Notch1 signaling plays a role in regulating precursordifferentiation during CNS remyelinationYueting Zhanga,b, Azeb Tadesse Argawa,b, Blake T. Gurfeina,b, Andleeb Zameera,b, Brian J. Snyderc, Changhui Ged,1,Q. Richard Lue, David H. Rowitchf, Cedric S. Raineg,h, Celia F. Brosnang,h, and Gareth R. Johna,b,h,2

aCorinne Goldsmith Dickinson Center for Multiple Sclerosis, bDepartments of Neurology and cNeurosurgery, Mount Sinai School of Medicine, New York, NY10029; eDepartment of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; fChildren’s Hospital at the Universityof California, San Francisco Medical Center, University of California, San Francisco, CA 94143; and dDepartments of Cell Biology, gNeuroscience,and hPathology, Albert Einstein College of Medicine, Bronx, NY 10461

Edited by Lynn T. Landmesser, Case Western Reserve University, Cleveland, OH, and approved September 16, 2009 (received for review March 17, 2009)

In the developing CNS, Notch1 and its ligand, Jagged1, regulateoligodendrocyte differentiation and myelin formation, but theirrole in repair of demyelinating lesions in diseases such as multiplesclerosis remains unresolved. To address this question, we gener-ated a mouse model in which we targeted Notch1 inactivation tooligodendrocyte progenitor cells (OPCs) using Olig1Cre and afloxed Notch1 allele, Notch112f. During CNS development, OPCdifferentiation was potentiated in Olig1Cre:Notch112f/12f mice. Im-portantly, in adults, remyelination of demyelinating lesions wasalso accelerated, at the expense of proliferation within the pro-genitor population. Experiments in vitro confirmed that Notch1signaling was permissive for OPC expansion but inhibited differ-entiation and myelin formation. These studies also revealed thatastrocytes exposed to TGF-�1 restricted OPC maturation viaJagged1-Notch1 signaling. These data suggest that Notch1 signal-ing is one of the mechanisms regulating OPC differentiation duringCNS remyelination. Thus, Notch1 may represent a potential ther-apeutical avenue for lesion repair in demyelinating disease.

autoimmunity � CNS repair � multiple sclerosis �oligodendrocyte progenitor � myelin

In demyelinating diseases of the CNS such as multiple sclerosis(MS), myelin loss is associated with conduction block in affected

axons and is linked to axonal transection in chronic lesions (1, 2).Conversely, remyelination is associated with return of conductionand functional recovery but often fails with disease progression (3,4). Myelin repair is known to be mediated by a pool of oligoden-drocyte progenitor cells (OPCs), and mechanisms regulating thenumber, recruitment, and differentiation of these cells are ofintense interest in MS research (5).

In the adult CNS, factors identified as controlling the OPCresponse following demyelination include the mitogens PDGF-AAand FGF-2 (6, 7) and regulators of differentiation such as insulin-like growth factor (IGF)-1, hyaluronan, and the Wnt–�-cateninmediator Tcf4 (8–10). Potential contributions of other pathwayshave also been discussed, including canonical Notch signaling(11–13), which restricts OPC maturation in the developing CNS andis permissive for proliferation (14–16). The canonical Notch path-way is mediated via binding of ligands, including Jagged1–2 andDelta1–4 to Notch1 and Notch2 receptors (17), and we have shownpreviously that in the lesions of MS, Jagged1 is expressed by reactiveastrocytes, whereas Notch1 and its effector Hes5 localize to oligo-dendrocytes (11). However, although expression of Jagged1 andNotch1 has been confirmed in demyelinating models, their role inremyelination remains unresolved (11–13).

Here, we have examined myelin repair in mice with selectiveinactivation of Notch1 throughout the oligodendrocyte lineage. Togenerate these animals, we paired Olig1Cre with Notch112f, aconditional Notch1 allele (18, 19). Olig1 is expressed at all stages ofOPC development, including early progenitors (19). To induce de-and remyelination in Olig1Cre:Notch112f/12f mice, we used stereo-tactic microinjection of lysolecithin into corpus callosum (20).

Notably, in the Olig1Cre:Notch112f/12f genotype, we found thatresolution of demyelinated lesions was accelerated, at the expenseof OPC proliferation. Collectively, these findings suggest thatcanonical Notch1 signaling is one of the pathways regulating OPCdifferentiation in demyelinating lesions in the adult CNS.

ResultsInhibition of Notch1 Restricts OPC Expansion and Potentiates Matu-ration and Myelination. To determine whether Notch1 signalingaffects OPC proliferation and maturation, we initially used siRNA(Fig. 1). In pilot studies in rat P1 A2B5�Ran2� OPC cultures (21),Notch1 and Notch2 localized to both CNPase� oligodendrocytes(Fig. 1 A and B) and A2B5� cells (results not shown). Jagged1 andDelta1 were not detected. In reporter studies, we observed basalNotch activity in OPC cultures that was blocked by �-secretaseinhibitors [see supporting information (SI) Methods].

OPCs were nucleofected with Notch1 siRNA or nontargetingcontrol and left to differentiate (Fig. 1 C–E). Notch1 siRNAinhibited expression of Notch1 but not Notch2 (Fig. 1C). In Notch1siRNA-nucleofected cultures, after 5 days, a higher percentage ofcells were mature O4�, CNPase�, and myelin basic protein (MBP)�

oligodendrocytes than in controls and a smaller percentage wereA2B5� OPCs or mitotically active (BrdU�) cells (Fig. 1D, t test, Pvalues shown; Fig. 1E, P � 0.01). To establish whether these effectswere associated with changes in myelin formation, we coculturednucleofected OPCs with dorsal root ganglion (DRG) neurons andinitiated myelination with TrkA-Fc (22) (Fig. 1 F–H). After 14 days,myelin segments were detected in all cultures by confocal micros-copy as MBP� profiles extending along neurofilament (NF)� axonsand were confirmed using electron microscopy (22) (Fig. 1 G andH). Importantly, cocultures containing Notch1 siRNA-nucleo-fected OPCs contained significantly more myelin segments thannontargeting siRNA controls (Fig. 1 F and G, P � 0.05). Segmentlength and G ratio (axon vs. fiber diameter) (23) were unaffected(results not shown). Segment numbers in controls were compatiblewith previous reports (24). These data demonstrated that inhibitionof Notch1 restricted OPC proliferation and potentiated maturationand myelin formation.

TGF-�1–Treated Human Astrocytes Restrict OPC Differentiation viaJagged-Notch Signaling. Previously, we have shown that Jagged1 isinduced in human astrocytes by TGF-�1 and that reactive astro-cytes express Jagged1 in MS plaques (11). To determine whether

Author contributions: G.R.J. designed research; Y.Z., A.T.A., B.T.G., A.Z., and B.J.S. per-formed research; C.G., Q.R.L., and D.H.R. contributed new reagents/analytic tools; Y.Z.,C.S.R., and C.F.B. analyzed data; and C.S.R., C.F.B., and G.R.J. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1Present address: Beijing Institute of Radiation Medicine, Beijing 100850, China.

2To whom correspondence should be addressed. E-mail: gareth.john@mssm.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0902834106/DCSupplemental.

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TGF-�1–treated astrocytes regulate OPC maturation via Jagged-Notch signaling, we used primary human cultures (24) (Fig. S1).

In human oligodendrocyte-enriched cultures, Notch1 immuno-reactivity localized to A2B5�, O4�, and CNPase� cells (Fig. S1A).Cultures nucleofected with the Hes1 reporter pGa981–6 (25) andplated onto HEK-293 cells stably expressing human Jagged1(pcDNA3hJag1) (26) displayed activation of Notch signaling (Fig.S1B, t test, P � 0.001). Cultures plated onto Jagged1-expressingcells also contained more A2B5� OPCs and fewer O4� andCNPase� oligodendrocytes than controls after 5 days (Fig. S1C, Pvalues shown). Interestingly, oligodendrocytes plated onto astro-cytes treated for 24 h with 10 ng/mL TGF-�1 followed by cytokinewashout displayed similarly restricted differentiation (Fig. S1 D andE, P values shown). To define the contribution of Jagged-Notchsignaling to this effect, we repeated the experiment using astrocytecultures transfected with Jagged1 siRNA or nontargeting controlbefore TGF-�1 treatment (Fig. S1 F and G). Inhibition of Jagged1induction was confirmed by immunoblotting (Fig. S1F). After 5days, oligodendrocytes plated onto astrocytes transfected withJagged1 siRNA before TGF-�1 treatment contained significantlymore O4� and CNPase� cells than nontargeting controls (Fig. S1G,t test). These results showed that TGF-�1–treated astrocytes re-stricted OPC differentiation and indicated that Jagged-Notch sig-naling contributed to this effect.

Precocious OPC Differentiation in Olig1Cre:Notch112f/12f Mice. Toinvestigate in vivo the relevance of these effects, we used a Cre-loxstrategy (Fig. 2 and Fig. S2). Mice carrying a floxed Notch1 allele(Notch112f) were crossed with Olig1Cre mice (Fig. S2A). InNotch112f mice, excision of Notch1 exons 6–8 generates an inactivereceptor lacking EGF repeats 8–12, which exhibits a Notch1 nullphenotype (18). In Olig1Cre mice, Cre is inserted into the Olig1 locusand is expressed throughout the oligodendrocyte lineage, includingearly OPCs (19). We confirmed recombination efficiency in pilotstudies using the ROSA26 reporter line (27). In 4-week-oldOlig1Cre:ROSA26�/fl mice, 71.3 � 2.3% Olig2� cells in corpuscallosum were lacZ�, compatible with previous reports (19).

Final intercrosses generated experimental Olig1Cre:Notch112f/12f

mice and controls (Olig1Cre:Notch1�/12f and non-Cre Notch112f/12f

and Notch1�/12f) in expected ratios. Neonatal Olig1Cre:Notch112f/12f

mice exhibited recombination at Notch1 in the CNS (Fig. S2 Band C) but not in other tissues tested (results not shown). Theydid not display neurological signs, although initial weight gainwas slower than in littermate controls (Fig. S2D). To determinewhether oligodendrocyte differentiation was altered inOlig1Cre:Notch112f/12f mice, we harvested litters at embryonalday 16.5 (E16.5) and postnatal day 14 (P14) and 12-week-oldadults (Fig. 2). Morphometric analyses of confocal Z-seriesprojections (see Materials and Methods) showed that lumbarspinal cord and corpus callosum of Olig1Cre:Notch112f/12f ani-mals and controls contained similar numbers of Olig2� oligo-dendrocyte lineage cells at E16.5 (Fig. 2 A and B) and P14 (Fig.2 E and G) and in adults (Fig. 2H). However, at E16.5 and P14,these populations were more mature in Olig1Cre:Notch112f/12f

mice than in controls. At E16.5, lumbar spinal cord ofOlig1Cre:Notch112f/12f mice contained more mature myelin as-sociated glycoprotein positive (MAG�) oligodendrocytes thancontrols (Fig. 2 A and C, P � 0.01, ANOVA plus Bonferroniposttest), although MBP immunoreactivity was absent in allgenotypes (data not shown). Oligodendrocyte maturation beginsin lumbar spinal cord and progresses rostrally, and at E16.5, fewMAG� cells were observed in corpus callosum in bothOlig1Cre:Notch112f/12f mice and controls (Fig. 2D). At P14,myelination appeared complete in spinal cord in all genotypes,but corpus callosum contained more CC-1� mature oligoden-drocytes in Olig1Cre:Notch112f/12f mice than in controls (Fig. 2 Fand G, P � 0.05), although no differences in MBP immunore-activity were observed (results not shown). In 12-week-oldadults, no differences in PDGF receptor (R)-�, CC-1, or MBPwere observed in corpus callosum or spinal cord (Fig. 2I). Thesestudies showed accelerated oligodendrocyte differentiation inOlig1Cre:Notch112f/12f mice.

Olig1Cre:Notch112f/12f Mice Show Accelerated Resolution of Demyeli-nating Lesions. We next examined whether repair of demyelinatinglesions was altered in Olig1Cre:Notch112f/12f adults. We used ste-reotactic microinjection of 1.5 �L of 1% lysolecithin (20) to inducefocal demyelination in corpus callosum of 12-week-old mice(Olig1Cre:Notch112f/12f) and controls (n � 140). Animals were killedat 7–28 days postinjection (dpi), and tissue was processed for

Fig. 1. Notch1 inhibition restricts OPC proliferation and potentiates differentiation and myelination. In rat P1 OPC cultures left to differentiate for 5 days,Notch1 and Notch2 localized to CNPase� oligodendrocytes (A and B) and A2B5� cells (results not shown). (C) Immunoblotting of rat OPCs nucleofected withNotch1 siRNA showed loss of full-length (fl) and transmembrane (tm) Notch1 (Left) at 5 days but no effect on Notch2 (Right). NT, nontargeting control. (D andE) In Notch1 siRNA-nucleofected cultures, a higher percentage of cells at 5 days were O4�, CNPase�, and MBP� oligodendrocytes and a smaller percentage wereA2B5� OPCs or mitotic (BrdU�) cells (D, t test, P values shown; E, P � 0.01). (F–H) Rat P1 OPCs were nucleofected with Notch1 siRNA or NT and plated onto E16.5rat DRG neurons; myelination was then induced using TrkA-Fc. After 14 days, myelin segments were observed as MBP� profiles (G, arrowheads) extending alongNF� axons, confirmed in electron microscopy studies (H). (F) Cocultures containing Notch1 siRNA-nucleofected OPCs contained significantly more segments thancontrols (P � 0.05). (Scale bars: A and B, 20 �m; G, 10 �m; H, 100 nm.) Data are representative of findings from at least 3 experiments.

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confocal imaging (Fig. 3, Fig. S3, and Table S1) or light and electronmicroscopy (Fig. 4, Fig. S3, and Table S2).

In confocal imaging experiments, serial 50-�m sections weretaken through cerebral cortices of Olig1Cre:Notch112f/12f mice andcontrols (at least 7 per genotype per time point) at 7 dpi (Table S1),14 dpi (Fig. 3 and Fig. S3), and 28 dpi (results not shown). Sectionswere immunostained for myelin (MBP), lineage markers (Olig2,GFAP, CD11b, and NF), and/or BrdU, and Z-series stacks werecaptured through each section. Examination of serial stacks ofMBP-stained samples rendered in 3 dimensions (Fig. 3A) and alsovisualized as projections (Fig. 3B and Fig. S3A) confirmed thatlysolecithin induced focal demyelination of corpus callosum (20).Immunostaining for lineage markers also confirmed that MBP�

lesions contained NF� axons, GFAP� astrocytes, and CD11b�

mononuclear phagocytes (Fig. S3B), and no differences in thesemarkers were detected between genotypes. At 7 dpi (Table S1),cross-sectional MBP� area and rostrocaudal lesion size (results notshown) were similar in all genotypes, but a smaller percentage ofOlig2� cells were mitotically active (BrdU�) in the lesion inOlig1Cre:Notch112f/12f mice than in controls (P � 0.05, ANOVAplus Bonferroni test). At 14 dpi, lesions remained hypercellular anddemyelinated (Fig. 3 A and B), but MBP� lesion area was smallerthan at 7 dpi (Fig. 3C) and a higher proportion of Olig2� cells wereBrdU� (Fig. 3D). Importantly, at 14 dpi, maximum cross-sectionalMBP� area in Olig1Cre:Notch112f/12f mice was significantly smallerthan in controls (Fig. 3 B and C, P � 0.05) and rostrocaudal lesion

size was also smaller (Fig. 3A). In addition, a smaller percentage ofOlig2� cells were BrdU� in lesions in Olig1Cre:Notch112f/12f mice(Fig. 3D, P � 0.01). BrdU� cells of other lineages were unchanged(Fig. 3E). By 28 dpi, repair was extensive in all genotypes (resultsnot shown). These studies showed that resolution of demyelinatedlesions was accelerated in Olig1Cre:Notch112f/12f mice.

To determine potential sources of Notch ligands in lysolecithinlesions, we used immunohistochemistry. Jagged1 was expressed byhypertrophic GFAP� astrocytes in hypercellular MBP� lesions inall genotypes (Fig. 3F), compatible with previous reports from MS(11) and animal models (12, 13). Jagged1 was not observed inunlesioned corpus callosum, and Delta1 was not detected (resultsnot shown).

Olig1Cre:Notch112f/12f Mice Display Potentiated Remyelination. Todefine the mechanism underlying these differences, we analyzedepoxy-embedded samples of corpus callosum from lysolecithin-injected mice (7, 14, and 28 dpi, at least 4 per time point pergenotype) and from unlesioned controls using light and electronmicroscopy (Fig. 4, Fig. S3, and Table S2). Morphometric quanti-tation of unlesioned samples (see Materials and Methods) showedthat myelin thickness and G ratio (axon vs. fiber diameter) (23) in12-week-old Olig1Cre:Notch112f/12f mice were similar to those incontrols (Table S2). Mean G ratio was between 0.53 and 0.58 in allgenotypes, within a range of 0.42 to 0.69.

Because confocal studies showed significant differences in lesion

Fig. 2. Precocious OPC differentiation in Olig1Cre:Notch112f/12f mice. Olig1Cre:Notch112f/12f mice plus littermate controls were killed at E16.5 (A–D), P14 (E–G),and 12 weeks (H and I) (at least 3 animals per genotype per time point). Morphometric analyses showed that in lumbar spinal cord and corpus callosum, numbersof Olig2� oligodendrocyte lineage cells were similar in all genotypes at E16.5 (A and B) and P14 (E and G) and in adults (H). (A and C) However, at E16.5, lumbarspinal cord of Olig1Cre:Notch112f/12f mice contained more mature MAG� oligodendrocytes than controls (ANOVA plus Bonferroni posttest, P � 0.01). Few maturecells were observed in corpus callosum at E16.5 (D), but at P14 (F and G), corpus callosum of Olig1Cre:Notch112f/12f mice contained more mature CC-1�

oligodendrocytes than controls (P � 0.05). Spinal cord was fully myelinated at P14 (results not shown). (H and I) In 12-week-old adults, no differences in PDGFR-��

OPCs or mature CC-1� cells were detected in corpus callosum (illustrated) or spinal cord. [Scale bars: A, 200 �m (Inset: 5 �m); G, 20 �m.]

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size in Olig1Cre:Notch112f/12f mice compared with controls at 14 dpi(Fig. 3), we focused on this time point in our analyses. Compatiblewith results of confocal imaging studies, light microscopy confirmedthat the lesion center in all genotypes was hypercellular anddemyelinated at 14 dpi (Fig. S3C) and contained hypertrophicastrocytes (Fig. S3D) and lipid-containing mononuclear phagocytes(Fig. S3E). Normal-appearing myelin was observed outside thelesion (Fig. S3F). Between the lesion center and normal-appearingwhite matter was a distinct border containing myelinated anddemyelinated axons (Fig. 4A) and axons wrapped by dispropor-tionately thin myelin characteristic of remyelination (1, 4) (Fig. 4B).

Examination of serial grids by electron microscopy confirmed thepresence of myelinated, demyelinated, and remyelinated axons atthe lesion border at 14 dpi in all genotypes (Fig. 4 C and D).However, remyelinated axons appeared more numerous inOlig1Cre:Notch112f/12f mice than in controls (Fig. 4C). A highfrequency of paranodal terminal loops (suggesting short internodes,and hence remyelination) (14) was also noted in these areas,particularly in Olig1Cre:Notch112f/12f mice (Fig. 4E). To quantifynumbers of remyelinated axons, we calculated myelin thickness andG ratio for individual fibers within the lesion border (at least 200per genotype at 14 dpi, see Materials and Methods). In all genotypes,the border contained axons with G-ratio values above the normalrange (G ratio �0.69), indicative of disproportionately thin myelin,and thus remyelination. These axons were significantly more abun-dant in Olig1Cre:Notch112f/12f mice than in littermate controls (Fig.4F; 22.2% of axons vs. 12.5–14.3% in other genotypes; P � 0.01,ANOVA plus Bonferroni posttest). These data showed that at 14dpi, remyelination was more extensive in Olig1Cre:Notch112f/12f

mice than in controls.

DiscussionIn this study, we have used a Cre-lox model to investigate the roleof Notch1 in remyelination in vivo. Because early OPCs areimportant targets of Notch signaling (14–16), we crossed micecarrying a floxed Notch1 allele (Notch112f) with animals carryingCre expressed under regulatory sequences of Olig1, known to beexpressed in these cells (18, 19). In Olig1Cre:Notch112f/12f mice,OPC differentiation in the developing CNS and remyelination inadults were accelerated, at the expense of OPC proliferation (Figs.2–4, Fig. S2, and Fig. S3). These studies were supported byexperiments in vitro, which confirmed that Notch1 signaling waspermissive for OPC proliferation and restricted differentiation andmyelin formation (Fig. 1). In addition, we found that astrocytesexposed to TGF-�1 restricted OPC maturation via Jagged1-Notch1signaling (Fig. S1).

In developing CNS in the Olig1Cre:Notch112f/12f genotype, theOPC population was more mature than in controls at both E16.5and P14, although similar to CnpCre:Notch1fl/fl mice, but (unlikeNotch1�/� mice) these effects were not associated with changes inMBP immunoreactivity (15, 16). At E16.5, lumbar spinal cord ofOlig1Cre:Notch112f/12f mice contained more MAG� oligodendro-cytes than in littermate controls, whereas the corpus callosum alsocontained greater numbers of mature CC-1� cells inOlig1Cre:Notch112f/12f animals at P14. This developmental pheno-type suggested 2 potential mechanisms for the accelerated lesionrepair detected in Olig1Cre:Notch112f/12f adults. In one, the alteredbalance between OPC differentiation and proliferation would favorOPC maturation following demyelination. In the other, potentiatedOPC differentiation during development would give rise to an

Fig. 3. Accelerated resolution of demyelinating lesions in Olig1Cre:Notch112f/12f mice. Experimental mice and controls (12 weeks old) were subjected tostereotactic microinjection of 1.5 �L of 1% lysolecithin into corpus callosum and then killed at 7–28 dpi (at least 7 mice per genotype per time point). Serial 50-�msections were immunostained for myelin (MBP), lineage markers, and BrdU. Serial Z-series stacks were rendered in 3 dimensions (A), also visualized as projectionsin 2 dimensions (B), and quantified as in the text. Results are shown for 14 dpi; data for 7 dpi are in Table S1. (A and B) Lysolecithin injection resulted indemyelination within corpus callosum. (B and C) At 14 dpi, measurements of maximum cross-sectional MBP� area were significantly smaller in Olig1Cre:Notch112f/

12f mice than in controls (C) (*, P � 0.05, ANOVA plus Bonferroni posttest). (A) Rostrocaudal lesion size was also smaller in Olig1Cre:Notch112f/12f mice. (D) Inaddition, a smaller percentage of Olig2� cells were mitotic (BrdU�) in the hypercellular lesion area in Olig1Cre:Notch112f/12f mice than in controls (D) (**, P �0.01). (E) No difference was observed in BrdU� GFAP� cells. (F) At 14 dpi, Jagged1 was expressed in MBP� areas of corpus callosum in all genotypes by GFAP�

reactive astrocytes. (Scale bars: A and B, 50 �m; E, 5 �m.)

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increased number of mature oligodendrocytes in adults, and hencegreater capacity for myelin repair. However, we found that thenumber and maturation state of oligodendrocytes in the CNS ofadult Olig1Cre:Notch112f/12f mice resembled those of other geno-types, suggesting that the developmental phenotype inOlig1Cre:Notch112f/12f animals left no lasting effect on the oligo-dendrocyte population in adults in the absence of challenge. Thus,the potentiated remyelination observed in Olig1Cre:Notch112f/12f

mice likely resulted from maturation within the OPC pool existingat the time of demyelination.

Olig1 is detected in murine spinal cord and ventral telencephalonfrom E8.5, preceding other oligodendrocyte precursor markers(19), and Olig1Cre was chosen for the current study for this reason.This approach differed from that of previous reports, which usedPLPCreERT2:Notch1fl/fl animals and did not observe strong effectson remyelination (12). Proteolipid protein (PLP) is the majorprotein of myelin, and ROSA26 studies have shown PLPCreERT2-driven recombination to be most widespread in regions undergoingmyelin formation (28), although a subpopulation of �-gal� cells isalso found in the laterobasal plate of diencephalon as early as E9.5

Fig. 4. Olig1Cre:Notch112f/12f mice display potentiated remyelination. (A and B) Light microscopy of epoxy-embedded sections stained with toluidine blueshowing representative 14 dpi lysolecithin lesions in an experimental Olig1Cre:Notch112f/12f mouse (Right) and an Olig1Cre:Notch�/[supi]12f littermate control(Left). (A) Between the demyelinated lesion center (Top) and myelinated white matter (Bottom), the lesion border (Middle) contained both myelinated anddemyelinated axons. Representative areas of the lesion border (A, rectangles) are illustrated at higher magnification (B, Upper). Compare with adjacent whitematter, outlined by squares in A and also shown in B (Lower). (B) Axons wrapped by disproportionately thin myelin characteristic of remyelination were observedat the lesion border in all genotypes (arrowheads) but appeared more abundant in Olig1Cre:Notch112f/12f mice. (C–E) Electron microscopy of serial grids of lesionsillustrated in A. Histological features of the lesion border included demyelinated axons (C, asterisks) and reactive astrocytes (A). (C) Axons wrapped by abnormallythin myelin characteristic of remyelination (arrowheads) appeared more abundant in Olig1Cre:Notch112f/12f mice. (D) Normally myelinated (Myel.) axons (Left)are compared with remyelinated (Remy.) axons (Right) from an Olig1Cre:Notch112f/12f mouse. (E) Illustration of a paranode (arrow) containing septate junctionsfrom an area of remyelination in Olig1Cre:Notch112f/12f at 14 dpi. Remyelinated internodes are disproportionately short (4); thus, paranodes are more frequentin remyelinated areas. (F) Results of morphometry to quantify remyelination at 14 dpi (see Materials and Methods). In all genotypes, the lesion border containedaxons with a G ratio above the range observed in unlesioned corpus callosum (G ratio �0.69; Table S2), indicative of remyelination. In Olig1Cre:Notch112f/12f mice,these axons were more abundant than in littermate controls of other genotypes (F) (**, P � 0.01, ANOVA plus Bonferroni posttest). [Magnification: A, �400;B, �1,000; C, �1,500; D, �4,000; E (Left), �8,000; E (Right), �20,000.]

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(29). Based on our findings, we suspect that inclusion of earlyoligodendrocyte progenitors as targets of Notch1 inactivation mayhave been significant in determining the phenotype we observedfollowing CNS demyelination.

Factors that include IGF-1 (8) and hyaluronan (9) are known toregulate OPC differentiation during remyelination, and recentwork has also shown that �-catenin–Tcf4–mediated Wnt signalinginhibits OPC maturation and impedes repair of demyelinatedlesions (10). Our results suggest that canonical Notch1 signaling isan additional mechanism that regulates OPC differentiation duringCNS remyelination. The downstream targets of these pathways andtheir potential interactions are notable areas for future research,with the relation between Wnt and Notch being of particularinterest. Canonical signaling via both the Wnt and Notch pathwaysrestricts OPC maturation during development and in adults (10, 15,30), and activation of both has been demonstrated in MS plaques(10, 11), whereas noncanonical Notch signaling in MS has also beenreported (31). The roles of Notch and Wnt in regulating thetranscription factors that control OPC maturation, and whethertheir effects converge, interact, or exist in parallel, are compellingquestions for future investigation.

Effective approaches for potentiating myelin repair require anunderstanding of mechanisms governing both the size and differ-entiation state of the OPC population in lesions of different types.In early MS, progenitors are believed to be present in significantnumbers in at least some lesions (1); thus, blockade of pathwaysinhibiting their maturation may promote myelin formation. Con-versely, later in the disease course when OPC numbers may belimiting (32), activation of these pathways may initially be beneficialto expand the progenitor pool for subsequent differentiation. Ourfindings suggest that Jagged1-Notch1 signaling is one of the path-ways regulating the balance between OPC proliferation and mat-uration in the adult CNS and, moreover, that this pathway issensitive to activation by inflammatory insult mediated specificallyvia the cytokine TGF-�1. In patients with demyelinating disease, wesuggest that manipulation of Notch1 signaling may represent ameans of regulating the characteristics of the OPC pool, and hencethe potential for remyelination.

Materials and MethodsMice. Olig1Cre mice were generated by Q. Richard Lu (University of TexasSouthwestern, Dallas, TX) (19), Charles Stiles (Harvard University, Cambridge,

MA), and David Rowitch (University of California, San Francisco, CA). Notch112f

mice were generated by Changhui Ge and Pamela Stanley (Albert EinsteinCollege of Medicine, Bronx, NY) (18). Olig1Cre:Notch1�/12f mice were crossedwith Notch12f/12f animals to generate experimental Olig1Cre:Notch112f/12f miceand controls. Genotyping was carried out as in Fig. S1 using primer sequencesavailable on request. ROSA26 reporter mice were generated by Philippe Soriano(Hutchinson Cancer Center, Seattle, WA) (27). CNS tissue sections (20 �m) from4-week-old Olig1Cre:ROSA26�/fl mice were stained with X-gal and counter-stained for Olig2.

Immunohistochemistry of Demyelinating Lesions. Resolution of demyelinatinglesions in 12-week-old mice was quantified in 10� Z-series stacks through thethickness of serial 50-�m cortical sections in at least 7 animals per genotype pertime point. Serial stacks were rendered in 3 dimensions using Volocity v4.0(Improvision) and also visualized as projections in 2 dimensions. Cross-sectionalMBP� area was measured in each stack using ImageJ v1.41 (National Institutes ofHealth) and rostrocaudal size of MBP� area estimated in serial stacks. Cellspositive for BrdU and lineage markers were quantified within the hypercellularlesion area.

Histopathology of Demyelinating Lesions. For light microscopy, 1-�m epoxysections of corpus callosum were stained with toluidine blue and examined andphotographed on a Zeiss Axioplan microscope. For electron microscopy, serialepoxy-embedded sections were placed on slotted or 200-mesh copper grids,contrasted with lead citrate and uranyl acetate, and scanned in a Hitachi HS600electron microscope. For quantitation of myelin thickness and G ratio (23),random fields of unlesioned corpus callosum or lysolecithin lesion border (seetext) were captured by light microscopy at �100 (at least 5 fields per animal andat least 4 mice per genotype) and axon and fiber diameter were measured for atleast 10 random axons per field by a blinded observer using ImageJ v1.41. Myelinthickness was calculated as (fiber diameter � axon diameter)/2, and G ratio wascalculated as (axon diameter)/(fiber diameter) (23). For further details, see SIMethods.

ACKNOWLEDGMENTS. We thank Dr. Brad Poulos Human Fetal Tissue Resourceat Albert Einstein College of Medicine (AECOM)] for tissue collection and Dr.Pamela Stanley at AECOM for helpful discussions. This study was supported byU.S. Public Health Service Grants NS046620 and NS056074 (to G.R.J.), NS011920and NS08952 (to C.S.R.), NS040511 (to D.H.R.), NS050389 (to Q.R.L), NCI95022 toPamela Stanley (C.G.), and CA095823 (to Mount Sinai School of Medicine Micros-copy). It was also supported by National Multiple Sslerosis Society Grants FG1739(to Y.Z.) and RG3874 (to G.R.J.); Grants RG1001-K11 (to C.S.R.), RG3827 (to C.F.B.),and RG3978 (to Q.R.L.); and Grant CA1022 (to D.H.R., C.F.B., C.S.R., and G.R.J.) andby the Jayne and Harvey Beker Foundation (to G.R.J) and Wollowick Foundation(to C.S.R).

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