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First published online 28 November 2007
doi: 10.1242/dev.004895
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1 Department of Physiology and Neurobiology, University of Connecticut, 75 North
Eagleville Road, Storrs, CT 06269-3156, USA.
2 Department of Neuroscience, Johns Hopkins University School of Medicine, 813
Wood Basic Science Building, 725 N. Wolfe Street, Baltimore, MD 21205,
USA.
* Author for correspondence (e-mail: akiko.nishiyama{at}uconn.edu)
Accepted 8 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: NG2 (CSPG4), Oligodendrocyte, Progenitor cell, Astrocyte, Myelin, Transgene, FACS
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Dawson et al., 2003;
Nishiyama et al., 2002;
Peters, 2004). In culture they
give rise to oligodendrocytes and are thus called oligodendrocyte precursor
cells (OPCs). The term `OPCs' is used instead of NG2 glia throughout this
manuscript to refer to cells that give rise to oligodendrocytes or to cells
whose identity had not been confirmed by NG2 immunolabeling.
Direct demonstration of the fate of NG2 glia in vivo has been difficult, as
NG2 expression is downregulated before they undergo terminal differentiation.
Indirect evidence that NG2 glia differentiate into oligodendrocytes has been
obtained from double immunolabeling for NG2 and oligodendrocyte antigens. A
small fraction of NG2-expressing cells in normal and demyelinated lesions also
express oligodendrocyte antigens (Levine
et al., 1993; Nishiyama et
al., 1996a; Trapp et al.,
1997; Polito and Reynolds,
2005). Demonstration by Reynolds and Hardy
(Reynolds and Hardy, 1997)
that all NG2-expressing cells in both gray and white matter of the mature rat
brain express the immature oligodendrocyte antigen O4 has provided strong
support for the notion that NG2 glia are committed precursors of the
oligodendrocyte lineage. Additional evidence that NG2 glia give rise to
oligodendrocytes was obtained by pulse-chase labeling with
5-bromo-2'-deoxyuridine (BrdU) combined with immunolabeling for cell
type-specific antigens at the age and location where the majority of the
proliferating cells are NG2 glia. In these areas, which include the juvenile
and adult spinal cord, BrdU-incorporated cells were seen to turn into mature
oligodendrocytes over time in normal and demyelinated tissues
(Bu et al., 2004;
Horner et al., 2000;
Watanabe et al., 2002;
McTigue et al., 2001). It
still remains unclear whether these cells go on to become myelinating
oligodendrocytes.
The question of whether NG2 glia can give rise to astrocytes has not yet
been entirely solved. It is well established that in culture, OPCs can give
rise to both oligodendrocytes and type-2 astrocytes
(Raff et al., 1983). The
search for an astrocytic fate of NG2 glia in vivo has not yielded positive
results because of the lack of evidence for the existence of cells that
co-express NG2 and astrocytic markers and the lack of evidence that
transplanted OPCs generate astrocytes in the CNS
(Reynolds et al., 2002;
Nishiyama et al., 2002;
Nishiyama et al., 2005;
Groves et al., 1993;
Espinosa de los Monteros et al.,
1993). Other studies, however, suggest that there may be glial
progenitor cells that can give rise to both oligodendrocytes and astrocytes.
Rao and Mayer-Proschel identified glial-restricted precursors that generate
astrocytes and oligodendrocytes but not neurons
(Rao and Mayer-Proschel,
1997), although the exact identity and fate potential of these
cells in vivo are not yet clear. The observation
(Franklin et al., 1995) that
transplanted OPCs generate astrocytes in an astrocyte-free environment is also
suggestive of an astroglial fate potential of NG2 glia.
In order to determine more directly the fate of NG2 glia, we have used the bacterial artificial chromosome (BAC) modification technique to generate transgenic mice that express the red fluorescent protein DsRed or the bacteriophage Cre recombinase specifically in NG2-positive (NG2+) cells. We show that NG2+ cells give rise to protoplasmic astrocytes as well as oligodendrocytes in vivo and in vitro.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Heintz, 2001) to generate
transgenic mice that express DsRed or Cre specifically in NG2-expressing cells
(see Fig. S1 in the supplementary material). We obtained a 208 kb BAC clone in
pBACe3.6 vector from the RPCI-23 C57BL/6J mouse BAC library that contained the
entire 33.97 kb Cspg4 (ng2) gene (BACPAC Resources; Oakland,
CA) in the center, flanked by 60,274 bp and 114,045 bp of additional sequences
on the 5' and 3' ends of the gene, respectively. DsRed.T1
(Bevis and Glick, 2002) (a gift
from Dr Benjamin Glick, University of Chicago, IL), which is a variant of the
original red fluorescent protein DsRed1 derived from the coral
Discosoma (Matz et al.,
1999) with excitation and emission peaks at 554 and 586 nm,
respectively, was inserted into the first exon of the ng2 gene in the
BAC clone as follows. An NG2-DsRed building vector was constructed in
pBluescript by ligating together the following DNA fragments from 5' to
3': 555 bp of the 5' NG2 homology arm, DsRed.T1 coding sequence,
535 bp of rabbit β-globin polyadenylation signal from pCAGGS (gift from
Dr Kenji Sakimura, Niigata University, Japan), and 603 bp of NG2 3'
homology arm. The 5' NG2 homology arm consisted of 542 bp upstream of
the translation initiation codon and 33 bp downstream of the ATG.
5'-RACE analysis revealed the transcription initiation site to be 127 bp
upstream from the translation initiation site (data not shown). DsRed.T1 was
inserted into the first exon of the ng2 gene after mutating the first
translation initiation ATG to AAG to prevent translation from the BAC
ng2 gene and to allow translation from the first ATG in the DsRed.T1
cDNA in the context of a strong Kozak consensus sequence. The 3'
homology arm consisted of sequences in the first exon starting 4 bp downstream
of the 5' homology arm and extending 533 bp into the first intron (see
Fig. S1A in the supplementary material).
The transgene was subcloned into pRecA.SV1 shuttle vector (provided by Dr
Nathaniel Heintz, Rockefeller University, New York) that contains
recA to allow homologous recombination
(Yang et al., 1997). The
resulting plasmid was used to transform DH10 competent cells harboring the
NG2BAC DNA. The shuttle vector pRecA.SV1 contained a temperature-sensitive
origin of replication and tetracycline resistance gene, which were used to
screen colonies for integration and subsequent resolution events. The
resulting modified BAC clones were analyzed by Southern blotting and PCR to
confirm the integrity of the transgene and were further tested for adequate
DsRed expression by transfecting N20.1 mouse oligodendrocyte precursor cells
(Verity et al., 1993). DNA
from a correctly modified clone was purified using the BAC Maxi kit (Clontech,
Palo Alto, CA), linearized with NotI, and microinjected into the
pronucleus of fertilized oocytes from B6SJL/F1 and C57BL/6J mice. Three
founders were identified by PCR using primers from DsRed sequence.
A similar approach was used to generate NG2creBAC transgenic mice (see Fig. S1B in the supplementary material). The nls-cre cDNA that contains the coding region of cre with a nuclear localization signal was obtained from Dr Kenji Sakimura (Brain Research Institute, Niigata, Japan). This was used to generate an NG2creBAC building vector that was identical to the NG2DsRedBAC building vector except that the DsRed coding region was replaced with the nls-cre coding region.
Z/EG (lacZ/EGFP) Cre reporter mice (see Fig. S1B in the supplementary
material) (Novak et al., 2000)
were kindly provided to us by Dr Caiying Guo (University of Connecticut Health
Center, Farmington, CT). Another Cre reporter mouse line, ROSA26R
(Soriano, 1999), was obtained
from the Jackson Laboratory (Bar Harbor, ME). These reporter lines were
maintained as homozygous lines and crossed to NG2creBAC mice.
Tissue processing
Mice were anesthetized and sacrificed by intracardiac perfusion with 2%
paraformaldehyde solution in phosphate buffer containing 0.01 M sodium
metaperiodate and 0.1 M lysine (paraformaldehyde-lysine-periodate fix)
(McLean and Nakane, 1974).
Brain, cerebellum and spinal cord tissues were removed and postfixed for 2
hours in the same fixative followed by washes in 0.2 M sodium phosphate buffer
(pH 7.4). Sections of 50 or 100 µm were cut on a Leica VT1000 Vibroslicer.
Embryos were fixed by immersion fixation in the same fixative immediately
after they were isolated from the uterus, rinsed in sodium phosphate buffer,
cryoprotected in 20% sucrose, and 20 µm sections were cut on a cryostat
(Microm HM 500M).
Fluorescence-activated cell sorting (FACS)
Cells were isolated from P2 or P3 wild-type (wt) and NG2DsRedBAC transgenic
mouse (tg) brains by dissociating the cerebral cortex, subcortical white
matter, and subventricular zone in S-MEM (Invitrogen, Carlsbad, CA) containing
0.1% trypsin and 50 µM kynurenic acid (Sigma, St Louis, MO) for 15 minutes
at 37°C. Trypsin was inactivated by the addition of an equal volume of
DMEM containing 10% fetal calf serum (FCS; Invitrogen). The cells were then
triturated in the presence of 0.05 mg/ml of DNase I (Sigma). On average
1.2x106 cells were obtained from each cerebrum. The
dissociated cells were subjected to FACS sorting using a Becton-Dickinson
FACScalibur Dual Laser Flow Cytometer. Cells from wt and tg cerebra were used
to establish the gate settings (Fig.
3). The electronic gain of the red channel was adjusted such that
the maximum intensity of wt cells was designated as 101 on the
x-axis of the flow cytometry data
(Fig. 3B,D). Cells from tg
cerebra were sorted at 1500-2500 cells per second. Sorted cells were collected
by centrifugation, and plated on glass coverslips coated with 100 µg/ml
poly-L-lysine. Cells were maintained in either neurobasal medium
containing B27 supplements (Invitrogen) and 10 ng/ml platelet-derived growth
factor (PDGF AA; R&D Systems, Minneapolis. MN), or neural stem cell medium
consisting of DMEM-F12 containing 0.6% glucose, 3 mM sodium bicarbonate, 2 mM
glutamine, 25 µg/ml insulin, 100 µg/ml transferrin, 20 nM progesterone,
60 µM putrescine, 30 nM sodium selenite 10 ng/ml FGF2 (fibroblast growth
factor 2; R&D Systems), and 20 ng/ml EGF (epidermal growth factor; R&D
Systems). In some experiments, cells from P3 subventricular zone (SVZ) or P5
cerebellum were dissociated as described above to obtain cultures of neural
stem cells or neurons.
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;
Bu et al., 2004). The source
and dilution of the antibodies are listed in
Table 1. Labeled sections were
mounted in Vectashield containing DAPI (Vector) and examined using a Leica DMR
epifluorescence upright microscope equipped with a digital camera (ORCA,
Hamamatsu, Japan) and the IP Lab image acquisition software (Scanalytics,
Fairfax, VA), Zeiss Axiovert 200M equipped with Axiocam and ApoTome, or with a
Leica TCS SP2 spectral confocal imaging system.
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Cell counts
For antigenic phenotyping of sorted cells, the number of cells that were
immunopositive for each antigen was counted and expressed as a percentage of
all DAPI+ cells. At least 150 cells were scored on each coverslip, and a
minimum of three coverslips from three independent sorting experiments were
analyzed. The numbers were expressed as means ± standard
deviations.
To assess regional differences in the number of astrocytes generated from NG2 glia, the number of cells that were immunoreactive for EGFP or S100β was counted in one hemisphere from three P14 NG2creBAC:Z/EG double tg brains. Using a 20x objective lens, the optical fields were systematically scanned from dorsal to ventral cortex or from dorsolateral striatum to the ventral midline, and the number of cells that were EGFP+ or S100β+ was counted. The density of cells was determined by dividing the total number of cells by the total area of the scanned fields. Area measurements were obtained using the Axiovision program on Zeiss Axiovert 200M.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The distribution of DsRed fluorescence in NG2DsRedBAC tg mice was compared with NG2 immunoreactivity in postnatal day 3 (P3) and P30 tg mice (Fig. 1). DsRed+NG2+ cells were distributed widely throughout the gray and white matter of the brain, cerebellum and spinal cord, with the exception of the external granule cell layer of the P3 cerebellum, which was devoid of DsRed+NG2+ cells (not shown). In addition to glial cells, vascular mural cells also expressed DsRed (see below). The distribution of DsRed+ and NG2-immunoreactive cells overlapped in all the regions examined at both developmental stages. DsRed fluorescence was sufficiently strong that it could be readily detected without using anti-DsRed antibodies.
The expression of DsRed was more closely compared with that of various cell
type-specific markers in the P30 brain. In the cerebral cortex and corpus
callosum, all NG2-immunoreactive cells contained DsRed fluorescence
(Fig. 2A,B). DsRed fluorescence
was most intense in the cell bodies but was also detected in distal processes
(Fig. 2D), suggesting that the
tetrameric DsRed.T1 mutant used in the current study
(Bevis and Glick, 2002) remains
soluble and does not aggregate as much as the original DsRed protein
(Yarbrough et al., 2001).
DsRed is expressed in cells of the oligodendrocyte lineage
In P30 tg mouse brain, all the DsRed+ non-vascular cells in both gray and
white matter expressed PDGFR
(Fig.
2C), an antigen known to be expressed by NG2+ OPCs
(Pringle et al., 1992;
Nishiyama et al., 1996a;
Nishiyama et al., 1996b).
DsRed+ vascular cells were negative for PDGFR. To determine whether
DsRed was expressed by mature oligodendrocytes, sections from P30 tg brains
were labeled with a monoclonal antibody to the adenomatosis polyposis
coli (Apc) gene product (clone CC1) which was previously shown
to be expressed by mature oligodendrocytes
(Bhat et al., 1996). In the
corpus callosum, APC+ oligodendrocytes were clustered in rows parallel to the
orientation of axons and had characteristic large oval cell bodies
(Fig. 2E). Strongly DsRed+
cells were negative for APC (Fig.
2E, arrows). However, there were some APC+ cells with typical
oligodendroglial morphology that had dim DsRed fluorescence
(Fig. 2E, arrowheads),
suggesting that DsRed expression persists in immature oligodendrocytes. Since
there was little overlap between NG2+ and APC+ cells (data not shown), it
appears that DsRed expression is downregulated after the disappearance of NG2,
which may reflect a longer half-life of DsRed protein compared to that of NG2.
We did not observe any myelin that had DsRed fluorescence.
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DsRed is present in mural cells of the cerebral vasculature
In addition to glial cells, DsRed was expressed on vascular cells, as
previously reported for NG2 (Ozerdem et
al., 2001). To investigate the expression of DsRed in the
vasculature, we used antibodies to CD31 (also known as PECAM1) to detect
endothelial cells (Vecchi et al.,
1994) and PDGFRβ to detect vascular mural cells, which
include pericytes and smooth muscle cells
(Ozerdem et al., 2001).
Labeling of P30 tg brain sections with antibodies to CD31 revealed the contour
of the capillaries. DsRed+ cells with long slender processes were seen along
the outer perimeter of the capillaries, and their processes seemed to surround
the surface of the blood vessels (Fig.
2J, arrowheads). All of the vascular DsRed+ cells in the P30 tg
brain were immunopositive for PDGFRβ
(Fig. 2K, arrowheads). DsRed+
vascular cells were morphologically different from parenchymal glial cells
(arrows in Fig. 2J,K). Vascular
DsRed+ cells had smaller, round cell bodies with fewer processes that appeared
thicker and `fuzzier' than those of DsRed+ glial cells. Vascular expression of
DsRed and NG2 was more prominent at P3
(Fig. 1A,C,E) than at P30
(Fig. 1B,D,F). The distribution
of DsRed+ cells described here demonstrates the highly tissue-specific
expression of DsRed in the NG2DsRedBAC tg mouse brain that is restricted to
NG2-expressing cells.
Fate of NG2+DsRed+ cells in vitro
Optimization of cell sorting conditions
Pilot experiments were performed to optimize the gating parameters for
sorting DsRed+ cells from NG2DsRedBAC tg brains. For plots of cells from wt
and tg brains, see Fig. S2 in the supplementary material. Gates for forward
and side scatter were adjusted to exclude small dead cells and large cells
with granular cytoplasm that were likely to be macrophages with
autofluorescence (see Fig. S2C in the supplementary material). Gates M1, M2
and M3 were set with increasing stringency to collect cells with relative
fluorescence intensity greater than 10, 50 and 100, respectively (see Fig. S2D
in the supplementary material). In wt cells the fluorescence intensity in the
red channel was less than 10. Sorted cells were plated on
poly-L-lysine-coated coverslips. The presence of DsRed fluorescence
in the sorted cells was scored 2 hours after plating using a Leica DMR upright
epifluorescence microscope. The percentage of DsRed+ cells among cells sorted
with gates M1, M2 and M3 were 87.4%, 91.3% and 97.8%, respectively. Upon close
inspection of the cells sorted with the M3 gate, all of the DsRed-negative
(DsRed-) cells had nuclei that were either apoptotic or were enlarged and
diffuse, without a well defined nuclear membrane. Such unhealthy-looking
DsRed-negative cells were not detected 4 hours after plating, suggesting that
they had not adhered to the coverslips and died during the additional 2 hours
of incubation. When the coverslips were scored 4 hours after sorting, 100% of
the DAPI+ cells sorted through the M3 gate exhibited visible DsRed
fluorescence (see Fig. S3 in the supplementary material). Therefore, all of
the subsequent sorting experiments were performed using the M3 gate.
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, the newly
differentiated oligodendrocyte antigen O1, GFAP and βIII-tubulin 4 hours
after plating (see Fig. S3 in the supplementary material). At this time point,
most of the cells had adhered, and short processes could be detected on some
cells, while the majority of the cells remained round. After NG2 and
PDGFR were cleaved from the cell surface during dissociation, punctate
immunoreactivity for these antigens was clearly discernible at the cell
surface by 4 hours after plating (see Fig. S3C,F in the supplementary
material). A small number of cells expressed O1 (see Fig. S3I in the
supplementary material). For the percentage of cells that were immunoreactive
for each antigen, see Fig. S4E in the supplementary material. More than 98% of
the cells expressed NG2 at the cell surface, and 1-5% of the cells were O1+.
Together, NG2+ and O1+ cells accounted for all of the cells, and a small
number of cells weakly expressed both NG2 and O1, as previously described for
secondary NG2 cell cultures from the cerebrum
(Nishiyama et al., 1996b) (see
also Fig. S4D in the supplementary material). The percentage of O1+ cells at 4
hours increased with the age of the mice from which the cells were isolated.
The majority of the sorted cells expressed PDGFR (see Fig. S3D-F in the
supplementary material), and none were PDGFRβ+ (data not shown),
indicating that the isolated cells were predominantly of the oligodendrocyte
lineage and not of vascular mural origin. None of the sorted cells expressed
the neuronal markers βIII-tubulin or NeuN.
Differentiation of sorted cells
To determine the differentiation potential of NG2-expressing cells, DsRed+
cells were sorted from P3 brains by FACS. The dissected material included
cerebral cortex, subcortical white matter, corpus callosum, subventricular
zone (SVZ), and hippocampus but did not include the basal ganglia or
diencephalon. Typically, three transgenic mouse brains were used for each FACS
experiment with a yield of 1
2x106 cells from each brain.
Sorting conditions were carefully established to yield 100% DsRed+ cells as
determined by fluorescence microscopy 4 hours after plating. Immunolabeling of
the sorted cells showed that NG2+ cells and O1+ cells accounted for all of the
sorted cells (see Figs S2, S3 in the supplementary material).
Oligodendrocyte lineage cells
Differentiation potential of the sorted cells was determined by culturing
them in neurobasal medium plus B27 and PDGF AA for 4 days, followed by
incubation in neurobasal medium plus B27 without PDGF AA for 7 days, and
examining their antigenic phenotype after 4, 7 and 11 days. For the percentage
of cells that were immunoreactive for each antigen after 4 and 11 days in
vitro (div), see Fig. S4E in the supplementary material. At 4 and 7 div, the
majority of the cells were NG2+ and PDGFR+ (see Fig. S4A,B in the
supplementary material), but there were also differentiated O1+
oligodendrocytes with expansive membranous processes (see Fig. S4C in the
supplementary material). The majority of the cells (>85%) were NG2+ even
after 11 div; some appeared relatively immature whereas others were multipolar
and resembled immature oligodendrocytes (see Fig. S4D in the supplementary
material). Under these conditions, O1+ cells constituted less than 20% of the
population in most experiments. Culturing the cells for longer periods of time
did not result in a significant increase in the number of O1+ cells above 15%.
There were signs of cell death among O1+ cells, suggested by the presence of
remnants of O1+ membranes that were unaccompanied by DAPI+ nuclei, and by
condensed nuclei. NG2+ cells and O1+ cells together accounted for the majority
of the cells at 4 and 11 days after plating.
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Neuronal lineage
To determine whether DsRed+ cells could be induced to differentiate into
neurons, sorted DsRed+ cells were cultured in neural stem cell (NSC) medium
previously used by Belachew et al.
(Belachew et al., 2003) to
achieve differentiation of neurons from cnp promoter-EGFP tg cells.
When DsRed+ cells from P3 brains were maintained in NSC medium in the presence
of FGF2 and EGF for 4 days, followed by 7 days of incubation in the absence of
growth factors, we did not detect any βIII-tubulin+ cells (see Fig. S4J
in the supplementary material). βIII-tubulin could be readily detected on
cerebellar granule neurons cultured from P5 mice (see Fig. S4K in the
supplementary material). These findings suggest that FACS-sorted NG2+DsRed+
cells are primarily of glial lineage and do not give rise to neurons under
these conditions.
Fate of NG2 cells in mice double transgenic for NG2creBAC and Z/EG
NG2creBAC transgenic mice
To determine whether NG2 glia give rise to astrocytes in vivo, we generated
NG2creBAC tg mice (see Fig. S1B in the supplementary material) by using the
same strategy that we had used to generate the NG2DsRedBAC tg mice, and
replaced DsRed cDNA with nls-cre cDNA, which encodes Cre with a nuclear
localization signal (obtained from Dr Kenji Sakimura; Brain Research
Institute, Niigata University). In the NG2creBAC tg mouse line, Cre
immunoreactivity was detected exclusively in NG2-expressing cells at all ages
examined (P1, P14 and P60) (Fig.
3) and was not detected in S100β+ or GFAP+ astrocytes or in
APC+ oligodendrocytes. To follow the progeny of NG2 glia, we crossed the
NG2creBAC tg mice with Z/EG reporter mice (see Fig. S1B in the supplementary
material) (Novak et al.,
2000). In NG2creBAC:Z/EG double tg mice, cells that have undergone
Cre-mediated excision can be distinguished by those that have not by the
expression of EGFP in the former and β-galactosidase in the latter. When
double-tg mice were analyzed at P1, P14 and P60, EGFP was expressed in the
majority of NG2+ cells throughout the brain at all time points examined. At
P14, 86% of NG2+ cells expressed EGFP in the double tg mice. A small
percentage of NG2+ cells expressed Cre but not EGFP, which indicates that the
efficiency of Cre-mediated recombination was less than 100%.
Myelinating oligodendrocytes are generated from NG2 glia
To determine whether NG2 glia generate myelinating oligodendrocytes in
vivo, we analyzed NG2creBAC:Z/EG double tg mice at P14, when myelination is
actively occurring in the forebrain. Oligodendrocytes and EGFP+ cells were
identified by antibodies to APC and GFP, respectively. In white matter tracts,
EGFP was detected not only in NG2+ cells but also in NG2-negative cells that
displayed a typical oligodendrocyte morphology, with large, round cell bodies,
which were also APC+. The distribution of such EGFP+ cells in the corpus
callosum and white matter of the striatum was similar to that of APC+
oligodendrocytes, and there was extensive colabeling of the same cells
(Fig. 4A-C). In the corpus
callosum and fiber tracts in the striatum, 77.8% of the APC+ oligodendrocytes
expressed EGFP, indicating that these APC+EGFP+ oligodendrocytes had been
generated from NG2 glia. A small number of APC+ oligodendrocytes were EGFP-.
EGFP was also detected in myelinating processes of the oligodendrocytes
(Fig. 4D-I). EGFP-labeled
myelin was seen more abundantly in the NG2creBAC:Z/EG double tg mice at P60
than at P14. These findings indicate that NG2 glia give rise to myelinating
oligodendrocytes in the postnatal brain.
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To determine the prevalence of NG2 glia-derived astrocytes, we counted the number of EGFP+ and S100β+ cells in different regions of 50 µm coronal sections from P14 NG2creBAC:Z/EG double tg mice. Cell counts were obtained from coronal sections through the anterior forebrain at the level of anterior commissure (Table 2A) and from sections through the posterior forebrain at the level of dorsal hippocampus (Table 2B). S100β was used as a marker to count astrocytes, since GFAP was not detectable in the majority of protoplasmic astrocytes. The number of EGFP+S100β+ astrocytes was determined by counting cells that were positive for both EGFP and S100β and displayed the typical morphology of protoplasmic astrocytes. There were some S100β+EGFP+ cells that were myelinating oligodendrocytes, and they were excluded from the counts. In ventral cortex of anterior forebrain (see diagram in Table 2 for definition of dorsal and ventral cortex used here), 18% of the S100β+ cells were EGFP+ astrocytes, and 22% of all the EGFP+ cells had astrocytic morphology. The percentage of EGFP+ astrocytes out of all S100β+ cells was two-fold greater in the ventral gray matter regions from posterior forebrain than in the corresponding ventral region of the anterior forebrain. More than one-third of S100β+ cells in posterior ventrolateral cortex were EGFP+. Considering that a small proportion of S100β+ cells were oligodendrocytes, the proportion of astrocytes in this region that were EGFP+ is likely to be higher. The density of EGFP+ astrocytes in dorsal cortex was 10- to 30-fold lower than that in ventral forebrain (see diagram in Table 2 for the designation of ventral and dorsal regions). Taken together, these findings indicate that NG2 glia differentiate into protoplasmic astrocytes primarily in the ventral gray matter of the forebrain, and that they do not generate fibrous astrocytes in white matter.
A similar pattern of distribution was found in P60 NG2creBAC:Z/EG double tg mice. EGFP+ astrocytes were also observed when another line of NG2creBAC transgenic mice was crossed to the Z/EG reporter line. In order to rule out the possibility that the ventral distribution of NG2 cell-derived protoplasmic astrocytes was due to the region-dependent expression of the reporter gene in Z/EG mice, we examined P14 Z/EG mice for the expression of the reporter and found that β-galactosidase was expressed similarly in both dorsal and ventral glial cells. Furthermore, when NG2creBAC tg mice were crossed to ROSA26R reporter mice in which the expression of the Cre reporter β-galactosidase is under an entirely different universally active promoter, a similar predominance of reporter expression in ventral telencephalic astrocytes was observed. These observations indicate that the higher frequency of NG2 cell-derived astrocytes in the ventral telencephalon is probably due to greater astrocyte differentiation from NG2 glia in ventral forebrain and not due to an uneven distribution of reporter expression.
At P1, some NG2-negative EGFP+ cells were already found in the ventral telencephalic gray matter of NG2creBAC:Z/EG double tg mice. The EGFP+NG2- cells did not express S100β or GFAP but expressed nestin and had multiple slender processes, and are likely to be immature astrocytes. Some EGFP+ cells that were weakly nestin+ had processes that appeared bushy in the distal regions. Weak NG2 expression was detected in such cells, especially at the tips of some of their processes (Fig. 6). Some of the EGFP+nestin+NG2+ `immature' astrocytes extended processes that terminated on blood vessels with a web-like ending (Fig. 6A, curved arrow), resembling the vascular end-foot of an astrocyte. At P14, a spectrum of different intensities of NG2 immunoreactivity was observed among the EGFP+ cells, and those with weaker NG2 labeling had bushier processes resembling those of protoplasmic astrocytes. These findings are indicative of a transition from NG2 glia to NG2-negative protoplasmic astrocytes in the ventral gray matter of the postnatal brain.
Such transitional forms were already evident at embryonic day 18 (E18). A cluster of EGFP+ cells was found in the ventral forebrain close to the ventral pial surface (Fig. 7B,E,F). The majority of the EGFP+ non-vascular cells expressed NG2 and had a relatively small number of long, slender processes. Some EGFP+ cells in the ventral forebrain had little or no detectable NG2 and expressed GLAST (glutamate aspartate transporter), known to be expressed by immature astrocytes (Fig. 7B,F). Such EGFP+GLAST+ cells were more abundant in the posterior (Fig. 7F) than anterior (Fig. 7B) ventral forebrain and were not detected in the dorsal forebrain at this age. They were found in close proximity to the cluster of EGFP+NG2+ cells. Some of the EGFP+GLAST+ cells, particularly those in the posterior ventral forebrain, had highly branched processes and morphologically resembled immature protoplasmic astrocytes seen in the early postnatal double tg brain (Fig. 7F, inset).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Mallon et al., 2002;
Gravel et al., 1998;
Yuan et al., 2002). We have
now generated several BAC tg mouse lines that express different genes using
the same BAC building vector. Although the level of tg expression varies from
line to line, the tissue specificity of tg expression in vivo is highly
reproduced in the different lines (R. Suzuki and A.N., unpublished
observations). The high degree of reproducibility of NG2 cell-specific pattern
of expression using the NG2 BAC building vector described here makes this a
powerful method for targeting various genes to NG2 cells.
Fate of NG2+DsRed+ cells in culture
The NG2DsRedBAC tg mice have enabled us to directly and exclusively select
for NG2 cells by FACS to study their fate potential. The sorting gate was set
stringently to ensure that 100% of the sorted cells were DsRed+, which
resulted in exclusion of some cells with low DsRed fluorescence. Under these
conditions, an average of 3.3% of the total cells from the cerebral cortex,
subcortical white matter and SVZ were sorted. NG2+ cells constituted more than
98% of the sorted cells, whereas O1+ oligodendrocytes accounted for 1-5%.
Cells that did not express NG2 are likely to be cells that had differentiated
into O1+ cells during the 5 hours between the time of sorting and staining, or
cells that had downregulated NG2 but still expressed DsRed due to the longer
half-life of the latter.
The majority of the sorted cells remained as NG2+ cells, and fewer than 20%
of the cells became oligodendrocytes. This is similar to the in vivo situation
where NG2 glia persist in large numbers in the mature brain and are not
depleted after oligodendrocyte differentiation. Cells that have cnp
promoter activity and express NG2 were shown to differentiate into neurons
(Belachew et al., 2003;
Aguirre et al., 2004;
Aguirre and Gallo, 2004;
Roy et al., 1999;
Nunes et al., 2003). In our
study, we did not observe neuronal differentiation from sorted DsRed+ cells
under conditions that had caused neuronal differentiation of SVZ cells. The
discrepancy may be attributed to differences in the sorted cell population.
The present study was conducted with cells that robustly expressed DsRed and
NG2 and excluded cells with weak or marginal expression of NG2.
In addition to oligodendrocyte lineage cells, GFAP+ cells emerged from
purified DsRed+ cells. They consisted not only of stellate type-2
astrocyte-like cells first described by Raff and colleagues
(Raff et al., 1983) but also
of flat pancake-shaped cells described by Miller and Szigeti
(Miller and Szigeti, 1991).
All the GFAP+ cells that appeared in cultures of sorted DsRed+ cells expressed
NG2 at 4 div and were detected in all six independent cultures of sorted
cells, which makes it unlikely that GFAP+ cells arose from contaminating cells
that were initially NG2-. Nor is it likely that NG2+GFAP+ cells were caused by
transgenic manipulation, since GFAP+NG2+ cells were also observed in secondary
glial cultures obtained from P3 wt mouse and rat brains by the shaking method
(McCarthy and de Vellis, 1980;
Yang et al., 2005), and many
of them expressed A2B5, although the proportion of NG2+ cells that expressed
A2B5 was greater in rat cells. The appearance of some GFAP+ cells that were
NG2-negative after 11 div suggests that GFAP+NG2+ cells give rise to GFAP+NG2-
cells.
Fate of NG2+Cre+ cells in vivo
We used NG2creBAC tg mice crossed to Z/EG reporter mice
(Novak et al., 2000) to
examine whether NG2 glia give rise to astrocytes in vivo. Cre immunoreactivity
was detected exclusively in NG2 cells. Not all NG2+ cells contained
immunohistochemically detectable levels of Cre. This may be due to a low level
of Cre in these cells below the threshold of antibody detection, or the
absence of cre transcription in a small subpopulation of NG2+ cells.
In NG2creBAC:Z/EG double tg mice, more than 85% of NG2+ cells were EGFP+. NG2+
cells that were EGFP-negative could represent NG2+ cells that failed to
express Cre and/or incomplete target excision in Cre-expressing cells.
NG2+Cre+ cells generate myelinating oligodendrocytes
In NG2creBAC:Z/EG double tg mice, EGFP was detected in 78% of
oligodendrocytes and myelin in the corpus callosum and striatal white matter
at P14 and later time points. EGFP+ oligodendrocytes with myelinating
processes were also found in the neocortex, indicating that NG2 glia generate
myelinating oligodendrocytes in both white and gray matter. These observations
provide direct evidence for the first time that endogenous NG2 glia give rise
to myelinating oligodendrocytes in both gray and white matter. The presence of
some oligodendrocytes that did not express EGFP may reflect incomplete
Cre-mediated excision in NG2 glia and/or heterogeneity of reporter expression
among oligodendrocytes. Although it is less likely, the existence of other
sources of oligodendrocytes besides NG2-positive precursors cannot be ruled
out.
NG2+Cre+ cells generate protoplasmic but not fibrous astrocytes
In addition to oligodendrocytes, we observed in NG2creBAC:Z/EG double tg
mice numerous EGFP+NG2- cells with highly branched, fine, bushy processes
resembling protoplasmic astrocytes (Bushong
et al., 2002). These EGFP+ astrocytes were most prevalent in the
gray matter of the ventral posterior forebrain, where they constituted nearly
half of S100β+ astrocytes, and some contained GFAP. Very few EGFP+
astrocytes were found in the dorsal cortex. Interestingly, there were no EGFP+
astrocytes in white matter. The observed regional difference in the prevalence
of EGFP+ astrocytes was not due to regional variation in the expression of the
reporter EGFP in the Z/EG mice, since β-galactosidase, which is expressed
in Z/EG mice prior to Cre-mediated excision, was detected in dorsal as well as
ventral cortical regions and in white matter. Furthermore, a similar ventral
distribution of Cre reporter expression was observed when NG2creBAC mice were
crossed to the ROSA26R reporter line
(Soriano, 1999). Curiously,
there seems to be a reciprocal distribution of EGFP+ astrocytes and
oligodendrocytes derived from Emx1-expressing cortical progenitor cells
(Kessaris et al., 2006). The
astrogliogenic potential of NG2 glia may depend on their site of origin.
Alternatively, environmental factors in the dorsal forebrain may be
restricting the manifestation of their astrogliogenic fate.
The presence of cells that appeared to be in transition from NG2 glia to
NG2-protoplasmic astrocytes provide additional support that NG2 glia
differentiate into protoplasmic astrocytes. These intermediate EGFP+ cells
were found as early as E18 and could be detected through P14 in NG2creBAC:Z/EG
double tg mice. They had faint NG2 immunoreactivity restricted to the distal
portions of their processes and had thicker proximal processes than the
typical NG2 glia. These faintly NG2+ astrocyte-like cells had broadened
web-like end-feet on blood vessels, a feature that is rarely seen in postnatal
NG2 glia. These vascular contacts of the intermediate cells resembled those
described for immature astrocytes by Zerlin and Goldman
(Zerlin and Goldman, 1997).
EGFP+ astrocytes increased in number in NG2creBAC:Z/EG double tg mice between
E18 and P14 and persisted through P60. They were often seen in clusters,
suggesting local proliferation of either newly generated astrocytes or NG2+
precursors. The presence of clusters of EGFP+ astrocytes in close proximity to
EGFP+ myelinating oligodendrocytes seems to suggest that both oligodendrocytes
and protoplasmic astrocytes can be generated from single NG2 cells, but clonal
analysis in vivo is needed to confirm this. Further studies using inducible
Cre in NG2 cells are currently under way to examine the timing of generation
of astrocytes from NG2 cells.
Radial glia and SVZ progenitors are known to generate telencephalic
astrocytes (Voigt, 1989;
Levison and Goldman, 1993;
Parnavelas, 1999), but the
origin of astrocytes in different forebrain regions is not clear. Since
neither NG2 nor EGFP was detected in radial cells or SVZ cells in the
embryonic forebrain of NG2creBAC:Z/EG double tg mice, it is unlikely that
transient activation of Cre in these early immature cells had caused reporter
expression in astrocytes. The clustered distribution of EGFP+ astrocytes near
the ventral pial surface at E18 is more indicative of a local differentiation
from parenchymal NG2+ cells into protoplasmic gray matter astrocytes. This is
further supported by the presence of a zone of EGFP+NG2+ cells lacking EGFP+
astrocytes that separated the ventral VZ/SVZ from the ventral subpial cluster
of EGFP+ astrocytes (Fig. 7F),
making it unlikely that EGFP+ astrocytes had been generated from Cre+ or GFP+
cells in the SVZ. Perhaps radial glia and SVZ progenitor cells generate
astrocytes in white matter and dorsal neocortex, whereas NG2 glia give rise to
astrocytes in ventral forebrain.
Transplanted OPCs have not been shown to generate astrocytes in the host
CNS (Groves et al., 1993;
Espinosa de los Monteros et al.,
1993) except under pathological astrocyte-free conditions
(Franklin et al., 1995). Based
on these observations, the astrocytic fate of OPCs [O-2A progenitor cells
(Raff et al., 1983)] has been
generally discarded as an in vitro artifact. However, since transplanted cells
preferentially migrate along white matter tracts, it is possible that these
studies had examined the fate of OPCs in regions where the environment does
not support astrocyte differentiation from these cells. Further studies are
needed to elucidate whether NG2 cell-derived astrocytes are functionally
distinct from other astrocytes that exist in the brain.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/1/145/DC1
|
ACKNOWLEDGMENTS |
|---|
,
Dr Joel Levine for rabbit antibody to NG2, and Dr Jacky Trotter for rat
anti-mouse NG2 (AN2). We thank Dr Caiying Guo (University of Connecticut
Health Center, Gene Targeting and Transgenic Facility) for the Z/EG mice, for
oocyte microinjections, and for her assistance with various transgenic mouse
techniques. We thank Drs Carol Norris and Michelle Barber for their assistance
with flow cytometry. We thank Youfen Sun for her assistance in maintaining and
genotyping transgenic mice. We thank Drs Ryusuke Suzuki and Mila Komitova for
critically reading the manuscript. This study was supported by grants from the
Wadsworth Foundation, National Multiple Sclerosis Society, National Science
Foundation, National Institutes of Health, and March of Dimes (D.E.B.). |
REFERENCES |
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