PTB Deficiency Causes the Loss of Adherens Junctions in the Dorsal Telencephalon and Leads to Lethal...

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PTB Deficiency Causes the Loss of Adherens Junctions in the Dorsal Telencephalonand Leads to Lethal Hydrocephalus

Takayuki Shibasaki1,2, Akinori Tokunaga1, Reiko Sakamoto1, Hiroshi Sagara3, Shigeru Noguchi4, Toshikuni Sasaoka5

and Nobuaki Yoshida1

1Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, 2Graduate School of FrontierScience, 3Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Tokyo, Japan 4Meiji Institute ofResearch and Development, Meiji Milk Products Company Limited, Tokyo, Japan and 5Department of Laboratory AnimalScience, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan

Address correspondance to N. Yoshida, Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, Instituteof Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Email: nobuaki@ims.u-tokyo.ac.jp

Polypyrimidine tract-binding protein (PTB) is a well-characterizedRNA-binding protein and known to be preferentially expressed inneural stem cells (NSCs) in the central nervous system; however,its role in NSCs in the developing brain remains unclear. To explorethe role of PTB in embryonic NSCs in vivo, Nestin-Cre–mediatedconditional Ptb knockout mice were generated for this study. In themutant forebrain, despite the depletion of PTB protein, neither ab-normal neurogenesis nor flagrant morphological abnormalities wereobserved at embryonic day 14.5 (E14.5). Nevertheless, by 10weeks, nearly all mutant mice succumbed to hydrocephalus (HC),which was caused by a lack of the ependymal cell layer in thedorsal cortex. Upon further analysis, a gradual loss of adherensjunctions (AJs) was observed in the ventricular zone (VZ) of thedorsal telencephalon in the mutant brains, beginning at E14.5. Inthe AJs-deficient VZ, impaired interkinetic nuclear migration andprecocious differentiation of NSCs were observed after E14.5.These findings demonstrated that PTB depletion in the dorsal tele-ncephalon is causally involved in the development of HC and thatPTB is important for the maintenance of AJs in the NSCs of thedorsal telencephalon.

Keywords: developing brain, ependymal cells, neural stem cell, precociousdifferentiation, region specific

Introduction

In the central nervous system (CNS), post-transcriptional generegulation, which includes processes such as pre-mRNAalternative splicing (Ule et al. 2005), mRNA localization(Bassell and Kelic 2004), turnover (Peng et al. 1998), andtranslation (Kuwako et al. 2010), all regulated by RNA-bindingproteins (RBPs), is involved in many aspects of developmentincluding the survival and function of neurons. For example,a recent study in knockout mice has revealed that Nova2, aneuron-specific RBP, is indispensable for the propermigration of neurons in the cortex and cerebellum via theregulation of an RNA splicing event, which controls the func-tion of the Dab1 protein (Yano et al. 2010). However, ingeneral, the roles of RBPs expressed in neural stem cells(NSCs) are still elusive.

Polypyrimidine tract-binding protein (PTB) is an RBP ex-pressed 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 maturemRNAs. PTB expression has been observed in various tissuesand cell lines including embryonic stem cells (ESCs; Lillevaliet al. 2001; Shibayama et al. 2009). In most mammals, 3 PTBparalogs, neural PTB (nPTB), regulator of differentiation 1(ROD1), and smooth muscle PTB (smPTB), are expressed in atissue-restricted manner. The paralog, nPTB, is expressedmainly in neurons (Polydorides et al. 2000), ROD1 is ex-pressed in hematopoietic cells (Yamamoto et al. 1999), andsmPTB is expressed in smooth muscle (Gooding et al. 2003).Interestingly, cross-regulation between PTB, nPTB, and ROD1has been reported (Spellman et al. 2007).

In the context of development, although the importance ofPTB in multiple biological processes in non-mammalianspecies has been reported (Hamon et al. 2004; Robida et al.2010), there are few reports of a role for PTB in mammaliandevelopment and organogenesis. Previously, we generatedPtb knockout mice and Ptb null ESCs and found that PTB isessential for early mouse development and important for theproliferation and differentiation of mouse ESCs (Shibayamaet al. 2009). Thus, it is expected that PTB would also haveimportant roles in tissue stem cells.

Due to its interesting expression pattern, PTB has beenwell studied in the nervous system. Its expression is observedpredominantly in NSCs and is lost in mature neurons (Lillevaliet 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 vitrostudy demonstrated that the knockdown of PTB leads towide-spread changes in alternative splicing events, similar tothe changes that occur upon neural differentiation (Boutzet al. 2007). PTB is also highly expressed in differentiatedcells including ependymal cells and choroid plexus (CP) epi-thelial cells (Lillevali et al. 2001), which are central to brainhomeostasis with respect to cerebrospinal fluid (CSF) circula-tion or metabolism (Banizs et al. 2005). Based on these find-ings, PTB might affect both neural differentiation in thedeveloping brain as well as brain homeostasis from late em-bryonic stage or later; however, to date, the role of PTB in thebrain remains unknown.

In this study, we inactivated the Ptb gene by employingCre-mediated conditional gene targeting systems and gener-ated 3 lines of mutant mice. Analyses of 3 types of mutantbrain revealed that PTB is required for the maintenance of ad-herens junctions (AJs) in embryonic NSCs in the dorsal tele-ncephalon and that defect of AJs maintenance in NSCs causes

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premature depletion of NSCs and lack of ependymal celllayer, resulting in postnatal development of hydrocephalus(HC). However, except for in the dorsal telencephalon, theloss of AJs was not observed in the developing mutant brain.Our data suggest a new regulatory mechanism mediated byPTB influencing the maintenance of AJs in NSCs in vivo.

In this paper, the term “NSCs” is used to indicate both neu-roepithelial cells and radial glial cells.

Materials and Methods

MiceAll mice were maintained on a mixed 129SV/J-C57BL/6 background.A floxed Ptb allele and a Neo cassette allele were generated byhomologous recombination (Shibayama et al. 2009). Nestin-Cre mice(Toshikuni Sasaoka et al., unpublished), Emx1-Cre mice (Iwasatoet al. 2004), and Nestin-CreERT2 mice (Imayoshi et al. 2006) werecrossed to Ptb+/neo mice to obtain Ptb+/neo; Nestin-Cre, Ptb+/neo;Emx1-Cre and Ptb+/neo; Nestin-CreERT2 mice. Mice, homozygous forthe Ptb floxed allele (Ptbfloxed/floxed) and Ptb+/neo; Nestin-Cre as well asPtb+/neo; Emx1-Cre and Ptb+/neo; Nestin-CreERT2 mice were bred togenerate Ptbfloxed/neo; Nestin-Cre, Ptbfloxed/neo; Emx1-Cre and Ptbfloxed/neo; Nestin-CreERT2 mice. All mouse work was performed in compli-ance with the guidelines of the Institutional Animal Care and UseCommittee of the University of Tokyo.

Primary AntibodiesThe 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 (SantaCruz, 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), mouseanti-N-cadherin (N-cad; BD, 610920; WB, 1:1000; IH, 1:500), mouseanti-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), mouseanti-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).

Western BlottingForebrains from embryos and pups were excised in phosphate-buffered saline (PBS), the meninges were removed and the vesicleshomogenized. After centrifuging, the samples were suspended in anappropriate amount of radio immunoprecipitation assay buffer (25mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycho-late, 0.1% sodium dodecyl sulfate [SDS]). Samples were diluted toprovide equal amounts of protein prior to SDS–polyacrylamide gelelectrophoresis. Western blot analysis was performed according to thestandard protocols, using the appropriate primary antibodies, andhorseradish peroxidase-conjugated goat IgG (GE Healthcare, 1:2000–1/4000) was used as the secondary antibody. Immunoreactivity wasdetected using an enhanced chemiluminescence kit (GE Healthcare)and X-ray film (Fuji Film).

Cresyl Violet Staining and ImmunohistochemistryBrains 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 in10 mM sodium citrate (pH 6.0) or at 100°C for 10 min in a microwave.Immunostaining was performed using a blocking reagent (1× block-ing reagent [Roche], 2% bovine serum albumin, 0.05% Tween-20,0.1% Triton X-100, 1× PBS), while primary and secondary antibodieswere diluted in the blocking reagent without Triton X-100. AlexaFluor (1:500; Molecular Probes) was used as secondary antibody.Nuclei were visualized using 2 μg/mL1, 40,6-diamino-2-phenylindole(DAPI; Sigma) diluted in the secondary antibody diluant, and sectionswere analyzed using a Keyence Biozero microscope or an Olympusfluorescence microscope.

Electron Microscopic AnalysisBrains of pups were fixed with 2% glutaraldehyde and 2% PFA in 0.1M phosphate buffer (PB; pH 7.4) for an hour, and then the ventralregion of the brain, the septum, and the hippocampus were removedand fixed overnight at 4°C in the same solution. Samples werewashed with 0.1 M PB 3 times on ice at 5-min intervals and postfixedwith 1% osmium tetroxide in 0.1 M PB for 2 h. The samples werethen washed with distilled water 5 times on ice at 5-min intervals, anddehydrated 3 times in an ethanol series. The ethanol was cleared withtert-butyl alcohol, and the samples were freeze-dried (ES-2030 FreezeDryer; Hitachi), vapor-deposited with an HPC-1S osmium coater(Vacuum Devices) and observed with a field-emission scanning elec-tron microscope (Model S-4200; Hitachi).

TUNEL AssayTerminal deoxynucleotidyl transferase-mediated dUTP nick end label-ing (TUNEL) analysis was performed on rehydrated paraffin sections(10 μm) using the In situ Cell Death Detection Kit (Roche) accordingto the manufacturer’s instructions. Sections were counterstained with2 μg/mL DAPI to assess the total cell number.

Tamoxifen Administration and BrdU LabelingTamoxifen was administrated by gavage to pregnant females once atE16.5 (200 mg per kg body weight). BrdU was injected intraperitone-ally into pregnant mice at E16.5 (50 mg per kg body weight). The in-terkinetic nuclear migration (INM) was measured by quantifying thenumber of BrdU-positive cells in each 20-μm zone, 30 min and 4 hafter a single BrdU injection. The cell cycle exit rate was measured byquantifying the number of BrdU+/ki67− cells 24 h after a single BrdUinjection.

Statistical AnalysisA log-rank test was performed to compare the survival rate. All othercomparisons were undertaken using the 1-way analysis of variance(ANOVA) with Tukey’s test. P-values were considered to be significantat P < 0.05.

Results

Subcellular Localization of PTB in Embryonic NSCsAlthough it is known that PTB undergoes nucleocytoplasmicshuttling in neurons (Ma et al. 2007), its subcellular localiz-ation in NSCs in vivo has not been described. Therefore, wemonitored the subcellular localization of PTB in NSCs in themouse telencephalon, which showed that PTB is concentratedin the nuclei and does not co-localize with either the NSCmarker, Nestin (predominantly localized to the cytoplasm, in-cluding both pial and apical processes), or with the AJsmarker, aPKC (localized to the termini of NSC apical process;

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Fig. 1B–E). We also found high PTB expression in basallylocated basal progenitor cells (BPCs) and postmitoticneurons, 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 oftarget mRNAs to the terminals of processes in NSCs understeady-state conditions and that PTB protein expression didnot abolish upon neuronal differentiation.

Generation of Nestin-Cre–Mediated Ptb cKO MiceTo 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 Ptballeles (Shibayama et al. 2009). Ptb+/floxed; Nestin-Cre(Control) mice were used as controls for most of the exper-iments in this report. Immunofluorescence analysis revealedthat the reduction in PTB protein levels is already underwayat E12.5 and that PTB expression is abolished in the cortexand ganglionic eminences (GE) by E14.5 (Fig. 2A–F),although western blot analysis revealed residual PTB proteinin the whole forebrain at E14.5 (Fig. 2G). Since PTB proteinlevels were barely detectable at E16.5, this residual proteinmust have originated from the caudal region of the telence-phalon, where Cre-mediated recombination does not occurbefore 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 celllines including HeLa, PAC1, and N2A, amongst others (Boutzet al. 2007; Spellman et al. 2007), and we observed a similarphenomenon in ESCs (Shibayama et al. 2009). In these celllines, nPTB expression is suppressed by PTB via nPTB exon10 suppression, which leads to nonsense-mediated decay.

However, as both PTB and nPTB are expressed in ventricularwalls in the embryonic mouse brain (Lillevali et al. 2001),there is a possibility that PTB does not suppress nPTBexpression in NSCs in the embryonic brain. Immunofluores-cence and western blot analyses were employed to test nPTBexpression in the Ptb cKO forebrain and, as expected, theresults showed that there is a little difference in the nPTBexpression level between the Ptb cKO forebrain and control atE14.5 and E16.5 (Fig. 2G, Supplementary Fig. S1). Theseresults suggested that, unlike several of the cell lines tested,PTB does not suppress nPTB expression at least in the em-bryonic forebrain.

PTB Depletion Results in the Development of ProgressiveHydrocephalusTo examine the effect of PTB depletion in embryonic NSCs,we assessed the expression patterns of cell-type–specificmarkers. Immunofluorescence analysis revealed that thepopulations and localizations of Pax6+ NSCs, Tbr2+ BPCs, andTuj1+ neurons, and the direction of Nestin+ fibers do not alterin the Ptb cKO cortex at E14.5 (Fig. 2H–O). In addition, glialcell marker, GFAP+ cells were not observed and there was nomorphological abnormality in the Ptb cKO cortex at E14.5(data not shown). These results revealed that abnormal neuro-genesis and ectopic gliogenesis does not occur before E14.5in the Ptb cKO cortex.

To analyze the effect of PTB depletion in brain homeosta-sis, the viability of Ptb cKO mice was monitored for the first 3weeks. As a result of genotyping 147 pups at postnatal day 21(P21), we found that Ptb cKO mice were born at the expectedMendelian ratio and were viable for at least 3 weeks (Fig. 3A).However, the heads of some Ptb cKO mice had a character-istic dome-like appearance at this stage (Fig. 3C,D) and nearly

Figure 1. PTB localizes to nuclei in NSCs in the telencephalon. (A–K) Coronal sections of E14.5 cortices stained with antibodies against PTB (A–G, J and K), Nestin (greenin D), aPKC (light blue in E), Tbr2 (H and J), and Tuj1 (I and K). (B–E), (F, H, and J), and (G, I, and K) correspond to the numbered boxed areas 1, 2, and 3, respectively, in A. PTBlocalized to nuclei but not to Nestin+ cytoplasm and aPKC+ terminal of apical process in NSCs and its expression is observed in BPCs and neurons. Lv, lateral ventricle. Scalebar: 20 μm in C and G.

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all Ptb cKO mice had died (90.9%) by 10 weeks (Fig. 3B). His-tological analyses revealed that all Ptb cKO brains (N = 15)had dilations of the lateral ventricles (Fig. 3E–H) with variableseverity, suggesting fully penetrant HC at P21. In addition, anincrease in the number of astrocytes, a common occurrence inHC, was also observed in the Ptb cKO brain at P21 (Sup-plementary Fig. S2A–E; Miller and McAllister 2007; Swegeret al. 2007). In the control brains (N = 13), such abnormalitieswere never observed. Taken together, these results clearlysuggested that PTB is important in brain homeostasis and thatHC is probably the direct cause of the early mortality of PtbcKO mice.

Region-Specific Disruption of Ependymal Cell Layerin the Ptb cKO BrainHC is a progressive degenerative disorder and is one of themost common abnormalities found in the CNS. It is character-ized by an excessive accumulation of CSF in the brain ventri-cle, and can be caused by an excess of CSF production, a lackof CSF reabsorption and impaired CSF flow.

To determine the cellular defect responsible for HC in PtbcKO mice, we initially looked for stenosis in the brain,because the most common cause of HC is stenosis (Sakaki-bara et al. 2002; Nechiporuk et al. 2007). Although ventriculardilation was observed in Ptb cKO brains at P10 (Figs 3I,J and4A–D), stenosis was not observed (Fig. 4A–F). Next, PTBexpression in the subcommissural organ (SCO) and CP wasexamined, as it is known that abnormalities in these 2 organslead to HC (Vio et al. 2000; Banizs et al. 2005). But there wasno difference in PTB expression in the SCO and CP betweenthe control and Ptb cKO brains at P10, which may be due toan absence or low-levels of Cre recombinase expression inthese 2 organs (Supplementary Fig. S3). Thus, these resultssuggested that the HC in the Ptb cKO brain is caused byanother mechanism.

The postnatal brain ventricles are lined by a layer of epi-thelial cells known as ependymal cells. Ependymal cells arederived from radial glia cells (RGCs) and bear dozens of ciliathat beat in a coordinated manner to facilitate the circulationof the CSF at the end of maturation. Abnormal ciliogenesiscompromises CSF dynamics without ventricular stenosis andleads to HC (Ibanez-Tallon et al. 2004; Lechtreck et al. 2008;Town et al. 2008).

To determine whether there were abnormalities in ependy-mal cilia and in ependymal cells themselves in the Ptb cKObrain, we initially performed histological analysis under lightmicroscopy and found that the ependymal cell layer looks se-verely disorganized on the dorsal wall of the lateral ventricles(Fig. 3K,L). Next, we examined the expression of the ependy-mal cell marker, S100β, GFAP, the cilia marker, Ac-tub andthe AJs markers, N-cad, and aPKC. Immunofluorescenceanalysis revealed the absence of Ac-tub+ cilia and an ependy-mal cells (S100β+, GFAP+, N-cad+, and aPKC+) in the dorsalwall of the lateral ventricles in postnatal Ptb cKO brains (Sup-plementary Figs S2F–K and Fig. 4I–N). By contrast, multici-liated ependymal cells were observed on the ventricular wallsof other regions in Ptb cKO brains at P10 (Fig. 4A–H).Because these observations strongly suggested that ependy-mal cells themselves are absent on the dorsal wall of thelateral ventricles in the Ptb cKO brain, we tried to reveal iden-tities of cells on the wall. Immunofluorescence analysis

Figure 2. Normal neurogenesis proceeds in the Ptb cKO cortex. (A–F) Coronalsections of embryonic telencephalons stained with antibodies against PTB. Theexpression of PTB was almost completely abolished at E14.5 (E and F) in the Ptb cKOcortex and GE. (G) Western blot analysis of PTB and nPTB in the whole forebrain atE14.5 and E16.5. Tuj1 and GAPDH were included as developmental stage control andloading control, respectively. (H–L) Coronal sections of E14.5 cortices stained withantibodies against Tuj1 (H and I), Tbr2 (J and K), Pax6 (L and M), and Nestin (N andO). Abnormal neurogenesis was not observed in the Ptb cKO cortex. Ctx, cortex andLv, lateral ventricle. Scale bar: 20 μm in B, D, and H; 100 μm in F.

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revealed that these cells are positive for the oligodendrocyteprogenitor marker Olig2, Tuj1, or the neuroblast marker Dcx(Supplementary Fig. S4) and suggested that cells normallylocate in the corpus callosum are exposed to the lateral ventri-cles due to the absence of ependymal cells in the Ptb cKObrain.

To confirm whether the absence of ependymal cells on thedorsal wall of the lateral ventricles and to check whetherthe absence of ependymal cells extended over the whole ofthe wall, we conducted scanning electron microscopic analy-sis at P10 and observed cilia as an indicator of ependymalcell. These results revealed that there was a complete lack ofcilia in the anterior region of the ventricle wall in contrast tothe striatal wall that did bear cilia (Fig. 5A–D). As expected,many cilia were observed on the posterior region of thedorsal 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 ependy-mal cell layer in the dorsal telencephalon was the cause of HCin the Ptb cKO brain, Ptbfloxed/neo; Emx1-Cre mice were gener-ated and the phenotype was analyzed. With the Emx1-Creallele, the Cre recombinase gene is inserted in front of theATG start codon of the endogenous Emx1 gene, which is ex-pressed exclusively in the dorsal telencephalon prior to theexpression of Nestin (Iwasato et al. 2004). Histological analysisat P10 revealed that the lateral ventricles were dilated inPtbfloxed/neo; Emx1-Cre mice (5 of 5 mice; Supplementary

Fig. S5A–C) and immunofluorescence analysis revealed anabsence of ependymal cilia in the whole of the dorsal telence-phalon including both the anterior and posterior regions inPtbfloxed/neo; Emx1-Cre brains (4 of 5 mice; SupplementaryFig. S5D–G). These results clearly indicated that the disruptionof the ependymal cell layer led to HC in the Ptb cKO brain.

Gradual Loss of AJs in the Developing DorsalTelencephalon in Ptb cKO MiceNext, we investigated the mechanism underlying the lack of anependymal cell layer in the Ptb cKO brain. Since most ependy-mal cells born before birth and postnatally maturate (Spasskyet al. 2005), we first analyzed whether PTB is required for matu-ration and survival of ependymal cells in vivo. To this end, wegenerated tamoxifen-inducible Ptb knockout (Ptbfloxed/neo;Nestin-CreERT2) mice. Although tamoxifen administration atE16.5 abolished PTB expression in Ptbfloxed/neo; Nestin-CreERT2at P0, mature ependymal cells were observed on the ventricularwall of the dorsal cortex at P10 (Supplementary Fig. S6). Thisresult clearly indicated that PTB is dispensable for maturationand survival of ependymal cells.

Since ependymal cells are derived from NSCs in the em-bryonic brain, we next hypothesized that PTB depletion actu-ally affected properties of the NSCs resulting in abnormalNSCs, which led to the lack of an ependymal cell layer. Immu-nofluorescence analysis revealed patches devoid of N-cadexpression on the apical surface of the dorsal telencephalon

Figure 3. PTB depletion in the brain causes progressive HC. (A) Genotypic analysis of pups at P21. Ptb cKO mice were viable at least for 3 weeks. (B) Survival rates of Ptb cKOmice aged between 3 and 10 weeks. Nearly all Ptb cKO mice (19 of 22) died by 10 weeks (*P<0.05, log-rank test). (C and D) Comparison of lateral view of pups at P21. Theheads of some Ptb cKO mice had a characteristic dome-like appearance (arrow in D). (E and F) Global brain view. Enlarged hemispheres and compressed olfactory bulbs(arrowhead) and cerebellum (arrow) were observed in Ptb cKO brains (F). (G–L) Coronal sections of P21 and P10 brains stained with Cresyl Violet. Extreme dilations of the lateralventricles (Lv, H) were observed in P21 cKO mice. The dilation of the lateral ventricles also observed at P10 cKO mice (8 of 8; I and J) and an ependymal cell layer of the dorsalwall were severely disorganized (K and L). Scale bar: 1 mm in F; 100 μm in J; 40 μm in L.

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in Ptb cKO brains at E14.5 (5 of 7 mice; Fig. 6A,B). BeforeE14.5, no such abnormality was detected (data not shown):However, by E15.5, these N-cad− patches were visible on theapical 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 junc-tion marker, ZO-1, and the AJs marker, Vinculin (Fig. 6E–L).Subsequently, the expression levels of AJs components in theforebrain were analyzed at E16.5. Western blot analysis re-vealed that the expression levels of N-cad, aPKC, and ZO-1showed no decrease in Ptb cKO forebrains compared withcontrols (Supplementary Fig. S7A). Taken together, theseresults suggested that PTB is required for the localization ofAJs components but does not regulate the expression levels ofthese proteins in NSCs in the dorsal telencephalon.

When the AJs-deficient ventricular zone (VZ) was examinedat E18.5, we found that the lack of NSCs that expressed Pax6and 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-cadexpression on the apical surface was observed (Fig. 6M,N).Because apoptosis was considered as a possible primarycause of the lack of NSCs in the VZ, a TUNEL assay wasconducted to detect apoptotic cells in the AJs-deficientpatches. However, apoptotic cells were not detected in theN-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, inthe dorsal telencephalon in Ptb cKO mice, the loss of AJs is

linked to the lack of an ependymal cell layer via the decreaseof NSCs in the VZ without apoptosis.

Precocious Differentiation Occurs in the AJs-DeficientVZRGCs, which act as stem cells, divide increasingly in an asym-metric 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 thedifferentiation of RGCs into BPCs and neurons, RGCs losetheir stem cell properties including AJs, apico-basal polarity,and INM. Several studies have demonstrated that these prop-erties are important for the precise regulation of the cell-fatedecision controlling the differentiation from RGCs to BPCsand neurons in the developing brain (Machon et al. 2003;Chae et al. 2004; Cappello et al. 2006; Costa et al. 2008; Tsaiet al. 2010). Thus, we hypothesized that precocious differen-tiation of RGCs occurred in the AJs-deficient VZ in Ptb cKObrains, and thus, we performed immunofluorescence analysisusing antibodies for several cell-type–specific markers andAJs markers.

There was no distinction between the expression pattern ofPax6 in the VZ in controls and the ZO-1− VZ in Ptb cKO cor-tices at E15.5. However, the expression patterns of Tbr2 andTuj1 were different (Fig. 7A–F). In the control cortices (N = 4),few Tbr2+ BPCs and Tuj1+ neurons were observed at the

Figure 4. Loss of ependymal cell layer in the dorsal telencephalon of the Ptb cKO brain. (A–H) Coronal sections at the level of the third ventricle (3v), aqueduct (Aq), spinal cord(Sc), and lateral ventricles (Lv) at P10 stained with antibodies against PTB (red) and Ac-tub (green). The ependymal cell layer was maintained and stenosis was not observed in3v, Aq, and Sc in Ptb cKO brains. (I–N) Coronal sections of the ventricular wall of the cortex at P10 stained with antibodies against PTB, GFAP (green in I and J), S100β (lightblue in I and J), Ac-tub (green in K and L), N-cad (light blue in M and N), and aPKC (red in M and N). The right-hand panel shows a higher magnification and single-color imagesof the boxed areas in the left column. Ependymal cells were not observed in the ventricular wall of the cortex in Ptb cKO brains. Ctx, cortex and Lv, lateral ventricle. Scale bar:40 μm in B, D, F, and H; 20 μm in J.

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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 radialaxis, were found in the center of the N-cad− VZ in Ptb cKOcortices (4 of 6 mice; Fig. 7G–H). Greater numbers (P < 0.05,1-way ANOVA with Tukey’s test) of BPCs, ectopically locatedat the apical side of the VZ, were observed in all parts of theN-cad− VZ in Ptb cKO cortices compared with the VZ incontrol and the N-cad+ VZ in Ptb cKO at E16.5 (Fig. 7I,J,M). Inaddition, accumulated neurons were observed along theradial axis of the N-cad− VZ, suggesting a possible defect inradial migration (Fig. 7K,L). Moreover, the cell cycle exit rate,which is an indicator of terminal differentiation (Buttitta andEdgar 2007), was increased in the N-cad− VZ (SupplementaryFig. S8A–H). Taken together, these results indicated that, inthe AJs-deficient VZ, there was not only a defect in radialmigration but also precocious differentiation from NSCs toBPCs and neurons.

In INM, the nuclei of newborn NSCs move away from theapical surface toward the basal lamina during G1 of the cellcycle, undergo S phase at a basal location, and return to theapical 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 zebrafish retina showed that a disturbance in INM solely affected thecell-fate decision of NSCs (Xie et al. 2007; Del Bene et al. 2008).

To investigate INM in the AJs-deficient VZ, labeling analysiswas conducted with the DNA base analog, BrdU, at E16.5.Cells were labeled with BrdU in S phase and their positionwas determined 30 min and 4 h after BrdU injection. After 30min, although most BrdU-labeled nuclei were located in thebasal part of the VZ in control cortices, BrdU-labeled nucleiwere scattered throughout the aPKC− VZ in Ptb cKO cortices(Fig. 7N,O,T; Supplementary S7B,C). When the cortices wereanalyzed 4 h after BrdU injection, approximately 30% ofBrdU-labeled nuclei reached an apical position (0–20 μm) andsome of these nuclei resided on the apical surface in controls,whereas relatively few BrdU-labeled nuclei (P < 0.05, 1-wayANOVA with Tukey’s test) reached an apical position and fewnuclei were found on the apical surface in the aPKC− VZ inPtb cKO cortices (Fig. 7P,Q,T). Since BrdU-labeled nucleiwere spread throughout the aPKC− VZ in Ptb cKO corticesafter 30 min, mitotic cells might also be distributed through-out in the AJs-deficient VZ of Ptb cKO cortices. Accordingly,mitotic nuclei were labeled by immunostaining usingphospho-histone H3 antibodies. The majorities were locatedat the apical surface in controls, while a relatively minorpopulation was found in the non-apical region, SVZ, and theintermediate zone. By contrast, in Ptb cKO cortices, only aminor portion of the mitotic nuclei was localized to the apicalsurface in the aPKC− VZ, with the majority found in non-apical regions of the VZ and SVZ (Fig. 7R,S,U). Next, we ver-ified whether the BrdU-labeled cells remaining at the apicalregion in Ptb cKO cortices after 30 min were stem cells or dif-ferentiated cells. Immunostaining analysis revealed thatalmost all of these cells were positive for the stem cell marker,Pax6 (Supplementary Fig. S7B,C). Thus, the INM defect pre-ceded the precocious differentiation of RGCs and might beresponsible for triggering the differentiation event. While theINM was completely normal in the AJs-positive VZ in Ptb cKOcortices, the loss of AJs from the VZ in Ptb cKO cortices wasdirectly responsible for the INM defect. Taken together, theseresults demonstrated that PTB indirectly affects the cell-fatedecision of NSCs.

Depletion of PTB Dose not Affect Over All LaminarStructure but Affects GliogenesisFinally, we analyzed laminar structure and gliogenesis in thedorsal telencephalon of Ptb cKO mice during P0 to P10,because premature depletion of NSCs and/or loss of AJs inthe dorsal telencephalon might affect both laminar structureand gliogenesis. Immunostaining analysis using several layermarkers revealed that over all laminar structure was normal inthe Ptb cKO cortex (Supplementary Fig. S9A–E). But wefound periventricular heterotopias in mutant mice at P0 (Sup-plementary Fig. S9F–T) and P10 (data not shown). These het-erotopias were located at the dorsal wall of lateral ventriclesor striato-cortical junction and consisted of neuronal cells,which are positive for Brn2. This observation confirmedmigration defect within affected AJ areas. Although overalllaminar structure was normal, generation of GFAP+ gilal cellwas affected in Ptb cKO mice. Before P2, few GFAP+ cellswere observed in the VZ of the dorsal cortex in the controlbrain, but a few GFAP+ cells were observed in the ZO-1− VZof the dorsal cortex in the cKO brain at P0 and quite a lot ofGFAP+ cells were observed at P2 (Supplementary Fig. S10).Because morphology of the Ptb cKO cortex seemed almost

Figure 5. Scanning electron microscopic (SEM) analysis confirmed theregion-specific absence of ependymal cilia in the Ptb cKO brain. (A and B) SEM viewof P10 dorsal wall of the anterior region of the lateral ventricles. (C and D) SEM viewof P10 ventricular wall of the striatum. (E and F) SEM view of P10 dorsal wall of theposterior region of the lateral ventricles. A region on the right side of broken line isthe original location of the hippocampus, which was removed in this analysis to makethe dorsal wall more visible. The presence of cilia on the ventricular wall of thestriatum and the dorsal wall of the posterior region of the lateral ventricles but not onthe dorsal wall of the anterior region of the lateral ventricles in Ptb cKO brains. Scalebar: 10 μm in B; 100 μm in E; 20 μm in F.

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normal until P5 (Supplementary Fig. S10C,F), these resultssuggested that PTB depletion and/or loss of AJs caused preco-cious differentiation of NSCs into glial cell. But total GFAP+

cell production significantly decreased at P5 probably due tothe premature depletion of NSCs in the dorsal cortex in thePtb cKO brain.

Discussion

This study demonstrated that conditional disruption of thePtb gene in the mouse brain leads to severe HC. A recentstudy showed that a partial loss and dysfunction of ependy-mal cell motile cilia in the dorsal wall of the lateral ventriclesis enough to cause lethal HC (Tissir et al. 2010). In Ptb cKObrains, there were no abnormalities such as ventricular steno-sis or morphological defects in the SCO and CP, which mightlead to HC, and the only anomaly was a loss of the ependymalcell layer in the ventricular wall of the dorsal telencephalon.Moreover, Ptbfloxed/neo; Emx1-Cre mice, in which thedepletion of PTB was restricted to the dorsal telencephalon,

showed a similar hydrocephalic phenotype to Ptb cKO mice.Taken together, these results demonstrated that the directcause of HC in Ptb cKO mice was the lack of an ependymalcell layer in the dorsal telencephalon. Although we could notexclude the possibility that abnormalities in differentiationfrom NSCs to ependymal cells also contributed to the lack ofan ependymal cell layer in Ptb cKO brains, present datasuggested that the lack of an ependymal cell layer was causedby the premature depletion of NSCs in the late embryonicstage and precocious differentiation of remaining NSCs toglial cells during postnatal first week.

The loss of AJs in Ptbfloxed/neo; Emx1-Cre mice was ob-served earlier than in Ptb cKO mice (data not shown). Thistime lag reflects a difference in Cre expression timingbetween the 2 Cre mouse lines and supports the notion thatPTB is important for the maintenance of AJs in NSCs in thedorsal telencephalon throughout the brain development. Theloss of AJs has been also observed in several knockout micein which genes that encode AJs component proteins are dis-rupted (Machon et al. 2003; Cappello et al. 2006; Imai et al.

Figure 6. The gradual loss of AJs leads to massive depletion of NSCs in the Ptb cKO brain. (A–D) Coronal sections of the E14.5 (A and B) and E15.5 (C and D) dorsaltelencephalon stained with antibodies against N-cad. Arrowheads (B and D) indicate the N-cad− patches on the apical surface of the dorsal telencephalon. (E–H) Highermagnification views of boxed areas in (C and D) are shown in (E and G) and (F and H). (I–L) Serial sections of (C and D) stained with antibodies against ZO-1 (I and J) andVinculin (K and L). AJs components were partially lost from the apical surface of the telencephalon in Ptb cKO brains. (M–P) Serial coronal sections of E18.5 dorsaltelencephalon stained with antibodies against N-cad (green in M and N), Pax6 (red in M and N), Nestin (green in O and P), and Tuj1 (red in O and P). (Q–T) Apoptotic cells weredetected using the TUNEL assay. Depletion of Pax6+ and Nestin+ NSCs was observed in the N-cad− VZ in E18.5 Ptb cKO brains (M–P). Cells undergoing apoptosis (arrowheadin Q and S) were not observed in the AJs-deficient patches of Ptb cKO brains (S and T). Ctx, cortex; Lv, lateral ventricle. Scale bar: 50 μm in B; 100 μm in D and N; 20 μm in F.

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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 andINM disturbance have been observed. The phenotypes ofthese mutant mice are similar but more severe than for PtbcKO mice. The difference in the severity of the brain malfor-mations may arise from the variable timing of Cre expressionand the biological stability of individual target proteins.However, in contrast to the various knockout mice describedabove, in which targeted AJs component proteins are de-pleted, the expression levels of AJs component proteins suchas N-cad, aPKC, and ZO-1 did not alter in the Ptb cKO mousebrain compared with the control mouse. Thus it is possiblethat the milder phenotype of Ptb cKO mice may also arise

from the unique function of PTB in NSCs, where it is involvedin the localization of AJs component protein(s) but may notbe involved in their expression. Loss of AJs has also been re-ported in Dlg5, Numb/Numbl, and α-Snap mutant mice (Chaeet al. 2004; Nechiporuk et al. 2007; Rasin et al. 2007).Although neither of these proteins are AJs components, theyco-localize with AJs at the apical surface of the VZ and are in-volved directly in the localization of AJs components. PTB, onthe other hand, is localized to NSC nuclei and is not found atthe apical surface of the VZ under steady-state conditions inthe developing brain. Although it does not appear to associatedirectly with AJs, PTB depletion results in the loss of AJs,suggesting a previously unknown mechanism of AJs mainten-ance by RBPs in the developing brain.

Figure 7. Differentiation and cell cycle-dependent INM is affected in the AJs-deficient VZ. (A–L) Coronal sections of the E15.5 (A–F) and E16.5 (G–H) dorsal telencephalonstained with antibodies against Pax6 (red in A, B, G, and H), Tbr2 (red in C, D, I, and J), Tuj1 (red in E, F, K, and L), ZO-1 (green in A–F), and N-cad (green in G and H). Dashedlines indicate the location of Pax6− cells in the VZ (H) and the corresponding areas (in J and K). (M) The relative number of Tbr2+ cells located in the basal region (between 50and 100 μm from the apical surface) and apical region (between 0 and 50 μm from the apical surface) of the dorsal telencephalon per 100 μm2 at E16.5 (error bars representstandard deviation [SD]. *P< 0.05, 1-way ANOVA with Tukey’s test, N= 4 for each sample). The number of BPCs increased in the AJs-deficient VZ in Ptb cKO brains comparedwith the VZ in control brains. (N–Q) INM analysis in coronal sections of E16.5 dorsal telencephalon stained with antibodies against BrdU (green) and aPKC (red). (R and S)Coronal sections of E16.5 dorsal telencephalon stained with antibodies against phospho-histone H3 (PH3) and aPKC. (T) Percentage of BrdU+ cells in each layer (20-μm thick)per total number of BrdU+ cells (error bars represent SD. *P< 0.05, 1-way ANOVA with Tukey’s test, N= 3 for each sample). INM was impaired at AJs-deficient patches inPtb cKO brains 30 min and 4 h after BrdU administration. (U) The number of PH3+ cells per 100 μm2 at the apical surface (As) and non-apical surface (nAS, >20 μm fromapical surface to intermediate zone; error bars represent SD. *P<0.05, 1-way ANOVA with Tukey’s test, N= 3 for each sample). PH3+ cells positioned on the nAS (arrowhead)increased at the cost of PH3+ cells positioned on the AS at AJs-deficient patches in Ptb cKO brains. LV, lateral ventricle. Scale bar: 50 μm in B, H, P, and T.

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Impact of PTB Depletion on AJs Maintenance in NSCsVaries in Different Regions of the BrainThe abnormalities observed in Ptb cKO brains describedabove were restricted to the dorsal telencephalon. In Ptb cKOmice, the expression of PTB was abolished, not only in thecortex but also in the GE of the developing forebrain;however, neither the loss of AJs nor the lack of the ependymalcell layer was observed in the GE and striatum. These resultsdemonstrated that the impact of PTB depletion on AJs main-tenance in NSCs varies in different regions of the brain. Onepossibility is that the requirement for PTB differs in differentlocations. A similar trend has also been reported in Msi1 andFMR2 RBP knockout mice (Sakakibara et al. 2002; Guo et al.2011). Although it is unclear whether a lack of Msi1 affectsNSCs in vivo, the disorganization of the ependymal cell layerwas restricted to the wall of the aqueduct in Msi1 knockoutmice. Moreover, in FMR2 knockout mice, adult neurogenesiswas affected in the hippocampus but not in the SVZ, despitethe fact that FMR2 expression was observed in NSCs, both inthe hippocampus and the SVZ in wild-type mice. Interest-ingly, in the case of FMR2, a target mRNA (Noggin) is ex-pressed in the NSCs of the hippocampus but not in those ofthe SVZ. Taken together, it appears that, in addition to theneed for particular transcription factors, post-transcriptionalregulation by certain RBPs, is required for specific NSC popu-lations in vivo, and it is thought that downstream target(s) ofPTB is a likely candidate for a specific regulator of AJs main-tenance in the dorsal telencephalon. Another possibility isthat the compensatory function of nPTB counteracts thedepletion of PTB in the maintenance of AJs in the GE. PTBand nPTB show a marked sequence similarity, especiallywithin their RNA recognition motif domains, which areresponsible for specific binding to target RNA molecules(Kikuchi et al. 2000), and both are expressed in NSCs in thecortex and GE in the developing mouse brain. Moreover, pre-vious in vitro microarray analysis revealed that these 2 pro-teins regulate the splicing of certain exons in the samedirection (Boutz et al. 2007). PTB and nPTB are presumed tohave redundant functions, although nPTB does not compen-sate for the function of PTB, at least in AJs maintenance in thedorsal telencephalon; however, it is possible that nPTB com-pensates for this function in the GE. Future experiments invol-ving the generation of PTB/nPTB double knockout mice willbe required to investigate the functional redundancy betweenPTB and nPTB in the developing mouse brain.

The Role of PTB in the Developing BrainPTB, which is highly expressed in NSCs, is thought to de-crease gradually during neural differentiation in the develop-ing dorsal telencephalon, based on the results of RNA in situhybridization analysis (Lillevali et al. 2001; McKee et al.2005). However, the immunofluorescence analysis performedfor this study revealed that PTB expression remained high inTuj1+ postmitotic neurons, while a previous report showedthat PTB was absent from NeuN+ mature neurons in the hip-pocampus and cerebellum of the adult mouse brain (Boutzet al. 2007). Thus, although Ptb mRNA might be abolished inTuj1+ immature neurons, the decrease in PTB protein levelsprogressed gradually during neural maturation, probably dueto the biological stability of the PTB protein. In this regard, it

is reasonable to assume that PTB depletion in NSCs does notlead directly to precocious differentiation.

Questions that still need to be answered are: “what is theidentity of the target regulated by PTB in NSCs?” and “how isthis target protein involved in AJs maintenance?” Previous invitro microarray analyses (Boutz et al. 2007; Makeyev et al.2007; Xue et al. 2009) and our observations of the subcellularlocalization of PTB have indicated that a possible molecularmechanism, responsible for the loss of AJs in the Ptb cKObrain, might be a disturbance in the regulation of alternativesplicing in NSCs. However, we were unable to detect signifi-cant changes in the alternative splicing of well known orpossible target exons of PTB, using RT-PCR analysis in PtbcKO cortices at E14.5, compared with controls (data notshown). This controversial result led us to focus on the differ-ences 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 begreatly affected by PTB depletion in alternative splicing regu-lation, and consistent with this idea, we were able to detectsignificant changes in the alternative splicing of severalexons, which are the same exons tested in Ptb cKO cortices,in Ptb null ESCs compared with wild-type ESCs. Thus furtherinvestigation, using microarrays, into the molecular mechan-isms responsible for the loss of AJs in the Ptb cKO brain isrequired to check the change in transcriptional regulation thatis a possible role of PTB (Brunel et al. 1996; Rustighi et al.2002; Motallebipour et al. 2010), and mRNA stability, as wellas alternative splicing. Answers to these questions willprovide novel insights into the mechanisms responsible forthe maintenance of AJs. AJs are also important for the main-tenance of the epithelial cell layer in other tissues and organsand for the maintenance of the stem cell niche in certaintypes of stem cells (Zhang et al. 2003; Nechiporuk et al. 2007;Smalley-Freed et al. 2010; Piven et al. 2011). Since PTB iswidely expressed in a variety of tissues, whether PTB is in-volved in the maintenance of the stem cell niche of tissuessuch as liver, intestine and the hematopoietic system isworthy of further investigation.

Supplementary MaterialSupplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

This work was supported by Grants from the Ministry of Edu-cation, Culture, Sports and Technology (MEXT), Japan (toN. Yoshida) and in part by the Global COE Program, “Centerof Education and Research for Advanced Genome-BasedMedicine – For personalized medicine and the control ofworldwide infectious diseases,” MEXT, Japan.

NotesWe thank S. Itohara for providing the Emx1-Cre mice andR. Kageyama for providing the Nestin-CreERT2 mice. Conflict of Inter-est: None declared.

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ReferencesBabic I, Sharma S, Black DL. 2009. A role for polypyrimidine tract

binding protein in the establishment of focal adhesions. Mol CellBiol. 29:5564–5577.

Banizs B, Pike MM, Millican CL, Ferguson WB, Komlosi P, Sheetz J,Bell PD, Schwiebert EM, Yoder BK. 2005. Dysfunctional cilia leadto altered ependyma and choroid plexus function, and result inthe formation of hydrocephalus. Development. 132:5329–5339.

Bassell GJ, Kelic S. 2004. Binding proteins for mRNA localization andlocal translation, and their dysfunction in genetic neurologicaldisease. Curr Opin Neurobiol. 14:574–581.

Boutz PL, Stoilov P, Li Q, Lin CH, Chawla G, Ostrow K, Shiue L, AresM, Jr, Black DL. 2007. A post-transcriptional regulatory switch inpolypyrimidine tract-binding proteins reprograms alternative spli-cing in developing neurons. Genes Dev. 21:1636–1652.

Brunel F, Zakin MM, Buc H, Buckle M. 1996. The polypyrimidinetract binding (PTB) protein interacts with single-stranded DNA ina sequence-specific manner. Nucleic Acids Res. 24:1608–1615.

Bushell M, Stoneley M, Kong YW, Hamilton TL, Spriggs KA,Dobbyn HC, Qin X, Sarnow P, Willis AE. 2006. Polypyrimidinetract binding protein regulates IRES-mediated gene expressionduring apoptosis. Mol Cell. 23:401–412.

Buttitta LA, Edgar BA. 2007. Mechanisms controlling cell cycle exitupon terminal differentiation. Curr Opin Cell Biol. 19:697–704.

Cappello S, Attardo A, Wu X, Iwasato T, Itohara S, Wilsch-Brauninger M,Eilken HM, Rieger MA, Schroeder TT, Huttner WB et al. 2006. TheRho-GTPase cdc42 regulates neural progenitor fate at the apicalsurface. Nat Neurosci. 9:1099–1107.

Chae TH, Kim S, Marz KE, Hanson PI, Walsh CA. 2004. The hyhmutation uncovers roles for alpha Snap in apical protein localiz-ation and control of neural cell fate. Nat Genet. 36:264–270.

Costa MR, Wen G, Lepier A, Schroeder T, Gotz M. 2008. Par-complexproteins promote proliferative progenitor divisions in the develop-ing mouse cerebral cortex. Development. 135:11–22.

David CJ, Chen M, Assanah M, Canoll P, Manley JL. 2010. HnRNP pro-teins controlled by c-Myc deregulate pyruvate kinase mRNA spli-cing in cancer. Nature. 463:364–368.

Del Bene F, Wehman AM, Link BA, Baier H. 2008. Regulation of neu-rogenesis by interkinetic nuclear migration through an apical-basal notch gradient. Cell. 134:1055–1065.

Gooding C, Kemp P, Smith CW. 2003. A novel polypyrimidine tract-binding protein paralog expressed in smooth muscle cells. J BiolChem. 278:15201–15207.

Guo W, Zhang L, Christopher DM, Teng ZQ, Fausett SR, Liu C,George OL, Klingensmith J, Jin P, Zhao X. 2011. RNA-bindingprotein FXR2 regulates adult hippocampal neurogenesis by redu-cing Noggin expression. Neuron. 70:924–938.

Hamon S, Le Sommer C, Mereau A, Allo MR, Hardy S. 2004. Polypyr-imidine tract-binding protein is involved in vivo in repression of acomposite internal/30 -terminal exon of the Xenopus alpha-tropomyosin Pre-mRNA. J Biol Chem. 279:22166–22175.

Ibanez-Tallon I, Pagenstecher A, Fliegauf M, Olbrich H, Kispert A,Ketelsen UP, North A, Heintz N, Omran H. 2004. Dysfunction ofaxonemal dynein heavy chain Mdnah5 inhibits ependymal flowand reveals a novel mechanism for hydrocephalus formation.Hum Mol Genet. 13:2133–2141.

Imai F, Hirai S, Akimoto K, Koyama H, Miyata T, Ogawa M, NoguchiS, Sasaoka T, Noda T, Ohno S. 2006. Inactivation of aPKClambdaresults in the loss of adherens junctions in neuroepithelial cellswithout affecting neurogenesis in mouse neocortex. Development.133:1735–1744.

Imayoshi I, Ohtsuka T, Metzger D, Chambon P, Kageyama R. 2006.Temporal regulation of Cre recombinase activity in neural stemcells. Genesis. 44:233–238.

Iwasato T, Nomura R, Ando R, Ikeda T, Tanaka M, Itohara S. 2004.Dorsal telencephalon-specific expression of Cre recombinase inPAC transgenic mice. Genesis. 38:130–138.

Kadowaki M, Nakamura S, Machon O, Krauss S, Radice GL, TakeichiM. 2007. N-cadherin mediates cortical organization in the mousebrain. Dev Biol. 304:22–33.

Kikuchi T, Ichikawa M, Arai J, Tateiwa H, Fu L, Higuchi K, YoshimuraN. 2000. Molecular cloning and characterization of a new neuron-specific homologue of rat polypyrimidine tract binding protein.J Biochem. 128:811–821.

Knoch KP, Bergert H, Borgonovo B, Saeger HD, Altkruger A, VerkadeP, Solimena M. 2004. Polypyrimidine tract-binding protein pro-motes insulin secretory granule biogenesis. Nat Cell Biol.6:207–214.

Kosinski PA, Laughlin J, Singh K, Covey LR. 2003. A complex contain-ing polypyrimidine tract-binding protein is involved in regulatingthe stability of CD40 ligand (CD154) mRNA. J Immunol.170:979–988.

Kuwako K, Kakumoto K, Imai T, Igarashi M, Hamakubo T, Sakaki-bara S, Tessier-Lavigne M, Okano HJ, Okano H. 2010. Neural RNA-binding protein Musashi1 controls midline crossing of precerebel-lar neurons through posttranscriptional regulation of Robo3/Rig-1expression. Neuron. 67:407–421.

Lechtreck KF, Delmotte P, Robinson ML, Sanderson MJ, Witman GB.2008. Mutations in Hydin impair ciliary motility in mice. J CellBiol. 180:633–643.

Lien WH, Klezovitch O, Fernandez TE, Delrow J, Vasioukhin V. 2006.alphaE-catenin controls cerebral cortical size by regulating thehedgehog signaling pathway. Science. 311:1609–1612.

Lillevali K, Kulla A, Ord T. 2001. Comparative expression analysis ofthe genes encoding polypyrimidine tract binding protein (PTB)and its neural homologue (brPTB) in prenatal and postnatalmouse brain. Mech Dev. 101:217–220.

Ma S, Liu G, Sun Y, Xie J. 2007. Relocalization of the polypyrimidinetract-binding protein during PKA-induced neurite growth.Biochim Biophys Acta. 1773:912–923.

Machon O, van den Bout CJ, Backman M, Kemler R, Krauss S. 2003.Role of beta-catenin in the developing cortical and hippocampalneuroepithelium. Neuroscience. 122:129–143.

Makeyev EV, Zhang J, Carrasco MA, Maniatis T. 2007. The MicroRNAmiR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol Cell. 27:435–448.

McKee AE, Minet E, Stern C, Riahi S, Stiles CD, Silver PA. 2005. Agenome-wide in situ hybridization map of RNA-binding proteinsreveals anatomically restricted expression in the developingmouse brain. BMC Dev Biol. 5:14.

Miller JM, McAllister JP, 2nd. 2007. Reduction of astrogliosis and mi-crogliosis by cerebrospinal fluid shunting in experimental hydro-cephalus. Cerebrospinal Fluid Res. 4:5.

Motallebipour M, Rada-Iglesias A, Westin G, Wadelius C. 2010. Twopolypyrimidine tracts in the nitric oxide synthase 2 gene: similarregulatory sequences with different properties. Mol Biol Rep.37:2021–2030.

Nechiporuk T, Fernandez TE, Vasioukhin V. 2007. Failure of epithelialtube maintenance causes hydrocephalus and renal cysts in Dlg5−/− mice. Dev Cell. 13:338–350.

Peng SS, Chen CY, Xu N, Shyu AB. 1998. RNA stabilization by theAU-rich element binding protein, HuR, an ELAV protein. EMBO J.17:3461–3470.

Piven OO, Kostetskii IE, Macewicz LL, Kolomiets YM, RadiceGL, Lukash LL. 2011. Requirement for N-cadherin-catenincomplex in heart development. Exp Biol Med (Maywood).236:816–822.

Polydorides AD, Okano HJ, Yang YY, Stefani G, Darnell RB. 2000. Abrain-enriched polypyrimidine tract-binding protein antagonizesthe ability of Nova to regulate neuron-specific alternative splicing.Proc Natl Acad Sci USA. 97:6350–6355.

Rasin MR, Gazula VR, Breunig JJ, Kwan KY, Johnson MB, Liu-Chen S,Li HS, Jan LY, Jan YN, Rakic P et al. 2007. Numb and Numbl arerequired for maintenance of cadherin-based adhesion and polarityof neural progenitors. Nat Neurosci. 10:819–827.

Robida M, Sridharan V, Morgan S, Rao T, Singh R. 2010. Drosophilapolypyrimidine tract-binding protein is necessary for spermatid in-dividualization. Proc Natl Acad Sci USA. 107:12570–12575.

Rustighi A, Tessari MA, Vascotto F, Sgarra R, Giancotti V, Manfioletti G.2002. A polypyrimidine/polypurine tract within the Hmga2

Cerebral Cortex 11

ownloaded from

minimal promoter: a common feature of many growth-relatedgenes. Biochemistry. 41:1229–1240.

Sakakibara S, Nakamura Y, Yoshida T, Shibata S, Koike M, Takano H,Ueda S, Uchiyama Y, Noda T, Okano H. 2002. RNA-bindingprotein Musashi family: roles for CNS stem cells and a subpopu-lation of ependymal cells revealed by targeted disruption and anti-sense ablation. Proc Natl Acad Sci USA. 99:15194–15199.

Sauer FC. 1935. Mitosis in the neural tube. J Comp Neurol.62:377–405.

Shibayama M, Ohno S, Osaka T, Sakamoto R, Tokunaga A, NakatakeY, Sato M, Yoshida N. 2009. Polypyrimidine tract-binding proteinis essential for early mouse development and embryonic stem cellproliferation. FEBS J. 276:6658–6668.

Smalley-Freed WG, Efimov A, Burnett PE, Short SP, Davis MA,Gumucio DL, Washington MK, Coffey RJ, Reynolds AB. 2010.p120-catenin is essential for maintenance of barrier functionand intestinal homeostasis in mice. J Clin Invest.120:1824–1835.

Spassky N, Merkle FT, Flames N, Tramontin AD, Garcia-Verdugo JM,Alvarez-Buylla A. 2005. Adult ependymal cells are postmitotic andare derived from radial glial cells during embryogenesis. J Neuro-sci. 25:10–18.

Spellman R, Llorian M, Smith CW. 2007. Crossregulation and func-tional redundancy between the splicing regulator PTB and itsparalogs nPTB and ROD1. Mol Cell. 27:420–434.

Sweger EJ, Casper KB, Scearce-Levie K, Conklin BR, McCarthy KD.2007. Development of hydrocephalus in mice expressing the G(i)-coupled GPCR Ro1 RASSL receptor in astrocytes. J Neurosci.27:2309–2317.

Takahashi T, Nowakowski RS, Caviness VS, Jr. 1993. Cell cycleparameters and patterns of nuclear movement in the neocorticalproliferative zone of the fetal mouse. J Neurosci. 13:820–833.

Tissir F, Qu Y, Montcouquiol M, Zhou L, Komatsu K, Shi D, FujimoriT, Labeau J, Tyteca D, Courtoy P et al. 2010. Lack of cadherinsCelsr2 and Celsr3 impairs ependymal ciliogenesis, leading to fatalhydrocephalus. Nat Neurosci. 13:700–707.

Town T, Breunig JJ, Sarkisian MR, Spilianakis C, Ayoub AE, Liu X,Ferrandino AF, Gallagher AR, Li MO, Rakic P et al. 2008. Thestumpy gene is required for mammalian ciliogenesis. Proc NatlAcad Sci USA. 105:2853–2858.

Tsai JW, Lian WN, Kemal S, Kriegstein AR, Vallee RB. 2010. Kinesin 3and cytoplasmic dynein mediate interkinetic nuclear migration inneural stem cells. Nat Neurosci. 13:1463–1471.

Ule J, Ule A, Spencer J, Williams A, Hu JS, Cline M, Wang H, Clark T,Fraser C, Ruggiu M et al. 2005. Nova regulates brain-specific spli-cing to shape the synapse. Nat Genet. 37:844–852.

Vio K, Rodriguez S, Navarrete EH, Perez-Figares JM, Jimenez AJ,Rodriguez EM. 2000. Hydrocephalus induced by immunologicalblockage of the subcommissural organ-Reissner’s fiber (RF)complex by maternal transfer of anti-RF antibodies. Exp Brain Res.135:41–52.

Wang C, Norton JT, Ghosh S, Kim J, Fushimi K, Wu JY, Stack MS,Huang S. 2008. Polypyrimidine tract-binding protein (PTB) differ-entially affects malignancy in a cell line-dependent manner. J BiolChem. 283:20277–20287.

Xie Z, Moy LY, Sanada K, Zhou Y, Buchman JJ, Tsai LH. 2007. Cep120and TACCs control interkinetic nuclear migration and the neuralprogenitor pool. Neuron. 56:79–93.

Xue Y, Zhou Y, Wu T, Zhu T, Ji X, Kwon YS, Zhang C, Yeo G, BlackDL, Sun H et al. 2009. Genome-wide analysis of PTB-RNA inter-actions reveals a strategy used by the general splicing repressor tomodulate exon inclusion or skipping. Mol Cell. 36:996–1006.

Yamamoto H, Tsukahara K, Kanaoka Y, Jinno S, Okayama H. 1999.Isolation of a mammalian homologue of a fission yeast differen-tiation regulator. Mol Cell Biol. 19:3829–3841.

Yano M, Hayakawa-Yano Y, Mele A, Darnell RB. 2010. Nova2 regu-lates neuronal migration through an RNA switch in disabled-1 sig-naling. Neuron. 66:848–858.

Zhang J, Niu C, Ye L, Huang H, He X, Tong WG, Ross J, Haug J,Johnson T, Feng JQ et al. 2003. Identification of the haematopoie-tic stem cell niche and control of the niche size. Nature.425:836–841.

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