Download Composition and Organization of the SCZ: A Large Germinal Layer

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
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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
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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
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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.
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