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 27, 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,20m; B,10m.)
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. 1B). MAP2 (Chemicon; diluted 1:200), GFAP (DAKO; diluted To separate proliferating GCPs from postmitotic GCPs, we 1:200), and brain lipid-binding protein (BLBP) (provided by N. used the HNK-1 antibody for sequential immunopanning. We Heintz, The Rockefeller University, New York; diluted 1:1,500). first collected the small cell fraction on a Percoll density Cells were washed with PBS and subsequently incubated with gradient. We then removed the microglial cells by negative fluorescein- or rhodamine-conjugated secondary antibodies [for selection with rabbit IgG and positively selected the HNK-1- detection of mouse and rabbit IgG (Jackson ImmunoResearch; positive cells, as described in Materials and Methods. We mea- diluted 1:100) or for detection of mouse IgM (Cappel; diluted sured the ability of the cells to incorporate BrdUrd when 1:100)] for 1 h. Propidium iodide (2 g͞ml) or TO-PRO-3 (2.5 stimulated by Shh-N, which has been shown to be a potent M; Molecular Probes) was used as a counterstain. Samples mitogen for GCPs (12–15). As shown in Fig. 1C, the HNK-1- were mounted by using Vectashield mounting medium (Vector positive population contained a higher proportion of BrdUrd- Laboratories) and visualized by using confocal laser scanning positive cells than the unsorted cells. microscopy (MRC-1024, Bio-Rad; or LSM 510 Meta, Zeiss). The HNK-1-positive population also contained a higher pro- For detection of 5-bromodeoxyuridine (BrdUrd) incorpora- portion of cyclin D1-positive, p27-negative, and NeuN-negative tion, cells were pulsed with 10 M BrdUrd (Roche Diagnostics) cells than the unsorted cells (data not shown), indicating that it for the last 24 h of culture. At the end of culture, cells were fixed was enriched for immature cells (15). When we cultured the with 4% paraformaldehyde for 10 min and incubated with HNK-1-positive GCPs on poly-D-lysine-coated slide glass in primary and fluorescein- or rhodamine-conjugated secondary serum-free medium in the absence of Shh-N, they stopped antibodies as described above. Cells were then postfixed with 4% dividing and differentiated into granule neurons within 2 days, paraformaldehyde for 5 min, incubated with 2 M hydrochloric strongly expressing -tubulin III (see Fig. 3A). Even when Shh-N acid for 20 min to denature DNA, and then neutralized with 0.1 was added to the culture medium, the HNK-1-positive GCPs M sodium tetraborate (pH 8.5). Cells were incubated with the rat stopped dividing within 1 wk and differentiated into granule monoclonal anti-BrdUrd antibody (Oxford; diluted 1:200) and neurons. then with fluorescein- or rhodamine-conjugated donkey anti-rat IgG antibodies (Jackson ImmunoResearch; diluted 1:100). Shh and BMP2 Increase GFAP-Positive Cells in GCP Cultures. To At least 500 cells were assessed in each sample, and determine whether the HNK-1-positive GCPs could differenti- the fraction of GFAP-positive or BrdUrd-positive cells was ate into astrocytes also, we cultured the cells under various determined. conditions and stained them with anti-GFAP antibodies. BMP2, which has been shown to induce oligodendrocyte precursor cells Results to differentiate into astrocytes (8, 16), failed on its own to induce Separation of Proliferating GCPs from Postmitotic GCPs. It has been the HNK-1-positive cells to express GFAP (Fig. 2A). Similarly, shown that oligodendrocyte precursor cells can be repro- Shh-N alone did not promote differentiation into GFAP-positive grammed by extracellular signals to resemble NSCs, which can cells (Fig. 2A). When we added Shh-N and BMP2 simulta- give rise to both neurons and glial cells (8). To examine whether neously, however, the number of GFAP-positive cells in the neuronal precursors have the potential to differentiate into glial culture increased substantially (Fig. 2 A and D), although most cells, we isolated immature GCPs from P7 mice. We used a of the cells still differentiated into  -tubulin III-positive neurons
1212 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307972100 Okano-Uchida et al. Downloaded by guest on September 27, 2021 Fig. 2. Shh-N and BMP2 dramatically increase GFAP-positive cells in GCP cultures. HNK-1-positive GCPs were cultured for 4 days without Shh-N or BMP2 (None), with 3 g͞ml Shh-N (Shh-N), with 80 ng͞ml BMP2 (BMP2), or with both Shh-N and BMP2 (Shh-N ϩ BMP2). BrdUrd was added during the last 24 h of culture. Cells were fixed and double stained with antibodies directed against GFAP (red) and BrdUrd (green). TO-PRO-3 (blue) was used as a counterstain. The percentage of GFAP-positive (A) or BrdUrd-positive (B) cells was counted, and results are shown as mean Ϯ SD of three areas. The number of total cells within the counted areas was 752 Ϯ 166 in the untreated culture and 819 Ϯ 202 in the culture treated with Shh-N and BMP2. The number of GFAP-positive cells was 1.7 Ϯ 1.2 in the untreated culture and 28.7 Ϯ 3.1 in the culture treated with Shh-N and BMP2. The results were confirmed in three additional experiments. A single, typical GFAP-positive cell found in BMP2-treated cultures is shown in C; note that it is BrdUrd-negative. GFAP-positive cells induced by Shh-N and BMP2 are shown in D; note that most of these cells are BrdUrd-positive. (Scale bar: 20 m.)
(Fig. 3B). Although we did not examine the survival rates under dose-dependent manner, with an effect at all concentrations Ͼ10 ͞ the various conditions, the number of total cells within the ng ml (data not shown). BMP4 and BMP7 had the same effect CELL BIOLOGY counted areas was not significantly different between the un- as BMP2 (data not shown). treated cultures and the Shh-N- and BMP2-treated cultures. Our immature GCP preparation contained Ͻ1% GFAP- Thus, the increase in proportion of GFAP-positive cells resulted positive cells when assessed at2hafterplating, and GFAP- from the net increase in the number of GFAP-positive cells (see positive cells remained Ͻ1% for Ͼ1 wk under all examined Fig. 2 legend for details). In the presence of a constant concen- conditions except when they were cultured with Shh-N and tration of Shh-N, BMP2 increased GFAP-positive cells in a BMP2. Most of these GFAP-positive cells had the typical morphological characteristics of astroglial cells (Fig. 2C) and expressed BLBP (Fig. 3D Inset), which is expressed in both astrocytes and Bergmann glia in developing mouse cerebellum (17). Because they also expressed nestin, a marker for NSCs, some of these GFAP-positive cells were probably developing Bergmann glia (18). Nestin has been reported to be expressed in immature Bergmann glia in vivo (19), and we confirmed this result by staining frozen sections from P7 mouse cerebellum with an antibody directed against nestin (data not shown). In contrast to these GFAP-positive cells, the GFAP-positive cells that developed in the presence of Shh-N and BMP2 had a distinctive morphology (mostly bipolar with fine processes) and did not express either nestin or BLBP (Fig. 3 C and D). Ciliary neurotrophic factor has been shown to induce GFAP- negative astrocyte precursor cells to differentiate into GFAP- positive astrocytes (20). In our cultures, however, ciliary neuro- trophic factor did not increase the number of GFAP-positive Fig. 3. GFAP-positive cells in GCP cultures treated with Shh-N and BMP2 do cells either in the absence or presence of Shh-N (data not not express nestin or BLBP. HNK-1-positive GCPs were treated with (B–D)or shown). In addition, although Shh has been reported to induce without (A) Shh-N and BMP2 for 2.5 days. Cells were fixed and double stained the expression of BLBP in immature glial cells (12), we did not  for GFAP (red in A, B, and C, green in D) and either -tubulin III (green in A and see an increase of BLBP-positive cells in our GCP cultures B), nestin (green in C), or BLBP (red in D). TO-PRO-3 (blue) was used as a counterstain. A GFAP-positive cell that also expresses nestin (an immature treated with either Shh-N or Shh-N and BMP2. Bergmann glia) is shown in C Inset; a GFAP-positive cell that also expresses When we prepared the astrocytes from P7 mouse cerebellum BLBP (an astroglial cell or an immature Bergmann glia) is shown in D Inset. according to the protocol by McCarthy and de Vellis (21) and (Scale bar: 50 m.) examined the effect of Shh-N on BrdUrd incorporation by these
Okano-Uchida et al. PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1213 Downloaded by guest on September 27, 2021 Fig. 4. BMP2 induces GFAP expression only if the GCPs are proliferating. In A and B, HNK-1-positive GCPs had been treated with Shh-N alone for various times before BMP2 addition. BMP2 was added to the cultures, and the cells were cultured for 2 additional days. BrdUrd was added during the last 24 h of culture. Cells were fixed and double stained for GFAP and BrdUrd and counterstained with TO-PRO-3. GFAP-positive (A) or BrdUrd-positive (B) cells were counted. In C, HNK-1-positive GCPs were treated with Shh-N alone for 2.5 days and then either BMP2, BMP2 and 2 M Ara-C (BMP2 ϩ Ara-C), or BMP2 and 2 g͞ml aphidicolin (BMP2 ϩ Aph) were added. After 2 additional days of culture, cells were fixed and stained for GFAP. GFAP-positive cells were counted. The results are shown as mean Ϯ SD of four to eight areas and were confirmed in two additional experiments.
astrocytes, Shh-N did not stimulate their BrdUrd incorporation for 2.5 days and then double stained them with antibodies either in the presence or absence of BMP2. directed against GFAP and TAG-1, a cell-surface glycoprotein Taken together, these results suggest that the GFAP-positive expressed by some CNS neurons (22). In frozen sections, TAG-1 cells induced by the combination of Shh-N and BMP2 did not appeared to be expressed transiently by postmitotic GCPs be- develop either from contaminating astrocytes or astrocyte pre- cause it was seen on cells in the deeper zone of EGL but not cursors or contaminating immature Bergmann glia but instead elsewhere in the EGL, in the molecular layer, or in the GL (Fig. developed from the immature GCPs themselves. 5D). When purified GCPs in culture were treated with Shh-N To determine whether the GFAP-positive cells could have and BMP2, some of the GFAP-positive cells also expressed developed from contaminating NSCs, we searched for nestin- TAG-1 (Fig. 5A). We further examined whether the GFAP- positive cells in our cultures. Except for contaminating immature positive cells expressed other neuronal markers. In frozen Bergmann glia, which were nestin-, BLBP-, and GFAP-positive sections of P7 mouse cerebellum, -tubulin III, NeuN, and from the start of the culture, as described above, all other cells MAP2 were all expressed strongly by GCPs in the deeper zone in our cultures in all conditions were nestin-negative, suggesting of the EGL, in the molecular layer, and in the GL (Fig. 5 E, F, that contaminating NSCs were not the source of the GFAP- and I, respectively). As was the case with TAG-1, when purified positive cells that developed in cultures treated with Shh-N and GCPs in culture were treated with Shh-N and BMP2, some of the BMP2. induced GFAP-positive cells expressed these three neuronal markers also (Fig. 5 B, C, G, and H). In these immunostaining BMP2 Acts on Proliferating GCPs to Induce GFAP Expression. Simul- studies, we used mouse monoclonal antibodies directed against taneous treatment with BMP2 and Shh-N significantly decreased the neuronal markers and rabbit antibodies directed against the percentage of BrdUrd-positive cells in our purified GCP GFAP. When we stained the cells with rabbit anti-MAP2 cultures compared with treatment with Shh-N alone (Fig. 2B; antibodies and a mouse monoclonal anti-GFAP antibody, the compare Shh-N with Shh-N ϩ BMP2). This finding suggested results were the same (Fig. 5H). We searched for the cells that that BMP2 opposed the mitogenic activity of Shh-N, thereby coexpressed neuronal and astroglial markers in our cultures in all helping to induce the terminal differentiation of GCPs into conditions and found these cells only in the cultures treated with GFAP-positive cells. To test this suggestion, we first cultured both Shh-N and BMP2; even in these cultures, such cells were GCPs with Shh-N alone and then added BMP2 at various times seen only in a narrow window between days 2 and 7. These results after plating. The later we added BMP2 to the Shh-N-treated cultures, the fewer GFAP-positive cells developed (Fig. 4A). The strongly suggest that the induced GFAP-positive cells developed decrease in the percentage of GFAP-positive cells paralleled a from immature GCPs. decrease in the percentage of BrdUrd-positive cells (Fig. 4; To confirm that the induced GFAP-positive cells went on to compare A and B), suggesting that BMP2 induced GFAP become bona fide astroglial cells, we continued the cultures for expression only in GCPs that were still proliferating. To confirm 7.5 days before staining them for other astroglial markers. The this suggestion, we used inhibitors of DNA synthesis, Ara-C or induced GFAP-positive cells cultured in Shh-N and BMP2 for aphidicolin. Simultaneous addition of either of these reagents 7.5 days had longer processes and stained more strongly for with BMP2 almost completely suppressed the appearance of GFAP than the induced cells after only 2.5 days of culture.  GFAP-positive cells in GCP cultures that had been treated with Furthermore, some of them also expressed S100- , a marker for Shh-N alone for 2 days (Fig. 4C). These reagents had no effect differentiated astroglial cells (Fig. 6B). None of GFAP-positive on cells that had already expressed GFAP (see Fig. 6B, and data cells at either time point expressed BLBP (data not shown). By not shown). As described above, treatment of fresh GCP cultures 7.5 days, all of the induced GFAP-positive cells had lost the with BMP2 alone did not increase GFAP-positive cells signifi- expression of all of the neuronal markers (data not shown). cantly (see Fig. 2A), even though immature GCPs had a high Taken together, these results suggest that immature GCPs proliferative rate at the start of culture in the absence of any treated with Shh-N and BMP2 differentiate first into the GFAP- added signaling molecule (data not shown, see ref. 15). Thus, and neuronal marker-positive cells and then finally into GFAP- Shh-N signaling apparently is required for BMP2 to induce positive, S100--positive, neuronal marker-negative, and differ- immature GCPs to differentiate into GFAP-positive cells. entiated astroglial cells.
Some ‘‘Switching Cells’’ Coexpress Neuronal and Astrocyte Markers Discussion Initially. To confirm that GFAP-expressing cells were derived BMPs and Shh are known to play crucial roles in the develop- from immature GCPs, we treated the cells with Shh-N and BMP2 ment of cerebellum. Alder et al. (2) demonstrated that BMPs
1214 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307972100 Okano-Uchida et al. Downloaded by guest on September 27, 2021 CELL BIOLOGY Fig. 5. Induced GFAP-positive cells coexpress various neuronal markers. HNK-1-positive GCPs were treated with Shh-N and BMP2 for 2.5 days, fixed, and double stained with rabbit antibodies directed against either GFAP (red in A–C and G) or MAP2 (red in H) and with mouse monoclonal antibodies directed against either TAG-1, -tubulin III, NeuN, MAP2, or GFAP (green in A–C, G, and H, respectively). TO-PRO-3 (blue) was used as a counterstain. Frozen sections from P7 mouse cerebella were stained with rabbit antibodies directed against GFAP (red in D–F and I) and mouse monoclonal antibodies directed against either TAG-1, -tubulin III, NeuN, or MAP2 (green in D–F and I, respectively). (Scale bars: A–C, G, and H,20m; D–F and I,50m.)
initiate the program of granule cell specification, and Angley et together to cultured GCPs. We find that a small proportion of al. (23) provided evidence that BMP4 might participate in the GCPs differentiate into astroglial cells in the presence of regulating postnatal granule cell and astroglial cell differentia- both BMPs and Shh but not in the presence of either protein tion. Several laboratories have shown that Shh produced and alone. released by Purkinje cells is a potent mitogen for GCPs (12–15, Is it possible that the GFAP-positive cells that develop in 24). Also, Dahmane and Ruiz-i-Altaba (13) reported that Shh response to BMPs and Shh are actually NSCs rather than bona fide astroglial cells? There is increasing evidence that at least induces the differentiation of Bergmann glia. Because both some NSCs express GFAP (25, 26). Furthermore, Kondo and BMPs (23) and Shh (12–14) are expressed in the postnatal Raff (8) reported that oligodendrocyte precursor cells can be cerebellum, we examined the effect of both factors administered reprogrammed by extracellular signals to resemble NSCs, and the first signals required for reprogramming include BMPs. We think it is unlikely, however, that the GFAP-positive cells induced by BMPs and Shh in our cultures are NSCs for the following reasons. First, when we remove Shh and BMP2 from our cultures and add basic fibroblast growth factor and͞or epidermal growth factor to promote NSC proliferation (1), the GFAP-positive cells do not divide or incorporate BrdUrd (data not shown). Second, when we remove Shh and BMP2 and add both platelet-derived growth factor and thyroid hormone to promote oligodendrocyte differentiation of NSCs (8, 27), we do Fig. 6. Induced GFAP-positive cells express S100-. HNK-1-positive GCPs were not see oligodendrocytes developing in our cultures (data not shown). Third, some of the GFAP-positive cells induced in our cultured in the presence (B) or absence (A) of Shh-N and BMP2 for 2.5 days.  Ara-C (2 M) was then added to the cultures to suppress the proliferation of cultures also expressed S100- , a marker of differentiated as- contaminating astroglia or immature Bergmann glia. The cells were then troglial cells. Together, these results strongly suggest that the cultured for an additional 5 days, fixed, and double stained for S100- (green) GFAP-positive cells are astroglial cells rather than NSCs. and GFAP (red). TO-PRO-3 (blue) was used as a counterstain. The results were It was thought originally that rapidly dividing cells in the EGL confirmed in two additional experiments. (Scale bar: 50 m.) give rise to various cell types in the cerebellar cortex, including
Okano-Uchida et al. PNAS ͉ February 3, 2004 ͉ vol. 101 ͉ no. 5 ͉ 1215 Downloaded by guest on September 27, 2021 granule cells, interneurons (basket cells and stellate cells), and reported that none of the progenitors in the white matter was some glial cells (24). Recent studies, however, using quail-chick doubly labeled with any combination of markers characteristic to chimeras (28, 29), transplantation of EGL cells (30), or retroviral different cell lineages (34), whereas the induced GFAP-positive labeling of EGL cells (31, 32) indicate that cells in the EGL give cells transiently express both neuronal and astroglial markers rise to granule cells exclusively in vivo. Here, we show that some (Fig. 5). EGL GCPs can differentiate into GFAP-positive glial cells in To our knowledge, this article is the first to show that neuronal vitro also. It is unknown whether some GCPs also develop into precursor cells can differentiate into glial cells and that they are, astroglial cells in vivo. Although it has been shown that both Shh therefore, not irreversibly committed to differentiate into neu- (12–14) and BMPs (23, 33) are expressed in Purkinje cells and rons. Our findings add to the expanding list of examples where in the EGL, it is not known whether the levels of these proteins precursor cells, or even differentiated cells, can be diverted from are sufficient to induce some GCPs to develop into astroglial their expected developmental fate by extracellular signals (for cells. Such differentiation could easily be missed because only a review, see ref. 35). It seems that such cells are specified rather small proportion of GCPs are induced to become astroglial cells than irreversibly committed or determined. in culture by Shh and BMPs. We cannot completely exclude the possibility that the induced We thank Professors B. Barres for providing the immunopanning GFAP-positive cells could have developed from the progenitors protocol and suggesting the use of the HNK-1 antibody for selection of in the white matter of the cerebellum, which are reported to give immature GCPs, N. Heintz for supplying the anti-BLBP antibody, and rise to cortical interneurons (stellate cells and basket cells in the P. Beachy for supplying the mouse Shh-N expression vector. We espe- molecular layer and Golgi cells in the GL), astroglial cells, and cially thank Professor M. Raff for helpful discussions and continuous oligodendrocytes (31, 32). We think that the possibility is encouragement. The TAG-1 hybridoma was obtained from the Devel- opmental Studies Hybridoma Bank, developed under the auspices of the unlikely, however, for the following two reasons. First, the National Institute of Child Health and Human Development and induced GFAP-positive cells express TAG-1. In developing maintained by the Department of Biological Sciences at the University cerebellum, only the cells in the deeper zone of the EGL express of Iowa, Iowa City. This work was supported by a Grant-in-Aid for TAG-1; the cells in the molecular layer or in the GL or in the Scientific Research from the Ministry of Education, Science, and Culture white matter do not express TAG-1 (Fig. 5D). Second, it is of Japan (to Y.I.).
1. Gage, F. H. (2000) Science 287, 1433–1438. 18. Lendahl, U., Zimmerman, L. B. & McKay, R. D. (1990) Cell 60, 585–595. 2. Alder, J., Lee, K. J., Jessell, T. M. & Hatten, M. E. (1999) Nat. Neurosci. 2, 19. Hockfield, S. & McKay, R. D. (1985) J. Neurosci. 5, 3310–3328. 535–540. 20. Mi, H. & Barres, B. A. (1999) J. Neurosci. 19, 1049–1061. 3. Wingate, R. J. (2001) Curr. Opin. Neurobiol. 11, 82–88. 21. McCarthy, K. D. & de Vellis, J. (1980) J. Cell Biol. 85, 890–902. 4. Hatten, M. E. & Heintz, N. (1995) Annu. Rev. Neurosci. 18, 385–408. 22. Dodd, J., Morton, S. B., Karagogeos, D., Yamamoto, M. & Jessell, T. M. (1988) 5. Altman, J. & Bayer, S. A. (1996) Development of the Cerebellar System: In Neuron 1, 105–116. Relation to its Evolution, Structure, and Functions (CRC Press, Boca Raton, FL). 23. Angley, C., Kumar, M., Dinsio, K. J., Hall, A. K. & Siegel, R. E. (2003) 6. Okano-Uchida, T., Himi, T., Komiya, Y. & Ishizaki, Y. (2003) Proc. Jpn. Acad. J. Neurosci. 23, 260–268. 79B, 223–226. 24. Altman, J. (1972) J. Comp. Neurol. 145, 353–397. 7. Hatten, M. E. (1985) J. Cell Biol. 100, 384–396. 25. Doetsch, F., Caille, I., Lim, D. A., Garcia-Verdugo, J. M. & Alvarez-Buylla, A. 8. Kondo, T. & Raff, M. (2000) Science 289, 1754–1757. (1999) Cell 97, 703–716. 9. Gao, W. O., Heintz, N. & Hatten, M. E. (1991) Neuron 6, 705–715. 26. Laywell, E. D., Rakic, P., Kukekov, V. G., Holland, E. C. & Steindler, D. A. 10. Baptista, C. A., Hatten, M. E., Blazeski, R. & Mason, C. A. (1994) Neuron 12, (2000) Proc. Natl. Acad. Sci. USA 97, 13883–13888. 243–260. 27. Johe, K. K., Hazel, T. G., Muller, T., Dugich-Djordjevic, M. M. & McKay, R. D. 11. Wernecke, H., Lindner, J. & Schachner, M. (1985) J. Neuroimmunol. 9, (1996) Genes Dev. 10, 3129–3140. 115–130. 28. Hallonet, M. E. & Le Douarin, N. M. (1993) Eur. J. Neurosci. 5, 1145–1155. 12. Wechsler-Reya, R. J. & Scott, M. P. (1999) Neuron 22, 103–114. 29. Hallonet, M. E., Teillet, M. A. & Le Douarin, N. M. (1990) Development 13. Dahmane, N. & Ruiz-i-Altaba, A. (1999) Development (Cambridge, U.K.) 126, (Cambridge, U.K.) 108, 19–31. 3089–3100. 30. Gao, W. Q. & Hatten, M. E. (1994) Development (Cambridge, U.K.) 120, 14. Wallace, V. A. (1999) Curr. Biol. 9, 445–448. 1059–1070. 15. Miyazawa, K., Himi, T., Garcia, V., Yamagishi, H., Sato, S. & Ishizaki, Y. 31. Zhang, L. & Goldman, J. E. (1996) J. Comp. Neurol. 370, 536–550. (2000) J. Neurosci. 20, 5756–5763. 32. Zhang, L. & Goldman, J. E. (1996) Neuron 16, 47–54. 16. Mabie, P. C., Mehler, M. F., Marmur, R., Papavasiliou, A., Song, Q. & Kessler, 33. Lin, J. C. & Cepko, C. L. (1998) J. Neurosci. 18, 9342–9353. J. A. (1997) J. Neurosci. 17, 4112–4120. 34. Milosevic, A. & Goldman, J. E. (2002) J. Comp. Neurol. 452, 192–203. 17. Feng, L., Hatten, M. E. & Heintz, N. (1994) Neuron 12, 895–908. 35. Raff, M. (2003) Annu. Rev. Cell Dev. Biol. 19, 1–22.
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