Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cerebral Cortex 2006;16:i103--i111 doi:10.1093/cercor/bhk027 Composition and Organization of the SCZ: A Large Germinal Layer Containing Neural Stem Cells in the Adult Mammalian Brain Two known germinal zones continue to generate new neurons and glia in the adult mammalian brain: the subventricular zone (SVZ), lining the lateral walls of the lateral ventricle, and the subgranular zone of the dentate gyrus. Here we describe a region we will refer to as the subcallosal zone (SCZ). The SCZ is a caudal extension of the SVZ that is no longer associated to an open ventricle. It lies between the hippocampus and the corpus callosum. Cells isolated from the SCZ and cultured as neurospheres behave as neural stem cells in vitro. Using electron and light microscopy, we describe the cell types present in this region and how their organization differs from that of the SVZ. Using retroviral labeling and homotypic-homochronic microtransplantation techniques, we show that the majority of cells born in the SCZ migrate into the corpus callosum to become oligodendrocytes in vivo. This study defines the organization and fate of cells born in a large germinal region of the adult forebrain. Keywords: adult brain, germinal zones, gliogenesis, neural stem cells, neurogenesis, oligodendrocytes, SCZ, ventricles Introduction Neural progenitor cells persist in restricted germinal layers in the adult vertebrate brain (Gage 2000; Temple 2001b; AlvarezBuylla and Lim 2004). These precursor cells continually produce young neurons and glial cells. Although the function of endogenous brain cell replacement is not understood, it is thought that adult neural stem cells (NSCs) could contribute to neural plasticity and repair (Barnea and Nottebohm 1996; Feng and others 2001; Shors and others 2001; Nakatomi and others 2002; Lichtenwalner and Parent 2006). Therefore, there is intense interest in understanding the organization of proliferative areas in the adult brain and the normal fate of the cells born within them. Two germinal regions have been extensively characterized in the adult mammalian brain: the subventricular zone (SVZ) on the lateral walls of the lateral ventricle and the subgranular zone (SGZ) in the dentate gyrus (DG) of the hippocampus (Altman and Das 1965; Altman 1969; Cameron and others 1993; Doetsch and others 1997; Seri and others 2004). Cells born in the SVZ migrate through a complex network of pathways parallel to the walls of the lateral ventricle (Doetsch and Alvarez-Buylla 1996) to join the rostral migratory stream that leads into the olfactory bulb (OB). Within the OB these young neurons mature into local granular and periglomerular interneurons (Carleton and others 2003; Lemasson and others 2005). In contrast to the SVZ, cells born in the SGZ only migrate a short distance and The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] B. Seri1,2, D.G. Herrera3, A. Gritti4, S. Ferron5, L. Collado5, A. Vescovi4, J.M. Garcia-Verdugo5 and Arturo Alvarez-Buylla1 1 Neurosurgery Department, University of California—San Francisco, San Francisco, CA 94143, USA, 2The Rockefeller University, Laboratory of Neurogenesis, New York, NY 10021, USA, 3Cornell Weill Medical College, Psychiatry Department, New York, NY 10021, USA, 4Institute for Stem Cell Research, Department of Biological and Technological Research (DIBIT), H. San Raffaele, Milan, Italy and 5Instituto Cavanilles, University of Valencia and Centro de Investigación Principe Felipe, Facultad de Ciencias Biológicas, 46100 Valencia, Spain differentiate into granule neurons in the DG (Altman and Das 1965; Kaplan and Bell 1984; Cameron and others 1993). Work in the neonatal rodent brain has shown that, in addition to neurons, oligodendrocytes and astrocytes are also formed in the SVZ (Levison and Goldman 1993; Luskin 1993). More recent work indicates that the adult SVZ may be a source of oligodendrocytes following a demyelinating lesion to the corpus callosum (Nait-Oumesmar and others 1999). In addition, new oligodendrocytes are also produced from the division of local progenitors within the brain parenchyma (Ffrench-Constant and Raff 1986; Wolswijk and Noble 1989; Wood and Bunge 1991; Levine and others 2001). Besides the SVZ and SGZ, a large number of proliferating cells are found between the hippocampus and the corpus callosum in the adult rodent brain (Altman and Das 1965; Reznikov 1991) (Fig. 1A). This lamina of dividing cells corresponds to the caudal and medial extension of the posterior horn of the lateral ventricle, a region where the ventricular walls collapse during development. This layer of dividing cells could be considered a caudal and dorsomedial extension of the SVZ (Doetsch and Alvarez-Buylla 1996). Unlike the SVZ, it is not associated to an overt open ventricular cavity but to isolated cavities filled with cerebrospinal fluid (CSF) (Figs 1C and 3A), which might be connected to the main compartment of the lateral ventricle. It is therefore not appropriate to refer to this region as a subependymal or subventricular germinal layer. Given its localization underlining the posterior corpus callosum, we will refer to this region as the subcallosal zone (SCZ). Unlike the SVZ or the SGZ, very little is known about the organization, cellular composition, or the fate of cells born in the SCZ. Interestingly, one study suggests that new neurons that contribute to the repair of the cornu ammonis 1 (CA1) pyramidal cell layer after ischemic lesion originate from the overlying proliferative zone that lies within the SCZ (Nakatomi and others 2002). Here we have used immunocytochemistry and electron microscopy (EM) to study the cellular composition and organization of the SCZ. Many cells in the SCZ appear to have similar characteristics to those observed in the SVZ. However, the organization of the SCZ is significantly different to that observed in the SVZ or SGZ. Using epidermal growth factor (EGF) and fibroblast growth factor (FGF), we found that the SCZ is a rich source of neurosphere-forming cells; these neurospheres can generate oligodendrocytes, neurons, and astrocytes. In vivo tracing experiments, using retroviral labeling and microtransplantation, in the normal brain, revealed that SCZ NSCs did not generate neurons but generate oligodendrocytes that migrate into the corpus callosum. perfused transcardially with 20 mL of 0.9% saline followed by 100 mL of 4% paraformaldehyde (PFA). The heads were removed and postfixed in the same fixative overnight. The next day the brains were removed from the skull and washed in 0.1 M phosphate buffer for 2 h. BrdU Quantification Brains were embedded in paraffin, cut into 7-lm sections, and stained with toluidine blue. One in every 12 sections was selected for BrdU staining as described before (Peterson and Jones 1993). A total of 10 sections were counted for each region. The SGZ, the SVZ, and the SCZ were used for the quantification. The area delimited by each region was traced, and the percentage of labeled cells was determined by counting the total number of cells and the number of BrdU-positive cells. Primary Cultures and Tissue Dissection Adult mice (3--8 months old, CD1 males or females) were deeply anesthetized with 4% chloral hydrate and decapitated, and whole brains were removed and placed in artificial CSF (124 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 0.1 mM CaCl2, 26 mM NaHCO3, and 10 mM D-glucose, pH 7.3). The brain was bisected longitudinally, and each hippocampal lobe was separated from the overlaying cortical white matter using the natural separation line along the alveus hippocampus. The fimbria and subiculum were removed. One-millimeter-wide corticocallosal ribbons containing the SCZ region were dissected longitudinally. The cortical surface was trimmed from each ribbon to remove the meninges and cortex allowing mostly corpus callosum to remain (callosal SCZ ribbons). Longitudinal (in the rostrocaudal direction) ribbons were obtained from the dorsal hippocampal surface underlying the SCZ region (hippocampal SCZ ribbons) and used as white matter controls for the neurosphere cultures. Figure 1. Location of the SCZ in the adult mammalian brain. (A) Schematic 3dimensional rendition from serial reconstructions of coronal sections of the SCZ in the adult rodent brain. Shaded areas show the location of regions in the SCZ with cavitations; likely remnants of the lateral ventricle that have not collapsed completely during development. The dotted line represents the boundaries of the SCZ region within the brain. The schematic shows the approximate location of the inferior horn of the lateral ventricle and the caudal SVZ with respect to the SCZ, and the long arrow indicates the approximate location of the RMS. Arroheads indicate the approximate anterior and caudal sites where homotypic grafts were transplanted. (B) Percentage of proliferating cells in 3 germinal regions of the adult brain. Percentage of BrdU+ cells over total number of cells ± standard error of mean. (C) Light micrograph of a coronal paraffin section through the dorsal hippocampus. Inset shows BrdU-labeled cells in the SCZ region at higher magnification. Scale bar in (C), 100 lm; in inset, 10 lm. CC, corpus callosum. Methods BrdU Injections Adult male mice, 3 months old, received 7 intraperitoneal (i.p.) injections of 5-bromo-29-deoxyuridine (BrdU), 50 lg/g body weight, once every 2 h. Two hours after the last injection, mice were deeply anesthetized with 4% chloral hydrate (15 lL/g body weight) and i104 SCZ: A Germinal Layer in the Adult Brain d Seri and others For Neurosphere Cultures Tissue dissected from the SCZ (the hippocampal and callosal sides were dissected separately), the SVZ of the anterior--lateral ventricle (Lois and Alvarez-Buylla 1993), and the hippocampus (without SVZ or SCZ tissue) were dissociated and cultured to form neurospheres as previously described (Gritti and others 1996). After 4--7 days in vitro (DIV), the number of spheres derived from each brain region was counted. To assess the self-renewal capacity of the neurospheres derived from the 4 different regions, cells in the primary cultures were dissociated and subcultured as single cells in matrigel (1:50) at a clonal density ( <1 cell/cm2) in the presence of EGF and FGF (both at 20 ng/mL). Serially passaged clonal spheres were plated onto polyornithine-coated glass coverslips and grown in growth factor--free media for 2 days, followed by the addition of fetal bovine serum for 5 DIV. Differentiated cultures were fixed (20 min) with 4% PFA and immunostained for cell-specific markers. The coverslips were then incubated for 90 min at 37 C in phosphate-buffered saline (PBS) containing 10% normal goat serum (NGS), 0.3% Triton X-100, and anti-bIII tubulin (TUJ1) 1:1250, Sigma, St. Louis, MO; antigalactocerebroside 1:50, Boehringer Mannheim, Indianapolis, IN; and rabbit antisera against glial fibrillary acidic protein (GFAP, ready to use, Incstar, Stillwater, MN). Coverslips were washed 3 times in PBS containing 10% NGS and incubated for 45 min (room temperature). Separate coverslips were processed for each marker in fluorescein isothiocyanate--conjugated or rhodamine isothiocyanate--conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (1:100; Boehringer Mannheim). Coverslips were rinsed 3 times in PBS, once in distilled water, and mounted on glass slides with Fluorsave (Calbiochem, La Jolla, CA). For quantitative analysis, after immunostaining, coverslips were counterstained with 49, 6-diamidino-2-phenylindole (DAPI). No labeling was observed in control experiments when the primary antibodies were omitted. SCZ Whole Mounts and Immunostaining Three CD1 mice between P60 and P90 were deeply anesthetized with a 1.3 mg/g of body weight i.p. injection of pentobarbital (Nembutal) and transcardially perfused with 10 mL of 0.9% normal saline solution. The brains were removed from the skull, bisected longitudinally, and each hippocampal lobe was separated from the overlaying cortical white matter as described above to expose the region of the SCZ. Brains were then fixed overnight in 3% PFA. Whole mounts were processed for polysialilated-neural cell adhesion molecule (PSA-NCAM) immunostaining as described in Doetsch, Garcia-Verdugo, and others (1999). Microtransplantations Two b-actin:GFP (green fluorescent protein) mice (N = 10 transplantation experiments) between P60 and P90 were decapitated, the brains removed from the skull, and 1-mm-wide corticocallosal ribbons containing the SCZ region were dissected longitudinally, as described above. This dissected tissue did not contain cortex, hippocampus, or SVZ. The ribbons were cut into small pieces ( <50 lm) and dissociated in 0.25% trypsin for 30 min at 37 C. Fifty thousand cells were resuspended into a 50-lL final volume and loaded into a glass micropipette with a 50-lm outer diameter and kept on ice. Thirty CD1 adult male mice received a unilateral microtransplantation of approximately 50 nL of the cell suspension into the SCZ at stereotaxic coordinates alkaline phosphatase (AP): 1.0, ML: 2.0, and DV: 1.25 for the anterior SCZ and AP: 2.3, ML: 2.6, and DV: 1.65 for the caudal SCZ (Bayer 1985). Mice were perfused, as previously described, 30 days after transplantation. Brains were cut with a vibrating microtome in 50-lm sagittal sections and processed for immunocytochemistry using an anti-GFP antibody (Quatum Biotech, Montreal, Canada). Retroviral Injections A total of 15 CD1 mice between P60 and P90 were stereotaxically injected with a retrovirus (2 3 107 cfu/mL) carrying the AP gene (w2DAP, American Type Cell Culture [ATCC], Manassas, VA) into the SCZ at stereotaxic coordinates. For anterior SCZ, antero-posterior (AP): 1.0, medio-lateral (ML): 2.0, and dorso-ventral (DV): 1.25, for medial SCZ, AP: 1.7, ML: 3.2, and DV: 1.55, and for caudal SCZ, AP: 2.3, ML: 2.6, and DV: 1.65 (Bayer 1985). Before injection, the virus was mixed with Polybrene (Sigma) to a final concentration of 8 lg/mL. One, 3, 15, 21, and 30 days after retroviral injection (n = 3 per survival DV time), animals were deeply anesthetized with an i.p. injection of pentobarbital (Nembutal, 1.3 mg/g of body weight) and transcardially perfused with 10 mL of 0.9% normal saline solution followed by 30 mL of 3% PFA. Brains were removed from the skull and fixed overnight in 3% PFA. Sagittal sections of 50 lm were cut using a vibrating microtome. Sections were incubated in PBS for 30 min at 65 C to inactivate endogenous AP activity and allowed to cool down to room temperature in fresh PBS, incubated in AP buffer (100 mM Tris--HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) for 10 min, and incubated in the dark in AP substrate: Nitroblue tetrazolium chloride (10 lL/mL of AP buffer) and 5Bromo-4-chloro-3-indolyl phosphate (2 lL/mL of AP buffer, Boehringer Mannheim). When enough precipitate developed (between 30 and 120 min), sections were rinsed 3 times in PBS and mounted on Aquamount (Polysciences, Warrington, PA). Results Cell Proliferation in the SCZ The SCZ in the adult mouse brain is a lamina containing a high density of cells compared with the surrounding brain parenchyma. Two hours after BrdU injections, many BrdU-labeled cells were observed throughout the SCZ from anterior to caudal regions (Fig. 1B,C). Some of the SCZ were found in mitosis (Fig. 1C). The above indicates that the SCZ is a region of active proliferation in the adult brain. We quantified the percentage of BrdU-labeled cells in the SCZ and compared it with that of the SVZ and the SGZ. Approximately 20% of SVZ cells, 5% of cells in the SGZ, and 15% of cells in the SCZ were labeled 2 h after the last BrdU injection (Fig. 1B). BrdU-positive cells were retained in the SCZ 30 days after a similar BrdU treatment. These labelretaining cells could correspond to NSCs (Potten and Loeffler 1990) or to cells that differentiated locally. The SCZ Contains NSCs To investigate if NSCs could be derived from the SCZ, we dissected the region excluding the DG and the walls of the lateral ventricle from the preparation (see Methods) and cultured the cells to determine if they could form neurospheres (Reynolds and Weiss 1992). Callosal and hippocampal SCZ ribbons were cultured separately. Neurospheres grew in the presence of FGF and EGF only from callosal SCZ tissue. Cells from these neurospheres could be passaged up to 20 times (n = 3 independent experiments) (Fig. 2A--C). Neurospheres, placed in mitogen-free media and allowed to attach to the culture dish and differentiate, generated GFAP-positive astrocytes, TUJ1positive neurons, and O4-positive oligodendrocytes (Fig. 2F--H). Furthermore, cells from primary neurospheres passaged and replated at clonal density--generated secondary neurospheres (Fig. 2D,E). These experiments suggest that the SCZ contains multipotent, self-renewing stem cells. Neurospheres were also grown from the SVZ as positive controls. SVZ and SCZ neurospheres were similar in size, but the number of primary neurospheres derived from SVZ was consistently higher than the number derived from the SCZ (Fig. 2A). Growth curves from proliferating cells derived from both the SVZ and the SCZ were very similar at lower and higher passages (Fig. 2B,C). No neurospheres originated from tissue dissected from the hippocampal side of the SCZ, from cortex, or from striatum (data not shown). These data suggest that the SCZ contains cells that behave as NSCs in vitro. Cellular Composition and Organization of the SCZ To better understand the overall organization of the SCZ, we made whole-mount preparations of this region separating the hippocampus from the corpus callosum. This provided an ‘‘onface’’ view of the cells within the SCZ. Both exposed walls were stained with antibodies for PSA-NCAM, which labels migrating cells in the SVZ (Rousselot and others 1995; Doetsch and Alvarez-Buylla 1996; Peretto and others 1997). On the laterodorsal wall, the one facing the corpus callosum, we found loosely interconnected clusters of PSA-NCAM--positive cells (Fig. 3G,I,K). Larger clusters of PSA-NCAM--positive cells were observed more laterally, at the edge of the SCZ, just where the ventricle opens to the region where the SCZ meets the SVZ. In this region, most PSA-NCAM--positive cells form chains that run caudorostrally and appear to correspond to the dorsal corridor of longitudinal chains destined for the OB (Fig. 3G, arrowheads). However, most of the PSA-NCAM--positive cell clusters in more lateral and caudal aspects of the SCZ were not connected to the SVZ or oriented in the direction of the OB. These loosely interconnected chains of PSA-NCAM--positive cells appear to be perpendicular to the rostrocaudal length of chains in the SVZ. We found almost no PSA-NCAM--positive cells in the medioventral wall (facing the hippocampus). Using light microscopy to further analyze the organization of the SCZ, we sectioned this region into 7-lm serial sections and stained them with toluidine blue (n = 15 mice). Serial reconstruction analysis provided a 3-dimensional perspective throughout the SCZ (Fig. 1A) revealing regional changes in cell density and areas containing open cavities (Fig. 1A, area within dotted line), likely remnants of the lateral ventricle. We mapped the position of these lacunae, and they appear to be isolated cavities surrounded by areas where both walls have fused. Interestingly, the pattern of the cavities in the SCZ between animals was similar. We then used EM to determine the cell types present in the SCZ and how these cells were organized. The SCZ contained cells similar to those previously defined in the SVZ (Doetsch and others 1997) including ependymal cells (E), astrocytes (B), migrating neuroblasts (A), and type C cells (C) (Fig. 3C,H). We confirmed with EM that cavities were covered by ependymal cells and formed a cuboidal pseudostratified layer (asterisks in Cerebral Cortex 2006, V 16 Supplement 1 i105 Figure 2. The SCZ contains cells with characteristics of stem cells in vitro. (A) Number of neurospheres produced in primary cultures from SVZ and SCZ. Total number of spheres ± standard error of mean after plating. (B, C) Growth curves for neurospheres obtained from SVZ and SCZ, sampled between passages 11 and 14 (B) and cells sampled between passages 19 and 23 (C); the growth was similar for cells derived from the 2 regions. (D, E) Clonal analysis of SCZ-derived neurospheres; SCZ cells were plated as single cells (D), from which neurospheres developed at 10 DIV (E). (F--H) Differentiation of clonally derived neurospheres from SCZ. Neurospheres plated under differentiation conditions generated O4-stained oligodendrocytes (F), TUJ1-positive neuroblasts, (G) and GFAP-positive astrocytes (H). Scale bars: (D, E), 10 lm; (F--H), 5 lm. Fig. 3B). Isolated and small groups of ependymal cells were also found throughout the SCZ (Fig. 3A--C), including in regions where there was no apparent cavity. All ependymal cells, including those not exposed to open cavities, had microvilli and were multiciliated with a 9 + 2 microtubule organization. Ependymal cells stained positive with antibodies against mCD24 (Fig. 3D) similar to ependymal cells present in the walls of the lateral ventricle. In contrast to the SVZ, where ependymal cells rarely contact axonal pathways, ependymal cells in the SCZ were frequently in contact with myelinated and nonmyelinated axons from the corpus callosum and hippocampus. SCZ astrocytes appear to be closely associated to each other through gap junctions and zonula adherens. They had large nuclei, few free ribosomes, and abundant intermediate filaments. They also contact myelinated and nonmyelinated axons. SCZ astrocytes stain with antibodies against GFAP and vimentin (Fig. 3E,F). Cells with migratory characteristics that stain positive for PSA-NCAM were also present. These cells were elongated and similar to the type A cells of the SVZ (Doetsch and others 1997) (Fig. 3G,I,K). These putative migrating cells were also identified using EM. These cells had an elongated cell body with 1 or 2 processes, abundant lax chromatin, and scant dark cytoplasm with numerous free ribosomes, microtubules, smooth contours, and intermittent intercellular spaces between them. They were more common in medial and rostral portions of the SCZ, consistent with observations from wholemount preparations. Interestingly, SCZ astrocytes did not completely ensheathe type A cells as they do in the SVZ. Therefore, SCZ type A cells sporadically contacted ependymal cells and were often in contact with myelinated axons of the corpus callosum (Fig. 3J). In addition, SCZ type A cells did not i106 SCZ: A Germinal Layer in the Adult Brain d Seri and others form a network of chains as in the SVZ but rather formed isolated clusters with astrocytes, other type A cells, and sporadically, a type C cell. We also found cells in the SCZ with the ultrastructural characteristics of type C cells, larger more spherical and electron lucent compared with type A cells, but more electron dense than type B cells. Type C cells were less common (~4% of the total) in the SCZ compared with the SVZ (~10% of the total) (Doetsch and others 1997). The nuclei of SCZ type C cells showed deep invaginations, mostly lax chromatin, large reticulated nucleoli, free ribosomes, a well-developed Golgi apparatus, and smooth contours (Fig. 3H). Their cytoplasm contains fewer ribosomes than type A cells and no bundles of intermediate filaments. The anterior portion of the SCZ has a more heterogeneous cell composition, and it is richer in astrocytes (Table 1). Clusters of cells containing astrocytes, type A cells, and type C cells were usually associated to the callosal side of the SCZ. Type A and type C cells with associated astrocytes were less frequent in the caudal SCZ. In the caudal SCZ, we found many ependymal cells, suggesting that this region corresponds mostly to ventricular walls that became occluded during development (Table 1). The ultrastructural data indicate that cell types in the SCZ have some characteristics similar to those in the SVZ; however, the distribution (Table 1) and arrangement of these cells appear to differ significantly. The proportions of type C and type A cells in the SCZ were significantly lower than that observed in the SVZ. The SVZ chain organization, with chains of type A cells ensheathed by astrocytes next to tight clusters of type C cells, is not observed in the SCZ. Interestingly, SCZ type A cells were frequently observed alone or in small groups, associated not only to astrocytes but also to other parenchymal elements including oligodendrocytes, ependymal cells, and white matter tracks. Figure 3. Cell types and immunocytochemical characterization of the SCZ region. (A) Light micrograph of a coronal semithin section through the dorsal hippocampus showing a cavity in the SCZ lined with ependymal cells. (B) Higher magnification of inset in panel A showing a region of the SCZ at the interface of an ependymal cavity and a fussed section of the SCZ. Notice how the cavity is lined with multiciliated ependymal cells (asterisks). (C) EM micrograph showing some of the cell types present in the SCZ: ependymal cells (E), astrocytes (B), and migrating cells (A). (D) Pre-embedding immunocytochemistry of a semithin coronal section of SCZ showing mCD24-labeled ependymal cells. (E) Pre-embedding immunocytochemistry of a semithin coronal section of SCZ showing GFAP-labeled astrocytes. (F) Pre-embedding Fate of SCZ Cells In Vivo To test the in vivo fate of SCZ cells, we microinjected a small volume (50--100 nL) of a murine retrovirus carrying AP as a reporter gene (w2-DAP) into the SCZ region of adult CD1 mice (n = 15). This virus becomes integrated in the DNA of dividing cells and labels their progeny (Cepko 1996). Three days after injection, astrocytes and migratory cells with morphology similar to that of type A cells were seen in the SCZ as well as in the neighboring roof of the SVZ (not shown). By 30 days, the majority of labeled cells had the morphology of oligodendrocytes, and most of these cells were found in the corpus callosum. Some astrocytes were also observed in the corpus callosum dorsal to the SCZ (Fig. 4E,F). In addition to oligodendrocytes and astrocytes in the SCZ and neighboring regions, we also observed a small number of AP+ granule and periglomerular neurons, which migrated to the OB (not shown). Some progenitors in the caudal SVZ at the level of the inferior horn of the lateral ventricle migrate rostrally and reach the OB (Doetsch and Alvarez-Buylla 1996). Due to viral spread after injections, we cannot exclude that the neurons we observed in the OB may have originated at the SVZ--SCZ border or in the neighboring caudal SVZ. To further define the fate of cells in regions within the SCZ and to avoid labeling cells in the borders or outside the SCZ, we performed homochronic, homotypic microtransplantations using donor SCZ cells from mice carrying the GFP gene under the b-actin promoter (b-actin:GFP) into the SCZ of adult CD1 mice. In one group of animals (n = 15), anterior SCZ grafts were placed in the anterior SCZ (AP: 1.0, ML: 2.0, and DV: 1.25). A second group (n = 15) received caudal SCZ grafts in the caudal SCZ (AP: 2.3, ML: 2.6, and DV: 1.65). These 2 regions have different cell composition as shown in Table 1. Transplanted cells were visualized by their green fluorescence and using a monoclonal antibody against GFP. One day after transplantation, GFP+ cells were found around the grafting site. Seven days after transplantation, GFP+ cells with the elongated morphology of migrating cells were observed as far away as 100 lm from the graft site. These cells had a round soma, a short leading process, and stained positive for PSA-NCAM and Rip-1 (Fig. 4A), an antibody that stains immature oligodendrocytes. Interestingly, GFP+ cells did not stain with antibodies against TUJ1 (not shown). Occasionally, GFP+ cells stained with antibodies against GFAP (Fig. 4C,D). The immunostaining suggested that the majority of transplanted SCZ cells were young migrating oligodendrocyte precursors, whereas some of the grafted cells were or became astrocytes (Fig. 4B--D). Thirty days after transplantation, GFP+ cells with a small round soma and extensive thin-branched processes were observed along the corpus callosum (Fig. 4B). These cells did not stain with antibodies against PSA-NCAM or TUJ1 but did stain positive with antibodies against Rip-1 (Fig. 4A), and immunocytochemistry of a semithin coronal section of SCZ showing vimentin-labeled astrocytes. (G--K) Whole-mount preparation of the SCZ region showing PSA-NCAM-positive cell clusters. These clusters (I) and (K) form short discontinuous chainlike structures. Arrowheads in (G) point to the boundary between the SCZ and the SVZ. (H) EM micrograph of type C cell in the SCZ. (J) EM micrograph showing an SCZ type A cell contacting myelinated fibers (arrows) in the corpus callosum. Scale bars: (A), 100 lm; (B), 10 lm; (C), 3 lm; (D--F), 15 lm; (G), 200 lm; (H), 3 lm; (J), 1 lm.CC, corpus callosum. Cerebral Cortex 2006, V 16 Supplement 1 i107 Table 1 Percent cell composition of the SCZ Anterior (305 cells) Posterior (311 cells) Ependyma Astrocytes Oligodendrocytes Type C Type A Microglia Unknown 21% (65) 49% (152) 39% (118) 29% (91) 7% (22) 6% (19) 6% (19) 2% (7) 24% (72) 11% (33) \1% (1) 1% (2) 3% (8) 2% (7) Note: Coronal semithin sections were processes for EM, and cell types were quantified for the anterior and posterior regions of the SCZ. In parenthesis is the number of cells counted using EM. occasionally, some cells with a round to polygonal soma and a thin-branched processes stained positive with antibodies against GFAP (Fig. 4C,D), whereas other cells with a round soma and multiple, thin branched processes did not stain with antibodies against GFAP (Fig. 4D, arrow). Grafts targeted to the anterior, medial, or the caudal SCZ produced similar types of glial cells. In grafts targeted to mid regions of the SCZ away from the SVZ, we found no evidence of neurons that migrated to the OB. Therefore, the transplantation experiments suggest that neurons that migrate into the rostral migratory stream (RMS) and OB after viral labeling do not originate from sites within the SCZ. These microtransplantation experiments suggest that these regions of the SCZ produce only oligodendrocytes and astrocytes, most of which migrate into the corpus callosum in the intact brain. We did not observe neurons derived from these microtransplantations in the underlying hippocampus (including the DG) or in cortex. Nevertheless, the SCZ extends over a large area between the hippocampus and corpus callosum, and we cannot exclude that some neurons may be born in certain regions of this large germinal zone. Discussion Here we describe the architecture, cellular composition, and fate of cells in the SCZ, an extensive germinal zone in the caudal adult murine telencephalon. This lamina of proliferating cells is located between the corpus callosum and the hippocampus sandwiched between large axonal bundles from these 2 regions, and it extends medial and caudal from the SVZ. Accordingly, this region has similarities with the SVZ, but it also possesses unique properties. We show that the SCZ contains cells that can be grown in vitro as neurospheres by the addition of EGF and FGF to the culture medium. Although fewer cells in the SCZ grew as neurospheres compared with the SVZ, SCZ neurospheres had similar characteristics to NSCs isolated from other regions of the developing or adult central nervous system (Reynolds and Weiss 1992; Gritti and others 1996; Morshead and others 1998). Because SCZ neurospheres could self-renew and generate differentiated progeny of oligodendrocytes, astrocytes, and neurons (Potten and Loeffler 1990; Van der Kooy and Wiess 2000; Temple 2001a), it appears to be a region containing cells that can generate NSCs in vitro. Although these experiments show that cells with this potential can be isolated from the adult SCZ, the behavior of individual primary progenitors in vivo is not known. Previous work in the SVZ indicates that the majority of neurospheres grown in EGF are derived from type C cells and only a minority is derived from type B cells, which function as primary precursors in vivo (Doetsch and others 2002). We also do not know which of the cells in the adult SCZ correspond to the progenitors giving rise to neurospheres in vitro. This region contains a smaller number of type C cells, and it is possible that this explains the reduced number of neurospheres isolated i108 SCZ: A Germinal Layer in the Adult Brain d Seri and others from the SCZ compared with the SVZ (Fig. 2A). We found that astrocytes in the SCZ divide and are retained for up to 30 days after BrdU treatment. This suggests that the primary progenitor cells in the SCZ could correspond to astrocytes as has been shown in other germinal zones of the adult brain (Doetsch, Caillé, and others 1999; Alvarez-Buylla and others 2001; Seri and others 2001; Imura and others 2003; Garcia and others 2004). However, the topography of the SCZ is complicated by the massive growth of the hippocampus, and the precise identification of primary progenitors in the SCZ and their origin during cortical development remains to be investigated. It would be interesting to determine if SCZ progenitors have a pallial origin and whether SCZ cells retain the expression of some pallial markers (e.g., Emx1/2, Pax6). A major difference between the SVZ and the SCZ is the organization of PSA-NCAM--stained progenitors. Whereas PSANCAM--positive cells in the SVZ form an extensive network of interconnected chains, most of which are aligned along the rostrocaudal plane (Doetsch and Alvarez-Buylla 1996), PSANCAM--positive cells in the SCZ form small clusters with few cells oriented orthogonal to the orientation of chains in the SVZ (Fig. 3G,I,K). Only, cells in the lateral and rostral border of the SCZ seem to join chains in the SVZ. The majority of PSANCAM--positive clusters within the SCZ are isolated and do not interconnect with other clusters or chains in the SVZ. This organization suggests that most of the cells born in the SCZ do not migrate tangentially into the SVZ but move into the overlying corpus callosum. Consistently, most of the proliferative activity observed in the SCZ was associated with its dorsal (callosal) side. Furthermore, using viral labeling and homotypic grafting of genetically labeled cells, we found that cells derived from the SCZ give rise to astrocytes and oligodendrocytes in the corpus callosum. Occasionally, after retroviral injection, we observed cells differentiate into oligodendrocytes in the striatum and in fimbria--fornix (Fig. 4). However, we cannot rule out that the spread of the virus may have infected local glial progenitors in the fimbria instead of infecting SCZ cells that migrated to this region because after microtransplantations, we did not observe GFP-positive cells in the fimbria. Ultrastructural data showed that type A cells in the SCZ are not surrounded by astrocytes, which is another major difference with the organization in the SVZ. Interestingly, SCZ cells form clusters that do not appear to reach into the network of chains in the SVZ. The direct contact between type A cells and myelinated axons in the corpus callosum (Fig. 3J) is also different from the organization observed in the SVZ and consistent with the observation that SCZ A cells migrate into the corpus callosum. Although the SCZ is in close proximity to the hippocampus, we found no evidence that cells in this region serve as precursors for the new neurons added to the adult DG (Kaplan and Hinds 1977; Cameron and others 1993; Kuhn and others 1996; Seri and others 2001). We also found no evidence that Figure 4. Cell types derived from the SCZ in vivo. (A--D) Cell fates after microtransplantations in the SCZ. (A, B) Macroglial cell types arising from homotypic GFP+ SCZ transplants. (A) Seven days after transplantation GFP+ (green)/Rip-1+ (red) cells were found around the transplant site in the anterior SCZ. (B) Thirty days after transplantation, GFP+ cells could be seen in the corpus callosum. Note the thinbranched processes of this small GFP+ cell. Thirty days after transplant, cells were visualized using an antibody against GFP. (C, D) Thirty days after transplantation, cells with a small soma and long thin branches (C) were observed as far as 100 lm away from the grafting site. Note that some of the GFP+ cells stain positive (arrow) with antibodies against GFAP (blue). (E, F) A retrovirus carrying the AP reporter gene was stereotaxically injected into the SCZ region. All panels show sagittal sections; anterior is right, and posterior is left. (E) Retrovirally labeled cells 30 days after viral infection. Inset in (E) shows 2 cells in dorsal striatum where they are localized within an axonal bundle. These cells have the morphology of immature oligodendrocytes. (F) Retrovirally labeled cells in the corpus callosum 30 days after infection. Cell in the inset shows the classical morphology of a mature oligodendrocyte. HP, hippocampus, LV, lateral ventricle. Scale bars: (A), 5 lm; (B--D), 10 lm; (E, F), 10 lm; inset, 5 lm. new neurons in Ammon’s horn originate in the SCZ, although it has been suggested that in some cases neurons may be recruited into the CA1 and CA2 regions of Ammon’s horn in the hippocampus (Rietze and others 2000; Nakatomi and others 2002). It has also been suggested that new neurons may be added to the lesioned neocortex of rodents (Magavi and others 2000) and that small neurons may be added to the lower cortex close to the corpus callosum in adult mice (Dayer and others 2005). Although we did not see any evidence for the incorporation of new cortical neurons in our experiments, the SCZ region has the potential to form new neurons, and it is located in direct contact with neocortical white matter. It is possible that following lesions, or under other conditions, the SCZ could be a source of cells that migrate into the neocortex and differentiate into neurons. This, however, remains to be clearly demonstrated. In rodents, the hippocampus dramatically increases in size during late fetal development (Altman and Bayer 1990; Reznikov 1991). A similar process occurs in the developing human brain (Arnold and Trojanowski 1996). This dramatic growth together with the early postnatal development of afferent and efferent fibers in the corpus callosum and alveus causes the space between cortex and the hippocampus to collapse. The walls of the lateral ventricle in this region become sequestered between the corpus callosum and the hippocampus, giving rise to the SCZ. Thus, the SCZ corresponds to 2 opposing walls of the lateral ventricle that collapsed following hippocampal growth and expansion. Consistent with this view, we observed remnants of ventricular cavity within the SCZ and regions containing ependymal cells (Fig. 3A--C). Ependymal cells in the mouse are born before birth (Bruni and others 1985; Spassky and others 2005) and become sequestered within regions of ventricular fusion (Kawamata and others 1995). Many ependymal cells in the SCZ are lost during postnatal development, but some remain even in regions where there is no evidence of ventricular cavity. Germinal regions that retain stem cells in the adult are of interest because of their role in endogenous brain plasticity, cellular homeostasis, and their potential use for brain repair. In addition, proliferative zones provide new experimental opportunities to study the regulation of neurogenesis and gliogenesis. Here we have described an extensive germinal region that continues to generate oligodendrocytes in the adult animal. It will be interesting to determine if SCZ progenitors are recruited to form new oligodendrocytes following demyelinating lesions as it has been shown for SVZ progenitors (Nait-Oumesmar and others 1999). During development, neuroepithelial progenitors appear to require a unique set of epigenetic signals to generate oligodendrocytes (Kuhn and Svendsen 1999; Anderson 2001; Rowitch 2004; Fogarty and others 2005). It will be important to determine if oligodendrocyte production is regulated in a similar manner in the adult brain and to investigate which cells within the SCZ or in the region immediately around it provide the epigenetic signals to create an oligodendrogenic niche. Interestingly, preliminary data from heterotypic microtransplantations of SVZ grafts into the SCZ revealed that a large proportion of cells from the grafts merged onto the RMS, migrated to the OB, and matured into granule neurons. A smaller proportion remained within the SCZ and became oligodendrocytes. Therefore, it will be interesting to characterize the specific markers that confer SCZ and SVZ cells their unique properties. The extensive size, unique localization between the hippocampus and cortex, and robust germinal capacity makes the SCZ an attractive region as a potential source of cells for brain repair. Cerebral Cortex 2006, V 16 Supplement 1 i109 Notes We are grateful to G. Rougon for the mCD24 and PSA-NCAM antibodies. We are thankful for the expert technical help of Sattie Haripal, Mario Soriano, and Daniela Ferrari and for Lawrence Reagan’s critical comments on the manuscript. BS was supported by National Institutes of Health (NIH) grant GM07524. Fundacio La Caixa supported JMGV. This work was supported by NIH grant NS28478. Conflict of Interest: None declared. Address correspondence to Arturo Alvarez-Buylla, Neurosurgery Department, University of California—San Francisco, Box 0520, 513 Parnassus, HSW1201F, San Francisco, CA 94143, USA. Email: abuylla@ itsa.ucsf.edu. References Altman J. 1969. Autoradiographic and histological studies of postnatal neurogenesis: IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J Comp Neurol 137:433--459. Altman J, Bayer S. 1990. Mosaic organization of the hippocampal neroepithelium and the multiple germinal sources of dentate granule cells. J Comp Neurol 301:325--342. Altman J, Das GD. 1965. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124:319--336. Alvarez-Buylla A, Garcı́a-Verdugo JM, Tramontin AD. 2001. A unified hypothesis on the lineage of neural stem cells. Nat Rev 2:1--17. Alvarez-Buylla A, Lim DA. 2004. For the long run: maintaining germinal niches in the adult brain. Neuron 41:683--686. Anderson DJ. 2001. Stem cells and pattern formation in the nervous system: the possible versus the actual. Neuron 30:19--35. Arnold SE, Trojanowski JQ. 1996. Human fetal hippocampal development: I. Cytoarchitecture, myeloarchitecture, and neuronal morphologic features. J Comp Neurol 367:274--292. Barnea A, Nottebohm F. 1996. Recruitment and replacement of hippocampal neurons in young and adult chickadees: an addition to the theory of hippocampal learning. Proc Natl Acad Sci USA 93:714--718. Bayer SA, editor. 1985. The rat nervous system. In: Hippocampal region. Sydney, Australia: Academic Press. p 335--354. Bruni JE, Del Bigio MR, Clattenburg RE. 1985. Ependyma: normal and pathological. A review of the literature. Brain Res 356:1--19. Cameron HA, Woolley CS, McEwen BS, Gould E. 1993. Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56:337--344. Carleton A, Petreanu LT, Lansford R, Alvarez-Buylla A, Lledo PM. 2003. Becoming a new neuron in the adult olfactory bulb. Nat Neurosci 6:507--518. Cepko CL. 1996. Transduction of genes using retrovirus vectors. Curr Protoc Mol Biol 2:9.9.1--9.14.3. Dayer AG, Cleaver KM, Abouantoun T, Cameron HA. 2005. New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors. J Cell Biol 168:415--427. Doetsch F, Petreanu L, Caille I, Garcia-Verdugo JM, Alvarez-Buylla A. 2002. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron 36:1021--1034. Doetsch F, Alvarez-Buylla A. 1996. Network of tangential pathways for neuronal migration in adult mammalian brain. Proc Natl Acad Sci USA 93:14895--14900. Doetsch F, Caillé I, Lim DA, Garcı́a-Verdugo JM, Alvarez-Buylla A. 1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703--716. Doetsch F, Garcia-Verdugo JM, 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. Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A. 1999. Regeneration of a germinal layer in the adult mammalian brain. Proc Natl Acad Sci USA 96:11619--11624. Feng R, Rampon C, Tang YP, Shrom D, Jin J, Kyin M, Sopher B, Miller MW, Ware CB, Martin GM, Kim SH, Langdon RB, Sisodia SS, Tsien JZ. 2001. i110 SCZ: A Germinal Layer in the Adult Brain d Seri and others Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. Neuron 32:911--926. Ffrench-Constant C, Raff MC. 1986. The oligodendrocyte-type-2 astrocyte cell lineage is specialized for myelination. Nature 323:335--338. Fogarty M, Richardson WD, Kessaris N. 2005. A subset of oligodendrocytes generated from radial glia in the dorsal spinal cord. Development 132:1951--1959. Gage FH. 2000. Mammalian neural stem cells. Science 287:1433--1438. Garcia AD, Doan NB, Imura T, Bush TG, Sofroniew MV. 2004. GFAPexpressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain. Nat Neurosci 7:1233--1241. Gritti A, Parati EA, Cova L, Frolichsthal P, Galli R, Wanke E, Faravelli L, Morassutti DJ, Roisen F, Nickel DD, Vescovi AL. 1996. Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci 16:1091--1100. Imura T, Kornblum HI, Sofroniew MV. 2003. The predominant neural stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain expresses GFAP. J Neurosci 23:2824--2832. Kaplan MS, Bell DH. 1984. Mitotic neuroblasts in the 9-day-old and 11-month-old rodent hippocampus. J Neurosci 4:1429--1441. Kaplan MS, Hinds JW. 1977. Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs. Science 197:1092--1094. Kawamata S, Stumpf WE, Bidmon HJ. 1995. Adhesion and fusion of ependyma in rat brain. Acta Anat (Basel) 152:205--214. Kuhn GH, Svendsen CN. 1999. Origins, functions, and potential of adult neural stem cells. Bioessays 21:625--630. Kuhn HG, Dickinson-Anson H, Gage FH. 1996. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 16:2027--2033. Lemasson M, Saghatelyan A, Olivo-Marin JC, Lledo PM. 2005. Neonatal and adult neurogenesis provide two distinct populations of newborn neurons to the mouse olfactory bulb. J Neurosci 25:6816--6825. Levine JM, Reynolds R, Fawcett JW. 2001. The oligodendrocyte precursor cell in health and disease. Trends Neurosci 24:39--47. Levison SW, Goldman JE. 1993. Both oligodendrocytes and astrocytes develop from progenitors in the subventricular zone of postnatal rat forebrain. Neuron 10:201--212. Lichtenwalner RJ, Parent JM. 2006. Adult neurogenesis and the ischemic forebrain. J Cereb Blood Flow Metab 26:1--20. Lois C, Alvarez-Buylla A. 1993. Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc Natl Acad Sci USA 90:2074--2075. Luskin M. 1993. Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11:173--189. Magavi SS, Leavitt BR, Macklis JD. 2000. Induction of neurogenesis in the neocortex of adult mice. Nature 405:951--955. Morshead CM, Craig CG, Van der Kooy D. 1998. In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain. Development 125:2251--2261. Nait-Oumesmar B, Decker L, Lachapelle F, Avellaneda-Adalid V, Bachelin C, Baron-Van Evercooren A. 1999. Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination. Eur J Neurosci 11:4357--4366. Nakatomi H, Kuriu T, Okabe S, Yamamoto S, Hatano O, Kawahara N, Tamura A, Kirino T, Nakafuku M. 2002. Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 110:429--441. Peretto P, Merighi A, Fasolo A, Bonfanti L. 1997. Glial tubes in the rostral migratory stream of the adult rat. Brain Res Bull 42:9--21. Peterson DA, Jones DG. 1993. Determination of neuronal number and process surface area in organotypic cultures: a stereological approach. J Neurosci Methods 46:107--120. Potten CS, Loeffler M. 1990. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties lessons for and from the crypt. Development 110:1001--1020. Reynolds BA, Weiss S. 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707--1710. Reznikov KY. 1991. Cell proliferation and cytogenesis in the mouse hippocampus. Adv Anat Embryol Cell Biol 122:1--83. Rietze RL, Polin P, Weiss S. 2000. Mitotically active cells that generate neurons and astrocytes are present in multiple regions of the adult mouse hippocampus. J Comp Neurol 424:397--408. Rousselot P, Lois C, Alvarez-Buylla A. 1995. Embryonic (PSA) N-CAM reveals chains of migrating neuroblasts between the lateral ventricle and the olfactory bulb of adult mice. J Comp Neurol 251:51--61. Rowitch DH. 2004. Glial specification in the vertebrate neural tube. Nat Rev Neurosci 5:409--419. Seri B, Garcia-Verdugo JM, Collado-Morente L, McEwen BS, AlvarezBuylla A. 2004. Cell types, lineage, and architecture of the germinal zone in the adult dentate gyrus. J Comp Neurol 478:359--371. Seri B, Garcia-Verdugo JM, McEwen BS, Alvarez-Buylla A. 2001. Astrocytes give rise to new neurons in the adult mammalian hippocampus. J Neurosci 21:7153--7160. Shors TJ, Miesegaes G, Beylin A, Ahao M, Rydel T, Gould E. 2001. Neurogenesis in the adult is involved in the formation of trace memories. Nature 410:372--376. Spassky N, Merkle FT, Flames N, Tramontin AD, Garcia-Verdugo JM, AlvarezBuylla A. 2005. Adult ependymal cells are postmitotic and are derived from radial glial cells during embryogenesis. J Neurosci 25:10--18. Temple S. 2001a. The development of neural stem cells. Nature 414:112--117. Temple S. 2001b. Stem cell plasticity—building the brain of our dreams. Nat Rev Neurosci 2:513--520. Van der Kooy D, Wiess S. 2000. Why stem cells? Science 287:1439-1441. Wolswijk G, Noble M. 1989. Identification of an adult-specific glial progenitor cell. Development 105:387--400. Wood PM, Bunge RP. 1991. The origin of remyelinating cells in the adult central nervous system: the role of mature oligodendrocytes. Glia 4:225--232. Cerebral Cortex 2006, V 16 Supplement 1 i111