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Previous Article | Next Article
The Journal of Neuroscience, October 15, 2001, 21(20):8091-8107
RNA-Binding Protein Musashi2: Developmentally Regulated Expression in Neural Precursor Cells and Subpopulations of Neurons in Mammalian CNS
Shin-ichi Sakakibara1, 2, Yuki Nakamura1, 3, Hitoshi Satoh4, and Hideyuki Okano1, 2, 3 1 Division of Neuroanatomy, Department of Neuroscience, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan, 2 Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan, 3 Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan, and 4 Division of Pathology, Department of Cancer Research, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Musashi1 (Msi1) is a mammalian neural RNA-binding protein highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic and postnatal CNS development. Here, we identified Musashi2 (Msi2), a novel mammalian RNA-binding protein that exhibits high sequence similarity to Msi1. The Msi2 transcript appeared to be distributed ubiquitously in a wide variety of tissues, consistent with the mRNA distribution of its Xenopus homolog, xrp1. However, the present study revealed cell type-specific and developmentally regulated expression of Msi2 in the mammalian CNS. Interestingly, Msi2 was expressed prominently in precursor cells in the ventricular zone and subventricular zone with the same pattern as Msi1 throughout CNS development. In the postnatal and adult CNS, this concurrent expression of Msi2 and Msi1 was seen in cells of the astrocyte lineage, including ependymal cells, a possible source for postnatal CNS stem cells. During neurogenesis, the expression of both Msi2 and Msi1 was lost in most postmitotic neurons, whereas Msi2 expression persisted in a subset of neuronal lineage cells, such as parvalbumin-containing GABA neurons in the neocortex and neurons in several nuclei of the basal ganglia. Msi2 may have a unique role that is required for the generation and/or maintenance of specific neuronal lineages. Furthermore, in vitro studies showed that Msi2 and Msi1 have similar RNA-binding specificity. These two RNA-binding proteins may exert common functions in neural precursor cells by regulating gene expression at the post-transcriptional level.
Key words: RNA-binding protein; Musashi2; Musashi1; CNS development; neural precursor cells; CNS stem cell; astrocyte; parvalbumin; GABAergic neuron
INTRODUCTION
A number of transcription factors that function in the proliferation and differentiation of neural precursor cells have been identified. However, the recent discovery of neural-specific RNA-binding proteins raises the possibility that the development of neural cells from the precursors may also be regulated at the post-transcriptional level, including by mRNA stabilization or translational control. We believe that two gene families are represented by the neural RNA-binding proteins found in both invertebrates and vertebrates (Okano, 1995
). One, the Elav family, possesses three sets of ribonucleoprotein (RNP)-type RNA recognition motifs (RRMs) and includes the Drosophila elav gene (Yao et al., 1993
During mammalian CNS development, neurons and glial cells are thought to be generated from common neural precursor cells (CNS stem cells) in the embryonic ventricular zone (VZ) (Davis and Temple, 1994
In the present study we identified Msi2, a novel member of the Msi family. Given their remarkable similarities in primary structure, RNA binding specificity, and expression profile during CNS development, Msi1 and Msi2 may play similar roles in neural precursor cells via post-transcriptional regulation.
MATERIALS AND METHODS
Cloning of mouse msi2 cDNA. A
gt 11 cDNA library obtained from adult mouse cerebellum (Sakakibara et al., 1996
Animals and tissue preparation. ICR (CD-1) mice, used for the preparation of tissue protein extracts, RNA, or tissue sections, were obtained from Charles River Japan Inc. (Kanagawa, Japan). The date of conception was established by the presence of a vaginal plug and recorded as E0, and the day of birth was designated as postnatal day (P) 0.
Northern blot analysis. Total RNA (20 µg) from each of the mouse tissues and embryos was isolated using Trizol (Life Technologies-BRL, Grand Island, NY) according to the manufacturer's instructions, fractionated by electrophoresis on 1% agarose-formaldehyde gels, and transferred onto Biodyne B nylon membranes (Pall, Port Washington, NY). 32P-labeled probe was prepared using a random-primed DNA labeling kit (Roche Diagnostics, Mannheim, Germany) of a 750 bp 3'-UTR fragment of the mouse msi2 cDNA and hybridized in 50% formamide, 6× SSPE, 5× Denhardt's solution, 0.5% SDS, and 200 µg/ml salmon sperm DNA at 42°C for 16 hr. The incubation was followed by stringent washes in 0.1× SSC, 0.1% SDS at 50°C, and filters were exposed for 48 hr on Kodak X-OMAT film (Kodak, Rochester, NY). The integrity of the transferred RNA samples was verified by reprobing each blot with a radiolabeled
-actin cDNA fragment (Clontech, Palo Alto, CA).
Production and purification of anti-Msi2 antibodies. A peptide corresponding to the 14 amino acid terminal sequence of the Msi2 protein (MEANGSPGTSGSAN) followed by a cysteinamide residue was synthesized for the purpose of antibody production. This peptide sequence does not overlap with that of the RNA-binding domain, RRM1 (see Fig. 1) and does not show any similarities to the corresponding N-terminal region of the Msi1 protein. Approximately 15 mg of the peptide was coupled through the cysteinamide residue to m-bromosuccinimide-treated keyhole limpet hemocyanin and used to immunize New Zealand White rabbits. For the affinity purification of the anti-Msi2 antisera, the synthesized peptide (5 mg) was coupled covalently to activated 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP) Cellulofine (Seikagaku Kogyo, Tokyo, Japan), according to the manufacturer's instructions. Ten milliliters of the filtered (0.45 µm) whole antisera were incubated with 3 ml of the peptide-FMP Cellulofine affinity resin, pre-equilibrated with TBS buffer (0.15 M NaCl, 20 mM Tris-HCl, pH 7.5) overnight at 4°C. The resin was then washed with 50 ml of 1 M NaCl, 1% Triton X-100, 20 mM Tris-HCl, pH 7.5, followed by 20 ml of 0.15 M NaCl, eluted with 4 ml of 100 mM glycine-HCl, pH 2.0, at 4°C, and immediately neutralized with 0.2 ml of 1 M Tris.
Recombinant Msi1 and Msi2 proteins. The 983 bp and 1072 bp BamHI-EcoRI fragments corresponding to the short- and long-form ORFs of the Msi2 protein were isolated by RT-PCR of the RNAs of E12 embryonic and adult brains and subcloned in-frame into the expression vector pRSET-A (Invitrogen, Carlsbad, CA) to construct pRSET-Msi2S (short form) and pRSET-Msi2L (long form) to generate fusion proteins containing six histidine residues at their amino termini. Expression vectors pRSET-Msi1 (Sakakibara et al., 1996
Protein phosphatase treatment and immunoblotting. Tissue lysate was prepared by homogenization in Buffer A [50 mM Tris-HCl, pH 7.6, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM dithiothreitol (DTT), 100 µg/ml phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml aprotinin, 5 µg/ml leupeptin], followed by centrifugation at 10,000 × g for 10 min. Bacterially expressed, purified recombinant proteins (50 ng per lane) or tissue lysate (30 µg of protein per lane) were resolved on 10% SDS-PAGE gels that were then electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA) with a semidry transfer apparatus. Equal loading of total protein from each tissue was determined by standard Bradford assay and verified by Coomassie blue staining of a duplicate gel. The chemiluminescent signals were detected by ECL (Amersham Pharmacia Biotech) with Kodak X-OMAT film according to the manufacturer's instructions. For the protein dephosphorylation assay, endogenous Msi2 protein was partially purified from embryonic brain lysate (E12.5). E12.5 brains (1.0 gm of wet weight) were homogenized in 5 ml of Buffer A and spun to pellet the nuclei (12,000 × g, 10 min, 4°C). The supernatant was removed and layered gently over a 1.5 ml sucrose (30% w/v in Buffer A) cushion and spun at 130,000 × g, 4°C for 2 hr, in a Beckman SW55Ti rotor. After removal of the S130 supernatant and sucrose cushion, the pelleted polysome fraction was rinsed and resuspended in 500 µl of Buffer A. A significant amount of Msi2 protein was recovered in this polysome fraction. For protein phosphatase treatment, 10 µg of protein from the cleared polysome fraction was incubated with 800 U of recombinant
In vitro transcription/translation and RNA-binding assays. The cDNA fragments corresponding to the coding region of the Msi2 long form (nucleotides 524-1564; GenBank accession no. AB056103) and Msi1 (nucleotides 64-1152; GenBank accession no. D49654) were isolated by PCR, using primers encoding a FLAG tag at the amino termini, and subcloned into pCDNA3 (Invitrogen) to construct the expression vectors pCDNA-msi2 and pCDNA-msi1. These plasmids were transcribed and translated in rabbit reticulocyte lysate (TNT T7 quick-coupled transcription/translation system; Promega, Madison, WI) in the presence of 0.4 mCi/ml of 35S-methionine (Amersham Pharmacia Biotech), according to the manufacturer's suggested conditions. Luciferase T7 control vector (Promega), which encodes a 61 kDa Luciferase protein, was also translated in vitro as described above. Binding of in vitro-translated proteins to RNA-homopolymers was performed essentially as described previously (Swanson and Dreyfuss, 1988
Immunohistochemistry. Pups and 8-week-old adults (anesthetized by ether inhalation) were perfused through the left ventricle with 4% paraformaldehyde (PFA) in 0.1 M PBS, pH 7.4. The embryos were removed by cesarean section and immersed in the same fixative. Brains and other tissues were dissected and post-fixed overnight at 4°C, cryoprotected in 30% sucrose in PBS overnight at 4°C, and embedded in O.C.T. compound (Tissue Tek; Miles, Elkhart, IN). Cryostat sections (12 µm) were cut and affixed to 3-aminopropyltriethoxysilane-coated glass slides (Matsunami Glass, Osaka, Japan). Sections were permeabilized in 0.4% Triton X-100 in PBS for 30 min, followed by blocking with 10% normal serum of the species in which the secondary antibody was raised, 1% skim milk, and 0.1% Triton X-100 in PBS for 1 hr at room temperature; subsequently, the sections were incubated in a mixture of primary antibodies overnight at 4°C. The mixture always included the affinity-purified rabbit anti-Msi2 polyclonal antibody, diluted 1:300 in 10% normal serum and 0.1% Triton X-100 in PBS, and one of the following antibodies: anti-Msi1 (14H1, rat monoclonal IgG2b, ascites) (Kaneko et al., 2000
Cell culture. Neurosphere cultures were prepared as described previously (Nakamura et al., 2000
Surgical procedures for brain lesions and bromodeoxyuridine incorporation. Brain injury experiments were performed as described previously (Sakakibara and Okano, 1997
Chromosomal mapping of mouse msi1 and msi2. Mouse 129 sv/J genomic DNA fragments of mouse msi1 (11.5 kb) and mouse msi2 (11 kb), each containing the ATG translation initiation codon, were subcloned into the pBluescriptII (Stratagene) and pZErO-1 (Invitrogen) plasmid vectors, respectively, and used for fluorescence in situ hybridization (FISH) as described previously (Satoh et al., 1993
GenBank accession numbers. The nucleotide sequence data reported in this paper have been deposited with the GSDB, DDBJ, EMBL, and NCBI under accession numbers AB056102 for msi2S and AB056103 for msi2L.
RESULTS
Identification of msi2 and characterization of its primary structure
To isolate the cDNA of a novel mammalian RNA-binding protein that was homologous to Msi1, a mouse nervous system cDNA library was screened using mouse msi1 and Xenopus xrp1 cDNA probes at reduced stringency. The Xenopus xrp1 gene (Good et al., 1993
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Figure 1. Primary structure and chromosomal localization of Msi2 and Msi1. A, The deduced amino acid sequence of mouse Msi2 protein. The two RNA recognition motifs are underlined. Each RRM includes two highly conserved sequences designated RNP-1 and RNP-2 (boxed sequences). The broken underlines indicate the regions that are missing in the form arising from alternative mRNA splicing. Asterisks denote the peptide sequence used as an immunogen to generate anti-Msi2 antibody. B, Domain structures of the mouse Msi2 and Msi1 proteins. The percentages of amino acid identity between Msi1 and Msi2 within each RRM and in the C-terminal half are shown. Bulged small boxes represent the regions arising from alternative splicing. C, Amino acid sequence alignment of mouse Msi2 with its known homologs, Xenopus RNA-binding protein (XRP1), and mouse Msi1. The two RRMs are boxed, and gaps in the alignment are indicated by dashes. Amino acid residues conserved in more than two proteins are shaded. D, Dendrogram showing a multiple sequence comparison of various mammalian RRM-containing RNA-binding proteins. Sequences derived from selected proteins were compared using the clustalW program on the DDBJ www server. The branch lengths are proportional to the differences between sequences. The accession numbers of each protein are given in Materials and Methods. E, Chromosomal localization of the mouse msi1 and msi2 genes by FISH. The left panels show metaphase chromosomes stained with DAPI for the identification of individual mouse chromosomes. Chromosomes 11 and 5 are numbered. The right panels represent the same metaphase chromosomes with the hybridization signals from the msi2 (top panels) and msi1 (bottom panels) genes (arrows indicate each signal). The closed bars on the right side of each ideogram indicate the possible location of the msi2 and msi1 genes.
Many RNA-binding proteins contain a conserved sequence of 80-90 amino acids, termed an RRM, that includes two short, highly conserved core domains called RNP-1 and RNP-2 (Burd and Dreyfuss, 1994
The relationship of the Msi2 and Msi1 proteins to other mammalian RRM-containing proteins was demonstrated by the pairwise comparisons presented in Figure 1D. Msi2 and Msi1 formed a unique subgroup within the hnRNP A/B class of proteins, the heterogeneous nuclear ribonucleoproteins that contain two copies of RRMs in addition to auxiliary domains. Among this class, hnRNP type A/B protein (Khan et al., 1991
To determine whether msi2 mRNA is expressed in a tissue-specific manner, we performed Northern blotting of the RNA from various tissues from adult mice, probing with a 750 bp 3'-UTR fragment of mouse msi2 cDNA (Fig. 2A). Substantial levels of a major msi2 transcript of 7.1 kb was detected in all tissues tested, with the highest expression observed in the cerebellum and smaller amounts in the liver and small intestine. A 1.5 kb short form of the msi2 transcript was detected only in the testis. Northern blots of RNA from different developmental stages indicated the continuous expression of the msi2 transcript in the CNS throughout development (Fig. 2B). This tissue distribution and developmental profile of the msi2 transcripts paralleled the distribution profile reported for Xenopus xrp1 mRNA (Good et al., 1993
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Figure 2. Tissue distribution and developmental expression of the msi2 transcripts. Total RNA (20 µg) from each mouse tissue was prepared, and the levels of msi2 mRNA were determined by Northern blot analysis. A, Tissue distribution of msi2 mRNA in adults. Expression of a 7.0 kb transcript can be seen in most lanes. A short transcript of ~1.5 kb is observed only in the testis. B, Changes in msi2 mRNA expression in the whole brain associated with development. Msi2 mRNA expression can be seen at E10, the earliest time point tested. The equal loading and quality of the RNAs were tested by performing the hybridization with a
On the basis of the striking similarity of both the predicted protein sequence and the expression pattern, we concluded that the msi2 gene was a mouse homolog of Xenopus xrp1. Msi1 and Msi2 seem to constitute a novel RNA-binding protein family (Msi family) in rodents, although they show tissue distributions that are different from each other.
Chromosomal localization of the msi1 and msi2 genes
Genomic Southern blot analysis using probes from msi1 and msi2 genomic fragments yielded the predicted single bands for several appropriate restriction enzymes, indicating that both are likely to be single-copy genes. The cytogenetic locations of the msi1 and msi2 genes were determined by FISH. As shown in Figure 1E, the msi1 and msi2 genes were mapped to chromosome 5qE3-F and 11qB5-C, respectively, by DAPI bands. Our previous FISH and somatic cell hybrid panel analyses (Good et al., 1998
RNA-binding properties of Msi2 protein
To test whether Msi2 could actually bind RNA, and if so, whether Msi1 and Msi2 recognize common RNA targets, we performed RNA-binding analysis in vitro. The full-length Msi2 and Msi1 proteins were prepared by in vitro transcription/translation, and their ability to bind RNA-homopolymers was determined. Both the Msi2 and Msi1 proteins showed strong binding to poly(U), weaker binding to poly(G), and no binding to poly(A) or poly(C), under normal stringency conditions in a buffer containing 100 mM NaCl (Fig. 3A). A 61 kDa luciferase polypeptide showed no binding to any of the homopolymers, confirming the RNA-binding activity of the Msi2 and Msi1 proteins. Furthermore, binding experiments performed under various degrees of stringency, up to 1.0 M NaCl, revealed that the Msi1 and Msi2 proteins both bound poly(U) and poly(G). The binding to poly(U) was stable in 250 mM NaCl, although the binding to poly(G) was only seen at 100 mM NaCl (Fig. 3B). The binding properties of Msi1 observed in this study were slightly different from our previous report, in which we found, using a Northwestern assay, that Msi1 binds poly(G) more efficiently than poly(U) (Sakakibara et al., 1996
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Figure 3. Similar RNA-binding properties of Msi2 and Msi1 proteins. A, Binding of the full-length Msi2 and Msi1 proteins to ribonucleotide homopolymers. 35S-methionine-labeled, in vitro-translated Msi2, Msi1, and Luciferase (as a control protein) were incubated with the indicated ribonucleotide homopolymer beads in buffer containing 100 mM NaCl. Bound proteins were visualized by SDS-PAGE and fluorography as described in Materials and Methods. Ribonucleotide homopolymers and proteins used in the assays are indicated at the top of each lane. Lanes marked Input contain each 35S-labeled protein used for binding reactions. The molecular sizes of markers are indicated on the left. Both Msi2 and Msi1 bound strongly to poly(U) and weakly to poly(G). Luciferase showed no binding to RNA homopolymers. B, Binding profile of Msi2 and Msi1 to RNA homopolymers under stringent conditions. Binding experiments of in vitro-translated Msi2 and Msi1 with poly(U) and poly(G) beads were performed as described above at the indicated NaCl concentrations. In the buffer containing 0.25 M NaCl, Msi2 and Msi1 binding to poly(G) RNA was greatly diminished, whereas that to poly(U) remained significant. Note that the binding properties of Msi2 to poly(U) and poly(G) in the given salt concentrations were almost identical to those of Msi1.
Specificity of the Msi2 antibody
To localize the Msi2 protein in tissues, polyclonal antiserum (anti-Msi2) was generated in rabbits using a peptide corresponding to the amino-terminal 14 amino acids of mouse Msi2, in which no obvious homology to Msi1 could be seen (Fig. 1A,C). As shown in Figure 4A, the anti-Msi2 antiserum recognized both recombinant full-length Msi2L and Msi2S proteins but did not cross-react with Msi1 protein. Conversely, the anti-Msi1 monoclonal antibody (14H1) (Kaneko et al., 2000
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Figure 4. Western blot analysis and cytoplasmic localization of Msi1 and Msi2 proteins. A, Specificity of the anti-Msi2 and anti-Msi1 antibodies. Bacterially expressed full-length Msi2S (lane 1), Msi2L (lane2), and Msi1 (lane 3) proteins were subjected to immunoblotting. Membranes were probed with either affinity-purified rabbit polyclonal anti-Msi2 or rat monoclonal anti-Msi1 (14H1) antibodies. The anti-Msi2 antibody specifically detected Msi2L and Msi2S and did not cross-react with Msi1 protein. Conversely, the anti-Msi1 antibody recognized only the Msi1 protein. The left panel shows the blot stained with Amido black dye, confirming the integrity and equal loading of each fusion protein. Lane M indicates protein size markers. B, Changes in Msi2 and Msi1 protein expression during brain development. A Western blot of protein extracts from E12 and P3 brains (30 µg protein per lane) was probed with anti-Msi1 or anti-Msi2 antibodies. The anti-Msi1 antibody recognized a doublet at 37-40 kDa (arrows). Msi2 was detected as two major proteins of 35 and 37 kDa (arrows), and each protein consisted of a closely spaced doublet. C, Msi2 is a phosphoprotein. Western blot analysis for Msi2 in the polysome fraction from E12.5 brain was treated with (+) or without ( ) protein phosphatase. Incubation with protein phosphatase produced a downward shift of the slower migrating form in each doublet. D, Confocal images of the neural cell line NG108 stained with anti-Msi1 (a) or anti-Msi2 (c), showing their cytoplasmic localization. b, d, The same fields, counterstained with Hoechst33342 dye to identify the individual nuclei, are shown. Scale bar (shown in d), 8 µm.
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Figure 5. Msi2 expression in neural precursor cells residing in the embryonic VZ and postnatal SVZ. Double-immunofluorescence labeling of coronal sections through embryonic, postnatal, and adult brains, with antibodies against Msi2 and Msi1, Ki67, Nestin, or MAP2. A/B, C/D, E/F, G/H, I/J, K/L, M/N, and O/P represent each pair of double-stained photomicrographs. The primary antibodies used are indicated in each panel. A-F, Telencephalon at E10. Msi2 and Msi1 are uniformly distributed in the Ki67- and Nestin-positive neuroepithelium. G/H, Cerebral cortex at E12. Insets indicate higher magnifications of the VZ cells. I-L, Cerebral cortex at E14. Coincidental expression of Msi2 and Msi1 is seen in the VZ of the telencephalon and ganglionic eminences. Note the absence of Msi2 expression in the intermediate zone and cortical plate, which are occupied by MAP2-positive immature neurons. M/N, The SVZ region surrounding the lateral ventricle in P7 postnatal forebrain. O/P, Adult ependyma and SVZ lining the lateral ventricle. Lateral is to the right, and dorsal is up. Many densely packed cells that are brightly immunostained with both Msi2 and Msi1 are observed in the P7 SVZ, adult SVZ, and ependymal layer. Small scattered astrocytes in the parenchyma are also labeled with anti-Msi2 and anti-Msi1 antibodies. The arrow in O/P indicates a Msi2-positive but Msi1-negative neuron-like cell. Scale bars: A-H, K, L, 50 µm; I, J, M, N, 90 µm; O, P, 25 µm; insets in G and H, 8 µm. Asterisks indicate lateral ventricle. cp, Cortical plate; vz, ventricular zone; lge, lateral ganglionic eminence; cc, corpus callosum; str, striatum.
Subcellular distribution of the Msi2 and Msi1 proteins
The Msi2 and Msi1 proteins were localized exclusively to the cytoplasm of several neural cell lines, including NG108 (Fig. 4D) and PC12 (data not shown) cells. Cytoplasmic expression of Msi2 and Msi1 was also observed in E10 neuroepithelial cells (Figs. 5G,H, insets, 6C). In addition, Msi2 was present in the cytoplasm of cells in non-neural tissues (data not shown) and in differentiated neurons, in which Msi2 protein was found in proximal regions of the somata rather than in the dendritic arborizations or axons (see Figs. 8G, 9G). During subcellular fractionation of E12.5 brain lysate, a significant amount of Msi2 was recovered in the polysome fraction (Fig. 4C). Consistently, our observations by subcellular fractionation (Imai et al., 2001
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Figure 6. Msi2 expression in the spinal cord. Double-immunofluorescence labeling of horizontal sections through the embryonic and adult lumbar spinal cord, with antibodies against Msi2 and Msi1, MAP2, or Hu. A/B, C/D, E/F, G/H, I/J, K/L, M/N, and O/P represent each pair of double-stained photomicrographs. The primary antibodies used are indicated in each panel. A/B, E10 spinal cord. Msi2 and Msi1 are uniformly distributed within the neural tube. C/D, Higher magnification of the ventricular zone of E10 spinal cord, showing the Msi2 expression in the Ki67-positive proliferating neuroepithelial cells. E/F, E12 spinal cord. G-L, E14 spinal cord. K/L, Higher magnification of the bracketed area in I/J. Msi2 and Msi1 are simultaneously expressed in the embryonic VZ lining the central canal. Outside the VZ, most of the MAP2-positive postmitotic neurons do not show immunoreactivity for Msi2, whereas a subset of migrating neurons that are sparsely distributed in the ventrolateral area of the mantle layer coexpress Msi2 and MAP2 (bracketed area in G). These Msi2-positive cells are negative for Msi1 and have a few processes, indicating that they are migrating out of the VZ (bracketed portion in I/J, and arrows in K/L). M/N, Adult spinal cord. O/P, Higher magnification of M/N. In the adult spinal cord, Msi2 and Msi1 are highly expressed in ependymal cells surrounding the central canal. Note the Msi2 expression in some, but not all, populations of neurons scattered within the parenchyma. Arrowheads in O and P indicate Hu-positive but Msi2-negative neurons. cc, Central canal. Scale bars: A, B, 100 µm; C, D, 12 µm; E-H, 50 µm; I, J, M, N, 45 µm; K, L, O, P, 10 µm.
Developmental expression of Msi2 in the CNS
Although the Msi1 protein was highly enriched in the embryonic CNS, the Msi2 protein level appeared to stay at a relatively constant level during brain development (Fig. 4B) in parallel with its mRNA levels (Fig. 2B). However, the following immunohistochemical results clearly demonstrated that the Msi2 expression was not ubiquitous, at least in various regions of the embryonic and postnatal CNS. Rather, Msi2 was expressed in a cell type-specific manner, and its expression appeared to be strongly regulated spatially and temporally during the development of the mouse brain and spinal cord. Notably, in the developing CNS, Msi2 was mainly detected in proliferating precursor cells in the VZ and SVZ with a pattern equivalent to that of Msi1.
VZ and SVZ
First, we examined the sites of Msi2 expression in the CNS at the time when active neurogenesis and cell differentiation are known to occur. The neural tube of the E10 mouse embryo is uniformly occupied with mitotic and undifferentiated neuroepithelial cells. At this stage, Msi2 expression was intense throughout the telencephalon and spinal cord (Figs. 5, 6). Most of these Msi2-positive cells were Msi1-positive (Figs. 5A,B, 6A,B), Ki67-positive (Figs. 5C,D, 6C,D), and nestin-positive (Fig. 5E,F) neuroepithelial cells. Ki67 is a marker for proliferating cells (Nakamura et al., 2000
To address whether Msi2 expression is restricted to the neural precursor cells/CNS stem cells in vivo, we double-stained the sections through the SVZ regions with the antibodies to GFAP, Hu, TuJ1, or PDGFR-
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Figure 7. Msi2 expression in neurospheres and absence of Msi2 in the immature neurons or oligodendrocyte precursor cells lying in the SVZ. A-C, Double immunostaining of neurosphere from E14.5 embryonic telencephalon, showing the coexpression of Msi2 (B, red) and Msi1 (C, green). Nuclei are counterstained with Hoechst dye (A, blue). D, Msi2 expression (red) in Ki67-positive proliferating cells (green) in a neurosphere. Inset shows the higher view of the neurosphere. Arrows point to the Msi2 and Ki67 double-positive cells. Note that Msi2 is mainly localized in the cytoplasm of the cells in neurosphere, whereas the antibody to Ki67 only stains the nuclei. E-G, Scanning confocal images of the coronal sections through the SVZ surrounding the lateral ventricle of the P2 mouse forebrain. E, GFAP (green) and Msi2 (red). Msi2 is expressed in the GFAP-negative densely packed SVZ cells. F, Hu (red) and Msi2 (green). Hu-positive immature neurons and Msi2-positive cells distribute intermingled within the dorsolateral area of the SVZ, but these two populations of cells hardly overlap with each other. G, PDGFR- (red) and Msi2 (green). Msi2 expression is never found in oligodendrocyte precursor cells that are positive for PDGFR-
Cerebrum
Neurons and glial cells, which originate in the embryonic VZ or the postnatal SVZ, respectively, migrate into appropriate layers through formative white matter, including the corpus callosum, to form the stratified adult cerebral cortex. Msi1 is known to be expressed in cells of the astrocyte lineage, which include both migrating GFAP-negative (protoplasmic) and GFAP-positive differentiated astrocytes (Miller and Raff, 1984
On the other hand, the Msi2 protein was also expressed in a population of large, round cells, distinct from the small, Msi1-positive astrocytes (Fig. 8A,B, arrows). These Msi2-positive cells were found predominantly within layers II-VI and overlapped with Hu immunoreactivity, indicating that they were neurons (Fig. 8C,D, arrows, G,H, asterisk). However, a comparison of the Hu and Msi2 expression clearly revealed that the vast majority of Hu-positive neurons were negative for Msi2 (Fig. 8C,D, and arrows in G,H). Consistent with the expression of Msi2 in a subset of neurons in the cerebrum, Msi2-positive neuron-like cells were seen in the embryonic spinal cord (bracketed area in Fig. 6G, H, arrows in Fig. 6K, L) and adult spinal cord (Fig. 6O,P) and striatum (Fig. 5O,P), as mentioned above. These data indicated that Msi2 expression was retained in a subset of cells of the neuronal lineage, accompanied by its expression in the astrocytes. Msi2 expression appeared to be absent or primarily downregulated by the very early stage of the oligodendrocyte lineage, as is the case for Msi1 (Sakakibara and Okano, 1997
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Figure 8. Distribution and cell type of Msi2-positive cells in adult cerebrum. Double immunofluorescence labeling of the somatosensory area of the adult neocortex, with antibodies against Msi2 and Msi1, or cell type-specific markers. A/B, C/D, E/F, G/H, I/J, and K/L represent each pair of double-stained photomicrographs. Primary antibodies used are indicated in each panel. A-D showing the expression of Msi2 in astrocytes scattered throughout the gray (layers II-III) and white matter (layer I) of the neocortex. Many small Msi2- and Msi1-positive astrocytes with small cell bodies are seen throughout the cortex (A, B). There is also a subset of neurons labeled with Msi2 (A/B, arrows) with larger cell bodies, which are found sparsely in layers II-III, and coexpress Hu antigens (C/D, arrows) but are never labeled with Msi1 (compare A/B with C/D). The pial surface is at the top. E-L, High-power photomicrographs of Msi2-expressing cells in the gray matter (layers II-III, E-H) and white matter (molecular layer, I/J; subcortical white matter, K/L). In the gray matter, concurrent expression of Msi2 and Hu is observed in a few round neuronal cells (asterisks in G/H), whereas numerous other Hu-positive neurons lack immunoreactivity for Msi2 (arrows in G/H). There is also a considerable number of Hu-negative but Msi2-positive cells, which have small oval cell bodies with elaborated multiple short processes (arrowhead in G/H). These cells may represent the population of GFAP-negative protoplasmic astrocytes that are frequently observed in deep gray matter and are positive for Msi1 (E/F). Fibrous astrocytes in the superficial molecular layer and near the pial surface show colocalization of Msi2 and GFAP (I/J), whereas CNPase-positive oligodendroglial cell bodies predominant in the subcortical white matter are Msi2 negative (arrows in K/L). Scale bars: A-D, 25 µm; E-L, 8 µm.
Msi2 expression in a subpopulation of GABA neurons
To determine the neuronal cell types that express Msi2 in the adult cerebral cortex, we performed double immunofluorescence staining with several markers for cholinergic (ChAT), dopaminergic (TH), and GABAergic neurons (parvalbumin, calbindin-D28K, GABA). The cerebral cortex of the adult mouse is composed of many types of neurons, including large pyramidal neurons within layers II, III, IV, and V and medium-sized nonpyramidal neurons distributed throughout the cortex. Two calcium-binding proteins, PV and CB, are known to be expressed in a small number of nonpyramidal neurons that contain GABA as a neurotransmitter (Hendry et al., 1989
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Figure 9. Msi2 expression in parvalbumin-positive GABAergic neurons in the adult neocortex. Double-immunofluorescence labeling with antibodies against Msi2 and parvalbumin (PV), calbindin-D28K (CB), GABA, choline acetyltransferase (ChAT), or tyrosine hydroxylase (TH) was performed on coronal sections of adult cerebral cortex, basal ganglia, and hippocampus. A/B, C/D, E/F, G/H, I/J, K/L, M/N, O/P, Q/R, and S/T represent each pair of double-stained photomicrographs. Primary antibodies used are indicated in each panel. A/B, Neocortex layers II-III, showing the coincidental expression of Msi2 and PV in GABAergic nonpyramidal neurons. Faint staining of Msi2 is also seen in the fine-branched processes and small cell bodies of astrocytes. C/D, Higher magnification of the Msi2- and PV-positive neurons in the deeper layer V. The somata of large pyramidal neurons surrounded by parvalbumin-positive multi-terminal endings express neither Msi2 nor PV (asterisks). E/F, Neocortex layers II-III stained with Msi2 and CB. Note that these two populations of neurons are intermingled but do not overlap with each other. Long arrows indicate Msi2-negative CB-positive neurons. Short arrows indicate the neurons that are Msi2 positive but CB negative. G/H, Higher magnification of neurons that express CB or Msi2 in layers II-III. I/J, K/L, Layers II-III stained with antibodies to Msi2 and GABA, showing the Msi2 expression in GABA-containing neurons. (I/J and arrowhead in K/L). Arrow in K/L points to a GABA-containing but Msi2-negative neuron. M/N, Gray matter of the neocortex, showing the absence of Msi2 expression from ChAT-positive cholinergic neurons (arrows). O/P, Hippocampal formation, showing the coexpression of Msi2 and PV in a small number of interneurons in the CA1 subfield. Numerous CA1 pyramidal neurons aligned in rows are not labeled with either Msi2 or PV. Q/R, S/T, Nuclei of the vertical limb of the diagonal band and the zona incerta, respectively. In the basal ganglia, Msi2 immunoreactivity was found in several ChAT-positive cholinergic neurons in the nuclei of diagonal bands (Ch2 and Ch3), in addition to the PV-positive neurons. Msi2 is also expressed in TH-positive dopaminergic neurons (A13) within the zona incerta in the subthalamic region. Scale bars: A, B, E, F, M-T, 25 µm; C, D, G, H, I-L, 8 µm. Rad, Stratum radiatum; Py, stratum pyramidale; Or, stratum oriens.
Cerebellum
A dynamic pattern of Msi2 expression was also observed in the developing cerebellum. In the cerebellar primordium at E14.5, Msi2 was expressed in the small tightly packed cells of the external granule cell layer (EGL) migrating out of the rhombic lip of the lateral recess and also in the VZ of the fourth ventricle (data not shown). During early postnatal development, the EGL covers the surface of the developing cerebellum and is composed exclusively of proliferating neuronal precursors. By P3, the expression of Msi2 could be detected in the EGL, although to a lesser degree than that of Msi1 (Fig. 10A,B). As neurogenesis progresses, two populations of cells appear in the EGL: cells proliferating in the upper portion of the EGL (EGLa) and cells undergoing the initial step of neuronal differentiation deeper in the EGL (EGLb) (Altman, 1972
The most striking difference between the Msi2 and Msi1 expression was seen in the neuronal population within the Purkinje cell layer (PCL) and deep cerebellar nuclei (Fig. 10A,B). In the developing and adult PCL, Msi2 expression was conspicuous in the large Purkinje neurons, which absolutely lacked Msi1 immunoreactivity (Fig. 10C,D,I,J, arrows). Msi2 expression in differentiated neurons was also observed within the deep cerebellar nuclei (Fig. 10B, asterisk) and in interneurons such as the stellate and basket cells residing in the molecular layer (Fig. 10D). We noticed, however, that Msi2 was not expressed in numerous granule neurons in the internal granule cell layer (IGL), which were positive for Hu (Fig. 10G). In the IGL, Msi2 was detectable only in Golgi interneurons (Fig. 10D, arrowhead). The sustained expression of Msi2 in the GABA neurons was also observed in the adult cerebellar cortex. Msi2 expression was confined to almost all the inhibitory GABA neurons, such as the stellate and basket cells in the molecular layer, the Purkinje cells, and the Golgi cells in the IGL (Fig. 10). Correspondingly, PV is known to be expressed in these cells (Celio, 1990
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Figure 10. Msi2 expression in the cerebellum. Double-immunofluorescence labeling of sagittal sections through the early postnatal and adult cerebellum, with antibodies against Msi2 and Msi1 or Hu. A/B, C/D, E/F, G/H, and I/J represent each pair of double-stained photomicrographs. Primary antibodies used are indicated in each panel. A/B, P3 cerebellum. Intense Msi1 staining is observed in neuronal precursor cells in the EGL (indicated by arrow), in addition to the cells in the PCL (arrowhead) and numerous glial precursor cells in the folia white matter. Msi2 expression is prominent in the PCL and is faint in EGL cells and glial precursor cells in the white matter. Deep cerebellar nuclei (asterisk) are labeled only with Msi2. E-H, P7 cerebellum. The EGL is toward the top part of the panels. At P7, a low level of Msi2 expression is seen in the EGL (E/F). In the PCL, Purkinje cells forming a single row show immunoreactivity for both Msi2 and Hu but not for Msi1, whereas many small cells residing in the vicinity of the Purkinje cells express both Msi2 and Msi1 (compare E/F with G/H). These cells are likely to be the developing Bergmann glia. Msi2 expression is absent from Hu-positive granule neurons in the IGL. C/D, Adult cerebellum. In adults, Msi2 is predominantly expressed in the Purkinje cells (arrows in C/D) and Bergmann glial cells, which have cell bodies located adjacent to the large cell bodies of Purkinje cells and extend their tangential processes into the ML. Arrowhead in C/D represents the Golgi neuron in the IGL. I/J, Higher magnifications of the adult PCL. Arrows in I/J indicate the Purkinje cells, and arrowhead in I/J points to the soma of a Bergmann glial cell. ML, Molecular layer; PCL, Purkinje cell layer; IGL, internal granule layer; EGL, external granule cell layer. Scale bars: A, B, 100 µm; C-H, 30 µm; I, J, 8 µm.
Other CNS regions
The persistent expression of Msi2 in certain subpopulations of neurons was also clearly observed in other brain regions. In the adult hippocampal formation, small, scattered Msi2-positive neurons were detected in the stratum pyramidale, stratum oriens, radiatum, and lacunosum-moleculare. A high-power view of the CA1 region showed intense expression of Msi2 in a small number of neurons along with diffuse expression in the astrocytes, which are uniformly distributed within this region (Fig. 9O,P). In the CA1 and CA3 subfields, ~20% of all the GABAergic neurons contain PV (Kosaka et al., 1987Changes in Msi2 expression after injury
A previous report (Sakakibara and Okano, 1997
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Figure 11. Induced expression of Msi2 and Msi1 in reactive astrocytes. Double-label fluorescence localization of Msi2 and Msi1, BrdU, or GFAP in the injured region of adult cerebral cortex immediately (day 0) (A, B) or 4 d (C-H) after lesioning. All photomicrographs correspond to cortex layers II-III, and the lesioned sites are toward the top in all panels. A, B, GFAP and Msi2 at day 0. The vicinity of the site of injury shows no immunoreactivity for GFAP. C, D, BrdU and Msi2 at 4 d after lesion. Msi2 is expressed in a population of BrdU-positive proliferating cells (arrows) that lie close to the injury site. E, F, Msi1 and Msi2 at 4 d after lesion. Simultaneous expression of Msi1 and Msi2 is evident in many enlarged reactive cells. G, H, GFAP and Msi2 at 4 d after lesion. Intense immunoreactivity of Msi2 is observed in an increased number of GFAP-positive reactive astrocytes; these cells exhibit enlarged, elongated cell bodies with multiple processes (arrows). Arrowhead indicates a rare Msi2-positive neuron-like cell that has an oval cell body and is immunonegative for GFAP. Scale bar (shown in H): A-H, 18 µm.
These observations demonstrated that Msi2 expression is induced at high levels in reactive astrocytes in the course of scar formation, with a profile similar to the expression of Msi1 (Sakakibara and Okano, 1997
DISCUSSION
Msi2 is an evolutionarily conserved RNA-binding protein found in different species of vertebrates (Xenopus, mouse, and human). On the basis of their striking sequence homology and similar RNA-binding preferences in vitro, Msi2 and Msi1 are likely to constitute a unique RNA-binding protein family (the Msi family). These two proteins are likely to play similar roles in the RNA metabolism that occurs in the cytoplasm, such as in the localization or stabilization of specific mRNAs, polyadenylation, or translational regulation (for review, see MacCarthy and Kollmus, 1995
Role of the Msi family in neural precursor cells/CNS stem cells
In the present study we demonstrated the cell type-specific and developmentally regulated expression of Msi2 in the mammalian CNS. In particular, proliferative cells in the embryonic VZ and SVZ showed a high level of Msi2 expression. As neurogenesis proceeded, Msi2 expression disappeared in most of the postmitotic or migrating neurons within the intermediate zone or cortical plate. Interestingly, our double-labeling results indicated that Msi2 expression was coincident with the expression of Msi1 in proliferative cells. Previous in vivo and in vitro analyses have shown that Msi1 is predominantly expressed in neural precursor cells/multipotent CNS stem cells (Sakakibara et al., 1996
In accordance with this idea, we have obtained results from preliminary in vitro experiments using neurospheres lacking expression of both the msi1 and msi2 genes that indicate that Msi1 and Msi2 are essential for the maintenance and/or proliferation of CNS stem cells (S. Sakakibara, unpublished results). The molecular basis for the function of the Msi family in CNS stem cells might be elucidated by identifying target mRNAs of the Msi1 and Msi2 proteins. We recently reported that Msi1 protein binds to the 3'-UTR of mouse numb mRNA, leading to repression of Numb expression (Imai et al., 2001
Msi2 in a subset of GABA neurons
During the development of the cerebral cortex, Msi2 expression is primarily downregulated in most of the cortical neurons except the GABA interneurons. Interneurons containing the GABA neurotransmitter are the major type of inhibitory neurons in the mammalian neocortex. These cells comprise 25% of all the cortical neurons, and exert powerful control over the local neuronal network by mediating both feedback and feedforward inhibition. Are there any significant differences between the developmental process of GABA interneurons and those of other types of neurons? It is well known that most differentiating cortical projection neurons migrate radially from the cortical VZ toward the pial surface and form the cortical plate, where they temporarily settle according to an inside-out gradient of positioning (Bayer and Altman, 1991
In adult neocortex, GABA interneurons are classified into two separate subpopulations: the CB-positive double-bouquet cells and the PV-positive chandelier and basket cells (Hendry et al., 1989
Intriguingly, the neuronal expression of Msi is not only a characteristic of vertebrates but occurs in invertebrates as well. In Drosophila, d-Msi is expressed in a small population of differentiated neurons, including the photoreceptor cells (Hirota et al., 1999
Msi2 and neurological diseases
Previous immunohistochemical studies have indicated a selective reduction in or functional impairment of PV-containing GABA neurons in the neocortex under certain pathological conditions, which include schizophrenia (Beasley and Reynolds, 1997
FOOTNOTES Received April 13, 2001; revised July 19, 2001; accepted July 27, 2001.
This work was supported by grants from the Japanese Ministry of Education, Japan Science and Technology Corporation (Core Research for Evolutional Science and Technology).
Correspondence should be addressed to Dr. Hideyuki Okano, Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: hidokano{at}sc.itc.keio.ac.jp.
S. Sakakibara's present address: Department of Histology and Neurobiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan.
REFERENCES
- Akamatsu W, Okano HJ, Osumi N, Inoue T, Nakamura S, Sakakibara S, Miura M, Matsuo N, Darnell RB, Okano H (1999) Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and peripheral nervous systems. Proc Natl Acad Sci USA 96:9885-9890
[Abstract/Free Full Text] .- Altman J (1972) Postnatal development of the cerebellar cortex in the rat. J Comp Neurol 145:353-398[Web of Science][Medline].
- Amano T, Hamprecht B, Kemper W (1974) High activity of choline acetyltransferase induced in neuroblastoma × glioma hybrid cells. Exp Cell Res 85:399-408[Web of Science][Medline].
- Barami K, Iversen K, Furneaux H, Goldman SA (1995) Early expression of Hu proteins by newly generated neurons of the adult avian forebrain. J Neurobiol 28:82-101[Web of Science][Medline].
- Bayer SA, Altman J (1991) In: Neocortical development. New York: Raven.
- Beasley CL, Reynolds GP (1997) Parvalbumin-immunoreactive neurons are reduced in the prefrontal cortex of schizophrenics. Schizophrenia Res 24:349-355[Web of Science][Medline].
- Berbel P, Marco P, Cerezo JR, DeFelipe J (1996) Distribution of parvalbumin immunoreactivity in the neocortex of hypothyroid adult rats. Neurosci Lett 204:65-68[Medline].
- Blaxall BC, Pellett AC, Wu SC, Pende A, Port JD (2000) Purification and characterization of
- Burd CG, Dreyfuss G (1994) Conserved structures and diversity of functions of RNA-binding proteins. Science 265:615-621
[Abstract/Free Full Text] .- Celio MR (1990) Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35:375-475[Web of Science][Medline].
- Davis AA, Temple S (1994) A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 372:263-266[Medline].
- DeFelipe J (1997) Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex. J Chem Neuroanat 14:1-19[Web of Science][Medline].
- DeMaria CT, Brewer G (1996) AUF1 binding affinity to A+U-rich elements correlates with rapid mRNA degradation. J Biol Chem 271:12179-12184
[Abstract/Free Full Text] .- Doetsch F, Caillé I, Lim DA, García-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703-716[Web of Science][Medline].
- Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in the adult human hippocampus. Nat Med 4:1313-1317[Web of Science][Medline].
- Fukuda T, Kosaka T (2000) The dual network of GABAergic interneurons linked by both chemical and electrical synapses: a possible infrastructure of the cerebral cortex. Neurosci Res 38:123-130[Web of Science][Medline].
- Gaiano N, Nye JS, Fishell G (2000) Radial glial identity is promoted by Notch1 signaling in the murine forebrain. Neuron 26:395-404[Web of Science][Medline].
- Good PJ, Rebbert ML, Dawid IB (1993) Three new members of the RNP protein family in Xenopus. Nucleic Acids Res 21:999-1006
[Abstract/Free Full Text] .- Good PJ, Yoda A, Sakakibara S, Yamamoto A, Imai T, Sawa H, Ikeuchi T, Tsuji S, Satoh H, Okano H (1998) The human Musashi homolog 1 (MSI1) gene encoding the homologue of Musashi/Nrp-1, a neural RNA-binding protein putatively expressed in CNS stem cells and neural progenitor cells. Genomics 52:382-384[Web of Science][Medline].
- Hendry SHC, Jones EG, Emson PC, Lawson DEM, Heizmann CW, Streit P (1989) Two classes of cortical GABA neurons defined by differential calcium protein immunoreactivities. Exp Brain Res 76:467-472[Web of Science][Medline].
- Hirota Y, Okabe M, Imai T, Kurusu M, Yamamoto A, Miyao S, Nakamura M, Sawamoto K, Okano H (1999) Musashi and Seven in absentia downregulate tramtrack through distinct mechanisms in Drosophila eye development. Mech Dev 87:93-101[Web of Science][Medline].
- Hutton M, Lendon CL, Rizzu P, Baker M, Froelich S, Houlden H, Pickering-Brown S, Chakraverty S, Isaacs A, Grover A, Hackett J, Adamson J, Lincoln S, Dickson D, Davies P, Petersen RC, Stevens M, de Graaff E, Wauters E, van Baren J (1998) Association of misssense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393:702-705[Medline].
- Imai T, Tokunaga A, Yoshida T, Hashimoto M, Mikoshiba K, Weinmaster G, Nakafuku M, Okano H (2001) The neural RNA-binding protein Musashi1 translationally regulates the m-numb gene expression by interacting with its mRNA. Mol Cell Biol 21:3888-3900
[Abstract/Free Full Text] .- Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisén J (1999) Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96:25-34[Web of Science][Medline].
- Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay RD (1996) Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes Dev 10:3129-3140
[Abstract/Free Full Text] .- Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, Ogawa Y, Toyama Y, Miyata T, Okano H (2000) Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev Neurosci 22:139-153[Web of Science][Medline].
- Kawaguchi Y, Katsumaru H, Kosaka T, Heizmann CW, Hama K (1987) Fast spiking cells in rat hippocampus (CA1 region) contain the calcium-binding protein parvalbumin. Brain Res 416:369-374[Web of Science][Medline].
- Kawashima T, Murakami AR, Ogasawara M, Tanaka KJ, Isoda R, Sasakura Y, Nishikata T, Okano H, Makabe KW (2000) Expression patterns of musashi homologues of the ascidians, Halocynthia roretzi and Ciona intestinalis. Dev Gene Evol 210:162-165[Web of Science][Medline].
- Khan F, Jaiswal AK, Szer W (1991) Cloning and sequence analysis of a human type A/B hnRNP protein. FEBS Lett 290:159-161[Web of Science][Medline].
- Kosaka T, Katsumaru H, Hama K, Wu J-Y, Heizmann CW (1987) GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus. Brain Res 419:119-130[Web of Science][Medline].
- Kuhar SG, Feng L, Vidan S, Ross ME, Hatten ME, Heinz N (1993) Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation. Development 117:97-104
[Abstract/Free Full Text] .- Leavitt BR, Hernit-Grant CS, Macklis JD (1999) Mature astrocytes transform into transitional radial glia within adult mouse neocortex that supports directed migration of transplanted immature neurons. Exp Neurol 157:43-57[Web of Science][Medline].
- Lendahl U, Zimmerman LB, McKay R (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585-595[Web of Science][Medline].
- Levison SW, Goldman JE (1993) Both oligodendrocytes and astrocytes develop from progenitors in the subventricular zone of postnatal rat forebrain. Neuron 10:201-212[Web of Science][Medline].
- Lewis PD (1968) A quantitative study of cell proliferation in the subependymal layer of the adult rat brain. Exp Neurol 20:203-207[Web of Science][Medline].
- Lois C, Alvarez-Buylla A (1993) Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc Natl Acad Sci USA 9:2073-2077.
- Luskin MB (1993) Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11:173-189[Web of Science][Medline].
- Lynch T, Sano M, Marder KS, Bell KL, Foster NL, Defendini RF, Sima AAF, Keohane C, Nygaard TG, Fahn S, Mayeux R, Rowland LP, Wilhelmsen KC (1994) Clinical characteristics of a family with chromosome 17-linked disinhibition-dementia-parkinsonism-amyotrophy complex. Neurology 44:1878-1884
[Abstract/Free Full Text] .- Malatesta P, Hartfuss E, Götz M (2000) Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage. Development 127:5253-5263[Abstract].
- Martina M, Vida I, Jonas P (2000) Distal initiation and active propagation of action potentials in interneuron dendrites. Science 287:295-300
[Abstract/Free Full Text] .- Marusich MF, Furneaux HM, Henion PD, Weston JA (1994) Hu neuronal proteins are expressed in proliferating neurogenic cells. J Neurobiol 25:143-155[Web of Science][Medline].
- McCarthy JE, Kollmus H (1995) Cytoplasmic mRNA-protein interactions in eukaryotic gene expression. Trends Biochem Sci 20:191-197[Web of Science][Medline].
- Miller RH, Raff MC (1984) Fibrous and protoplasmic astrocytes are biochemically and developmentally distinct. J Neurosci 4:585-592[Abstract].
- Miyake T, Okada M, Kitamura T (1992) Reactive proliferation of astrocytes studied by immunohistochemistry for proliferating cell nuclear antigen. Brain Res 590:300-302[Web of Science][Medline].
- Morshead CM, Reynolds BA, Craig CG, McBurney MW, Staines WA, Morassutti D, Weiss S, van der Kooy D (1994) Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13:1071-1082[Web of Science][Medline].
- Morshead CM, Craig CG, van der Kooy D (1998) In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain. Development 125:2251-2261[Abstract].
- Mukonoweshuro W, Griffiths PD, Blaser S (1999) Neurofibromatosis type 1: the role of neurology. Neuropediatrics 30:111-119[Medline].
- Munoz-Garcia D, Ludwin SK (1984) Classic and generalized variants of Pick's disease: a clinicopathological, ultrastructural, and immunocytochemical comparative study. Ann Neurol 16:467-480[Web of Science][Medline].
- Nakamura M, Okano H, Blendy J, Montell C (1994) Musashi, a neural RNA-binding protein required for Drosophila adult external sensory organ development. Neuron 13:67-81[Web of Science][Medline].
- Nakamura Y, Sakakibara S, Miyata T, Ogawa M, Shimazaki T, Weiss S, Kageyama R, Okano H (2000) The bHLH gene Hes1 as a repressor of the neuronal commitment of CNS stem cells. J Neurosci 20:283-293
[Abstract/Free Full Text] .- Nishiyama A, Lin XH, Giese N, Helden CH, Stallcup WB (1996) Co-localization of NG2 proteoglycan and PDGF
- Noctor SC, Flint AC, Weissman TA, Dammerman RS, Kriegstein AR (2001) Neurons derived from radial glial cells establish radial units in neocortex. Nature 409:714-720[Medline].
- Ohtsuka T, Ishibashi M, Gradwohl G, Nakanishi S, Guillemot F, Kageyama R (1999) Hes1 and Hes5 as Notch effectors in mammalian neuronal differentiation. EMBO J 18:2196-2207[Web of Science][Medline].
- Okabe M, Imai T, Kurusu M, Hiromi Y, Okano H (2001) Translational repression determines a neuronal potential in Drosophila asymmetric cell division. Nature 411:94-98[Medline].
- Okano H (1995) Two major mechanisms regulating cell-fate decisions in the developing nervous system. Dev Growth Differ 37:619-629[Web of Science].
- Okano HJ, Darnell RB (1997) A hierarchy of Hu RNA binding proteins in developing and adult neurons. J Neurosci 17:3024-3037
[Abstract/Free Full Text] .- Palmer TD, Takahashi J, Gage FH (1997) The adult rat hippocampus contains primordial neural stem cells. Mol Cell Neurosci 8:389-404[Web of Science][Medline].
- Petersen RB, Tabaton M, Chen SG, Monari L, Richardson SL, Lynches T, Manetto V, Lanska DJ, Markesbery WR, Currier RD, Autilio-Gambetti L, Wilhelmsen KC, Bambetti P (1997) Familial progressive subcortical gliosis: presence of prions and linkage to chromosome 17. Neurology 45:1062-1067
[Abstract/Free Full Text] .- Pincus DW, Keyoung H, Restelli C, Goodman RR, Fraser RAR, Edgar M, Sakakibara S, Okano H, Nedergaad M, Goldman SA (1998) FGF2/BDNF-responsive neuronal progenitor cells in the adult human subependyma. Ann Neurol 43:576-585[Web of Science][Medline].
- Reynolds BA, Weiss S (1996) Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 175:1-13[Web of Science][Medline].
- Richter K, Good PJ, Dawid IB (1990) A developmentally regulated, nervous system-specific gene in Xenopus encodes a putative RNA-binding protein. New Biol 2:556-565[Medline].
- Roper SN, Eisenschenk S, King MA (1999) Reduced density of parvalbumin- and calbindin D28k-immunoreactive neurons in experimental cortical dysplasia. Epilepsy Res 37:63-71[Web of Science][Medline].
- Sakakibara S, Okano H (1997) Expression of neural RNA-binding proteins in the postnatal CNS: implications of their roles in neuronal and glial cell development. J Neurosci 17:8300-8312
[Abstract/Free Full Text] .- Sakakibara S, Imai T, Hamaguchi K, Okabe M, Aruga J, Nakajima K, Yasutomi Y, Nagata T, Kurihara Y, Uesugi S, Miyata T, Ogawa M, Mikoshiba K, Okano H (1996) Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev Biol 176:230-242[Web of Science][Medline].
- Satoh H, Nagai F, Homma H, Mori S, Matsui M (1993) Regional assignment of rat androsterone UDP-glucuronosyltransferase gene (UGT2B2) to chromosome 14p21.2-p22. Cytogenet Cell Genet 62:49-51[Web of Science][Medline].
- Scheffler B, Horn M, Blumcke I, Laywell ED, Coomes D, Kukekov VG, Steindler DA (1999) Marrow-mindedness: a perspective on neuropoiesis. Trends Neurosci 22:348-357[Web of Science][Medline].
- Spillantini MG, Geodert M (1998) Tau protein pathology in neurodegenerative diseases. Trends Neurosci 21:428-433[Web of Science][Medline].
- Sprinkle TJ (1989) 2' 3'-Cyclic nucleotide 3'-phosphodiesterase: an oligodendrocyte-Schwann cell and myelin-associated enzyme of the nervous system. Crit Rev Neurobiol 4:235-301[Web of Science][Medline].
- Stinehelfer S, Vruwink M, Burette A (2000) Immunolocalization of mGluR1
- Streubel B, Latta E, Kehrer-Sawatzki H, Hoffmann GF, Fonatsch C, Rehder H (1999) Somatic mosaicism of a greater than 1.7-Mb deletion of genomic DNA involving the entire NF1 gene as verified by FISH: further evidence for a contiguous gene syndrome in 17q11.2. Am J Med Genet 87:12-16[Web of Science][Medline].
- Swanson MS, Dreyfuss G (1988) Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA binding proteins. Mol Cell Biol 3:2237-2241.
- Tamamaki N, Fujimori KE, Takauji R (1997) Origin and route of tangentially migrating neurons in the developing neocortical intermediate zone. J Neurosci 17:8313-8323
[Abstract/Free Full Text] .- Tan SS, Kalloniatis M, Sturm K, Tam PPL, Reese BE, Faulkner-Jones B (1998) Separate progenitors for radial and tangential cell dispersion during development of the cerebral neocortex. Neuron 21:295-304[Web of Science][Medline].
- Thomas WE (1992) Brain macrophages: evaluation of microglia and their functions. Brain Res Rev 17:61-74[Medline].
- van Hooft JA, Giuffrida R, Blatow M, Monyer H (2000) Differential expression of group I metabotropic glutamate receptors in functionally distinct hippocampal interneurons. 20:3544-3551.
- Wakamatsu Y, Maynard TM, Jones SU, Weston JA (1999) NUMB localizes in the basal cortex of mitotic avian neuroepithelial cells and modulates neuronal differentiation by binding to NOTCH-1. Neuron 23:71-81[Web of Science][Medline].
- Whittington MA, Traub RD, Jefferys JG (1995) Synchronized oscillations in interneuron networks driven by metabotropic glutamate receptor activation. Nature 373:612-615[Medline].
- Yao K-M, Samson M-L, Reeves R, White K (1993) Gene elav of Drosophila melanogaster: a prototype for neuron-specific RNA binding protein gene family that is conserved in flies and humans. J Neurobiol 24:723-739[Web of Science][Medline].
- Yoda A, Sawa H, Okano H (2000) MSI-1, a neural RNA-binding protein, is involved in male mating behavior in Caenorhabditis elegans. Genes Cells 5:1-12[Abstract].
- Zhang W, Wagner BJ, Ehrenman K, Schaefer AW, DeMaria CT, Crater D, DeHaven K, Long L, Brewer G (1993) Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1. Mol Cell Biol 13:7652-7665
[Abstract/Free Full Text] .
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