Polypyrimidine tract-binding protein (PTB) is a well-characterized RNA-binding protein and known to be preferentially expressed in neural stem cells (NSCs) in the central nervous system; however, its role in NSCs in the developing brain remains unclear. To explore the role of PTB in embryonic NSCs in vivo, Nestin-Cre–mediated conditional Ptb knockout mice were generated for this study. In the mutant forebrain, despite the depletion of PTB protein, neither abnormal neurogenesis nor flagrant morphological abnormalities were observed at embryonic day 14.5 (E14.5). Nevertheless, by 10 weeks, nearly all mutant mice succumbed to hydrocephalus (HC), which was caused by a lack of the ependymal cell layer in the dorsal cortex. Upon further analysis, a gradual loss of adherens junctions (AJs) was observed in the ventricular zone (VZ) of the dorsal telencephalon in the mutant brains, beginning at E14.5. In the AJs-deficient VZ, impaired interkinetic nuclear migration and precocious differentiation of NSCs were observed after E14.5. These findings demonstrated that PTB depletion in the dorsal telencephalon is causally involved in the development of HC and that PTB is important for the maintenance of AJs in the NSCs of the dorsal telencephalon.
In the central nervous system (CNS), post-transcriptional gene regulation, which includes processes such as pre-mRNA alternative splicing (Ule et al. 2005), mRNA localization (Bassell and Kelic 2004), turnover (Peng et al. 1998), and translation (Kuwako et al. 2010), all regulated by RNA-binding proteins (RBPs), is involved in many aspects of development including the survival and function of neurons. For example, a recent study in knockout mice has revealed that Nova2, a neuron-specific RBP, is indispensable for the proper migration of neurons in the cortex and cerebellum via the regulation of an RNA splicing event, which controls the function of the Dab1 protein (Yano et al. 2010). However, in general, the roles of RBPs expressed in neural stem cells (NSCs) are still elusive.
Polypyrimidine tract-binding protein (PTB) is an RBP expressed in NSCs that regulates mRNA stability (Kosinski et al. 2003; Knoch et al. 2004), internal ribosome entry site-dependent translation (Bushell et al. 2006), mRNA localization (Ma et al. 2007; Babic et al. 2009), and alternative splicing (Xue et al. 2009; David et al. 2010) by interacting with polypyrimidine-rich sequences of target pre- and mature mRNAs. PTB expression has been observed in various tissues and cell lines including embryonic stem cells (ESCs; Lillevali et al. 2001; Shibayama et al. 2009). In most mammals, 3 PTB paralogs, neural PTB (nPTB), regulator of differentiation 1 (ROD1), and smooth muscle PTB (smPTB), are expressed in a tissue-restricted manner. The paralog, nPTB, is expressed mainly in neurons (Polydorides et al. 2000), ROD1 is expressed in hematopoietic cells (Yamamoto et al. 1999), and smPTB is expressed in smooth muscle (Gooding et al. 2003). Interestingly, cross-regulation between PTB, nPTB, and ROD1 has been reported (Spellman et al. 2007).
In the context of development, although the importance of PTB in multiple biological processes in non-mammalian species has been reported (Hamon et al. 2004; Robida et al. 2010), there are few reports of a role for PTB in mammalian development and organogenesis. Previously, we generated Ptb knockout mice and Ptb null ESCs and found that PTB is essential for early mouse development and important for the proliferation and differentiation of mouse ESCs (Shibayama et al. 2009). Thus, it is expected that PTB would also have important roles in tissue stem cells.
Due to its interesting expression pattern, PTB has been well studied in the nervous system. Its expression is observed predominantly in NSCs and is lost in mature neurons (Lillevali et al. 2001; McKee et al. 2005; Boutz et al. 2007). The down-regulation of PTB is regulated, in part, by the nervous system-specific miRNA, miR-124 (Makeyev et al. 2007). An in vitro study demonstrated that the knockdown of PTB leads to wide-spread changes in alternative splicing events, similar to the changes that occur upon neural differentiation (Boutz et al. 2007). PTB is also highly expressed in differentiated cells including ependymal cells and choroid plexus (CP) epithelial cells (Lillevali et al. 2001), which are central to brain homeostasis with respect to cerebrospinal fluid (CSF) circulation or metabolism (Banizs et al. 2005). Based on these findings, PTB might affect both neural differentiation in the developing brain as well as brain homeostasis from late embryonic stage or later; however, to date, the role of PTB in the brain remains unknown.
In this study, we inactivated the Ptb gene by employing Cre-mediated conditional gene targeting systems and generated 3 lines of mutant mice. Analyses of 3 types of mutant brain revealed that PTB is required for the maintenance of adherens junctions (AJs) in embryonic NSCs in the dorsal telencephalon and that defect of AJs maintenance in NSCs causes premature depletion of NSCs and lack of ependymal cell layer, resulting in postnatal development of hydrocephalus (HC). However, except for in the dorsal telencephalon, the loss of AJs was not observed in the developing mutant brain. Our data suggest a new regulatory mechanism mediated by PTB influencing the maintenance of AJs in NSCs in vivo.
In this paper, the term “NSCs” is used to indicate both neuroepithelial cells and radial glial cells.
Materials and Methods
All mice were maintained on a mixed 129SV/J-C57BL/6 background. A floxed Ptb allele and a Neo cassette allele were generated by homologous recombination (Shibayama et al. 2009).,Nestin-Cre mice (Toshikuni Sasaoka et al., unpublished), Emx1-Cre mice (Iwasato et al. 2004), and Nestin-CreERT2 mice (Imayoshi et al. 2006) were crossed to Ptb+/neo mice to obtain Ptb+/neo; Nestin-Cre, Ptb+/neo; Emx1-Cre and Ptb+/neo; Nestin-CreERT2 mice. Mice, homozygous for the Ptb floxed allele (Ptbfloxed/floxed) and Ptb+/neo; Nestin-Cre as well as Ptb+/neo; Emx1-Cre and Ptb+/neo; Nestin-CreERT2 mice were bred to generate Ptbfloxed/neo; Nestin-Cre, Ptbfloxed/neo; Emx1-Cre and Ptbfloxed/neo; Nestin-CreERT2 mice. All mouse work was performed in compliance with the guidelines of the Institutional Animal Care and Use Committee of the University of Tokyo.
The following primary antibodies were used for western blotting (WB) and immunohistochemistry (IH): mouse anti-PTB (Zymed, 324800; WB, 1:2000), goat anti-PTB (Santa Cruz, sc-16547; IH, 1:400), mouse anti-nPTB (Abnova, H0058155-A01; WB, 1:1000; IH, 1:500), rabbit anti-Pax6 (Millipore, AB2237; IH, 1:500), mouse anti-Nestin (R&D, MAB2736; IH, 1:250), rabbit anti-Tuj1 (Covance, PRB-435P; WB, 1:5000; IH, 1:500), rabbit anti-Tbr2 (Abcam, ab23345; IH, 1:500), rabbit anti-Tbr1 (Abcam, ab31940; IH, 1:500), goat anti-DCX (Santa Cruz, sc-8066; IH, 1:500), rabbit-GFAP (glial fibrillary acidic protein; Dako, Z0334; WB, 1:1000; IH, 1:500), goat anti-GFAP (Santa Cruz, sc-6170; IH, 1:500), goat anti-Olig2 (R&D, AF2418; IH, 1:500), mouse anti-N-cadherin (N-cad; BD, 610920; WB, 1:1000; IH, 1:500), mouse anti-ZO-1 (Invitrogen, 339100; WB, 1:1000; IH, 1:500), rabbit anti-aPKC (atypical protein kinase C; Santa Cruz, sc-216; WB, 1:2000; IH, 1:250), mouse anti-Vinculin (Sigma, V9131; IH, 1:500), mouse anti-S100β (Sigma, S2532; IH, 1:250), mouse anti-acetylated α-tubulin (Ac-tub; Sigma, T6793; IH, 1:500), mouse anti-phospho-histone H3 (Cell Signaling, 9706; IH, 1:500), rat anti-BrdU (bromodeoxyuridine; Abcam, ab6326; IH, 1:250), rabbit anti-Ki67 (Novacastra, NCL-Ki67p; IH, 1:500), rat anti-Ctip2 (Abcam, ab18465; IH, 1:500), goat anti-Brn2 (Santa Cruz, sc-6029; IH, 1:250), and mouse anti-GAPDH (Millipore, MAB374; WB, 1:4000).
Forebrains from embryos and pups were excised in phosphate-buffered saline (PBS), the meninges were removed and the vesicles homogenized. After centrifuging, the samples were suspended in an appropriate amount of radio immunoprecipitation assay buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]). Samples were diluted to provide equal amounts of protein prior to SDS–polyacrylamide gel electrophoresis. Western blot analysis was performed according to the standard protocols, using the appropriate primary antibodies, and horseradish peroxidase-conjugated goat IgG (GE Healthcare, 1:2000–1/4000) was used as the secondary antibody. Immunoreactivity was detected using an enhanced chemiluminescence kit (GE Healthcare) and X-ray film (Fuji Film).
Cresyl Violet Staining and Immunohistochemistry
Brains of pups and embryos were fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C for paraffin-embedded sectioning (10 μm). Paraffin sections were rehydrated and stained using 0.05% Cresyl Violet. Rehydrated sections were heated at 121°C for 10 min in 10 mM sodium citrate (pH 6.0) or at 100°C for 10 min in a microwave. Immunostaining was performed using a blocking reagent (1× blocking reagent [Roche], 2% bovine serum albumin, 0.05% Tween-20, 0.1% Triton X-100, 1× PBS), while primary and secondary antibodies were diluted in the blocking reagent without Triton X-100. Alexa Fluor (1:500; Molecular Probes) was used as secondary antibody. Nuclei were visualized using 2 μg/mL1, 4′,6-diamino-2-phenylindole (DAPI; Sigma) diluted in the secondary antibody diluant, and sections were analyzed using a Keyence Biozero microscope or an Olympus fluorescence microscope.
Electron Microscopic Analysis
Brains of pups were fixed with 2% glutaraldehyde and 2% PFA in 0.1 M phosphate buffer (PB; pH 7.4) for an hour, and then the ventral region of the brain, the septum, and the hippocampus were removed and fixed overnight at 4°C in the same solution. Samples were washed with 0.1 M PB 3 times on ice at 5-min intervals and postfixed with 1% osmium tetroxide in 0.1 M PB for 2h. The samples were then washed with distilled water 5 times on ice at 5-min intervals, and dehydrated 3 times in an ethanol series. The ethanol was cleared with tert-butyl alcohol, and the samples were freeze-dried (ES-2030 Freeze Dryer; Hitachi), vapor-deposited with an HPC-1S osmium coater (Vacuum Devices) and observed with a field-emission scanning electron microscope (Model S-4200; Hitachi).
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis was performed on rehydrated paraffin sections (10 μm) using the In situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions. Sections were counterstained with 2 μg/mL DAPI to assess the total cell number.
Tamoxifen Administration and BrdU Labeling
Tamoxifen was administrated by gavage to pregnant females once at E16.5 (200 mg per kg body weight). BrdU was injected intraperitoneally into pregnant mice at E16.5 (50 mg per kg body weight). The interkinetic nuclear migration (INM) was measured by quantifying the number of BrdU-positive cells in each 20-μm zone, 30 min and 4 h after a single BrdU injection. The cell cycle exit rate was measured by quantifying the number of BrdU+/ki67− cells 24 h after a single BrdU injection.
A log-rank test was performed to compare the survival rate. All other comparisons were undertaken using the 1-way analysis of variance (ANOVA) with Tukey's test. P-values were considered to be significant at P < 0.05.
Subcellular Localization of PTB in Embryonic NSCs
Although it is known that PTB undergoes nucleocytoplasmic shuttling in neurons (Ma et al. 2007), its subcellular localization in NSCs in vivo has not been described. Therefore, we monitored the subcellular localization of PTB in NSCs in the mouse telencephalon, which showed that PTB is concentrated in the nuclei and does not co-localize with either the NSC marker, Nestin (predominantly localized to the cytoplasm, including both pial and apical processes), or with the AJs marker, aPKC (localized to the termini of NSC apical process; Fig. 1B–E). We also found high PTB expression in basally located basal progenitor cells (BPCs) and postmitotic neurons, as detected by double staining with the BPC marker, Tbr2, and the postmitotic neuron marker, Tuj1 (Fig. 1A,F–K). These observations suggested that PTB acts mainly in nuclei, and therefore, might not be involved in the localization of target mRNAs to the terminals of processes in NSCs under steady-state conditions and that PTB protein expression did not abolish upon neuronal differentiation.
Generation of Nestin-Cre–Mediated Ptb cKO Mice
To assess the role of PTB in the developing mouse brain, Nestin-Cre–mediated conditional Ptb knockout (Ptbfloxed/neo; Nestin-Cre, cKO) mice were generated using 2 targeted Ptb alleles (Shibayama et al. 2009). Ptb+/floxed; Nestin-Cre (Control) mice were used as controls for most of the experiments in this report. Immunofluorescence analysis revealed that the reduction in PTB protein levels is already underway at E12.5 and that PTB expression is abolished in the cortex and ganglionic eminences (GE) by E14.5 (Fig. 2A–F), although western blot analysis revealed residual PTB protein in the whole forebrain at E14.5 (Fig. 2G). Since PTB protein levels were barely detectable at E16.5, this residual protein must have originated from the caudal region of the telencephalon, where Cre-mediated recombination does not occur before E14.5 (Imai et al. 2006).
It has been reported that the knockdown of PTB up-regulates the expression of the nPTB protein in certain cell lines including HeLa, PAC1, and N2A, amongst others (Boutz et al. 2007; Spellman et al. 2007), and we observed a similar phenomenon in ESCs (Shibayama et al. 2009). In these cell lines, nPTB expression is suppressed by PTB via nPTB exon 10 suppression, which leads to nonsense-mediated decay. However, as both PTB and nPTB are expressed in ventricular walls in the embryonic mouse brain (Lillevali et al. 2001), there is a possibility that PTB does not suppress nPTB expression in NSCs in the embryonic brain. Immunofluorescence and western blot analyses were employed to test nPTB expression in the Ptb cKO forebrain and, as expected, the results showed that there is a little difference in the nPTB expression level between the Ptb cKO forebrain and control at E14.5 and E16.5 (Fig. 2G, Supplementary Fig. S1). These results suggested that, unlike several of the cell lines tested, PTB does not suppress nPTB expression at least in the embryonic forebrain.
PTB Depletion Results in the Development of Progressive Hydrocephalus
To examine the effect of PTB depletion in embryonic NSCs, we assessed the expression patterns of cell-type–specific markers. Immunofluorescence analysis revealed that the populations and localizations of Pax6+ NSCs, Tbr2+ BPCs, and Tuj1+ neurons, and the direction of Nestin+ fibers do not alter in the Ptb cKO cortex at E14.5 (Fig. 2H–O). In addition, glial cell marker, GFAP+ cells were not observed and there was no morphological abnormality in the Ptb cKO cortex at E14.5 (data not shown). These results revealed that abnormal neurogenesis and ectopic gliogenesis does not occur before E14.5 in the Ptb cKO cortex.
To analyze the effect of PTB depletion in brain homeostasis, the viability of Ptb cKO mice was monitored for the first 3 weeks. As a result of genotyping 147 pups at postnatal day 21 (P21), we found that Ptb cKO mice were born at the expected Mendelian ratio and were viable for at least 3 weeks (Fig. 3A). However, the heads of some Ptb cKO mice had a characteristic dome-like appearance at this stage (Fig. 3C,D) and nearly all Ptb cKO mice had died (90.9%) by 10 weeks (Fig. 3B). Histological analyses revealed that all Ptb cKO brains (N = 15) had dilations of the lateral ventricles (Fig. 3E–H) with variable severity, suggesting fully penetrant HC at P21. In addition, an increase in the number of astrocytes, a common occurrence in HC, was also observed in the Ptb cKO brain at P21 (Supplementary Fig. S2A–E; Miller and McAllister 2007; Sweger et al. 2007). In the control brains (N = 13), such abnormalities were never observed. Taken together, these results clearly suggested that PTB is important in brain homeostasis and that HC is probably the direct cause of the early mortality of Ptb cKO mice.
Region-Specific Disruption of Ependymal Cell Layer in the Ptb cKO Brain
HC is a progressive degenerative disorder and is one of the most common abnormalities found in the CNS. It is characterized by an excessive accumulation of CSF in the brain ventricle, and can be caused by an excess of CSF production, a lack of CSF reabsorption and impaired CSF flow.
To determine the cellular defect responsible for HC in Ptb cKO mice, we initially looked for stenosis in the brain, because the most common cause of HC is stenosis (Sakakibara et al. 2002; Nechiporuk et al. 2007). Although ventricular dilation was observed in Ptb cKO brains at P10 (Figs 3I,J and 4A–D), stenosis was not observed (Fig. 4A–F). Next, PTB expression in the subcommissural organ (SCO) and CP was examined, as it is known that abnormalities in these 2 organs lead to HC (Vio et al. 2000; Banizs et al. 2005). But there was no difference in PTB expression in the SCO and CP between the control and Ptb cKO brains at P10, which may be due to an absence or low-levels of Cre recombinase expression in these 2 organs (Supplementary Fig. S3). Thus, these results suggested that the HC in the Ptb cKO brain is caused by another mechanism.
The postnatal brain ventricles are lined by a layer of epithelial cells known as ependymal cells. Ependymal cells are derived from radial glia cells (RGCs) and bear dozens of cilia that beat in a coordinated manner to facilitate the circulation of the CSF at the end of maturation. Abnormal ciliogenesis compromises CSF dynamics without ventricular stenosis and leads to HC (Ibanez-Tallon et al. 2004; Lechtreck et al. 2008; Town et al. 2008).
To determine whether there were abnormalities in ependymal cilia and in ependymal cells themselves in the Ptb cKO brain, we initially performed histological analysis under light microscopy and found that the ependymal cell layer looks severely disorganized on the dorsal wall of the lateral ventricles (Fig. 3K,L). Next, we examined the expression of the ependymal cell marker, S100β, GFAP, the cilia marker, Ac-tub and the AJs markers, N-cad, and aPKC. Immunofluorescence analysis revealed the absence of Ac-tub+ cilia and an ependymal cells (S100β+, GFAP+, N-cad+, and aPKC+) in the dorsal wall of the lateral ventricles in postnatal Ptb cKO brains (Supplementary Figs S2F–K and Fig. 4I–N). By contrast, multiciliated ependymal cells were observed on the ventricular walls of other regions in Ptb cKO brains at P10 (Fig. 4A–H). Because these observations strongly suggested that ependymal cells themselves are absent on the dorsal wall of the lateral ventricles in the Ptb cKO brain, we tried to reveal identities of cells on the wall. Immunofluorescence analysis revealed that these cells are positive for the oligodendrocyte progenitor marker Olig2, Tuj1, or the neuroblast marker Dcx (Supplementary Fig. S4) and suggested that cells normally locate in the corpus callosum are exposed to the lateral ventricles due to the absence of ependymal cells in the Ptb cKO brain.
To confirm whether the absence of ependymal cells on the dorsal wall of the lateral ventricles and to check whether the absence of ependymal cells extended over the whole of the wall, we conducted scanning electron microscopic analysis at P10 and observed cilia as an indicator of ependymal cell. These results revealed that there was a complete lack of cilia in the anterior region of the ventricle wall in contrast to the striatal wall that did bear cilia (Fig. 5A–D). As expected, many cilia were observed on the posterior region of the dorsal walls of the lateral ventricles in Ptb cKO brains (Fig. 5E,F). This might be due to the late onset of Cre-mediated recombination in this region.
To confirm whether the specific disruption of the ependymal cell layer in the dorsal telencephalon was the cause of HC in the Ptb cKO brain, Ptbfloxed/neo; Emx1-Cre mice were generated and the phenotype was analyzed. With the Emx1-Cre allele, the Cre recombinase gene is inserted in front of the ATG start codon of the endogenous Emx1 gene, which is expressed exclusively in the dorsal telencephalon prior to the expression of Nestin (Iwasato et al. 2004). Histological analysis at P10 revealed that the lateral ventricles were dilated in Ptbfloxed/neo; Emx1-Cre mice (5 of 5 mice; Supplementary Fig. S5A–C) and immunofluorescence analysis revealed an absence of ependymal cilia in the whole of the dorsal telencephalon including both the anterior and posterior regions in Ptbfloxed/neo; Emx1-Cre brains (4 of 5 mice; Supplementary Fig. S5D–G). These results clearly indicated that the disruption of the ependymal cell layer led to HC in the Ptb cKO brain.
Gradual Loss of AJs in the Developing Dorsal Telencephalon in Ptb cKO Mice
Next, we investigated the mechanism underlying the lack of an ependymal cell layer in the Ptb cKO brain. Since most ependymal cells born before birth and postnatally maturate (Spassky et al. 2005), we first analyzed whether PTB is required for maturation and survival of ependymal cells in vivo. To this end, we generated tamoxifen-inducible Ptb knockout (Ptbfloxed/neo; Nestin-CreERT2) mice. Although tamoxifen administration at E16.5 abolished PTB expression in Ptbfloxed/neo; Nestin-CreERT2 at P0, mature ependymal cells were observed on the ventricular wall of the dorsal cortex at P10 (Supplementary Fig. S6). This result clearly indicated that PTB is dispensable for maturation and survival of ependymal cells.
Since ependymal cells are derived from NSCs in the embryonic brain, we next hypothesized that PTB depletion actually affected properties of the NSCs resulting in abnormal NSCs, which led to the lack of an ependymal cell layer. Immunofluorescence analysis revealed patches devoid of N-cad expression on the apical surface of the dorsal telencephalon in Ptb cKO brains at E14.5 (5 of 7 mice; Fig. 6A,B). Before E14.5, no such abnormality was detected (data not shown): However, by E15.5, these N-cad− patches were visible on the apical surface in all Ptb cKO brains (5 of 5 mice; Fig. 6C,D) and these patches grew bigger as the development proceeds. These patches also lacked expression of aPKC, the tight junction marker, ZO-1, and the AJs marker, Vinculin (Fig. 6E–L). Subsequently, the expression levels of AJs components in the forebrain were analyzed at E16.5. Western blot analysis revealed that the expression levels of N-cad, aPKC, and ZO-1 showed no decrease in Ptb cKO forebrains compared with controls (Supplementary Fig. S7A). Taken together, these results suggested that PTB is required for the localization of AJs components but does not regulate the expression levels of these proteins in NSCs in the dorsal telencephalon.
When the AJs-deficient ventricular zone (VZ) was examined at E18.5, we found that the lack of NSCs that expressed Pax6 and Nestin was observed in part of the VZ in Ptb cKO brains (6 of 6 mice; Fig. 6M–P). In all of these VZ, the loss of N-cad expression on the apical surface was observed (Fig. 6M,N). Because apoptosis was considered as a possible primary cause of the lack of NSCs in the VZ, a TUNEL assay was conducted to detect apoptotic cells in the AJs-deficient patches. However, apoptotic cells were not detected in the N-cad− patches of Ptb cKO brains at E16.5 (data not shown) or at E18.5 (N = 6, Fig. 6Q–T). These results indicated that, in the dorsal telencephalon in Ptb cKO mice, the loss of AJs is linked to the lack of an ependymal cell layer via the decrease of NSCs in the VZ without apoptosis.
Precocious Differentiation Occurs in the AJs-Deficient VZ
RGCs, which act as stem cells, divide increasingly in an asymmetric manner to self-renew and generate BPCs and neurons. BPCs migrate from the VZ to the subventricular zone (SVZ) and divide symmetrically to generate 2 neurons. During the differentiation of RGCs into BPCs and neurons, RGCs lose their stem cell properties including AJs, apico-basal polarity, and INM. Several studies have demonstrated that these properties are important for the precise regulation of the cell-fate decision controlling the differentiation from RGCs to BPCs and neurons in the developing brain (Machon et al. 2003; Chae et al. 2004; Cappello et al. 2006; Costa et al. 2008; Tsai et al. 2010). Thus, we hypothesized that precocious differentiation of RGCs occurred in the AJs-deficient VZ in Ptb cKO brains, and thus, we performed immunofluorescence analysis using antibodies for several cell-type–specific markers and AJs markers.
There was no distinction between the expression pattern of Pax6 in the VZ in controls and the ZO-1− VZ in Ptb cKO cortices at E15.5. However, the expression patterns of Tbr2 and Tuj1 were different (Fig. 7A–F). In the control cortices (N = 4), few Tbr2+ BPCs and Tuj1+ neurons were observed at the apical end of the VZ; however, in Ptb cKO cortices (N = 5), BPCs and neurons accumulated at the apical end of the ZO-1− VZ. At E16.5, Pax6− cells, ectopically located along the radial axis, were found in the center of the N-cad− VZ in Ptb cKO cortices (4 of 6 mice; Fig. 7G–H). Greater numbers (P < 0.05, 1-way ANOVA with Tukey's test) of BPCs, ectopically located at the apical side of the VZ, were observed in all parts of the N-cad− VZ in Ptb cKO cortices compared with the VZ in control and the N-cad+ VZ in Ptb cKO at E16.5 (Fig. 7I,J,M). In addition, accumulated neurons were observed along the radial axis of the N-cad− VZ, suggesting a possible defect in radial migration (Fig. 7K,L). Moreover, the cell cycle exit rate, which is an indicator of terminal differentiation (Buttitta and Edgar 2007), was increased in the N-cad− VZ (Supplementary Fig. S8A–H). Taken together, these results indicated that, in the AJs-deficient VZ, there was not only a defect in radial migration but also precocious differentiation from NSCs to BPCs and neurons.
In INM, the nuclei of newborn NSCs move away from the apical surface toward the basal lamina during G1 of the cell cycle, undergo S phase at a basal location, and return to the apical surface during G2 for the next mitosis (Sauer 1935; Takahashi et al. 1993). Although INM is linked to AJs and apico-basal polarity, studies in the mouse cerebral cortex and zebra fish retina showed that a disturbance in INM solely affected the cell-fate decision of NSCs (Xie et al. 2007; Del Bene et al. 2008).
To investigate INM in the AJs-deficient VZ, labeling analysis was conducted with the DNA base analog, BrdU, at E16.5. Cells were labeled with BrdU in S phase and their position was determined 30 min and 4 h after BrdU injection. After 30 min, although most BrdU-labeled nuclei were located in the basal part of the VZ in control cortices, BrdU-labeled nuclei were scattered throughout the aPKC− VZ in Ptb cKO cortices (Fig. 7N,O,T; Supplementary S7B,C). When the cortices were analyzed 4 h after BrdU injection, approximately 30% of BrdU-labeled nuclei reached an apical position (0–20 μm) and some of these nuclei resided on the apical surface in controls, whereas relatively few BrdU-labeled nuclei (P < 0.05, 1-way ANOVA with Tukey's test) reached an apical position and few nuclei were found on the apical surface in the aPKC− VZ in Ptb cKO cortices (Fig. 7P,Q,T). Since BrdU-labeled nuclei were spread throughout the aPKC− VZ in Ptb cKO cortices after 30 min, mitotic cells might also be distributed throughout in the AJs-deficient VZ of Ptb cKO cortices. Accordingly, mitotic nuclei were labeled by immunostaining using phospho-histone H3 antibodies. The majorities were located at the apical surface in controls, while a relatively minor population was found in the non-apical region, SVZ, and the intermediate zone. By contrast, in Ptb cKO cortices, only a minor portion of the mitotic nuclei was localized to the apical surface in the aPKC− VZ, with the majority found in non-apical regions of the VZ and SVZ (Fig. 7R,S,U). Next, we verified whether the BrdU-labeled cells remaining at the apical region in Ptb cKO cortices after 30 min were stem cells or differentiated cells. Immunostaining analysis revealed that almost all of these cells were positive for the stem cell marker, Pax6 (Supplementary Fig. S7B,C). Thus, the INM defect preceded the precocious differentiation of RGCs and might be responsible for triggering the differentiation event. While the INM was completely normal in the AJs-positive VZ in Ptb cKO cortices, the loss of AJs from the VZ in Ptb cKO cortices was directly responsible for the INM defect. Taken together, these results demonstrated that PTB indirectly affects the cell-fate decision of NSCs.
Depletion of PTB Dose not Affect Over All Laminar Structure but Affects Gliogenesis
Finally, we analyzed laminar structure and gliogenesis in the dorsal telencephalon of Ptb cKO mice during P0 to P10, because premature depletion of NSCs and/or loss of AJs in the dorsal telencephalon might affect both laminar structure and gliogenesis. Immunostaining analysis using several layer markers revealed that over all laminar structure was normal in the Ptb cKO cortex (Supplementary Fig. S9A–E). But we found periventricular heterotopias in mutant mice at P0 (Supplementary Fig. S9F–T) and P10 (data not shown). These heterotopias were located at the dorsal wall of lateral ventricles or striato-cortical junction and consisted of neuronal cells, which are positive for Brn2. This observation confirmed migration defect within affected AJ areas. Although overall laminar structure was normal, generation of GFAP+ gilal cell was affected in Ptb cKO mice. Before P2, few GFAP+ cells were observed in the VZ of the dorsal cortex in the control brain, but a few GFAP+ cells were observed in the ZO-1− VZ of the dorsal cortex in the cKO brain at P0 and quite a lot of GFAP+ cells were observed at P2 (Supplementary Fig. S10). Because morphology of the Ptb cKO cortex seemed almost normal until P5 (Supplementary Fig. S10C,F), these results suggested that PTB depletion and/or loss of AJs caused precocious differentiation of NSCs into glial cell. But total GFAP+ cell production significantly decreased at P5 probably due to the premature depletion of NSCs in the dorsal cortex in the Ptb cKO brain.
This study demonstrated that conditional disruption of the Ptb gene in the mouse brain leads to severe HC. A recent study showed that a partial loss and dysfunction of ependymal cell motile cilia in the dorsal wall of the lateral ventricles is enough to cause lethal HC (Tissir et al. 2010). In Ptb cKO brains, there were no abnormalities such as ventricular stenosis or morphological defects in the SCO and CP, which might lead to HC, and the only anomaly was a loss of the ependymal cell layer in the ventricular wall of the dorsal telencephalon. Moreover, Ptbfloxed/neo; Emx1-Cre mice, in which the depletion of PTB was restricted to the dorsal telencephalon, showed a similar hydrocephalic phenotype to Ptb cKO mice. Taken together, these results demonstrated that the direct cause of HC in Ptb cKO mice was the lack of an ependymal cell layer in the dorsal telencephalon. Although we could not exclude the possibility that abnormalities in differentiation from NSCs to ependymal cells also contributed to the lack of an ependymal cell layer in Ptb cKO brains, present data suggested that the lack of an ependymal cell layer was caused by the premature depletion of NSCs in the late embryonic stage and precocious differentiation of remaining NSCs to glial cells during postnatal first week.
The loss of AJs in Ptbfloxed/neo; Emx1-Cre mice was observed earlier than in Ptb cKO mice (data not shown). This time lag reflects a difference in Cre expression timing between the 2 Cre mouse lines and supports the notion that PTB is important for the maintenance of AJs in NSCs in the dorsal telencephalon throughout the brain development. The loss of AJs has been also observed in several knockout mice in which genes that encode AJs component proteins are disrupted (Machon et al. 2003; Cappello et al. 2006; Imai et al. 2006; Lien et al. 2006; Kadowaki et al. 2007). In these mice, gross abnormalities in the neuroepithelial tissue architecture, including the invasion of differentiated neuronal cells and INM disturbance have been observed. The phenotypes of these mutant mice are similar but more severe than for Ptb cKO mice. The difference in the severity of the brain malformations may arise from the variable timing of Cre expression and the biological stability of individual target proteins. However, in contrast to the various knockout mice described above, in which targeted AJs component proteins are depleted, the expression levels of AJs component proteins such as N-cad, aPKC, and ZO-1 did not alter in the Ptb cKO mouse brain compared with the control mouse. Thus it is possible that the milder phenotype of Ptb cKO mice may also arise from the unique function of PTB in NSCs, where it is involved in the localization of AJs component protein(s) but may not be involved in their expression. Loss of AJs has also been reported in Dlg5, Numb/Numbl, and α-Snap mutant mice (Chae et al. 2004; Nechiporuk et al. 2007; Rasin et al. 2007). Although neither of these proteins are AJs components, they co-localize with AJs at the apical surface of the VZ and are involved directly in the localization of AJs components. PTB, on the other hand, is localized to NSC nuclei and is not found at the apical surface of the VZ under steady-state conditions in the developing brain. Although it does not appear to associate directly with AJs, PTB depletion results in the loss of AJs, suggesting a previously unknown mechanism of AJs maintenance by RBPs in the developing brain.
Impact of PTB Depletion on AJs Maintenance in NSCs Varies in Different Regions of the Brain
The abnormalities observed in Ptb cKO brains described above were restricted to the dorsal telencephalon. In Ptb cKO mice, the expression of PTB was abolished, not only in the cortex but also in the GE of the developing forebrain; however, neither the loss of AJs nor the lack of the ependymal cell layer was observed in the GE and striatum. These results demonstrated that the impact of PTB depletion on AJs maintenance in NSCs varies in different regions of the brain. One possibility is that the requirement for PTB differs in different locations. A similar trend has also been reported in Msi1 and FMR2 RBP knockout mice (Sakakibara et al. 2002; Guo et al. 2011). Although it is unclear whether a lack of Msi1 affects NSCs in vivo, the disorganization of the ependymal cell layer was restricted to the wall of the aqueduct in Msi1 knockout mice. Moreover, in FMR2 knockout mice, adult neurogenesis was affected in the hippocampus but not in the SVZ, despite the fact that FMR2 expression was observed in NSCs, both in the hippocampus and the SVZ in wild-type mice. Interestingly, in the case of FMR2, a target mRNA (Noggin) is expressed in the NSCs of the hippocampus but not in those of the SVZ. Taken together, it appears that, in addition to the need for particular transcription factors, post-transcriptional regulation by certain RBPs, is required for specific NSC populations in vivo, and it is thought that downstream target(s) of PTB is a likely candidate for a specific regulator of AJs maintenance in the dorsal telencephalon. Another possibility is that the compensatory function of nPTB counteracts the depletion of PTB in the maintenance of AJs in the GE. PTB and nPTB show a marked sequence similarity, especially within their RNA recognition motif domains, which are responsible for specific binding to target RNA molecules (Kikuchi et al. 2000), and both are expressed in NSCs in the cortex and GE in the developing mouse brain. Moreover, previous in vitro microarray analysis revealed that these 2 proteins regulate the splicing of certain exons in the same direction (Boutz et al. 2007). PTB and nPTB are presumed to have redundant functions, although nPTB does not compensate for the function of PTB, at least in AJs maintenance in the dorsal telencephalon; however, it is possible that nPTB compensates for this function in the GE. Future experiments involving the generation of PTB/nPTB double knockout mice will be required to investigate the functional redundancy between PTB and nPTB in the developing mouse brain.
The Role of PTB in the Developing Brain
PTB, which is highly expressed in NSCs, is thought to decrease gradually during neural differentiation in the developing dorsal telencephalon, based on the results of RNA in situ hybridization analysis (Lillevali et al. 2001; McKee et al. 2005). However, the immunofluorescence analysis performed for this study revealed that PTB expression remained high in Tuj1+ postmitotic neurons, while a previous report showed that PTB was absent from NeuN+ mature neurons in the hippocampus and cerebellum of the adult mouse brain (Boutz et al. 2007). Thus, although Ptb mRNA might be abolished in Tuj1+ immature neurons, the decrease in PTB protein levels progressed gradually during neural maturation, probably due to the biological stability of the PTB protein. In this regard, it is reasonable to assume that PTB depletion in NSCs does not lead directly to precocious differentiation.
Questions that still need to be answered are: “what is the identity of the target regulated by PTB in NSCs?” and “how is this target protein involved in AJs maintenance?” Previous in vitro microarray analyses (Boutz et al. 2007; Makeyev et al. 2007; Xue et al. 2009) and our observations of the subcellular localization of PTB have indicated that a possible molecular mechanism, responsible for the loss of AJs in the Ptb cKO brain, might be a disturbance in the regulation of alternative splicing in NSCs. However, we were unable to detect significant changes in the alternative splicing of well known or possible target exons of PTB, using RT-PCR analysis in Ptb cKO cortices at E14.5, compared with controls (data not shown). This controversial result led us to focus on the differences in PTB expression levels between NSCs and cell lines. In several cell lines, including ESCs, and in several tumors, high-level PTB expression has been observed (Wang et al. 2008; David et al. 2010). These cell lines are likely to be greatly affected by PTB depletion in alternative splicing regulation, and consistent with this idea, we were able to detect significant changes in the alternative splicing of several exons, which are the same exons tested in Ptb cKO cortices, in Ptb null ESCs compared with wild-type ESCs. Thus further investigation, using microarrays, into the molecular mechanisms responsible for the loss of AJs in the Ptb cKO brain is required to check the change in transcriptional regulation that is a possible role of PTB (Brunel et al. 1996; Rustighi et al. 2002; Motallebipour et al. 2010), and mRNA stability, as well as alternative splicing. Answers to these questions will provide novel insights into the mechanisms responsible for the maintenance of AJs. AJs are also important for the maintenance of the epithelial cell layer in other tissues and organs and for the maintenance of the stem cell niche in certain types of stem cells (Zhang et al. 2003; Nechiporuk et al. 2007; Smalley-Freed et al. 2010; Piven et al. 2011). Since PTB is widely expressed in a variety of tissues, whether PTB is involved in the maintenance of the stem cell niche of tissues such as liver, intestine and the hematopoietic system is worthy of further investigation.
This work was supported by Grants from the Ministry of Education, Culture, Sports and Technology (MEXT), Japan (to N. Yoshida) and in part by the Global COE Program, “Center of Education and Research for Advanced Genome-Based Medicine – For personalized medicine and the control of worldwide infectious diseases,” MEXT, Japan.
We thank S. Itohara for providing the Emx1-Cre mice and R. Kageyama for providing the Nestin-CreERT2 mice. Conflict of Interest: None declared.