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Originally published In Press as doi:10.1074/jbc.M512669200 on March 16, 2006

J. Biol. Chem., Vol. 281, Issue 19, 13374-13381, May 12, 2006
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The Sox2 Regulatory Region 2 Functions as a Neural Stem Cell-specific Enhancer in the Telencephalon*

Satoru Miyagi{ddagger}§, Masazumi Nishimoto{ddagger}1, Tetsuichiro Saito, Mikiko Ninomiya||**, Kazunobu Sawamoto||**, Hideyuki Okano||, Masami Muramatsu{ddagger}, Hideyuki Oguro§, Atsushi Iwama§, and Akihiko Okuda{ddagger}{ddagger}{ddagger}2

From the {ddagger}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 {ddagger}{ddagger}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
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sox2 is expressed at high levels in neuroepithelial stem cells and persists in neural stem/progenitor cells throughout adulthood. We showed previously that the Sox2 regulatory region 2 (SRR2) drives strong expression in these cells. Here we generated transgenic mouse strains with the beta-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 beta-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
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neural stem cells possess the remarkable ability to self-renew and to differentiate into neurons or glia. They can be found during development of the CNS3 as well as in regions of the adult brain where neurogenesis persists (1-4).

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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—The SRR2-beta-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 beta-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 Mice—Transgenic 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 Culture—Neurospheres 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 beta-geo-positive neurospheres.

X-Gal Staining—Whole 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.

Antibodies—The 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-beta-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).

Immunostaining—Immunocytochemistry 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'; beta-actin, 5'-GGTCAGAAGGACTCCTATGT-3' and 5'-ATGAGGTAGTCTGTCAGGTC-3'; and c-Myc, 5'-GACGCTTGGCGGGAAA-3' and 5'-CTCTGCACACACGGCTCTT-3'.

Bisulfite Sequencing Analyses—1 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.


Figure 1
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  		FIGURE 1.
beta-Galactosidase activity in SRR2-beta-geo transgenic embryos at midgestation. A, 10.5-dpc embryo. B, 12.5-dpc embryo. C, 14.5-dpc embryo. D, brain sections from a 14.5-dpc embryo. E, brain sections from a 16.5-dpc embryo. F and G, a brain section from SRR2-beta-geo embryo at 10.5 dpc was stained with the antibodies as indicated at the bottom of each panel. Bars in A and B correspond to 1 mm, and those in D and E correspond to 0.5 mm. beta-gal, beta-galactosidase.

 
Injury Model—Adult SRR2-beta-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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The SRR2 Enhancer Element Drives Reporter Gene Expression in the Ventricular Zone of the Telencephalon—Our recent in vitro experiments have demonstrated that the Sox2 enhancer SRR2 (+3300 to +3677, the adenine nucleotide of the translation initiation codon is set to +1) drives transcription in neural stem/progenitor cells (17). To examine the spatiotemporal activity of SRR2, we generated SRR2-beta-geo transgenic mice and examined beta-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 beta-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 beta-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 beta-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-beta-geo reporter gene expression at this early stage of brain development (data not shown). beta-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 beta-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 beta-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 beta-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 Brain—To determine whether the beta-galactosidase-positive cells in the VZ are neural stem/progenitor cells, we performed double antibody staining against beta-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 beta-galactosidase and Nestin are overlapping in VZ cells of the developing telencephalon (Fig. 2, A-F), although the expression domains of beta-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 beta-galactosidase (Fig. 2, J-L). Although some cells are clearly positive for both beta-galactosidase and Nestin, a substantial number of cells that are positive for Nestin are negative for beta-galactosidase (Fig. 2, C and F). This apparent lack of colocalization may not represent the presence of Nestin+/beta-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 beta-galactosidase-positive cells are also positive for Nestin, whereas only 9.3 ± 2.1% (n = 3) of beta-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 beta-Galactosidase under the Control of the SRR2 Element Include Neural Stem Cells—Because Nestin marks lineage-restricted progenitor cells in addition to neural stem cells, we performed clonogenic analyses in order to determine whether SRR2-beta-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-beta-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 beta-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 beta-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 beta-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-beta-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-beta-geo cells could be passaged and expanded in G418-containing media for at least 2 months (data not shown).


Figure 2
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  		FIGURE 2.
The SRR2 enhancer functions in Nestin-positive neural stem/progenitor cells in the embryonic brain. A-L, brain sections from SRR2-beta-geo embryos at 14.5 dpc were stained with the antibodies indicated at the top of each panel. M-T, cells were recovered from embryonic brain, plated on a coverslip, and immunostained. These cells were also counterstained with 4,6-diamidino-2-phenylindole to visualize nuclei. D-F, magnified views of A-C. Dorsal is on top, and medial is to the right. Arrows in Q and R indicate cells that are positive for beta-galactosidase (beta-gal) but negative for MAP-2. lv, lateral ventricle. Bars in A and J, 100 µm; bars in D and G, 50 µm; bar in M, 20 µm.

 


Figure 3
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  		FIGURE 3.
SRR2-positive cells possess self-renewing and multipotent properties. A, cells from forebrain or mid/hindbrain regions of 12.5 dpc mouse embryonic brains were subjected to primary neurosphere formation in the presence (+) or absence (-) of G418, and generated colonies were counted. B, primary neurospheres were stained with X-gal. C, self-renewal of SRR2-beta-geo+ cells. Primary neurospheres with a diameter of about 0.2 mm were chosen from cultures of forebrain cells either with or without added G418. These neurospheres were then individually dissociated and subjected to secondary neurosphere formation in the absence of G418. The number of spheres generated was counted under a microscope. D, multipotency of SRR2-beta-geo+ cells. Primary neurospheres were plated on a laminin-coated coverslip and were induced to differentiate with medium containing fetal bovine serum and retinoic acid. Cells were stained with anti-GFAP and anti-MAP2 antibodies and were counterstained with 4,6-diamidino-2-phenylindole (DAPI). The bar corresponds to 20 µm.

 
Next, in order to examine the differentiation potential of SRR2-beta-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-beta-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). beta-Galactosidase expression in olfactory bulb neurons, in which Sox2 is not normally expressed, is likely due to perdurance of the protein. beta-Galactosidase expression was also detected in the SGL of the dentate gyrus, albeit very weakly (data not shown). To further characterize the beta-galactosidase-positive cells in the SVZ, we stained sections from SRR2-beta-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 beta-galactosidase expression was analyzed following injury. Data from three independent experiments indicate that the number of beta-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 beta-galactosidase expression was evident in the ventricular wall. Immunostaining using anti-GFAP antibody suggests that the increased beta-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, beta-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 Cord—In 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).


Figure 4
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  		FIGURE 4.
The SRR2 functions in adult neurogenic regions. A, beta-galactosidase staining of subventricular zone from an adult SRR2-beta-geo transgenic mouse (left panel) and immunostaining of the equivalent portion of the mouse brain with anti-Sox2 antibody (right panel). B, beta-galactosidase (beta-gal) staining of RMS. C-L, brain sections from SRR2-beta-geo transgenic mice were doubly stained with anti-beta-galactosidase and anti-GFAP antibodies (C-G) or anti-beta-galactosidase and anti-PSA-NCAM antibodies (H-L). E-G, magnified views of C and D and merged image of E and F. J-L, magnified views of H and I and merged image of J and K. M and Q, beta-galactosidase staining of brain sections from needle-injured (Q) and sham-operated (M) SRR2-beta-geo transgenic mice. N and R, magnified views of M and Q. O and P, a section from sham-operated SRR2-beta-geo mouse stained with anti-beta-galactosidase (O) and anti-GFAP antibodies (P). S-V, a section from injured SRR2-beta-geo mouse stained with anti-beta-galactosidase (S) and anti-GFAP antibodies (T). U, merged image of S and T. V-X, magnified views of S, T, and U. Str, lv, Cc, and OB represent striatum, lateral ventricle, corpus callous, and olfactory bulb, respectively, and an arrow with a dotted line indicates the injured position with the needle insertion. Bar in A, 200 µm; bar in V, 20 µm.

 


Figure 5
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  		FIGURE 5.
Expression patterns of Sox2, Brn1, and Brn2 in the developing brain. Brain sections were immunostained with the indicated antibodies. A-C, forebrain from 11.5-dpc embryo. D-F, forebrain from 14.5-dpc embryo. G-I, forebrain from 16.5-dpc embryo. J-L, spinal cord from 11.5-dpc embryo. M and N, diencephalon from 16.5-dpc embryo. lv, lateral ventricle; 3rd, third ventricle; CC, central canal. Bars in A, D and G, 100 µm; bars in J and M, 50 µm.

 
Together with the previously documented cooperative activation of the SRR2 enhancer by Sox2 and Brn, these observations may account for the inactivity of the SRR2 during early stages of neurogenesis (9.5 dpc). However, because we also observed expression of Brn1 and Brn2 in the spinal cord and diencephalon (Fig. 5, J-N), where Sox2 is expressed but SRR2 is inactive, we examined whether Sox2 and Brn can bind to SRR2 in vivo by a chromatin immunoprecipitation assay. We immunoprecipitated chromatin from the forebrain and spinal cord of 12.5 dpc embryos with anti-Sox2, Brn1, or Brn2 antibody, and we amplified the precipitated DNA by PCR using primers specific to the SRR2. We also examined the Nestin 2nd intronic enhancer and a portion of the beta-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 beta-actin could not be amplified from immunoprecipitated DNA from forebrain or spinal cord (Fig. 6A).


Figure 6
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  		FIGURE 6.
Sox2, Brn1, and Brn2 bind to the SRR2 sequence in the forebrain but not in the spinal cord. A, chromatins prepared from forebrain and spinal cord of 12.5 dpc embryonic brains were immunoprecipitated with the indicated antibodies, and recovered chromatin DNAs were subjected to PCR to amplify either the SRR2 enhancer, the Nestin enhancer, or the beta-actin gene. B, chromatin DNA was recovered as in A, using forebrain-derived primary neurospheres as the starting material. C, ChIP analysis with anti-Brn1 and anti-Sox2 antibodies was performed using mouse embryonic fibroblasts as the source of chromatin DNA.

 
Although neural stem/progenitor cells are the major cell type in the forebrain at 12.5 dpc, post-mitotic neurons do exist at this stage. Therefore, it was possible that the above data reflected the binding of Sox2, Brn1, and Brn2 proteins to the SRR2 in post-mitotic neurons. Therefore, to further support the interaction of Sox2-POU complexes with the SRR2 in neural stem/progenitor cells, we prepared primary neurospheres from the forebrain of 12.5 dpc mouse embryos to enrich these cell populations, and we then performed ChIP as above. As shown in Fig. 6B, we obtained comparable results as when embryonic forebrain tissue was used (Fig. 6A), suggesting that the SRR2 enhancer recruits Sox2 and Brn factors in neural stem and progenitor cells, although our data did not exclude the possibility that binding also occurs in post-mitotic neurons. As a negative control, we also performed ChIP with mouse embryonic fibroblasts and found that none of the regulatory regions were amplified (Fig. 6C), confirming the specificity of our immunoprecipitation reactions. These data suggest that regional specificity of SRR2 activation is because of the forebrain-specific recruitment of Sox2, Brn1, and Brn2 onto the SRR2.

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.


Figure 7
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  		FIGURE 7.
Chromatin status at the SRR2 in telencephalon and spinal cord. A, chromatins were recovered from forebrain and spinal cord with anti-dimethylated histone H3-K4 (Met) or anti-acetylated histone H3 (Ac) antibody. Recovered chromatin DNAs were subjected to PCRs to amplify either the SRR2 enhancer, the Nestin enhancer, or the c-Myc gene promoter. B, methylation status of a region around the SRR2 core sequence in telencephalon, spinal cord, and liver cells. The unmethylated (open circle) and methylated (solid circle) statuses of seven CpG sites are shown schematically. Numbers represent the positions where the adenine nucleotide of the translation initiation codon is set to +1. Oct and Sox indicate octamer and Sox-binding site-like sequences, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
During neurogenesis, Sox2 expression is first detected uniformly in the neural tube. As development proceeds, expression is detected all along the rostrocaudal axis of the nervous system, including the telencephalon, diencephalon, and spinal cord, but it is restricted to the VZ, which is occupied by neural stem/progenitor cells. Sox2 is also expressed in adult neurogenic regions, suggesting that Sox2 represents a common marker of neural stem and progenitor cells. We previously demonstrated mainly through in vitro tissue culture experiments that the SRR2 enhancer, which is located downstream of the Sox2 coding sequence, drives strong transcription in neural stem/progenitor cells (17), suggesting that it makes a significant contribution to the above in vivo Sox2 expression pattern.

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 beta-galactosidase-positive domains of SRR2-beta-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 beta-galactosidase expression in the adult SRR2-beta-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
 
* This work was supported in part by the Ministry of Education, Science, Sports, and Culture. This work was performed as part of the Rational Evolutionary Design of Advanced Biomolecules Project, the Saitama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, supported by the Japan Science and Technology Agency. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Radioisotope Experimental Laboratory, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan. Back

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-beta-D-galactopyranoside; VZ, ventricular zone; SVZ, subventricular zone; SGL, subgranular layer; RMS, rostral migrating stream; PBS, phosphate-buffered saline; GFAP, glial fibrillary acidic protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Tatsunori Seki for providing us with the anti-PSA-NCAM antibody. We also thank Dr. Toshihide Yamashita and Dr. Hidetoshi Ino for help with the brain injury experiments. We are also indebted to the "Laboratory Animal Resource Center, University of Tsukuba" for generation of transgenic mice. The Saitama Medical University Research Center for Genomic Medicine was the recipient of a grant from the Ministry of Education, Science, Sports, and Culture.



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 RESULTS
 DISCUSSION
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M. Cavallaro, J. Mariani, C. Lancini, E. Latorre, R. Caccia, F. Gullo, M. Valotta, S. DeBiasi, L. Spinardi, A. Ronchi, et al.
Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants
Development, February 1, 2008; 135(3): 541 - 557.
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