray bars) or Apc2-deficient (dark bars) mice. *p < 0.05, **p < 0.01. PC, Purkinje cell layer. C, Sagittal sections of P30 hippocampus stained with anti-NeuN (green). Right, Enlarged images of regions surrounded by dashed lines in left panels. Arrows indicate ectopically distributed pyramidal cells, and arrowheads indicate broadening of dentate gyrus granule cells. D, Coronal sections of P30 olfactory bulb stained with anti-NeuN (green) and DAPI (blue). Bottom, Enlarged images of regions surrounded by dashed lines in upper panels. Arrowheads indicate diffusely distributed mitral cells. Scale bars: A, B, D, 50 μm; C, 100 μm.
- Figure 5.
Enhanced apoptosis in the Apc2-deficient cortex. A, Apoptotic cells were detected in sagittal sections of E16.5 cerebral cortex by TUNEL staining. Sections were counterstained with Hoechst 33342. TUNEL-positive cells are indicated by arrowheads. B, Apoptotic cells in sagittal sections of P4 cerebral cortex. Bottom, Enlarged images of the region surrounded by dashed lines in upper left panels, in which TUNEL-positive cells are clearly visible by fluorescence. Scale bars, 50 μm.
- Figure 6.
Abnormal distributions of cortical layer markers in the Apc2-deficient cortex. Sagittal sections of the cortex at P30 immunostained for layer markers. A, Anti-CDP/CUX1 antibody (red, a layer II–IV-specific marker). B, Anti-Ctip2 antibody (red, a layer V-specific and VI-specific marker). C, Anti-FoxP2 antibody (red, layer VI-specific marker). Also, all sections were coimmunostained with an anti-NeuN antibody (green). Genotypes are indicated at the top of the panels. The cortices were divided into 10 equal areas (1–10 in each panel), and the percentage of marker-positive cells in each area was determined. Results were plotted as histograms for wild-type (gray bars) or Apc2-deficient (dark bars) mice. Anti-FoxP2 signals in layers 9 and 10 in the wild-type cortex in C are nonspecific, which are not positive for NeuN. Scale bars, 50 μm.
- Figure 7.
Impaired neuronal migration in the Apc2-deficient cortex. A, B, In vivo BrdU labeling. Wild-type and Apc2-deficient mouse brains were labeled with BrdU by injection intraperitoneally at E13.5 (A) or E16.5 (B), and fixed at P30. Sagittal sections were stained with anti-BrdU (red) and anti-NeuN (green) antibodies. Quantification was performed by measuring the distribution of BrdU-labeled cells in each bin of 10 areas. The percentage of BrdU-labeled cells in each area was plotted as histograms for wild-type (gray bars) or Apc2-deficient (dark bars) mice. C, D, Normal neuronal proliferation in the Apc2-deficient cortex. C, Immunostaining of sagittal sections of E13.5 cortex with an anti-BrdU antibody. Embryos were labeled with BrdU for 3 h before fixation. D, Immunostaining of sagittal sections of E13.5 cortex with an anti-Ki67 antibody. Scale bars, 50 μm.
- Figure 8.
Disorganization of the subplate in the Apc2-deficient cortex revealed by the altered distribution of TBR1-positive neurons. A, Sagittal brain sections at the indicated stage immunostained with anti-NeuN (green) and anti-TBR1 (red, a marker for early-born neurons). NeuN-staining revealed that the subplate (SP) was poorly formed in Apc2-deficient brains compared with wild-type brains. There is no difference in the distribution of TBR1-positive neurons at E14.5, but the distribution increased afterward in Apc2-deficient mice. B, Disorganization of the subplate. Sagittal sections of the cortex at E15.5 stained with an anti-chondroitin sulfate (CS) antibody (red) and DAPI (blue). In the wild-type cortex, a CS-positive subplate was clearly observed beneath the cortical plate (CP). In contrast, the Apc2-deficient cortex displayed diffuse and disorganized CS staining, accompanied by hypoplasia of the cortical plate. C, Immunostaining of sagittal sections of E17.5 cortex with an anti-Reelin antibody. There is no visible difference in the Reelin expression (red) in the marginal zone between wild-type and Apc2-deficient mice. Scale bars: A, E14–P0, 100 μm; P4–P20, 250 μm; B, C, 50 μm.
- Figure 9.
Migration defects of Apc2-deficient cerebellar granule cells. Wild-type and Apc2-deficient mice were labeled with BrdU at P10, and fixed after 24 (A), 72 (B), 120 (C), or 240 (D) h. Sagittal sections were immunostained with anti-BrdU (red) and anti-NeuN (green) antibodies. Quantification was performed by measuring the distribution of BrdU-labeled cells in the EGL, ML, and IGL in the cerebellum. The percentage of BrdU-labeled cells in each layer was plotted as histograms of wild-type (gray bars) or Apc2-deficient (dark bars) mice. PC, Purkinje cell layer. Scale bars, 50 μm.
- Figure 10.
Normal scaffold of radial glial fibers in Apc2-deficient brains. A, Immunostaining of sagittal sections of E14.5 cortex with an anti-Nestin antibody. There is no visible difference in the Nestin-immunoreactive radial glial fibers (green) between wild-type and Apc2-deficient mice. B, Immunostaining of sagittal sections of P20 cerebella with an anti-GFAP antibody. There is no visible difference in the GFAP-immunoreactive Bergmann glial fibers between the two. Scale bars: A, 100 μm; B, 250 μm.
- Figure 11.
Impaired rotarod performance and increased foot-stride length in the Apc2-deficient mouse. A, Behavior of wild-type (n = 7) and Apc2-deficient (n = 7) mice at ∼P70 in the rotarod test. The longest latency to fall off and the number of falls within a 3 min period at 36 rpm were measured (left and right, respectively). Values are shown as the mean ± SEM Mann–Whitney U tests showed significant differences between wild-type and mutant mice. *p < 0.05. B, Footprints and hind-limb stride lengths of wild-type (n = 7) and Apc2-deficient (n = 5) mice at ∼P70. Left, Representative footprints. Right, Distribution of hind-limb stride length. The mutant mice showed increased stride length (2.58 ± 0.63 mm in wild-type mice vs 3.07 ± 0.72 in mutant mice; the mean ± SD). Student's t test showed significant differences between wild-type and mutant mice (*p < 0.001). Scale bars, 30 mm.
- Figure 12.
Normal in vitro migration of Apc2-deficient cerebellar granule cells. A, Neuronal migration and neurite outgrowth from P4 EGL. EGL microexplants were cultured in vitro for 72 h. Migrated granule cells were detected with DAPI, and neurite outgrowth was analyzed by anti-Tuj1 staining. Scale bars: top, middle, 500 μm; bottom, 100 μm. B, Summary of the distribution of migrating granule cells. Values are shown as the mean ± SEM C, Summary of neurite outgrowth from EGL microexplants. Values are shown as the mean ± SEM.
- Figure 13.
Impaired response of Apc2-deficient CGCs to a BDNF or Slit2 gradient. Purified wild-type and Apc2-deficient CGCs were grown for 16 h in Boyden chambers with or without 30 ng/ml BDNF or 50 ng/ml Slit2. The factors were added to both compartments (uniform) or only to the lower compartment (gradient). CGCs that migrated through the porous membrane into the lower chamber were stained with DAPI and quantified. A, Representative images of migrated CGCs under distinct conditions. Scale bars, 200 μm. B, Quantification was performed by counting migrated cells per high-powered field (cells/HPF ± SEM) in 10 fields from replicate wells. Gray bars, wild-type CGCs; black bars, Apc2-deficient CGCs. The asterisk indicates a significant difference between the two values by Student's t test (*p < 0.05). C, Western blot analyses of TrkB, Robo, and GAPDH in extracts of purified CGCs.
- Figure 14.
Distribution of APC2 along microtubules and actin fibers and destabilization of microtubules in Apc2-deficient CGCs. A, B, Distribution of APC2 in cultured CGCs examined by a confocal laser scanning microscope (CLSM). APC2 was well colocalized with microtubules both in the cell soma and at the leading edge (A). On the other hand, APC2 partially colocalized with F-actin in the leading edge (B). C, D, Distribution of APC2 at the leading edge of cultured CGCs examined by a TIRFM. Signals for APC2 were observed along microtubules in the shaft of the leading edge (C). On the other hand, APC2 was well colocalized with F-actin at the periphery of the leading edge (D). E, TIRFM observation of a HEK293T cell coexpressing APC2-mCherry and Lifeact-EGFP, which visualizes F-actin. Scale bars, 5 μm. F, Western blot analyses of cytoskeletal proteins in extracts of wild-type and Apc2-deficient CGCs. Signal intensities were quantified and summarized. Values are shown as the mean ± SEM. The asterisk indicates a significant difference between the two genotypes by Student's t test (*p < 0.05).
- Figure 15.
Abnormal TrkB distribution and actin reorganization in Apc2-deficient CGCs. A, Distribution of F-actin (green) and microtubule (red). Before or after stimulation with a BDNF-gradient for 1 h, CGCs were stained with an anti-Tuj1 antibody and phalloidin. Quantification was performed by measuring the fluorescent intensity in the leading edge. s, cell soma; ld, leading edge. B, Western blot analyses of GTP-bound forms of Rac1 and Cdc42, and total Rac1 and Cdc42 in extracts of CGCs with or without 1 h BDNF-gradient stimulation. Signal intensities were quantified and summarized. C, Distribution of TrkB (red) and phosphorylated TrkB (green). Before or after stimulation with a BDNF-gradient for 1 h, CGCs were immunostained with anti-TrkB and anti-phosphorylated TrkB antibodies. Quantification was performed by measuring the fluorescent intensity in the leading edge. D, Quantification of immunohistochemical staining of TrkB and pTrkB in the cell soma. Values are shown as the mean ± SEM E, Ratio of TrkB fluorescent intensity at the leading edge to the total cellular TrkB fluorescent intensity. Values are shown as the mean ± SEM. The asterisk indicates a significant difference between the two values by Student's t test (A, C–E) or ANOVAs (B) (*p < 0.05, **p < 0.01). Scale bars, 5 μm.
- Figure 16.
Distribution of pTrkB in the inner EGL of cerebellar sections from P10 wild-type and Apc2-deficient mice. Sections were stained with an anti-pTrkB antibody (red) and DAPI (blue). Bottom, Enlarged images of the regions surrounded by dashed lines in upper panels. Arrows and arrowheads indicate representative CGCs with and without pTrkB polarization, respectively. Quantification was performed by calculating percentage of CGCs with pTrkB polarization. Values are shown as the mean ± SEM. The asterisk indicates a significant difference between the two values by Student's t test (**p < 0.01). Scale bar, 25 μm.
