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TISSUE-SPECIFIC STEM CELLS

Epidermal Growth Factor Signaling Mediated by Grb2 Associated Binder1 Is Required for the Spatiotemporally Regulated Proliferation of Olig2-Expressing Progenitors in the Embryonic Spinal Cord

^aDepartment of Neurobiology, Graduate School of Medicine, Gifu University, Gifu, Japan;
^bDepartment of Physiology, Keio University School of Medicine, Tokyo, Japan;
^cLaboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan;
^dInstitute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan;
^eLaboratory for Developmental Immunology, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan;
^fCore Research for Evolutional Science Technology, Solution-Oriented Research for Science and Technology, Japan Science and Technology Agency, Saitama, Japan

Key Words. Epidermal growth factor • Grb2 associated binder1 • Olig2 • Proliferation

Correspondence: Takuya Shimazaki, Ph.D., Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Telephone: +81-3-5363-3747; Fax: +81-3-3357-5445; e-mail: shimazak{at}sc.itc.keio.ac.jp; or Hideyuki Okano, M.D., Ph.D., Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Telephone: +81-3-5363-3747; Fax: +81-3-3357-5445; e-mail: hidokano{at}sc.itc.keio.ac.jp

Received September 19, 2006; accepted for publication February 18, 2007.
First published online in STEM CELLS EXPRESS   March 1, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Gab1 (Grb2 associated binder1) has been identified as an adaptor molecule downstream of many growth factors, including epidermal growth factor (EGF), fibroblast growth factor, and platelet-derived growth factor, which have been shown to play crucial roles as mitotic signals for a variety of neural progenitor cells, including stem cells, both in vitro and in vivo. Here, we show that Gab1 deficiency results in a reduction in the number of Olig2-positive (Olig2⁺) progenitor cells in the developing mouse spinal cord after embryonic day 12.5 (E12.5), when gliogenesis starts in the pMN domain where the EGF receptor (EGFR) is expressed predominantly. Our in vitro analysis further revealed that Gab1 is essential for EGF-dependent proliferation of Olig2⁺ progenitor cells derived from the E12.5 ventral and E14.5 dorsal but not ventral spinal cord, whereas Gab1 is always required for the activation of Akt1 but not of ERK1/2. Moreover, we found that the action of the Gab1/Akt pathway is context-dependent, since constitutively active Akt1 could rescue the proliferation defect only in the E12.5 spinal cord of the Gab1-deficient mouse in vitro. Finally, we demonstrated that EGFR-deficient mice and Gab1-deficient mice showed a similar reduction in the number of Olig2⁺ progenitor cells in the developing spinal cord. These findings indicate that EGFR-mediated signaling through Gab1/Akt contributes to the sufficient expansion of Olig2⁺ progenitor cells in a spatiotemporally regulated manner, which represents the origin of glial cells in the developing spinal cord.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Specification of progenitor cells in the embryonic spinal cord has been intensively studied [1]. Distinct types of cells are generated along the dorsoventral (D-V) axis of the neural tube, where progenitor domains are defined by a combination of homeodomain and basic helix-loop-helix (bHLH) transcription factors, whose expressions are precisely regulated by bone morphogenetic proteins, Wnt proteins secreted from the roof plate, and sonic hedgehog (SHH) derived from the notochord and floor plate [2–4]. Olig2 has been identified as a bHLH transcription factor expressed in the pMN domain where motoneurons and glia are sequentially generated under the control of the SHH signal [5–8]. More recently, it has been shown that a subpopulation of Pax7 (a paired homeodomain transcription factor)-expressing progenitor cells located in the dorsal half of the ventricular zone (VZ) of the mouse embryonic spinal cord also starts to express Olig2 after embryonic day 14.5 (E14.5) and may subsequently differentiate into oligodendrocytes [9, 10]. Analysis of mice lacking Olig2 has confirmed the essential role of this factor in the development of both motoneurons and oligodendrocytes [11–14]. Despite extensive analysis of the roles of Olig2, the effects of extracellular signalings other than SHH on Olig2⁺ progenitors remain poorly understood.

In the present study, we tried to examine the roles of epidermal growth factor (EGF) signaling mediated by Grb2 associated binder1 (Gab1) upon the proliferation of Olig2⁺ progenitors in the embryonic spinal cord. Gab1, originally identified as a Grb2 (growth factor receptor bound protein 2) binding protein [15], is a member of the Gab/daughter of sevenless family of adaptor molecules. It is ubiquitously expressed and plays a role as a common intracellular signaling mediator for various growth factor and cytokine receptors, including EGF receptor (EGFR), fibroblast growth factor receptors (FGFRs), gp130 (a common receptor subunit for the IL-6 cytokine family), platelet-derived growth factor (PDGF) receptor, and insulin-like growth factor-I receptor [15, 16]. Depending on the ligand stimulus, Gab1, which is recruited to activated receptors, assembles multimeric signaling complexes containing Grb2, SHP2, p85 phosphatidylinositol 3-kinase (PI3K), and phospholipase C- and serves as a signal "amplifier" [17]. These signals activate the Ras-mitogen-activated protein kinase (Ras/MAPK) and PI3K pathways, mainly through their association with Gab1 [18, 19]. It has been shown that Gab1 is actually involved downstream of various cytokine and growth factor signals in the proliferation and differentiation of various types of cells [20–22]. However, the involvement of Gab1 in the development of the central nervous system (CNS) has not yet been thoroughly studied, although the roles of Gab2, another member of the Gab family of proteins, in the fibroblast growth factor (FGF)2-dependent proliferation of neural stem cells and survival of differentiating neurons have recently been shown [23]. In this study, we attempted to elucidate the roles of Gab1 in CNS development by focusing on progenitor cell proliferation in the developing mouse spinal cord and found that EGF/Gab1/Akt pathway plays essential roles in the expansion of Olig2⁺ progenitors in a context-dependent manner.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Mice and Genotyping
Wild-type ICR (CD-1) mice were purchased from CLEA Japan, Inc. (Tokyo, http://www.clea-japan.com) or Japan SLC (Shizuoka, Japan, http://www.jslc.co.jp). Gab1-mutant mice were maintained in a C57BL6 background (>F5) and genotyped as described in a previous report [24]. Egfr-mutant mice were purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) and maintained in an ICR background (>F10) and genotyped as described in a previous report [25]. Noon of the day on which the vaginal plug was found was termed E0.5.

Primary Cultures
Spinal cords from E12.5 or E14.5 mouse embryos were dissociated mechanically and then cultured in a media hormone mix medium [26] in the presence or absence of growth factors (supplemental online Materials and Methods). Cells were seeded at 5 x 10⁵ cells per 0.75 cm² on 10-mm diameter coverslips (Matsunami, Osaka, Japan, http://www.matsunami-glass.co.jp/e-index.html) coated with poly-L-ornithine (30 µg/ml; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and laminin (10 µg/ml; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) in a 48-well culture plate (Corning, Corning, NY, http://www.corning.com).

Immunohistochemistry and Immunocytochemistry
Serial 12-µm cryosections of the mouse embryo trunk were prepared and then processed for immunohistochemistry. Sections were incubated overnight at 4°C or at room temperature with primary antibodies (supplemental online material) followed by incubation with Alexa dye-conjugated secondary antibodies (1:1,000; Molecular Probes, Carlsbad, CA, http://probes.invitrogen.com) or the combination of biotinylated secondary antibodies (1:500; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com), the VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), and the visualization by using the TSA Fluorescence System (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). The cultured cells were fixed for 15 minutes in 4% paraformaldehyde in phosphate-buffered saline and processed for immunocytochemistry (ICC) as described above. Hoechst 33258 (Sigma-Aldrich) was used to detect cell nuclei for the quantitative experiments. Immunostained specimens were examined with either a universal fluorescence microscope (Axiophot 2) or a confocal laser scan microscope (LSM510) (Carl Zeiss, Jena, Germany, http://www.zeiss.com).

Immunoblot Analysis
Cells were grown on 6-well culture plates (Corning) coated with poly-L-ornithine and laminin in the presence of 20 ng/ml FGF2 or EGF for 72 hours and then stimulated with 20 ng/ml EGF or FGF2 for 15 minutes. After the stimulation, the cells were lysed and subjected to immunoblot analysis. The immune complexes were visualized with a chemiluminescence system (ECL Western Blotting Detection Reagents; GE Healthcare Bio-Sciences Corp., Piscataway, NJ, http://www.gelifesciences.com) and analyzed in an LAS-3000mini image analyzer (FUJIFILM, Kanagawa, Japan, http://www.fujifilmlifescienceusa.com).

Statistical Analysis
The statistical significance of the variations was evaluated using an unpaired two-tailed Student's t test.

	RESULTS

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Reduction of Progenitor Cell Proliferation in the Developing Spinal Cord of Gab1-Deficient Mice After the Neurogenic Period
Pattern formation and cytogenesis in the vertebrate spinal cord have been extensively analyzed [3, 4, 27]. To elucidate the role of Gab1 in CNS development, we conducted a study focusing on the proliferation of progenitor cells in the developing spinal cord in Gab1-deficient mice. Until E11.5, the development of the spinal cord in the Gab1^–/– mice was indistinguishable from that in the wild-type (wt) mice (supplemental online Fig. S1). In contrast, at E12.5, proliferative activity of progenitor cell, as determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation and phosphohistone H3 (pH3) expression, was significantly lower in the Gab1^–/– mice than that in the wt mice (Fig. 1A, 1B, 1E, 1F, 1S). Moreover, in the pMN domain, where glial progenitors begin to appear at this stage [12, 28, 29], a significant reduction in the number of Olig2⁺ progenitor cells (43.5%) was observed in the Gab1^–/– mice (Fig. 1I, 1M, 1S). The numbers of Olig1⁺ and Mash1⁺ cells, which are subpopulations of Olig2⁺ cells in the pMN domain [30], were also decreased by 27.2% and 40.1%, respectively (Fig. 1J, 1L, 1N, 1P, 1S). In contrast to the dramatic changes in the pMN domain at this gliogenic stage, no differences in the number of Nkx2.2⁺ cells were observed in either the p3 domain or the marginal zone (MZ) between the Gab1^–/– and the wt mice (Fig. 1K, 1O; data not shown). Interestingly, no changes in the numbers of Pax7⁺ cells and Mash1⁺ cells in the dorsal segment of the spinal cord were observed either in the Gab1-deficient mice (Fig. 1C, 1D, 1G, 1H; data not shown), despite the reduction in progenitor cell proliferation throughout the D-V axis. We did not detect any increase in cellular apoptosis as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and the presence of pyknotic nuclei (Fig. 1Q, 1R; data not shown). These findings indicate that Gab1 is involved in the proliferation of spinal cord progenitor cells, in particular that of Olig2⁺ progenitor cells in the pMN domain after gliogenesis begins. Therefore, in the subsequent experiments, we focused on the roles of Gab1 in the proliferation of Olig2⁺ progenitors.

View larger version (54K): [in this window] [in a new window]   Figure 1. Significant reduction in the number of Olig2+ progenitors in the E12.5 Gab1–/– spinal cord. Cross sections of E12.5 wt and Gab1–/– embryos were immunostained with antibodies directed against BrdU ([A, E]; red), pH3 ([B, F]; red), Pax7 ([C, G]; green), Mash1 ([D, H, L, P]; red), Olig2 ([I, M]; green), Olig1 ([J, N]; green), Nkx2.2+ ([K, O]; red), and TUNEL labeled ([Q, R]; red). Panels (D) and (H) represent higher-magnification micrographs of the dorsal spinal cord. Panels (I–P) represent higher-magnification micrographs of the ventral spinal cord. Quantification of the cells is shown in panel (S). Five sections (12 µm) from each embryo were counted (three embryos for each genotype). The absolute numbers of immunoreactive cells are represented as mean ± SEM (blue bars: wt, red bars: Gab1–/–; *, p < .05; **, p < .01 vs. wt). Scale bars: 50 µm (shown in [G] for [A–C] and [E–G] and in [R] for [D, H–R]). Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; D, dorsal; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; V, ventral.

View larger version (52K): [in this window] [in a new window]   Figure 2. Deficiency of Gab1 resulted in a reduction of the EGF-dependent proliferation of Olig2+ progenitors in vitro. Whole spinal cord cells from E12.5 Gab1 mutant mice were cultured for 72 hours in the absence of any growth factor or presence of 20 ng/ml EGF, 20 ng/ml FGF2, or 10 ng/ml PDGF-AA and then processed for double immunocytochemistry (ICC) for Olig2/O4 or Olig2/BrdU. Representative micrographs show the results of Olig2/BrdU ([A, B]: Olig2, green; BrdU, red; Hoechst, blue) and Olig2/O4 ICC ([C]: Olig2, green; O4, red) in cells grown in the presence of EGF or GF-. The absolute numbers of Olig2+ cells (green bars) and the total number of cells (blue bars) are shown at the bottom in panels (A). The percentages of Olig2+/BrdU+ cells (yellow bars) and BrdU+ cells (red bars) and of Olig2+/O4+ cells (yellow bars) and Olig2+ cells (green bars) are shown at the bottom in panels (B) and (C), respectively. Ten visual fields in each condition were randomly chosen and counted. Each bar represents mean ± SEM (n 3 embryos for each genotype; *, p < .05; **, p < .01 vs. wt). (D): Developmental changes in the EGF receptor expression in the mouse spinal cord. Confocal micrographs of Olig2 (green) and EGFR (red) immunohistochemistry in the thoracic spinal cord at E12.5, E13.5, and E14.5 are shown. Right panels are higher-magnification pictures corresponding to the squares (dotted lines) in left panels. Expression of EGFR was not detected until E11.5. At E12.5 and E13.5, EGFR expression was detected predominantly in the Olig2+-pMN domain. At E14.5, EGFR expression was also detected in the dorsal progenitor domain, including Olig2+ cells. Scale bars: 50 µm (shown in the bottom left panel for all left panels); 10 µm (upper left panel and shown in bottom right panel for both bottom and middle right panels). Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; E, embryonic day; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; GF–, without growth factor; PDGF, platelet-derived growth factor.

The fact that the proportion of Olig2⁺/Nkx2.2⁺ OPCs within Olig2⁺ cells derived from Gab1-deficient E12.5 spinal cords after 72 hours of culture in the presence of EGF was not different from that in GF– cultures and was similar to that in GF– cultures of wt spinal cord (supplemental online Fig. S4) suggests that Gab1 is required not only for proliferation but also for self-renewal of those Olig2⁺ progenitors. Alternatively, a certain level of mitotic activity may be essential for the maintenance of undifferentiated state of Olig2⁺/Nkx2.2^– progenitors. In fact, the proportion of Nestin⁺ cells within Gab1-deficient Olig2⁺ cells in EGF or GF– cultures was also the same level as that in wt GF– cultures. Again, in case that Olig2⁺/Nkx2.2⁺ OPCs are mainly originated from Nkx2.2-single positive cells in these cultures, the result that EGF did not induce the reduction in the proportion of Olig2⁺/Nkx2.2⁺ OPCs within Olig2⁺ cells in Gab1^–/– cultures may be simply reflecting the reduction of proliferation of Olig2⁺/Nkx2.2^– progenitors.

Spatiotemporal Differences in the Requirement of Gab1 for the Expansion of Olig2⁺ Progenitors
In the course of this study, we found that EGFR expression, which is initially restricted to the Olig2⁺ pMN domain in the E12.5 spinal cord, then spreads to the dorsal progenitor domain, including Olig2⁺ cells at E14.5 (Fig. 2D). Therefore, we analyzed Gab1-deficient mice to clarify the roles of Gab1 as a mediator of EGFR signaling in spinal cord histogenesis. Since Gab1^–/– embryos can hardly survive beyond E15 [24], we analyzed these embryos as far as E14.5.

At E13.5, when some of the Olig2⁺ progenitors in the pMN domain start to express Nkx2.2 and migrate away from the VZ as OPCs [28, 29, 41], the progenitor cell proliferative activity, as defined by the expression of pH3, was still lower in the Gab1^–/– spinal cords than in the wt spinal cords, throughout the D-V axis (Fig. 3A, 3G, 3M). Similarly, the number of Olig2⁺ progenitors in the VZ was also significantly lower, although the difference between the wt and Gab1^–/– was not as significant at this stage as that at E12.5 (Fig. 3A, 3G, 3Q). The number of Olig2⁺/Nkx2.2⁺ OPCs in the Gab1^–/– spinal cords was also significantly lower in the absence of any significant difference in the total number of Nkx2.2⁺ cells (Fig. 3B, 3H, 3N, 3R). At E14.5, when the subpopulation of Pax7⁺ dorsal progenitors starts to express Olig2 [9, 10], no significant difference in the number of Olig2⁺ cells was observed in the ventral VZ, in contrast to the E13.5 spinal cord. However, the number of these cells in the ventral MZ was significantly lower in the Gab1^–/– mice than in the wt mice (Fig. 3C, 3I, 3Q). The difference in the number of Olig2⁺/Nkx2.2⁺ OPCs, which had been detectable at E13.5, was also lost at E14.5 (Fig. 3C, 3I, 3O, 3S). These results indicate that Gab1 transiently contributes to the expansion of Olig2⁺ progenitors in the pMN domain between E12.5 and E13.5. In the dorsal segment, the total number of Olig2⁺ cells, which may also include some populations migrating from the ventral segment [9], Pax7⁺/Olig2⁺ cells, and EGFR⁺/Olig2⁺ cells were significantly lower in the Gab1^–/– mice, in the absence of any significant differences in the total number of Pax7⁺ cells and EGFR⁺/Pax7⁺ cells (Fig. 3D–3F, 3J–3L, 3P, 3T), indicating that Gab1 is required for the expansion of dorsally originating Olig2⁺ progenitors. Similar to the observation in the E12.5 spinal cord, there was no detectable increase in the number of apoptotic cells as recognized by the presence of pyknotic nuclei in the Gab1^–/– spinal cord at these stages either (data not shown).

View larger version (65K): [in this window] [in a new window]   Figure 3. Analysis of E13.5 and E14.5 spinal cords of Gab1-deficient mice. Cross-sections of E13.5–E14.5 wt and Gab1–/– embryos were analyzed by immunohistochemistry for Olig2 ([A–E, G–K], green), pH3 ([A, G], red), Nkx2.2 ([B, C, H, I], red), EGFR ([E, F, K, L], red), and Pax7 ([D, J], red; [F, L], green). Quantification of the cells is shown in panels (M–T). Counting was conducted in five sections (12 µm) from each embryo counted. The absolute numbers of immunoreactive cells are represented as mean ± SEM (blue bars: wt; red bars: Gab1–/–; n 3 embryos for each genotype; *, p < .05; **, p < .01 vs. wt). Scale bars: 50 µm (shown in [I] for [A, C, G, I], in [H] for [B, H], and in [L] for [D–F, J–L]). (U–X): Regional differences in the requirement of Gab1 for epidermal growth factor (EGF)-dependent proliferation of Olig2+ progenitors in vitro. Cells from the dorsal and ventral segments of the spinal cord prepared separately from E14.5 Gab1-deficient mice were cultured for 2 or 72 hours in the presence of 1 µM BrdU and in the presence or absence of 20 ng/ml of EGF, followed by triple immunocytochemistry for Olig2, Pax7, and BrdU. Representative micrographs show the Olig2 (red)/Pax7 (green)/BrdU (blue) to cells grown for 72 hours in the presence of EGF (U). The numbers of immunoreactive cells in ten randomly chosen fields (>800 total cells) were counted. Expansion of Olig2+ and/or Pax7+ cells between 2 and 72 hours after plating is shown in relative terms (fold) (blue bars: wt; red bars: Gab1–/–) (V). Percentages of Olig2+ and/or Pax7+ cells relative to the total number of cells in each condition (W) and the time point (2 and 72 hours after plating, respectively) are shown (Olig2+ cells, red bars; Olig2+/Pax7+ cells, yellow bars; Pax7+cells, green bars). Percentages of BrdU+ and BrdU+/Olig2+ (including Olig2+/Pax7+) cells relative to the total number of cells after 72 hours culture in the presence of EGF are shown (BrdU+ cells: blue bars; Olig2+/BrdU+cells: pink bars) (X). Each value represents mean ± SEM (n 3 embryos; *, p < .05; **, p < .01 vs. wt). In panel (W), the asterisks for single Olig2+ or Pax7+ cells and Olig2+/Pax7+cells are shown in the same color as the respective bars. In panel (X), the asterisks for the total number of BrdU and Olig2+/BrdU+ cells are shown in blue and pink, respectively. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; C2h, 2 hours after plating without growth factor; C72h, 72 hours after plating without growth factor; D, dorsal cultures; dP1–P2, dP1 domain to P2 domain; E, embryonic day; E72h, 72 hours after plating in epidermal growth factor; EGFR, epidermal growth factor receptor; MZ, marginal zone; Olig2D, Olig2+ progenitors in the dorsal segment; V, ventral cultures; VZ, ventricular zone; wt, wild-type.

It has been suggested that Olig2⁺ progenitor cells derived from the dorsal Pax7⁺ progenitors generate OPCs just like the corresponding cells from the ventral cord [9, 10]. On the other hand, the requirement of Gab1 for the EGF-dependent proliferation of Olig2⁺ progenitor cells is context-dependent (i.e., spatially and temporally regulated as described above). Since the timing of differentiation of dorsally derived OPCs is likely to be later than that of the ventrally derived OPCs [9, 10], it is possible that differences in the cellular context depending on the differentiation status cause the difference in Gab1 requirement. To clarify whether the differentiation status of EGF-responsive Olig2⁺ progenitor cells is regionally different in the E14.5 spinal cord, we compared the cellular phenotype of EGF-responsive Olig2⁺ progenitors derived from the dorsal segment with that of the cells derived from the ventral segment. We carried out a similar in vitro analysis to that described in Table 1 and supplemental online Figure S3. We cultured cells from the dorsal and ventral halves of E14.5 spinal cord separately, followed by double immunocytochemistry for Olig2 with Nestin, NG2, Nkx2.2, O4, or Sox10, which is another OPC marker [42] (Table 2 and supplemental online Fig. S5). Along with the expansion of Olig2⁺ cells in the presence of EGF, significant differences between the dorsal and ventral cultures appeared in the expressions of NG2, Sox10, Nkx2.2, and O4. The proportions of cells expressing these markers within the population of Olig2⁺ cells in the presence of EGF were significantly lower in the dorsal cultures than those in the ventral cultures, whereas most of the Olig2⁺ cells in either of the two cultures coexpressed Nestin. In particular, the majority of Olig2⁺ cells in the dorsal cultures showed no expression of Sox10 or NG2 (also expressed by OPCs) [43] (70.1% and 89.1%, respectively), whereas a small proportion (0.8%) was also O4⁺, indicating that these dorsally derived EGF-responsive Olig2⁺ progenitor cells were more undifferentiated or specified to nonoligodendrocytic lineage than the corresponding ventrally derived cells.

View larger version (64K): [in this window] [in a new window]   Figure 4. Reduction in the number of Olig2+ progenitors in the epidermal growth factor receptor-deficient mouse. Cross-sections of E13.5–E14.5 wt and Egfr–/– embryos were analyzed by double immunohistochemistry for Olig2 ([A, B, E, F, I, J, M, N], green) and pH3 ([A, B], red), Nkx2.2+ ([E, F, I, J], red), or Pax7 ([M, N], red). Quantification of the cells is shown in panels (C, D, G, H, K, L, O). The absolute numbers of immunoreactive cells in the five sections (12 µm) counted are represented as mean ± SEM (blue bars: wt; red bars: Egfr–/–; n 3 embryos for each genotype; *, p < .05; **, p < .01 vs. wt). Scale bars: 50 µm (shown in [B] for [A, B], in [J] for [I, J], and in [N] for [E, F, M, N]). Abbreviations: dP1–P2, dP1 domain to P2 domain; E, embryonic day; MZ, marginal zone; Olig2D, Olig2+ progenitors in the dorsal segment; VZ, ventricular zone.

View larger version (45K): [in this window] [in a new window]   Figure 5. Involvement of Akt downstream of Gab1. (A): Immunoblot analysis of activation of known intracellular signaling molecules in EGF and FGF2 signalings. Wt and Gab1–/– cells from E12.5 whole, E14.5 dorsal, or E14.5 ventral spinal cords were grown for 72 hours in the presence of 20 ng/ml FGF2 (E12.5 whole and E14.5 dorsal) or 20 ng/ml EGF (E14.5 ventral) and then treated with 20 ng/ml of EGF or FGF2 for 15 minutes. The cells were lysed and subjected to immunoblot analysis using the indicated antibodies. (B–D): In vitro rescue of the Gab1–/– phenotype in relation to the EGF-dependent proliferation of Olig2+ progenitors by the introduction of constitutively active Akt1. Wt and Gab1–/– cells from E12.5 whole and E14.5 dorsal spinal cord were infected with lentiviruses expressing Venus (control) or the active form of Akt1 and Venus and cultured for 72 hours in the presence of EGF followed by double immunocytochemistry for Olig2 with Venus (using GFP antibody). The infection efficiency as recognized by the expression of Venus was 40%–60% throughout the experiment. Counting was conducted in ten randomly chosen visual fields (>300 Venus+ cells). In E12.5 cultures, expression of mAkt1 in the Gab1–/– cells significantly increased the number of Olig2+ cells within the Venus+ cell population as compared with that in cells infected with control viruses, and to the same level as that in the wt cells; on the other hand, no such increase was observed in the wt cells (B, C) (*, p < .05, n = 4 embryos). In E14.5 dorsal spinal cord cell cultures, unlike E12.5 cultures, mAkt1 expression did not rescue the Gab1–/– phenotype (B, C) (n = 5 embryos). Each value (blue bars: wt; red bars: Gab1–/–) represents the mean ± SEM. Expression and function of mAkt1 were confirmed by immunoblot analysis of E12.5 spinal cord cells (wt) proliferating in the presence of EGF with antibodies against pAkt1 (Thr-308), which recognized truncated mAkt1 (47 kDa) and phosphorylated Akt substrates (D). Cells were lysed 30 minutes after washing out of the EGF. Abbreviations: E, embryonic day; EGF, epidermal growth factor; FGF, fibroblast growth factor; GF–, without growth factor; IB, immunoblot; mAkt1, active form of Akt1.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Regulation of Progenitor Proliferation by Growth Factor Signalings in the Developing Spinal Cord
In the developing spinal cord, after the cessation of motoneuron generation, Gab1 deficiency resulted in a reduction in the number of Olig2⁺ progenitors in the pMN domain, followed subsequently by a reduction in the subpopulation of Pax7⁺ dorsal progenitors expressing EGFR, without any detectable increase of apoptosis (Figs. 1, 3; data not shown); the cells also showed failure of EGF-dependent proliferation in vitro (Figs. 2, 5). In contrast, the proliferation of not only Olig2⁺ progenitors, but also that of Olig2^– progenitors in response to FGF2 stimulation in vitro, was maintained to the same extent in the Gab1-deficient cultures as that in the wild-type cultures (Fig. 2; data not shown), despite the fact that Gab1 mediates signals to the Akt pathway in FGF signaling in mouse embryonic fibroblasts (MEFs) [46]. It has been shown that FGF receptor substrate 2 (FRS2), another adaptor protein belonging to the common insulin receptor substrate family, functions as a key mediator in FGF signaling in other types of cells, including cortical progenitor cells [47, 48]. Moreover, Gab2 mediates Akt activation by FGF2 during retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells [23]. In our study, however, FGF2 stimulation scarcely activated Akt1 in spinal cord progenitors (Fig. 5; data not shown); this suggests that the effect of FGF2 on progenitor cell proliferation may be mediated mainly by the FRS/MAPK pathway. Alternatively, another signaling pathway, such as integrative nuclear FGFR1-signaling [49], may regulate the FGF2-dependent proliferation. Reduced progenitor proliferation outside of EGFR-expressing pMN domain, particularly in the dorsal segment, in the E12.5 and 13.5 spinal cord of Gab1^–/– mice (Figs. 1, 3), suggests the existence of mitotic signal other than EGF and FGF2 utilizing Gab1. On the other hand, there was no reduction in the number of Pax7⁺ dorsal progenitors, even at E14.5 (Figs. 1, 3), suggesting that cell death, which may be scarcely detectable because of the rapid clearance of the cell corpses after apoptosis [50] and/or differentiation of the progenitors, is also suppressed in the Gab1^–/– mice.

Downstream of Gab1
Signal transduction through Gab1, downstream of EGF signaling, also depends on the cellular context. Previous reports showed reduction of EGF-induced activation of MAPK in Gab1^–/– MEFs [24] and MAPK-kinase in HEK293 cells transfected with Gab1 small interfering RNA [51]. However, in this study, MAPK activation was maintained in Gab1^–/– spinal cord progenitors, whereas activation of Akt1 was significantly reduced (Fig. 5). This discrepancy between these two results may be explained by the existence of a preference for the signaling cascade downstream, depending on the cell types, for the same ligand [52]. Alternatively, there may be differences in the signaling crosstalk between MAPK and PI3K/Akt pathways downstream of Gab1. It has been suggested that Gab1 mediated PI3K/Akt activation extends duration of Ras/MAPK signaling [51]. The contribution magnitude of Akt1 to the EGF-dependent proliferation of Olig2⁺ progenitors is also context-dependent. Expression of an active form of Akt1 could rescue the defect in the E12.5 but not E14.5 spinal cord of Gab1^–/– mice (Fig. 5). It has been shown that the balance of PI3K/Akt and MAPK determines the phenotypes of smooth muscle cells [53]. Therefore, an imbalance between the two signaling pathways rather than a mere reduction of the absolute level of active Akt1 might cause the defect of proliferation in the Gab1^–/– mice. If that is indeed the case, the critical balance should be different in different types of cells. Indeed, Olig2⁺ progenitors derived from the ventral spinal cord of the E14.5 Gab1^–/– mouse proliferate normally in response to EGF, despite the significantly lower level of active Akt1 than that in the wt mouse (Figs. 3, 5). Alternatively, Olig2⁺ progenitors from E14.5 dorsal spinal cord may require unknown Gab1 targets other than or in addition to Akt and/or MAPK for their EGF-dependent proliferation. In the ventricular zone of the embryonic mouse cortex, a small population of cells shows high levels of expression of Akt1. Enhancement of Akt1 expression with a retrovirus vector induced increased phosphorylation of Akt1 at Thr-308 and the number of EGF-responsive neurospheres [54]. This report is consistent with our results, at least those obtained in the E12.5 spinal cord. Although our analysis of the activation of signaling molecules in cells expanded in the presence of FGF2 (Fig. 5A) might not totally reflect actual signal response in EGF-responsive Olig2⁺ progenitors originating in the developing spinal cord because it has been reported that FGF2 induces Olig2⁺ progenitors from Olig2^– progenitors [38, 55] and EGFR expression in EGFR-negative progenitors [56], taken together with the results from in vitro rescue experiments (Fig. 5B–5D), it would be reasonable to surmise that the Gab1/Akt cascade is essential, depending on the cellular context, for the proliferation of Olig2⁺ progenitors in response to EGF.

Specific Roles of EGF Signaling
EGFR-mediated signaling has been shown to be a mitotic signal for neural progenitor cells, including stem cells [32, 57–60]. Therefore, the functions of EGFR could be, at least in a part, embodied in the functions of EGF-responsive NSPCs. In the developing spinal cord, EGFR expression begins to be detected predominantly in the Olig2⁺-pMN domain of the spinal cord after the cessation of motoneuron generation, and subsequently extends to the dorsal segment (Fig. 2D). Moreover, we demonstrated that EGF specifically maintains both the mitotic and undifferentiated state of Olig2⁺ progenitors derived from the E12.5 ventral spinal cord and E14.5 dorsal spinal cord of the mouse in vitro. Finally, analysis of EGFR-deficient mice confirmed the contribution of the EGF signal to the expansion of Olig2⁺ progenitors in vivo. Since Olig2⁺ progenitors located in these regions are major sources of oligodendrocytes and astrocytes in vivo [8–13, 61], the spatiotemporal regulation of the expression of EGFR could be important for the supply of pools of NSPCs for the spatiotemporally regulated generation of glia.

	CONCLUSION

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In normal CNS development, generation of specific types of cells is properly regulated in relation to their number and timing. Proliferation of various types of neural progenitors is a major part of such regulation and is dependent on the integration of multiple signaling pathways, including growth factor signaling. Here, we demonstrated the function of Gab1, which is a common signaling mediator for several growth factor signals involved in CNS development. We provide evidence suggesting that Gab1 contributes to the proliferation of Olig2-expressing neural progenitors downstream of EGF signaling in a spatiotemporally regulated manner in the developing spinal cord. We further demonstrated a context-dependent change in the utilization of Akt1 as a downstream target of Gab1 in the EGF-dependent proliferation of Olig2-expressing progenitors. These findings suggest that, in addition to the differential expression of ligands and receptors, differential utilization of intercellular signaling components is integrated into the regulation of progenitor proliferation to complete the CNS histogenesis by growth factor signals.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
We are grateful to Dr. M. Nakafuku and Dr. H. Takebayashi for supplying us with the antibodies and T. Miyao for assistance with the mouse breeding. We also thank the other members of Okano laboratory, in particular M. Yano and Y. Okada, for their useful advice and encouragement. This study was supported by CREST/SORST-JST (H.O.), grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology in Japan (T.S. and H.O.), and a grant-in-aid for JSPS Fellows (Y.Y.).

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