INTRODUCTION

Oligodendrocytes are the myelinating macroglial cellsdistributed in all regions of the central nervous system (CNS).Despite their wide distribution throughout the entire CNS,recent studies have indicated that oligodendrocytes are derivedfrom a restricted domain of neuroepithelium in the ventral CNS(Warf et al., 1991; Noll and Miller, 1993) under the influenceof sonic hedgehog (Shh) signaling (Trousse et al., 1995; Poncetet al., 1996; Pringle et al., 1996; Orentas et al., 1999). In thespinal cord region, expression of early oligodendrocyte genes,such as Pdgfra, Sox10,Olig1, Olig2, Nkx2.2, Plp and O4antigen, is initially confined to the ventral neuroepitheliumadjacent to the Shh-expressing floor plate (Pringle andRichardson, 1993; Yu et al., 1994; Spassky et al., 1998; Onoet al., 1995; Xu et al., 2000). Soon after oligodendrocyteprogenitor cells (OLPs) are generated from the ventralventricular zone, they migrate into the surrounding gray and

white matter regions where they undergo rapid proliferationprior to their terminal differentiation (Barres and Raff, 1994;Ono et al., 2001; Xu et al., 2000).

Despite the consensus view on the ventral origin ofoligodendroyctes, the precise site of oligodendrocyte generationin the spinal cord remains under intense investigation. In therodent spinal cord, expression of the oligodendrocyte markergene Pdgfra is initially mapped to the lower region of the Pax6gradient but dorsal to the Nkx2.2domain (Sun et al., 1998). Thisdomain corresponds to the motoneuron precursor domain (pMNdomain) which lies dorsal to the Nkx2.2+p3 domain but ventralto the Irx3+Nkx6.1+p2 domain (Briscoe et al., 1999; Briscoeet al., 2000). Thus, it is believed that motoneurons andoligodendrocytes are generated from the same neuroepithelialdomain but during different time windows (Richardson et al.,1997; Richardson et al., 2000; Spassky et al., 2000). In supportof this hypothesis, expression of two novel oligodendrocyte-specific genes, Olig1and Olig2, also appears to be mapped to

681Development 129, 681-693 (2002)Printed in Great Britain © The Company of Biologists Limited 2002DEV4587

In this study, we have investigated the relationship ofOlig2+ and Nkx2.2+oligodendrocyte progenitors (OLPs)by comparing the expression of Olig2 and Nkx2.2 inembryonic chicken and mouse spinal cords before andduring the stages of oligodendrogenesis. At the stages ofneurogenesis, Olig2 and Nkx2.2 are expressed in adjacentnon-overlapping domains of ventral neuroepithelium.During oligodendrogenesis stages, these two domainsgenerate distinct populations of OLPs. From the Olig2+motoneuron precursor domain (pMN) arise the Olig2+/Pdgfra+ OLPs, whereas the Nkx2.2+p3 domain give riseto Nkx2.2+ OLPs. Despite their distinct origins, bothpopulations of OLPs eventually appear to co-express Olig2and Nkx2.2 in the same cells. However, there is a species

difference in the timing of acquiring Nkx2.2expression bythe Olig2+/Pdgfra+OLPs. The co-expression of Nkx2.2andOlig2 in OLPs is tightly associated with myelin geneexpression in the normal and PDGFA–/– embryos,suggesting a cooperative role of these transcription factorsin the control of oligodendrocyte differentiation. In supportof this suggestion, inhibition of expression of thesetwo transcription factors in culture by antisenseoligonucleotides has an additive inhibitory effect on OLPdifferentiation and proteolipid protein (PLP) geneexpression.

Key words: Oligodendrocyte progenitors, Embryonic origins,Nkx2.2, Olig2, Expression, Pdgfamutant, Antisense inhibition

SUMMARY

Dual origin of spinal oligodendrocyte progenitors and evidence for the

cooperative role of Olig2 and Nkx2.2 in the control of oligodendrocyte

differentiation

Hui Fu 1,*, Yingchuan Qi 1,*, Min Tan 1,*, Jun Cai 1, Hirohide Takebayashi 2, Masato Nakafuku 3,William Richardson 4 and Mengsheng Qiu 1,†

1Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, KY 40292, USA2Department of Pathology and Tumor Biology Graduate School of Medicine, Kyoto University Konoe-chou, Sakyo-ku, Kyoto 606-8501, Japan3Department of Neurobiology, University of Tokyo, Graduate School of Medicine, 7-3-1 Hongo, Bunkyoku, Tokyo 113-0033, Japan4Wolfson Institute for Biomedical Research, The Cruciform Building, University College London,Gower Street, London WC1E 6AE,UK*These authors contributed equally to this work†Author for correspondence (e-mail: m0qiu001@louisville.edu)

Accepted 1 November 2001

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the pMN domain at early stages (Lu et al., 2000; Zhou et al.,2000; Takebayashi et al., 2000).

In recent studies of the developing chicken spinal cord, otherinvestigators have found results that are not entirely supportiveof this hypothesis, and a different site of origin ofoligodendrocytes has been suggested. In the chicken embryos,expression of early markers of the oligodendrocyte lineage, suchas Pdgfra and O4 antigen, was initially detected in the Nkx2.2+neuroepithelium (Xu et al., 2000; Soula et al., 2001). The OLPsthat migrate away from the ventricular zone retain the Nkx2.2expression and gradually acquire expression of lateoligodendrocyte markers such as GalC and proteolipid protein(PLP) (Xu et al., 2000). Based on these observations, it wasproposed that oligodendrocyte progenitors could originate fromthe Nkx2.2+p3 domain (Xu et al., 2000; Soula et al., 2001), andthat oligodendrocytes and motoneurons may not share the samelineage in embryonic chicken spinal cord (Soula et al., 2001).

These contradictory observations in mouse and chickenspinal cords have raised several important possibilities on theorigin and lineage of oligodendrocyte progenitors in the spinalcord. One possibility is that oligodendrocyte progenitors mayarise from different neuroepithelial domains in rodents andbirds. It is conceivable that OLPs originate from the pMNdomain in mammals, but from the more ventral p3 domainin avians. The second possibility is the dual origin ofoligodendrocytes, i.e. that two distinct populations ofoligodendrocyte progenitors arise from distinct sites ofneuroepithelium in the same spinal cord tissue. It is possiblethat the Olig2+progenitor cells might originate from the pMNdomain, whereas the Nkx2.2+ progenitors could be generatedfrom the ventral p3 domain. Finally, it is also possible that theOlig2+ progenitor cells and the Nkx2.2+ progenitors mayrepresent the same population of progenitors that arise from amerged region of the Nkx2.2and Olig2domains at later stagesof spinal cord development.

To investigate these possibilities, including the relationshipof Olig2+ and Nkx2.2+ OLPs, we first compared theexpression of Olig2,Nkx2.2and other oligodendrocyte markersin embryonic chicken and mouse spinal cords. Our expressionstudies revealed that at early stages of spinal cord development,the Olig2+, Pdgfra+ and Sox10+ OLPs originate from thepMN domain of the ventral neuroepithelium in both mouse andchicken. Interestingly, this population of OLPs gains Nkx2.2expression before their migration in chicken, but aftermigration in mouse. In addition, the Nkx2.2+p3 domain canalso produce OLPs which are initially Nkx2.2+/Olig2–,but appear to gain Olig2 expression as they migrate anddifferentiate. At later stages of embryogenesis, nearly all OLPcells in the spinal cord parenchyma co-express the Nkx2.2andOlig2 transcription factors. The co-expression of Nkx2.2and Olig2 in OLPs precedes, and is necessary for, OLPdifferentiation and myelin gene expression. Inhibition ofexpression of these two transcription factors in dissociatedculture by antisense oligonucleotides has an additive inhibitoryeffect on OLP differentiation.

MATERIALS AND METHODS

Materials Fertilized chick eggs (White Horn, SPAFAS) were incubated at 38°C

in a humidified incubator and embryos were staged according to thecriteria set by Hamburger and Hamilton (Hamburger and Hamilton,1951). Anti-Nkx-2.2hybridoma supernatants were obtained from theDevelopmental Studies Hybridoma Bank (University of Iowa, IowaCity). The Alexa-488 or Alexa-594 conjugated secondary antibodieswere obtained from Molecular Probes.

In situ RNA hybridization Embryos from various stages of chicken development were fixed in4% paraformaldehyde at 4°C overnight. Tissue preparation and in situhybridization with digoxigenin-labeled riboprobes were performedaccording to Schaeren-Wiemers and Gerfin-Moser (Schaeren-Wiemers and Gerfin-Moser, 1993) with minor modifications.

Immunofluorescence and immounohistochemistrySpinal cord tissues from the thoracic or brachial regions were isolatedfrom day 3-12 chicken embryos, fixed in 4% paraformaldehyde andsectioned on a cryostat. For immunofluorescence, slides wereincubated with anti-Olig2polyclonal antibody (1:3000 dilution), anti-Nkx2.2 (1:10) or anti-GalC (5 µg/ml from Boeringer Mannheim)overnight at 4°C. Sections were then washed five times withphosphate-buffered saline (PBS), incubated with Alexa-488- orAlexa-594-conjugated secondary antibodies (50 µg/ml). Fluorescentimages were collected by Nikon epifluorescence microscope. Thecombined immunohistochemistry and in situ hybridization waspreviously described in Schaeren-Wiemers and Gerfin-Moser(Schaeren-Wiemers and Gerfin-Moser, 1993)

Antisense treatment in dissociated chicken spinal cordculture Spinal cord tissues were isolated from day 5 chicken embryos in1×PBS, minced and then physically dissociated by repeated pipettingwith fine-tip plastic pipettes. The dissociated cells were subsequentlygrown on poly-L-Lysine coated cover-glass in DMEM + 5%fetal bovine serum +N2 supplement (Gibco) for 4-5 hours at 37°Cfollowed by oligonucleotide treatment, which PAGE-purified sense(for Nkx2.2) or antisense (for Nkx2.2or Olig2) phosphorothiateoligodeoxynucleotides (p-ON, from Intergrated DNA Science) wasadded to culture medium at the final concentration of 1 µM each. Theoligonucleotide sequences are as follows: Nkx2.2 sense (5′-GCTGTTCAGACGCTGCCT-3′), Nkx2.2antisense (5′-AGGCAGC-GTCTGAACAGC-3′) and Olig2 antisense (5′-TCATCTGCTTC-TTGTCCT-3′).

Five days after treatment, cells were fixed for 10 minutes in 4%paraformaldehyde, washed twice with PBS and blocked with 5% goatserum. The coverslips were then incubated overnight with anti-GalChybridoma supernatant overnight at 4°C. After several washes withPBS, Alexa-594-conjugated goat secondary antibodies and DAPIwere applied for 1 hour at room temperature. The coverslips were thenwashed three times with PBS and mounted for immunofluorescentdetection. The GalC+cells and total cell numbers were scored underNikon epifluorescent microsope. For each score, three coverslips havebeen used and at least 10 fields and about 2000-3000 cells have beencounted for each coverslip. Only the relative number of GalC+ cellsfrom various treatments was plotted, with the Nkx2.2sense control as100.

The effects of antisense treatments on PLP expression wereassessed by in situ hybridization with Plp riboprobe as described bySchaeren-Wiemers and Gerfin-Moser (Schaeren-Wiemers and Gerfin-Moser, 1993). PLP+ cells were counted under light microscope andthe number of positive cells from each 10 field (n=3) was plotted.

Dissociated cell culture of mouse embryonic spinal cordE13.5 mouse spinal cords were bisected into the dorsal and ventralexplants which were dissociated and cultured separated in NEP basalwith 35 ng/ml basic fibroblast growth factor (FGF) for 2 days or 5days. Cells were then fixed in 2% paraformaldehyde and processed

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683Nkx2.2 and Olig2 in oligodendrocyte progenitors

for double immunostaining with anti-Nkx2.2 monoclonal antibody(1:10) and anti-Olig2(1:3000) (Takebayashi et al., 2000) polyclonalantibody as previously described (Rao et al., 1998; Qi et al., 2001).

RESULTS

Pdgfra+ and Sox10+ oligodendrocytes arise from theOlig2 domain in chicken Previous studies in rodents have demonstrated that OLPs areproduced from Olig2+pMN domain which is dorsal to theNkx2.2+p3 domain in the spinal cord (Sun et al., 1998; Luet al., 2000; Zhou et al., 2000; Takebayashi, 2000). However,oligodendrocytes appear to be generated from the moreventral Nkx2.2+p3 domain in chicken (Xu et al., 2000; Soulaet al., 2001). To investigate this potential species differencein the origin of oligodendrogenesis, we examined in detailthe expression of two early oligodendrocyte marker genes,Sox10 and Pdgfra, in relation to that of Olig2and Nkx2.2inchicken.

During neurogenesis, Olig2 is precisely expressed in thepMN domain dorsal to the Nkx2.2+ p3 domain duringneurogenesis (Fig. 1A). In E3 and E4 chick spinal cord, theMNR+ motoneurons are exclusively produced from the entireOlig2+ domain of neuroepithelium (Fig. 1B,C). Comparisonof the expression of Olig2 withSox10 or Pdgfra onimmediately adjacent sections from E7-9 chick spinal cordrevealed that Sox10+and Pdgfra+OLPs are situated within orimmediately adjacent to the Olig2domain of neuroepithelium(Fig. 1D-G). Thus, the Pdgfra+and Sox10+oligodendrocyteprogenitors originate from the Olig2+motoneuron precursordomain in chicken.

Upregulation of Nkx2.2 in Olig2domain in embryonic chickenspinal cordIntriguingly, when E7 chicken spinalcord was simultaneously stained withNkx2.2and Sox10or Pdgfra, we found

that expression of these two OLP markers falls into theNkx2.2+ neuroepithelium (Fig. 1H,I), instead of being dorsalto the Nkx2.2+ cells. One possible explanation is that Nkx2.2expression is upregulated in the Olig2 domain beforeoligodendrogenesis. To examine this possibility, weperformed double labeling experiments with Olig2and Nkx2.2in chicken spinal cord during the crucial stages ofoligodendrogenesis in chick. Spinal cord sections preparedfrom E3 to E12 chicken embryos were subjected to Olig2insitu hybridization followed by anti-Nkx2.2immunohistochemistry. At E3-4, Olig2and Nkx2.2expressionis restricted in adjacent non-overlapping domains ofneuroepithelial cells, with each domain spanning a width offour to five cell bodies (Fig. 1A, Fig. 2A). At E5, Nkx-2.2expression appears to expand dorsally into the Olig2domain,whereas both the width and the position (relative to the floorplate) of the Olig2domain remain relatively unchanged (Fig.2B). By E6, the entire Olig2domain gained a weak expressionof Nkx2.2. A few Nkx2.2+but Olig2-progenitor cells start tomigrate away from the Nkx2.2+/Olig2-domain (p3 domain),first ventrally and then dorsally (Fig. 2C). From E7 to E10,the p3 domain gradually moves down along the floor plate asthe central canal decreases and the floor plate elongates (Fig.2D-G). As a result, the original pMN domain is concomitantlydragged downwards along the central canal (compare Fig. 2D-G with the p3 domain). At E10, cells in both the pMN domainand p3 domain are rapidly decreased. In the ventricle, only asmall number of Olig2+/Nkx2.2+neuroepithelial cells remainassociated with the reduced central canal (Fig. 2G). By E12,expression of Olig2and Nkx2.2is nearly completely absent inthe ventricular cells of the spinal cord (Fig. 2H).

Fig. 1.Pdgfra+ and Sox10+OLPs aregenerated from the Olig2+pMN domain ofventral neuroepithelium in chicken. (A) E3chicken spinal cords were double-labeledwith anti-Nkx2.2(brown) and Olig2(blue).(B,C) E3-4 chicken spinal cords were double-stained with anti-Mnr2(brown) and Olig2(blue). Mnr+motoneurons are produced fromthe Olig2+domain dorsal to the Nkx2.2domain. (D-G) Immediately adjacent chickenspinal cord sections from E7 (D), E8 (E,G)and E9 (F) were subjected to in situhybridization with Pdgfra, Sox10or Olig2riboprobes. The ventral half from one side ofthe stained spinal cord was aligned with thatfrom the same side of the adjacent cord.(H,I) Spinal cord from E7 chicken embryoswere double-labeled with anti-Nkx2.2(brown) and Sox10(blue in H) or Pdgfra(blue in I). Sox10and Pdgfraexpression islocated in the dorsal region of the Nkx2.2+neuroepithelium (arrows).

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Co-expression of Nkx-2.2 and Olig2 in migratorychicken oligodendrocyte progenitorsSince the Olig2domain appears to gain expression of Nkx2.2before the onset of migration of Sox10+, Pdgfra+and Olig2+progenitors, it is expected that these migratory OLP cells in thesurrounding regions would co-express Nkx2.2. Starting aroundE6-E7, a few Olig2+OLP start to migrate ventrolaterally (Fig.2C,D). As development proceeds, an increasing number ofOlig2+ migratory OLP cells was detected in the surroundinggray and white matter. Closer examination revealed that atthese stages, all migratory Olig2+progenitors in thesurrounding regions co-express Nkx2.2. However, many of theNkx2.2+ progenitors in the gray matter, especially thoseadjacent to the ventricle and the floor plate, do not expressOlig2 (Fig. 2C,D,F,G,J). These Nkx2.2+/Olig2–progenitorsare generated slightly earlier than Olig2+OLPs and migratefirst ventrally and then dorsolaterally (Fig. 2C-D). Thispopulation of OLPs is likely to be produced from the p3domain.

Interestingly, in the gray and white matter region furtheraway from the ventral ventricular zone, the proportion ofNkx2.2+/ Olig2– is decreased with time, whereas thepercentage of Nkx2.2+/Olig2+cells is increased. By E12,nearly all Nkx2.2+progenitor cells are positive for Olig2(Fig.2H). Moreover, the staining intensity of Olig2 also appearsto become stronger at E8 and later stages (Fig. 2I-L). Theincreasing intensity of Olig2expression, together with the

observation that an increasing percentage of Nkx2.2+progenitors express Olig2, strongly suggests that the Nkx2.2+OLPs may gain expression of Olig2during the process ofmigration and maturation.

Upregulation of Olig2 in Nkx2.2 oligodendrocyteprogenitors in embryonic chicken hindbrainIn search of evidence for the capability of migratory Nkx2.2+OLPs to gain Olig2 expression, we performed a similar doublelabeling experiment on other regions of the CNS, includingthe hindbrain, midbrain and forebrain, at the critical stages ofoligodendrogenesis. Transverse sections of the brain tissuesprepared from E6-8 chicken embryos were double-labeledwith Nkx2.2and Olig2. We found that in the hindbrain region,many Nkx2.2+OLPs acquired Olig2expression during theirmigration process. Intriguingly, in this region, only Nkx2.2 isinitially expressed in the ventral neuroepithelium at the onsetof oligodendrogenesis (Fig. 3A-D). Olig2 is not expressed inthe ventricular zone dorsal to the Nkx2.2domain as is seen inthe spinal cord (Fig. 3A-D). However, Olig2 expression isclearly detected in groups of Nkx2.2+cells in thesubventricular zone and in individual migratory cells at E6-E7(arrows in Fig. 3B,D). By E8, Nkx2.2+cells are dispersed intosurrounding regions, where many Nkx2.2 cells co-expressOlig2. However, a few Nkx2.2+ migratory cells adjacent tothe ventral midline remain Olig2negative (arrowheads in Fig.3F). These results provide further evidence that Nkx-2.2+

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Fig. 2.Double labeling of Olig2 and Nkx-2.2from E4 to E12. Spinal cord sections from E4 (A), E5 (B), E6 (C), E7 (D), E8 (E,I), E9 (F,J,K),E10 (G,L) and E12 (H) were subjected to Nkx-2.2immunohistochemical staining (in brown) followed by in situ hybridization (in blue) withOlig2 riboprobe. Only the ventral half of spinal cords are shown. In A, anti-Nkx2.2immunostaining and Olig2 in situ hybridization wereperformed on immediately adjacent slides, and half of the ventral cord is aligned closely for comparison. In B, the overlapping region of theOlig2 domain and Nkx2.2domain is indicated. The Olig2+/Nkx2.2+cells and Olig2–/Nkx2.2+ cells are represented by arrows and arrowheads,respectively. I is a higher power view of F. J,K are higher power views of F. L is a higher power view of G.

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oligodendrocyte progenitors could acquire Olig2 expressionduring the process of migration and proliferation.

Pdgfra+ oligodendrocyte progenitors arise from theOlig2 domain in mouse To investigate whether the origin of oligodendrogenesis isconserved between mouse and chicken, we re-examinedthe expression relationship of early OLP markers withOlig2, Nkx2.2 and other neural identity genes. Consistentwith previous suggestions (Takeyabashi et al., 2000),we confirmed that during neurogenesis stages, Olig2 isprecisely expressed in the pMN domain, flanked ventrally byNkx2.2 expression and dorsally by Irx3expression (Fig.4A,B) (Briscoe et al., 2000). However, unlike in chicken,Nkx2.2 is not upregulated in the Olig2 domain at the onsetstage of oligodendrogenesis. At E12.5, Olig2 and Nkx2.2arestill expressed in the adjacent domains of ventralneuroepithelium with no or little overlapping (Fig. 4C). Atthis stage, Pdgfra+ OLPs are exclusively born from theOlig2+ domain, as expression of Pdgfrais confined withinor immediately adjacent to, the Olig2+neuroepithelium(Fig. 4D-E), dorsal to the Nkx-2.2+neuroepithelial cells(Fig. 4F).

Nkx2.2 domain and Olig2domain are merged in mouse atlater stages ofoligodendrogenesis Although Nkx2.2is not upregulated inthe Olig2+ neuroepithelium at theearly stage of oligodendrogenesis, it ispossible that these two domains aremerged at later stages. To examine thispossibility, we performed detailedexpression studies with Nkx2.2andOlig2 on spinal cord sections preparedfrom E12.5 to E14.5 mouse embryosby in situ hybridization. As describedabove, at E12.5, Olig2 and Nkx2.2areexpressed in adjacent non-overlappingdomains. At this stage, a few Nkx2.2+cells start to migrate out of the p3domain into the adjacent gray matter(Fig. 5A,B). At E13.5, the Olig2+OLPs start to migrate away from thepMN domain into the dorsal andventral spinal cord. The number ofNkx2.2+ OLPs in the ventral graymatter surrounding the p3 domain is

also increased (Fig. 5C,D). At the same time, the Nkx2.2expression in the neuroepithelial cells starts to expand dorsallyinto the Olig2domain, similar to the observations in embryonicchicken spinal cord. At E14.5, Olig2 domain and Nkx2.2domain are almost completely merged (Fig. 5E,F). Thus, thedorsal expression of Nkx2.2into the pMN domain occurs abouttwo days after OLPs start to migrate out.

Upregulation of Nkx2.2 expression in the Olig2+migratory OLPs in embryonic mouse spinal cordContrary to the embryonic chicken spinal cord, Nkx2.2is notco-expressed in the Olig2+migratory OLPs in mouse duringthe early stages of oligodendrogenesis. By E14.5 when thepMN domain expresses Nkx2.2, Olig2+progenitor cells havealready spread into the entire spinal cord (Fig. 5C-F). AlthoughNkx2.2is initially not expressed in these Olig2+OLPs, it isconceivable that this population of OLPs could acquire Nkx2.2expression at later stages of oligodendrocyte development, assuggested by the co-expression of Olig2 and Nkx2.2in chickenOLPs that are derived from the pMN domain. To examine thispossibility, we performed Nkx2.2in situ hybridization onpostnatal day one (P1) mouse spinal cord sections followed byanti-Olig2 immunohistochemistry. As shown in Fig. 5G,H,

Fig. 3.Double staining for Olig2andNkx2.2in the hindbrain. Sections from E6(A,B), E7 (C,D) and E8 (E,F) hindbraintissues were subjected to anti-Nkx-2.2immunostaining (in brown) followed byOlig2 in situ hybridization (in blue). Olig2is only expressed in groups of migratoryNkx2.2+ OLP cells, but not in theventricular cells. The representativedouble-positive cells are indicated byarrows, whereas the Nkx2.2+/Olig2-cellsare represented by arrowheads.

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most, if not all, of the Olig2+ cells are also positive forNkx2.2(arrows), although expression of Nkx2.2in someOlig2+ cells is fairly weak (arrowheads), probably becausethey are in the early phase of gaining Nkx2.2expression.Similarly, all Nkx2.2+cells are also immunoreactive foranti-Olig2.

To test further whether Olig2+OLPs can gain Nkx2.2expression after migration, spinal cords from E13.5 mouseembryos were bisected and the dorsal halves were used fordissociated cell culture. At this stage, Nkx2.2+ OLPs arerestricted to the ventral half (Fig. 5C,D), but the Olig2+OLPs have already migrated into the dorsal cord. Two hoursafter dissociation, no Olig2+/Nkx2.2+double positive OLPcells were observed from the dorsal culture (data notshown). However, after 2 days in vitro (2 DIV) culture,about 45% of Olig2+ cells (n=60) became immunoreactiveto Nkx2.2although Nkx2.2expression in many of these cellswas relatively weak (Fig. 6A-C). By 5 DIV, the percentageof Nkx2.2+ cells in Olig2+ OLPs increased to 85%(n=130), and the intensity of Nkx2.2immunostaining alsoappeared to be increased (Fig. 6D-F). These experimentsprovide direct evidence that in rodents, the Olig2+ OLPscan acquire Nkx2.2expression after they migrate away fromthe ventricular zone.

To summarize our expression analyses, we foundthat Pdgfra+, Sox10+ and Olig2+ OLPs are producedexclusively from the Olig2+pMN domain in both mouseand chicken. The Nkx2.2+p3 domain can also produce

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Fig. 4.Pdgfra+ OLPs are generated fromthe Olig2+pMN domain in mouse.(A,B) Sections from E10.5 mouse spinalcord were subjected to in situ hybridizationwith Nkx2.2/Olig2(B) or Olig2/Irx3(A)probes. Nkx2.2expression is flanked byNkx2.2ventrally and Irx3dorsally. (C)Double labeling of E12.5 spinal cord withanti-Nkx2.2(green) and anti-Olig2(red)antibodies by immunofluorescence.(D-F) E12.5 spinal cord were double-labeledby in situ hybridization with Olig2/Pdgfra(D,E) or Nkx2.2/Pdgfra(F). D,E are fromtwo separate embryos. Pdgfra expression isindicated by arrows.

Fig. 5.(A-E) Merging of the Olig2domain and Nkx2.2domain inmouse after the onset of emigration of Olig2+OLPs. Sectionsfrom E12.5 (A,B), E13.5 (C,D) and E14.5 (E,F) were labeled byNkx2.2(left half in A,C,E), Olig2(right half in A,C,E) or both(B,D,F). Nkx2.2expression in the ventricular zone starts to expanddorsally at E13.5 and almost overlaps with the entire Olig2domainat E14.5. (G-H) Co-expression of Olig2 and Nkx2.2in OLPs in P1mouse spinal cord. Spinal cord sections were double-stained withanti-Olig2(in brown) and Nkx2.2(in blue). Although double-labeled cells are present throughout the entire cord, pictures weretaken only from the lateral (G) and ventral (H) regions. Olig2+cells with strong Nkx2.2staining are represented by arrows, whilethose with weak Nkx2.2expression are represented by arrowheads.

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OLPs which are initially Nkx2.2+/Olig2-, but might gain Olig2expression as they migrate. The pMN domain gains Nkx2.2expression during oligodendrogenesis but at different timewindow in chicken and mouse. In chicken, Nkx2.2is upregulatedin the pMN domain before the migration of Olig2+ and Pdgfra+OLPs; however, in the mouse the upregulation occurs 2 daysafter the onset of OLP migration. Thus, the Olig2+ OLPs gainNkx2.2expression before their migration in chicken, but aftermigration in mouse.

Co-expression of Olig2 and Nkx2.2 is intimatelyassociated with myelin gene expression in thenormal and Pdgfa mutant animals Based on the dynamic expression of Olig2 and Nkx2.2inneuroepithelium and OLP cells, we hypothesize that there aretwo separate populations of OLPs at early stages of mousespinal cord development. The Olig2+/Pdgfra+/Nkx2.2-OLPsare produced from the pMN domain, whereas the Nkx2.2+/Olig2–/Pdgfra– OLPs are generated from the more ventralp3 domain. If our hypothesis on the dual origins of OLPs istrue, the number of these two OLP populations wouldbe differentially affected by the mutation of PDGFA, anoligodendrocyte mitogen required for proliferation of Pdgfra+OLPs (Fruttiger et al., 1999).

To examine this possibility, adjacent spinal cord sectionsfrom E13.5 embryos to P7 pups were examined for theexpression of Pdgfra, Olig2and Nkx2.2. At E13.5, numerousPdgfra+ and Olig2+ progenitor cells have already spread intothe ventral and dorsal spinal cord in the wild-type embryos (seeFig. 9A,B). However, only few Pdgfra+ and Olig2+cells aredetected within or adjacent to the ventricular zone in themutants (Fig. 7D,E). The drastic reduction in the number of

Pdgfra+ and Olig2+ OLPs in the mutants is also detectedthroughout the later stages of animal development (Fig. 7, Fig.8). The parallel delay and reduction of the Pdgfra+ cells andOlig2+ cells is consistent with our hypothesis that theyrepresent the same population of OLPs, and that Pdgfra islikely to be transiently expressed in Olig2+cells.

By contrast, the number and distribution of Nkx2.2+ cells arenot affected in the mutants at early stages. At E13.5 and E16.5,similar patterns of Nkx2.2expression in the ventral gray matterare observed in the wild-type and mutant embryos (Fig.7C,F,I,M). At P0, Nkx2.2expression in the ventral gray matteris not significantly affected in the mutants (Fig. 8C,D). However,the number of Nkx2.2+cells in the white matter is dramaticallyreduced (Fig. 8D). The parallel reduction of Pdgfra+/Olig2+OLPs and Nkx2.2+ OLPs in the white matter is consistent withour hypothesis that Olig2+/Pdgfra+ cells can acquire Nkx2.2expression after migration (Fig. 8C,G). At P7, expression ofNkx2.2is greatly reduced in the entire spinal cord, especially inthe white matter (Fig. 8K,O), similar to that of Pdgfraand Olig2.

We next investigated how the differential reduction oftwo populations of OLPs in the PDGFA mutants affectsoligodendrocyte differentiation and distribution using myelinbasic protein (MBP) as a marker. MBP expression can beobserved in the ventral gray matter of both normal and mutantembryos as early as E16.5 (Fig. 7J,N). The PDGFA mutationdoes not reduce MBP expression at this stage. In the wild-typeP0 animal, MBP is expressed in both gray matter and to a largerextent in the white matter (Fig. 8D). In the mutants, MBPexpression in the gray matter is more or less the same as in thewild type, whereas in the white matter, it is drastically reduced(Fig. 8H), similar to the preferential reduction of Nkx2.2expression in the white matter (Fig. 8H). By P7, expression of

Fig. 6.Co-expression of Olig2and Nkx2.2in dissociated spinal cord culture. The dorsal halves of E13.5 mouse spinal cords were isolated,dissociated and cultured on coverslips for 2 days (A-C) or 5 days (D-F). Cells were then simultaneously stained with anti-Nkx2.2(green) andanti-Olig2(red) by double immunofluorescence. The Olig2+ cells with strong Nkx2.2expression are indicated by arrows, while those withweak Nkx2.2expression are represented by arrowheads.

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MBP in the mutants is decreased in both the gray and whitematter (Fig. 8L,P).

Comparison of expression of MBP, Nkx2.2and Olig2in bothwild-type and mutant animals revealed that the expressionpattern of MBP appears to closely follow the overlapping regionof Nkx2.2 andOlig2, especially from E16.5 to P0. At P7, therelatively large number of MBP+ oligodendrocytes in themutants could be due to the slow but steady differentiation andaccumulation of oligodendrocytes in the white matter after birth.

Suppression of Nkx2.2 and Olig2 expression has anadditive inhibitory effect on oligodendrocytedifferentiationThe co-expression of Nkx2.2and Olig2 in OLPs in bothchicken and mouse suggests their important and perhapscollaborative role in the control of oligodendrocytedifferentiation. To examine this possibility, we tested theeffects of inhibition of their expression on oligodendrocytedifferentiation in culture by antisense approach. Dissociatedcells prepared from E5 chicken spinal cord were plated oncover slips at the same density and cultured for 5 days in thepresence of synthetic sense or antisense phosphorothiateoligonucleotides (at the final concentration of 1 µM for each)

derived from the chicken Nkx2.2or Olig2 sequences. Cellswere then examined for GalC expression (Ranscht et al., 1982;Bansal et al., 1989) by immunofluorescence or PLP expression(Dubois-Dalcq et al., 1986; Knapp et al., 1987) by in situRNA hybridization as indicators of oligodendrocytedifferentiation.

After 5 days in vitroculture, a significant decrease in thenumber of GalC+and PLP+oligodendrocytes was observedin dissociated spinal cord following Olig2 or Nkx2.2antisensetreatment compared with the Nkx2.2sense control group (Fig.9). When the Nkx2.2and Olig2antisense oligonucleotides wereapplied together, we detected a further decrease of GalC+andPLP+ oligodendrocytes, indicating an additive inhibitoryeffect on GalC and MBP expression. Under the sameconditions, Nkx2.2 expression in dissociated culture issimilarly decreased in the cells treated with Nkx2.2antisenseoligonucleotides, but not with the Olig2 antisense (data notshown), indicating the efficiency and specificity of antisensetreatment. These observations, together with the fact that Olig2and Nkx2.2are co-expressed in OLPs before oligodendrocytedifferentiation, strongly suggest that these two transcriptionfactors may cooperate to control oligodendrocytedifferentiation.

H. Fu and others

Fig. 7.Expression of Pdgfra, Olig2and Nkx2.2in the Pdgfamutants at E13.5 (A-F) and E16.5 (G-N). (A-F) Immediately adjacent sections fromE13.5 wild-type (A-C) and mutant (D-F) spinal cords were probed for Pdgfra(A,D), Olig2 (B,E) and Nkx2.2(C,F) by in situ hybridization. (G-N)Adjacent sections from E16.5 wild-type (G-J) and mutant (K-N) spinal cords were stained with Pdgfra(G,K), Olig2(H,L), Nkx2.2(I,M) and MBP(J,N). At these two stages, expression of Pdgfraand Olig2is delayed and reduced in the mutants, whereas expression of Nkx2.2and MBP is notaffected. Note the similar patterns of expression of Nkx2.2and MBP (I-J,M-N).

689Nkx2.2 and Olig2 in oligodendrocyte progenitors

DISCUSSION

Dual origin of spinal oligodendrocytes In this study, we investigated the relationship of Olig2+andNkx2.2+ oligodendrocyte progenitors by comparing theexpression pattern of Olig2and Nkx2.2in the embryonic chickenand mouse spinal cords during the critical stages ofoligodendrogenesis. At the early stages of oligodendrogenesis,Olig2+, Pdgfra+ and Sox10+ OLPs migrate out from theOlig2+ pMN domain in both chicken and mouse (Fig. 1, Fig.4). Pdgfra+ cells, Olig2+ cells and Sox10+cells are likely torepresent the same population of OLPs, as suggested by theparallel delay and reduction of production of these cell types inPDGFA mutants (Fig. 7, Fig. 8; data not shown). Soon after theyare generated, this population of OLPs rapidly spreads into allregions of the spinal cord and later differentiates into matureoligodendrocytes (Hall et al., 1996; Kuhlbrodt et al., 1998; Luet al., 2000; Zhou et al., 2000).

At the same time or slightly earlier stages, many Nkx2.2+butOlig2– cells also migrate out into the surrounding gray matterarea. In the chicken embryos, these Nkx2.2+cells quickly

migrate dorsally and laterally into both gray and white matter(Fig. 2D-F). Our previous studies have indicated that all Nkx2.2+cells are OLPs, but not astrocytes or neurons (Xu et al., 2000).Many Nkx2.2+ cells from the p3 domain start to express O4before they migrate away from the ventricular zone (Soula et al.,2001). A recent study further confirmed that someNkx2.2+/Olig2–OLPs are indeed immunoreactive to O4 antigen(Zhou et al., 2001). In the mouse, the Nkx2.2+ cells that aregenerated from the p3 domain migrate relatively slowly andremain in the ventral gray matter until at least E16.5. Theevidence to indicate that these Nkx2.2+/Olig2–cells to becomeoligodendrocyte is not as strong in mouse as it is in chicken. Onepiece of evidence is that all Nkx2.2+cells in rodents expressoligodendrocyte markers, but not neuronal or astrocytic markers,both in vivo and in vitro (Qi et al., 2001) (Fig. 5G,H).Furthermore, in the dissociated cell culture from the Nkx2.2-richventral halves of the E13.5 mouse spinal cord, every Nkx2.2+cell became immunoreactive to anti-Olig2after 2 days in vitro(data not shown).

Based on the present and previous studies, we propose thatoligodendrocytes can be generated from both the pMN domain

Fig. 8.Expression of Pdgfra, Olig2and Nkx2.2in the PDGFA mutants at P0 and P7. (A-H) Immediately adjacent sections from P0 wild-type(A-D) and mutant (E-H) spinal cords were probed with Pdgfra(A,E), Olig2(B,F), Nkx2.2(C,G) and MBP (D,H) by in situ hybridization.(I-P) Adjacent sections from P7 wild-type (I-L) and mutant (M-P) spinal cords were stained with Pdgfra(I,M), Olig2 (J,N), Nkx2.2(K,O) andMBP (L,P). At these two stages, expression of Pdgfraand Olig2is delayed and reduced in the mutants, whereas expression of Nkx2.2and MBPis mostly affected in the white matter. Note the similar patterns of expression of Nkx2.2and MBP, especially in the gray matter, at P0 (C-D,G-H).

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and p3 domain of ventral neuroepithelium during the early stagesof oligodendrogenesis in both mouse and chicken. Thehypothetical model is proposed in Fig. 10. In this model, the

pMN domain gives rise to the well-characterized Olig2+/Pdgfra+/Sox10+OLPs, whereas the p3 domain gives rise toNkx2.2+/Olig2–/Pdgfra– OLPs. The generation of the Olig2+,

H. Fu and others

Fig. 9. Inhibition of GalC+ cells and PLP+ cells in dissociated spinal cord culture by Nkx2.2and Olig2antisense oligonucleotide treatment.Dissociated culture from E5 spinal cords were treated with sense control (from Nkx2.2) or antisense oligonucleotides treatment (anti-Nkx2.2,anti-Olig2, or both) for 5 days. The relative numbers of GalC+cells from different treatments were plotted, with the sense control as 100. ForPLP+ cells, the average number of positive cells from each ten-field group (three groups from each treatment) was plotted.

Fig. 10. Hypothetical model on the embryonic origin and dynamic gene expression profile of oligodendrocyte progenitors in embryonic spinalcord. (A) During neurogenesis stages, the ventral neuroepithelium can be divided into five domains, and each domain produces a distinct classof ventral interneuron (V0-V3) or motoneurons (MN) (adapted from Briscoe et al. (Briscoe et al., 2000). At this stage, Olig2 and Nkx2.2areexpressed in adjacent pMN domain and p3 domain, respectively. (B) Oligodendrogenesis in chicken. OLPs can be generated from both thepMN domain and p3 domain. Right before OLPs are produced from the pMN domain, expression of Nkx2.2is dorsally expanded into the pMNdomain. The OLPs that arise from the p3 domain appear to gain Olig2 expression before their terminal differentiation (see text).(C,D) Oligodendrogenesis in mouse. At the early stage of oligodendrogenesis, Nkx2.2expression is not dorsally expanded and the Olig2+OLPs acquire Nkx2.2expression after migration. At later stages of oligodendrogenesis, Nkx2.2expression is also upregulated in the pMNdomain, similar to the situation in the chicken spinal cord. The p3-derived Nkx2.2+OLPs might gain Olig2 expression after migration.

691Nkx2.2 and Olig2 in oligodendrocyte progenitors

Pdgfra+ and Sox10+OLPs from the pMN domain in both mouseand chicken indicates that the lineage relationship of somaticmotoneurons and oligodendrocytes is evolutionally conserved(Richardson et al., 1997; Richardson et al., 2000). This is incontrast to the recent interpretation of distinct origin sites foroligodendrocytes and somatic motoneurons in the chicken(Soula et al., 2001).

The Olig2+ OLPs acquire Nkx2.2 expression atdifferent stages in chicken and mouse Although the Olig2+OLPs arise from the pMN domain dorsalto the Nkx2.2+p3 domain, this population of OLPs can acquireNkx2.2expression either before migration in chicken or aftermigration in rodents. Before oligodendrogenesis, Olig2 andNkx2.2are expressed in adjacent non-overlapping domains inboth chicken and mouse (Fig. 1, Fig. 4) (Lu et al., 2000; Zhouet al., 2000). Interestingly, at stages when neurogenesis isswitched to gliogenesis, the expression boundary of Olig2 andNkx2.2 breaks down and the Nkx2.2 expression expandsdorsally into the Olig2domain. In chick, Nkx2.2expansionoccurs between E5 and E7, right before generation ofOlig2+/Pdgfra+ OLPs from the pMN domain (Fig. 2). Thus,all migratory Olig2+and Pdgfra+OLPs in chicken are alsoNkx2.2+at the beginning of their migration. However, mergingof these two domains occurs relatively late in mouse, about 2days after OLPs have already migrated out into the surroundingregions. Therefore, many of the Olig2+OLPs at the earlystages of oligodendrogenesis (before E16.5) do not expressNkx2.2. These observations could certainly explain theapparent species difference in the pattern of Nkx2.2expressionduring early stages of spinal cord development (Xu et al., 2000;Qi et al., 2001). In the chicken, Nkx2.2is expressed in OLPsderived from both the pMN domain and the p3 domain fromthe beginning. However, Nkx2.2 expression is only initiallyrestricted to the ventral region where the OLPs derived fromthe p3 domain.

Although the Olig2+/Pdgfra+ OLPs originating from thepMN domain is initially Nkx2.2-negative in rodents, thispopulation of OLPs appears to acquire Nkx2.2expression aftertheir emigration into the spinal cord parenchyma based on thefollowing observations. First, around the time of birth, nearly allthe Olig2+ OLPs in white matter co-express Nkx2.2, althoughthe expression level in some cells is relatively low (Fig. 5G,H).Second, the OLPs derived from E13.5 dorsal spinal cordgradually gain Nkx2.2 expression with time in dissociatedculture (Fig. 6). Similarly, the percentage of Nkx2.2+ cells inimmunopurified A2B5+ cells also increases with time in vitro(Qi et al., 2001) (data not shown). Third, in the PDGFA mutantembryos, reduction of Olig2+/Pdgfra+ OLPs in white matter isaccompanied by a decrease in Nkx2.2expression at P0, althoughNkx2.2expression in ventral gray matter is not affected (Fig. 8).

Nkx2.2+ OLPs that originate from the p3 domain mayacquire Olig2 expression during migration anddifferentiation The OLPs that originate from the p3 domain initially expressNkx2.2, but not Olig2 andPdgfra (Fig. 2, Fig. 7), which couldexplain why the number of Nkx2.2+OLPs in the ventral graymatter is not reduced in the Pdgfamutants at P0 and earlierstages. There is some good evidence to suggest that thispopulation of OLPs might gain Olig2 expression during or after

migration. In chicken spinal cord, the p3 domain activelyproduces Nkx2.2+/Olig2–OLPs between E6 and E9. As theseOLPs are dispersed into the surrounding gray and white matterregion, the percentage of Nkx2.2+/Olig2–OLPs decreases fromthe ventral ventricular zone to the white matter at E8-10 (Fig.2). Similarly, the percentage of Olig2+cells in the white matterincreases with time between E8 and E10. Further evidencefor the capability of Nkx2.2+migratory OLPs to gain Olig2expression comes from the subventricular expression of Olig2directly beneath the Nkx2.2+neuroepithelial cells in thedeveloping hindbrain (Fig. 3). However, we are also aware ofthe alternative explanation that the increase of Olig2+cells inthe embryonic spinal cord results from preferential proliferationof the Olig2+/Nkx2.2+population that arises from the pMNdomain.

In the mouse, the p3-derived Nkx2.2+ OLP cells mightsimilarly gain Olig2expression after they migrate out into thegray matter. There is only some weak evidence to support thishypothesis. First, the Olig2expression in the Nkx2.2+region ishigher than the rest of the spinal cord at E16.5 in both the wild-type and Pdgfamutant embryos (Fig. 7). At birth, all the Nkx2.2+cells in this region, and in the white matter as well, are positivefor Olig2 expression (Fig. 5H, data not shown). Second, in thedissociated cell culture with the ventral halves of the E13.5 mousespinal cord, every Nkx2.2+cell is immunoreactive for anti-Olig2after two days in vitro (data not shown). However, the evidencefor the acquisition of Olig2expression by this population of OLPsis still indirect, and further immunological and genetic labelingstudies are required to further confirm this hypothesis.

Synergistic role of the Nkx2.2 and Olig2 genes inoligodendrocyte differentiationSide-by-side comparison of expression of Olig2, Nkx2.2andMbp revealed that expression of MBP closely follows that ofNkx2.2during oligodendrocyte differentiation. In E16.5 mousespinal cord, MBP expression is detected only in the ventral graymatter where Nkx2.2is expressed. At P0, expression of bothNkx2.2and MBP is observed in the white matter. Reduction ofNkx2.2expression in the white matter in the Pdgfamutants isaccompanied by the reduction of MBP expression in this region.As nearly every Nkx2.2+cell is co-labeled with Olig2 at thesestages (Fig. 5G,H; data not shown), it is reasonable to speculatethat myelin gene expression and oligodendrocyte differentiationmay be initiated by the interaction of Nkx2.2and Olig2. Thereare at least two lines of evidence to support this concept. First,terminal differentiation of oligodendrocytes appears to requiresimultaneous expression of these two transcription factors, assuggested by the additive inhibitory effects of the Nkx2.2andOlig2 antisense treatments on PLP gene expression (Fig. 9).Inhibition of either Nkx2.2or Olig2 expression is accompaniedby a smaller but significant reduction of GalC+ and PLP+ cellsin culture. The inhibition of oligodendrocyte differentiation byNkx2.2antisense treatment is in agreement with our previousfindings that mutation of the Nkx2.2gene can cause a dramaticreduction of MBP and PLP expression (Qi et al., 2001). Second,expression of either Olig2or Nkx2.2alone is not sufficient foroligodendrocyte differentiation in vivo (Zhou et al., 2001) (J. C.,H. F. and M. Q., unpublished). However, co-transfection ofNkx2.2 and Olig2 can result in ectopic and precociousoligodendrocyte differentiation in embryonic chicken spinalcord (Zhou et al., 2001). Based on these observations, it appears

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that co-expression of the Olig2and Nkx2.2is both necessary andsufficient for oligodendrocyte differentiation and myelin geneexpression.

If co-expression of Olig2and Nkx2.2is directly responsiblefor myelin gene expression, we would predict a speciesdifference in the expression of MBP as Nkx2.2expression isupregulated much earlier in the pMN-derived OLPs in thechicken than in the mouse. Our preliminary results confirm thisspecies difference. In the chick, MBP expression starts relativelyearly (E8-9) and is initially detected in white matter and to alesser extent in the pMN domain (H. F. and M. Q., unpublished)in which Nkx2.2expression is upregulated (Fig. 2). In mouse,MBP+ cells are initially observed in the ventral gray matter, theregion where Olig2and Nkx2.2are co-expressed (Fig. 7). Thus,MBP expression closely follows the co-expression of Olig2 andNkx2.2in the chicken, as in the mouse. It is conceivable that thepace and pattern of myelin gene expression could be regulatedduring evolution by controlling the timing and location of theco-expression of these two transcription factors.

It is worthwhile mentioning that co-expression of Olig2 andNkx2.2is not required for the initial specification of OLPs. Inthe embryonic mouse spinal cord, OLP production from thepMN and p3 domain occurs prior to merging of these twodomains. In the hindbrain of chicken embryo, Olig2 is notexpressed in the ventricular zone when Nkx2.2+OLPs are born(Fig. 5). Moreover, in the Nkx2.2mutants, production ofOlig2+/Pdgfra+ OLPs is normal or even slightly increased,although their terminal differentiation is greatly reduced anddelayed (Qi et al., 2001). The residual expression of Mbp andPlp in the Nkx2.2mutants might imply that the function ofNkx2.2could be weakly compensated by a related unidentifiedtranscription factor.

We thank Drs David Anderson, Chuck Stiles and C.C. Hui forproviding cDNA probes. We are particularly grateful to Dr DavidAnderson for providing chicken Olig2 before publication. We alsothank Dr David Stapp and the anonymous reviewers for their insightfulcomments and suggestions. The anti-Nkx2.2 hybridoma cells wereobtained from the Developmental Studies Hybridoma Bank developedunder the auspices of the NICHD and maintained by The University ofIowa, Iowa City. This study is supported by NSF (IBN-9808126), NIH(NS 37712) and National Multiple Sclerosis Society.

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