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Bone Morphogenetic Protein Signaling and Olig1/2 Interact to

Regulate the Differentiation and Maturation of Adult

Oligodendrocyte Precursor Cells

Xiaoxin Chenga,b, Yaping Wanga,b,d, Qian Hea,b, Mengsheng Qiua,c, Scott R.

Whittemorea,b,c, and Qilin Caoa,b

aKentucky Spinal Cord Injury Research Center, University of Louisville School of Medicine,

Louisville, Kentucky, USA

bDepartment of Neurological Surgery, University of Louisville School of Medicine, Louisville,

Kentucky, USA

cDepartment of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine,

Louisville, Kentucky, USA

dDepartment of Anesthesiology, Second Xian-Ya Hospital of South Central University, Changsha,

Hunan, People's Republic of China

Abstract

Promotion of remyelination is an important therapeutic strategy for the treatment of the

demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which

normally reside quiescently in the adult central nervous system (CNS), become activated and

proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited

because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the

demyelinated CNS. Understanding the molecular mechanisms that regulate the differentiation of

adult OPCs could lead to new therapeutic strategies to treat these disorders. In this study, weestablished a stable culture of adult spinal cord OPCs and developed a reliable in vitro protocol to

induce their sequential differentiation. Adult OPCs expressed bone morphogenetic protein (BMP)

type Ia, Ib, and II receptor subunits, which are required for BMP signal transduction. BMP2 and 4

promoted dose-dependent astrocyte differentiation of adult OPCs with concurrent suppression of OL

differentiation. Treatment of OPCs with BMP2 and 4 increased ID4 expression and decreased the

expression of olig1 and olig2. Overexpression of olig1 or olig2 blocked the astrocyte differentiation

of adult OPCs induced by BMP2 and 4. Furthermore, overexpression of both olig1 and olig2, but

not olig1 or olig2 alone, rescued OL differentiation from inhibition by BMP2 and 4. Our results

demonstrated that downregulation of olig1 and olig2 is an important mechanism by which BMP2

and 4 inhibit OL differentiation of adult OPCs. These data suggest that blocking BMP signaling

combined with olig1/2 overexpression could be a useful therapeutic strategy to enhance endogenous

remyelination and facilitate functional recovery in CNS demyelinated disorders.

Correspondence: Qilin Cao, M.D., Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, MDR 616, 511South Floyd Street, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA. Telephone: 502-852-0284; Fax:502-852-5148; [email protected].

Disclosure of Potential Conflicts of Interest: The authors indicate no potential conflicts of interest.

Supplementary Material: Supplementary material can be found at: http://www.StemCells.com/cgi/content/full/2007-0284/DC1

NIH Public AccessAuthor ManuscriptStem Cells. Author manuscript; available in PMC 2009 September 14.

Published in final edited form as:

Stem Cells. 2007 December ; 25(12): 3204–3214. doi:10.1634/stemcells.2007-0284.

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Keywords

Adult oligodendrocyte precursor cell; Spinal cord; Bone morphogenetic protein; Transcription factor;

Differentiation Remyelination

Introduction

In spite of the ubiquitous distribution in the mature central nervous system (CNS), the majority

of oligodendrocytes (OLs) originate from relatively discrete ventral regions along the neural

axis during early embryogenesis. They subsequently migrate laterally and dorsally to populate

all parts of the developing CNS before differentiating into myelin-forming OLs [1,2]. The

secreted glycoprotein Sonic Hedgehog (Shh), released from the notochord and floor plate, is

primarily responsible for the initial specification of oligodendrocyte precursor cells (OPCs) in

the ventral spinal cord [3–5]. More recent studies show that a small number of OLs are

generated from the dorsal spinal cord or forebrain during later developmental stages in a Shh-

independent manner [6–8]. Irrespective of their ventral or dorsal origin, however, two basic-

helix-loop-helix (bHLH) transcription factors, olig1 and olig2, are ultimately required for OL

development, since OLs completely fail to develop in olig1 and olig2 double-null mice [9,

10]. During CNS development, bone morphogenetic proteins (BMPs), members of the

transforming growth factor- β protein family of extracellular ligands, repressoligodendrogenesis while enhancing the development of astrocytes [11,12]. Implantation of

noggin-producing cells into the early developing chicken spinal cord or anti-BMP antibody

coated beads into developing Xenopus promotes the subsequent appearance of OL progenitors

in dorsal neural tube [13,14], suggesting that endogenous dorsally expressed BMPs inhibit

oligodendrogenesis. Conversely, elevated BMP expression inhibits the appearance of ventral

OL progenitors [13,14], and overexpression of BMP4 enhances astrocytic lineage commitment

in vivo and significantly inhibits the generation of OLs [15]. In vitro, BMP signaling promotes

astrocytic differentiation at the expense of OLs from embryonic or postnatal multipotential

NSCs [16–20] or OPCs [21,22]. However, the mechanism(s) by which BMP signaling inhibits

OL development remain to be elucidated.

OPCs reside in the white matter of the adult CNS [23,24]. Although these cells remain relatively

quiescent in the normal CNS, they become activated and proliferate in response to CNS injury,including demyelination [25–28]. More importantly, these cells have the potential to

differentiate into mature OLs and myelinate axons both in vitro and in vivo [29–31]. However,

endogenous remyelination by adult OPCs is very limited in a number of demyelinated

disorders, including multiple sclerosis [32,33] and spinal cord injury (SCI) [34,35]. The failure

of endogenous remyelination may be due to the inhibition of adult OPC differentiation and

maturation into myelinating OLs. Although OPCs are present in the demyelinated adult CNS

and even contact demyelinated axons, they fail to differentiate into mature OLs that remyelinate

these axons [28,36–38]. Understanding the molecular mechanism(s) that regulate the

differentiation of adult OPCs may lead to new therapeutic strategies for demyelinated

disorders. Most previous studies investigating the response of OPCs to environmental factors

present within demyelinating lesions have used embryonic and neonatal progenitor cells.

However, the proliferation and differentiation of adult and perinatal OPCs in response to

growth factors are different [39–41]. The molecular mechanism(s) that control their

differentiation may also be different. For example, olig1 is not required for OL generation and

myelination during CNS development but is essential for remyelination following

demyelinating lesions in adult mice [42].

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In the present study, we established an in vitro model to directly study the proliferation and

differentiation of adult spinal cord OPCs. We provide evidence that the interaction between

BMP signaling and olig1 and olig2 regulates the differentiation of adult OPCs.

Materials and Methods

All animal care and surgical interventions were undertaken in strict accordance with the Public

Health Service Policy on Humane Care and Use of Laboratory Animals and with the approvalof the University of Louisville Institutional Animal Care and Use Committee and the

Institutional Biosafety Committee.

Isolation of OPCs from Adult Spinal Cord

OPCs were immunopanned from adult spinal cords of Fischer rats (3–4 months old) with an

O4 antibody using a protocol modified from a previous study [39]. Briefly, the dissected spinal

cords were minced into 1-mm3 pieces and incubated in Hanks' balanced salt solution containing

0.1% papain, 0.1% neutral protease, and 0.01% DNase for 30 minutes at 37°C. The digestion

was stopped by the addition of an equal volume of Dulbecco's modified Eagle's medium

(DMEM) containing 20% fetal bovine serum (FBS). Tissues were dissociated by repeated

trituration with fire-polished Pasteur pipettes and were filtered through 70- μm nylon mesh.

The cells were incubated at 37°C on an anti-RAN-2 antibody-coated dish for 30 minutes to

deplete type 1 astrocytes and meningeal cells and then transferred to an O4-coated dish for 45minutes to select OPCs. The purified OPCs were removed with trypsin and cultured in poly-

L-lysine/laminin (P/L)-coated dishes with DMEM/Ham's F-12 medium containing 1 × N2 and

1 × B27 supplements, 20 ng/ml fibroblast growth factor 2 (FGF2), 10 ng/ml platelet-derived

growth factor aa (PDGFaa), 5 μg/ml insulin, and 0.1% bovine serum albumin. Cells were fed

with fresh growth medium every other day. In all cases, an aliquot of cells was analyzed

immunohistochemically or by fluorescence-activated cell sorting the next day to determine the

efficiency of the immunopanning. Only those cell preparations in which >95% of the bound

cells expressed O4 were used in the subsequent experiments.

OL Differentiation and Maturation of Adult OPCs

Passage 4–7 adult OPCs were plated on P/L-coated 60-mm culture dishes for Western blot or

24-well plates for immunohistochemical analyses. The following day, adult OPCs wereinduced to differentiate by withdrawal of FGF2 and PDGFaa from the growth medium for 3

days with or without BMP2 and 4 (R&D Systems Inc., Minneapolis,

http://www.rndsystems.com). To examine the effects of BMP2 and 4 on OL maturation of

adult OPCs, cells were allowed to differentiate into O1+ OLs by withdrawal of FGF2 and

PDGFaa for 2 days and then cultured for 3 more days with or without BMPs. The concentration

of BMP2 and 4 was 10 ng/ml except where otherwise indicated.

Olig1 and Olig2 Overexpression

Olig1, olig2, and enhanced green fluorescence protein (EGFP) cDNAs were cloned into the

LZRS retroviral vector [43]. To generate high titer virus, Φ NX cells (provided by Dr. Gary

Nolan, Stanford University) were transfected using GenePorter II (Gene Therapy Systems,

Inc., San Diego, http://www.genlantis.com), which routinely gave transfection efficiencies of

50%–65%. Selection with 2 μg/ml puromycin began 48 hours later. To harvest viral

supernatant, medium was changed to the appropriate serum-free medium overnight and

harvested the next day. We routinely obtained titers of 5 × 105 to 5 × 106 transforming units/

ml. Cells were treated with 1 μg/ml polybrene for 1 hour, followed by a wash and subsequent

replacement of half of the medium with viral stock. After incubation at 37°C for 6 hours, the

medium was changed to the appropriate growth medium. With this protocol, approximately

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50% of the cells were labeled. Two days after infection, adult OPCs were induced to

differentiate and mature with or without BMP2 and 4, as described above.

olig1 and olig2 Small Interference RNA

A retrovirus vector carrying small interference RNAs (siRNAs) targeting the mouse olig1 or

olig2 gene was prepared using the BD Knockout RNAi System (Clontech, Palo Alto, CA,

http://www.clontech.com). The polyacrylamide gel electrophoresis-purified complementary

oligonucleotide pair for the hairpin siRNA was synthesized to target the coding region of mouseolig1 or olig2 mRNA as follows: 5′-gatccGCCACGAGTACAAACATCAATT-

CAAGAGATTGATGTTTGTACTCGTGGTTTTTTACGCGTg-3 ′, 5′-

aattcACGCGTAAAAAACCACGAGTACAAACATCAATC-

TCTTGAATTGATGTTTGTACTCGTGGCg-3′, and 5′-gatccGT-

ACAAAGATTGACTCCTTTTCAAGAGAAAGGAGTCAATCTT-

TGTACTTTTTTACGCGTg-3′, 5′ -aattcACGCGTAAAAAAGTA-

CAAAGATTGACTCCTTTCTCTTGAAAAGGAGTCAATCTTT-GTACg-3 ′, respectively.

A complementary pair of oligonucleotides for a sense-only control RNA was also designed.

These oligonucleotide pairs containing BamHI and EcoRI overhangs were annealed and ligated

to a linearized RNAi-Ready pSIREN-RetroQ-ZsGreen vector digested with BamHI and EcoRI

(Clontech). The RNAi-Ready pSIR EN-RetroQ-ZsGreen vector is a self-inactivating retroviral

expression vector designed to express a small hairpin RNA using the human U6 promoter. The

resultant constructs were amplified, purified, and sequenced. High-titer retrovirus vectors

expressing olig1 or olig2 siRNAs were produced in Φ NX cells as described above. The

ZsGreen fluorescent marker yield s a bright green fluorescence, permitting direct monitoring

of the infection efficiency in adult OPCs.

Immunofluorescence In Vitro

To detect surface membrane antigens, cells cultured on 24-well plates were incubated with the

primary antibodies A2B5, O4, and O1 (hybridoma supernatant, undiluted; American Type

Culture Collection, Manassas, VA, http://www.atcc.org) at 4°C for 45 minutes. After fixation

with 4% paraformaldehyde (PFA), cells were incubated in fluorescein isothiocyanate- or Texas

Red-conjugated donkey anti-mouse IgM for 1 hour at room temperature (RT). For recognition

of other antigens, cells cultured on 24 well plates were fixed with 4% paraformaldehyde (PFA).

Mouse monoclonal antibodies against 2′,3′-cyclic nucleotide phosphodiesterase (CNPase;1:800; Sternberger Monoclonal, Covance, Princeton, NJ, http://www.covance.com), myelin

basic protein (MBP; 1:50; Chemicon, Temecula, CA, http://www.chemicon.com), glial

fibrillary acidic protein (GFAP; 1:400; Sigma-Aldrich, St. Louis,

http://www.sigmaaldrich.com), or proteolipid protein (PLP; 1:200; Serotec, Serotec, Raleigh,

NC, http://www.serotec.com) or rabbit polyclonal antibodies against olig1 (1:100; Chemicon),

Olig2 (1:2,000; a generous gift from Dr. Charles Stiles, Harvard University), or chicken

polyclonal antibody anti-EGFP (1:500; Chemicon) were applied overnight at 4°C. Then, the

appropriate fluorophore-conjugated secondary antibodies (1:200; Jackson Immunoresearch

Laboratories, West Grove, PA, http://www.jacksonimmuno.com) were applied, and the nuclei

were counterstained with 4,6-diamidino-2-phenylindole. Negative control experiments

performed with the appropriate species-specific IgG or with inappropriate secondary

antibodies showed negligible background. Total cellular counts for each experimental well

were obtained in 10 fields with a ×20 objective from three independent culture wells. The resultfor each experimental condition was verified a minimum of three times.

Western Blot Experiments

Cells were harvested in ice-cold lysis buffer. Equivalent amounts of total protein extract from

each sample were mixed with sample buffer, boiled, and loaded onto SDS polyacrylamide gels.

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Electrophoretic separation of the extracts was typically performed on 7.5%–15% (depending

on the molecular weight of the protein of interest) discontinuous acrylamide gels under

denaturing conditions. Proteins were then transferred to nitrocellulose membranes and probed

with monoclonal-specific antibodies raised against GFAP (Sigma-Aldrich), MBP, PLP, or

olig1 or with polyclonal antibodies raised against olig2, and phosphated Smad 1/5/8. In

addition, an antibody against β -actin (Sigma-Aldrich) was used as a loading control.

Appropriate secondary horseradish peroxidase-conjugated antibodies were used for detection

with chemiluminescence ECL reagents (Amersham Biosciences, Piscataway, NJ,http://www.amersham.com).

Spinal Cord Injury and Immunohistochemistry for Tissue

After anesthetization with Nembutal (50 mg/kg, i.p.), adult female Fischer 344 rats received a

dorsal laminectomy at the ninth thoracic vertebral level (T9) to expose the spinal cord, and

then a 150 kdyn contusive SCI using the Infinite Horizons (Lexington, KY) impactor as

described previously [44,45]. After surviving 1 week or 1 month, animals were perfused

transcardially with 0.01 M phosphate-buffered saline, pH 7.4, followed by 4% PFA in 0.1 M

phosphate buffer. The injured spinal cords were removed, cryoprotected in 30% sucrose buffer

overnight at 4°C, and then transversely sectioned at 20 μm on a cryostat. After blocking with

10% donkey serum in Tris-buffered saline containing 0.3% Triton X-100 (TBST) for 1 hour

at RT, the sections were incubated in TBST containing 5% donkey serum, polyclonal rabbit

anti-GFAP (1:500; Dako, Carpinteria, CA, http://www.dako.com)/monoclonal mouse anti-

BMP2 or anti-BMP4 (1:200; Chemicon), monoclonal mouse anti-olig1 (1: 100; a generous

gift from Dr. Charles Stiles, Harward University)/polyclonal anti-NG2 (1:200; Chemicon), or

monoclonal mouse anti-platelet-derived growth factor receptor α (1:25; BD Pharmingen, San

Diego, http://www.bdbiosciences.com/index_us.shtml)/polyclonal rabbit anti-olig2 (1:2,000)

overnight at 4°C. Then, the appropriate fluorophore-conjugated secondary antibodies (1:200;

Jackson Immunoresearch Laboratories) were applied. A Nikon C1 confocal microscope

(Nikon, Tokyo, http://www.nikon.com) was used to capture representative images.

Photomicrographs were assembled using Adobe Photoshop and Adobe Illustrator software

(Adobe Systems Inc., San Jose, CA, http://www.adobe.com).

Results

Characterization of Adult OPC

In the presence of FGF2 and PDGFaa, O4+ OPCs proliferated and could be passaged many

times. Proliferating OPCs displayed a characteristic adult OPC morphology, with several small

short processes emanating from a small round cell body (Fig. 1A), as shown in the intact adult

spinal cord by NG2 immunostaining [23,46]. More than 95% of these cells were stained by

antibodies directed against O4 (Fig. 1B, 1F), A2B5 (Fig. 1C, 1F), or NG2 (Fig. 1D, 1F). Fewer

than 5% of the adult OPCs expressed the more mature OL-specific proteins O1 or CNPase and

they did not express MBP, PLP, GFAP, or vimentin (data not shown). To examine their

proliferative capacity, the adult OPCs were cultured in serum-free medium containing FGF2

and PDGFaa for 5 days, and then 10 μM 5-bromo-2′-deoxyuridine (BrdU) was added overnight.

More than 50% of adult OPCs were BrdU+ (Fig. 1B, 1C), indicating that they were mitotically

active. After proliferating for 2–8 passages, the percentage of adult OPCs expressing A2B5,

NG2, or O4 did not significantly change (Fig. 1F), indicating that adult OPCs can proliferateextensively in vitro without phenotypic drift. When cultured in serum-free medium containing

insulin and ciliary neurotrophic factor but lacking FGF2 and PDGFaa for 3 days, more than

85% of cells differentiated to express O1 and developed the typical process-bearing

morphology of OLs (Fig. 1G). Moreover, after 6 days of differentiation, more than 80% of the

cells developed into CNPase+ (Fig. 1H) or MBP+ (Fig. 1I) mature OLs with obvious membrane

sheet. No GFAP+ astrocytes were observed (data not shown). In contrast, when cultured in the

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same differentiation medium but containing 10% FBS, more than 60% of the cells

differentiated into GFAP+ astrocytes that coexpressed A2B5 with typical stellate morphology

of type 2 astrocytes (Fig. 1J). These results showed that adult spinal cord OPCs were bipotential

and differentiated into OLs in the absence of serum and into type 2 astrocytes in its presence.

Expression of BMP Receptors by OPCs and OLs

OL differentiation in cell culture can be roughly divided into three stages: A2B5+/O4+ OPCs,

O1+ immature OLs, and MBP+ mature OLs. As described above, adult OPCs readilydifferentiated into O1+ and MBP+ OLs after withdrawal of FGF2 and PDGFaa for 3 and 5

days, respectively. To determine whether cells in the three stages of OL development can

respond to BMP, we examined their expression of BMP receptors Ia, Ib, and II using

immunohistochemistry and Western blot. Adult OPCs expressed BMP receptors BMPR Ia

(Fig. 2Aa), BMPR Ib (Fig. 2Ab), and BMPR II (Fig. 2Ac). With slight downregulation,

immature O1+ and mature MBP+ OLs still persistently expressed these receptors (Fig. 2Ad).

These findings suggest that OPCs in the different stages of OL differentiation all have the

potential to respond to BMP.

BMP2 and BMP4 Suppress OL Differentiation and Promote Astrocyte Differentiation by Adult

OPCs

Previous studies showed that either BMP2 or BMP4 increased astrocyte differentiation and inhibited OL differentiation of neural stem cells or OPCs isolating from embryonic or postnatal

developing CNS [17–19,21,22]. Whether BMPs have similar effects on adult OPCs has not

been determined. When adult OPCs were differentiated without BMP2 or BMP4 for 3 days,

almost all of the cells differentiated into O1+ OLs. No astrocyte differentiation was observed

(Fig. 2Ba). After addition of BMP2, BMP4, or both in the differentiation medium, the number

of OPCs that differentiated into O1+ OLs dramatically decreased, with concurrent increases

of GFAP+ astrocytes (Fig. 2Bb–2Bd). The number of OPCs that differentiated into mature

MBP+ OLs also decreased (Fig. 2Be–2Bh). To determine dose-dependent effects on the

differentiation of adult OPCs, increasing concentrations of BMP2, 4, or both 2 and 4 were

added to the medium. As shown in Figure 2C, when differentiated for 3 days in the presence

of BMP2 at concentrations of 1, 2, 5, or 10 ng/ml, the percentage of adult OPCs that

differentiated into GFAP+ astrocytes significantly increased from 0.4% at control to 14%, 18%,

25%, and 46%, respectively ( p < .001 at all four concentrations). The percentage of O1+ OLsdecreased from 77% to 60%, 45%, 34%, and 19% ( p < .001), respectively, and MBP+ OLs

also were suppressed from 58% to 60%, 45%, 34%, and 19% ( p < .01), respectively. BMP4

was more potent than BMP2 in the inhibition of OL differentiation and promotion of astrocyte

differentiation. In the presence of BMP4 at 1, 2, 5, and 10 ng/ml, the number of OPCs that

differentiated into GFAP+ astrocytes were 35%, 44%, 66%, and 75%, respectively, all being

significantly increased compared with 0.4% in control cultures (all p < .001). The number of

O1+ and MBP+ OLs were 20%, 13%, 6%, 1% and 21%, 9%, 2%, 1%, respectively, all

significantly decreased compared with 77% and 58% (all p < .001). In the presence of BMP2

and 4 at the same concentrations, further increases in the number of GFAP+ astrocytes and

decreases of O1+ or MBP+ OLs were observed (Fig. 2C). These findings were confirmed by

Western blot experiments (Fig. 2D). Moreover, we found that the expression of pSmad1/5/8

and ID4, two intracellular downstream targets of BMP signaling, was also increased in

proportion with the dosage of BMP2, 4, or 2 and 4, suggesting that BMP signaling inhibits OLdifferentiation and promotes astrocyte differentiation of adult OPCs through Smad-dependent

pathways.

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BMP2 and BMP4 Downregulate the Expression of o lig1 and o lig2 During Adult OPC

Differentiation

To determine the potential mechanism through which BMPs affect the differentiation of adult

OPCs, we examined the expression of olig1 and olig2, two bHLH transcription factors

important for OL development. When differentiating for 3 days in the absence of BMP2 and

4, the majority of cells developed into OLs (Fig. 3Aa) that expressed olig1 (Fig. 3Ab) and olig2

(Fig. 3Ac). Addition of BMP2 and 4, however, significantly decreased the number of olig1-

expressing cells from 79% to 15% ( p < .001) and olig2-expressing cells from 76% to 15% ( p< .001) in addition to dramatically increasing the number of GFAP+ astrocytes and decreasing

O1+, CNPase+, or MBP+ OLs (Fig. 3Ad–3Af, 3B). Western blot experiments confirmed that

addition of BMP2 and 4 in the differentiating medium activated Smad1/5/8 to inhibit the

expression of olig1, olig2, and MBP and to promote the expression of GFAP (Fig. 3C).

Previous studies have shown that adult OPCs change their responses to growth factors after

culturing for a long time in the presence of FGF2 and PDGFaa [41]. To address this possibility,

we tested the effects of BMP2 and 4 on differentiation of freshly isolated OPCs from adult

spinal cord. As shown in supplemental online Figure 1, all OPCs expressed A2B5

(supplemental online Fig. 1A) and O4 (supplemental online Fig. 1B). After differentiation

without FGF2 and PDGFaa for 3 days, most OPCs differentiated into OLs expressing O1

(supplemental online Fig. 2A), CNPase (supplemental online Fig. 2C), and MBP (supplemental

online Fig. 2E). No GFAP+ astrocytes were observed (supplemental online Fig. 2A). Additionof BMP2 and 4 dramatically increased the number of GFAP+ astrocytes (supplemental online

Fig. 2B) and simultaneously decreased the number of O1+ (supplemental online Fig. 2B),

CNPase+ (supplemental online Fig. 2D), or MBP+ (supplemental online Fig. 2E) OLs. The

number of cells expressing Olig2 (supplemental online Fig. 2C, 2D) or Olig1 (supplemental

online Fig. 2E, 2F) was also significantly decreased by the treatment of BMP2 and 4. These

data indicate that the effects of BMP2 and 4 on the differentiation of freshly isolated adult

OPCs are identical to their effects on OPCs of passages 4–7, which we used in most of our

present study. Thus, exposure to FGF2 and PDGFaa in vitro does not change the responses of

adult OPCs to BMP2 and 4.

BMP2 and 4 Inhibit OL Maturation

The findings that immature O1+ OLs expressed BMP receptors (Fig. 2) and that the number of MBP+ OL decreased after the treatment with BMP2 and 4 suggested that BMP might also

inhibit the maturation of OL from immature O1+ to more mature MBP+ stages. To determine

the effects of BMP2 and 4 on OL maturation, adult OPCs were allowed to differentiate into

O1+ OLs by withdrawing FGF2 and PDGFaa for 2 days and then continued to mature for 3

more days in the absence or presence of BMP2 and 4. As shown previously, when

differentiating for 2 days after withdrawal of FGF2 and PDGFaa, more than 75% of adult OPCs

developed into O1+ OLs; after 3 more days, more than 95% of cells became O1+ OLs (Fig.

4Aa), and 90% of OPCs matured into CNPase+ (Fig. 4Ab) or MBP+ (Fig. 4Ac) OLs. When

10 ng/ml BMP2 and 4 were added for the last 3 days of differentiation, a significant decrease

in the number of OLs expressing either MBP+ (from 95% to 64%) or CNPase (from 93% to

58%; p < .001) was observed (Fig. 4Ad–4Af, 4B). However, the number of O1+ OLs (from

98–89%; p > .05) or GFAP+ astrocytes (from 1.1% to 5.6%; p > .05) did not significantly

change (Fig. 4Aa, 4Ad, 4B). These results showed that BMP2 and 4 inhibited the maturationof O1+ into MBP+ or CNPase+ OLs.

We further examined the expression of olig1 and olig2 during the maturation of adult OPCs.

When differentiating in the presence of BMP2 and 4, the number of olig1+ cells significantly

decreased from 79% at 5 days after differentiation without BMP2 and 4 to 49% ( p < .01) (Fig.

4Ab, 4Ae, 4B, 4C). The number of olig2+ cells was also decreased from 80% at control to

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45% ( p < .05) (Fig. 4Ac, 4Af, 4B, 4C). BMP2 and 4 also inhibited the development of both

CNPase- or MBP-expressing mature OLs, as shown by both immunohistochemistry (Fig. 4A,

4B) and Western blot (Fig. 4C). Our results showed that BMP2 and 4 specifically decreased

the expression of both olig1 and olig2 and subsequently inhibited OL differentiation and

maturation of adult OPCs.

Overexpression of olig1 and ol ig2 Blocks the BMP-Mediated Inhibition of Adult OPC

DifferentiationWhen differentiating in the presence of BMP2 and 4 for 3 days, 70% of EGFP-expressing

OPCs became GFAP+ astrocytes (Fig. 5A, 5D). Astrocyte differentiation in olig1- or olig2-

expressing OPCs was dramatically decreased to 25% ( p < .001) and 32% ( p < .001),

respectively (Fig. 5B–5D). However, the percentage of O1+ OLs derived from olig1- or olig2-

expressing OPCs (22% and 26%, respectively) after 3 days of differentiation in the presence

of BMP2 and 4 was slightly but not significantly increased compared with 17% of EGFP-OPCs

(both p > .05) (Fig. 5E–5I). These results indicate that overexpression of olig1 or olig2 is

sufficient to block the astrocyte differentiation of adult OPCs induced by BMP2 and 4.

However, overexpression of either olig1 or olig2 failed to block the inhibition of BMP2 and 4

on OL differentiation of adult OPCs. Since BMP2 and 4 downregulate the expression of both

olig1 and olig2 in the adult OPCs, overexpression of both olig1 and olig2, not olig1 or olig2

alone, may be necessary to overcome the inhibition of BMP2 and 4 on OL differentiation of

adult OPCs. To test this hypothesis, we infected adult OPCs with retroviruses encoding both

olig1 and olig2 and then differentiated the cells for 3 days in the presence of BMP2 and 4. As

shown in Figure 5H and 5I, the percentage of O1+ OLs in olig1/olig2-expressing adult OPCs

was 63%, which was significantly increased compared with 17%, 22% or 26% in EGFP-,

olig1-, or olig2-expressing adult OPCs, respectively (all p < .001).

Expression of Both o lig1 and olig2 Is Necessary for OL Differentiation o f Adul t OPCs

To further elucidate the role(s) of olig1 and olig2 in the differentiation of adult OPCs, we tested

whether downregulation of olig1 or olig2 by specific siRNA would inhibit OL differentiation

of adult OPCs. We first tested whether siRNAs were able to efficiently downregulate the

expression of olig1 or olig2 in the adult OPCs. Expression of either olig2 (Fig. 6A–6C) or olig1

(Fig. 6D–6F) in adult OPCs infected with retroviruses encoding control scrambled siRNA did

not change. However, the expression of olig2 was dramatically decreased in these adult OPCsinfected with retroviruses encoding olig2 siRNA (Fig. 6A′ –6C′). Expression of olig1, however,

did not change in these olig2 siRNA OPCs (data not shown). Similarly, adult OPCs expressing

olig1 siRNA significantly downregulated its expression of olig1 (Fig. 6D′ –6F′) but not olig2

(data not shown). These results demonstrated that olig1 or olig2 siRNA specifically knocked

down the expression of olig1 and olig2 in adult OPCs, respectively. We then examined whether

OL differentiation of adult OPCs was inhibited following the downregulation of olig1 or olig2

with specific siRNA. Adult OPCs infected with retroviruses encoding either control scrambled,

olig1, or olig2 siRNA were differentiated for 3 days. In the control group, more than 90% of

infected adult OPCs differentiated into O1+ OLs (Fig. 6G, 6J). O1+ OLs, however,

significantly decreased in olig1 siRNA OPCs (35%; p < .001) (Fig. 6I, 6J) or in olig2 siRNA

OPCs (59%; p < .05) (Fig. 6H, 6J). These results showed that both olig1 and olig2 were

necessary for OL differentiation of adult OPCs. No GFAP+ astrocyte differentiation was

observed in either EGFP, olig1, or olig2 siRNA OPCs after 3 days differentiation withoutBMP2 and 4 (data not shown).

Expression of BMP2 and 4 and olig1 and 2 in the Injured Spinal Cord

Obvious expression of BMP2 or BMP4 was not observed in the normal adult spinal cord

(supplemental online Fig. 3A, 3D). At 7 days after contusion, expression of BMP2 and BMP4

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was significantly increased in the reactive hypotrophic astrocytes in the injured spinal cord

(supplemental online Fig. 3B, 3E). Their expression in the astrocytes continued to increase in

the spinal cord at 1 month after contusion (supplemental online Fig. 3C, 3F). These results

showed that expression of BMP2 and BMP4 was significantly increased after SCI.

Olig1 was localized in the nuclei of NG2+ OPCs in the normal spinal cord, and almost all NG2

+ OPCs expressed olig1 (supplemental online Fig. 4A, inset, arrows). At 1 week (supplemental

online Fig. 4B) and 1 month (supplemental online Fig. 4C) after the contusion, the number of NG+ OPCs significantly increased in the injured spinal cord, but the number of olig1+ cells

decreased. Most of NG2+ OPCs did not express olig1 (supplemental online Fig. 4B, 4C, inset,

arrowheads), whereas some OPCs remained olig1-positive (supplemental online Fig. 4B, 4C,

inset, arrows). OPCs in the adult spinal cord also expressed olig2 in their nuclei (supplemental

online Fig. 4D, inset, arrow). At 1 week postinjury, the number of olig2+ cells increased in the

injured spinal cord, and its expression in some cells was upregulated (supplemental online Fig.

4E). However, many adult OPCs did not express olig2 (supplemental online Fig. 4E, inset,

arrowheads). The number of olig2+ cells and its expression level decreased in the injured spinal

cord at 1 month postinjury compared with 1 week postinjury (supplemental online Fig. 4F).

More OPCs did not express olig2 (supplemental online Fig. 4F, inset, arrowheads).

Discussion

OPCs in the Adult Spinal Cord

We obtained highly purified OPCs from the adult rat spinal cord that share many fundamental

properties with OPCs from perinatal and adult optic nerve or subcortical white matter. They

express similar phenotype-specific antigens, O4, A2B5, and NG2. OPCs cultured from adult

rat optical nerve [47] or human cortical white matter [48,49] express O4. Approximately 80%

of acutely isolated cycling progenitor cells in the adult mammalian white matter are O4+

[50]. OPCs in the adult spinal cord also express A2B5 and NG2, which is in accord with

previous results from adult optic nerve [39,47]. In the intact adult spinal cord, more than 70%

of BrdU+ cycling precursor cells express NG2 [23]. Therefore, the purified A2B5+/NG2+/O4

+ OPCs in this study are the most actively proliferative precursor cells in the normal adult

spinal cord.

Consistent with present data with adult spinal cord OPCs, OPCs from postnatal seven days ratoptic nerve can proliferate for many passages without differentiation in the presence of PDGFaa

and in the absence of thyroid hormone [51,52]. FGF2 can inhibit the differentiation of OPCs

and promote their proliferation [49,53] and can convert slowly dividing adult OPCs to rapidly

dividing cells with characteristics of perinatal OPCs [41]. Moreover, the adult spinal cord OPCs

differentiate into OLs in the serum-free medium and into type 2 astrocytes in medium

containing serum, a property of OPCs from other CNS regions [54,55]. The fact that adult

spinal cord OPCs can be induced to differentiate into different developmental stages of OLs

by withdrawing FGF2 and PDGFaa for certain times (Fig. 1) establishes a stable adult spinal

cord OPC model to investigate potential molecular mechanism(s) by which adult OPCs

proliferate and differentiate in vitro.

BMP Signaling Inhibits Oligodendrogenesis and Promotes Astrogliogenesis from Adult

OPCs

In vitro, BMP signaling enhances astrocyte differentiation and inhibits OL differentiation from

cultured neural stem cells or OPCs isolated from embryonic or perinatal CNS [16,17,21,22,

56]. In vivo, BMP signaling also represses OL development [11,13,57]. Furthermore,

overexpression of BMP4 in transgenic mice under the control of the neuron-specific enolase

promoter results in a significant decrease in OLs, with a concurrent increase in astrocytes

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[15]. The present study extends these previous findings to show that BMP signaling also

inhibits the differentiation and maturation of adult OPCs, suggesting that BMP signaling may

play an important role in the regulation of remyelination of the adult CNS following traumatic

or demyelinating injuries.

Importantly, present data document the molecular mechanism(s) by which BMP signaling

inhibits OPC differentiation into mature OLs. One possible mechanism is the inhibition of

olig1/2 activity by ID4 (Fig. 2), as suggested by a previous study [20]. Although ID4 and ID2are expressed in proliferating OPCs, their expression decreases progressively as OPCs

differentiate and mature [20,58,59]. Overexpression of ID2 or ID4 in OPCs inhibits OL

differentiation, whereas downregulation of their expression induces premature differentiation

in vitro [20,58,59]. ID2 and ID4 bind olig1 and olig2 in the cytoplasm of OPCs to prevent their

translocation to nucleus, thereby preventing olig1 and olig2 from binding target DNA [20].

The present study further suggests that an additional mechanism by which BMP signaling

inhibits OL differentiation from adult OPC is the downregulation of olig1 and olig2 (Figs. 2–

5). That overexpression of olig1 and olig2 reverses the inhibitory effects of BMP signaling on

OL differentiation of OPCs further confirms the suggestion that BMP signaling inhibits

oligodendrogenesis from OPCs by downregulating olig1 and olig2. Therefore, downregulation

of olig1 and olig2 expression and inhibition of olig1/2 activity by increased ID4 may work

synergistically to inhibit OL differentiation and to enhance astrogliogenesis (Fig. 7).

Olig1 and olig2 in the Differentiation o f Adult OPCs

Transcriptional regulation orchestrates oligodendrogenesis during CNS development [1,2,

60]. Olig1 and olig2 play important roles in generating OLs during embryogenesis [9,10,61,

62]. Olig2 is required for OL lineage specification during CNS development, whereas olig1

contributes more to OL differentiation and maturation [9,10,63,64]. However, olig2

compensates for the function of olig1 during OL development, since OL differentiation and

myelination are delayed but eventually reach normal level in olig1-null mice [9,10]. In contrast

to embryonic development, adult OPCs require both olig1 and olig2 for their differentiation

and maturation. Downregulation of either olig1 or olig2 by siRNA inhibits adult OPCs

differentiation into OLs (Fig. 6). Overexpression of olig1 or olig2 alone fails to overcome the

inhibition of BMP to promote OL differentiation since BMP signaling decreases the expression

of both olig1 and olig2. However, overexpression of both olig1 and olig2 enhances OL

differentiation even in the presence of BMP2 and 4 (Fig. 5). These results are consistent with

data from Arnett et al. [42], which show that olig1 is required not for the development of OLs

but for remyelination in adult mice following demyelination. Taken together, these data

indicate that olig1 is required for the differentiation and maturation of adult OPCs and

endogenous remyelination after demyelination in adult CNS. Whether olig2 plays an important

role in the differentiation and maturation and myelination of OLs during development is not

known, since olig2-null mice die during the perinatal period. Interestingly, olig2 expression is

retained during OL maturation, suggesting its possible role in OL differentiation. Consistent

with this idea, our in vitro analyses show that olig2 is also required for OL differentiation and

maturation from adult OPCs. These data also suggest that it may have a potential role during

remyelination. Conditional olig2 knockout mice will be very useful to study its effects on

remyelination in vivo.

In addition to OL development, olig1 and olig2 also play an important role in astrogliogenesis.

Overexpression of olig1 or olig2 significantly inhibits astrocyte differentiation of adult OPCs

induced by BMP signaling, suggesting that either olig1 or olig2 is sufficient to repress

astrogliogenesis. These results are in good accord with the previous observation of Lu et al.

[10], which show that the null mutation of either olig1 or olig2 alone is not sufficient to increase

astrogliogenesis in the spinal cord. The double-null mutation of olig1 and olig2, however,

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results in an apparent increase in astrogliogenesis, with a complete failure of OL development

[9]. Thus, downregulation of both olig1 and olig2 is required for OPCs to differentiate into

astrocytes since olig1 and olig2 could complement the functions of one another sufficient to

inhibit astrocyte differentiation. Consistent with this notion, we observe that both olig1 and

olig2 are dramatically downregulated when astrocyte differentiation from adult OPCs is

induced by BMP signaling. Our results further show that downregulation of olig1 or olig2

alone by siRNA fails to result in enhanced astrocyte differentiation even though OL

differentiation is inhibited, suggesting that repression of OL differentiation alone is notsufficient to cause the astrocyte differentiation from adult OPCs if BMP signaling is not present.

BMP signaling activates Smad proteins, which translocate into the nucleus to form a complex

with p300 to directly activate the GFAP promoter [65,66]. Olig2 interacts with p300 to interrupt

the complex of p300 and Smad1 and thereby represses the astrocyte-specific GFAP promoter

[67]. Because of their structural similarity, it is possible that olig1 may use the same mechanism

to inhibit astrocyte differentiation. This would suggest that BMP signaling is able to induce

adult OPCs to differentiate into astrocytes through the activation of GFAP promoter by the

Smad-p300 complex only when olig1 and olig2 are also down-regulated (Fig. 7).

Downregulation of both olig1 and olig2 is required, but not sufficient, for astrocyte

differentiation from adult OPCs.

Implications for Remyelination

OPCs reside quiescently in the white matter of the adult CNS [23,24]. They become activated

and proliferate in response to CNS injury, including demyelination [25–28]. However, adult

OPCs fail to differentiate and mature into OL capable of remyelinating demyelinated axons in

multiple sclerosis lesions even though endogenous OPCs are present in the demyelinated areas

and some even closely contact the affected axons [36,38,68–70]. Inhibitory signals that prevent

OPCs from differentiation and maturation are present in the demyelinated lesions [71,72]. We

suggest that BMP signaling may be an important factor that inhibits OL differentiation and

maturation in the injured CNS. In fact, expression of BMPs dramatically increases after CNS

injury [73,74]. Moreover, neuronal and OL differentiation from engrafted neural stem cells in

the injured spinal cord is increased after blocking BMP signaling by expressing noggin [73].

Consistent with those findings, we also observed a significant increase of BMP2 and 4 in the

hypertrophic astrocytes after traumatic SCI (supplemental online Fig. 3), and expression of

olig1 and olig2 in the OPCs of the injured spinal cord decreased (supplemental online Fig. 4).Manipulation of BMP signaling may provide a new therapeutic strategy to enhance

remyelination from endogenous or/and grafted OPCs after multiple sclerosis or SCI.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

This work was supported by National Center for Research Resources Grant 5P20 RR 015576 (to Q.C. and S.R.W.),

NIH R01 NS37717 (to M.Q.), the Kentucky Spinal Cord and Head Injury Research Trust (to Q.C. and S.R.W.), Norton

Healthcare, the Commonwealth of Kentucky Research Challenge for Excellence Trust Fund, and The Christopher

Reeve Paralysis Foundation (to S.R.W.). We thank George Harding of the Anatomical Sciences and Neurobiology,

University of Louisville School of Medicine, for expert technical help.

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Figure 1.

Isolation and differentiation of adult oligodendrocyte precursor cells (OPCs) in vitro. Adult

OPCs were purified from the spinal cord of adult rats by immunopanning with the O4 antibody.The proliferating precursors displayed a characteristic adult OPC morphology with several

small short processes emanating from a small round cell body (A). All cells expressed O4

(B), A2B5 (B), and NG2 (D, E). In the presence of fibroblast growth factor 2 (FGF2) and

platelet-derived growth factor aa (PDGFaa), adult OPCs divided and are readily labeled by

BrdU (B, C). These OPCs proliferated for multiple passages without changing phenotypes

(F). Data in (F) were the mean ± SD of four independent experiments. Three days after

withdrawal of FGF2 and PDGFaa, OPCs constitutively differentiated into OLs expressing O1



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(G). Five days after differentiation, the majority of adult OPCs differentiated into mature OLs

expressing CNPase (H) and MBP (I). When differentiated in the presence of 10% fetal bovine

serum, more than 60% of the cells differentiated into GFAP+ astrocytes (J) that also expressed

A2B5 and had a typical stellate morphology of type 2 astrocytes (J). Scale bars = 25 μm (A-

C, G, J), 10 μm (D), 20 μm (E, I), and 50 μm (H). Abbreviations: BrdU, 5-bromo-2′-

deoxyuridine; CNPase, 2′,3′-cyclic nucleotide phosphodiesterase; GFAP, glial fibrillary acidic

protein; MBP, myelin basic protein.



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Figure 2.

Inhibition of oligodendrocyte (OL) differentiation and promotion of astrocyte differentiation

of adult oligodendrocyte precursor cells (OPCs) by BMP2 and 4. Adult OPCs expressed BMPR Ia (Aa), Ib (Ab), and II (Ac). Although they were slightly downregulated, all three BMPRs

were persistently expressed in immature O1+ and more mature MBP+ OLs after Dif3d and

Dif5d, respectively (Ad). Treatment with BMP2, 4, or both decreased the number of O1+ OLs,

with concurrent increases in the number of GFAP+ astrocytes (Ba – Bd). The number of MBP

+ OLs was also decreased (Be – Bh). Quantification of cells immunostained for GFAP, O1, or

MBP showed a dose-dependent increase in the number of GFAP+ astrocytes and decrease of

O1+ or MBP+ OLs differentiated from adult OPCs by BMP2, 4, or both, respectively (C). Data



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represent the mean ± SD (n = 4; one and two stars represent p < .05 and 0.01, respectively).

Western blot experiments confirmed the dose-dependent activation of pSmad1/5/8, ID4, the

intracellular downstream target of BMP signaling with the increasing and decreasing

expression of GFAP and MBP, respectively, by BMP2, 4, or both (D). Scale bar = 20 μm (Aa–

Ac) and 25 μm (Ba–Bh). Abbreviations: BMP, bone morphogenetic protein; BMPR, bone

morphogenetic protein receptor; DAPI, 4,6-diamidino-2-phenylindole; Dif3d, differentiation

for 3 days; Dif5d, differentiation for 5 days; GFAP, glial fibrillary acidic protein; MBP, myelin

basic protein; Undif, undifferentiated.



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Figure 3.

BMP2 and BMP4 inhibit adult oligodendrocyte precursor cell (OPC) differentiation into

oligodendrocytes (OLs) in vitro by downregulating olig1 and olig2. Three days after

differentiation in vitro, almost all adult OPCs differentiated into OLs expressing O1 (Aa) or

CNPase (Ab). A few cells expressed MBP (Ac). Addition of 10 ng/ml BMP2 and 4 dramatically

decreased the number of O1-, CNPase-, or MBP-expressing OLs while significantly increasing

the number of GFAP+ astrocytes (Ad–Af ). BMP2 and 4 significantly decreased the number of

cells immunostained for olig1 and olig2 (B). Data represent the mean ± SD (n = 4; stars indicate

p < .001). Western blot experiments confirmed the immunohistochemical results and further

showed that BMPs activate pSmad1/5/8 in adult OPCs (C). Similar results were obtained in

four independent experiments. Scale bar = 100 μm (Aa–Af ). Abbreviations: BMP, bone

morphogenetic protein; CNPase, 2′,3′-cyclic nucleotide phosphodiesterase; DAPI, 4,6-

diamidino-2-phenylindole; Dif3d, differentiation for 3 days; GFAP, glial fibrillary acidic

protein; MBP, myelin basic protein; Undif, undifferentiated.



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Figure 4.

BMP2 and BMP4 inhibit oligodendrocyte (OL) maturation in vitro. Differentiated for 5 days,

almost all cells matured into CNPase or MBP OLs expressing olig1 and olig2 (Aa–Ac, B). If

they were differentiated for 2 days without BMPs to allow adult oligodendrocyte precursor

cell to become O1+ OLs and then differentiated for 3 more days with BMPs, the number of

cells expressing O1 did not change (Ad, B). The number of cells expressing CNPase, MBP,

olig1, and olig2, however, significantly decreased (Ae–Af, B). No obvious GFAP+ astrocyte

differentiation was observed with or without BMPs (Aa, Ad, B). Data represent the mean ±

SD (n = 4; stars indicate p < .01). Western blot experiments confirmed the decreasing

expression of CNPase, MBP, olig1, and olig2 with increasing pSMAD1/5/8 after BMP

treatment (C). Scale bar = 25 μm (Aa–Af ). Abbreviations: BMP, bone morphogenetic protein;

CNPase, 2′,3′-cyclic nucleotide phosphodiesterase; DAPI, 4,6-diamidino-2-phenylindole;

GFAP, glial fibrillary acidic protein; MBP, myelin basic protein.



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Figure 5.

Overexpression of olig1 and olig2 inhibits astrocyte differentiation and promotes OL

differentiation of adult oligodendrocyte precursor cells (OPCs). Adult OPCs were infected withretroviruses encoding EGFP, Olig1, Olig2, or Olig1 and 2 and then differentiated for 3 days

in the presence of 10 ng/ml BMP2 and 4. Many adult OPCs, including EGFP-infected ones

(A, D) differentiated into GFAP+ astrocytes. Overexpressing Olig1 (B, D), or Olig2 (C, D)

significantly decreased the number of adult OPCs differentiating into astrocytes (D). Data

represent the mean ± SD (n = 4; stars indicate p < .001). The number of adult OPCs

differentiating into O1+ OLs was repressed in the presence of BMP2 and 4 (E, I).

Overexpression of olig1 (F, I) or olig2 (G, I) failed to result in a significant increase of O1+



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OLs. Overexpression of olig1 and olig2, however, overcame the inhibition of BMP signaling

to significantly increase OL differentiation from adult OPCs (H, I). Data represent the mean

± SD (n = 4; star indicates p < .01). Scale bars = 25 μm. Abbreviations: DAPI, 4,6-diamidino-2-

phenylindole; EGFP, enhanced green fluorescence protein; GFAP, glial fibrillary acidic

protein; OL, oligodendrocyte.



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Figure 6.

Inhibition of adult oligodendrocyte precursor cell (OPC) differentiation into oligodendrocytes

(OLs) following downregulation of Olig2 and Olig1. Olig2 and Olig1 siRNA downregulated

the expression of Olig2 and Olig1 in adult OPCs. Adult OPCs were infected with retrovirusesthat express either scrambled negative control (A–F), Olig2 (A′–C′), or Olig1 (D′–F′) siRNA.

Two days later, control adult OPCs did not alter their expression of Olig2 ([A–C], arrows) or

Olig1 ([D–G], arrows). However, adult OPCs infected with retroviruses expressing either

Olig2 or Olig1 siRNA downregulated their expression of Olig2 ([A′–C′], arrows) or Olig1

([D′–G′], arrows), respectively. Adult OPCs were differentiated for 3 days following infection

with retroviruses encoding negative control, Olig2, or Olig1 siRNA. Uninfected OPCs ([G–



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I], not green cells) and OPCs infected with scrambled control siRNA ([G], green) differentiated

into OLs expressing O1 ([G–I], red). Adult OPCs expressing Olig2 siRNA ([H], green cells)

or Olig1 siRNA ([I], green cells) did not differentiate into O1+ OLs. Data represent the mean

± SD (n = 4; one and two stars indicate p < .05 and 0.01, respectively). Scale bar = 25 μm (A–

F, A′–F′), 20 μm (G, H), and 10 μm (I). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole;

EGFP, enhanced green fluorescence protein; siRNA, small interference RNA.



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Figure 7.

A schematic illustration of the regulation of adult oligodendrocyte precursor cell (OPC)

differentiation by the interaction of BMP signaling and olig1/2. BMPs upregulate or downregulate the expression of ID4 and olig1/2, respectively, by Smad-dependent or -

independent pathways. Increasing the expression of ID4, which blocks the translocation of

olig1 and 2 to the nucleus, works synergistically with the downregulation of olig1 and 2 to

inhibit oligodendrocyte (OL) differentiation and permit and/or potentiate astrocyte

differentiation following BMP signaling. Olig1 and 2 are necessary to promote OL

differentiation and sufficient to inhibit astrocyte differentiation by binding p300 to interrupt

the formation of the Smad/p300 complex, which activates the GFAP promoter. Therefore,

overexpression of olig1 and 2 overcomes the effects of BMP signaling on adult OPCs to

promote OL differentiation and inhibit astrocyte differentiation. Abbreviations: BMP, bone

morphogenetic protein; BMPR, bone morphogenetic protein receptor.



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