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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.

^* Author for correspondence (e-mail: ohnos{at}med.yokohama-cu.ac.jp)

Accepted 16 February 2006

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKC, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKC knockout mice, neuroepithelial cells of the neocortical region lost aPKC protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKC is indispensable for the maintenance of adherens junctions and may function in the regulation of adherens junction integrity upon differentiation of neuroepithelial cells into neurons. In spite of the loss of adherens junctions in the neuroepithelium of conditional aPKC knockout mice, neurons were produced at a normal rate. Therefore, we concluded that, at least in the later stages of neurogenesis, regulation of cell cycle exit is independent of adherens junctions.

Key words: aPKC, Cell polarity, Adherens junction, Neurogenesis, Brain

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the central nervous system of mammals, neuroepithelial cells serve as neural progenitors, differentiating into either neurons or glial cells (Ever and Gaiano, 2005; Gotz and Huttner, 2005; Haubensak et al., 2004; Miyata et al., 2001; Noctor et al., 2001). In this paper, we use the term `neuroepithelial cells' to indicate both actual neuroepithelial cells and radial glial cells. In mouse neocortex, neurogenesis precedes gliogenesis and commences around embryonic day 11 (E11), peaks around E15 and finishes around birth (Jacobsen, 1991). Newly generated neurons migrate towards the pial side, forming the six-layered neocortex (Gupta et al., 2002; Marin and Rubenstein, 2003). Neuroepithelial cells form a well-packed cell layer called the ventricular zone and line the lateral ventricles. Although the ventricular zone looks like multiple cell layers, most cells face both the ventricular/apical and pial/basal surfaces of this cell layer with processes extending from the cell body. Basal side cellular processes are elongated towards the pial surface of the telencephalic vesicles, serving as a radial scaffold for the migration of newly generated neurons. However, apical side cellular processes are elongated towards the ventricular surface to form adherens junctions (Aaku-Saraste et al., 1996; Astrom and Webster, 1991).

Once neurogenesis starts, neuroepithelial cells undergo asymmetric cell division producing daughter cells with a different cell fate; one keeps the characters of neuroepithelial cells, while the other differentiates into a post-mitotic neuron or an intermediate progenitor, which produces two neurons after the next cell division in the subventricular zone (Haubensak et al., 2004; Miyata et al., 2004; Noctor et al., 2004). Importantly, post-mitotic neurons and intermediate progenitors lose adherens junctions and move out from the ventricular zone. Thus, the loss of either adherens junctions or the apical domain defined by those junctions could represent a cue for the differentiation of neuroepithelial cells (Kosodo et al., 2004; Wodarz and Huttner, 2003). In support of this notion, asymmetric inheritance of the components of adherens junctions is occasionally observed with cells dividing at the apical surface of the ventricular zone (Chenn et al., 1998; Manabe et al., 2002). However, neither the importance of adherens junctions to differentiation nor the molecular mechanisms for differentiation-dependent regulation of the integrity of adherens junctions in neuroepithelial cells has been elucidated.

A subgroup of protein kinase C (PKC), atypical PKC (aPKC) regulates cell polarity in several organisms (Macara, 2004; Ohno, 2001). aPKC forms a complex with cell polarity proteins PAR3 and PAR6, localizes predominantly at tight junctions in mammalian epithelial cells, and its kinase activity is required for the establishment of apicobasal cell polarity and the formation of tight junctions (Hirose et al., 2002; Suzuki et al., 2001; Yamanaka et al., 2001). Drosophila genetic studies have shown that aPKC, along with PAR3 and PAR6 are responsible for junction formation in epithelial cells and for cell fate determination in neuroblasts (Kuchinke et al., 1998; Petronczki and Knoblich, 2001; Rolls et al., 2003; Wodarz et al., 2000; Wodarz et al., 1999). In mammalian neural tissues, aPKC localizes with PAR3 and PAR6 at the adherens junctions of embryonic neuroepithelial cells (Manabe et al., 2002). However, the role of aPKC during mammalian neurogenesis remains unknown.

To explore the role of aPKC in the development of mouse neocortex, we inactivated the gene for aPKC, one of two aPKC isotypes, by employing a Nestin promoter and an intronic enhancer-driven Cre-mediated conditional gene targeting system. The phenotype of the mutant mice indicates that aPKC is indispensable for neuroepithelial cells to form adherens junctions and maintain cell polarity in the neuroepithelium. However, aPKC is not required for cell cycle exit and the subsequent radial migration in developing neocortex.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice
Mice harboring a floxed aPKC gene in which exon 5 was flanked by loxP sequences were generated by homologous recombination (Hashimoto et al., 2005; Koike et al., 2005; Matsumoto et al., 2003). Nestin-Cre mice (T.S., unpublished) harboring tandem arrays of the Cre gene driven by a rat nestin promoter (Zimmerman et al., 1994), the internal ribosome entry site (IRES) sequence, the lacZ gene and a nestin neuron-specific enhancer (Zimmerman et al., 1994).

Detection of ß-galactosidase activity
Any ß-galactosidase expressed from Rosa26R gene that lost the suppressor neo-cassette following cre-mediated recombination was detected in frozen sections fixed using 4% PFA for 10 minutes by staining with X-gal according to the standard protocol. The Nestin-Cre transgene bears the lacZ gene; however, the expression of ß-galactosidase from this lacZ gene was negligible compared with that from the Rosa26R gene (data not shown).

Western blot analysis
Telencephalic vesicles from E13.5 or E15.5 embryos were cut out in ice-cold PBS, the meninges removed and vesicles homogenized in 1 ml of SDS-PAGE sample buffer. Samples were appropriately diluted to provide equal protein amounts and used for SDS-PAGE. Western blot analysis was performed according to standard protocols using the following antibodies: anti-aPKC antibody 1/1000 (clone 23, BD); anti-aPKC antibody 1/1000 (rabbit, Santa Cruz); and anti-ß-actin 1/2000 (AC-15, Sigma). For secondary antibodies, horseradish peroxidase was conjugated with anti-rabbit or - mouse IgG 1/2000 (goat, Amersham). Enzyme activity was detected using an ECL system (Amersham) and luminescence was quantified using a FUJI Las 3000 luminescence image analyzer (Fuji Photo Film, Tokyo). All images were arranged and labeled using Photoshop 7.0 (Adobe Systems).

Immunostaining
Embryos were fixed with 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for paraffin wax-embedded sectioning (5-6 µm). When required, BrdU was injected intraperitoneally into pregnant mice at the time points indicated in the text. Paraffin sections were hydrolyzed and heated at 120°C for 20 minutes in 10 mM sodium citrate (pH 6.0). For staining with MAP2, CSPG neurogenin 2 antibody, embryos were frozen directly in OCT compound and sectioned at 8 µm. Frozen sections were then fixed with a methanol/acetone 1:1 mix at -20°C for 10 minutes and air-dried. Immunostaining was performed according to standard protocols using 10% normal goat serum in TBST [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20] as a blocking reagent, and primary and secondary antibodies were diluted in 0.1% BSA/1.5% normal goat serum/TBST, as follows. Primary antibodies: affinity purified anti-aPKC rabbit antibody 1/1000 (3) (Suzuki et al., 2001); anti-N-cadherin 1/1000 (clone 32, BD); anti-ß-catenin 1/1000 (clone 14, BD); anti ZO-1 1/1000 (clone ZO1-1A12, Zymed); anti-PAR-6ß 1/500 (BC32AP) (Yamanaka et al., 2003); anti-ASIP/PAR-3 1/1000 (C2-3) (Hirose et al., 2002); anti--tubulin 1/500 (rabbit, Sigma); anti-BrdU 1/1000 (clone 2B1, MBL); anti-phospho-histone H3 1/1000 (rabbit, Upstate); anti-ßIII-tubulin 1/2000 (clone TuJ1, Babco); anti-Ki67 1/200 (rabbit, Novocastra); anti-CSPG 1/1000 (clone CS-56, Sigma); anti-MAP2 (clone HM-1; Sigma) and anti-neurogenin 2 1/50 (clone 5C6) (Lo et al., 2002). Secondary antibodies: Cy3-conjugated anti-rabbit or -mouse IgG 1/2000 (goat, Amersham); Alexa488-conjugated anti-rabbit or -mouse IgG (goat, Molecular Probes); and TOPRO-3 1/100 (Molecular probes). DAPI (2.5 µg/ml, Sigma) was included in the final wash buffer (TBST) for nuclear staining. For immunohistochemistry, biotinylated secondary anti-mouse IgG antibodies 1/1000 (goat, Vector) were detected using a Vectastain Elite ABC kit (Vector). Images were captured using a BX50 fluorescent microscope (Olympus) equipped with a CCD camera (Photometrics) or an LSM510 (Zeiss). All images were arranged and labeled using Photoshop 7.0 (Adobe Systems). Some paraffin sections were stained using Carazzi's hematoxylin (Muto Pure Chemicals) and Eosin B (Sigma) or with 0.5% Cresyl Violet.

Electron microscopy
Samples were fixed in 1% glutaraldehyde/4% PFA in 0.1 M sodium cacodylate buffer overnight at 4°C, post-fixed with 2% osmium in 0.1 M sodium cacodylate buffer for 2 hours at 4°C, and processed for Epon embedding. Sections were examined at 75 kV using an H-7500 transmission electron microscope (Hitachi).

Slice culture analysis
Coronal slices of E15.5 embryonic telencephalon (200-300 µm) were prepared and cultured in collagen gel, as previously described (Miyata et al., 2004). To examine cell shapes, dissected telencephalon was immersed for 10 seconds in DiI suspension (1 mg/ml culture medium), DiI was deposited on the pial surface prior to making the slices and the dye was left to diffuse for more than 2 hours. Time-lapse photographs were taken by hand.

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nestin-Cre-mediated conditional disruption of the aPKC gene
Homozygous aPKC allele lacking exon 9 (PKC^9/9) mice show lethality at an early embryonic stage (Soloff et al., 2004). To assess the function of aPKC in neocortical neurogenesis, we generated Nestin-Cre-mediated conditional aPKC knockout mice (aPKC cKO). In the conditional aPKC allele (PKC^floxed), exon 5 of the aPKC gene is flanked by loxP sequences. As exon 5 is 86 bp long, deletion results in a frame-shift mutation upstream of the kinase domain-coding sequence (Hashimoto et al., 2005; Koike et al., 2005; Matsumoto et al., 2003). This conditional allele can thus be converted into a strong loss-of-function allele (PKC⁵) by Cre recombinase-mediated recombination. With the Nestin-Cre allele, Cre recombinase express under the control of the rat Nestin promoter and intronic enhancer (T.S., unpublished). To generate aPKC cKOs carrying the aPKC floxed allele in homo and Nestin-Cre allele in hetero (PKC^{floxed/floxed}; Nestin-Cre), we bred mice carrying the aPKC floxed allele in homo (PKC^{floxed/floxed}) and mice carrying the aPKC floxed allele in hetero and Nestin-Cre allele in hetero (PKC^floxed/+; Nestin-Cre). PKC^floxed/+; Nestin-Cre mice found as littermates of the aPKC cKO mice are viable, fertile and show no obvious abnormalities, similar to the heterozygous aPKC mutant (PKC^5/+) mice (data not shown) that were used as controls for most of the experiments presented in this manuscript. Genotypes and numbers of newborn pups obtained from this breeding program were 21 for aPKC cKO (PKC^{floxed/floxed}; Nestin-Cre), 13 for PKC^{floxed/floxed}, 25 for PKC ^floxed/+; Nestin-Cre and 19 for PKC floxed/+, most fitting the expected ratio of 1:1:1:1. Although for some unknown reason the number of PKC ^{floxed/floxed} pups was somewhat lower than the expected value, comparable numbers of aPKC cKO (PKC ^{floxed/floxed}; Nestin-Cre) and control (PKC^floxed/+; Nestin-Cre) pups were obtained; indicating that the most aPKC cKO mice can survive during embryonic and early postnatal days. These aPKC cKO mice were healthy and indistinguishable from littermates up to 5 days after birth, although after this point growth retardation started to be evident. Thereafter, all aPKC cKO mice died within 1 month of birth and all displayed hydrocephalus (21/21, 100%).

To test the tissue specificity of the Nestin-Cre-mediated recombination, Nestin-Cre transgenic mice were crossed with Rosa26R reporter mice in which a floxed neo cassette interrupts the ubiquitous expression of the lacZ gene (Soriano, 1999). In Rosa26R mouse embryos carrying the Nestin-Cre transgene, Cre-mediated recombination monitored by the expression of ß-galactosidase activity was specifically detected in the central nervous system of E15.5 embryos, including in the telencephalon, diencephalon, mesencephalon, metencephalon, myeloncephalon and spinal cord. In the telencephalon, recombination was detected in the ganglionic eminence and rostral neocortial region, but no recombination was evident at this stage in the caudal neocortical region, including the hippocampus (Fig. 1A).

We then tested if aPKC protein was abolished in embryonic telencephalon of aPKC cKO mice. Western blot analysis using a specific antibody against aPKC demonstrated a substantial reduction of aPKC protein from telencephalic vesicles at E15.5, while the protein was still present at E13.5 (Fig. 1B, upper). Remnant aPKC protein would have originated from the caudal region of the telencephalon, where Cre-mediated recombination did not proceed even at E15.5 (Fig. 1A). As the activity of Cre recombinase monitored by lacZ expression from the modified Rosa locus had been detected in a large part of E13.5 brain (data not shown), the subtle decrease in the amount of aPKC protein at E13.5 might indicate the relatively low susceptibility of the aPKC locus to Cre recombinase or the long half-life of aPKC protein.

As previously reported, aPKC protein was highly concentrated in the apical ridge of the ventricular zone, predominantly in the adherens junctions of neuroepithelial cells (Manabe et al., 2002). When localization of aPKC was examined under immunofluorescent microscopy, aPKC protein was undetectable in the anterior neocortical region of aPKC cKO mouse embryos at E15.5, while apical localization of this protein was obvious in control embryos (Fig. 5A,B).

View larger version (48K): [in this window] [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.

Loss of aPKC causes disruption of neuroepithelial tissue architecture in the telencephalon
To examine the effects of aPKC disruption on the gross morphology of the neocortical region, histological analysis was initially performed under light microscopy. At E13.5, when the expression of aPKC protein was not severely impaired, no morphological abnormalities were observed in the neocortical regions of aPKC cKO embryos (data not shown). Abnormalities became evident at E15.5, when aPKC protein in this region decreased to undetectable levels. In control embryos, the ventricular zone comprised uniformly packed neuroepithelial cells and had a smooth ventricular surface (Fig. 2A). By contrast, the ventricular surface in aPKC cKO embryos was rough and neuroepithelial cells were loosely packed. The ventricular zone was thus barely distinguishable from the subventricular zone (Fig. 2E). Nevertheless, the subplate and cortical plate were comparable with these layers in control embryos, and little abnormality was apparent in the ventricular zone at the caudal neocortical region where apical localization of aPKC protein was maintained (Fig. 5E, data not shown). Morphological abnormalities became more evident at E16.5. The ventricular and subventricular zones were severely disorganized, and the ventricular, subventricular and intermediate zones were difficult to distinguish (Fig. 2C,G). Gross death of neuroepithelial cells was considered unlikely as the primary cause of this morphological abnormality, as no significant increase in the number of cells undergoing apoptosis was observed (see Fig. S1 in the supplementary material) and the total number of proliferating cells as assessed by the number of cells in S-phase was not significantly changed in the neocortical region of aPKC cKO embryos (Fig. 3C). Instead, a dispersion of neuroepithelial cells into the outer layers, and the subventricular and intermediate zones might have caused these abnormalities. This possibility is supported by the unusual distribution of TuJ1-positive differentiated neurons. In control embryos, TuJ1-positive cells were for the most part excluded from the ventricular and subventricular zones (Menezes and Luskin, 1994). However, in aPKC cKO embryos, TuJ1 staining was found in all layers of the neocortical region (Fig. 2H). To identify the somata of TuJ1-positive differentiated neurons, we stained sections with an antibody for the proliferation marker Ki67 and TuJ1. In such staining, Ki67-negative nuclei surrounded by TuJ1 staining represented somata of differentiated neurons found in the most apical region in aPKC cKO embryos (Fig. 2H'). In contrast to the severe disorganization of ventricular-side layers of the neocortical region, pial-side layers comprising the subplate and cortical plate were unaffected, and staining with chondroitin sulfate proteoglycans and MAP2, as markers for the subplate and cortical plate, respectively, yielded a pattern indistinguishable from that in control embryos (Fig. 2C,G; see Fig. S2 in the supplementary material). The abnormality was more prominent in the ganglionic eminence, where the loss of aPKC took place earlier than in the neocortical region. Typically, fusion of the right and left medial ganglionic eminences was observed at E13.5, and at E16.5 irregular protrusions formed at the ventricular surface and the anterior horn of the lateral ventricle was filled with neuroepithelial cells and neurons (Fig. 2F, see Fig. S3 in the supplementary material). These results suggest that aPKC is indispensable for neuroepithelial cells to form pseudostratified epithelium in the ventricular zone.

View larger version (91K): [in this window] [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.

Another common feature of cells in pseudostratified epithelium is the presence of basal and apical cellular processes extending to the pial and ventricular surfaces, respectively (Astrom and Webster, 1991; Miyata et al., 2001). Although the length of each process changes depending on the interkinetic nuclear migration, centrosomes are always anchored at the tip of the apical process, except in mitotic round-up cells, so centrosomes are apically localized in pseudostratified epithelium (Astrom and Webster, 1991). In aPKC cKO embryos, centrosomes were dispersed throughout the ventricular zone and subventricular zone with minor apical localization, indicating a considerable degree of cell-shape changes (Fig. 4A). When neuroepithelial cells in control embryos were labeled applying DiI from the pial surface, most cells displayed cellular processes extending to both the pial and ventricular surfaces (left panels in Fig. 4B,C). In aPKC cKO embryos, most apical cellular processes were shortened and detached from the ventricular surface (right panels in Fig. 4B,C).

View larger version (50K): [in this window] [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 this window] [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.

Disruption of aPKC results in the disappearance of neuroepithelial adherens junctions

Disruption of aPKC does not affect neurogenesis and radial migration
Previous studies have shown that aPKC is involved in the cell fate determination of neuroblasts in the Drosophila central nervous system (Wodarz et al., 2000, Rolls et al., 2003). In mammals, loss of adherens junctions presumably causes the loss of polarity cue in neuroepithelial cells, and, depending on asymmetric cell division, affects neurogenesis (Wodarz and Huttner et al., 2003). In the neocortex of aPKC cKO mice at postnatal day (P) 3, lateral ventricles were swollen as in the hydrocephalus, but no serious abnormalities in the laminated structure of the neocortex were found (Fig. 6B). The apparently normal structure of the neocortex at P3 suggests that neurogenesis and the radial migration that follows are unaffected by the loss of aPKC. To further test this possibility, neurons produced at around E15.5 or E17.5 were labeled with BrdU, and the position of these neurons in the neocortex was examined at P3. In both control and aPKC cKO neocortex, neurons labeled at E17.5 were located in the outermost layer of the neocortex adjacent to the marginal zone (layers II or III of mature neocortex) at P3, and neurons labeled at E15.5 were located slightly closer (Fig. 6C,D). These results indicate that radial migration was unaffected in aPKC cKO mice; moreover, no obvious differences were identified in the numbers of labeled cells, possibly reflecting the rate of neurogenesis at E15.5 or E17.5. However, directly comparing numbers of labeled neurons was difficult because of the enlarged shape of the aPKC cKO mouse telencephalon. To clarify this point, the rate of neurogenesis was monitored by measuring the cell cycle exit rate (Chenn and Walsh, 2002), as follows. BrdU was loaded at E15.5 and brains were fixed at E16.5 for immunostaining using antibodies against BrdU and Ki67, a protein marker for proliferating cells. Ki67-negative cells in the BrdU-positive cell cohort thus correspond to cells that have exited the cell cycle and differentiated in the period between E15.5 and E16.5 (Fig. 7A,B). The ratio of Ki67-negative/BrdU-positive cells to all BrdU-positive cells was about 50% in the neocortical region of both control and aPKC cKO embryos (Fig. 7C). We then compared the expression of neurogenin 2, an early differentiation marker, which is essential for the commitment to neuronal linage (Kageyama et al., 2005; Ross et al., 2003). Again, no significant changes in the ratio of neurogenin 2-positive nuclei were observed among the total number of nuclei in the ventricular zone and subventricular zone of E15.5 aPKC cKO and control embryos (Fig. 7D-F). Notably, the number of GFAP-positive glial cells in the neocortex at E15.5, E17.5 and P3 remained unchanged with the loss of aPKC (data not shown). Moreover, neuroepithelial cells from the neocortical region of aPKC cKO embryos at E15.5 or E16.5 proliferated in suspension culture forming neurospheres, and these cells retained the potential to differentiate into neurons and glia (data not shown). Taken together, these results indicate that aPKC is not essential for the neurogenesis of neocortical development, at least in the later stages after E15.5.

View larger version (77K): [in this window] [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 this window] [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 this window] [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.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Adherens junctions is not required for mammalian neurogenesis
The loss of adherens junctions is a natural cellular event observed when neuroepithelial cells differentiate into neurons or a certain cohort of progenitor, intermediate progenitor. Recent reports have described that adherens junction-free progenitors dividing at the subventricular zone or intermediate zone undergo symmetric cell divisions producing two neurons in the most cases (Miyata et al., 2004; Noctor et al., 2004). Adherens junctions play a crucial role in the maintenance of proliferative status in a variety of stem cells, including Drosophila germinal cells, mouse hematopoietic stem cells and epidermal cells by maintaining the stem cell niche (Fuchs et al., 2004; Song et al., 2002; Yamashita et al., 2003). For example, expression levels of adherens junction components are important for generating hair follicles and the inactivation of -catenin impairs hair follicle morphogenesis and adherens junctions (Jamora et al., 2003; Vasioukhin et al., 2001). This has been predicted to remain true for neuroepithelial cells, the neural stem cells. However, if adherens junctions play a crucial role in the proliferative status in neuroepithelial cells, loss of adherens junctions may cause temporal overproduction of neurons, resulting in a considerable decrease in neuroepithelial cell numbers. In fact, loss of adherens junctions has also been reported in conditional ß-catenin knockout and Lgl1 knockout mice (Klezovitch et al., 2004; Machon et al., 2003). In such cases, decreases or increases in cell proliferation rate have been reported in addition to the disorganization of the neuroepithelium in the neocortical region or ganglionic eminence. However, the present results indicate that adherens junctions are not required for either maintenance of proliferative status or cell cycle exit in the mouse neocortex, at least at the late stage of neurogenesis. This is supported by three observations. First, the total number of BrdU-labeled S-phase cells was not altered in neocortical regions where adherens junctions were lost. Second, the cell cycle exit rate reflecting the rate of neural cell differentiation and the neurogenin 2-positive cell rate were also unaffected. Third, neurons produced 2 days after the loss of adherens junctions (E17.5) were found in normal positions in the laminated neocortex after birth. These different effects of the loss of adherens junctions on cell proliferation may reflect the function of ß-catenin and Lgl1, rather than the maintenance of the adherens junction structure, as ß-catenin acts as a component of the transcription factor required for induction of Wnt signaling-dependent cell proliferation (Chenn and Walsh, 2002), and Lgl is characterized as a tumor suppressor gene (Bilder et al., 2000). Adherens junctions were unaffected until E14.5 in the neocortical region of aPKC cKO embryos, as aPKC protein was still present until this time. Thus, adherens junctions might play essential roles in the maintenance of proliferative status or cell cycle exit of neuroepithelial cells at stages earlier than E14.5.

aPKC regulates integrity of adherens junctions
Although the presence of adherens junctions does not affect neurogenesis as discussed above, the integrity of adherens junction may be regulated by a mechanism that accords to cell fate determination. One possibility is that the orientation of the cleavage plate regulates adherens junction integrity. When the cleavage plate is oriented parallel to the ventricular surface, a daughter cell located distal to the surface may inherit few or no adherens junctions (Chenn et al., 1998; Manabe et al., 2002). However, recent studies have revealed that this type of cell division is rather rare in developing mouse neocortex (Haydar et al., 2003) and that only one daughter cell might inherit adherens junctions even when the cleavage plate is oriented perpendicular to the ventricular surface (Kosodo et al., 2004). In this case, differentiation of neuroepithelial cells into intermediate progenitors or neurons may trigger the destruction of adherens junctions. Our finding that the inactivation of aPKC causes a loss of adherens junctions suggests the involvement of this protein kinase in such a differentiation-dependent regulation of adherens junctions.

The molecular mechanisms underlying the regulation of adherens junctions by aPKC are largely unknown. In epithelial cells, aPKC regulates the integrity of tight junctions with its binding partners, PAR3 and PAR6 (Macara, 2004; Ohno, 2001). PAR6 binds to Pals1, Lgl1 and Lgl2, and last two are the substrate for aPKC (Hurd et al., 2003; Plant et al., 2003; Yamanaka et al., 2003). PAR3 is also phosphorylated by aPKC, with the phosphorylation of these proteins being a crucial step for the establishment and probably also for the maintenance of tight junctions in epithelial cells (Hirose et al., 2002; Nagai-Tamai et al., 2002; Yamanaka et al., 2003). Although neuroepithelial cells lose tight junctions in their early embryonic stages, the remaining adherens junctions retain many of these aPKC-binding proteins (Aaku-Saraste et al., 1996; Astrom and Webster, 1991). Some common molecular mechanisms may therefore act in the establishment of epithelial tight junctions and in the maintenance of neuroepithelial adherens junctions. This notion is in part supported by the observation that Lgl1, Pals1 and PAR3, as well as aPKC play crucial roles in the maintenance of adherens junctions in the neuroepithelium of the mouse telencephalon or zebrafish retina (Klezovitch et al., 2004; Wei et al., 2004; Wei and Malicki, 2002).

The role of aPKC on mammalian neurogenesis
In Drosophila neuroblasts, aPKC regulates cell fate by modifying cell polarity and proliferation (Rolls et al., 2003; Wodarz et al., 2000). As Drosophila neuroblasts lose adherens junctions upon delamination from the neuroectoderm, these functions of aPKC are independent of the maintenance of adherens junctions. Mouse aPKCs could also display such adherens junction-independent functions to regulate the differentiation in neuroepithelial cells. As aPKC cKO embryos exhibited no significant alteration of neurogenesis as described above, the argument could be made that another aPKC member protein, aPKC, may display such functions. However, a major form of aPKC expressed in neural tissue of both aPKC cKO and control embryos is PKM, which lacks the N-terminal PB1 domain essential for interaction with PAR6 (Hirano et al., 2005; Suzuki et al., 2001; Wilson et al., 2003). The functions of PKM might thus be more restricted than intact aPKC or aPKC. In any case, a definitive answer for the issue of whether aPKCs regulate neural cell differentiation in mammals will be provided by analyzing the phenotype of aPKC/aPKC double-knockout mice.

Radial migration was not significantly affected in aPKC cKO embryos, and the radial glia scaffold comprising the basal cellular processes of neuroepithelial cells was normally aligned, even though the apical process was retracted (Fig. 4B,C). In contrast to the apical processes, in which integrity was totally dependent on adherens junctions, basal processes were probably maintained by interactions with the basal lamina at the pial surface and with differentiated neurons located in the subplate and cortical plate. Conversely, some reports have indicated the involvement of aPKCs in neural cell migration (Jossin et al., 2003; Solecki et al., 2004). Although the present data show little contribution of aPKC to radial migration, a signaling pathway to regulate cell migration could be driven by PKM and residual amounts of aPKC found in the aPKC cKO embryonic brain.

The loss of aPKC eventually causes brain malformation characterized by hydrocephalus and loss of the ependymal layer along with most of the striatum, which becomes prominent at P3 or later (Fig. 6B). The overall structure of the neocortex was relatively well maintained even in the absence of the ependymal layer, probably because neural fiber enriched layers such as cortical plate, subplate and intermediate zone defend neocortical cell layers against the gross disorganization observed in the striatum. In other words, brain malformation in aPKC cKO mice indicates that the maintenance of neuroepithelial tissue architecture by adherens junctions is essential for mechanical support of the developing brain, although the process is largely dispensable for neural cell differentiation and migration in the late embryonic stages. Moreover, appropriate regulation of aPKC activity could contribute to morphogenesis in brain ontogeny.

	ACKNOWLEDGMENTS

 
We thank David Anderson for the neurogenin 2 antibody; Noriko Ohsumi for the neurogenin 2 staining; Tomonori Hirose, Masa-aki Nakaya and Tomoyuki Yamanaka for their helpful comments; and Atsumi Kawaguchi and Yumi Bamba for animal care. F.I. was supported by Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists. This work was also supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.O.).

	Footnotes

 
Supplementary material

Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/9/1735/DC1

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