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First published online 29 March 2006
doi: 10.1242/dev.02330

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Inactivation of aPKC results in the loss of adherens junctions in neuroepithelial cells without affecting neurogenesis in mouse neocortex

¹ Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan.
² College of Nursing, Yokohama City University Graduate School of Medical Science, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan.
³ Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan.
⁴ Laboratory for Cell Culture Development, Brain Science Institute, RIKEN, Saitama 351-0198, Japan.
⁵ Pharmaceutical Development Department, Meiji Dairies Co., 540 Naruda, Odawara, Kanagawa 250-0862, Japan.
⁶ National Institute for Basic Biology, National Institute of Natural Sciences Laboratory of Neurochemistry, Center for Transgenic Animals and Plants, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
⁷ Department of Molecular Genetics, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8575, Japan.

View larger version (48K): [in a new window]   Fig. 1. Nestin-Cre mediated conditional disruption of aPKC gene. (A) Activity of ß-galactosidase in Nestin-Cre;Rosa26R embryo on a sagittal section at E15.5 is detected by X-gal staining to show tissue specificity of Cre-recombinase activity. Cre activity is relatively low in the caudal neocortical region (arrowhead indicates caudal and rostral boundary). (B) Proteins extracted from the telencephalon at E13.5 and E15.5 were analyzed by western blotting using an aPKC-specific antibody (upper panel) or antibody recognizing all three aPKC members: aPKC, aPKC and PKM (middle panel). Positions of aPKC (70 kDa), aPKC (70 kDa) and PKM (55 kDa) are indicated by arrowheads. Signals detected with ß-actin antibody served as internal controls for equal protein loading (lower panel). Te, telencephalon; Di, diencephalon; Me, mesencephalon; Mt, metencephalon; My, myelencephalon; Sc, spinal cord; nc, neocortical region; ge, ganglionic eminence. Scale bar: 1 mm.

View larger version (91K): [in a new window]   Fig. 2. Loss of aPKC disrupts neuroepithelial pseudostratified structure. Coronal sections of telencephalon in control embryos (A-D) and aPKC cKO embryos (E-H) were stained using Hematoxylin and Eosin (A-C,E-G), with anti-ßIII tubulin antibody (TuJ1, green) and Ki67 (red) (D,H), or with TuJ1 (green) and DAPI (magenta) (D',H'; arrow indicates TuJ1 staining surrounding and Ki67-negative differentiated neuron). Brains were dissected at E15.5 (A,E) or 16.5 (B-D,F-H). Higher magnification view of boxed areas in B and F are shown in C and G. aPKC cKO embryos display abnormal protrusions in the ganglionic eminence (arrowheads in F) and lack the anterior horn of the lateral ventricle (arrow in F). Tuj1-positive cells locate at the ventricular surface (arrows in H). Scale bars: 100 µm in A,C,E,G; 1 µm in B,F; 10 µm in D,H.

View larger version (50K): [in a new window]   Fig. 3. Loss of aPKC impairs cell cycle-dependent nuclear positioning. (A,B) Nuclei of cells in S-phase of cell cycle are labeled using BrdU antibody (red), and mitotic cells are labeled with phospho-histone H3 antibody (green) on confocal sections of the neocortical region. Brains were dissected from control (A) and aPKC cKO (B) embryos at E15.5, 2 hours after administration of BrdU. Scale bars: 10 µm. (C-E) Distributions of BrdU positive or mitotic cell nuclei in each layer were quantified and graphed. Total numbers of BrdU-positive nuclei in neocortical layers are not significantly changed by loss of aPKC (C). The large majority of BrdU-positive nuclei in aPKC cKO and control embryos are located in the subventricular zone and ventricular zone (SVZ/VZ) (D). With the SVZ/VZ divided into three layers, differences in distribution of BrdU-labeled nuclei in each layer are obvious between control and aPKC cKO embryos (D). *P<0.01. Cells dividing in non-surface areas are greatly increased in aPKC cKO embryos (E).

View larger version (85K): [in a new window]   Fig. 4. Loss of aPKC causes retraction of apical process in neuroepithelial cells. (A) Immunofluorescence for -tubulin (red) and nuclei (blue) on confocal sections of the neocortical region in control (left panel) and aPKC cKO embryos (right panel) at E15.5. (B) Cell shape of neuroepithelial cells was examined by DiI labeling. Slice cultures were prepared with telencephalic vesicles of control (left panels) and aPKC cKO embryos (right panels) at E15.5. Phase-contrast images of slice cultures are also shown (phase). Broken white lines indicate pial surface of slice culture, and the ventricular surface is shown by broken black lines. Asterisks indicate pial end of cell process with DiI mass. Arrowheads indicate position of cell body, and an arrow indicates retracted apical cellular process in slice culture prepared from an aPKC cKO embryo. (C) Quantitation of neuroepithelial cell morphology. Most cells attach to the ventricular surface in control embryos; however, these cells are rare in aPKC cKO embryos. (D) Time-lapse image of a DiI-labeled neuroepithelial cell in a slice culture prepared from aPKC cKO embryo at E15.5. Apical tip of a cellular process indicated by arrows is detached from the ventricular surface and shortened progressively. LV, lateral ventricle. Scale bars: 10 µm.

View larger version (77K): [in a new window]   Fig. 5. Loss of aPKC results in disappearance of neuroepithelial adherens junctions. (A-D) Immunofluorescence for aPKC (red), ß-catenin (green) and nuclei (blue) on confocal sections of the neocortical region in control (A,C) and aPKC cKO embryos (B,D) at E15.5. Immunofluorescence of ß-catenin alone is shown in C and D. Dot-like signals of ß-catenin are constantly seen in control embryos (A,C; arrowheads), but are rare in aPKC cKO embryos (B,D; arrow). (E-H) Electron micrographs of the ventricular surface of neuroepithelium in E15.5 aPKC cKO embryos. (G,H) High-magnification views. Adherens junctions (electron dense lines indicated by arrowheads) are constantly observed in the caudal neocortical region where aPKC is still retained (E,G), while only fragmented adherens junctions (arrow) are rarely observed in the rostral region where aPKC is predominantly lost (F,H). LV, lateral ventricle. Scale bars: 10 µm in A,B; 1 µm in E,F.

View larger version (125K): [in a new window]   Fig. 6. Loss of aPKC does not severely alter radial migration. (A,B) Nissl staining of coronal sections from rostral telencephalon in control (A) and aPKCl cKO mice (B) at P3. (C,D) Birth-date analysis of cortical neurons. BrdU was administrated at E15.5 (C) or E17.5 (D) and sections were made at P3. No obvious differences in distribution of BrdU-labeled cells (brown) are observed between control embryos (left panel) and aPKCl cKO embryos (right panel). Scale bars: 250 mm in A,B; 100 µm in C,D.

View larger version (96K): [in a new window]   Fig. 7. Loss of aPKC does not severely alter neurogenesis. (A,B) Coronal section of E16.5 cortex from control (A) and aPKC cKO embryos (B) are stained with antibodies against Ki67 (green) and BrdU (red). BrdU was administrated 24 hours before fixation at E15.5. Arrowheads indicate cell cycle exit cells (BrdU-positive/Ki67-negative). Arrows indicate re-entry cell cycle cells (BrdU-positive/Ki67-negative). (C) The cell cycle exit rate was calculated by dividing the number of BrdU-positive/Ki67-negative cells by the total number of BrdU-positive cells. Results from two independent experiments are shown. Cell cycle exit rate at E15.5 are not significantly affected by the loss of aPKC. (D,E) Coronal section of E16.5 cortex from control (D) and aPKC cKO (E) embryos are stained with antibodies against neurogenin 2 (green) and DAPI (blue). (F) The population of neurogenin 2-positive cells in VZ, SVZ, IZ and SP were quantified and graphed. The rate of neurogenin 2-positive cells is not significantly changed by loss of aPKC. Scale bars: 100 µm in A,B; 25 µm in D,E.