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First published online 18 March 2008
doi: 10.1242/jcs.025064
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Research Article |
1 Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
2 Bridgestone Laboratory of Developmental and Regenerative Neurobiology, Keio University School of Medicine, Tokyo 160-8582, Japan
3 Solution Oriented Research for Evolutional Science and Technology (SORST), Japan Science and Technology Agency (JST), Saitama 332-0012, Japan
4 Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, Saitama 351-0198, Japan
5 Laboratory for Nanosystems Physiology, Research Institute for Electronic Science, Hokkaido University, Hokkaido 060-0812, Japan
6 Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
7 Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan
* Author for correspondence (e-mail: hidokano{at}sc.itc.keio.ac.jp)
Accepted 24 January 2008
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Summary |
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Key words: Intermediate filament, Phosphorylation, Radial glia, POU, SOX, Cell cycle
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Introduction |
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), thickens as newly generated neurons accumulate between the ventricle and the pia. There are marked stage-dependent changes in the morphology of the progenitor cells in the developing cerebral cortex during the time that brain-wall thickening is most prominent (Takahashi et al., 1993) (summarized in Fig. 5A). Initially, in embryonic day (E) 9 to E11 mice, progenitor cells, called neuroepithelial cells, span the cerebral wall, crossing distances of up to 100 µm. As development proceeds, the progenitor cells adopt a radially elongated morphology connected to the ventricular and pial surfaces (also called `radial glial' cells) (Rakic, 2003). Their extended processes span 200-300 µm by E13-E14 and 400-500 µm at E15-E16.
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) or is connected to the pial surface, depending on whether or not it inherited the pial process from the parent cell (Miyata et al., 2001; Noctor et al., 2001). The daughter lacking the pial process (Fig. 5B, arrowhead ii) elaborates a new one, mainly during G1 phase, and subsequently adopts a bipolar `radial glial' morphology by the time it reaches S phase (Miyata et al., 2001; Noctor et al., 2002). The daughter that inherited the pial process (Fig. 5B, arrowhead i) must also elongate its process to span the thickening cerebral wall, and must do so within a single cell cycle (Takahashi et al., 1993). Although the elongated processes of neural progenitor cells have long been known to serve as a scaffold for migrating newborn neurons (Rakic, 1972), how their polarized and elongated morphology is established during brain development remains unclear. Here we ask, what is the `trick' that enables a progenitor cell to undertake two completely different jobs (elaborating a process and dividing) that must be carefully coordinated?
To approach this question, we focused on the transcriptional pattern of the nestin gene (Nes) for two reasons. First, nestin is strongly expressed in neural progenitor cells (Hockfield and McKay, 1985). Second, Nes encodes an intermediate filament (IF) protein (Lendahl et al., 1990), which comprises one of the three major cytoskeletal systems. Furthermore, a recent report showed that IFs are involved in the establishment of the bipolar morphology and also the twisting of the pial process that influences the kinetics of neural progenitor nuclear movement (Miyata and Ogawa, 2007). Moreover, several papers have shown that nestin is important in regulating the assembly or disassembly of the IF network (Sahlgren et al., 2001; Chou et al., 2003), and it has been proposed to function as a crossbridge with other cytoskeletal systems (Marvin et al., 1998). Thus, we predicted that the transcription of Nes might be coordinated with the stage- or cell-cycle-dependent morphological alteration of neural progenitor cells. To elucidate the mechanism for regulating Nes expression, we observed its transcription pattern in vivo, relative to cell development and the cell cycle.
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). EGFP is stable for too long to be used in evaluating changes that occur within such a short period as a single cell cycle. Therefore, we generated transgenic mice expressing destabilized Venus (dVenus) under the control of the same driver – the Nes second intronic enhancer (E/nestin:dVenus; Fig. 1A). Venus is a GFP derivative made from YFP and contains mutations that greatly accelerate the maturation of the fluorescent protein at 37°C. It also shows 10- to 100-fold stronger fluorescence than YFP in vitro (Nagai et al., 2002). In the dVenus construct, a PEST amino acid sequence from mouse ornithine decarboxylase is fused to the C-terminus of Venus, thus targeting the protein for rapid, cell-cycle-independent intracellular degradation (Li et al., 1998; Corish and Tyler-Smith, 1999). Therefore, the dVenus reporter cassette allows the strict on-off enhancer-promoter activity to be detected as a fluorescent signal (Kohyama et al., 2005).
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We first observed coronal sections of the developing forebrain of E/nestin:dVenus transgenic mice. The dVenus fluorescence was localized strictly to the ventricular zone (VZ) at all developmental stages (supplementary material Fig. S1). Because our focus was on the regulation of Nes transcription, we also studied the expression patterns of molecules from candidate regulator genes. The cis-element of Nes used to drive dVenus expression has been extensively studied, and the evolutionarily conserved region in the 3' half of the second intron contains an enhancer that is synergistically regulated by group B1 SOXs (SOX1-SOX3) and class III POU (Pit-Oct-Unc) transcription factors (Zimmerman et al., 1994; Lothian and Lendahl, 1997; Josephson et al., 1998; Tanaka et al., 2004). We found that the expression of both transcription factors roughly paralleled that of the dVenus expression during neurogenesis (supplementary material Fig. S1).
Usefulness of E/nestin:dVenus mice and correlation between fluorescence intensity and Nes mRNA expression
To confirm that the dVenus expression pattern of our novel transgenic mice faithfully reproduced the expression pattern of endogenous nestin, we first compared the dVenus expression from our transgenic mice with the EGFP pattern of E/nestin:EGFP mice, in which the EGFP sequence is driven by the same regulatory sequence as in the dVenus-expressing transgenic mice (Kawaguchi et al., 2001). To evaluate the transgenic lines under the same conditions, we mated E/nestin:dVenus with E/nestin:EGFP transgenic mice and generated a double-transgenic embryo. Because dVenus is a modified YFP, their emission spectra were close (Fig. 1B). To separate the signals from the same section, we used the linear unmixing function of the confocal laser-scanning microscope LSM510 META (Zeiss). Brains from E14.5 embryos expressing both transgenes were sectioned and counterstained with an antibody to β-tubulin III, an early neuronal marker (Fig. 1C,D). We found that the dVenus-expressing cells were entirely included within the population of EGFP-positive cells in the VZ, and there was no overlap between dVenus expression and β-tubulin-III-expressing neurons (Fig. 1D). However, some EGFP-positive cells were double-labeled for β-tubulin III, and the EGFP signal persisted in cells in the intermediate zone, even after their neuronal differentiation (Fig. 1D, arrowheads). These results indicated that the E/nestin:dVenus transgenic mice expressed dVenus only in cells that had not differentiated into neurons, showing that the dVenus fluorescence was more faithful to the nestin expression pattern than the EGFP fluorescence driven by the same enhancer.
To determine the correlation between dVenus expression and the differentiation state, we sorted cerebral wall cells from dVenus transgenic embryos by fluorescence-activated cell sorting (FACS). Cells were fractionated into dVenus-positive (F2) and -negative (F1) populations, following the profile of the negative control from wild-type mouse brain (Fig. 1E). We characterized the cells in each fraction using antibodies against nestin and β-tubulin III. As expected, nestin-expressing cells were abundant (96.4±1.2%) in the F2 fraction, and made up less (26.9±1.0%) of the F1 fraction. The relative abundance of the β-tubulin-III-positive cells was reciprocal to that of the nestin-expressing cells: they made up 0.7±1.0% of the cells in F2 and 80.3±6.4% of the cells in F1 (Fig. 1F).
To confirm the accuracy of the sorting process, we also used an anti-GFP antibody to label dVenus. We found that the dVenus-expressing cells were reliably sorted into the F2 fraction (94.7±2.5%) and were almost absent from the F1 fraction (1.0±0.3%) (Fig. 1F). Moreover, cells from the F2 fraction formed self-renewable and multipotent neurospheres at a much higher rate (16±1 cells/500 cells) than those from the F1 fraction (0.2±0.4 cells/500 cells) (supplementary material Fig. S2B,C). These observations confirmed that our new dVenus reporter transgenic mice were more useful than the E/nestin:EGFP transgenic mice for characterizing neural stem/progenitor cells.
To determine the correlation between Nes mRNA expression and dVenus fluorescence intensity, we subdivided the dVenus-positive fraction (F2) into equal subfractions containing the high (F4) and low (F3) transgene-expressing-cells, based on fluorescence intensity (Fig. 1E). The level of Nes mRNA in the dVenus (and largely nestin)-positive progenitor populations (F3 and F4) and the dVenus-negative fraction (F1) was determined by real-time quantitative PCR. The fluorescence intensity of the E/nestin:dVenus cells exhibited a positive correlation with the amount of Nes mRNA (Fig. 1G), indicating that the reporter expression reflects the actual expression of Nes mRNA. Therefore, we monitored the expression level of Nes mRNA via the fluorescence intensity of dVenus in vivo.
Cell cycle-dependent expression of Nes mRNA
Because EGFP is a stable protein, the level of fluorescence produced by E/nestin:EGFP was uniform throughout the embryonic VZ. By contrast, owing to the short half-life of dVenus, the fluorescence in the VZ of E/nestin:dVenus embryos showed scattered fluorescent cells (Fig. 1D). This observation was consistent with our idea that the enhancer activity of Nes might be regulated by the cell cycle in neural progenitors, particularly because the VZ contains proliferating cells in various cell cycle phases. Therefore, we focused on the relationship between dVenus expression and the cell cycle.
Each cell cycle phase was discriminated by labeling the cells for differing periods of time before sacrifice with thymidine analogues (Fig. 2A; see Materials and Methods for details). The cells that expressed dVenus were counted at each cell cycle phase. To label cells in S phase, IdU was injected into pregnant dams 30 minutes before sacrifice (Fig. 2B purple). A high proportion of cells in S phase expressed dVenus (Fig. 2B arrowheads; 63.3±5.2%). However, cells in G2-M phase, which were labeled by injecting IdU 3 hours before sacrifice (Fig. 2C red), showed hardly any dVenus expression (Fig. 2C arrows; 9.3±1.4%). dVenus expression was stronger (Fig. 2D arrowheads; 47.4±7.9%) in G1 phase cells, which were labeled by injecting IdU 14.5 hours before sacrifice (Fig. 2D red). To distinguish cells in S phase, BrdU was injected 30 minutes before sacrifice in all trials (Fig. 2B-D purple). These data strongly support the hypothesis that Nes expression is regulated in a cell-cycle-dependent manner within the VZ, with expression being downregulated during G2-M phase (Fig. 2E).
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), we next explored the relationship between Nes expression and the three-dimensional (3D) morphology of the neural progenitor cells, especially the radial glial cells. To visualize live radial glial cells, we placed DiI on the pial surface of the cerebrum and performed time-lapse imaging of cerebral wall slices. Radial glial cells are known to retain the radial process during mitosis, and the fiber is inherited by one of the daughter cells (Miyata et al., 2001; Noctor et al., 2001). Fig. 3A shows a series of time-lapse imaging of a pair of daughter cells 4 hours after their birth at the ventricular surface. The daughter cell that did not inherit the radial fiber (Fig. 3A, arrowhead ii) began to extend a new pial process, and its sister cell (Fig. 3A, arrowhead i) thickened and extended its inherited fiber 20 µm according to the brain wall thickening within this period (Fig. 3A, *). The strong expression of dVenus observed in both of the newly born daughter cells (Fig. 3A) confirmed that the expression level increased during G1 phase, as we had observed in the BrdU/IdU labeling assay (Fig. 2). This observation also demonstrated that the dynamic activation of the Nes enhancer correlates with the 3D growth of cortical progenitor cells.
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Because the nuclear movement towards the ventricular surface (adventricular migration) is characteristic of G2 phase progenitor cells (Hayes and Nowakowski, 2000), we next asked whether progenitor cells whose nucleus was translocating toward the ventricular surface would show downregulated dVenus expression, as the BrdU/IdU labeling suggested (Fig. 2). As expected, dVenus was consistently undetectable in these G2-phase cells (Fig. 3B) (n=4). Immunostaining for phospho-histone H3 further demonstrated the lack of dVenus expression in M-phase cells (Fig. 3C). Thus, Nes expression is high in G1 phase during process extension, and it is lost in G2-M phase, which is characterized by the adventricular nuclear movement and rounding up of the soma.
Phosphorylation of Brn2 on Ser362 reduces Brn2 binding to DNA and lowers the expression of Nes during G2-M
We next addressed the underlying molecular mechanism of the cell-cycle-dependent regulation of Nes. We first investigated whether the responsible transcription factors were expressed in a cell-cycle-dependent manner. Immunohistochemical analysis for SOX1-SOX3 and class III POU transcription factor (Pou3f2 or Brn2) showed them to be expressed ubiquitously throughout the VZ (Fig. 4A), indicating that dVenus was expressed only in a subset of these SOX- or POU-expressing cells. This result led us to consider whether post-translational modification of the transcription factors might contribute to the cyclic changes in dVenus expression.
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; Segil et al., 1991; Caelles et al., 1995), and the phospho-acceptor site is highly conserved in all POU-domain proteins. This site corresponds to Ser362 in Brn2. Therefore, we next asked whether the phosphorylation of Brn2 increased during G2-M in vitro. 293T cells expressing Flag-tagged Brn2 or Brn2 S362A, in which Ser362 is replaced by alanine, were synchronized at G1, S and G2-M phases (supplementary material Fig. S3), and labeled with [32P]orthophosphate (GE Healthcare) for the 6 hours preceding harvest. The level of Brn2 phosphorylation was low in G1 (Fig. 4B lane 1) and S (Fig. 4B lane 2) phases, but it increased as the cells progressed through the cycle to G2-M phase (Fig. 4B lane 3). By contrast, the phosphorylation level of Brn2 S362A remained constant, showing essentially the same level in G2-M (Fig. 4B lane 5) as in G1 (Fig. 4B lane 4). These results indicate that Brn2 is phosphorylated on Ser362, in a cell-cycle-dependent manner, during G2-M phase.
To investigate the relationship between the phosphorylation of Brn2 and Nes expression, we next introduced a phosphorylation-mimicking point mutation that substituted Asp for Ser at Brn2 Ser362. We first used this construct in luciferase reporter assays (Fig. 4C). An octamerized 30-mer nestin core enhancer (Nes30) (Tanaka et al., 2004), which includes the SOX- and POU-binding sites, was fused to the rabbit β-globin minimal promoter and inserted into the pGL3 basic luciferase reporter vector. When SOX2 was expressed alone, Nes30 was weakly activated (4.5-fold). The expression of Brn2 alone led to stronger activation (8.6-fold). By contrast, Brn2 S362D increased reporter expression only slightly, to twice the basal level. When SOX2 and Brn2 were expressed together, Nes30 activity increased dramatically (24.5-fold). This synergistic action, consistent with a previous report (Tanaka et al., 2004), remarkably reduces when Brn2 was replaced with Brn2 S362D in the same experiment. The Nes30 activity increase (5-fold) was the same as for SOX2 alone (Fig. 4C).
This effect of expressing phospho-mimicking Brn2 on Nes30 activity could be caused by a decreased binding affinity for DNA or by instability of the phosphorylated Brn2. We performed an electrophoretic mobility shift assay (EMSA) to discriminate between these possibilities, using the Nes30 sequence probe and whole-cell lysates of 293T cells overexpressing Flag-tagged Brn2 or Brn2 S362D. The amount of Brn2 S362D binding complex was significantly reduced (Fig. 4D black arrow; lane 3), compared with that of wild-type Brn2 (Fig. 4D black arrow; lane 2). To ascertain whether the binding to Nes30 was Brn2 specific, an anti-Flag antibody was added to the binding reactions in a super-shift assay, which showed specific binding of the Flag-tagged Brn2 (Fig. 4D black arrowhead; lanes 4, 5). No binding reaction was observed in lysates of untransfected 293T cells (Fig. 4D lane 1), and no significant difference was seen between the stability of Brn2 and phospho-mimicking Brn2 S362D (Fig. 4E lane 2, 3), normalized to the expression of 
-tubulin (Fig. 4E, *), consistent with the uniform expression pattern of Brn2 in the VZ in vivo (Fig. 4A). These results strongly suggested that the reduction of Nes expression during G2-M phase is due to the decreased binding affinity of phosphorylated Brn2 for the Nes core enhancer element. Therefore, we conclude that the phosphorylation of POU transcription factors downregulates the cell-cycle-dependent expression of Nes.
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), the fluorescent signal was still detectable after cells had differentiated into neurons (Fig. 1D). Therefore, the novel transgenic mouse expressed dVenus in the desired pattern, but with a half-life that restricted its expression to neural progenitors. Our novel transgenic mice also allowed us to prospectively isolate self-renewable and multipotent neural stem cells using flow cytometry (supplementary material Fig. S2), and real-time quantitative PCR analysis showed that the dVenus reporter fluorescence intensity accurately reflected the relative expression levels of Nes mRNA (Fig. 1G). These results confirmed the utility of this reporter system. We then used this system to trace changes in the transcription of Nes in vivo during the cell cycle.
Relationship between SOX-POU signaling and the maintenance of neural progenitor cells
The SOX family of HMG-box transcription factors is a key regulator of embryonic development and cell-fate determination. In certain cases, SOX1-SOX3 interact with various partner transcription factors and participate in defining distinct cell states that depend on the partner factors, such as Pax6 in lens differentiation, Oct3/4 in establishing the epiblast/ES cell state and Brn2 in the neural primordium (Kamachi et al., 2000; Tanaka et al., 2004). Although SOX1-SOX3 are known to suppress neurogenesis by maintaining neural progenitor cells in an undifferentiated state in the vertebrate CNS (Bylund et al., 2003), the responsible target gene has not been identified. Since Nes is a target gene of SOX and POU transcription factors (Tanaka et al., 2004), we monitored its expression pattern to learn more about the contribution of SOX proteins to the maintenance of the neural progenitor cell state. We found that the expression of Nes, as represented by the fluorescence intensity of dVenus, reflected its activation by a SOX-POU complex (supplementary material Fig. S1). We also found that transcription by the SOX-POU complex was inactivated during G2-M (Figs 2, 3) by the cell-cycle-dependent phosphorylation of the POU transcription factor (Fig. 4). Our data suggest that the cell-fate choice to remain an undifferentiated neural progenitor cell, regulated by the SOX-POU complex, is completed during G1 to S phase. Interestingly, the activation of Notch1 (indicated by its nuclear localization), which maintains neural progenitor cells in the undifferentiated state by inducing Hes1/5 expression (Gaiano and Fishell, 2002), also takes place in G1 to S phase (Tokunaga et al., 2004). However, it is still unclear whether cells that have been stimulated with SOX-POU during the early G1 phase can change their cell fate decision and differentiate into neurons without further progression of the cell cycle. Therefore, future research needs to address the timing of the cell-fate decision during G1 phase of daughter cells. Our present study demonstrates that the target genes of the SOX-POU transcription factor complex are regulated in a more complicated manner than expected (Tanaka et al., 2004), and it provides a model for further understanding the contribution of SOX transcription factors to the maintenance of neural progenitor cells in an undifferentiated state.
Cell-cycle-dependent expression of nestin
By allowing mouse cells to incorporate thymidine analogues at different times before sacrifice, we confirmed that Nes expression was regulated in a cell-cycle-dependent manner in vivo (Fig. 2). Moreover, in vitro experiments suggested that the weak Nes expression during G2-M could be attributed to the cell-cycle-dependent phosphorylation of Brn2 (Fig. 4B). This phosphoacceptor site at Brn2 Ser362 is highly conserved in all POU-domain proteins, and other POU proteins, such as Oct1 and GHF1 (Pit1), are phosphorylated on the same site during G2-M (Kapiloff et al., 1991; Segil et al., 1991; Caelles et al., 1995). In agreement, our data showed a marked decrease in reporter activity from the Nes core enhancer when Asp was substituted for Brn2 Ser362 (Fig. 4C). By EMSA, we demonstrated that the reduced transcription was due to a dramatic drop in Brn2 S362D binding of the Nes core enhancer, compared with wild-type Brn2 (Fig. 4D). Our hypothesis that cell-cycle-dependent Brn2 phosphorylation regulates Nes expression is based on in vitro studies, and we have not characterized the kinase responsible for this regulation. However, because Cdc2 mitotic kinase, which is ubiquitously expressed in all cells during G2-M phase, is a candidate kinase for the cell-cycle-dependent phosphorylation of Oct1 and GHF1 (Segil et al., 1991; Caelles et al., 1995), it is reasonable to expect that Brn2 is subject to the same regulatory mechanism in the developing brain in vivo.
Physiological relevance of nestin in neural progenitor cells in vivo
In spite of the numerous IF family members, the expression of IFs exhibits a high degree of cell-type specificity (Herrmann and Aebi, 2004). The unique physical properties and interaction capabilities of these distinct IF molecules with accessory proteins mediate the generation of a highly dynamic and interconnected cell-type-specific cytoarchitecture. In the CNS, undifferentiated progenitor cells express nestin and vimentin. During neural differentiation, nestin is downregulated and displaced with the expression of neurofilament (NF) proteins and/or peripherin in neurons, and GFAP in astrocytes (Lariviere and Julien, 2003). Although many of the gene-targeting approaches of the neural IF genes demonstrate mild phenotypes, substantial developmental loss of motor axons was detected in mice lacking NF-L and in NF-M–NF-H double knockout mice. Mice lacking NF-L also had a scarcity of IF structures and exhibited a severe axonal hypotrophy (Lariviere and Julien, 2003). In GFAP and vimentin double-deficient mice, the post-traumatic glial scarring was looser and less organized (Pekny et al., 1999). This evidence supports a role for IFs in cytogenesis and stabilization.
Nestin cannot form filaments of its own, but it can readily form copolymer IFs when combined with vimentin both in vivo and in vitro (Marvin et al., 1998; Steinert et al., 1999). A complex of nestin-vimentin heterodimers is less stable than vimentin homopolymers when subjected to increasing concentrations of urea in vitro (Steinert et al., 1999). Therefore, nestin can retain the flexibility of the IF network in neural progenitor cells. It has been reported that the pial process of neural progenitor cells exhibits a coiled or hairpin-loop structure that produces a spring-like force, which functions to propel the cell soma away from the proliferating VZ (Miyata and Ogawa, 2007). Since the IF network has been shown to have a vital role for this twisting of the process (Miyata and Ogawa, 2007), nestin can enable the neural progenitor cell to possess this complicated architecture. This assumption is supported by the fact that the IF network depolymerizes during M phase, requiring the twisting process to take place during G1 phase when the Nes transcription is most prominent (Fig. 2).
Furthermore, nestin has been reported as a potential target of cyclin-dependent kinase 5 (Cdk5) and phosphorylation may target it for subsequent ubiquitylation and degradation outside the VZ (Sahlgren et al., 2003). In the Cdk5-deficient cortex, nestin expression persisted in cortical neurons (Cicero and Herrup, 2005), suggesting that the post-translational modification fine-tunes the expression of nestin. Since Cdk5 is predominantly observed outside the VZ, phosphorylation of nestin must take place after the transcriptional shutdown (Fig. 1D). Interestingly, transcription of Nes is also downregulated in G2-M (Fig. 2) when Cdc2 phosphorylates nestin (Sahlgren et al., 2001; Chou et al., 2003), suggesting that the transcription and post-translational modification is precisely orchestrated.
Correlation between Nes expression and morphological change in neural progenitor cells in vivo
The reduction in the binding activity of transcription factors during G2-M might be a general event that frees chromatin during mitosis. However, we also found that the molecular regulation of Nes correlated with changes in the 3D morphology of the neural progenitor cell in vivo. During the transition from M to G1, the process of the progenitor cell becomes extremely thin. Simultaneously, mitosis-specific phosphorylation promotes disassembly of the IF network (Yasui et al., 2001; Sahlgren et al., 2001; Noctor et al., 2002; Chou et al., 2003). Although the thin fiber is retained during M phase and inherited by only one of the daughter cells (Fig. 3A), both daughter cells must reorganize their cytoskeleton during G1 to S phase, either to extend a new process or to thicken the inherited one and elongate it to match the increasing width of the cerebral wall. Even if the nestin protein is stable and is divided equally between the daughter cells, it must double in quantity to effect this network-remodeling step. In addition, this cyclical regulation of Nes transcription provides nestin concomitant with the elongation of the radial fiber in G1 to S phase (Fig. 2 and Fig. 3A), which must happen at each division because of the continuous thickening of the cerebral wall (Takahashi et al., 1993). Moreover, it is interesting that the length of G1 phase extends during development (Takahashi et al., 1995), simultaneously with the requirement for a longer process, as the cerebral wall thickens (Takahashi et al., 1993).
In addition to nestin being supplied when needed for process elongation, the dramatic decline in Nes expression during G2 phase, when neural progenitor cells possess a thick and matured process, is also crucial for normal morphological dynamics (Fig. 2 and Fig. 3B,C). It is known that nestin disassembles the IFs through phosphorylation during the transition from M to G1 (Sahlgren et al., 2001; Chou et al., 2003). However, an extremely high dosage of nestin can also disassemble the IFs physically (Steinert et al., 1999), irrespective of its phosphorylation state. Thus, the reduction of Nes transcription in G2-M phase is essential, once the progenitor cell obtains a mature IF network, for preserving the appropriate timing of IF disassembly. Furthermore, an attractive hypothesis is that the expression of Nes correlates with the cell-cycle-dependent migration of the nuclei, described as interkinetic nuclear movement (Sauer and Walker, 1959; Fujita, 1962). Recently, several groups proposed that microtuble- and actin-dependent mechanisms are important for the adventricular nuclear movement in G2 phase. Knockdown of LIS1 (Tsai et al., 2005), myosin II (Tsai et al., 2007), Cep120 and transforming acid coiled-coil proteins (TACCs) (Xie et al., 2007) impaired the migration of the nuclei towards the ventricular surface without affecting the bipolor morphology of the neural progenitor cell. By contrast, the mechanism of migrating away from the ventricular surface in G1 phase, which is slower than the adventricular migration (Takahashi et al., 1995), remains unknown. Because of the synchronization of Nes transcription and the nuclear movement, nestin could be involved not only in the extension of the progenitor cell, but also the nuclear movement in G1 phase. Building on previously reported results showing that IF depolymerization creates abnormal neural progenitor cell morphology (Miyata and Ogawa, 2007), our present study, which accurately maps the expression profile of Nes, strongly suggests that not only post-translational modification but also precise transcriptional regulation are vital for (1) maintaining the 3D morphology of neural progenitor cells and (2) their mitotic cycle, through remodeling of the cytoskeletal network in vivo (Fig. 5).
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) was subcloned into the AseI-BamHI site of pd4EGFP-N1 (BD Biosciences, San Jose, CA). The gene for Venus was inserted into this plasmid in place of that for EGFP. The purified transgene was microinjected into the pronucleus of fertilized mouse eggs. Four independent lines showing identical expression patterns were generated in a C57BL/6 background. The data presented are from experiments using heterozygous mice from a single line. All procedures were performed in accordance with institutional guidelines. The date of conception was established by the presence of a vaginal plug and recorded as E0.5.
Immunohistochemistry and immunocytochemistry
Post-fixed mouse embryo brains were embedded in 3% LMP Agarose (Invitrogen, Carlsbad, CA) and sectioned at 50 µm on a vibratome (VT1000; Leica, Heidelberg, Germany). Free-floating sections were washed twice with 0.1 M phosphate-buffered saline, pH 7.4 (PBS) and blocked with blocking solution (PBS containing 5% normal donkey serum and 0.3% Triton X-100; Sigma-Aldrich, St Louis, MO) for 30 minutes. Subsequently, sections were incubated overnight at 4°C in a mixture of the primary antibodies described above, in blocking solution. After washing three times with PBS, sections were reacted with secondary antibodies at room temperature for 2 hours, washed three more times, and then coverslipped. Immunostained sections were observed with a confocal laser-scanning microscope LSM510 META (Zeiss, Jena, Germany). dVenus fluorescence was readily detectable in fixed sections. However, when the counterstaining antibody required some pretreatment of the sections, dVenus was immunostained with a rabbit anti-GFP antibody. Sorted cells were spun down immediately onto poly-L-ornithine (15 µg/ml) and fibronectin (1 µg/ml) coated coverslips and immunostained by standard procedures. Post-stained cells were observed under a fluorescence microscope (Axiophoto; Zeiss). Primary antibodies against nestin (Rat401; Developmental Studies Hybridoma Bank of Iowa University), β-tubulin III, GFAP (Sigma), O4 (Chemicon, Temecula, CA), GFP (MBL, Woburn, MA), Brn2 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-histone H3 (Cell Signaling Technology, Danvers, MA) and Group B1 SOX [SOX1-3; a kind gift from H. Kondoh, Ohsaka University (Tanaka et al., 2004)] were used according to the manufacturers' protocols.
Flow cytometry
The cerebral wall of E14.5 transgenic or wild-type mouse embryos was dissected out into a chilled Hank's buffered saline solution, and suspended by brief mechanical treatment. One µg/ml of propidium iodide (PI) was added and the cells were filtered through a nylon mesh. Cell sorting and analysis were performed using MoFlo (Cytomation, Fort Collins, CO) with Summit software. Cells were analyzed for forward scatter, side scatter, PI fluorescence and dVenus or EGFP fluorescence with an argon laser (488 nm, 100 mW). Dead cells were excluded by gating on forward and side scatter, and by eliminating PI-positive events. Viable cells from the transgenic mice were sorted into ice-cold culture medium. Cells harvested from wild-type mice were used to set the background fluorescence.
Determination of cell-cycle kinetics
Timed-pregnant female mice bearing transgenic embryos expressing dVenus were given thymidine analogues (50 µg/g body weight of the pregnant mouse) by intraperitoneal injection. The thymidine analogues, 5-bromo-2'-deoxyuridine (BrdU) and 5-iodo-2'-deoxyuridine (IdU) (Sigma-Aldrich), are incorporated into cells during the S phase of the cell cycle. The length of each cell-cycle stage in the dorsomedial region of the E14.5 mouse embryo was reported previously (Takahashi et al., 1995). On the basis of this report, we identified the cells that were labeled with IdU injected 3 hours before sacrifice as being in the G2-M phase. To overcome potential confusion from the long half-life of IdU, and to eliminate the labeling of cells remaining in G2-M phase, cells in the G1 phase were labeled with IdU 14.5 hours before sacrifice, when all the labeled cells had left the ventricular surface. Cells in the S phase were labeled by injecting BrdU 30 minutes before sacrifice in each trial. To detect BrdU and IdU, sections were pretreated with 2 M HCl for 5 minutes at room temperature, neutralized with 0.1 M Tris-HCl (pH 8.8) for 10 minutes, washed three times with PBS, and treated as described above for immunological staining from the blockage step. To detect IdU, an antibody that crossreacts with BrdU (Invitrogen) was used. The BrdU signal was distinguished from the IdU signal with an anti-BrdU-specific antibody (Abcam, Cambridge, MA).
Reporter assays
An octamerized Nes30 sequence was fused to rabbit β-globin minimal promoter and inserted into the pGL3-Basic (Promega, Madison, WI) reporter vector. For reporter assays, NIH3T3 cells were cotransfected with 0.6 µg reporter, 10 ng effecter and reference pEF-LacZ (0.6 µg) using Lipofectamine Plus (Invitrogen). The amount of transfected DNA was equalized with empty vectors.
Electrophoretic mobility shift assay
Flag-tagged Brn2 and Brn2 S362D were overexpressed in 293T cells. After 2 days, whole-cell lysates were prepared in a lysis buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 30 mM sodium phosphate, 50 mM sodium fluoride, 20 mM β-glycerophosphate, 1% Triton X-100 and protease inhibitor mixture (Complete; Roche Applied Science, Basel, Switzerland). The sense and antisense Nes30 sequences were annealed and labeled with [
-32P]ATP (GE Healthcare, Piscataway, NJ) as a probe. The binding reaction was performed in a total volume of 25 µl binding buffer (50 mM HEPES-KOH pH 7.8, 250 mM KCl, 5 mM EDTA pH 8.0, 25 mM MgCl2, 50% glycerol, and 25 mM DTT). Cell lysates were incubated for 30 minutes at room temperature with the labeled oligonucleotides. For the supershift assay, the binding product was incubated with an antibody against Flag (Sigma) for another 30 minutes. The samples were separated on native 8% polyacrylamide gels at 20 mA in Tris-borate EDTA buffer.
Slice culture and DiI labeling
Slice culture and DiI labeling were performed as reported previously (Miyata et al., 2004). The cerebral cortex of E14.5 E/nestin:dVenus transgenic mouse embryos was labeled with DiI from the pial surface and sliced into coronal sections. Sections were embedded in collagen gel for time-lapse recording. The cultured slices were fixed in 4% paraformaldehyde for 15 minutes, vibratome sectioned and subjected to confocal microscopy to observe the dVenus expression in DiI-labeled cells.
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