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Cerebellar Granule Cell Precursors Can Differentiate Into Astroglial Cells

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Cerebellar Granule Cell Precursors Can Differentiate Into Astroglial Cells Cerebellar granule cell precursors can differentiate into astroglial cells Takayuki Okano-Uchida*, Toshiyuki Himi†, Yoshiaki Komiya*, and Yasuki Ishizaki*‡ *Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, 3-39-22 Showamachi, Maebashi 371-8511, Japan; and †Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan Communicated by Setsuro Ebashi, National Institute for Physiological Sciences, Okazaki, Japan, December 1, 2003 (received for review November 11, 2003) During CNS development, multipotent neural stem cells give rise human natural killer 1 (HNK-1) antibody. Cerebella from first to various kinds of specified precursor cells, which proliferate postnatal day 7 (P7) mice were cut into small pieces and extensively before terminally differentiating into either neurons or incubated at 37°C for 30 min in papain solution (16.5 units/ml glial cells. It is still not clear, however, whether the specified papain͞200 ␮g/ml L-cysteine͞0.008% DNase). Tissue was rinsed precursor cells are irreversibly determined to differentiate into in Dulbecco’s PBS containing 1.5 mg͞ml ovomucoid, 1.5 mg͞ml their particular cell types. In this study, we show that isolated BSA, and 0.008% DNase and triturated in the same solution mouse cerebellar granule cell precursors from the outermost, containing rabbit anti-mouse macrophage antibodies to obtain a proliferative zone of the external germinal layer can differentiate single-cell suspension. Cells were centrifuged at 1,000 rpm for 10 into astroglial cells when exposed to sonic hedgehog (Shh) and min at room temperature and suspended in Dulbecco’s PBS bone morphogenetic proteins. These induced cells initially ex- containing 10 mg͞ml ovomucoid and 10 mg͞ml BSA and pressed both glial fibrillary acidic protein and neuronal markers, centrifuged again. Cells were resuspended in panning buffer but they then lost their neuronal markers and acquired S100-␤,a (Dulbecco’s PBS containing 0.02% BSA and 5 ␮g͞ml insulin) marker of differentiated astroglial cells. These results indicate that and passed through a cell strainer (Falcon). To obtain a fraction at least some granule cell precursors are not irreversibly committed enriched in GCPs (7), the cell suspension was loaded onto a step to neuronal development but can be induced to differentiate into gradient of 35% and 60% Percoll (Amersham Biosciences) and astroglial cells by appropriate extracellular signals. centrifuged at 3,000 rpm for 20 min at room temperature. GCPs were recovered from the 35%͞60% interface and washed twice lthough neural stem cells (NSCs) are studied intensely by in panning buffer. To remove contaminating microglial cells, the Aresearchers interested in either regenerative medicine or cell suspension was plated onto a 100-mm tissue culture dish developmental neurobiology, the detailed pathways by which precoated with affinity-purified goat anti-rabbit IgG antibodies NSCs give rise to neurons, astrocytes, or oligodendrocytes are (Jackson ImmunoResearch) and incubated for 20 min at room still uncertain (1). It is generally accepted, for example, that temperature. The dish was shaken vigorously, and then nonad- NSCs first give rise to neuronal precursors, which proliferate and herent cells were plated onto a Petri dish precoated with then terminally differentiate into postmitotic neurons. It is still affinity-purified goat anti-mouse IgM antibodies (Jackson Im- CELL BIOLOGY not clear, however, whether such neuronal precursors are irre- munoResearch) and supernatant from HNK-1 hybridoma versibly committed to become neurons or whether they have the (American Type Culture Collection) for 30 min. The dish was ability to differentiate into glial cells or even to revert to NSCs. rinsed with Dulbecco’s PBS to remove nonadherent cells com- In this study, we have examined whether granule cell precur- pletely, and strongly adherent cells (i.e., HNK-1-positive imma- sors (GCPs) are irreversibly determined to differentiate into ture GCPs) were harvested by trypsinization (0.125% trypsin granule cells, the most abundant class of CNS neurons. GCPs solution; Sigma). Immature GCPs were suspended in neurobasal ͞ ͞ arise from the rhombic lip, the dorsal part of the neural tube at medium (GIBCO BRL) containing 100 units ml penicillin, 100 ␮ ͞ the boundary of the mesencephalon and the metencephalon (2, g ml streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, ͞ ␮ ͞ 3). They migrate rostrally up the lip and onto the surface of 2% B-27 (all obtained from GIBCO BRL), 5 g ml insulin, 100 ␮ ͞ ␮ ͞ ͞ cerebellar anlage, where they form the external germinal layer g ml apotransferrin, 100 g ml BSA, 62 ng ml progesterone, ␮ ͞ ͞ ␮ (EGL) (3, 4). In rodents, GCPs proliferate rapidly in the EGL 16 g ml putrescine, 40 ng ml sodium selenite, and 30 M for 2–3 wk after birth. They then exit the cell cycle, extend axons, N-acetyl cysteine (all obtained from Sigma) and plated on 8-well ␮ ͞ and migrate inward to their final destination in the granule layer slide glasses (Matsunami, Osaka) precoated with 10 g ml ϫ 4 Ϸ ϫ 4 (GL) (4, 5). We show that some GCPs in the EGL are not poly-D-lysine at a density of 3 10 cells per well ( 6 10 cells irreversibly determined to differentiate into granule cells; when per cm2). treated with sonic hedgehog (Shh) and bone morphogenetic To identify glial fibrillary acidic protein (GFAP)-positive cells proteins (BMPs) in culture, a proportion of the GCPs lose their that coexpressed neuronal markers, immature GCPs were plated ϫ 3 neuronal markers and differentiate into astroglial cells (6). at a low density (6 10 cells per well) on poly-D-lysine-coated 8-well slide glasses. Materials and Methods Animals and Materials. C57BL͞6 mice were purchased from SLC Immunostaining. Cells were fixed with 4% paraformaldehyde for (Hamamatsu, Japan). The recombinant N-terminal-active frag- 20 min, washed with PBS, and then incubated for 30 min in ͞ ment of mouse Shh (Shh-N) and recombinant human BMP2 blocking buffer [Tris-buffered saline (50 mM Tris 150 mM were purchased from Genzyme. Shh-N was expressed also in Escherichia coli transformed with GST-Shh-N plasmid (provided by P. A. Beachy, The Johns Hopkins University, Baltimore). The A preliminary report of this work has been published (6). Abbreviations: Ara-C, 1-␤-D-arabinofuranosylcytosine; BLBP, brain lipid-binding protein; DNA synthesis inhibitors aphidicolin and 1-␤-D-arabinofurano- BMP, bone morphogenetic protein; EGL, external germinal layer; GCP, granule cell pre- sylcytosine (Ara-C) were purchased from Wako Pure Chemical cursor; GFAP, glial fibrillary acidic protein; GL, granule layer; HNK-1, human natural killer (Osaka, Japan) and Sigma, respectively. 1; MAP2, microtubule-associated protein 2; NeuN, neuronal nuclei; NSC, neural stem cell; Pn, postnatal day n; Shh, sonic hedgehog; Shh-N, N-terminal-active fragment of mouse Shh. Preparation of Immature GCPs by Immunopanning. Immature GCPs ‡To whom correspondence should be addressed. E-mail: [email protected]. were prepared by immunopanning methods using the anti- © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307972100 PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1211–1216 Downloaded by guest on September 25, 2021 Fig. 1. Immature GCPs strongly express HNK-1 both in vivo and in vitro.(A) Frozen sections from P7 mouse cerebellum were stained with the HNK-1 monoclonal antibody, followed by FITC-conjugated goat-anti-mouse IgM (green). The sections were counterstained with propidium iodide (red). (B) GCPs were cultured for 3 days in the presence of 3 ␮g͞ml Shh-N. BrdUrd was added during the last 24 h of culture. Cells were fixed and double stained with antibodies directed against HNK-1 (green) and BrdUrd (red). Cells were counterstained with TO-PRO-3 (blue). (C) HNK-1-positive and unsorted GCPs were cultured for 3 days in the presence (Shh-N) or absence (None) of Shh-N. BrdUrd was added during the last 24 h of culture. Cells were fixed and stained with an anti-BrdUrd antibody. BrdUrd-positive cells were counted, and results are shown as mean Ϯ SD of three areas. (Scale bars: A,20␮m; B,10␮m.) NaCl, pH 7.4) containing 50% goat serum, 1% BSA, 100 mM protocol developed by Hatten and coworkers (7, 9, 10), which L-lysine, and 0.04% sodium azide]. For intracellular antigens, exploits the small size of GCPs to separate them from the other blocking buffer containing 0.4% Triton X-100 was used to cell types in the developing cerebellum. This protocol does not, permeabilize cells. The cells were incubated with primary anti- however, separate proliferating immature GCPs from postmi- body for1hatroom temperature. Primary antibodies used for totic differentiating GCPs. To do this, we used the HNK-1 immunostaining included anti-HNK-1 (as described above), monoclonal antibody, which has been reported to label cells in anti-TAG-1 (hybridoma obtained from the Developmental the outermost, proliferative zone of the EGL (11). Studies Hybridoma Bank, Iowa City), mouse monoclonal anti- First, we stained frozen sections of developing mouse cere- bodies directed against neuronal nuclei (NeuN), microtubule- bellum with the HNK-1 antibody and confirmed that it labeled associated protein 2 (MAP2), and GFAP (all obtained from cells strongly in the outer zone of the EGL and labeled cells only Chemicon; diluted 1:100, 1:400, and 1:400, respectively), ␤-tu- weakly in the deeper, postmitotic zone (Fig. 1A). As shown by bulin class III (Research Diagnostics, Flanders, NJ; diluted Wechsler-Reya and Scott (12), the antibody also strongly labeled 1:200), S100-␤ (Sigma; diluted 1:100), nestin (BD Biosciences; most of the proliferating cells (BrdUrd-positive cells) in cultures diluted 1:100), rabbit polyclonal antibodies directed against of P7 cerebellar cells (Fig.
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