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Genes to Cells (2007) 12, 983-996. doi:10.1111/j.1365-2443.2007.01113.x
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Pax6 transcription factor is required for the interkinetic nuclear movement of neuroepithelial cells

 Hiroshi Tamai1, Hiroshi Shinohara1, Takaki Miyata2,3, Kanako Saito2, Yuji Nishizawa2, Tadashi Nomura1 and Noriko Osumi1,3,*

1 Division of Developmental Neuroscience, Center for Translational and Advanced Animal Research, Tohoku University School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
2 Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University, 65, Turumai-machi, Shouwa-ku, Nagoya 466-8550, Japan
3 CREST, Japan Science and Technology Corporation (JST), 4-1-8, Honmachi, Kawaguchi, 332-0012, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The mammalian cerebral cortex develops from proliferative neuroepithelial cells that exhibit a cell cycle-dependent nuclear movement (interkinetic nuclear migration; INM). Pax6 transcription factor plays pivotal roles in various aspects of corticogenesis. From live observation using cultured cortical slices from the Pax6 mutant rat, we identified the premature descent of S phase cells, the unsteady ascent or descent of G2 phase cells, and ectopic cell division within the basal side of the ventricular zone (VZ). The centrosome normally stayed at the most apical side, apart from the nucleus, in the neuroepithelial cell during the S to G2 phase, while the Pax6 mutant showed unstable movement of the centrosome associated with an abnormal INM. Our results suggest the possibility that Pax6 regulates the INM by stabilizing the centrosome at the apical side.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
During development, the cortical primordium consists of neural progenitor cells exhibiting a bipolar shape with apical and basal processes spanning from the ventricular surface to the pial surface of the brain (we refer herein to these neural progenitor cells as "neuroepithelial cells," which include "radial glial cells"). In the most apical (ventricular) side, the neuroepithelial cells are joined to one another by a subcellular structure called the adherens junction (Chenn et al. 1998), which is associated with cell polarity proteins such as atypical protein kinase C (aPKC), partitioning-defective protein-6 (Par6) and partitioning-defective protein-3 (Par3) (Manabe et al. 2002; Henrique & Schweisguth 2003). Another subcellular component found at the apical side of the neuroepithelial cell is a pair of centrosomes (Hinds & Ruffett 1971; Shoukimas & Hinds 1978). In the cortical primordium, cell proliferation occurs in two distinct proliferation regions; one is the ventricular zone (VZ) that lies immediately adjacent to the ventricle, and the other is a region that consists of the subventricular zone (SVZ) and intermediate zone (IM) that covers the VZ. One of the intriguing features of the neuroepithelial cells is cell cycle-dependent nuclear movement (interkinetic nuclear migration; INM). The majority of the neuroepithelial cells in the VZ divide at the ventricular (luminal) surface, after pulling down the nucleus in the G2 phase from the basal side of the VZ where the nucleus of the S phase exists. This was first hinted at by Sauer (1935), who histologically examined the neuroepithelial cells of vertebrate embryos and raised the concept of nuclear migration to and from the ventricular surface at each cell division, which was subsequently confirmed by other researchers who applied autoradiography (Sauer & Walker 1959; Fujita 1962). Although this unique INM phenomenon was described almost 70 years ago, the underlying mechanism remains to be defined at subcellular and molecular levels.

The centrosome is the major center of microtubule organization, and extensive research has been conducted into the role of the centrosome and cytoskeletal components in nuclear locomotion during neuronal migration (see review by Marin et al. 2006). Therefore, the centrosome is considered a good target to focus on as one of the subcellular components functioning in INM, although the position of the centrosome is somehow different from general types of cells; the centrosome of neuroepithelial cells locates apart from the nucleus during the interphase.

Pax6 transcription factor is expressed in the VZ of the presumptive cerebral cortex (Stoykova & Gruss 1994), predominantly in neuroepithelial cells dividing at the ventricular surface rather than in basal progenitors dividing in the SVZ (Englund et al. 2005). We and other groups previously showed the abnormal laminar formation of the cortex in Small eye mice and rats that lack Pax6 function due to spontaneous mutations in the Pax6 gene (Stoykova et al. 1996; Caric et al. 1997; Fukuda et al. 2000). Interestingly, Pax6 dysfunction in the developing cerebral cortex produces cell kinetic abnormalities, such as a wider distribution of S phase cells and mis-position of M phase cells (Estivill-Torrus et al. 2002; Quinn et al. 2007). These findings prompted us to explore the molecular and subcellular mechanisms of INM.

In the present study, we first monitored INM by time-lapse video microscopy, and detected a mitotic mis-position resulting from abnormal nuclear movement during the S to M phase in the cortical primordium of the Pax6 homozygous mutant rat (rSey2/rSey2; Osumi et al. 1997). Moreover, immunostaining with an anti-{gamma}-tubulin antibody identified many mis-localized centrosomes in the rSey2/rSey2 cortex. We also performed live imaging studies using an expression vector that targeted the centrosome, and found in the wild-type that during the descendent process of nuclear movement (S to G2 phase), the centrosome stayed at the ventricular surface away from the nucleus, a phenomenon different from that observed in migrating neurons. On the other hand, in rSey2/rSey2, the centrosome frequently changed its position during nuclear migration. The results of direct imaging of nuclear and centrosomal behaviors by time-lapse video microscopy suggest that Pax6 is required for normal INM during the S to M phase via proper apical localization of the centrosome.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Ectopic localization of S and M phase cells in the rSey2/rSey2 cerebral cortex

Pax6 transcription factor was strongly expressed in the VZ of the rat cortical primordium at E17 (corresponding to E15 in mice; Fig. 1A). To explore the molecular and cellular mechanisms of INM, we first examined the cell cycle kinetics of neuroepithelial cells of the cortical primordium in both the wild-type and the rSey2/rSey2 by using bromodeoxyuridine (BrdU) pulse labeling (for 15 min) and immunostaining with an antibody against a mitotic cell marker, phosphorylated histone H3 (PH3).


Figure 1
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  		Figure 1  Ectopic localization of S and M phase cells in the rSey2/rSey2 cortex. Immunoreactivity of Pax6 (A), BrdU (B, C) and PH3 (D, E) in the cortical primordium of E17 wild-type (A, B, D) and rSey2/rSey2 (C, E) taken by fluorescent microscopy. BrdU-incorporating cells are increased in number, and scattered in the VZ and SVZ, and cell division in ectopic positions is observed in rSey2/rSey2. The histogram shows the distribution of cells in the S phase (F) and the mitosis phase (G) in the cortical primordium of the wild-type (blue bar) and rSey2/rSey2 (red bar). We counted all BrdU positive cells (970 cells in the wild-type; 1357 cells in the rSey2/rSey2) and all PH3 positive cells (168 cells in the wild-type; 278 cells in the rSey2/rSey2) from 0 µm (the ventricular surface) to the basal area within 450 µm. Compared with the wild-type, the number of BrdU-labeled cells increased, PH3 positive cells at the surface decreased in number and non-surface dividing cells conversely increased in the rSey2/rSey2. The number of BrdU (F) and PH3 (G) positive cells was calculated from 0 µm (the ventricular surface) to the basal area within 450 µm and quantified as a percentage of the labeled cells against the total number of cells in each area. Data from three sections from three animals in each experiment were averaged to give the histograms. Means were compared statistically using the Student's t-test (*P < 0.05, **P < 0.01). VZ, ventricular zone; SVZ, subventricular zone; IM, intermediate zone; CP, cortical plate. Bar = 20 µm.

 
In the cortical primordium of the E17 wild-type rat, most BrdU-incorporating nuclei were located in the basal half of the VZ (Fig. 1B). The majority of the mitotic figures were located at the ventricular surface, though a small number of cells divided in the SVZ (Fig. 1D). In the rSey2/rSey2 cortex, we have previously reported that the VZ seems to expand in the rSey2/rSey2 cortex from general histology (Fukuda et al. 2000). In the present pulse labeling, BrdU-incorporating cells increased and scattered within the VZ and SVZ (Fig. 1C,F), and many mitotic cells were identified in the SVZ and in the basal side of the VZ (Fig. 1E). These abnormal features of cell kinetics are similar to those reported in previous mouse studies (Estivill-Torrus et al. 2002).

Quantification of the distribution of the BrdU-positive cells and PH3-positive cells provided a more comprehensive representation of S and M phase patterns throughout the developing cortex. In the wild-type, a large number of BrdU-labeled cells (about 25%) were identified in the ventricular surface, while the remaining labeled cells (about 75%) were found in the basal side of the VZ and in the SVZ (Fig. 1F). In contrast, a large number of dividing cells (about 55%) were identified in the ventricular surface, while the remaining dividing cells (about 45%) were found in the basal side of the VZ and in the SVZ (Fig. 1G). In the rSey2/rSey2 cortex, the number of BrdU-labeled cells at the basal side of the VZ and in the SVZ dramatically increased (Fig. 1F). Moreover, the number of dividing cells at the ventricular surface decreased to about 20%, while the dividing cells in the VZ and SVZ conversely increased to about 80%, especially in the middle and basal sides of the VZ (Fig. 1G). We measured the distribution of the BrdU-labeled cells and PH3-positive cells in three adjacent sections at three different anterior–posterior positions in the wild-type and rSey2/rSey2 cortex, and found no significant differences in each region. These results clearly indicate increased and mis-localized S phase and M phase cells in the rSey2/rSey2 cortex, suggesting abnormal nuclear movement during the S to M phase. We also immunostained wild-type and rSey2/rSey2 rat embryos at E16 and E18 in addition to E17. The data obtained at E16 and E18 were similar to those in the E17 wild-type and rSey2/rSey2 (data not shown).

Abnormal INM in the rSey2/rSey2 cerebral cortex

To monitor the patterns of nuclear movement during the cell cycle, we took advantage of time-lapse video microscopy using DiI-labeled cortical slice cultures (Miyata et al. 2001). In the E17 wild-type (Fig. 2A), the nucleus of the cell which divided at the ventricular surface remained still in the VZ during the S phase (about 4 h), but moved rapidly toward the ventricular surface during the G2 phase (about 1–1.5 h). This nuclear migration was a G2 phase-specific phenomenon, and did not occur during the S phase (Fig. 2B). Mitosis occurred immediately upon arrival of the nucleus to the ventricular surface (Fig. 2A, Supplementary Movie S1). All cells observed in the wild-type cortex showed this migration pattern during the S to M phase, which we considered as the normal pattern (Fig. 2F,I). A small number of cells divided in the SVZ (Fig. 1D). It was previously reported that non-surface dividing cells exhibit another type of a nuclear migration pattern, in which the apical process of the cell collapsed before mitosis (Miyata et al. 2004). In the present study, we did not observe non-surface cell division by time-lapse video microscopy in the wild-type.


Figure 2
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  		Figure 2  Abnormal nuclear migration patterns in the rSey2/rSey2 cortex. Time-lapse fluorescent micrographs of DiI-labeled neuroepithelial cells (30-min intervals). In the wild-type (A, B), the nucleus stays in the basal half of VZ during the S phase, then descends rapidly during the G2 phase, and divides at the ventricular surface (A, Supplementary Movie S1). (B) BrdU treatment for 30 min is performed during the nuclear descent (black arrowed in fluorescent micrographs). White bar = 5 µm. The high magnification confocal micrograph of the BrdU treated cell revealed that descending (G2-phase) cells (DiI labeled; red) normally did not incorporate BrdU (green), indicating that the nuclear descent is a G2-phase specific phenomenon and does not occur during the S-phase. In rSey2/rSey2 (C, D), descent of nucleus is slower and sometimes incomplete, leading to division away from the surface (C), or the nucleus shows a rebound motion even though mitosis occurs at the ventricular surface (D, Supplementary Movie S3). (E) Premature descent is detected in BrdU-incorporating (S-phase) rSey2/rSey2 cells (DiI, red; BrdU, green), indicating dissociation between cell cycling and nuclear movement (black arrowed). White bar = 5 µm. (F–H) Migratory behaviors are categorized into the normal (F), Type I (G, Supplementary Movie S3) and Type II (H, Supplementary Movie S2) (see text for detail). (I) Summary of S-G2-M nuclear migration patterns observed in the wild-type and rSey2/rSey2 slices. All wild-type cells exhibited the normal pattern. In contrast, only 60% of the rSey2/rSey2 cells showed the normal pattern (Supplementary Movie S4), whereas the remaining 40% exhibited abnormal (Type I or Type II) patterns.

 
In contrast, abnormal patterns of the nuclear migration during the S to M phase were often observed in the rSey2/rSey2 cortex (Fig. 2C–E). For example, the nucleus did not reach the ventricular surface, dividing ectopically in the basal side of the VZ (Fig. 2C, Supplementary Movie S2), or tottered up and down during the S to G2 phase (Fig. 2D, Supplementary Movie S3). These nuclear migration patterns were never observed in the wild-type and were not similar to the nuclear migration of non-surface dividing cells observed in the wild-type (Miyata et al. 2004). Furthermore, the nucleus tended to start migration prematurely during the S phase (Fig. 2E) compared with the wild-type (Fig. 2B). We categorized these nuclear migration patterns during the cell cycle by the combination of behavior during the S to G2 phase and the position of mitosis: (i) normal (see above, Fig. 2F), (ii) Type I: involving abnormal behavior during the S to G2 phase and normal mitosis at the ventricular surface (Fig. 2G); and (iii) Type II: involving abnormal behavior during the S to G2 phase and ectopic mitosis in the VZ/SVZ (Fig. 2H). In the rSey2/rSey2 cortex, although 120/199 (60.3%) cells showed the normal pattern (Fig. 2I, Supplementary Movie S4), 62/199 (31.2%) cells showed the Type I pattern and 17/199 (8.5%) cells showed the Type II pattern (Fig. 2I). These findings suggest that the loss of Pax6 function is associated with irregular INM behaviors during the S to M phase in about 40% of neuroepithelial cells in the rSey2/rSey2 cortex. In the time-lapse imaging, there was no difference in the behavior of cells between the rostral and caudal cortex or between the dorsal and lateral cortex in the wild-type and rSey2/rSey2 cortex (data not shown).

Normal apical adhesion apparatus in the rSey2/rSey2 cerebral cortex

Next, we investigated the subcellular components of neuroepithelial cells to obtain more insight into INM mechanisms. During time-lapse imaging, loss of the apical process was often observed in the rSey2/rSey2 (Fig. 2C), which led us to suspect weakening or disruption of the apical adhesion apparatus attaching neighboring cells in the rSey2/rSey2, resulting in the abnormal nuclear migration. It has been reported that neuroepithelial cells form the adherens junction at the most ventricular side but lack the tight junction (Astrom & Webster 1991). We thus examined the expression of adherens junction proteins such as N-cadherin, ß-catenin and F-actin in the cerebral cortex of the wild-type and rSey2/rSey2.

In the wild-type, N-cadherin, ß-catenin and F-actin were highly concentrated at the most ventricular side of neuroepithelial cells (Fig. 3A,C,E). In the rSey2/rSey2 cortex, the patterns of expression of these proteins were similar on the ventricular surface (Fig. 3B,D,F). Moreover, electron microscopy revealed no difference in the apical junction between the wild-type (Fig. 3G) and rSey2/rSey2 (Fig. 3H). Therefore, the apical adherens junctions of neuroepithelial cells are regarded to be almost normal in rSey2/rSey2.


Figure 3
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  		Figure 3  Normal adherens junction in the rSey2/rSey2 cortex. Immunoreactivity of the adherens junction protein, N-cadherin (A, B), ß-catenin (C, D) and F-actin (E, F) and electron micrographs of adherens junction (G, H) in the E17 wild-type (A, C, E, G) and rSey2/rSey2 (B, D, F, H) cortex. Expression patterns of these adherens junctional proteins and the ultrastructure of the adherens junction in the rSey2/rSey2 appears normal compared with those of the wild-type. Bar in (A)–(F) = 10 µm. Bar in (G)–(H) = 100 nm.

 
Abnormal localization of the centrosome in the rSey2/rSey2 cerebral cortex

The centrosome is the main microtubule-organizing center in animal cells (Rieder et al. 2001), and plays a key role in translocation of the nucleus during neuronal migration (see reviews by Rouvroit & Goffinet 2001; Marin et al. 2006). For better understanding of the mechanisms of INM, we next focused on the centrosome of neuroepithelial cells. We first examined the distribution of the centrosome in neuroepithelial cells of both the wild-type and rSey2/rSey2 cortex at E17 by staining with an anti-{gamma}-tubulin antibody, and counted the number of the centrosomes localized within 20 µm from the ventricular surface.

In the wild-type cortex, 81% (537/662) of the centrosomes were localized within 1 µm from the ventricular surface (Fig. 4A,C). The rest of the centrosomes (19%, 125/662) were found in the more basal side (Fig. 4A, white broken circles, white arrowheads). The majority of these basally-located centrosomes were observed in mitotic cells, which were confirmed by nuclear staining with 4', 6-diamino-2-phenylindole (DAPI, Fig. 4A, white broken circles). In the rSey2/rSey2 cortex, however, the number of the centrosomes localized within 1 µm from the ventricular surface was reduced to 67% (415/616), and those localized in the areas more than 1 µm from ventricular surface increased in number (33%, 201/616) (Fig. 4B,C, white arrowheads). The number of the basally-located centrosomes in the rSey2/rSey2 cortex were particularly increased to 12% (71/616) at the position of 1–2 µm from the ventricular surface, whereas the number only accounted for 5% (32/662) in the wild-type (Fig. 4C). These patterns of centrosomal localization in the rSey2/rSey2 cortex were significantly different from that in the wild-type (P < 0.01 in the Mann–Whitney's U test).


Figure 4
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  		Figure 4  Mis-localization of the centrosome in neuroepithelial cells of the rSey2/rSey2 cortex. Immunoreactivity of {gamma}-tubulin in the VZ of the E17 wild-type (A) and rSey2/rSey2 (B). White broken circles indicate mitotic cells and white arrowheads indicate centrosomes ectopically localized in the VZ. We counted centrosomes in the area 0–20 µm from the ventricular surface for the wild-type (n = 662) and in the rSey2/rSey2 (n = 616). Compared with the wild-type, the number of centrosomes in the areas more than 2 µm from the ventricular surface dramatically increased in the rSey2/rSey2. (C) Quantitative analysis of centrosomal position in the E17 wild-type and the rSey2/rSey2. The number of centrosomes localized at 1 µm from the ventricular surface decreased and that of the centrosomes localized in the basal area conversely increased (especially in areas 2–4 µm from the ventricular surface) in the rSey2/rSey2. Electron micrographs of the centrosome in the E17 wild-type (D, E) and rSey2/rSey2 (F–I). A pair of centrosomes is indicated by black arrowheads, and the membrane vesicle is indicated by black arrows. Most centrosomes are localized at the most ventricular side and associated with the membrane vesicle at their apical side. In rSey2/rSey2, the centrosomes are often mis-localized in the apical process and associated with the membrane vesicle at their basal side (G, H', I'). (H, I) Black boxes showing the position of the centrosomes in the apical process are enlarged in H' and I'. Bar in (A) and (B) = 10 µm. Bar in (D)–(G), (H') and (I') = 200 nm. Bar in (H) and (I) = 1 µm.

 
The results seen in the immunostaining were confirmed by using transmission electron microscopy. In the wild-type, a pair of centrosomes were localized at the most ventricular side (Fig. 4D, E-black arrowheads), and were rarely observed in the apical process compared with the rSey2/rSey2 (data not shown). The centrosomes were localized at the most ventricular side associated with the membrane vesicle at its apical side (5/5 wild-type neuroepithelial cells; Fig. 4D, E-black arrow). A previous study suggested that the membrane vesicle derived from the Golgi complex plays an important role in forming the primary cilia and is normally formed at the apical side of the centrosome (Cohen et al. 1988; Alieva & Vorobjev 2004). In the eleven rSey2/rSey2 neuroepithelial cells analyzed extensively by transmission electron microscopy, we found six cells had a centrosome in their apical endfeet (Fig. 4F,G), as seen in the wild-type cells, whereas the centrosomes in the remaining five cells were found less apically (Fig. 4H,I). Moreover, five rSey2/rSey2 neuroepithelial cells showed an inverse centrosome–vesicle orientation, that is, the membrane vesicle was situated basally to the centrosome (Fig. 4G, I'-black arrow). In fact, the number of mature primary cilia decreased in the rSey2/rSey2 (14.3 ± 1.0/100 µm2) compared with the wild-type (17.7 ± 0.7/100 µm2) as observed with a scanning electron microscope (data not shown).

These quantitative immunohistochemical data and qualitative electron microscopic observations suggest that the loss of Pax6 function is involved in abnormal positioning of the centrosome and the centrosome-associated membrane vesicle in neuroepithelial cells of rSey2/rSey2, which may lead to the impaired maturation of the primary cilia.

Abnormal behavior of the centrosome in the rSey2/rSey2 cerebral cortex

To obtain further insight into the relationship between abnormalities of the centrosome and INM, we monitored the centrosomal behavior of neuroepithelial cells during the S to G2 phase by in utero electroporation and the live cortical slice culture technique. It was reported previously that the C-terminal region of AKAP450, a kinase anchoring protein, was sufficient to confer its centrosomal localization by using a GFP reporter (Gillingham & Munro 2000). Thus, we used a GFP-AKAP450 expression vector (Supplementary Fig. S1A) to visualize the centrosome together with pCAGGS-RFP to envisage the cell behavior in live imaging. When GFP-AKAP450 was co-injected with pCAGGS-RFP, a single neuroepithelial cell and its centrosome could be labeled with both expression vectors (Supplementary Fig. S1B, Supplementary Movie S5). It was confirmed that electroporation and expression of GFP-AKAP450 and pCAGGS-RFP had no effect on normal INM (Fig. 5A).


Figure 5
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  		Figure 5  Unstable centrosomal behavior in neuroepithelial cells of the rSey2/rSey2 cortex. Time-lapse fluorescent micrographs of GFP/RFP-labeled neuroepithelial cells (50-min intervals). GFP-AKAP450 and pCAGGS-RFP were used to visualize the centrosome and the individual neuroepithelial cells, respectively. White broken lines indicate the ventricular surface. In the wild-type (A–C), the centrosome remains at the most ventricular side during the S to early M phase but migrates toward the nucleus during the M phase (B, C). In rSey2/rSey2 (D–F), the neuroepithelial cells exhibit three types of centrosomal behaviors: Type A in which the centrosome stayed at the most ventricular side for a while (during S to G2 phase) and then two centrosomes were observed, at the ventricular side, in both individual dividing daughter cells clearly labeled with RFP, as observed in the wild-type (D, G); Type B in which a GFP-labeled centrosome was detected after a while basally within the very thin apical process labeled with RFP (E, H); and Type C with the centrosome changing its position frequently during time-lapse observation (F, I) (see text for detail). (J) S–G2–M centrosomal behaviors observed in the wild-type and rSey2/rSey2 slices. Most wild-type cells (82.9%) exhibited Type A centrosomal behavior, whereas the remaining 17.1% exhibited Type B behavior. In contrast, only 50.0% of rSey2/rSey2 cells exhibited Type A centrosomal behavior, whereas 33.3% exhibited Type B behavior, and the remaining 16.7% exhibited Type C behavior.

 
In the wild-type, the centrosome remained at the most ventricular side during the S to early M phase (Fig. 5B,C) as expected from the immunostaining and electron microscopic data (Fig. 4A,C,D,E). When the nucleus reached the ventricular surface, two centrosomes labeled with GFP were observed at the ventricular surface in both individual dividing daughter cells clearly labeled with RFP, which started moving basally toward the nucleus, detached from the ventricular surface (Fig. 5B,C), probably preparing for chromosomal division. This centrosomal behavior, here defined as Type A, was observed in cells showing the normal INM pattern with ventricular surface division (29/35 cells; Fig. 5G,J). Another centrosomal behavior was observed in wild-type neuroepithelial cells in which the centrosome labeled with GFP was detected basally within the very thin apical process (almost the same width as the centrosome) labeled with RFP. This GFP-labeled centrosome is considered to be of cells (probably neurons or basal progenitors) migrating away from the ventricular surface without showing cell division and apical process retraction (Fig. 5H). We designated this behavior as Type B, which was observed in 6/35 wild-type cells (Fig. 5J).

In the rSey2/rSey2, the centrosome of 18/36 neuroepithelial cells showed Type A behavior during the S to M phase (Fig. 5D,G,J). This Type A behavior was observed in neuroepithelial cells showing normal INM (see Fig. 2F). In contrast, the centrosome of 12/36 neuroepithelial cells exhibited Type B behavior (Fig. 5E,H,J). In this case, some centrosomes migrated in accompany with the apical process retraction (Fig. 5E). In addition, another abnormal centrosomal behavior was observed in rSey2/rSey2 neuroepithelial cells that showed an abnormal INM pattern (Fig. 2G). In this case, the centrosome frequently changed its position, moving up and down near the ventricular surface during the S to G2 phase (Fig. 5F). We designated this behavior as Type C, which was observed in 6/36 rSey2/rSey2 cells (Fig. 5I,J) but was never detected in the wild-type.

These results obtained by live imaging clearly indicate that the neuroepithelial cells of the rSey2/rSey2 cortex show a 40% decrease of Type A centrosomal behavior and a 95% increase of Type B behavior. Moreover, 20% of rSey2/rSey2 neuroepithelial cells exhibited abnormal Type C centrosomal behavior that was never observed in the wild-type. Thus, loss of Pax6 function is associated with abnormal behavior of the centrosome during the S to M phase, presumably leading to the abnormal INM.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Pax6 affects nuclear movement during the S to G2 phase

Previous studies on fixed specimens have revealed abnormal distribution of BrdU-labeled cells and increased proportions of progenitors dividing in ectopic positions in the neuroepithelium of Pax6 mutant mice, suggesting an impaired INM (Estivill-Torrus et al. 2002; Quinn et al. 2007). These observations are similar to those in the rSey2/rSey2 (Fig. 1; Fukuda et al. 2000), implying that these defects in the cortical neuroepithelium are due to the loss of Pax6 function. However, the precise cellular mechanism of INM remains unclear from these static observations.

To confirm the correlation between Pax6 and INM, the present study was designed to determine whether nuclear migration itself is abnormal, and whether linkage between cell cycling and nuclear migration is abnormal in the Pax6 deficient condition. Our live observations of the behavior of neuroepithelial cells during and before their division at the apical surface clearly demonstrated that: (i) their abventricular migration during G2 phase is abnormal in the Pax6 mutant, with slow and incomplete descent or to- and fro- movement which often leads to ectopic division; and that (ii) cell cycling and nuclear migration is decoupled in the Pax6 mutant, as shown in the premature descent during the S phase. The INM pattern of the cells divided ectopically in the Pax6 mutant was different from that of the non-surface dividing cells in the wild-type (Miyata et al. 2004). Although non-surface dividing cells in the wild-type never exhibited a nuclear descending movement toward the ventricular surface, ectopically dividing cells in the Pax6 mutant exhibited a nuclear descending movement toward the ventricular surface during the S to G2 phase. These direct observations explain the reasons behind both the increased non-surface mitosis and wider (more diffuse) distribution of S-phase cells in the VZ of the Pax6 mutant. Thus, our results suggest that Pax6 controls stable positioning of the nucleus during the S phase and descent migration of the nucleus during the G2 phase in E17 rat embryos.

Recent reports have demonstrated that progenitor cells that divide in the SVZ (sometimes referred as "basal progenitors") generate two types of neurons (Haubensak et al. 2004; Miyata et al. 2004; Noctor et al. 2004). Pax6 is strongly expressed in apically dividing progenitors but is nearly negative in SVZ-dividing progenitors (Englund et al. 2005). In the present study we demonstrated that loss of the Pax6 function resulted in a decrease of the ventricular surface division of progenitor cells and conversely an increase in non-surface division. It is assumed that the decrease in surface division at the mid-stage of corticogenesis may result in a reduced number of progenitors that would continue cell cycling, thereby causing production of a smaller number of neurons (Fukuda et al. 2000; Estivill-Torrus et al. 2002; Quinn et al. 2007). Therefore, although Pax6 is widely known to promote neurogenesis under culture conditions (Götz et al. 1998), it is also important for maintaining the surface-dividing progenitor cells. How such a fate decision may couple with cellular behavior is a challenging question that will be investigated in the future.

Although an abnormal INM was clearly observed under the loss of Pax6 condition, it was detected only in a subset of the neuroepithelial cells all of which expressed Pax6 in normal development. The reason for such insufficient effects remains enigmatic at the moment, but there may be certain heterogeneity in the neuroepithelial cells at the stage we observed. For example, the neuroepithelial cells are almost all RC2 immunoreactive, but are different in their GLAST and BLBP (FABP7) content (Hartfuss et al. 2001). Moreover, while all RC2-positive precursor cells in the cerebral cortex express Pax6 only a subset of them express bHLH transcription factors of the proneural gene family (Götz et al. 1998). Thus, the neuroepithelial cells exhibit heterogeneity in characteristics during neurogenesis, which might exert an influence on INM phenotypes under the loss of Pax6 condition.

The centrosome as an apical component of INM

To identify the type of subcellular components responsible for the abnormal descent of the nucleus during the S to M phase, we first focused on the adherence junction because loss of the apical process was often observed in the rSey2/rSey2. In addition, expression of R-cadherin, a cell adhesion molecule, is down-regulated in the Pax6 mutant cortex (T. Inoue and N. Osumi, unpublished results in the rat). However, we could not detect any significant difference in the adherens junction in the rSey2/rSey2 cortex based on the immunostaining and electron microscopy findings. Therefore, although it is still possible that there are some minor defects in the apical adhesion apparatus, it is more likely that loss of the Pax6 function affects subcellular components other than the adherens junction.

The next target is the centrosome that is localized at the most ventricular side of the neuroepithelium during the inter phase in the mitotic process (Hinds & Ruffett 1971; Chenn et al. 1998). The centrosomes were frequently spotted in the abventricular area in the rSey2/rSey2 (Fig. 4B,C), and this was confirmed by electron microscopy (Fig. 4H,I). Furthermore, the direction of the membrane vesicle associated with the centrosome sometimes changed even if it was localized at the most ventricular side (Fig. 4G). This prompted us to monitor the centrosomal behavior during the S to M phase in relation to INM.

Our live imaging in the wild-type cortical slice confirmed the stable positioning of the centrosome at the most ventricular side during the S to G2 phase and its basally-oriented migration during the M phase (Type A). This centrosomal behavior was seen in the majority of the G2 phase progenitor cells (about 80%) that divide at the ventricular surface showing the normal INM. The centrosomal behavior in the neuroepithelium identified in our study is quite different to that in migrating neurons; during neuronal migration, the centrosome first advances into the leading process in front of the nucleus, but is rapidly followed by nucleokinesis in the direction of the centrosome (Solecki et al. 2004), while the centrosome of the neuroepithelial cell is stably localized far away from the nucleus during the S to G2 descent nuclear migration, a unique and novel phenomenon.

The centrosome of wild-type neuroepithelial cells showed another type of behavior, that is, basally-oriented migration without mitosis (Type B, 17.1%). It was suggested previously that neuronal differentiating cells detach the apical process from the ventricular surface concurrently with centrosomal migration (Shoukimas & Hinds 1978). However, our live observation in the wild-type did not reveal that Type B centrosomal migration was accompanied by detachment of the apical process. As mentioned above, there are two types of neural progenitor division, that is, ventricular surface division and non-surface division in the basal side of the VZ (Fig. 1D; Miyata et al. 2004). Therefore, it is likely that the centrosomes of neural progenitor cells that divide in the SVZ show Type B behavior prior to detachment of the apical process.

In the rSey2/rSey2 neuroepithelial cells, we found a 40% decrease of Type A centrosomal behavior and conversely a 95% increase of Type B behavior. However, the latter type of migration did not always accompany detachment of the apical process. In addition, 16.7% of the cells showed Type C behavior; the centrosome continually changed its position near the ventricular surface during the S to G2 phase. This abnormal behavior, which was never observed in the wild-type, was observed in neuroepithelial cells that exhibited the abnormal INM pattern, that is, instability in the S to G2 phase and surface division. Since Type B centrosomal behavior in the rSey2/rSey2 (33.3%) is considered to include the normal behavior of SVZ-dividing progenitors (17.1%) and abnormal behavior of SVZ-dividing progenitors (16.2%), the ratio of abnormal behavior of the centrosome (16.2% + 16.7% = 32.9%) is roughly in proportion to that of abnormal INM (40%). Our ultrastructural studies also revealed centrosome-related abnormalities in half of the rSey2/rSey2 neuroepithelial cells. When considered together, these results suggest that the centrosome is one of the players in the regulation of INM and that the Pax6 function is essential for the stable localization of the centrosome to the most ventricular (i.e. apical) side of neuroepithelial cells (Fig. 6).


Figure 6
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  		Figure 6  A model of INM and the position of the centrosome in the wild-type and rSey2/rSey2. In the wild-type, the centrosome is stably localized at the most ventricular side during the S to G2 phase and regulates the nuclear descent by microtubular organization (A). In contrast in the rSey2/rSey2, the centrosome frequently changes its localization because of the loss of Pax6 and this centrosomal behavior results in abnormal INM in the S to M phase (B).

 
When all our data are combined, the findings in the present study suggest a possible involvement of the centrosome in INM behavior and of Pax6 in the centrosomal localization. However, the molecular mechanisms by which Pax6 regulates the centrosomal position and INM are still unclear. One of the candidate molecules is the cell polarity protein. The neuroepithelial cell is a highly polarized cell, and previous studies suggest that polarity of the neuroepithelial cell is established by cell polarity proteins such as aPKC, Par6 and Par3 (Manabe et al. 2002; see review by Etienne-Manneville & Hall 2003; Henrique & Schweisguth 2003). Both Par6 and Par3 are PSD95/Discs-Large/ZO1 (PDZ) domain proteins and these two proteins and aPKC form a complex at the apical region of the neuroepithelial cell (see review by Henrique & Schweisguth 2003). These apical polarity proteins are recruited in the adherens junction of neuroepithelial cells (Manabe et al. 2002). Moreover, it has been previously reported that the Par6/aPKC complex plays pivotal roles in radial migration of neurons via reorganization of microtubule polarity (see review by Henrique & Schweisguth 2003; Solecki et al. 2004). Thus, it is possible that cell polarity proteins are also associated with centrosomal localization. Interestingly, a previous study suggests that loss of aPKC{lambda} results in an increased number of ectopic cell divisions in the zebrafish retina (Horne-Badovinac et al. 2001), like the rSey2/rSey2 mutant cortical neuroepithelial cells. Moreover, in conditional aPKC{lambda} knockout mice, neuroepithelial cells of the neocortical region demonstrated a loss of adherens junctions, retraction of apical processes and impaired INM (Imai et al. 2006). In addition, it was previously reported that aPKC-phosphorylated FEZ1 protein regulates axonal outgrowth and fasciculation through interaction with microtubules (Okumura et al. 2004). A recent study further showed that FEZ1 can bind to kinesin, a plus end-directed microtubule motor protein, and thus may regulate kinesin-mediated transport pathways (Suzuki et al. 2005). Furthermore, we have found down-regulation of FEZ1 in our microarray analyses using the wild-type and rSey2/rSey2 rat neural tubes at E11.5 (K. Numayama-Tsuruta, U. Fukuzaki and N. Osumi, unpublished data) and a quantitative RT-PCR analysis using the wild-type and rSey2/rSey2 rat cortex primordium at E17. FEZ1 is thus another good candidate molecule that regulates stable localization of the centrosome. To elucidate the molecular mechanism through which the centrosome participates in the descent migration of the nucleus during the S to G2 phase is our next challenge.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Animals

Pregnant Sprague–Dawley (SD) rats were purchased from Japan Charles River (Tokyo, Japan). The Pax6 heterozygous mutants on SD background rats (rSey2) (Osumi et al. 1997) were intercrossed in our laboratory to obtain rSey2/rSey2 embryos, which are distinguished from the wild-type and heterozygous littermates by an eyeless phenotype. All animal experiments were carried out in accordance with the National Institute of Health guidelines for the care and use of laboratory animals, and were approved by The Committee for Animal Experiments in Tohoku University School of Medicine.

Immunostaining

Procedures were basically those described in previous studies (Osumi et al. 1997). On E17 (corresponding to E15 mouse embryos), wild-type and rSey2/rSey2 rat embryos were perfused transcardially with 4% paraformaldehyde (PFA) in 0.01 M PBS (sodium phosphate buffer, pH 7.4), and the cortex primordium was dissected out, fixed again in 4% PFA for 20 min, and sectioned with a cryostat (CM3050, Leica, Heerbrugg, Switzerland). The sections were immersed in 2% goat serum in TBST (Tris-buffered saline plus 1% Triton X-100) for 1 h, and were incubated with anti-Pax6 (rabbit polyclonal; Inoue et al. 2000), anti-PH3 (rabbit polyclonal, Upstate Biotechnology, Lake Placid, NY) anti-{gamma}-tubulin (mouse monoclonal, Sigma Chemical Co., St. Louis, MO), anti-N-cadherin (rabbit polyclonal, Takara Bio, Ohtsu, Japan) anti-ß-catenin (mouse monoclonal, Transduction Laboratories, San Jose, CA), or anti-BrdU (mouse monoclonal, Becton Dickinson Immunocytometry Systems, San Jose, CA) antibodies and Rhodamine-Phalloidin (Molecular Probes, Eugene, OR) at 4 °C overnight. A Cy3-conjugated and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit or anti-mouse IgG antibody (Jackson Immunoresearch Laboratories, West Grove, PA) and a Texas Red dye-conjugated anti-mouse IgG antibody (Jackson Immunoresearch, Laboratories) were used as the secondary antibodies. The sections were examined under the fluorescent microscope (Axioplan-2, Zeiss, Jena, Germany) equipped with a CCD camera (AxioCam, Zeiss). Confocal images were acquired with a Zeiss LSM5 PASCAL confocal microscope, and three-dimensional images were constructed with Zeiss confocal software.

BrdU labeling

Timed pregnant rats received single intraperitoneal injection of BrdU (Sigma) at 14 mg/100 g body weight (10 mg/mL stock, dissolved in 0.9% saline), and were sacrificed at 15 min after a single injection. The obtained 10-µM frozen sections were incubated in 2N HCl for 10 min at 37 °C, and washed in TBST. Incorporated BrdU was detected using the method described previously (Takahashi & Osumi 2002).

Slice culture

Procedures were basically conducted according to the methods described previously (Miyata et al. 2001). Wild-type and rSey2/rSey2 brains were dissected out from E17 embryos, and the pia mater was removed. The isolated brains were transferred to DMEM/F12 medium with or without fine crystals of fluorescent dye DiI (Molecular Probes) to label neuroepithelial cells. The cortex labeled either with DiI or by electroporation was cut manually into slices, which were embedded in collagen gel. The behavior of the labeled cells was observed under an inverted fluorescent microscope (Axiovert 135, Zeiss) equipped with a CCD camera (SenSys, Bad Saarow/OT Neu Golm, Germany). We focused on cells that exhibited descending behavior toward the ventricular surface. Cells exhibiting an unusually high level of fluorescence or faintly labeled cells were excluded. Images were captured manually at 30- or 50-min intervals for 10 h. For automatic recording, a fluorescent inverted microscope (IX70, Olympus, Tokyo, Japan) equipped with a CCD camera (CoolsnapHQ, Roper Scientific, Duluth, GA) was used and imaging was controlled by METAMORPH4.0 software (Universal Imaging, Buckinghamshire, UK).

Gene transfer by in utero electroporation

In utero electroporation was performed as described previously (Takahashi et al. 2002). Timed pregnant wild-type or rSey2/rSey2 rats at E16 were anesthetized with 97% 2,2,2-Tribromoethanol in tert-Amyl alcohol. After cleaning the abdomen with 70% ethanol, a midline laparotomy of about 3-cm was performed, and the uterus was pulled out. To visualize the centrosome, we used a GFP-AKAP450 vector (kindly provided by Dr Munro; Gillingham & Munro 2000). One to three microliters of DNA solution in PBS was injected into the lateral ventricle with a mouth-controlled glass capillary pipette, and square pulses (35 V, 50 ms, five pulses at 1 s intervals) were delivered into the embryos with tweezer-type electrodes, which consist of a pair of round platinum plates of 1 cm diameter (Unique Medical Imada, Miyagi, Japan) and by using an electroporator (CUY-21, NEPPAGENE, Tokyo, Japan). We co-injected a pCAGGS-RFP vector (kindly provided by Dr Masanori Uchikawa; Campbell et al. 2002) to visualize individual neuroepithelial cells. To record centrosomal behavior, we carefully chose GFP/RFP double positive cells.

Electron microscopy

Procedures were basically conducted according to the methods described previously (Uematsu et al. 2005). On E17, wild-type and rSey2/rSey2 rat embryos were perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M PB (phosphate buffer, pH 7.4), and the cortex primordium was dissected out, fixed again in 2% PFA and 2% glutaraldehyde in 0.1 M PB for 2 h. After washing in the same buffer, they were post-fixed with 1% OsO4, dehydrated in a graded ethanol series and embedded in Epon. Thin sections were stained with 5% uranyl acetate in 50% ethanol followed by 0.4% lead citrate and observed with a JEM 1200EX electron microscope at 100 kV.

Statistical analysis

Statistical analysis was performed using the Student's t-test in the analysis of results shown in Fig. 1 and the Mann–Whitney's U test for those in Fig. 4.


   Acknowledgements
 
We thank Dr Sean Munro for the GFP-AKAP450 plasmid vector, and Dr Masanori Uchikawa for the pCAGGS-RFP plasmid vector. We also thank Drs Wieland Huttner, Shigeo Ohno, Shuichi Hirai, Yoshio Wakamatsu and Masanori Takahashi for valuable suggestions on our work, and Ms Michi Otonari, Ms Ayumi Ogasawara and Ms Sayaka Makino for maintenance of the rSey2/rSey2 colony. We are grateful for the encouragement by all other members of our laboratory. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas "Molecular Brain Science" from MEXT and CREST from the Japanese Science and Technology Corporation (to N.O.). H.T. was a fellow of the 21st Century COE Program "Future Medical Engineering Based on Bio-Nanotechnology."


   Footnotes
 
Communicated by: Masayuki M. Yamamoto

* Correspondence: E-mail: osumi{at}mail.tains.tohoku.ac.jp


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 Discussion
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Received: 16 February 2007
Accepted: 28 May 2007




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