RBP-J Promotes Neuronal Differentiation and Inhibits Oligodendroglial Development in Adult Neurogenesis

Developmental Biology 332 (2009) 339–350

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Developmental Biology

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RBP-J promotes neuronal differentiation and inhibits oligodendroglial development in adult neurogenesis

Motoaki Fujimoto a,b,1, Yasushi Takagi b,1, Kazue Muraki a,1, Kazuhiko Nozaki b, Norio Yamamoto d, Masayuki Tsuji d, Nobuo Hashimoto b, Tasuku Honjo d, Kenji Tanigaki a,⁎ a Research Institute, Shiga Medical Center, Moriyama 5-4-30, Shiga 524-8524, Japan b Department of Neurosurgery, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan d Department of Immunology and Genomic Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan article info abstract

Article history: Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms Received for publication 29 September 2008 supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of Revised 1 June 2009 RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the Accepted 1 June 2009 olfactory bulbs (OBs) with concomitant increase in Olig2+ oligodendroglial progenitors, although generation Available online 6 June 2009 of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J- fl + fi Keywords: oxed mice indicated that Olig2 cells in the OBs can be generated from RBP-J-de cient SVZs, although no RBP-J oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to Subventricular zone oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by Adult neurogenesis in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the Olig2 maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. © 2009 Elsevier Inc. All rights reserved.

Introduction regulation is crucial for neuronal differentiation. Overexpression of Olig2 leads to the inhibition of neurogenesis and promotes oligoden- The most prominent region of neurogenesis in the adult droglial differentiation (Hack et al., 2005; Lee et al., 2005; Marshall mammalian brain is the subventricular zone (SVZ) of the lateral et al., 2005). ventricle wall (Doetsch et al., 1999). Olfactory bulb (OB) interneurons, Notch/RBP-J signaling has been known to play pivotal roles in continuously generated throughout adulthood, originate from astro- various aspects of neural cell fate speciﬁcation. Notch is a membrane- glia-like stem cells (B cells) in the SVZ. These stem cells divide slowly bound receptor. Ligand binding to the Notch receptor leads to its and give rise to type C cells, which are rapidly-dividing, transient- proteolytic processing to release the intracellular domain (IC) that amplifying multipotent precursors. Type C cells in turn generate type translocates to the nucleus and interacts with a DNA-binding protein, A cells, which are neuroblasts, migrate to the OB through the rostral RBP-J (Tamura et al., 1995). In the absence of Notch IC, RBP-J forms a migratory stream (RMS). In the OB, they differentiate into interneur- complex with co-repressors such as SMRT, N-CoR and MINT to repress ons, such as granule cells and periglomerular neurons (Doetsch et al., the transcription of its target genes (Hsieh and Hayward, 1995; Kao 1999; Lois and Alvarez-Buylla, 1994). In adults, SVZ cells generate OB et al., 1998; Kuroda et al., 2003). Notch IC competes with co-repressors neurons but not OB oligodendroglial cells (Lois and Alvarez-Buylla, for RBP-J to activate RBP-J-mediated transcription. Notch/RBP-J 1994; Menn et al., 2006; Spassky et al., 2001; Suzuki and Goldman, signaling inhibits neuronal differentiation and supports neural stem 2003), although they have the potential to differentiate into cells (Androutsellis-Theotokis et al., 2006; de la Pompa et al., 1997; oligodendroglial cells in corpus callosum, striatum and ﬁmbria cornix Ohtsuka et al., 2001). In spinal cord oligodendroglial development, (Menn et al., 2006). Olig2 is a transcriptional factor expressed in type Notch/RBP-J signaling is essential for the commitment of oligoden- C cells in the SVZ, which is essential for the potential of transient- droglial progenitors (Park and Appel, 2003), but inhibits the amplifying cells (Hack et al., 2005, 2004). However, its down- maturation of oligodendroglial progenitors (Genoud et al., 2002; Wang et al., 1998). In adult neurogenesis, loss of Jagged1, a Notch ligand, or Notch1 blocks neural stem cell proliferation but does not effect cell lineage commitment between neurons, astrocytes and ⁎ Corresponding author. Fax: +81 77 582 6041. E-mail address: [email protected] (K. Tanigaki). oligodendroglial cells in vitro (Nyfeler et al., 2005). However, the role 1 Contributed equally to this work. of Notch signaling in vivo remains to be elucidated.

Here we showed that loss of RBP-J leads to the decrease of granule was 1.2 mm anterior and 1.3 mm lateral to bregma at a depth of cells in the OB with concomitant increase of SVZ-derived oligoden- 1.6 mm and 0.7 mm anterior and 0.9 mm lateral to bregma at a depth droglial progenitor cells. RBP-J-deficient neural progenitor cells of 1.9 mm. All procedures were performed according to the guidelines showed defects in neuronal differentiation and preferential differ- on animal experiments of Kyoto University and Shiga Medical Center. entiation to oligodendroglial cells. Knockdown of Olig2 rescued the defects in neuronal differentiation of RBP-J-deficient SVZ neural Neural progenitor cell culture progenitor cells. Our experiments indicate the pivotal roles of Notch/RBP-J Neural progenitor cell cultures were established from the lateral signaling to maintain stable adult neurogenesis in vivo. RBP-J ventricular walls of newborn control or RBP-Jf/f mice. Tissues were promotes neurogenesis by inhibiting oligodendroglial differentiation dissociated by trituration with a fire-polished Pasteur pipette. Cells at least in part through the regulation of Olig2 transcription. were plated at 8000 cells/cm2 in neurosphere medium (DMEM/F12 containing 0.025 mg/ml apo-transferrin, 0.015 mg/ml insulin, 20 nM Materials and methods progesterone, and 30 nM Na selenite) supplemented with EGF (20 ng/ ml) and FGF2 (10 ng/ml). After 2 to 4 passages, cells were dissociated Transgenic lines from neurospheres and seeded on a Matrigel-coated slide chamber (Nunc). For analysis of progenitor differentiation, cells were infected RBP-J-floxed mice (Han et al., 2002; Tanigaki et al., 2002), CamKII- with Cre-expressing adenoviruses and RBP-J or EEF-expressing Cre (line 93) transgenic mice (Rios et al., 2001), and ROSA26R-LacZ Cre lentiviruses one day after passage and maintained for 4 days in reporter (Soriano, 1999) mouse lines have been described. neurosphere medium supplemented with 5% FCS. Cells were then fixed with 4% PFA for 15 min and processed for immunohistochem- BrdU labeling analysis istry. OHT-inducible-Notch IC-expressing adult hippocampal progenitor cells (LHCTM-transduced AHPs) were described in a previous We injected 9- to 12-week-old mice intraperitoneally with BrdU study (Tanigaki et al., 2001). (100 mg/kg) once at day 0 for the pulse chase experiment or every 12 h for continuous BrdU treatment. Mice were transcardially Reporter assay perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS (PFA) 2, 72 or 168 h after the first administration of BrdU. Brains were 4× RBP-J-binding sites and SV40 promoter were subcloned into fixed for 12 h in 4% PFA and were frozen in tissue-tek O.C.T. compound pGL3 and Venus was substituted with luciferase gene to generate RBP- (Sakura Finetechnical, Tokyo, Japan) and cut at 40 μm thickness. BrdU J-Venus. Mouse Olig2 promoter (−1832 to 0) was subcloned into staining was performed as described previously (Handler et al., 2000). pGL4 (Promega) by PCR (Olig2-luc). We introduced mutation into RBP-J binding sites of these constructs using site-directed mutagen- Immunohistochemistry of tissue sections esis to abolish RBP-J binding, based on a strategy used in a previous study (Barolo et al., 2000). Reporter plasmid Olig2-luc was cotrans- Mice were perfused and sectioned, as above. Immunohistochem- fected with pRL-EF to SVZ-derived neural progenitor cells using ical staining was performed with primary antibodies for 48 h at 4 °C Lipofectamine 2000 (Invitrogen). The total amount of DNA was kept after blocking for 1 h at room temperature with 4% skim milk (Becton constant with pBS (Stratagene). After 24 h incubation in neurosphere Dickinson). Then the sections were incubated for 1 h at room medium, cells were subjected to luciferase assay using a luminometer temperature with secondary antibodies (Molecular Probes). The (GlowMax Luminometer, Promega). Normalized luciferase activity primary antibodies used were anti-LacZ (Promega and Cappel), anti- (firefly luciferase/sea urchin luciferase ratio) was then compared in BrdU (BU1/75, Oxford Biotechnology), anti-GFP (Molecular Probes), each experiment, samples were analyzed in triplicate, and experi- anti-PSA-NCAM (Chemicon), anti-Dlx-2 (Chemicon and Santa Cruz), ments were repeated at least three times. anti-GFAP (DAKO), anti-NeuN (Chemicon), anti-Olig2 (IBL), anti- PDGFRα (Cell Signaling), anti-Nestin (Chemicon), anti-O4 (Chemi- RNA interference con), anti-Mash1 (R&D) and anti-Tuj1 (Sigma) antibodies. The TUNEL assay was performed using the Apoptag Fluorescein In Situ Apoptosis The target region in Olig2 was identified by the BLOK-iT RNAi Detection Kit (Chemicon). Slides were examined with an Olympus Designer Program (Invitrogen). The sequence used to generate shRNA confocal laser scanning microscope (FV-300, Olympus). Numbers of was GGGAGTCGAATATATGGGAACCGAA. Synthetic duplexes were cells positive for BrdU or cell type-specific markers were system- subcloned into the pcDNA6.2GW/EmGFP/MiR vector (Invitrogen), atically counted in every fifth sagittal section covering the complete which co-expresses the shRNA surrounded by miR155-flanking OB. Analysis of LacZ activity was performed by X-gal staining, as sequences with EmGFP. This vector was used to transfer the expression previously described (Zinyk et al., 1998). cassette to the lentiviral vector, pLenti6/V5-DEST vector (Invitrogen), by Gateway recombination following the kit instructions. Immunoblotting Results Cells were lysed for 30 min in 0.1 ml of ice-cold lysis buffer that contained 1% Triton X. After centrifugation, cell lysates were subjected Reduced neuronal differentiation in RBP-J-deficient adult OB to 15% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Hybond-P, Amersham Pharmacia, Uppsala, Sweden). In the adult SVZ, astroglia-like stem cells (B cells) in the SVZ The blot was incubated with anti-RBP-J (T6709, Institute of Immunol- express glial fibrillary acidic protein (GFAP). These stem cells generate ogy), anti-Olig2 (IBL), and anti-β-actin (Sigma) antibodies and type C cells, which express Dlx2 but not polysialylated form of neural visualized by the ECL-plus detection system (Amersham Pharmacia). cell adhesion molecules (PSA-NCAM). Next, type C cells differentiate into type A cells, which express PSA-NCAM. To examine the functions Stereotaxic injection of RBP-J in adult neurogenesis, we utilized adult CamKII-Cre×RBP-J f/f (RBP-J f/f ×Cre) mice. In these transgenic mice, Cre expression begins Injections of adenoviruses or lentiviruses were performed stereo- at C cell stage in the SVZs of the lateral ventricles, as confirmed by taxically in 9- to 12-week-old mice. The coordinate of the injection site immunohistochemical analysis of the SVZs of R26R-LacZ Cre reporter M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350 341 mice crossed with these CamKII-Cre transgenic mice (Fig. 1A, 519.6 cells/mm2: RBP-Jf/f ×Cre mice, 5463.1±884.2 cells/mm2: Supplementary Fig. 1). Cre expression was observed in 2.7±1.3% of n=7745, 13 hemispheres, P=0.0062 (Student's t test)) (Figs. 2A, B). GFAP+ cells, 77.7±8.0% of C cells (PSA-NCAM−, Dlx2+ cells), and In contrast, RBP-J deficiency enhanced generation of oligodendroglial 92.4±6.0% of A cells (PSA-NCAM+, Dlx2+ cells) of the SVZs (n=1777, progenitors expressing Olig2 and platelet-derived growth factor 12 hemispheres). Cre expression began earlier than postnatal day 10 receptor α (PDGFRα) (Olig2: RBP-Jf/+ ×Cre mice, 161.1±25.3 cells/ (Supplementary Fig. 1). In the absence of RBP-J, the density of mm2, RBP-Jf/f ×Cre mice, 268.3±46.9 cells/mm2, n=3196, 39 hemi- Neuronal Nuclei (NeuN)+ granule cells in the OB GCL was reduced spheres, Pb0.0001 (Student's t test); PDGFRα: RBP-Jf/+ ×Cre mice, (densities of NeuN+ granule cells: control RBP-Jf/+ ×Cremice, 6986.3± 196.6±49.7 cells/mm2, RBP-Jf/f ×Cre mice, 276.6±48.3 cells/mm2,

Fig. 1. Enhanced generation of A cells from RBP-J-deficient SVZ cells. (A) Schematic drawings of a coronal plane section (left) showing a neurogenic area of the brain. Immunofluorescent analysis for Dlx2 (green, middle panel), PSA-NCAM (red, middle panel), GFAP (red, right panel) and LacZ (blue) in the SVZs of CamKII-cre×R26R-LacZ mice. Scale bar=20 μm. (B) Immunofluorescence analysis for Dlx2 (green), PSA-NCAM (red), and DAPI staining of nuclei (blue) in RBP-J f/+ ×Cre and RBP-J f/f ×Cre SVZs. Scale bar=20 μm. (C) Quantitative analysis of distributions of C cells (PSA-NCAM−, Dlx2+ cells), and A cells (PSA-NCAM+, Dlx2+ cells) in RBP-J f/+ ×Cre (white bar) and RBP-J f/f ×Cre (black bar) SVZs. Values are the mean±S.D. for 5 to 12 mice. ⁎Pb0.05, ⁎⁎Pb0.01 (Student's t test). (D) Density of BrdU+ A cells (PSA-NCAM+, Dlx2+ cells) in the SVZs of RBP-J f/+ ×Cre (white bar) and RBP-J f/f ×Cre mice (black bar). Single dose of BrdU (100 mg/kg) was administered 2 h before sacrifice. Values are the mean±S.D. for 3 to 4 mice. ⁎Pb0.05 (Student's t test). 342 M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350

Fig. 2. RBP-J deficiency caused defects in neuronal differentiation with concomitant increase of oligodendroglial progenitors in OBs. (A) Immunostaining for NeuN, Olig2, and PDGFRα in the granule cell layer of the OBs of RBP-J f/+ ×Cre and RBP-J f/f ×Cre mice. Scale bar=100 μm. Density of marker+ cells in the GCLs of RBP-J f/+ ×Cre (white bars) and RBP-J f/f ×Cre OBs (black bars). Values are the mean±S.D. for 6 to 20 mice. ⁎⁎Pb0.01 (Student's t test). (C, D) The kinetics of BrdU+ Olig2+ cells in the GCLs from RBP-J f/+ ×Cre and RBP-J f/f ×Cre OBs at various time points. BrdU was administrated once for a pulse chase experiment (D) or continuously every 12 h (C). The data is the mean±S.D. from 3 to 8 mice. ⁎Pb0.05, ⁎⁎Pb0.01 (Student's t test). n=1825, 20 hemispheres, P=0.0021 (Student's t test)) (Figs. 2A, B). deoxyuridine (BrdU) labeling experiments. Two hours after admin- Next, we examined expressions of other oligodendroglial markers in istration of BrdU, the number of BrdU+ Olig2+ cells increased 2.5 RBP-Jf/f ×Cre mice (Supplementary Fig. 2). The expressions of PLP and times in the SVZs of RBP-Jf/f ×Cre mice compared with that of control Olig2 but not CNPase or MBP increased in the absence of RBP-J, which RBP-Jf/+ ×Cre mice (control RBP-Jf/+ ×Cre mice, 6.2±4.0 cells/mm2: confirmed the enhanced generation of oligodendroglial cells caused by RBP-Jf/f ×Cre mice, 15.2±5.1 cells/mm2: 12 hemispheres, P=0.030 RBP-J deficiency. (Student's t test)) (Fig. 2C), which suggests that the local proliferation of oligodendroglial progenitors in the OB is enhanced in the absence of The kinetics of generation of Olig2+ cells in the absence of RBP-J RBP-J. After continuous BrdU treatments for 72 h, the difference of the ratio of BrdU+ Olig2+ cells drastically increased from 2.5 times to 4.3 The enhancement of oligodendroglial progenitor generation can be times (control RBP-Jf/+ ×Cre mice, 29.4±6.4 cells/mm2: RBP-Jf/f ×Cre caused by a local increase of the proliferation rate of oligodendroglial mice, 125.7±22.4 cells/mm2: 12 hemispheres, P=0.01 (Student's t progenitors in the OB, or by an increase of immigration of test)) (Fig. 2C). Next, we performed BrdU pulse chase experiments. oligodendroglial progenitors to the OB from other brain regions. To RBP-J deficiency caused much higher labeling rate of Olig2+ cells in discriminate between these two possibilities, we performed bromo- the OBs 3 days after one BrdU administration (control RBP-Jf/+ ×Cre M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350 343 mice, 4.8±3.2%: RBP-Jf/f ×Cre mice, 20.8 ±3.3%: 6 hemispheres, determine the rate of generation of A cells, we performed BrdU P=0.013 (Student's t test)) (Fig. 2D). These data suggested that labeling experiment. Two hours after injection of BrdU, we analyzed enhanced immigration to the OB as well as enhanced proliferation of the incorporation of BrdU in A cells in RBP-J deficient SVZ. We oligodendroglial progenitors caused increased density of Olig2+ cells observed enhanced generation of A cells in the absence of RBP-J (6 in the OB in the absence of RBP-J. hemispheres, P=0.021 (Student's t test)) (Fig. 1D). TUNEL analysis To examine the process of development of RBP-J-deficient SVZ demonstrated that the absence of RBP-J did not affect cell death in neural progenitor cells, we performed immunofluorescence studies of adult neurogenesis (data not shown). These findings showed that the composition of C, and A cells in the SVZ and RMS. In the RBP-J- RBP-J deficiency in SVZ enhanced generation of immature neurons but deficient SVZ, the percentage of A cells, which are immature led to impairment of neuronal maturation to granule cells in the OBs neuroblasts, increased from 60.9±8.5% to 77.8±5.5% at the expense with concomitant increase of Olig2+ oligodendroglial progenitor cells. of C cells (11 hemispheres, P=0.0035 (Student's t test)) (Figs. 1B, C), whereas in the RMS A cells decreased (RMS: 21 hemispheres, Ectopic expression of Olig2 in PSA-NCAM+ cells in the absence of RBP-J P=0.034 (Student's t test)), with concomitant increase in Olig2+ cells (RMS: 11 hemispheres, P=0.012 (Student's t test)) (Figs. To examine which cells express Olig2 in the absence of RBP-J, we 3A–C, E). In addition, the Olig2 expression level of RMS cells was performed immunohistochemical characterization of Olig2+ cells in slightly enhanced in the absence of RBP-J, although the difference was the SVZs and the RMSs. It has been reported that Olig2 is exclusively not statistically significant (P=0.09 (Student's t test)) (Figs. 3D, E). To expressed in C cells and that Mash1+ cells are C cell-like cells and small

Fig. 3. The increased number of Olig2+ cells in the absence of RBP-J. (A) Schematic drawings of a sagittal plane section showing a neurogenic area of the brain. (B, C) Quantitative analysis of distributions of A cells (PSA-NCAM+ and Dlx2+ cells) (B) and Olig2+ cells (C). Dots represent the mean for 5 to 12 mice, with S.D. shown as bars. ⁎Pb0.05, ⁎⁎Pb0.01 (Student's t test). (D, E) Quantification of the expression level of Olig2 (D) by measuring immuno-fluorescent intensity of Olig2 in the RMSs of RBP-J f/+ ×Cre and RBP-J f/f ×Cre mice (E). Scale bar =20 μm. 344 M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350 fraction of Mash1+ cells expresses PSA-NCAM, which is expressed in A SVZs by stereotaxic injection of Cre-expressing adenoviruses into the cells (Hack et al., 2005; Hack et al., 2004; Parras et al., 2004). Large SVZ of RBP-Jf/+ or RBP-Jf/f mice with R26R-LacZ Cre reporter (Fig. 5A). fraction of RBP-J-deficient Olig2+ cells was Mash1-negative and This Cre reporter enabled labeling of the progeny of Cre-expressing expressed PSA-NCAM (Mash1: SVZ P=0.028, RMS P=0.042; PSA- adenovirus-infected SVZ cells with LacZ. Cre-mediated LacZ expres- NCAM: SVZ P=0.007, RMS P=0.021 (Student's t test), 6 hemispheres) sion was observed in GFAP+ cells, C cells, and A cells 24 h after (Figs. 4A, B). This data might suggest that some of prospective injection (GFAP+ cells: 24.5±2.3%, Olig2+ cells: 32.4±3.5%, C cells: neuroblasts ectopically express Olig2 in the absence of RBP-J. 15.4±0.4%, A cells: 49.2±6.6% of LacZ+ SVZ cells (n=444, 10 hemispheres)). Two weeks after infection, RBP-J deficiency caused a Ectopic generation of OB Olig2+ cells from RBP-J-deficient SVZ drastic reduction in astroglia-like GFAP+ cells in the SVZs (control RBP-Jf/+ ×R26R-LacZ mice, 35.9±7.6%; RBP-Jf/f ×R26R-LacZ mice, To trace how RBP-J-deficient SVZ neuronal progenitors differenti- 15.7±2.9%: n=1426, 17 hemispheres, P=0.00019 (Student's t test)) ate in vivo, we induced deletion of the RBP-J allele specifically in the (Figs. 5B, C) but did not affect the migration of LacZ+ cells to the OBs. TUNEL analysis demonstrated that the absence of RBP-J did not affect cell death of the adeno-infected SVZ cells (control RBP-Jf/+ ×R26R- LacZ mice, 2.2±0.9%; RBP-Jf/f ×R26R-LacZ mice, 1.7±0.9%: n=412, 6 hemispheres, P=0.485 (Student's t test)). In control mice, the LacZ+ cells in the OB were exclusively NeuN+ neurons (two weeks after infection, 94.5±0.9%: three weeks after infection, 99.6±0.5%), consistent with previous reports (Lois and Alvarez-Buylla, 1994; Menn et al., 2006; Suzuki and Goldman, 2003). In contrast, in the absence of RBP-J, the percentage of NeuN+ cells among LacZ+ cells decreased to 68.9 ±10.4% (n = 1274, 16 hemispheres, P b0.0001 (Student's t test)), whereas LacZ+ Olig2+ cells increased to 9.9± 4.1% (two weeks after infection) compared with 0.5±1.0% (two weeks after infection) and 0% (three weeks after infection) of control mice (n=1056, 16 hemispheres, Pb0.0001 (Student's t test)) (Fig. 5D). LacZ+ Olig2+ cells slightly increased also in the RMSs but not in the SVZ, which are not statistically significant (RMS P=0.17:SVZP=0.49 (Student's t test), 7 hemispheres) (Fig. 5E). Taken together, these findings indicate that the reduction of mature granule neurons in RBP-J-deficient mice is correlated with the increase in the number of ectopic Olig2+ cells in the OBs.

Preferential differentiation of SVZ-derived neural progenitor cells to oligodendroglial cells in the absence of RBP-J

To determine the functions of RBP-J in promoting neuronal maturation, we utilized SVZ neural progenitor culture from the lateral ventricular walls of RBP-Jf/f or RBP-Jf/+ mice at birth. After 2–4 passages, Cre was introduced with adenoviruses and cultivated in differentiating conditions for four days. Cre-mediated deletion efficiency was estimated using R26R-LacZ Cre reporter (Fig. 6D). LacZ expression from R26R-LacZ Cre reporter was observed in all SVZ neural progenitor cells infected by Cre-expressing adenoviruses. Loss of RBP-J in infected SVZ neural progenitors was confirmed by western blotting (Fig. 6E). Immunohistochemical analysis with a post-mitotic neuronal marker, TujI, and an oligodendroglial marker, O4 and real- time RT-PCR analysis of a oligodendroglial marker, PLP showed that RBP-J deficiency in the SVZ neural progenitor culture resulted in the reduction of mature neurons with preferential differentiation to oligodendroglial cells (TujI+ cells (N): control RBP-Jf/+ culture, 8.9± 1.6%; RBP-Jf/f culture, 5.0±1.7%, Pb0.05 (Student's t test), O4+ cells (O): control RBP-Jf/+ culture, 17.7±1.1%; RBP-Jf/f culture, 38.2±3.9%, Pb0.001 (Student's t test)) (Figs. 6A–C). These observations confirmed that RBP-J deficiency in SVZ neural progenitor cells also leads to impairment of neuronal maturation in vitro. RBP-J functions as a transcriptional repressor in the absence of Notch activation (Hsieh and Hayward, 1995; Kao et al., 1998; Kuroda et al., 2003)(Fig. 7C). To test whether the repressor activity of RBP-J is required for promotion of neuronal maturation, we performed rescue experiments with lentiviral-mediated expression of RBP-J or RBP-J EEF259AAA mutant, a mutant ineffective in mediating transcriptional Fig. 4. Increased expression of Olig2 in RBP-J-deficient A cells. (A) Immunostaining for suppression (Tani et al., 2001; Zhou and Hayward, 2001)(Figs. 7A, B). PSA-NCAM, Mash1, and Olig2 in the SVZs of RBP-J f/+ ×Cre and RBP-J f/f ×Cre mice. RBP-J re-expression enhanced the maturation of TujI+ neurons from Scale bar=20 μm. (B) Percentages of marker+ cells in the Olig2+ cells of the SVZs and RMSs of RBP-J f/+ ×Cre (white bars) and RBP-J f/f ×Cre mice (black bars). Values are the SVZ neural progenitors. In contrast, the RBP-J EEF259AAA mutant mean±S.D. for 3 to 5 mice. ⁎Pb0.05, ⁎⁎Pb0.01 (Student's t test). failed to rescue the defects in neuronal maturation and decreased the M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350 345

Fig. 5. Reduced OB neuronal differentiation from RBP-J-deﬁcient SVZ neural progenitors in vivo. (A) Schematic drawings illustrating the site of adenoviral injection (an arrow). CC, corpus callosum; LV, lateral ventricle. (B, C) LacZ staining (B) and graphs (C) illustrating the cell types of adenovirus-infected LacZ+ cells in the SVZs of RBP-J f/+ ×R26R-LacZ and RBP-J f/f ×R26R-LacZ mice 14 days after injection of Cre-expressing adenoviruses into the SVZs. Values are the mean for 6 to 10 mice. Scale bar =250 μm. (D) Immunoﬂuorescence analysis for LacZ (green), NeuN (red, upper left panel), and Olig2 (red, upper right panel) of LacZ+ cells in the OBs of RBP-J f/f ×R26R-LacZ mice 14 days after injection of Cre-expressing adenoviruses into the SVZs. These cells were derived from adenovirus-infected SVZ cells. Scale bar=20 μm. Lower graphs show percentages of NeuN+ or Olig2+ cells in the OBs of RBP-J f/+ ×R26R-LacZ (white bars) and RBP-J f/f ×R26R-LacZ mice (black bars) 14 or 21 days after injection of Cre-expressing adenoviruses into the SVZs. Values are the mean±S.D. for 5 to 10 mice. ⁎Pb0.001 (Student's t test). (E) The percentages of Olig2+ cells in the adenovirus-infected cells of RBP-J f/+ ×R26R-LacZ and RBP-J f/f ×R26R-LacZ mice 14 days after injection of Cre-expressing adenoviruses into the SVZs. Values are the mean±S.D. for 3 to 5 mice. ⁎Pb0.01 (Student's t test).

number of TujI+ neurons, suggesting that RBP-J-mediated transcrip- fluorescence caused by loss of RBP-J is dependent on Notch activation or tional repression is important for neuronal maturation (TujI+ cells: not, we shut down Notch activation using γ-secretase inhibitor, DAPT. GFP, 22.3±6.6%; RBP-J, 37.7±3.69%; EEF, 9.9±2.8%, Pb0.05 (Student's DMSO was used as a negative control. The number of SVZ neural t test)) (Figs. 7A, B). The percentage of TujI+ neurons in lentivirus- progenitor cells with high Venus-fluorescent intensity drastically infected cells is higher than that of differentiated RBP-J-deficient SVZ decreased by the treatment with DAPT, which showed that high neural progenitor cells (Figs. 6A, B). This is caused by the preference of Venus fluorescence in SVZ neural progenitor depends on Notch lentiviruses to infect neuronal cells, which might bias the interpreta- activation (Fig. 8E). In contrast, RBP-J deficiency caused increased tion of this data. number of cells with high fluorescent signal (Fig. 8F). Taken together, these results suggest that RBP-J mediated transcriptional repression RBP-J-mediated transcriptional repression in SVZ neural progenitor cells exists in SVZ neural progenitor cells and the function is independent of Notch activation. The increase of fluorescent signal in RBP-J-deficient To visualize RBP-J-mediated transcription in SVZ neural progenitor cells induced by DAPT might suggest that the transcriptional repression cells, a reporter gene expressing Venus-fluorescent protein (Nagai et al., activity of RBP-J may be higher in differentiating neurons than in neural 2002) under the control of 4 tandem RBP-J binding sites and mutated progenitor cells, because DAPT treatment causes premature neural RBP-J binding sites was constructed (RBP-J-Venus/mRBP-J-Venus) differentiation of neural progenitor cells. (Barolo et al., 2000; Mizutani et al., 2007)(Fig. 8A). The specificity of this reporter was assessed by transient transfection assay using a Regulation of Olig2 transcription by RBP-J constitutive active mutant form of Notch1 (RAMIC) and mRBP-J-Venus. Notch activation by RAMIC enhanced Venus expression from RBP-J- Persistent expression of Olig2 is known to interfere with neuronal Venus in SVZ neural progenitor cells (Fig. 8B), whereas mRBP-J-Venus differentiation and promote oligodendroglial cell differentiation construct did not show any responses to RAMIC (Fig. 8C). These data (Hack et al., 2005; Lee et al., 2005; Marshall et al., 2005), which led demonstrated that Venus-fluorescent intensity is Notch-responsive us to test whether Olig2 is a direct target of RBP-J-mediated and specific. transcriptional regulation. Analysis of the nucleotide sequence of the Next, we examined the effects of loss of RBP-J induced by Cre- Olig2 promoter region revealed two binding sites (CGTGAGAA/ expressing adenovirus infection. RBP-J deficiency decreased the number TGTGAGAA) for RBP-J, one of them conserved between mice and of cells with low Venus-fluorescent intensity (Fig. 8D), suggesting leaky humans (Fig. 9A). We first examined the activation of Olig2 promoter expression by de-repression. Finally, to examine whether this increased by activation of Notch signaling. When a constitutive active mutant 346 M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350

Fig. 6. The preferential differentiation of RBP-J-deficient neural progenitor cells to oligodendroglial cells associated with reduced neuronal differentiation. (A) Immunofluorescent study of Nestin, Tuj1, O4 and GFAP (green) and nuclei stained with DAPI (blue). The SVZ neural progenitor cells were isolated from RBP-Jf/+ and RBP-Jf/f mice and infected by Cre- expressing adenoviruses. Immunofluorescent staining was performed four days after adenoviral infection. Scale bar=100 μm. (B) Quantification of neural stem cells (S, Nestin+ cells), neurons (N, Tuj1+ cells), oligodendroglial cells (O, O4+ cells) and astrocytes (A, GFAP+ cells) in a 4-day culture of the SVZ neural progenitor cells from RBP-Jf/+ (white bars) and RBP-Jf/f mice (black bars) infected by Cre-expressing adenoviruses. The data is the mean±S.D. from 3 mice. ⁎Pb0.05, ⁎⁎Pb0.01 (Student's t test). (C) Time course of change in gene expression in RBP-J-deficient SVZ neural progenitors. Quantitative real-time RT-PCR analysis of gene expression in SVZ neural progenitors during the 84 h period following Cre-expressing adenovirus infection. Values have been normalized to β-actin abundance. ⁎Pb0.05, ⁎⁎Pb0.01 (Student's t test). (D) LacZ staining of SVZ neural progenitors of CamKII-cre×R26R-LacZ mice 24 h after infection by Cre-expressing adenoviruses. Scale bar=100 μm. (E) Immunoblot analysis of RBP-J in RBP-Jf/+ and RBP-Jf/f SVZ neural progenitors infected by Cre-expressing adenoviruses.

form of Notch1, RAMIC, was transfected with the Olig2-promoter nated LHCTM. LHCTM-transduced AHPs were treated with OHT. 24 h (−1832-0)-luc into SVZ-derived neural progenitor cells, transactiva- Notch activation induced Olig2 expression in LHCTM-transduced AHP tion of Olig2 promoter was observed (Fig. 9B). To examine whether cells (Fig. 9E). In SVZ neural progenitor cells, RBP-J deﬁciency also led Notch activation induces endogenous Olig2, we utilized 4-hydro- to a slight increase in endogenous Olig2 expression (Fig. 6C). These xytamoxifen (OHT)-inducible Notch activation system of adult results suggest that Olig2 may be a target of Notch/RBP-J signaling. hippocampus-derived neural progenitors (AHPs) (Tanigaki et al., To examine the roles of the RBP-J binding sites in the transcriptional 2001). The constitutive active form of Notch1 is fused to the human regulation of Olig2, we introduced mutations in RBP-J binding sites in estrogen receptor and introduced to retrovirus vector, LHCX, desig- Olig2 promoter (mOlig2-luc). These mutations abolished the response M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350 347

Fig. 7. Repressor activity of RBP-J is required for promotion of neuronal differentiation from adult SVZ neural progenitors. (A, B) Immunohistochemical analysis of EGFP (green), O4, or TujI (red) and nuclei stained with DAPI (blue). SVZ neural progenitors from RBP-Jf/f mice were infected with Cre-expressing adenoviruses and lentiviruses that express RBP-J IRES EGFP (RBP-J) or RBP-J-EEF259AAA IRES EGFP (EEF). Immunofluorescence staining was performed four days after infection. Scale bar=50 μm. Bar graphs (B) showing quantification of percentages of O4- or Tuj1+ cells among EGFP+ lentivirus-infected cells. Values are the mean±S.D. for 3 mice. ⁎Pb0.05 (Student's t test). (C) Models for RBP-J function in the regulation of target gene transcription. In the absence of Notch signaling activation, RBP-J recruits co-repressors (CoR) and represses target gene expression. After Notch activation, intracellular domain of Notch (IC) translocates to nucleus, transactivates RBP-J-mediated transcription by displacement of and recruitment of coactivators (CoA). EEF259AAA (EEF), a mutant form of RBP-J without transcriptional repression activity, sometimes fails to suppress the expression of target genes, leading to ectopic target gene expression. of Olig2 promoter to Notch activation and slightly increased luciferase et al., 1998; Kuroda et al., 2003), we cotransfected RBP-J EEF259AAA basal expression (Figs. 9C, D), which might represent release from mutant with Olig2-promoter-luc (Tani et al., 2001; Zhou and Hayward, RBP-J-mediated repression of Olig2 promoter, although it was not 2001). Overexpression of RBP-J EEF mutant de-repressed the expres- statistically significant (P=0.069 (Student's t test)) (Fig. 9C). Next, to sion of Olig2 gene (Fig. 9B), suggesting that transcription of Olig2 is examine whether RBP-J acts as a transcriptional repressor of Olig2 gene actively suppressed by RBP-J in the process of development of SVZ in the absence of Notch activation (Hsieh and Hayward, 1995; Kao neural progenitor cells. 348 M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350

Fig. 8. Reporter systems for RBP-J-mediated transcription in SVZ neural progenitors. (A) Schematic representation of RBP-J-mediated transcription reporter construct. (B–F) SVZ neural progenitor cells from RBP-Jf/f mice were transfected with 4× RBP-J (B-F)or mRBP-J (C) -Venus and with (B) or without Notch RAMIC expression vector(C–F) in the presence (E, F) or absence of γ-secretase inhibitor, DAPT(1 μM) (B–D). Loss of RBP-J was induced by Cre-expressing adenovirus infection (D, F). Results are the mean±S.D. of triplicate data points from a representative experiment. ⁎Pb0.05, ⁎⁎Pb0.01 (Student's t test).

De-repression of Olig2 inhibits neuronal maturation in the absence repression of Olig2 expression, although we could not exclude the of RBP-J possibility of indirect effects caused by Olig2 reduction.

To further examine the functional importance of RBP-J-mediated Discussion Olig2 regulation, we assessed the effects of simultaneous depletion of Olig2 in RBP-J-deficient SVZ neural progenitors, using RNA inter- In this study, we demonstrated that RBP-J plays pivotal roles in ference. We transduced microRNA155-fused short hairpin RNA neuronal maturation in adult neurogenesis. In the absence of RBP-J, (shRNA) against Olig2 (MiR-Olig2) with lentivirus vectors, which generation of granule cells from SVZ neural progenitor cells was co-cistronically express emerald GFP (EmGFP) to enable the identi- decreased with concomitant increase in Olig2+ and PDGFRα+ fication of shRNA-introduced cells. Introduction of MiR-Olig2 oligodendroglial progenitors. The maturation of RBP-J-deficient SVZ decreased the level of ectopically-expressed Olig2 in 293 cells and neuronal progenitors to TujI+ post-mitotic neurons was also delayed endogenous Olig2 in SVZ neural progenitors (Figs. 9F, G). Next, MiR- in vitro. Our in vitro findings suggest that transcriptional repression by Olig2 expressing lentiviruses were stereotaxically injected to the SVZs RBP-J is required for the maturation of neurons, and one of candidate of RBP-Jf/+ ×Cre or RBP-Jf/f ×Cre mice. Lentivirus-infected EmGFP+ RBP-J target genes may be Olig2. RNA interference experiments show cells appeared in OBs 14 days after infection. Immunohistochemical that knockdown of Olig2 partially rescues defects in neuronal analysis of EmGFP+ cells in OBs showed that reduction of Olig2 differentiation caused by RBP-J deficiency. expression rescued neuronal differentiation in OBs from RBP-J- Reduction in number of OB granule cells in the absence of RBP-J deficient SVZ neural progenitors (EmGFP+ NeuN+ cells/EmGFP+ may result from several mechanisms: (1) RBP-J may be required for cells in OBs: MiR-Control, 70.1±2.2%; MiR-Olig2, 80.7±4.0%, Pb0.05 the generation of immature neuroblasts, A cells, (2) migration of (Student's t test)) (Fig. 9H). These findings suggest that the defect in granule cells to the OB may depend on RBP-J or (3) RBP-J may be maturation of RBP-J-deficient neurons is due at least in part to de- required for the survival of granule cells. In the RBP-J-deficient SVZ, M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350 349

Fig. 9. Enhancement of neuronal differentiation from RBP-J-deficient SVZ neuronal progenitors by knockdown of Olig2 expression. (A) Conservation of a RBP-J binding site in the promoter region of Olig2 between mice and humans. Asterisks denote identical bases. RBP-J binding sites are boxed. Arrowheads indicated the single base changed (from G to C) in mutated sites; this point mutation abolishes RBP-J binding. (B–D) SVZ neural progenitor cells were transfected with Olig2-promoter-luc (B,C), mOlig2-promoter-luc (C,D) and with (B, D) or without increasing amounts of Notch1 RAMIC or RBP-J-EEF259AAA mutant (C). Results are the mean±S.D. of triplicate data points from a representative experiment. ⁎Pb0.05 (Student's t test). (E) Increased expression of endogenous Olig2 activation in AHPs by Notch activation. RNA was isolated from OHT-inducible Notch IC-expressing AHPs (LHCTM) or control AHPs (LHCX) cultured for 24 h in the presence or absence of 50 nM OHT and analyzed by RT-PCR. (F) Immunoblot analysis of Olig2 in whole-cell lysates of 293 cells transfected with Olig2-expression vector (Olig2) and various doses of microRNA155-fused shRNA against Olig2 (MiR-Olig2) and control microRNA155 (MiR-control). (G) Reduced expression of Olig2 in SVZ neural progenitors 18 h after infection of lentiviruses that express shRNA targeting microRNA155-fused shRNA against Olig2 (white bar) or control microRNA155 (MiR-control) (black bar). Level of expression was quantified by real-time RT-PCR and normalized to β-actin abundance. ⁎Pb0.05 (Student's t test). (H) Quantification of NeuN+ cells in the EmGFP+ OB cells of RBP-J f/+ ×Cre and RBP-J f/f ×Cre mice. Lentiviruses that express microRNA155-fused shRNA against Olig2 (MiR-Olig2) (white bar) or control microRNA155 (MiR-control) (black bar) with co-cistronic EmGFP were injected into the SVZs of RBP-J f/+ ×Cre and RBP-J f/f ×Cre mice. Immunohistochemical analysis were performed 14 days after injection. Values are the mean±S.D. for 3 mice. ⁎Pb0.05 (Student's t test). generation of A cells was enhanced at the expense of C cells. OB (Parras et al., 2004). Olig2 is required for oligodendroglial cell Adenoviral lineage tracing experiments revealed normal migration generation (Lu et al., 2002; Zhou and Anderson, 2002), and over- of RBP-J-deficient SVZ-derived cells to the OBs and abnormal expression of Olig2 in SVZ neural progenitor cells leads to preferential generation of OB oligodendroglial progenitor cells from RBP-J- differentiation to oligodendroglial cells and inhibition of neuronal deficient SVZs. We found ectopic expression of Olig2 in A cells but differentiation (Buffo et al., 2005; Hack et al., 2005; Marshall et al., did not observe any evidence of increased apoptosis of A cells. These 2005). The effects of loss of RBP-J are similar to those of Olig2 findings suggest that RBP-J deficiency affects the maturation process overexpression in SVZ neural progenitor cells, although the change in of granule cells. Olig2 expression caused by loss of RBP-J is slight. Our analysis of Olig2 Notch signaling has recently been reported to promote neural enhancer suggested the possibility of RBP-J-mediated Olig2 regulation. lineage commitment of ES cells (Lowell et al., 2006; Nemir et al., It has been reported that Notch signaling promotes commitment of 2006). However, loss of Notch1 or a Notch ligand, Jagged1, caused only oligodendroglial progenitor cells in the embryonic spinal cord (Park loss of self-replication by neural stem cells but no abnormality in and Appel, 2003). This is inconsistent with our findings, since loss of neuronal cell lineage commitment in adult SVZ neurogenesis both in RBP-J enhanced differentiation of OB oligodendroglial progenitor cells. vivo and in vitro (Nyfeler et al., 2005).These findings suggest that The existence of dual roles of RBP-J could account for this apparent Notch-independent activity of RBP-J might be responsible for deficits discrepancy. RBP-J might upregulate Olig2 expression as a positive in neuronal maturation and preferential differentiation to an regulator with Notch IC in the embryonic spinal cord, and act as a oligodendroglial fate in the absence of RBP-J. negative regulator of Olig2 transcription for neuronal differentiation In addition to neuronal maturation defects of RBP-J-deficient SVZ in adult neurogenesis. This hypothesis is also supported by the neural progenitor cells, our data indicated that RBP-J-deficient SVZ findings in Notch1- or RBP-J-deficient spinal cord development. In neural progenitor cells preferentially differentiate to oligodendroglial neural progenitor-specific Notch1-deficient spinal cord, an increase in cells. Several transcriptional factors have been reported to be involved neuronal differentiation and a decrease in number of Olig2+ in oligodendrogliogenesis in the SVZ. Loss of Mash1 caused reduced progenitors at E11.5 have been found (Yang et al., 2006), whereas generation of both oligodendroglial cells and neurons in the neonatal neural-progenitor-specific RBP-J deficiency has no effects on the 350 M. Fujimoto et al. / Developmental Biology 332 (2009) 339–350 number of Olig2+ cells in E11.5 spinal cord and enhances the during the development of the bristle sensory organ precursor cell of Drosophila. + Development 130, 1973–1988. generation of Olig2 cells in later developmental stages (Taylor et Kuroda, K., Han, H., Tani, S., Tanigaki, K., Tun, T., Furukawa, T., Taniguchi, Y., Kurooka, H., al., 2007). In Drosophila, Su(H), the RBP-J counterpart, also functions Hamada, Y., Toyokuni, S., Honjo, T., 2003. Regulation of marginal zone B cell as a Notch-independent transcriptional repressor, and the function of development by MINT, a suppressor of Notch/RBP-J signaling pathway. Immunity 18, 301–312. it is required for the differentiation of SOP cells to neurons as well as Lee, S.K., Lee, B., Ruiz, E.C., Pfaff, S.L., 2005. Olig2 and Ngn2 function in opposition eye photoreceptor development (Koelzer and Klein, 2003; Tsuda et al., to modulate gene expression in motor neuron progenitor cells. Genes Dev. 19, 2006). 282–294. Notch/RBP-J signaling regulates the process of neural develop- Lois, C., Alvarez-Buylla, A., 1994. Long-distance neuronal migration in the adult mammalian brain. Science 264, 1145–1148. ment in context-dependent fashion. RBP-J not only mediates Notch Lowell, S., Benchoua, A., Heavey, B., Smith, A.G., 2006. Notch promotes neural lineage signaling to support stem cell populations, but also promotes neuronal entry by pluripotent embryonic stem cells. PLoS Biol. 4, e121. maturation, which might be mediated at least in part through Lu, Q.R., Sun, T., Zhu, Z., Ma, N., Garcia, M., Stiles, C.D., Rowitch, D.H., 2002. Common fi developmental requirement for Olig function indicates a motor neuron/oligoden- regulation of Olig2 in the adult SVZ. The new ndings of the present drocyte connection. Cell 109, 75–86. study on the regulation of neurogenesis by Notch/RBP-J signaling will Marshall, C.A., Novitch, B.G., Goldman, J.E., 2005. Olig2 directs astrocyte and provide new insights into the molecular mechanisms supporting the oligodendrocyte formation in postnatal subventricular zone cells. J. Neurosci. 25, 7289–7298. maturation of neuron-committed progenitors, and will be of para- Menn, B., Garcia-Verdugo, J.M., Yaschine, C., Gonzalez-Perez, O., Rowitch, D., Alvarez- mount importance for efficient induction of neuronal differentiation Buylla, A., 2006. Origin of oligodendrocytes in the subventricular zone of the adult from adult neural stem cells in regeneration therapy. brain. J. Neurosci. 26, 7907–7918. Mizutani, K., Yoon, K., Dang, L., Tokunaga, A., Gaiano, N., 2007. Differential Notch signalling distinguishes neural stem cells from intermediate progenitors. Nature Acknowledgments 449, 351–355. Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K., Miyawaki, A., 2002. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological We thank R. Jaenisch for providing CamKII-Cre transgenic mice. applications. Nat. Biotechnol. 20, 87–90. This work was supported by Grants-in-Aid for Young Scientists (A) Nemir, M., Croquelois, A., Pedrazzini, T., Radtke, F., 2006. Induction of cardiogenesis (17689014) and (B) (20790244) from the Ministry of Education, in embryonic stem cells via downregulation of Notch1 signaling. Circ. Res. 98, – Culture, Sports, Science and Technology (MEXT), Mochida Memorial 1471 1478. Nyfeler, Y., Kirch, R.D., Mantei, N., Leone, D.P., Radtke, F., Suter, U., Taylor, V., 2005. Foundation for Medical and Pharmaceutical Research, Takeda Science Jagged1 signals in the postnatal subventricular zone are required for neural stem Foundation, Ichirou Kanehara Foundation, Sumitomo Foundation, the cell self-renewal. EMBO J. 24, 3504–3515. – General Insurance Association of Japan, the Shiga Medical Science Ohtsuka, T., Sakamoto, M., Guillemot, F., Kageyama, R., 2001. Roles of the basic helix loop–helix genes Hes1 and Hes5 in expansion of neural stem cells of the developing Association for International Cooperation, and RIKEN Bioresource brain. J. Biol. Chem. 276, 30467–30474. Center (RIKEN BRC). Park, H.C., Appel, B., 2003. Delta-Notch signaling regulates oligodendrocyte specification. Development 130, 3747–3755. Parras, C.M., Galli, R., Britz, O., Soares, S., Galichet, C., Battiste, J., Johnson, J.E., Nakafuku, Appendix A. Supplementary data M., Vescovi, A., Guillemot, F., 2004. Mash1 specifies neurons and oligodendrocytes in the postnatal brain. EMBO J. 23, 4495–4505. Rios, M., Fan, G., Fekete, C., Kelly, J., Bates, B., Kuehn, R., Lechan, R.M., Jaenisch, R., 2001. Supplementary data associated with this article can be found, in Conditional deletion of brain-derived neurotrophic factor in the postnatal brain the online version, at doi:10.1016/j.ydbio.2009.06.001. leads to obesity and hyperactivity. Mol. Endocrinol. 15, 1748–1757. Soriano, P., 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71. References Spassky, N., Heydon, K., Mangatal, A., Jankovski, A., Olivier, C., Queraud-Lesaux, F., Goujet-Zalc, C., Thomas, J.L., Zalc, B., 2001. Sonic hedgehog-dependent emergence Androutsellis-Theotokis, A., Leker, R.R., Soldner, F., Hoeppner, D.J., Ravin, R., Poser, S.W., of oligodendrocytes in the telencephalon: evidence for a source of oligodendrocytes Rueger, M.A., Bae, S.K., Kittappa, R., McKay, R.D., 2006. Notch signalling regulates in the olfactory bulb that is independent of PDGFRalpha signaling. Development stem cell numbers in vitro and in vivo. Nature 442, 823–826. 128, 4993–5004. Barolo, S., Walker, R.G., Polyanovsky, A.D., Freschi, G., Keil, T., Posakony, J.W., 2000. A Suzuki, S.O., Goldman, J.E., 2003. Multiple cell populations in the early postnatal notch-independent activity of suppressor of hairless is required for normal subventricular zone take distinct migratory pathways: a dynamic study of glial and mechanoreceptor physiology. Cell 103, 957–969. neuronal progenitor migration. J. Neurosci. 23, 4240–4250. Buffo, A., Vosko, M.R., Erturk, D., Hamann, G.F., Jucker, M., Rowitch, D., Gotz, M., 2005. Tamura, K., Taniguchi, Y., Minoguchi, S., Sakai, T., Tun, T., Furukawa, T., Honjo, T., 1995. Expression pattern of the transcription factor Olig2 in response to brain injuries: Physical interaction between a novel domain of the receptor Notch and the implications for neuronal repair. Proc. Natl. Acad. Sci. U. S. A. 102, 18183–18188. transcription factor RBP-J kappa/Su(H). Curr. Biol. 5, 1416–1423. de la Pompa, J.L., Wakeham, A., Correia, K.M., Samper, E., Brown, S., Aguilera, R.J., Tanigaki, K., Nogaki, F., Takahashi, J., Tashiro, K., Kurooka, H., Honjo, T., 2001. Notch1 and Nakano, T., Honjo, T., Mak, T.W., Rossant, J., Conlon, R.A., 1997. Conservation of Notch3 instructively restrict bFGF-responsive multipotent neural progenitor cells the Notch signalling pathway in mammalian neurogenesis. Development 124, to an astroglial fate. Neuron 29, 45–55. 1139–1148. Tanigaki, K., Han, H., Yamamoto, N., Tashiro, K., Ikegawa, M., Kuroda, K., Suzuki, A., Doetsch, F., Caille, I., Lim, D.A., Garcia-Verdugo, J.M., Alvarez-Buylla, A., 1999. Nakano, T., Honjo, T., 2002. Notch-RBP-J signaling is involved in cell fate Subventricular zone astrocytes are neural stem cells in the adult mammalian determination of marginal zone B cells. Nat. Immunol. 3, 443–450. brain. Cell 97, 703–716. Tani, S., Kurooka, H., Aoki, T., Hashimoto, N., Honjo, T., 2001. The N- and C-terminal Genoud, S., Lappe-Siefke, C., Goebbels, S., Radtke, F., Aguet, M., Scherer, S.S., Suter, U., regions of RBP-J interact with the ankyrin repeats of Notch1 RAMIC to activate Nave, K.A., Mantei, N., 2002. Notch1 control of oligodendrocyte differentiation in transcription. Nucleic. Acids. Res. 29, 1373–1380. the spinal cord. J. Cell. Biol. 158, 709–718. Taylor, M.K., Yeager, K., Morrison, S.J., 2007. Physiological Notch signaling promotes Hack, M.A., Sugimori, M., Lundberg, C., Nakafuku, M., Gotz, M., 2004. Regionalization gliogenesis in the developing peripheral and central nervous systems. Development and fate specification in neurospheres: the role of Olig2 and Pax6. Mol. Cell. 134, 2435–2447. Neurosci. 25, 664–678. Tsuda, L., Kaido, M., Lim, Y.M., Kato, K., Aigaki, T., Hayashi, S., 2006. An NRSF/REST- Hack, M.A., Saghatelyan, A., de Chevigny, A., Pfeifer, A., Ashery-Padan, R., Lledo, P.M., like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Gotz, M., 2005. Neuronal fate determinants of adult olfactory bulb neurogenesis. Drosophila. EMBO J. 25, 3191–3202. Nat. Neurosci. 8, 865–872. Wang, S., Sdrulla, A.D., diSibio, G., Bush, G., Nofziger, D., Hicks, C., Weinmaster, G., Barres, Handler, M., Yang, X., Shen, J., 2000. Presenilin-1 regulates neuronal differentiation B.A., 1998. Notch receptor activation inhibits oligodendrocyte differentiation. during neurogenesis. Development 127, 2593–2606. Neuron 21, 63–75. Han, H., Tanigaki, K., Yamamoto, N., Kuroda, K., Yoshimoto, M., Nakahata, T., Ikuta, K., Yang, X., Tomita, T., Wines-Samuelson, M., Beglopoulos, V., Tansey, M.G., Kopan, R., Shen, Honjo, T., 2002. Inducible gene knockout of transcription factor recombination J., 2006. Notch1 signaling influences v2 interneuron and motor neuron develop- signal binding protein-J reveals its essential role in T versus B lineage decision. Int. ment in the spinal cord. Dev. Neurosci. 28, 102–117. Immunol. 14, 637–645. Zhou, Q., Anderson, D.J., 2002. The bHLH transcription factors OLIG2 and OLIG1 couple Hsieh, J.J., Hayward, S.D., 1995. Masking of the CBF1/RBPJ kappa transcriptional neuronal and glial subtype specification. Cell 109, 61–73. repression domain by Epstein–Barr virus EBNA2. Science 268, 560–563. Zhou, S., Hayward, S.D., 2001. Nuclear localization of CBF1 is regulated by interactions Kao, H.Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C.R., Evans, with the SMRT corepressor complex. Mol. Cell. Biol. 21, 6222–6232. R.M., Kadesch, T., 1998. A histone deacetylase corepressor complex regulates the Zinyk, D.L., Mercer, E.H., Harris, E., Anderson, D.J., Joyner, A.L., 1998. Fate mapping of the Notch signal transduction pathway. Genes Dev. 12, 2269–2277. mouse midbrain–hindbrain constriction using a site-specific recombination Koelzer, S., Klein, T., 2003. A Notch-independent function of Suppressor of Hairless system. Curr. Biol. 8, 665–668.