| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Division of Developmental Neuroscience, Center for Translational and Advanced Animal Research (CTAAR), Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
2 Mitsubishi Kagaku Institute of Life Sciences (MITILS), Minamiooya 11, Machida, Tokyo 194-8511, Japan
3 Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi-machi, Kodaira, Tokyo 187-8502, Japan
4 CREST, Japan Science and Technology Corporation (JST), 4-1-8 Honmachi, Kawaguchi, 332-0012, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
In hippocampal neurogenesis, dividing precursor cells give rise to daughter cells, which migrate away from the SGZ and start to differentiate into neurons (Seki & Arai 1993; Kuhn et al. 1996; Kempermann et al. 2003). Several lines of evidence have suggested that there are distinct subtypes of neural progenitor cells in the SGZ. In electron microscopy analysis, there are two types of mitotically active SGZ cells; type B cells that have ultrastructural features of astrocytes with light cytoplasm containing GFAP and multiple processes, rapidly convert into type D cells that are small electron-dense cells and GFAP negative (Seri et al. 2001). Another paper (Fukuda et al. 2003) has shown two distinct progenitor cells based on morphology, molecular expression, and electrical features: GFAP+ type I cells with lower input resistance (IR) and PSA-NCAM+ type II cells with higher IR. Roughly speaking, type I cells correspond to B cells, while type II cells to D cells. In this paper, we use GFAP+ early progenitors and PSA-NCAM+ late progenitors as clearer definition.
Although adult and embryonic neurogenesis differs in some aspects, there indeed is similarity. In the adult hippocampus, GFAP+ early progenitors have the appearance of radial glial cells, and not only do they produce neurons but also provide scaffolding for migration of newly born neurons (Forster et al. 2002). These features are quite similar to embryonic radial glial cells that constitute the ventricular zone (Gotz 2003; Tramontin et al. 2003). The finding that radial glia-like cells in the adult brain have stem cell properties (Doetsch et al. 1997, 1999; Seri et al. 2001) leads us to search for intrinsic molecular mechanisms that commonly govern pre- and postnatal neurogenesis.
A transcription factor Pax6 is strongly expressed during brain development in the discrete regions such as the dorsal telencephalon and the ventral hindbrain and serves as one of the key factor for patterning the central nervous system (see reviews by Osumi 2001; Simpson & Price 2002). Specific Pax6 expression is observed in the nucleus of the ventricular zone cells, which are most likely radial glial cells (Gotz 2003). In a spontaneous mutant Small eye (Sey) mouse that lacks functional Pax6, radial glial cells are less in number and show a distorted morphology, altered gene expression patterns and abnormal cell cycle characteristics (Stoykova et al. 1997; Gotz et al. 1998; Estivill-Torrus et al. 2002). A similar observation that less PCNA-positive cells constitute the thinner ventricular zone in Small eye rat mutant (rSey2) has also been reported (Fukuda et al. 2000). Curiously enough, Pax6 is expressed not only in the embryonic neuroepithelium but also in the adult brain including the SGZ and the SVZ (Stoykova & Gruss 1994; Nakatomi et al. 2002; Hack et al. 2004, 2005); the above-mentioned regions which are known as the places that neurogenesis persists in adulthood. All these lines of evidences have prompted us to examine the role of Pax6 in postnatal neurogenesis.
Here, we show that Pax6-expressing cells in the SGZ of the hippocampus have a neural progenitor-like character at the molecular and the cellular levels. Detailed BrdU pulse/chase experiments have revealed that the ratio of GFAP+ early progenitor cells in total BrdU+ cells decreased, and instead, PSA-NCAM+ late progenitors increased in the Pax6-deficient rSey2/+ rat. Therefore Pax6 is necessary for the maintenance of the GFAP+ early progenitor cells in the SGZ. We further searched for Pax6 downstream molecules that are relevant for the proliferation of neural progenitor cells in postnatal hippocampus, and found that expressions of Wnt ligands Wnt7a and Wnt7b and downstream effecter Dvl1 are changed in the DG of the rSey2/+. Our results suggest that a genetic cascade of Pax6–Wnt is critical for postnatal hippocampal neurogenesis especially at the early step.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Previous studies have reported that Pax6 is expressed in discrete regions of the postnatal brain such as the cerebellum and the limbic system including the olfactory bulb, olfactory cortex, and hippocampus (Stoykova & Gruss 1994; Nakatomi et al. 2002; Hack et al. 2004). In the present study, we focused on Pax6 expression in the DG of which knowledge on adult neurogenesis has been gathered.
In the DG of the wild-type rat at 4 weeks when the architecture of the hippocampus is established, many Pax6+ cells were observed in unique distribution patterns (Fig. 1A). A majority of Pax6+ cells were located in the SGZ, sometimes in clusters of five to eight cells (Fig. 1A,C). A much smaller number of Pax6+ cells were detectable in the hilus and molecular layer, whereas Pax6+ cells were scarcely found in the granule cell layer (GCL). To elucidate the character of Pax6+ cells, we performed double labeling with various markers for neural progenitor cells, neurons, and astrocytes (Fig. 1B,C). Many of Pax6+ cells co-expressed GFAP, a marker for astrocytes or early progenitors marker (59.0%, 85/144 cells; Fig. 1B,C), and a majority of Pax6/GFAP double-positive cells had processes oriented radially into the GCL of the DG (Fig. 1C,C'; also see Fig. 5E). Pax6+ cells also expressed neural stem cell markers nestin (34.4%, 20/58 cells) and Musashi1 (68.7%, 22/32 cells) (Fig. 1B). About a third of Pax6+ cells co-expressed a late progenitor marker PSA-NCAM (31.5%, 41/130 cells) (Fig. 1B). Contrastingly, Pax6+ cells scarcely expressed neuronal marker NeuN (1.9%: 12/652 cells) (Fig. 1B). These results suggest that Pax6+ cells exhibited the neural progenitor cell-like character.
|
|
Defects in the DG of rSey2/+ rats
In order to elucidate the role of Pax6 in hippocampal neurogenesis, we first observed the DG of Pax6-deficient rat (rSey2). rSey2 is a spontaneous mutant that has a nonsense mutation in the Pax6 gene (Osumi et al. 1997), although truncated Pax6 protein is undetectable in the homozygote (our unpublished observation). Since homozygous Pax6 mutant rats die at birth, heterozygotes (rSey2/+) were examined in this study. In the DG of rSey2/+ at 4 weeks, we found that the number of Pax6+ cells reduced and that an expression level of Pax6 also decreased (Fig. 2A). We could not observe an apparent difference in the architecture of the DG in rSey2/+ rats at this stage.
|
Decreased cell proliferation in the DG of rSey2/+ rats
To address the question whether postnatal neurogenesis is affected in the SGZ of Pax6-deficient rats, we first compared the total number of BrdU+ cells in the DG between the wild type and rSey2/+ at 4, 12, and 20 weeks. Rats were intraperitoneally injected with BrdU three times a day for 3 days and sacrificed at 24 h after the last BrdU injection (Kempermann & Gage 1999). In the DG of the wild type, the total number of BrdU-labeled cells in the SGZ per hemisphere decreased as the stage proceeded (Fig. 3A,B). Interestingly, a significant decrease in the total number of BrdU-labeled cells was observed in rSey2/+ at 4 weeks (33.3% decrease), 12 weeks (31.6%), and 20 weeks (26.2%) (Fig. 3B). These data clearly indicate that the number of proliferating progenitor cells is considerably reduced in the SGZ of Pax6-deficient rat.
|
In a BrdU labeling study for a longer period (2 weeks), the ratio of BrdU+/Pax6+ cells in the wild type was increased up to fivefold (35%) comparing with the samples labeled for a short period (30 min) (7.7% at 4 week; 6.3% at 6 week). This may imply that a population of cells expressing Pax6 contain neural stem cells (or quiescent GFAP+ early progenitor cells) whose cell cycle is longer than that of GFAP+ early progenitor cells. We also found that the number of Pax6+/GFAP+ double-positive cells in rSey2/+ was 22% less than that in the WT at 4 week (WT, 482 578 ± 33 757 cells/mm3; rSey2/+, 374 981 ± 21 527 cells/mm3; n = 4, P < 0.01). Furthermore, the number of BrdU+/Pax6+ double-positive cells was 27% decreased in rSey2/+ rats (WT, 238 208 ± 27 545 cells/mm3; rSey2/+, 174 921 ± 13 478 cells/mm3; n = 4, P < 0.01) in BrdU labeling study for a longer period (2 weeks). All these results consistently suggest that Pax6 is essential for proliferation of neural progenitor cells, thereby keeping the size of the progenitor pool.
The number of GFAP+ early progenitors decreased in the SGZ of rSey2/+
To further elucidate the role of Pax6 in hippocampal neurognesis, we investigated at which step a transition of neurogenesis is impaired by detailed BrdU pulse/chase experiments combined with immunostaining with progenitor markers at 4 weeks. At the beginning, we re-examined the character of Pax6+ cells in combination with BrdU labeling. Remarkably, more than 90% of total BrdU-incorporated cells in the SGZ expressed Pax6 at 30 min after BrdU injection (Fig. 4A,B). This result strongly suggests that Pax6 is vital for the cell proliferation in postnatal hippocampal neurogenesis. The fact that Pax6+ cells are highly proliferative may also explain why they were often seen in clusters in the SGZ (Fig. 1A,C).
|
The same analyses were then performed on the DG of the rSey2/+ rat. The percentage of Pax6+ cells in total BrdU+ cells slightly decreased but was statistically unchanged between the wild type and rSey2/+ (Fig. 4B). In contrast, we found a significant decrease (38.4% decrease at 24 h; 49.6% decrease at 72 h, P < 0.03) in the frequency of GFAP+ cells in total BrdU+ cells (Fig. 4C) and an opposite increase (15.1% increase at 24 h; 13.5% increase at 72 h, P < 0.03) in PSA-NCAM+ cells (Fig. 4D). These data clearly indicate that maintenance of GFAP+ early progenitor cells is extremely impaired in the DG of rSey2/+.
We performed BrdU pulse labeling study with a short survival period (5 days) to examine whether Pax6 accelerates the neuronal differentiation from dividing PSA-NCAM+ late progenitor cells to NeuN expressing immature neurons. The ratio of NeuN+/BrdU+ cells in this study was unchanged between WT and rSey2/+ (4w WT, 63.4%; 4w rSey2/+, 66.6%; n = 4, P = 0.36). Therefore, Pax6 may not be involved in neuronal differentiation but in maintenance of the GFAP+ early progenitor cells by regulating their proliferation.
We further examined how the character of neural progenitors in the DG was different between the wild type and rSey2/+ at 4 weeks. As described previously, 31.5% of Pax6+ cells co-expressed a marker for the late progenitor, PSA-NCAM (Figs 1B and 5C). Quite interestingly, Pax6+/PSA-NCAM+ cells dramatically increased up to 55.5% in rSey2/+ rats (45/81 cells; 76% increase than that of wild type; Fig. 5B,C). Moreover, GFAP and PSA-NCAM double-positive cells were scarcely detected in the DG of WT rats, while such GFAP+/PSA-NCAM+ cells were quite often observed in the DG of rSey2/+ rats (Fig. 5D). These results may imply that premature neuronal differentiation occurs in the DG of the rSey2/+. As seen in 16-week rats (Fig. 2C,C'), the number of GFAP+ cells was much less in the SGZ and hilus, and GFAP+ cells have thin and underdeveloped processes in rSey2/+ rats (Fig. 5A,E). Quantitatively, the level of GFAP expression was 16% less in the mutant hippocampus as judged from real-time polymerase chain reaction (PCR) (data not shown). These results suggest that hippocampal neurogenesis is quite abnormal in Pax6-deficient rat. All the findings consistently suggest a pivotal role of Pax6 in maintenance of the GFAP+ early progenitor cells in the postnatal hippocampus.
Wnt signaling is impaired in the DG of rSey2/+
What kinds of molecules then regulate cell proliferation under the control of Pax6 transcription factor? Among various candidate factors, we focused on Wnt signaling molecules because their expressions are reported in the postnatal DG (Shimogori et al. 2004) and also because we ourselves have shown down-regulation of a Wnt ligand expression in rSey2/rSey2 rat embryos (Osumi et al. 1997; Takahashi et al. 2002).
We first searched expression patterns of various Wnt signaling molecules by performing in situ hybridization of genes encoding Wnt ligands, Frizzled receptors, and a downstream molecule Dvl1. Among them, Wnt7a, Wnt7b, Fz3, and Dvl1 showed interesting expression patterns in the DG for 3–4 weeks (Fig. 6). Wnt7a, a Wnt ligand, was preferentially expressed in the hilus and along the SGZ of the blades of the DG. Another Wnt ligand, Wnt7b, was detected in the SGZ and the GCL, but Wnt7b-expressing cells did not morphologically seem to be granule cells in the GCL. Weak expression of Wnt3a was also detected in the SGZ at 2 weeks, but almost diminished by 4 weeks in the rat (data not shown). These expression patterns of Wnt ligands hint us to imagine that they are expressed in the progenitors themselves or other cells that may constitute a niche for keeping the undifferentiated state of the progenitor cells. In the DG of rSey2/+, the number of Wnt7a-expressing cells significantly decreased (687 cells in the wild type; 576 cells in rSey2/+; 16% decrease), and the number of Wnt7b-expressing cells also decreased (607 in the wild type; 544 in rSey2/+; 11% decrease). We could not observe any difference in expression of Wnt3a in the DG of rSey2/+. Expression of a Wnt receptor Fz3 was detected mainly in the GCL, and unchanged in the DG of rSey2/+. Contrastingly, the expression level of Dvl1 was increased in the DG of rSey2/+ (Fig. 6). Taken altogether, Wnt signaling is impaired in the DG under the Pax6 deficient condition, which may result in reduced proliferation of GFAP+ early progenitor cells in rSey2/+.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Previous papers suggest that adult hippocampal neurogenesis originates from precursor cells in the DG and results in new granule neurons through multiple steps from the GFAP+ early progenitor cells to the PSA-NCAM+ late progenitor cells (Seri et al. 2001; Fukuda et al. 2003; Kempermann et al. 2004). In the present study, we revealed that Pax6+ cells frequently co-expressed GFAP and Musashi1, sometimes expressed nestin and PSA-NCAM, but scarcely co-expressed NeuN in the SGZ of the postnatal DG (Fig. 1B). That is, Pax6 is considered to be expressed mostly in the GFAP+ early progenitor cells.
The ratio of nestin-positive cells among BrdU+ was less than that in a previous study using nestin-EGFP reporter mice (Filippov et al. 2003; Fukuda et al. 2003). This may be due to difference in sensitivity of anti-nestin antibody and duration of nestin-promoter-driven EGFP. More importantly, a large number of Pax6+/GFAP+ cells had early progenitor-like morphology with a long radial process (Fig. 1C'), and Pax6+/nestin+ mostly showed a GFAP+ early progenitor shape (Fig. 1B) and sometimes a PSA-NCAM+ late progenitor cell shape. Pax6+ cells sometimes co-expressed PSA-NCAM, but such Pax6+/PSA-NCAM+ cells always exhibited the late progenitor cell-like morphology. From immuno-electron microscopy, we found that the majority of Pax6+ cells showed features corresponding to type B cells, and that a small number of Pax6+ cells had characters corresponding to type D cells, while Pax6+ cells never showed phenotypes of granule cells (Fig. 1D,D', E,E'). Therefore, it is concluded that more than half of Pax6+ cells have the character of the GFAP+ early progenitor cells in the SGZ of the hippocampus (Fig. 7).
|
The role of Pax6 in postnatal hippocampus
As discussed above, Pax6+ cells have the character of neural stem cells and GFAP+ early progenitor cells in the DG of postnatal hippocampus. It is thus expected that Pax6 is involved in cell proliferation and/or cell differentiation in hippocampal neurogenesis.
There are some papers where Pax6 is involved in cell proliferation in developing cortex (Warren et al. 1999). We found from BrdU labeling analyses that cell proliferation was dramatically reduced in the Pax6-deficient DG (Fig. 2A). In addition, more than 90% of total BrdU+ cells were Pax6-positive at 30 min after BrdU injection in the SGZ of adult hippocampus (Fig. 4A,B). These data strongly suggest that Pax6 is vital for the cell proliferation in the hippocampal neurogenesis. Then, for which steps of the neurogenesis is Pax6 required in cell proliferation?
Previous papers report that both GFAP+ early progenitor cells and PSA-NCAM+ late progenitor cells are transit amplifying cells. Rapid transition from GFAP+ early progenitors/type B cells to PSA-NCAM+ late progenitors/type D cells occurs between 2 and 24 h after the BrdU single injection (Seri et al. 2001; Fukuda et al. 2003). In the similar experiment, we found dramatic decrease in the number of GFAP+/BrdU+ cells and an inverse increase in the number of PSA-NCAM+/BrdU+ cells in the SGZ of the rSey2/+ (Fig. 4C,D). Moreover, the morphology of GFAP+ cells was altered in rSey2/+ (Figs 2C', 5A, 5E). Our findings suggest that the maintenance of the GFAP+ early progenitor cells is perturbed in the DG of the rSey2/+, presumably resulting in a more rapid shift from the GFAP+ early progenitors to the PSA-NCAM+ late progenitors (Fig. 7).
There are some reports that Pax6 promotes neuronal differentiation in the developing cortex and adult SVZ (Heins et al. 2002; Hack et al. 2004, 2005). To test the possibility that Pax6 is involved in neuronal differentiation in the postnatal hippocampus, we examined the ratio of NeuN+/total BrdU+ cells at 5 days time point after the BrdU injection in rSey2/+. The ratio of NeuN+ /BrdU+ cells in rSey2/+ was not different from that in the WT, even though there is a more rapid shift from the GFAP+ early progenitor cells to the PSA-NCAM+ late progenitor cells in rSey2/+. Therefore, such abnormally differentiated PSA-NCAM+ late progenitor cells did not effectively contribute to produce neurons. In fact, these PSA-NCAM+ late progenitor cells exhibited abnormal characters; they often retained GFAP expression, which is hardly observed in the WT, and did not line up at the SGZ but colonized in disorganized positions (Fig. 5B,D). Since we observed increased cell death in rSey2/+ (M.M. and N.O., unpublished observation), such functionally abnormal PSA-NCAM+ late progenitor cells may eventually die off in the SGZ of the rSey2/+. Taken altogether, Pax6 functions in cell proliferation rather than differentiation in the DG.
We found a marked decrease of the percentage of GFAP+ in BrdU+ cells in rSey2/+ compared with the wild type, while no significant difference was detected in the percentage of NeuN+ in all BrdU+ cells at 4 weeks after the BrdU injection (Fig. 3C,D). However, since the total number of BrdU+ cells dramatically decreased in rSey2/+, newly generated granule cells were markedly reduced in rSey2/+ rat. Eventually, at 16 weeks, the GCL became thinner in the DG of rSey2/+ rats than that of the wild type (Fig. 2B). Therefore, it is suggested that Pax6 primarily functions to maintain the progenitor pool in the hippocampus; if the size of the progenitor pool is reduced by Pax6 haplo-insufficiency, the subsequent production of neurons is severely impaired.
The number of Pax6+/GFAP+ cells, BrdU+ cells (BrdU labeling three times a day for 3 days) and BrdU+/Pax6+ cells (BrdU labeling two times a day for 2 weeks) were already more reduced in rSey2/+ rats than in WT rats at the earliest time point we observed (4 weeks). Therefore, it is possible that Pax6 is necessary for the production of GFAP+ early progenitor cells during the initial formation of the hippcampus. This is quite reasonable because we observed that extremely less GFAP+ cells (including not only those mature astrocytes but also neural stem/progenitor cells) were produced in the DG of rSey2/+ rats (Fig. 3C,D). Taken all together, it is concluded that Pax6 is necessary for keeping a good balance between cell proliferation and differentiation in the hippocampal neurogenesis (Fig. 7).
Pax6–Wnt pathway in hippocampal neurogenesis
Because Pax6 is a transcription factor, its influence on the production of GFAP+ early progenitor cells is naturally brought by transcriptional regulation of other genes. Although several secreted molecules such as EGF, FGF2, BDNF, and Shh have been known to regulate adult neurogenesis (Craig et al. 1996; Kuhn et al. 1997; Tropepe et al. 1997; Zigova et al. 1998; Machold et al. 2003), we could not find any difference in expression of these molecules in the DG of rSey2/+ rats (not shown). However, we have found dramatically different expression patterns of genes involved in Wnt signaling pathway.
Wnt genes encode secreted proteins that regulate fate decisions of various cells depending on the context. The functions of Wnt signaling are studied intensively in many aspects of embryogenesis such as anterior–posterior axis formation, cell type specification, cell proliferation, and axonal growth (Patapoutian & Reichardt 2000; Wang & Wynshaw-Boris 2004; Zou 2004). Although Wnt signaling in the postnatal brain has been comparatively little investigated, a recent report describes remarkably patterned gene expressions of Wnt signaling components in the postnatal mouse brain including the hippocampus (Shimogori et al. 2004). In addition, Wnt signaling has already been reported to be altered in embryonic brains of Pax6 mutant mice and rats (Grindley et al. 1997; Osumi et al. 1997; Warren & Price 1997; Kim et al. 2001; Takahashi et al. 2002). We found specific expression of Wnt7a and Wnt7b in the wild-type SGZ, and marked reduction of Wnt7a and Wnt7b expressions in the DG of the rSey2/+. Conversely, Dv1 was up-regulated in the DG of the rSey2/+ (Fig. 6). Therefore, Wnt signaling is altered in the Pax6-deficient DG.
During cortical development, Wnt signaling has multiple and stage-specific roles. In early embryonic stages, Wnt7a, 7b, and stabilized ß-catenin promote self-renewal of neural precursor cells and suppress neural differentiation (Chenn & Walsh 2002; Viti et al. 2003). On the other hand, it is reported that the Wnt/ß-catenin pathway directs neuronal differentiation of the cortical precursor cells at later developmental stages (Hirabayashi et al. 2004). Curiously, in adult hippocampus, lithium facilitates proliferation and differentiation of progenitor cells to a specific neural cell type by perturbing functions of GSK3ß, a pivotal player not only in the PI3 kinase pathway but also in Wnt/ß-catenin pathway (Chen et al. 2000; Kim et al. 2004). In the present study, we found that specific expressions of Wnt ligands in the SGZ and that Wnt signaling is altered in the DG of Pax6-deficient rat (Fig. 6) suggest an intriguing possibility that impaired Wnt signaling may perturb the production of GFAP+ early progenitor cells in postnatal hippocampus. Expression patterns of Wnt7a and 7b may also support the idea that cells expressing Wnt ligands constitute an environment as a stem cell niche to maintain neural progenitor cells. It would be important to elucidate how Pax6-Wnt signaling coordinately regulates proliferation of neural stem cells/GFAP+ early progenitor cells in the SGZ.
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Large colonies of heterozygous Pax6 mutant rats (rSey2/+) and wild-type Sprague–Dawley (SD) rats (littermates of rSey2/+ rats) were maintained at Tohoku University School of Medicine and National Institute of Neuroscience. The genotype of rSey2/+ rats was distinguishable based on the presence of eye defects. All animal experiments were carried out in accordance with the National Institute of Health guidelines for the care and use of laboratory animals and were approved by the Committee for Animal Experiments in the aforementioned organizations.
Tissue preparation
Rats were deeply anesthetized with diethyl ether or pentobarbital sodium before sacrifice. Brains were perfused transcardially with 4% paraformaldehyde (PFA) in 0.01 M PBS (sodium phosphate buffer, pH 7.4) or 4% PFA and 0.5% picric acid in 0.01 M PBS, or 2% PFA and 2.5% glutaraldehyde in 0.01 M PBS, for immunohistochemistry, immuno-electron microscopic analysis, and conventional electron microscopic analysis, respectively. The brains were incubated in the same fixative for 2 h at 4 °C and cut into 70 µm coronal sections with a vibratome (Leica) or cut by a cryostat (Leica) into 14 µm sagittal sections.
Immunohistochemistry
Procedures were basically according to the previous reports (Osumi et al. 1997; Fukuda et al. 2003). Detailed information will be provided on request. Antibodies against Musashi, nestin, and PSA-NCAM are kind gifts from Drs Hideyuki Okano, Masaharu Ogawa, and Tatsunori Seki (Miyata & Ogawa 1994; Seki & Arai 1999; Kaneko et al. 2000). Fluorescent signals were detected using a confocal laser-scanning microscope (Leica) or a fluorescent microscope (Axioplan-2, Zeiss).
BrdU labeling analyses
Four-week-old rats received single intraperitoneal injections of 5-bromo-2-deoxyuridine (BrdU) (Sigma, St. Louis, MO) at 50 µg/kg body weight (10 mg/mL stock, dissolved in 0.9% saline), and were sacrificed at 30 min, 24 h, and 72 h after the injection (Seri et al. 2001; Kempermann et al. 2004). For cell fate analyses, 4-, 12- or 20-week-old rats received similar injections of BrdU three times a day for 3 days, and were sacrificed at day 1 or 4 weeks later (Kempermann & Gage 1999). For quiescent stem cell analysis, 4-week-old rats received injections of BrdU twice a day for 14 days, and were sacrificed 1 day later (Magavi et al. 2000). Seventy micrometers free-floating sections were cut and incubated in 2 N HCl for 1 h at room temperature, and washed in 0.01 M PBS (Saegusa et al. 2004). Otherwise, 14 µm frozen sections were boiled in 0.01 M citric acid and incubated in 2 N HCl for 10 min at 37 °C, and washed in 0.01 M PBS.
Quantification
For BrdU pulse/chase examination, percentages of Pax6+, GFAP+, or PSA-NCAM+ in total BrdU+ cells were calculated in three sections per hemisphere. For quantification analysis, sampling of BrdU-positive cells was performed throughout the DG in its rostrocaudal extension. Every sixth section (14 µm) was used for counting, and the total number was obtained by multiplying the value by 6 (Kempermann & Gage 1999). For the fate analysis, BrdU+/NeuN+ in total BrdU+ cells and BrdU+/GFAP+ in total BrdU+ cells were counted in three adjacent sections in the same rostrocaudal regions of a DG (Kempermann & Gage 1999). For the quantification of the number of GFAP+/Pax6+ double-positive cells and Pax6+/BrdU+ double-positive cells, we counted these cells within the limited range in six adjacent sections and calculated the density. The number of these cells was counted in the blind manner.
Electron microscopy
Procedures were basically according to the previous reports (Yuasa et al. 1996; Saegusa et al. 2004). Detailed information will be provided on request. These ultrathin sections were stained with lead citrate and uranyl acetate, and observed under a Hitachi H-7000 electron microscope.
In situ hybridization
Procedures were basically according to the previous reports (Osumi et al. 1997; Takahashi et al. 2002). Wnt7a- and Wnt7b- expressing cells were counted on three adjacent sections in the same rostrocaudal region of a DG.
Statistical analysis
Statistical analyses were performed with Microsoft Excel (Office 98), and ANOVA or two-sided t-test was applied when appropriate.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
* Correspondence: E-mail: osumi{at}mail.tains.tohoku.ac.jp
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Alvarez-Buylla, A., Seri, B. & Doetsch, F. (2002) Identification of neural stem cells in the adult vertebrate brain. Brain Res. Bull. 57, 751–758.[CrossRef][Medline]
Chen, G., Rajkowska, G., Du, F., Seraji-Bozorgzad, N. & Manji, H.K. (2000) Enhancement of hippocampal neurogenesis by lithium. J. Neurochem. 75, 1729–1734.[CrossRef][Medline]
Chenn, A. & Walsh, C.A. (2002) Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science
297, 365–369.
Craig, C.G., Tropepe, V., Morshead, C.M., et al. (1996) In vivo growth factor expansion of endogenous subependymal neural precursor cell population in the adult mouse brain. J. Neurosci.
16, 2649–2658.
Doetsch, F., Caille, I., Lim, D.A., Garcia-Verdugo, J.M. & Alvarez-Buylla, A. (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97, 703–716.[CrossRef][Medline]
Doetsch, F., Garcia-Verdugo, J.M. & Alvarez-Buylla, A. (1997) Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J. Neurosci.
17, 5046–5061.
Estivill-Torrus, G., Pearson, H., van Heyningen, V., Price, D.J. & Rashbass, P. (2002) Pax6 is required to regulate the cell cycle and the rate of progression from symmetrical to asymmetrical division in mammalian cortical progenitors. Development 129, 455–466.
Filippov, V., Kronenberg, G., Pivneva, T., et al. (2003) Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes. Mol. Cell. Neurosci. 23, 373–382.[CrossRef][Medline]
Forster, E., Tielsch, A., Saum, B., et al. (2002) Reelin, Disabled 1, and beta 1 integrins are required for the formation of the radial glial scaffold in the hippocampus. Proc. Natl. Acad. Sci. USA
99, 13178–13183.
Fujita, S. (2003) The discovery of the matrix cell, the identification of the multipotent neural stem cell and the development of the central nervous system. Cell Struct. Funct. 28, 205–228.[CrossRef][Medline]
Fukuda, S., Kato, F., Tozuka, Y., et al. (2003) Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus. J. Neurosci.
23, 9357–9366.
Fukuda, T., Kawano, H., Osumi, N., Eto, K. & Kawamura, K. (2000) Histogenesis of the cerebral cortex in rat fetuses with a mutation in the Pax-6 gene. Brain Res. Dev. Brain Res. 120, 65–75.[CrossRef][Medline]
Gage, F.H. (2000) Mammalian neural stem cells. Science
287, 1433–1438.
Gotz, M. (2003) Glial cells generate neurons—master control within CNS regions: developmental perspectives on neural stem cells. Neuroscientist 9, 379–397.[Abstract]
Gotz, M., Stoykova, A. & Gruss, P. (1998) Pax6 controls radial glia differentiation in the cerebral cortex. Neuron 21, 1031–1044.[CrossRef][Medline]
Grindley, J.C., Hargett, L.K., Hill, R.E., Ross, A. & Hogan, B.L. (1997) Disruption of PAX6 function in mice homozygous for the Pax6Sey-1Neu mutation produces abnormalities in the early development and regionalization of the diencephalon. Mech. Dev. 64, 111–126.[CrossRef][Medline]
Hack, M.A., Saghatelyan, A., de Chevigny, A., et al. (2005) Neuronal fate determinants of adult olfactory bulb neurogenesis. Nature Neurosci.
Hack, M.A., Sugimori, M., Lundberg, C., Nakafuku, M. & Gotz, M. (2004) Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6. Mol. Cell. Neurosci. 25, 664–678.[CrossRef][Medline]
Heins, N., Malatesta, P., Cecconi, F., et al. (2002) Glial cells generate neurons: the role of the transcription factor Pax6. Nature Neurosci. 5, 308–315.[CrossRef][Medline]
Hirabayashi, Y., Itoh, Y., Tabata, H., et al. (2004) The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development
131, 2791–2801.
Kaneko, Y., Sakakibara, S., Imai, T., et al. (2000) Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev. Neurosci. 22, 139–153.[CrossRef][Medline]
Kempermann, G. & Gage, F.H. (1999) Experience-dependent regulation of adult hippocampal neurogenesis: effects of long-term stimulation and stimulus withdrawal. Hippocampus 9, 321–332.[CrossRef][Medline]
Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M. & Gage, F.H. (2003) Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice. Development
130, 391–399.
Kempermann, G., Jessberger, S., Steiner, B. & Kronenberg, G. (2004) Milestones of neuronal development in the adult hippocampus. Trends Neurosci. 27, 447–452.[CrossRef][Medline]
Kim, A.S., Anderson, S.A., Rubenstein, J.L., Lowenstein, D.H. & Pleasure, S.J. (2001) Pax-6 regulates expression of SFRP-2 and Wnt-7b in the developing CNS. J. Neurosci. 21, RC132.
Kim, J.S., Chang, M.Y., Yu, I.T., et al. (2004) Lithium selectively increases neuronal differentiation of hippocampal neural progenitor cells both in vitro and in vivo. J. Neurochem. 89, 324–336.[Medline]
Kuhn, H.G., Dickinson-Anson, H. & Gage, F.H. (1996) Neurognesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci.
16, 2027–2033.
Kuhn, H.G., Winkler, J., Kempermann, G., Thal, L.J. & Gage, F.H. (1997) Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J. Neurosci.
17, 5820–5829.
Machold, R., Hayashi, S., Rutlin, M., et al. (2003) Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron 39, 937–950.[CrossRef][Medline]
Magavi, S.S., Leavitt, B.R. & Macklis, J.D. (2000) Induction of neurogenesis in the neocortex of adult mice. Nature 405, 951–955.[CrossRef][Medline]
Miyata, T. & Ogawa, M. (1994) Developmental potentials of early telencephalic neuroepithelial cells: a study with microexplant culture. Dev. Growth Differ. 36, 319–331.[CrossRef]
Nakatomi, H., Kuriu, T., Okabe, S., et al. (2002) Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 110, 429–441.[CrossRef][Medline]
Osumi, N. (2001) The role of Pax6 in brain patterning. Tohoku J. Exp. Med. 193, 163–174.[CrossRef][Medline]
Osumi, N., Hirota, A., Ohuchi, H., et al. (1997) Pax-6 is involved in the specification of hindbrain motor neuron subtype. Development 124, 2961–2972.[Abstract]
Patapoutian, A. & Reichardt, L.F. (2000) Roles of Wnt proteins in neural development and maintenance. Curr. Opin. Neurociol. 10, 392–399.
Saegusa, T., Mine, S., Iwasa, H., et al. (2004) Involvement of highly polysialylated neural cell adhesion molecule (PSA-NCAM)-positive granule cells in the amygdaloid-kindling-induced sprouting of a hippocampal mossy fiber trajectory. Neurosci. Res. 48, 185–194.[Medline]
Seki, T. & Arai, Y. (1993) Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat. J. Neurosci. 13, 2351–2358.[Abstract]
Seki, T. & Arai, Y. (1999) Different polysialic acid-neural cell adhesion molecule expression patterns in distinct types of mossy fiber boutons in the adult hippocampus. J. Comp. Neurol. 19, 115–125.
Seri, B., Garcia-Verdugo, J.M., McEwen, B.S. & Alvarez-Buylla, A. (2001) Astrocytes give rise to new neurons in the adult mammalian hippocampus. J. Neurosci.
21, 7153–7160.
Shimogori, T., VanSant, J., Paik, E. & Grove, E.A. (2004) Members of the Wnt, Fz, and Frp gene families expressed in postnatal mouse cerebral cortex. J. Comp. Neurol. 473, 496–510.[CrossRef][Medline]
Simpson, T.I. & Price, D.J. (2002) Pax6; a pleiotropic player in development. Bioessays 24, 1041–1051.[CrossRef][Medline]
Stoykova, A., Gotz, M., Gruss, P. & Price, J. (1997) Pax6-dependent regulation of adhesive patterning, R-cadherin expression and boundary formation in developing forebrain. Development 124, 3765–3777.[Abstract]
Stoykova, A. & Gruss, P. (1994) Roles of Pax-genes in developing and adult brain as suggested by expression patterns. J. Neurosci. 14, 1395–1412.[Abstract]
Takahashi, M., Sato, K., Nomura, T. & Osumi, N. (2002) Manipulating gene expressions by electroporation in the developing brain of mammalian embryos. Differentiation 70, 155–162.[CrossRef][Medline]
Tramontin, A.D., Garcia-Verdugo, J.M., Lim, D.A. & Alvarez-Buylla, A. (2003) Postnatal development of radial glia and the ventricular zone (VZ): a continuum of the neural stem cell compartment. Cereb. Cortex.
13, 580–587.
Tropepe, V., Craig, C.G., Morshead, C.M. & van der Kooy, D. (1997) Transforming growth factor-alpha null and senescent mice show decreased neural progenitor cell proliferation in the forebrain subependyma. J. Neurosci.
17, 7850–7859.
Viti, J., Gulacsi, A. & Lillien, L. (2003) Wnt regulation of progenitor maturation in the cortex depends on Shh or fibroblast growth factor 2. J. Neurosci.
23, 5919–5927.
Wang, J. & Wynshaw-Boris, A. (2004) The canonical Wnt pathway in early mammalian embryogenesis and stem cell maintenance/differentiation. Curr. Opin. Genet. Dev. 14, 533–539.[CrossRef][Medline]
Warren, N., Caric, D., Pratt, T., et al. (1999) The transcription factor, Pax6, is required for cell proliferation and differentiation in the developing cerebral cortex. Cereb. Cortex.
9, 627–635.
Warren, N. & Price, D.J. (1997) Roles of Pax-6 in murine diencephalic development. Development 124, 1573–1582.[Abstract]
Yuasa, S., Kawamura, K., Kuwano, R. & Ono, K. (1996) Neuron-glia interrelations during migration of Purkinje cells in the mouse embryonic cerebellum. Int. J. Dev. Neurosci. 14, 429–438.[CrossRef][Medline]
Zigova, T., Pencea, V., Wiegand, S.J. & Luskin, M.B. (1998) Intraventricular administration of BDNF increase the number of newly generated neurons in the adult olfactory bulb. Mol. Cell. Neurosci. 11, 234–245.[CrossRef][Medline]
Zou, Y. (2004) Wnt signaling in axon guidance. Trends Neurosci. 27, 528–532.[CrossRef][Medline]
Received: 13 March 2005
Accepted: 11 July 2005
This article has been cited by other articles:
|
|
 |
|
  M. A. Gomez-Climent, E. Castillo-Gomez, E. Varea, R. Guirado, J. M. Blasco-Ibanez, C. Crespo, F. J. Martinez-Guijarro, and J. Nacher A Population of Prenatally Generated Cells in the Rat Paleocortex Maintains an Immature Neuronal Phenotype into Adulthood Cereb Cortex, October 1, 2008; 18(10): 2229 - 2240. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Osumi, H. Shinohara, K. Numayama-Tsuruta, and M. Maekawa Concise Review: Pax6 Transcription Factor Contributes to both Embryonic and Adult Neurogenesis as a Multifunctional Regulator Stem Cells, July 1, 2008; 26(7): 1663 - 1672. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sakurai and N. Osumi The Neurogenesis-Controlling Factor, Pax6, Inhibits Proliferation and Promotes Maturation in Murine Astrocytes J. Neurosci., April 30, 2008; 28(18): 4604 - 4612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Hodge, T. D. Kowalczyk, S. A. Wolf, J. M. Encinas, C. Rippey, G. Enikolopov, G. Kempermann, and R. F. Hevner Intermediate Progenitors in Adult Hippocampal Neurogenesis: Tbr2 Expression and Coordinate Regulation of Neuronal Output J. Neurosci., April 2, 2008; 28(14): 3707 - 3717. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||
