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J. Biol. Chem., Vol. 281, Issue 19, 13374-13381, May 12, 2006
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From the
Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, the
Department of Cellular and Molecular Medicine and the ¶Department of Developmental Biology, Graduate School of Medicine, Chiba University, Chiba 260-8670, the ||Department of Physiology and **Bridgestone Laboratory of Developmental and Regenerative Neurobiology, Keio University School of Medicine, Shinjyuku-ku, Tokyo 160-8582, and 
REDS Group, Saitama Small Enterprise Promotion Corp., Skip City, Kawaguchi, Saitama 333-0844, Japan
Received for publication, November 28, 2005 , and in revised form, March 16, 2006.
| ABSTRACT |
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-geo reporter gene under the control of the SRR2 in order to examine the spatiotemporal function of this regulatory region. We show that the SRR2 functions specifically in neural stem/progenitor cells. However, unlike Nestin 2nd intronic enhancer, the SRR2 shows strong regional specificity functioning only in restricted areas of the telencephalon but not in any other portions of the central nervous system such as the spinal cord. We also show by in vitro clonogenic assay that at least some of these SRR2-functioning cells possess the hallmark properties of neural stem cells. In adult brains, we could detect strong
-geo expression in the subventricular zone of the lateral ventricle and along the rostral migrating stream where actively dividing cells reside. Chromatin immunoprecipitation assays reveal interactions of POU and Sox factors with SRR2 in neural stem/progenitor cells. Our data also suggest that the specific recruitment of these proteins to the SRR2 in the telencephalon defines the spatiotemporal activity of the enhancer in the developing nervous system. | INTRODUCTION |
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Sox2 encodes an SRY-related high mobility group box transcription factor and is expressed in at least three types of stem cells, i.e. neural stem cells (NSC), embryonic stem (ES) cells, and trophoblast stem cells, but not in their differentiated derivatives (5-12). Gene targeting experiments have revealed a cell-autonomous requirement for Sox2 in multipotent cells in early embryonic development (8), suggesting that Sox2 may also be involved in the maintenance of the stem cell state of NSCs. However, because Sox2 null mutant mice failed to develop beyond implantation, the role of this protein in brain could not be examined by conventional knock-out analysis. Notwithstanding, studies from several laboratories have demonstrated the importance of Sox2 for NSC identity. Graham et al. (13) demonstrated that constitutive expression of Sox2 results in maintenance of the neural stem/progenitor cell state and blocks neuronal differentiation. In addition, Bylund et al. (14) showed that the Sox2 protein plays an important role in maintaining neural progenitor identity by counteracting the function of the basic helix-loop-helix-containing proneural transcription factors. Most intriguingly, recent knock-down analyses with RNA interference technology have demonstrated a pivotal role for Sox2 in the conversion of oligodendrocyte progenitor cells into NSC-like cells (15), suggesting that Sox2 function is not limited to maintenance of stem cell state but is also involved in acquisition of stem cell identity.
Because Sox2 appears to play such an important role in NSCs, we have been interested in unraveling the regulatory mechanisms that control its transcription. We previously identified a Sox2 enhancer, termed Sox2 regulatory region 2 (SRR2), that is specific to ES cells in the pluri-potent state (16). We subsequently showed that the SRR2 also functions in neural stem/progenitor cells (17).
Here we characterize the SRR2 function in the developing brain, and we show that it functions in neural stem/progenitor cells in the telencephalon but not in other regions of the CNS, such as the hindbrain and spinal cord. We also show by chromatin immunoprecipitation assay using developing brain that Sox2 and class III POU proteins, such as Brn1 and Brn2, interact with the SRR2 enhancer specifically in the telencephalon, implicating that specific recruitment of Sox2-class III POU protein complexes on the SRR2 in the telencephalon is the key determinant for the regional specificity of SRR2 in embryonic brain.
| EXPERIMENTAL PROCEDURES |
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-geo reporter construct was generated as follows. SRR2 was amplified as a BglII/BamHI PCR fragment using the following oligonucleotides: 5'-TAGGATCCCGGGGCGCTTTCTGCCCTTCAG-3' and 5'-GCAGATCTAGCGCTGCAGTTTATCAAAGCA-3'. This fragment was ligated to the BamHI/NcoI tk promoter from the tk-Luc reporter plasmid (16), and the resulting BglII/NcoI fragment was isolated. Intronic sequence and a poly(A) signal sequence were isolated as an XbaI/SpeI PCR fragment, using the pGL2 basic plasmid (Promega) as a template. These two DNA fragments were mixed with an NcoI/ XbaI fragment from pGT1.81IresBgeo that bears the entire
-geo coding region (18) and then subcloned into a modified pBluescript II KS+ vector with a BglII site inserted between the KpnI and EcoRI sites. Generation of Transgenic MiceTransgenic mice were generated by pronuclear DNA injection into fertilized oocytes from C57BL6 mice (19). Four lines (lines 16, 29, 43, and 54) showed reporter gene expression in the ventricular zone of the telencephalon at 10.5 days post-coitum (dpc) and 12.5 dpc and were propagated through breeding with C57BL6 mice.
Neurosphere CultureNeurospheres were generated from 12.5 dpc embryonic mouse brains as described previously (17). Dissociated brain cells were cultured at clonal density (1,000 cells per well). Cells were cultured in medium with 200 µg/ml G418 when selecting
-geo-positive neurospheres.
X-Gal StainingWhole embryos (10.5 or 12.5 dpc) and floating sections (14.5 or 16.5 dpc) were stained with X-gal according to Zappone et al. (20). Adults were perfused with 1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA, 0.02% Nonidet P-40 in PBS, and the brain was dissected, fixed for 90 min in the same fixative, and sectioned at 100 µm using a vibratome. Sections were post-fixed for 30 min, washed, and stained in the dark in PBS containing 5 mM K3Fe(CN)6, 5 mM K4(CN)6, 2 mM MgCl2, 0.02% Nonidet P-40, and 1 mg/ml X-gal for 4 h.
AntibodiesThe following antibodies were used in this study: anti-Sox2 (rabbit IgG; Chemicon); anti-phosphohistone H3 (clone 6G3; Cell Signaling Technology); anti-MAP2 (clone HM-2; Sigma); anti-GFAP (clone GA5; Sigma); anti-PSA-NCAM (mouse IgM from Dr. Seki, University of Juntendo, Japan (21)); anti-Nestin (clone RAT401; Pharmingen); anti-
-galactosidase (rabbit IgG; Biogenesis); anti-Brn1 (goat IgG, Santa Cruz Biotechnology); anti-Brn2 (goat IgG, Santa Cruz Biotechnology); anti-acetylated histone H3 (rabbit IgG, Upstate); and anti-dimethylated histone H3-K4 (rabbit IgG, Abcam).
ImmunostainingImmunocytochemistry was performed according to Hirabayashi et al. (22). For sections of embryonic brain tissue, dissected brains were fixed overnight in 4% paraformaldehyde in PBS, cryoprotected in 30% sucrose, frozen, and sectioned at 12 µm. Frozen sections were immunostained as described previously (17). For sections of adult brain, mice were perfused with 4% paraformaldehyde in PBS. Brains were dissected, post-fixed overnight in the same fixative as used for embryonic tissue, and sectioned at 50 µm using a vibratome. Free-floating sections were blocked with 10% normal goat serum, 0.1% Triton X-100 in PBS for 2 h and incubated for 16 h at 4 °C with primary antibodies, followed by incubation with the appropriate Alexa Fluor dye-conjugated secondary antibodies (Invitrogen). Staining was visualized using confocal microscopy (Leica Microsystems).
Chromatin Immunoprecipitation Assay (ChIP)ChIP was performed according to Saba et al. (23). Immunoprecipitated DNA was amplified using the following primers: SRR2, 5'-TCATTTCAGGTGTAGAGTTGG-3' and 5'-CCTATGTGTGAGCAAGAACTG-3'; Nestin enhancer, 5'-TTGTCTGTCACCAGCTCTGG-3' and 5'-TTCGATCAGACTCCTCAGATC-3';
-actin, 5'-GGTCAGAAGGACTCCTATGT-3' and 5'-ATGAGGTAGTCTGTCAGGTC-3'; and c-Myc, 5'-GACGCTTGGCGGGAAA-3' and 5'-CTCTGCACACACGGCTCTT-3'.
Bisulfite Sequencing Analyses1 µg of genomic DNA isolated from forebrain or spinal cord was digested with EcoRI and then subjected to bisulfite treatment, in which unmethylated cytosines are converted to uracil residues, as described by Clark et al. (24). The bisulfite-modified DNA was then used as a template for Nested PCR to amplify a region containing SRR2. The primers used for PCRs were as follows: 1st PCR, 5'-TTATTCAGTTCCCAATCCAAAC-3' and 5'-TCAAATATATACCAAACCCCTTCC-3'; 2nd PCR, 5'-CAATCCAAACTAAACAAATTCC-3' and 5'-ACATATACATTTTATACTATCC-3'. The PCR products were subcloned into pGEM-T easy vector (Promega) and analyzed by sequencing.
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-geo mice (10 weeks old) were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) and placed in a stereotaxic frame as described by Johansson et al. (25). The needle (Hamilton, 26-gauge) was inserted at 0.5 mm anterior and 1.3 mm lateral to bregma and 3.5 mm below the dura mater and then withdrawn. The animals were sacrificed after 4 days post-surgery. | RESULTS |
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-geo transgenic mice and examined
-galactosidase activity during development. To avoid any potential effects of chromosomal integration site on gene expression, we used a larger fragment (+3300 to +4124) that showed similar activity as the smaller fragment when introduced into E13.5 mouse brains by in utero electroporation (data not shown).
Of 13 transgenic founder males tested, four (lines 16, 29, 43, and 54) generated progeny with
-galactosidase expression in the telencephalon at 10.5 and 12.5 dpc (data not shown, but see Fig. 1, A and B for reference), but no expression was detected in other parts of the nervous system. No
-galactosidase-expressing progenies were obtained from the remaining nine founders. Detailed examination of stable transgenic lines generated from the two founders (lines 16 and 54) exhibited essentially the same pattern of
-galactosidase expression at any developmental stages and adult stage (data not shown); therefore, we show only data obtained from line 16 (Fig. 1).
At 8.5 dpc, endogenous Sox2 expression is evident in the neuroepithelium, which is largely composed of multipotent NSCs (8, 26-28). However, we could not detect SRR2-
-geo reporter gene expression at this early stage of brain development (data not shown).
-Galactosidase expression became evident only after 10.5 dpc and was confined to the telencephalon (Fig. 1A). This pattern of expression persisted through later stages (Fig. 1, B and C). Sections of brains from 14.5 dpc (Fig. 1D) and 16.5 dpc (Fig. 1E) embryos revealed that SRR2 functioning domains resembled the expression profile of the gene, BF-1 encoding one of fork head type of transcription factors (29, 30), i.e. strong expression in the ganglionic eminence and most of the cortex. Although
-galactosidase expression covered almost the whole cortex at the rostral levels, it was excluded from the medial and dorsal cortex at the caudal levels. Strong
-galactosidase staining was detected in the ventricular zone (VZ), where undifferentiated neural stem/progenitor cells reside. However, expression was weak in the intermediate zone and was completely absent in the cortical plate (Fig. 1, D and E). Fig. 1, F and G, shows double
-galactosidase and Sox2 antibody staining of a sectioned 10.5 dpc brain, demonstrating extensive colocalization of SRR2 activity and endogenous Sox2.
The SRR2 Functions in Neural Stem/Progenitor Cells but Not in Post-mitotic Neurons in the Developing BrainTo determine whether the
-galactosidase-positive cells in the VZ are neural stem/progenitor cells, we performed double antibody staining against
-galactosidase and either Nestin or MAP-2, which are markers for neural stem/progenitor cells and post-mitotic neurons, respectively, using forebrain from 14.5 dpc transgenic embryos. Expression domains of
-galactosidase and Nestin are overlapping in VZ cells of the developing telencephalon (Fig. 2, A-F), although the expression domains of
-galactosidase and MAP2 are mutually exclusive (Fig. 2, G-I). Immunostaining with anti-phosphohistone H3, a specific marker for mitotic cells, revealed that most phosphohistone H3-positive cells are also positive for
-galactosidase (Fig. 2, J-L). Although some cells are clearly positive for both
-galactosidase and Nestin, a substantial number of cells that are positive for Nestin are negative for
-galactosidase (Fig. 2, C and F). This apparent lack of colocalization may not represent the presence of Nestin+/
-galactosidase- cells but differences in the subcellular distribution of these two proteins. To determine whether or not the proteins are colocalized more clearly, forebrain tissue from transgenic embryos was dissociated into single cells and plated onto coverslips for antibody staining. Representative examples are shown in Fig. 2, M-T. We found that 95 ± 11.3% (n = 3) of
-galactosidase-positive cells are also positive for Nestin, whereas only 9.3 ± 2.1% (n = 3) of
-galactosidase-positive cells express MAP2. Taken together, these results strongly suggest that the SRR2 enhancer functions mainly in Nestin-positive neural stem/progenitor cells but not in MAP2-positive post-mitotic neurons in the developing telencephalon.
Cells Expressing
-Galactosidase under the Control of the SRR2 Element Include Neural Stem CellsBecause Nestin marks lineage-restricted progenitor cells in addition to neural stem cells, we performed clonogenic analyses in order to determine whether SRR2-
-geo cells in the telencephalon include bona fide neural stem cells. Dissociated cells from 12.5 dpc embryonic mouse brains were cultured at clonal density (1,000 cells/well) in the absence or presence of G418, which selects for SRR2-
-geo cells, and were permitted to develop into primary neurospheres. Under these conditions, more than 95% of the neurospheres generated were clonal, as revealed by mixing experiments using enhanced green fluorescent protein- or enhanced cyan fluorescent protein-positive cells (data not shown). As shown in Fig. 3, A and B, when cultured in G418-containing medium, neurospheres could be efficiently produced only from forebrain- and not mid/hindbrain-derived cells. Moreover, without G418 selection, neurospheres with strong
-galactosidase expression could be recovered only from the forebrain, although all neurospheres from the mid/hindbrain showed weak or no X-gal staining. These results indicate that regional specificity of the SRR2 element in the developing brain persists even in vitro. It is likely that the
-galactosidase-negative (or weakly positive) neurospheres in Fig. 3B, upper-left panel (asterisks), were derived from SRR2-negative cells in other parts of the telencephalon, such as the dorsal region (Fig. 3B). Neurospheres generated in the absence of G418 were composed exclusively of either
-galactosidase-positive or -negative cells, but never a mixture of the two, confirming that the neurospheres were indeed clonal. To assess the self-renewal capacity of SRR2-
-geo-positive cells, the efficiency of secondary neurosphere formation was compared between neurospheres obtained under and without G418 selection. We observed that primary neurospheres obtained under G418 selection show higher self-renewing ability than neurospheres obtained without G418 selection, although the difference is not prominent. Moreover, we found that neurospheres from SRR2-
-geo cells could be passaged and expanded in G418-containing media for at least 2 months (data not shown).
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-geo cells, G418-selected primary spheres were induced to differentiate in medium containing fetal bovine serum and retinoic acid and then stained with antibodies against neural cell markers. As shown in Fig. 3D, both neurons and glia, which are marked by MAP2 and GFAP, respectively, were detected in the progeny of single SRR2-
-geo cells. We conclude from these results that the SRR2-functioning neurosphere cell population contains at least equivalent or even higher amount of cells that possess the self-renewal and multipotent properties of neural stem cells compared with neurosphere cells obtained without G418 selection.
The SRR2 Element Acts as an Enhancer in Adult Neurogenic Regions New neurons are continually generated also in the adult vertebrate brain (1, 2). However, unlike the embryonic stages, neurogenesis in the adult brain usually occurs only in restricted areas. In mammals, the subventricular zone (SVZ) of the lateral ventricle and the subgranular layer (SGL) of the hippocampal dentate gyrus are known to be involved in neurogenesis at adult stages. Specifically, the slowly dividing SVZ astrocytes (also termed type B cells) have stem cell character and are able to generate neuroblasts (type A cells) via the transit amplifying cells (type C cells). These newly formed neuroblasts migrate through glial tunnels formed by SVZ astrocytes (rostral migrating stream (RMS)) and then differentiate into olfactory bulb neurons (31, 32). Because Sox2 is known to be expressed in these neurogenic regions (26, 33) (also see Fig. 4A, right panel for reference), we examined SRR2 activity in the adult brain by X-gal staining. We were able to detect stained cells in the periventricular cells of the lateral ventricle (Fig. 4A) and along the RMS, which reaches to the olfactory bulb (Fig. 4B).
-Galactosidase expression in olfactory bulb neurons, in which Sox2 is not normally expressed, is likely due to perdurance of the protein.
-Galactosidase expression was also detected in the SGL of the dentate gyrus, albeit very weakly (data not shown). To further characterize the
-galactosidase-positive cells in the SVZ, we stained sections from SRR2-
-geo mouse brains with antibodies against GFAP and PSA-NCAM, which mark type B and A cells, respectively. As expected, most LacZ-positive cells also expressed either GFAP (Fig. 4, C-G) or PSA-NCAM (Fig. 4, H-L). These results suggest that the SRR2 element functions to up-regulate Sox2 expression in both type A and B cells.
From the analyses of Nestin gene expression, it has been demonstrated that the numbers of reactive astrocytes increase in adult brain in response to injury (25). Therefore, we characterized the function of the SRR2 enhancer in the injured adult brain. With the aid of a stereotaxic frame, the brains of SRR2 transgenic mice were subjected to needle injury, and
-galactosidase expression was analyzed following injury. Data from three independent experiments indicate that the number of
-galactosidase-positive cells in the striatum close to the needle scar was about 3-fold higher than observed in the same region in sham-operated control mice (sham operated mice, 99 ± 21.2 versus injured mice, 289 ± 78.2 in 0.4 x 0.3 mm) (Fig. 4, M and Q), although no obvious change in
-galactosidase expression was evident in the ventricular wall. Immunostaining using anti-GFAP antibody suggests that the increased
-galactosidase-positive cells in number were reactive astrocytes (Fig. 4, O, P, and S-X) that proliferate as part of an injury response. Collectively, our data demonstrate that the SRR2 enhancer regulates Sox2 expression in neural stem/progenitor cells in adult neurogenic regions and reactive astrocytes in the striatum, and at least,
-galactosidase-positive reactive astrocytes respond to CNS injury.
The SRR2 Enhancer Recruits Sox2 and Class III POU Proteins in the Telencephalon but Not in the Spinal CordIn our previous studies, we demonstrated that an octamer- and Sox-binding sequence plays a pivotal role in mediating transcriptional activation via SRR2 in ES cells and the developing brain, and multimerization of this sequence is sufficient to drive reporter gene expression in neural stem/progenitor cells. Moreover, we showed that, like Oct-3/4, class III POU proteins such as Brn1 and Brn2, which are known to be expressed in neural stem and progenitor cells, can cooperate with Sox2 to enhance SRR2 activity in COS cells (17). To further substantiate these data, we first examined the expression of these transcription factors in the developing brain. As expected, Sox2 was expressed in the VZ of the developing telencephalon (Fig. 5, A, D, and G). Brn1 and Brn2 were also expressed in VZ cells of the telencephalon. However, unlike Sox2, which is already detectable in the neural plate at early stages (for details, see Ref. 12), Brn1 expression was evident only after 11.5 dpc, with expression persisting until 14.5 dpc (Fig. 5, B and E) and dropping off by 16.5 dpc (Fig. 4H). Brn2 was strongly expressed in the VZ cells of the telencephalon only after 16.5 dpc, with only a weak signal detected at earlier stages (Fig. 5, C, F, and I).
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-actin gene. The Nestin enhancer is known to function in both the forebrain and spinal cord (34) and, like the SRR2, is controlled by Sox-POU complexes (35). Consistent with these reports, the Nestin enhancer DNA was amplified efficiently from immunoprecipitated chromatin from both forebrain and spinal cord tissue. In contrast, the SRR2 DNA was amplified from immunoprecipitated chromatin from forebrain tissue but not from spinal cord tissue. As a negative control, we confirmed that
-actin could not be amplified from immunoprecipitated DNA from forebrain or spinal cord (Fig. 6A).
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The above data suggest that the chromatin structure at the SRR2 element may allow access to Sox2-Brn complexes in the telencephalon but not other regions of the CNS. To test this idea, we performed ChIP analyses with antibodies against acetylated histone H3 and dimethylated H3-K4, which mark active chromatin. However, no significant differences were evident in either histone H3 acetylation or methylation levels between the telencephalon and spinal cord (Fig. 7A). Moreover, as shown in Fig. 7B, we found that CpG methylation patterns of SRR2 in the spinal cord were essentially the same as those in the telencephalon. Therefore, at present, we have no evidence supporting the above hypothesis, although our results do not rule out the possibility that differences in chromatin structure account for differential activity of the SRR2 element. Another possibility is that a telencephalon-specific factor is required for SRR2 to recruit Sox2-Brn complexes. Alternatively, a molecule that specifically prevents binding of Sox2-Brn complexes to SRR2 is present everywhere in the brain except the telencephalon.
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| DISCUSSION |
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In this study, we addressed this issue directly by transgenic analysis. Although our data demonstrated that the SRR2 enhancer indeed functions in neural stem/progenitor cells during development, we also found that it shows strong regional specificity for restricted areas of the telencephalon, with no activity in other portions of the CNS, such as the spinal cord. These data contrast with those obtained with the Nestin 2nd intronic enhancer, which displays its activity in neural stem/progenitor cells present in entire portions of the CNS (33). Our data also suggested that the inactivity of the SRR2 in regions outside of the telencephalon is because of the failure to recruit Sox2-Brn complexes in these regions.
SRR2 is not the only Sox2 regulatory element that functions specifically in telencephalic neural stem/progenitor cells. Zappone et al. (20) have shown that a region upstream of Sox2 directs such expression. However, this regulatory region mediates expression rather specifically in the dorsal part of the telencephalon, which is mostly complementary to
-galactosidase-positive domains of SRR2-
-geo embryos, suggesting that endogenous Sox2 expression in the telencephalon reflects the combined actions of these two regulatory regions. The SRR2 also exerts its activity in the adult brain. The SRR2 element drives strong reporter gene expression in the SVZ as well as the RMS. However, only weak activity is evident in the SGL of the hippocampal dentate gyrus. Although the origin of the neural stem/progenitor cells of these adult neurogenic regions is not known, it is assumed based on anatomical location that stem cells in the SVZ are derived from the ganglionic eminence in the telencephalon, although those in the SGL originate from the medial telencephalon. If this is indeed the case, the
-galactosidase expression in the adult SRR2-
-geo mouse is rather expected, based on the embryonic expression pattern.
It is known that neurospheres derived from different parts of the brain are heterogeneous with respect to gene expression (36). However, Sox2 appears to be a marker common to all cells within these heterogeneous populations (10, 12, 28, 37). Therefore, it would be reasonable to assume that Sox2 expression is maintained by a single regulatory enhancer acting in all of these cells. However, data from many laboratories (20, 38) as well as those described in this study have shown that this is not the case; pan-neural Sox2 expression results from the combined actions of many regulatory enhancers, each functioning in a specific area of the brain.
Studies in chick embryos showed that most of the regulatory regions involved in Sox2 expression in the brain have a Sox-binding sequence, indicating the existence of an autoregulatory loop of Sox2 expression in the brain. The SRR2 also contains a functional Sox-binding site whose mutation results in complete loss of transcriptional stimulating activity (17). Thus, this autoregulatory mechanism is at least in part conserved between chicken and mouse.
To our knowledge, the sequence identified by Zappone et al. (20) and the SRR2 element are the only Sox2 enhancers that function specifically in the telencephalon in mammals and are useful tools for marking and isolating neural stem/progenitor cells in the telencephalon. They will be especially useful for isolating telencephalic neural stem/progenitor cells generated during ES cell differentiation, because the majority of neuronal cells obtained by this method possess characteristics of midbrain dopaminergic neurons, whereas cells with telencephalic character represent only a minor population (39). It has been shown that neuronal cells derived from mouse ES cells, when engrafted in 6-hydroxydopamine-treated Parkinsonian rats, are able to reverse the behavioral deficits significantly (39, 40). The range of clinical applications for a pure telencephalic neural stem/progenitor cell population is tremendously large, because these cells could be used for treatment of major brain diseases such as those coupled to stroke. We assume that cell sorting using telencephalon-specific Sox2 regulatory regions will be one of effective means to enrich the telencephalic cells differentiated from ES cells. We also hope that these regulatory regions will be useful for elucidating the molecular mechanisms of gene regulation, as well as the cell fate decisions made by telencephalic neural stem/progenitor cells.
| FOOTNOTES |
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1 Present address: Radioisotope Experimental Laboratory, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan. ![]()
2 To whom correspondence should be addressed: Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan. Tel.: 81-42-985-7268; Fax: 81-42-985-7264; E-mail: akiokuda{at}saitama-med.ac.jp.
3 The abbreviations used are: CNS, central nervous system; SRR2, Sox2 regulatory region 2; ChIP, chromatin immunoprecipitation; NSC, neural stem cells; ES, embryonic stem; dpc, days post-coitum; X-gal, 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside; VZ, ventricular zone; SVZ, subventricular zone; SGL, subgranular layer; RMS, rostral migrating stream; PBS, phosphate-buffered saline; GFAP, glial fibrillary acidic protein. ![]()
| ACKNOWLEDGMENTS |
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| REFERENCES |
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