The adult mammalian brain and spinal cord contain glial precursors that express platelet-derived growth factor receptor ? subunit
spinal cord during experimental autoimmune encephalomyelitis (EAE)—a demyelinating condition that models some aspects of mul-
expression in PDGFRA/NG2 cells and their differentiated progeny. We subsequently induced EAE and observed a large (?4-fold)
increase in the local density of YFP?cells, ?90% of which were oligodendrocyte lineage cells. Many of these became CC1-positive,
NG2-negative differentiated oligodendrocytes that expressed myelin markers CNP and Tmem10/Opalin. PDGFRA/NG2 cells generated
Oligodendrocytes (OLs) synthesize CNS myelin, which is re-
tiple sclerosis (MS), OLs die, possibly through autoimmune
attack, and demyelination results. During this and other demy-
elinating diseases there is spontaneous regeneration of lost OLs
and myelin. The replacement OLs are believed to originate from
adult oligodendrocyte precursors (OLPs), which are widespread
and abundant in the adult CNS (ffrench-Constant and Raff,
1986; Wolswijk and Noble, 1989; Pringle et al., 1992; Butt et al.,
2005; Wilson et al., 2006). Adult OLPs, also known as “NG2
cells,” coexpress platelet-derived growth factor receptor ? sub-
unit (PDGFRA) and the NG2 proteoglycan (Nishiyama et al.,
early postnatal period in vivo (Raff et al., 1983; Hall et al., 1996;
Zhu et al., 2008; Guo et al., 2009). They also generate a subset of
protoplasmic astrocytes during perinatal development (Zhu et
al., 2008; Guo et al., 2009). Adult OLPs continue to divide and
generate new myelinating OLs in the healthy adult mouse CNS
ing rate with age (Lasiene et al., 2009; Psachoulia et al., 2009). They
this remains controversial (Aguirre and Gallo, 2004; Dayer et al.,
Several studies have indicated that adult NG2 cells can gener-
ate remyelinating OLs following experimental demyelination in
wine and Armstrong, 1998; Watanabe et al., 2002; Polito and
tial evidence that NG2 cells generate remyelinating OLs during
ever, neither in rodents nor in humans has it been demonstrated
chronic demyelination. Moreover, it is not known whether the
reported multilineage differentiation potential of NG2 cells is
realized—or augmented—in the abnormal environment of the
injured CNS. To establish the differentiation fates of PDGFRA/
NG2 cells during MS-like pathology we administered tamoxifen
(Tam) to adult Pdgfra-CreERT2:Rosa26R-YFP double-transgenic
mice to induce YFP labeling of PDGFRA-expressing cells, then in-
L.E.R. was supported by a collaborative studentship from the Biotechnology and Biological Sciences Research
TheJournalofNeuroscience,December1,2010 • 30(48):16383–16390 • 16383
lomyelitis (EAE) by immunizing with
emulsified myelin oligodendrocyte glycop-
rotein (MOG) peptide. This caused wide-
spread demyelination along the neuraxis.
We subsequently identified YFP-labeled
progeny by immunohistochemistry. Our
lineage tracing study provides direct evi-
dence that PDGFRA/NG2 cells generate
very few astrocytes and practically no neu-
labeled cells could not be identified with a
battery of antibodies against neurons, glia,
neural stem/progenitor cells, or vascular or
We also report that Tam pretreatment
resulted in significantly reduced locomotor
disability in female but not male mice with
Induction of EAE. All animal work conformed
to local ethical committee guidelines and the Animals (Scientific Proce-
dures) Act 1986 and was specifically approved by the United Kingdom
Government Home Office. Pdgfra-CreERT2BAC transgenic mice have
tion of C57BL/6-CBA F1 hybrids and maintained on the Rosa26R-YFP
dissolved in corn oil (Sigma, C8267), by sonication for 45 min at 30°C.
Adult mice were given 300 mg/kg body weight by oral gavage on 4 con-
secutive days starting 14 d before EAE induction (Fig. 1). Our Pdgfra-
CreERT2line expresses Cre exclusively in PDGFRA-immunoreactive
precursors (Rivers et al., 2008) but not in differentiated OLs, which do
not express PDGFRA (Hall et al., 1996; Butt et al., 1997). Cre-mediated
in the spinal cord for at most 10 d following Tam induction (Psachoulia
et al., 2009). The efficiency of Cre recombination (proportion of PDG-
FRA?cells that became YFP?) in the adult spinal cord was ?30%, and
this fraction remained stable between 14 and 42 d post-Tam. This was
slightly lower than we found previously in the adult forebrain (?45–
50%) (Rivers et al., 2008).
EAE was induced in 14- to 18-week-old [postnatal day ?110
(?P110)] male and virgin female Pdgfra-CreERT2:R26R-YFP mice by
with Freund’s adjuvant [1 mg/ml MOG peptide, 2.5 mg/ml Mycobacte-
PBS], injected subcutaneously on days 0 and 7 (i.e., 14 and 21 d post-
Tam). In addition, 0.1 ml of Pertussis toxin (300 ng/ml) was injected
same inoculum without MOG peptide. The time line of the experiments
is illustrated in Figure 1A. We analyzed three groups of mice that had
received (1) Tam in corn oil followed by mock-EAE immunization
(“Tam-only”), (2) corn oil followed by EAE inoculum (“EAE-only”),
and (3) Tam followed by EAE inoculum (“Tam?EAE”).
disability on a 7 point scale (supplemental Table S1, available at www.
jneurosci.org as supplemental material). Mice showing severe spasticity
or a score ?5 were killed immediately by a humane method.
Tissue processing. Mice were perfused intracardially with 4% (w/v)
paraformaldehyde (PFA) in PBS at room temperature (?20°C). Spinal
cords were dissected and postfixed in 4% PFA overnight at 4°C. Tissue
was cryoprotected in 20% (w/v) sucrose at 4°C overnight, embedded in
OCT compound, frozen, and stored at ?80°C until sectioning. Spinal
nominal thickness and collected by floating on the surface of PBS.
Immunocytochemistry and microscopy. Floating spinal cord sections
were pretreated with blocking solution [10% (v/v) sheep serum, 0.1%
antibodies are given in supplemental Table S2 (available at www.
jneurosci.org as supplemental material). Solochrome cyanine dye was
used to visualize myelin. The following secondary antibodies were used
in blocking solution: Alexa Fluor 488 goat anti-rat IgG (Invitrogen,
1:500), Alexa Fluor 567 goat anti-mouse IgG1, Alexa Fluor 647 goat
anti-rabbit IgG (Invitrogen, 1:1000), and Cy3-conjugated donkey anti-
guinea pig IgG (Millipore Bioscience Research Reagents, 1:500).
Fluorescein-conjugated isolectin B4 from Bandeiraea (Griffonia) sim-
plicifolia (ILB4, Vector Labs, 1:100) was used to label microglia and en-
dothelial cells. Cell nuclei were visualized by poststaining with Hoescht
and examined in a PerkinElmer Ultraview confocal microscope.
Two sections from each level (cervical, thoracic, and lumbosacral) of
lapping sample fields were counted in the white matter and six fields in
the gray matter of every section (Fig. 1B) and data were pooled per
lapping with an area of 0.11 mm2(Fig. 1B). However, if a WM box
straddled WM and GM—which sometimes happened in lumbosacral
spinal cord—the cells in GM were easily excluded since GM and WM
could be visually distinguished. The general strategy was to place at least
one WM box in each of the dorsal, dorsolateral, lateral, and ventral
funiculi and to place two GM boxes in each of the dorsal horn, ventral
horn, and intermediate GM areas. Since EAE results in multiple diffuse
decided to take this nonselective approach to field selection. Data were
plotted and statistical analyses performed using GraphPad Prism 5.0
software. All data are plotted as mean ? SEM.
Locomotoranalysisof Pdgfra-CreERT2:R26R-YFP mice
16384 • J.Neurosci.,December1,2010 • 30(48):16383–16390Tripathietal.•PDGFRACellsMainlyGenerateNewOligodendrocytesinEAE
at www.jneurosci.org as supplemental material). A progressive
worsening of locomotion was observed following the onset of
symptoms (Fig. 2A), with a few (9 of 32) mice showing a
to show earlier onset of symptoms, but this did not reach statis-
Since it was necessary to administer Tam to our mice to fate-
map PDGFRA/NG2 cells during EAE, we asked whether Tam
itself might have an effect on disease progression (Tam?EAE
the locomotor scale), which was never seen in the control group
that received corn oil without Tam (EAE-only males) (Fig. 2B).
Moreover, all of the Tam?EAE males had to be humanely killed
before 28 d post-EAE immunization because of the severity of
their symptoms; this curtailed the experiment before a statisti-
cally significant difference between Tam?EAE and EAE-only
males was established. Nevertheless, there was a trend toward
more severe outcome in Tam?EAE male mice (Fig. 2B). In con-
trast, Tam treatment decreased the severity of EAE symptoms in
mice displayed any locomotor disability.
immunization with MOG peptide caused demyelination in our
chrome cyanine) and myelin basic protein (MBP) immunohisto-
chemistry revealed demyelinated plaques/lesions all along the
To determine the fates of PDGFRA cells following EAE im-
munization, we examined spinal cord tissue on 14 d postimmu-
nization (14 dpi), when most animals had begun to show
locomotor symptoms, and also on 28 dpi (females) or 24 dpi
(males). No significant differences were noted among these time
points (nor between males and females) by any of our immuno-
labeling criteria, so data for all time points and both sexes were
In Tam?EAE mice there was an ?4.5-fold increase in YFP?
cells in the white matter and an ?2-fold increase in the gray
matter of the spinal cord (Figs. 3E,F, 4I). These are average fig-
ures; there was variation among individual mice—from 2- to
cells in gray matter to begin with, this resulted ultimately in a
roughly even distribution of YFP?cells in the gray and white
matter of Tam?EAE animals (Fig. 4I). Most YFP?cells in
cord levels. Also, massive inflammation marked by CD45 (C) and ILB4 (D) labeling indicated
dpi in Tam?EAE animals unless indicated otherwise. Sections B–F are counterstained with
MOG immunization causes inflammation and demyelination. A–F, Following
Tripathietal.•PDGFRACellsMainlyGenerateNewOligodendrocytesinEAEJ.Neurosci.,December1,2010 • 30(48):16383–16390 • 16385
Tam?EAE spinal cords were OLIG2?, identifying them as OL
lineage cells (92 ? 2% in white matter, 96 ? 2% in gray) (Fig.
4A–C,G). In Tam-only animals 98 ? 1% of YFP?cells were also
OLIG2?in white matter, compared with 92 ? 1% in gray (Fig.
4G). A large fraction of YFP?cells was also NG2?, both in
Tam-only mice (78 ? 2% in white, 80 ? 6% in gray) (Fig.
Approximately 2% of all YFP?cells in the white matter of
(18/970) were GFAP?(Fig. 4J). In gray matter, ?1% (9/898) of
YFP?cells were GFAP?in Tam?EAE mice, compared with
at a low level or not at all in protoplasmic astrocytes in gray
matter, we also coimmunolabeled for YFP and S100?, which is
expressed by astrocytes and a subset of oligodendrocyte lineage
cells. We did not detect YFP?, S100??double-labeled cells in
TAM?EAE mice, even in or around lesions. It was originally
ture astrocytes (Malatesta et al., 2003) but we have found that
both fibrous and protoplasmic astrocytes are strongly labeled in
P50 Fgfr3-CreERT2:Rosa26-YFP animals (Young et al., 2010).
Hence the use of the Rosa26-YFP reporter does not preclude our
ability to detect astrocytes. Approximately 1.4% (10/716) of
YFP?cells in Tam-only spinal cords were NeuN?and ?0.2%
(4/2147) in Tam?EAE cords (supplemental Fig. S1, available at
new neurons generated from the SVZ have been previously re-
ported in a subpopulation of multiple sclerosis lesions (Chang et
al., 2008). However it is unclear whether these were generated
from NG2 cells. Together, the evidence indicates that PDGFRA/
NG2 glia generate very few, if any, astrocytes or neurons in the
EAE spinal cord.
In agreement with previous data (Ligon et al., 2006), we did not
detect any NG2?cells that were not also OLIG2?in Tam?EAE
or Tam-only spinal cords (data not shown). Since more YFP?
cells were OLIG2?than NG2?(e.g., 92% versus 71%, respec-
tively, in Tam?EAE white matter; Fig. 4G,H), it follows that a
subset of OLIG2?cells (?21% in this example) were NG2-
only white matter (?20%). Since there were ?4.5 times more
Tam-only white matter, it is clear that many new differentiated
OLs are generated in response to EAE induction.
To look for direct evidence of remyelinating OL production,
we double-immunolabeled Tam?EAE spinal cords for YFP and
dendrocytes: adenomatous polyposis coli (APC, recognized by
monoclonal CC1), 2?,3?-cyclic nucleotide phosphodiesterase
(CNP), MBP, Tmem10/Opalin or Ermin/Juxtanodin. APC is
found in oligodendrocyte cell bodies and proximal processes,
CNP and MBP in cell bodies, processes and myelin sheaths.
Tmem10/Opalin is expressed at the onset of myelination in the
Ermin/Juxtanodin is an oligodendrocyte cytoskeletal-related
protein that is found in the cytoplasmic tongue processes and
terminal loops of compact myelin (Zhang et al., 2005; Brock-
schnieder et al., 2006). Its temporal expression profile closely
follows that of MBP. YFP is a large cytoplasmic protein that is
physically excluded from compact myelin, so it is difficult to
correlate YFP expression with myelin wraps. Nevertheless, we
16386 • J.Neurosci.,December1,2010 • 30(48):16383–16390Tripathietal.•PDGFRACellsMainlyGenerateNewOligodendrocytesinEAE
found many YFP?cell bodies and processes that also colabeled
estimated that in Tam?EAE animals 35 ? 12% of 404 YFP?
cells, and in Tam-only animals, 21 ? 7% of 183 YFP?cells were
also CC1?(mean ? SEM, 30 sections from three mice). Both
YFP?/Opalin?and YFP?/Ermin?cells could be found in close
apposition to Neurofilament-positive profiles (Fig. 6A,B,F–H)
but these were not numerous, presumably because YFP is ex-
cluded from the myelin sheaths. Consequently, we cannot be
certain that the new OLs all form myelin; it is possible that some
because of the lack of suitable axons or because the pathological
conditions of EAE inhibit myelination. To address this issue we
would need a reporter transgene that specifically labels myelin
sheaths but such a reporter is not yet available.
of YFP?cells that was not accounted for by immunolabeling for
NG2, OLIG2, GFAP or NeuN. These unidentified cells were rare
numerous during EAE (?13% of YFP?cells) (Fig. 4G). In gray
matter, ?13% of YFP?cells in Tam-only mice and ?9% in
Tam?EAE mice were unaccounted for (Fig. 4G). This contrasts
with our previous study, in which we could colabel essentially all
YFP?cells in the gray and white matter of the normal adult
forebrain with antibodies against OLIG2, NG2, or NeuN (Rivers
might have failed to detect 100% of OL lineage cells with our
OLIG2 antibody for technical reasons.
However, we found that the YFP?,
OLIG2?cells also failed to label for
SOX10 in triple-label experiments (see
supplemental Fig. S2, available at www.
jneurosci.org as supplemental material),
arguing against a purely technical prob-
lem and suggesting that they might be
non-OL lineage cells.
We tried, unsuccessfully, to identify
these OLIG2-negative cells with a battery
of antibodies against blood-borne cells
(T-cells, B-cells, monocytes, macrophages,
neutrophils and granulocytes), vascular
cells (endothelial cells and pericytes),
tal Fig. S3, available at www.jneurosci.org
as supplemental material). A few YFP?
cells could be immunolabeled for the
Schwann cell myelin marker Protein zero
(P0) but these were rare relative to the
OLIG2?YFP?cells (supplemental Fig.
S4, available at www.jneurosci.org as sup-
plemental material). Moreover, they were
almost always found close to the pial sur-
face whereas most of the OLIG2?, YFP?
cells were in the interior of the cord. The
YFP?, OLIG2?cells therefore remain
The main purpose of our study was to in-
vestigate the fates of NG2 cells in a demy-
elinating/remyelinating disease model, EAE. Using Cre-lox
methodology in transgenic mice, we found that there was a sig-
nificant increase (?4-fold) in the overall number of OL lineage
cells (YFP?, OLIG2?) in the demyelinating cord and a parallel
?4-fold increase in the number of differentiated OLs (YFP?,
OLIG2?, NG2?; Fig. 4G,H). This is an underestimate of the
increase that occurs within lesion areas, since our counting
method did not discriminate between lesions and adjacent
normal-appearing white matter, thus “diluting” the increase
within lesions. New YFP?OLs could be coimmunolabeled with
ber of YFP?, CC1?OLs (?35% of YFP?cells) in Tam?EAE
mice (40 d post-Tam) exceeded the number of YFP?, OLIG2?,
expression in newly differentiating OLs. Nevertheless, there
Tam?EAE mice. We presume that the newly formed OLs are
engaged in remyelinating axons that had become demyelinated
during the course of EAE. A generally accepted hallmark of re-
wraps) than developmentally generated OLs. To detect myelin
directly would require electron microscopy (EM), but we have
been unable to identify YFP-labeled myelin sheaths unambigu-
ously by EM immunohistochemistry, because YFP is physically
excluded from compact myelin (Rivers et al., 2008).
Even in Tam-only spinal cord ?20% of YFP?, OLIG2?cells
in white matter were NG2-negative at 40 d post-Tam and a sim-
single-labeled YFP?cells. The CC1 images were taken from 24 dpi tissue and the CNP images from 28 dpi tissue. Sections are
Tripathietal.•PDGFRACellsMainlyGenerateNewOligodendrocytesinEAEJ.Neurosci.,December1,2010 • 30(48):16383–16390 • 16387
of YFP?cells coimmunolabeled with an-
tibody CC1 in Tam-only spinal cord. We
and others have previously shown that
new myelinating OLs continue to be
formed for an extended period after birth
(at least 8 months) in the forebrain gray
ers et al., 2008; Lasiene et al., 2009; Psa-
choulia et al., 2009).
Apart from remyelinating OLs, many
hypertrophic “reactive” astrocytes appear
cord (supplemental Fig. S3, available at
www.jneurosci.org as supplemental ma-
terial). These GFAP?astrocytes tend to
accumulate at the periphery of demyeli-
“scar.” This could be a barrier to inward
hibit repair, especially after multiple epi-
sodes of demyelination at the same locus
such as occurs in relapsing-remitting MS.
The origin of reactive astrocytes has been
controversial, some reports suggesting
that they are generated from NG2 cells
et al., 2006; Leoni et al., 2009). However,
the data presented here do not support
that view, for we found that only small
numbers of YFP?, GFAP?astrocytes
were generated from Pdgfra-CreERT2-
expressing cells, either in the normal
healthy spinal cord or after EAE induc-
tion. This accords with another recent
study of cell generation in a mouse model
of acute focal demyelination (stereotaxic
injection of ethidium bromide or lysolec-
ithin into spinal cords of Pdgfra-CreERT2:
R26R-YFP mice) (Zawadzka et al., 2010).
The latter study also demonstrated exten-
sive oligodendrocyte regeneration from
PDGFRA/NG2 cells but little reactive as-
trocyte production. However, Zawadzka
surrounding gliotoxin-induced lesions
renchymal astrocytes and ependymal zone (EZ) cells but not
PDGFRA/NG2 cells are labeled (Young et al., 2010). Therefore it
is likely that most reactive astrocytes in and around areas of de-
myelination are formed by activation and migration of EZ stem
cells and/or by multiplication of preexisting parenchymal astro-
cytes. Further fate mapping experiments with astrocyte- and EZ-
specific Cre lines would be required to distinguish these
The lack of significant astrocyte production from PDGFRA/
NG2 glia in our current study and that of Zawadzka et al. (2010)
2007; Sellers et al., 2009; Zhao et al., 2009). However, all of the
latter studies involved stab or cut injury models, not demyelina-
tion. The fates of stem/precursor cells will be influenced by the
on the nature of the insult. It is possible that we might have
underestimated astrocyte production specifically within lesions,
for the same reason that we underestimated OL production (the
dilution effect of our counting method, described above). How-
ever, note that the study of Zawadzka et al. (2010) did not suffer
from this dilution effect because they counted cells specifically
within focal lesions. Another potential source of disparity be-
tween our study and others who reported astrocyte production
from NG2 cells is in the experimental approach or its interpreta-
tion. Some previous studies have relied on coexpression of anti-
gens to infer lineage relationships and have concluded, on the
basis of coexpression of NG2 and GFAP, that NG2 glia start to
express GFAP on their way to generating astrocytes. However, it
also seems possible that GFAP?astrocytes start to express NG2
of which were found closely apposed to Neurofilament (NF) immunolabeling (A, B). Since Opalin is expressed in late-stage,
found entwined with NF?processes (I–K). Arrows indicate double-labeled cells. Images A, B, and F–K are from longitudinal
16388 • J.Neurosci.,December1,2010 • 30(48):16383–16390Tripathietal.•PDGFRACellsMainlyGenerateNewOligodendrocytesinEAE
and/or OLIG2, perhaps as part of a dedifferentiation program,
before going on to generate more astrocytes.
In our own study, only ?30% of PDGFRA?cells in the spinal
cords of Pdgfra-CreERT2:R26R-YFP mice recombined and ex-
are not representative of the population as a whole and that the
low level of astrocyte production is an artifact of this selectivity.
We have tried to discover whether Cre recombination marks a
neuroepithelium. We have shown that the OLPs that undergo
Cre recombination are equally likely to fall into any of these
experiments is simply a function of the relatively low activity of
CreERT2(compared with constitutive Cre), the narrow window
of opportunity for recombination following Tamoxifen treat-
promoter (discussed by Young et al., 2010). Inefficient cell label-
ing is not unusual with CreER lines (Dimou et al., 2008; Guo et
Unexpectedly, we found a significant minority (2–10%) of
YFP?cells in the spinal cords of healthy as well as EAE mice that
did not colabel with antibodies against OLIG2, SOX10 or NG2.
These cells therefore seem not to belong to the OL lineage. They
also did not label with any of a battery of reagents selected to
identify resident or infiltrating immune system cells (microglia,
cells (endothelial cells or pericytes), astrocytes or neurons. Each
of these cell types could be detected in the EAE spinal cord but
tery” cells. Therefore, the YFP?non-OL lineage cells remain un-
identified. Given the presence of some of these mystery cells in
An unexpected finding was that Tam administration reduced
Perhaps Tam produces a long-lasting effect on the immune sys-
tem that moderates subsequent autoimmune attack. Alterna-
Acute Tam treatment is known to be protective during experi-
treatment, not binding of Tam to estrogen receptors, and is evi-
dent at a very low dose of Tam compared with that used in our
current study (a single dose of 5 mg/kg body weight, versus four
doses of 300 mg/kg) (Zhang et al., 2007). Low dose Tam (5 mg/
kg) has recently been reported to be beneficial following a spinal
cord contusion injury in male rats (Tian et al., 2009). The bene-
in males Tam appeared to have a damaging effect, advancing the
onset of locomotor deficits and accelerating subsequent deterio-
ration. Therefore, the action of Tam is likely to be complex, in-
cluding both short and long term effects and mediated via
estrogen receptors as well as other routes.
In conclusion, we have shown that in a MOG-induced EAE
model of demyelinating disease, PDGFRA/NG2 cells in the adult
including differentiated—presumably remyelinating—OLs, but
very few astrocytes or neurons.
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