Regulation of FGF receptors in the oligodendrocyte lineage

Molecular and Cellular Neuroscience 7, 263–275 (1996)

Article No. 0020 MCNRegulation of FGF Receptors in theOligodendrocyte Lineage

Rashmi Bansal,* Madhur Kumar,* Kerren Murray,*Richard S. Morrison,† and S. E. Pfeiffer**Department of Microbiology, University of Connecticut School of Medicine, Farmington,Connecticut 06030-3205; and †Department of Neurological Surgery,University of Washington, Seattle, Washington 98195

Fibroblast growth factors (FGFs) affect a broad spectrum sis, in response to FGF-2, FGF receptor-1 transduces sig-nals that stimulate the prolonged proliferation and migra-of developmentally regulated cellular responses involved

in the control of growth and differentiation. To identify tion of early progenitors, FGF receptor-3 promotes theproliferation and arrest of differentiation of late progeni-specific FGF receptor forms involved in these responses,

we have characterized FGF receptor transcript expres- tors, and FGF receptor-2 transduces signals for terminaldifferentiation, but not proliferation, in mature oligoden-sion, and its modulation by FGF-2, as enriched popula-

tions of oligodendrocyte progenitors differentiate into drocytes.mature oligodendrocytes. The data demonstrate that thelevels of mRNA expression for FGF high-affinity receptors-1, -2, and -3 are differentially regulated during lineage pro- INTRODUCTIONgression: FGF receptor-1 expression increases with lin-eage progression, FGF receptor-2 is predominantly ex-pressed by terminally differentiated oligodendrocytes, The interrelationship between proliferation and differ-and FGF receptor-3 reaches a peak level of expression in entiation is a cornerstone of developmental regulation.late progenitors and then declines upon further differenti- Fibroblast growth factors (FGFs) are a structurally re-ation; FGF receptor-4 expression was not detected in oli- lated family of nine peptides acting at this interface. Theygodendrocytes. Distinct patterns of alternatively spliced target a broad spectrum of cell types derived from meso-variants of FGF receptor-1 and -2 transcripts are ex- derm and neuroectoderm, inducing a variety of biologi-pressed: the predominant FGF receptor-1 transcripts con-

cal activities that include either inhibition or stimulationtain three Ig-like domains (FGF receptor-1a), whereas theof differentiation, proliferation, and chemotaxis (re-FGF receptor-2 transcripts contain two Ig-like domainsviewed in Eckenstein, 1994). The corresponding high-(FGF receptor-2b2) and this form is up-regulated as oligo-affinity FGF receptors (FGFRs) are a diverse family ofdendrocytes differentiate. In addition, the expression oftyrosine kinases for which four separate genes (FGFR 1–these receptors is differentially regulated by the ligand,

FGF-2: FGF receptor-1 mRNA expression is up-regulated 4) have been identified (reviewed in Johnson and Wil-in early progenitors, and FGF receptor-2 mRNA expres- liams, 1993). The basic structure of FGFRs consists of ansion is down-regulated in mature oligodendrocytes. Fi- extracellular ligand-binding region with three immuno-nally, astrocytes express FGF receptor-1, -2, and -3 tran- globulin (Ig)-like domains, a transmembrane region, andscripts, but at different levels and with different exon utili- a cytoplasmic tyrosine kinase domain. Some FGFRs bind,zation (FGF receptor-1b, FGF receptor-2b1/b2) compared and are activated by, multiple FGFs, whereas others haveto oligodendrocytes. To our knowledge this is the first a higher degree of specificity (Ornitz and Leder, 1992;report that demonstrates that the mRNA expression of

Partanen et al., 1991; Johnson and Williams, 1993). Multi-these three FGF receptor types is differentially regulatedple-splice variants of FGFR-1, -2, and -3 can have dramat-in primary cells as they differentiate along a lineage fromically altered ligand specificities and binding affinitiesprogenitors to terminally differentiated cells. We propose(Miki et al., 1992; Werner et al., 1992; Johnson et al., 1991).that this pattern of expression provides a molecular basisFGF receptor expression has differential spatial distribu-for the developmentally varying response of cells to a

common ligand. For example, according to this hypothe- tion during embryogenesis (Peters et al., 1992; Orr-Ur-

1044-7431/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved. 263

AID MCN 0547 / 6p09$$$101 05-19-96 22:02:03 mcnal AP: MCN

264 Bansal et al.

treger et al., 1991) and in adult tissues (Asai et al., 1993; ple FGF high-affinity receptor transcripts in specific alter-natively spliced isoforms are expressed in a developmen-Yazaki et al., 1994). In addition, a second class of cell

surface components essential for FGF signaling have tally regulated manner. These results, combined with ourobservations that multiple putative FGF coreceptorsbeen identified, a family of low-affinity (Kd 2–20 nM ),

high-capacity heparan sulfate proteoglycans (Yayon et (syndecans 1–4, glypican; Bansal et al., 1996) are alsodevelopmentally regulated in these cells, suggest thatal., 1991; Rapraeger et al., 1991). With at least nine similar

but distinct ligands, four receptors, and their multiple there is a changing repertoire of these signaling mole-cules on the cell surface as they progress through thevariant isoforms, plus an extended family of coreceptors,

it is clear that a cell has ample opportunity to modulate developmental lineage. In addition, the data show thatFGF-2 itself regulates the mRNA levels of these receptorsits response to FGF(s) by regulating the combinatorial

complexity of a diverse set of cell surface receptors. at specific stages during development.Oligodendrocytes (OLs) are emerging as a prominent

model in which to examine key issues of cell biology ina developmental context (Pfeiffer et al., 1993). OLs form RESULTSmyelin in the central nervous system through an elabora-tion of their plasma membranes which wrap around neu- Phenotypic Characterization of Enrichedronal axons to form compact, multilamellar sheaths. The Populations of Cells of theprogression of OL progenitors along a carefully regu- Oligodendrocyte Lineagelated lineage, characterized by changes in their morphol-

Highly enriched populations of OL-lineage cells wereogy and proliferative and migratory capacity, and theprepared at three stages of differentiation. The transitionordered expression of OL and myelin-specific proteinsfrom O2A to Pro-OL was marked by a change in mor-and lipids, can be formalized as:phology from bipolar to multipolar cells, followed byfurther process and membrane elaboration as the cells[early progenitors (O2A)] rdifferentiated into postmitotic, mature OLs (Fig. 1B). Thephenotypic compositions are shown (Fig. 1A). In the ab-[late progenitors (Pro-OLs)] r [mature OLs (OLs)].sence of FGF-2 (0FGF), the ‘‘early progenitor’’ popula-tions were a mixture of Ç70% early progenitors andEarly progenitors are proliferative and migratory cells;Ç30% late progenitors (see Fig. 1 legend) andÇ1% OLs;late progenitors are proliferative but nonmigratory cells;the ‘‘late progenitor’’ populations were a mixture ofmature OLs are postmitotic, terminally differentiatedÇ80% late progenitors and Ç5% OLs; the ‘‘mature OL’’cells expressing myelin proteins and lipids. Purified OLpopulation was Ç75% O1/ andÇ54% MBP/. In the pres-progenitors grown in culture follow a developmentalence of FGF-2 (/FGF), because of the synchronizing effectpathway consistent with lineage progression in situof FGF-2 on developmental progression, progenitor pop-(Warrington and Pfeiffer, 1992). OL developmental lin-ulations with enhanced homogeneity were obtained.eage is regulated by a variety of growth factors, including‘‘Early progenitor’’ populations were Ç95% early pro-FGF-2 (basic FGF). However, the nature of the responsegenitors and only Ç5% late progenitors; ‘‘late progeni-to FGF-2 varies markedly as a function of the stage of thetor’’ populations were Ç80% late progenitors and Ç1%OL lineage. FGF-2 up-regulates platelet-derived growthOLs; on the other hand, ‘‘mature OL’’ populationsfactor (PDGF-a) receptors on early progenitors and, intreated with FGF-2 for 2 days were reduced in homoge-combination with PDGF, supports their long-term prolif-neity to Ç40% O1/ and Ç15% MBP/ cells due to aneration (McKinnon et al., 1990; Bogler et al., 1990); it isapparent induction of phenotypic reversion (Introduc-mitogenic for late progenitors and reversibly blocks theirtion). Astrocytes constituted 1–6% of the cell popula-terminal differentiation (Gard and Pfeiffer, 1993; McKin-tions, depending on conditions of growth.non et al., 1990; Mayer et al., 1993; Bansal and Pfeiffer,

1994); it causes an apparent phenotypic reversion of ma-ture OLs (Grinspan et al., 1993; Fressinaud et al., 1995). Oligodendrocytes Express Multiple FGF Receptor

We hypothesized that multiple FGF receptors and Types during Differentiation in Culturecoreceptors are expressed in a stage-specific manner, ac-counting in part at least for the variety of responses of We evaluated by Northern blot analyses the expres-

sion of individual FGF receptor mRNA by OLs as theyOL-lineage cells to FGF-2. In order to test this hypothesis,we have investigated the developmental expression of undergo growth and terminal differentiation (Figs. 2A

and 2B, black bars, 0FGF). Since members of the FGFRthese receptors and its ligand-mediated regulation. Thedata demonstrate that during lineage progression, multi- family are encoded by genes that show an overall amino


265Regulation of FGF Receptors in Oligodendrocytes

FIG. 1. Phenotypic characterization of enriched populations of OL-lineage cells grown in the absence or presence of FGF-2. (A) Quantitativedistribution of phenotypes within cell populations. Cells were characterized by immunostaining with stage-specific markers. The percentage oftotal cells that were immunostained with antibodies O4 (Pro-OL / OL marker, black columns), O1 (OL marker, striped columns), anti-MBP (moremature OL marker, spotted columns), or anti-GFAP (astrocyte marker) is shown. 97 –98% of total cells were stained with antibody A2B5 at thetwo progenitor stages (not shown). A2B5/O40 Å O2A early progenitors; O4/O10 Å Pro-OL late progenitors; O4/O1/MBP/ Å mature OLs. O2A(0FGF) (*) indicates weakly O4/ cells still retaining an early progenitor morphology and may represent a transitional stage to late progenitors.Error bars, standard errors of the mean (N Å 3–6). (B) Morphology of cells analyzed by immunofluorescent microscopy at three stages of the OLlineage used to generate the data in A. Bar, 25 mm.

acid sequence homology of 60 –70%, specific cDNA of Ç30 and 50%, respectively. Enriched OL populationsshowed a 2.3-fold increase in message level over thatprobes and hybridization conditions were used that pre-

vent cross-hybridization of the different receptors. The in P2 brain. We conclude that FGFR-1 transcripts areexpressed throughout the OL lineage.specificity of these probes for the specific receptor type

has been established previously (Mansukhani et al., 1990, Hybridization with an FGFR-2 probe yielded the ex-pected major band at 4.4 kb (Dionne et al., 1990). FGFR-21992; Partanen et al., 1991; Ornitz and Leder, 1992).

Hybridization with an FGFR-1 probe revealed the ex- expression was barely detectable in early and late progeni-tors, but was up-regulated (ú10-fold) as OLs entered termi-pected 4.2-kb species (Templeton and Hauschka, 1992).

FGFR-1 mRNA was expressed at the highest levels in nal differentiation. Expression of FGFR-2 was detected inP2 and P5 (not shown) brain at a much lower level thanmature OLs and in early and late progenitors at levels


266 Bansal et al.

AID MCN 0547 / 6p09$$0547 05-19-96 22:02:03 mcnal AP: MCN


in OLs. A second RNA band of Ç1 kb was repeatedly main, or contain deletions and insertions within the ex-tracellular or intracellular domains (reviewed in Johnsondetected only in mature OLs at a level of Ç30% of the

major band that could in principle represent a message for and Williams, 1993). We determined by nonquantitativeRT-PCR if OL-lineage cells expressed variant transcriptsa soluble form of the receptor (Johnson and Williams, 1993).

We conclude that FGFR-2 is primarily a mature OL recep- of FGF receptors not distinguishable on Northern blots.For RT-PCR amplification of FGFR-1 mRNA, we usedtor and that its developmental expression parallels that of

the other major myelin-specific proteins and lipids. a 5*-primer located in the signal sequence and a 3*-primerlocated between Ig-domains I and II, beyond the ‘‘acidHybridization with a probe for FGFR-3 demonstrated

the expected 4.2-kb transcript (Keegan et al., 1991). Unlike box’’ (Fig. 3). This primer set should identify receptortranscripts that encode for isoforms including eitherFGFR-1 and -2 transcripts that were maximally expressed

upon terminal differentiation, FGFR-3 reached peak lev- three (FGFR-1a) or two (FGFR-1b) Ig-like domains, pro-ducing fragments of 450 and 184 bp, respectively. Ampli-els of expression at the late progenitor stage, and then

declined upon further terminal differentiation. Expres- fication of cDNA prepared from early or late progenitors,mature OLs, and brain all produced prominent 450-bpsion of FGFR-3 was also detected in adult brain at some-

what higher levels than in OLs. We conclude that FGFR-3 fragments (a very faint band under the FGFR-1a isoformis nonspecific) (Fig. 4A).transcripts are maximally expressed by late progenitors.

Hybridization with a probe for FGFR-4 resulted in the The expression of transcripts for FGFR-2a and -2b iso-forms was examined by RT-PCR (Fig. 4B) and Southernexpected band of Ç3.2 kb in lung and liver (Partanen et

al., 1991), but not in OL-lineage cells or brain, consistent blot analysis using radiolabeled internal sequence oligo-nucleotides (Fig. 4Da, b). The 5*-primer was located inwith in situ hybridization studies showing a restricted

spatial expression of FGFR-4 in the brain (Yazaki et al., the untranslated region and a 3*-primer in Ig-like domainIII (Fig. 3). These primers should amplify three FGFR-1994) (although GAPDH controls were not available for

the example shown in Fig. 2, subsequent analysis of this 2-specific fragments of 1100 bp (FGFR-2a, three Ig-likedomains), 900 bp (FGFR-2b1, two Ig-like domains), andblot with other probes demonstrated that the RNA and

blot were of high quality; other independent blots with 800 bp (FGFR-2b2, two Ig-like domains, but lacking theacid box) (Yamaguchi et al., 1994). In the early and lateglyceraldehyde-3-phosphate dehydrogenase (GAPDH)

controls produced the same results). The FGFR-4 probe progenitors, we detected low levels of FGFR-2a, -2b1,and -2b2 isoforms in the ratio 1:1:2.5 (this was clear upondid hybridize to another smaller RNA band, the identity

of which is not known, in all lineage stages of OLs and longer exposure of the autoradiograms). In contrast, ma-ture OLs expressed transcripts for FGFR-2a and -2b2 inin brain, but not in liver or lung. We conclude that OL-

lineage cells do not express the 3.2-kb FGFR-4 transcript. the ratio 1:35, whereas evidence for FGFR-2b1 was ab-sent (confirmed by the absence of FGFR2-b1 in Southernblot hybridizations; Fig. 4Db). These FGFR-2a, -b1, and

Oligodendrocytes Express Alternatively Spliced -b2 fragments were also amplified from adult rat brainVariants of FGF Receptors cDNA (Fig. 4B) and a positive control cDNA (Fig. 4B,

4Db). In addition, we detected in mature OLs a PCRFGFR-1, -2, and -3 can be expressed in multiple variantfragment of Ç950 bp that may represent another variantforms arising from alternative splicing of mRNA tran-form of FGFR-2 (Fig. 4B, asterisk) (confirmed by South-scripts (Fig. 3). Receptor variants lack Ig-like domain I,ern hybridization with an internal sequence oligonucleo-exhibit differences in exon III utilization (exons IIIa, b,

c) in the carboxy-terminal half of the third Ig-like do- tide specific for FGFR-2 under stringent washing condi-

FIG. 2. Developmental expression and regulation by FGF-2 of FGF receptor 1–4 mRNAs at three stages of the OL lineage analyzed by Northernblotting. (A) Representative blots for each receptor are shown. Total RNA was extracted from populations (see Fig. 1 for phenotypic characterization)of early progenitors, late progenitors, or mature OLs grown in the absence (0) or presence (/) of FGF-2, enriched astrocytes (Ast), and P2 wholebrain (P2 BR) or adult telencephalon (Ad Tel). Total RNA (20 mg) was loaded per lane and the blots were hybridized with cDNA probes specificfor each of the receptor types. Blots were then rehybridized with a probe for GAPDH. Note that the order of minus and plus FGF addition isreversed for FGFR-1 and -2 relative to the bar graphs. (B) The mRNA levels were quantified and normalized for variations in RNA loading(GAPDH), and the values were expressed as relative mRNA levels (black bars, 0FGF; hatched bars, /FGF; the highest level in each group wasset at 100%). Since the FGFR cDNA probes have different specific activities, the values can be used only to compare relative mRNA levels amongthe three stages for a given receptor type. Quantification for FGFR-4 is not shown since no bands were seen at the known size for FGFR-4 in theOL-lineage cells (but seen in positive controls of lung or liver [arrow]; the identity of the lower bands is not known). The probe for FGFR-2 alsorecognized a lower band in the OL (0FGF)-stage population; the quantification does not include this band, which is Ç30% of the known, 4.4-kbFGFR-2 band (arrow). Error bars, standard errors of the mean (N Å 3– 6) or ranges of duplicates.


268 Bansal et al.

FIG. 3. Diagrammatic representation of an FGF receptor, showing alternatively spiced variants and the positions of RT-PCR primers used(indicated by arrows). R-1, FGFR-1; R-2, FGFR-2; R-3, FGFR-3; a, 3*-primer; b, 5*-primer; UTR, untranslated region; SP, signal peptide; I, II, III, Ig-like domains; AB, acid box; TK1 and TK2, tyrosine kinase domains; KI, kinase insert; CT, cytoplasmic tail. The ligand-binding specificationsdetermined by the use of exon IIIb and IIIc are indicated.

tions; Fig. 4Db, asterisk). TheÇ950-kb fragment was also We conclude that multiple isoforms of FGFR mRNAare expressed by OL-lineage cells and that a poten-detected in rat adult brain (Fig. 4B), albeit more weakly.

We therefore compared areas of adult rat brain that were tially novel FGFR-2 mRNA transcript is expressed bymature OLs.either enriched (brain stem) or relatively low (forebrain

gray matter) in oligodendrocytes and myelin. An ampli-fied band was readily detected by Southern blot analysis

The Expression of FGF Receptors Is Regulatedin the material from brain stem but not from gray matter

by FGF-2(Fig. 4Da, asterisk).

Since the choice of exon IIIb or IIIc determines ligand- In order to determine whether the expression of thereceptors is regulated by FGF-2 we exposed the cells tobinding specificity (Miki et al., 1992; Werner et al., 1992),

we examined the expression of exon IIIc in the FGFR-2 FGF-2 for 2 days prior to RNA extraction and Northernblot analysis (Figs. 2A and 2B, hatched bars,/FGF). FGF-and -3 transcripts. Since the primers chosen for RT-PCR

(Fig. 3) will identify exon IIIc utilization, the appearance 2 induced an increase in the expression of FGFR-1 mRNAin both early and late progenitors to a common levelof amplified products for FGFR-2 (Fig. 4B) and -3 (Fig.

4C; 145 bp) indicates that they did in fact contain exon comparable to the level exhibited by mature OLs withor without growth factor. The expression of FGFR-2IIIc sequences. Based on previous analyses of exon III-

mediated ligand-binding specificity, these data suggest mRNA by early and late progenitors was unaffected byFGF-2, but was strongly down-regulated in mature OLs.that FGFR-2 expressed by OLs is capable of binding both

FGF-1 and -2 with equally high affinity (Fig. 3), and that The expression of FGFR-3 mRNA was not affected byFGF-2. FGFR-4 mRNA expression was not induced byFGFR-3 can bind FGF-1 at higher affinity than FGF-2

(Ornitz and Leder, 1992). The presence of FGFR-3 was FGF-2. Therefore, although early progenitors expressedboth FGFR-1 and -3 transcripts, FGF-2 regulated the ex-further confirmed in OL-lineage cells and astrocytes by

amplification of its transcripts utilizing a second set of pression of only FGFR-1 in these cells; similarly, althoughmature OLs expressed all three FGF receptor genes, onlyPCR primers spanning a region of Ig-like domain II and

interdomain region II/III (data not shown). the level of FGFR-2 message was regulated by FGF-2.



FIG. 4. Qualitative RT-PCR analyses of the expression of variant forms: (A) FGFR-1, (B, D) FGFR-2, and (C) FGFR-3. Total RNA was obtainedfrom cultures of O2A and Pro-OL progenitor populations exposed to FGF-2, mature OLs grown in the absence of FGF-2 (see Fig. 1 for culturecharacterization), and highly enriched astrocytes (Astro), adult rat brain (Ad Br), S45Y cells used as a positive control (Control), brain stem (Da,1), and forebrain gray matter (Da, 2). (Da,b) For FGFR-2, RT-PCR was followed by Southern blot analyses (shown for separate gels) usingradiolabeled internal sequence oligonucleotides. Primers specifically detect fragments representing the following structural variants: (A) FGFR-1a(450 bp) and -1b (184 bp); (B, D) FGFR-2a (1100 bp), -2b1 (900 bp), and -2b2 (800 bp); (C) FGFR-3, exon IIIc (145 bp). A control for the RT-PCRreaction was included in which the template was omitted from the reaction (No Templ). Representative experiments are shown. For FGFR-2, notethe fragment of Ç950 bp (asterisk) between a and b1 in adult rat brain and mature OLs (B, Db) and brain stem (Da, 1). Since the RT-PCR analyseswere not quantitative, the levels of expression among cell populations should not be compared to the quantitative Northern results.

We conclude that during OL growth and differentiation, cell cultures did not contribute to the receptor isoformexpression observed by RT-PCR in these cells. Whetherthe transcripts of individual FGF receptors are selectively

modulated by FGF-2. FGF receptors are differentially expressed in astrocytesduring development, as in OLs, and whether a mecha-nism of autocrine regulation of these receptors exists,

Astrocytes Express FGF Receptors will be of interest since astrocytes also synthesize theligand.The expression of FGF receptor mRNA by cultured

astrocytes was analyzed, and the relative levels of ex-pression were compared to that of OL-lineage cells. Al-ternately spliced isoforms of each receptor were deter-mined by RT-PCR. Astrocytes expressed mRNA tran-scripts of the predicted sizes for FGFR-1, -2, and -3 (Fig.2A) but not FGFR-4 (data not shown). The relative levelsof expression of FGFR-1 and -3 were 1.5- and 1.8-foldhigher, respectively, in astrocytes compared to OL-lin-eage cells, whereas the levels of FGFR-2 were 2.5-foldhigher in OLs (Fig. 5). Astrocytes express predominantlythe FGFR-1b isoform (Fig. 4A) and FGFR-2a, -2b1, and-2b2 in the ratio 1:20:20 (Figs. 4B and 4Db). In summary,although astrocytes and OLs express the same threetypes of FGF receptors, the levels of mRNA expressionand the isoform patterns of each receptor are different FIG. 5. Comparison of the relative FGF receptor mRNA expressionbetween the two cell types. This also shows that the low- between astrocytes (hatched bars) and the highest levels of expression

observed during the OL lineage (black bars).level astrocyte contamination (1–6%) in the OL-lineage


270 Bansal et al.

sion of FGF receptors by cells at distinct stages in a singleDISCUSSIONdevelopment lineage of primary cells had not been re-ported. Because these studies are difficult to perform inTo examine molecular mechanisms governing sequen-vivo, we have taken advantage of a culture system thattial developmental responses to FGF, we have studiedhas allowed us to study FGF receptor expression inthe regulation of FGF receptor expression by cells as theyhighly purified and developmentally synchronized pop-differentiate through a lineage, using the well-definedulations of primary cells, at specific, well-defined stagesOL system. The results demonstrate four main points.of differentiation.First, cells of the OL lineage express FGF high-affinity

Among the three types of high-affinity FGF receptorsreceptor transcripts -1, -2, and -3, but not -4. Second,(FGFR-1, -2, and -3) expressed during the OL-lineagethe expression of these transcripts is developmentallyprogression, FGFR-1 was the only one that was up-regu-regulated; specifically, the expression of mRNA forlated by its ligand. This effect was restricted to the pro-FGFR-1 is increased during lineage progression, forgenitor stages. In early progenitors, FGF-2 also up-regu-FGFR-3 is transiently up-regulated in the late progenitorlates PDGFa receptors (McKinnon et al., 1990). Therefore,stage, and for FGFR-2 is dramatically up-regulated as thethe enhanced coexpression of these receptors by FGF-2progenitors differentiate into mature OLs. Third, FGF-2may be instrumental in the stimulation of mitotic andup-regulates its own receptor gene expression; for exam-migratory capacity that these cells experience in responseple, FGF-2 up-regulates the expression of FGFR-1 mRNAto the combined administration of PDGF plus FGF-2in progenitors, but down-regulates expression of FGFR-(Bogler et al., 1990). Ligand-mediated up-regulation of2 mRNA in mature OLs. Fourth, distinct patterns of alter-FGFR-1 mRNA expression has previously been observednatively spliced variants of FGFR-1 and -2 are expressed;for FGF-2 in tumor cells (Saito et al., 1991) and for otherspecifically, the predominant FGFR-1 transcript containsgrowth factors (Earp et al., 1988). Based on in situ hybrid-three Ig-like domains, whereas the FGFR-2 transcriptsization studies, it has been suggested that neurons arepredominantly contain two Ig-like domains, which arethe main sites of FGFR-1 expression in the brain (Wanakaup-regulated as OLs differentiate.et al., 1991; Asai et al., 1993; Yazaki et al., 1994; Peters etThe principle conclusion that emerges from these re-al., 1992; Lai and Lemke, 1991), although some expressionsults is that OLs differentially express specific high-af-was also detected in adjacent nonneuronal cells uponfinity FGF receptor transcripts as they undergo lineageablation of the somatosensory cortex (Yuguchi et al.,progression, leading to a developmentally changing rep-1994) and in astrocytomas (Yamaguchi et al., 1994). In theertoire of these signaling molecules, thus providing apresent study, we detected FGFR-1 in highly enrichedmolecular basis for the lineage stage-specific responsespopulations of OLs, perhaps leading to an enhancementof OLs to the common ligand, FGF-2. To our knowledge,of the signal.this is the first study that has demonstrated that the ex-

The pattern of FGFR-2 expression during OL develop-pression of individual FGF receptors is regulated duringment is particularly interesting. In contrast to the down-lineage progression of primary cells, both as a functionregulation of FGFR-1 transcripts upon the differentiationof the developmental stage and the availability of theof developing myoblasts (Templeton and Hauschka,ligand. We predict that these principles will prove to be1992) and chondrocytes (Iwamoto et al., 1991), and ofgenerally used during the differentiation of other lin-PDGF a-receptor transcripts in OLs (Hart et al., 1989),eages as well.the present data show that FGFR-2 mRNA expression isdramatically up-regulated upon terminal differentiation(mirroring the expression of OL differentiation markersDevelopmental Patterns of Expression of FGFsuch as myelin basic protein). This is consistent withReceptor Typesthe observation that FGFR-2 mRNA is localized in themyelinated fiber tracts of adult rodent brain (Asai et al.,Individual FGF receptors have distinct patterns of

gene expression during early embryonic development 1993). In addition, FGF-2 has little or no mitogenic effecton mature OLs, but stimulates their process outgrowthand organogenesis and in adult tissue (Partanen et al.,

1991; Orr-Urtreger et al., 1991; Peters et al., 1992, 1993; (Yong et al., 1994; Gogate et al., 1994; Fressinaud et al.,1993; Bansal et al., unpublished), perhaps via its stimula-Yazaki et al., 1994). In in situ hybridization studies of

brain, the identity of the cells expressing a particular tion of protein phosphorylation of the actin-bindingMARKS protein and GAP-43 (Deloulme et al., 1992), pro-receptor has been based on their spatial location, mor-

phology, and intensity of Nisel staining, but no distinc- teins involved in the organization of the cytoskeleton.However, an apparent paradox exists, for FGF-2 alsotion was made between the two major glial cell types,

OLs and astrocytes. In addition, changes in the expres- down-regulates the expression of FGFR-2 mRNA (these



data) as well as of myelin-specific genes in these cells selective expression by mature OLs, is involved in themodulation of differentiation rather than proliferation,(Grinspan et al., 1993; Fressinaud et al., 1995; Bansal et

al., unpublished). The ‘‘paradox’’ may be resolved by the leading to cytoskeletal alterations.The study of the signaling pathways initiated by eachpossibility that instead of FGF-2, the adhesion molecules

NCAM, L1, and N-cadherin may be involved. These mol- receptor type is currently an emerging field of investiga-tion. However, differences have been observed in theecules are expressed in the vertebrate nervous system

where they are involved in neural cell –cell adhesion abilities of FGFR-1 and -4 to induce tyrosine phosphory-lation (Wang et al., 1994; Shaoul et al., 1995). Our model(Rutishauser and Jessell, 1988). Recently they have been

shown to bind to FGFR-1 and -2 via specific sequences implies that differences in the responses to FGF-2 aredue to the sequential activation of different signalingand stimulate neurite outgrowth (Williams et al., 1994).

Therefore, they could in principle be the preferred li- cascades, depending on the predominating receptor typeat each stage of OL differentiation. However, sincegands for OLs at this point of differentiation and a mech-

anism similar to that in neurons could occur during OL FGFR-1 and -3 are expressed throughout the lineage (al-beit at varying levels), it is possible that a given receptordifferentiation and myelination. Additionally, the down-

regulation of myelin genes is of potential clinical interest, activates a series of different pathways, depending ontheir stage-dependent properties.since it could in principle provide a mechanism by which

FGF-2 can drive differentiated OLs to dedifferentiate inpathological situations by inhibiting the gene expression Several Isoforms of FGF Receptors Are Expressedof its own receptor. In summary, we suggest that FGFR-

by Oligodendrocytes2 has its principle role in the onset and regulation ofterminal differentiation of OLs, and/or the maintenance As FGF-responsive progenitors migrate through a se-

ries of microenvironments en route to their final destina-of the structure and function of mature myelin, and thatcell adhesion molecules and/or FGF-2 may modulate tions, they are likely to be sequentially exposed to multi-

ple members of the FGF family, which in the brain in-these processes via regulation of the level of FGFR-2 ex-pression and/or second messenger metabolism induced clude FGF-1, -2, -3, -5, and -9 (Eckenstein, 1994). The four

FGF receptors and their isoforms exhibit redundancy asby activation of this receptor.FGFR-3 is predominantly expressed in cells with glial well as significant differences in their binding specifici-

ties and affinities for this family of related ligands. Thus,morphology in the embryonic and postnatal CNS (Peterset al., 1993; Yazaki et al., 1994). The ventral to dorsal mechanisms that regulate either preferential expression

of different receptor genes or alternative splicing coulddevelopmental expression of FGFR-3 mRNA in rodentspinal cord coincides well with the origin and subse- allow a cell to achieve developmentally regulated, selec-

tive responsivity to different FGFs. We therefore exam-quent migration route suggested for some OL progeni-tors (discussed in Ono et al., 1995). Here we have shown ined exon utilization as a function of OL development.

Variants of FGFRs are generated by alternative splic-that FGFR-3 mRNA is maximally expressed in late pro-genitors, making FGFR-3 a likely candidate for transduc- ing of a single exon, resulting in isoforms containing

either all three (a form) or only two (b form, missinging signals from FGF-2 that reversibly block terminaldifferentiation by late progenitors. FGFR-3 is the only Loop-I) Ig-like domains in the extracellular region (John-

son et al., 1990), while the inclusion or exclusion of theFGF receptor whose expression is not modulated byFGF-2 at any stage of OL development. Since FGFR-3 ‘‘acid box’’ generates isoforms b1 and b2, respectively.

Loop-I and the sequences between Loop-I and Loop-IIhas a much higher affinity for FGF-1 than for FGF-2 (Or-nitz and Leder, 1992), FGF-1 may be the preferred ligand (which include the acid box) are not needed for ligand-

binding activity (Johnson et al., 1990), although their ex-for FGFR-3 in vivo. Studying the effect of FGF-1 on FGFR-3 expression will provide further insight. clusion from FGFR-1 (b isoform) significantly increases

the binding affinities for FGF and heparin ligands (WangBased on the available data, we propose the followinghypothetical model to explain the varying response of et al., 1995b). The a and b isoforms of FGFR-1 and -2 are

coexpressed in a variety of cell lines in proportions thatOLs as a function of lineage progression (Fig. 6). At theearly progenitor stage, FGF-2 up-regulates both FGFR-1 generally vary in a tissue-specific manner (Johnson et al.,

1990; Werner et al., 1992). Only the a isoform of FGFR-(these data) and PDGFa receptor (McKinnon et al., 1990)mRNA expression, which together transduce mitogenic 3 and -4 has been reported to date (Jaye et al., 1992).

Here we have shown that in terminally differentiatedand migratory signals in these early progenitors; FGFR-3 mRNA is maximally expressed by late progenitors, OLs, FGFR-1 is expressed primarily as the a isoform

transcript, whereas FGFR-2 is expressed primarily as thewhere its activation signals these cells to proliferate andmaintain this developmental stage; FGFR-2, unique in its b2 isoform transcript. Since the b isoform of FGFR-1


272 Bansal et al.

FIG. 6. Hypothetical model for the regulation of OL-lineage cells by FGF receptors. Red line, FGFR-1. Yellow line, FGFR-2. Blue line, FGFR-3.Vertical dashed lines with arrowheads, up- or down-regulation of FGFR mRNA expression by FGF-2. The ‘‘Receptor Expression’’ only comparesrelative mRNA levels among the three stages for a given receptor type, not the abundance of the different receptors relative to each other.

binds both FGF and heparin with greater affinity than affinity binding of FGF-2 to FGFR-1 and -2, but low-affinity binding to FGFR-3 (Fig. 3) (Werner et al., 1992;the a isoform (see above), and assuming that the pattern

of ligand specificity for FGFR-2 is similar, FGFR-2 may Miki et al., 1992; Ornitz and Leder, 1992).In order to determine the ligand-binding specificity ofbe the more active receptor at this stage of OL differentia-

tion. In addition, a third apparent FGFR-2 isoform tran- the FGF receptors expressed by OLs, we examined themRNA expression of exon IIIc isoforms of FGFR-2 andscript was detected in mature OLs, the identity of which

is under investigation. -3. We found that both receptors utilized exon IIIc andpredict, therefore, that in OLs FGFR-2 binds both FGF-A second type of variant results from differences in

the carboxy-terminal of Ig-like domain III. This domain 1 and FGF-2 with high affinity, and that FGFR-3 bindsFGF-1 with high affinity but FGF-2 with low affinity.can be coded by exon IIIa, IIIb, or IIIc, generating alterna-

tively spliced isoforms of FGFR-1, -2, and -3 (reviewed Whether OLs express, in addition, transcripts for the IIIaand IIIb isoforms of FGFR-1 and -2 is not known; bothin Johnson and Williams, 1993). The replacement of exon

IIIc by IIIb dramatically alters both the FGF binding af- of these forms have been reported in the brain in lowerproportions than the IIIc isoform (Werner et al., 1992).finity and its specificity to that of the ‘‘minimal ligand-

binding region’’ of the receptor (Wang et al., 1995a). For Further defining the expression of FGFR variant formsduring growth and differentiation will lead to additionalexample, the IIIb and IIIc variants of FGFR-1, -2, and -3

all bind FGF-1 with high affinity; the IIIb variant of insights regarding the identity of the preferred ligandsand functions in vivo.FGFR-2 binds FGF-7 with high affinity, but FGF-2 with

lower affinity. Conversely, the IIIc variant leads to high- Knowledge of the functional specificity of the FGF re-



ceptors and their isoforms is still limited. Nevertheless, labeling cells with a panel of antibodies (Bansal and Pfeif-fer, 1994) (see Results). Astrocytes isolated from mono-our finding that OLs and their progenitors exhibit a vari-

ety of FGF receptor types, in various alternatively spliced layer cultures left after releasing OL progenitors frommixed primary cultures (above) (Gard et al., 1993) wereforms, the expression of which changes with differentia-

tion, indicates that a large variety of ligand–receptor in- ú99% positive for the astrocytic marker glial fibrillaryacidic protein.teractions is possible. In addition, the levels of expression

of these receptors are selectively regulated by the ligand.Further, we have found that heparan sulfate proteogly-

RNA Isolation and Northern Blottingcans belonging to the syndecan and GPI-linked families(putative FGF coreceptors) are also differentially regu- Total cellular RNA was isolated by acid guanidium

thiocyanate–phenol–chloroform extraction (Chomczyn-lated as a function of development and by FGF-2 (Bansalet al., 1996). The combinatorial complexity created by this ski and Sacchi, 1987). For Northern blots (Ausubel et

al., 1991), radioactive probes were prepared by randomchanging pattern of expression of the multiple compo-nents of the FGF signaling system during growth and priming (Prime-It II; Stratagene), purified on NucTrap

push-columns (Stratagene), and hybridized in the pres-differentiation is reflected in, and may in fact be responsi-ble for, the varied response of cells to FGF-2 as they ence of salmon sperm DNA. Membranes were washed

twice (21 SSC/0.1% SDS, 5 min, room temperature; 21progress through their lineage.SSC/1% SDS, 30 min, 627C; 0.11 SSC, 30 min, room tem-perature). Blots were quantified with the Packard InstantImager 2024 (Packard Canberra) and exposed (24–72 h,EXPERIMENTAL METHODS0707C) to Kodak X-Omat film with intensifying screen.Blots were stripped (0.11 SSC, 1% SDS, 1007C, 15 min)Cell Culture and Phenotypic Characterizationand reprobed for GAPDH to normalize for RNA loading.cDNA probes used were mouse FGFR-1 (1.3-kb EcoRI–OL progenitors obtained from mixed primary cultures

from neonatal rat telencephalon by overnight shaking BamHI fragment; Mansukhani et al., 1990), mouse FGFR-2 (1.2-kb PvuII fragment; Mansukhani et al., 1992), mouse(McCarthy and DeVellis, 1980) were further enriched

(§97%) by differential adhesion and complement lysis FGFR-3 (430-bp EcoRI–HindIII fragment; Ornitz andLeder, 1992), and mouse FGFR-4 (1-kb EcoRI fragment;and were grown in a serum-free, defined medium (mN2)

(Gard et al., 1993; Bansal and Pfeiffer, 1994). Enriched Partanen et al., 1991).populations of cells under six different experimental con-ditions were obtained by the use of the following growth

RNA Polymerase Chain Reactionconditions: O2A (without FGF stimulation), cells weremitotically expanded for 1 day in mN2 supplemented First-strand cDNA synthesis used 5 mg total RNA

(cDNA synthesis kit and oligo(dT) primers from Stra-with PDGF-BB (Upstate Biotechnology; 10 ng/ml); earlyprogenitors (with FGF stimulation), cells were expanded tagene). Primers (Fig. 3): FGFR-1, 5* nt 50–70, 3* nt 480 –

500 (Yazaki et al., 1994); FGFR-2, 5* nt 113–136, 3* ntand arrested at this stage by growth in PDGF plus FGF-2 (Upstate Biotechnology; 10 ng/ml) for 2 days (Bogler et 1196–1217 (Dionne et al., 1990; Yamaguchi et al., 1994);

FGFR-3, 5* nt 970–990, 3* nt 1095–1115 (Chellaiah et al.,al., 1990); Pro-OLs (without FGF stimulation), following 2days of expansion in PDGF/FGF-2 (above), cells were 1994; P. Prinos and P. Tsipuroas, University of Connecti-

cut). RT-PCR was performed (GenAmpPCR kit; Perkingrown in mN2 alone for 1–2 days to allow them to differ-entiate to the late progenitor stage; late progenitors (with Elmer) (Yamaguchi et al., 1994). As a control, PCR ampli-

fication of GAPDH cDNA was performed on each sam-FGF stimulation), after the initial expansion of the earlyprogenitor population in PDGF/FGF-2 (above), cells ple using the same amount and batch of cDNA and con-

ditions (primers: 5* nt 27–46, 3* nt 238–257). RT-PCRwere grown for an additional 3–4 days in FGF-2, whichblocks lineage progression at the late progenitor stage products were separated on 1.5% agarose gels and the

band positions were aligned with a control cDNA (S45Y(McKinnon et al., 1990; Bansal and Pfeiffer, 1994); matureOLs (without FGF stimulation), early progenitors were cells for FGFR-2) and molecular weight markers. Each

sample was assayed at least three times, resulting in theexpanded in PDGF/FGF-2 (above), then grown in mN2

without growth factors for 5–7 days to allow them to amplification of the same fragments. For FGFR-2, thePCR-amplified products were further analyzed by South-differentiate into mature OLs; mature OLs (with FGF

stimulation), mature OLs (above) were grown in 10 ng/ ern blot hybridization using a radiolabeled internal se-quence oligonucleotide (nt 192–212). Signal intensityml FGF-2 for 2 days before harvesting. Cell purity and

phenotypic characteristics were determined by immuno- was measured by phosphorimaging, and data were nor-


274 Bansal et al.

stimulated by basic fibroblast growth factor and inhibited by oligo-malized to similarly analyzed GAPDH levels for FGFR-dendrocyte-type 2 astrocyte precursors. Dev. Biol. 158: 317– 329.2 (data not shown).

Fressinaud, C., Vallat, J. M., and Labourdette, G. (1995). Basic fibroblastgrowth factor down-regulates myelin basic protein gene expressionand alters myelin compaction of mature oligodendrocytes in vitro. J.Neurosci. Res. 40: 285– 293.ACKNOWLEDGMENTS

Gard, A. L., and Pfeiffer, S. E. (1993). Glial cell mitogens bFGF andPDGF differentially regulate development of O4/GalC0 oligodendro-Supported by grants from the NIH (NS10861) and the National Mul-cyte progenitors. Dev. Biol. 159: 618– 630.tiple Sclerosis Society (RG2182B-6). We thank Janice Seagren for word

Gard, A. L., Pfeiffer, S. E., and Williams, W. C., II (1993). Immunopan-processing, Ezinna Anosike for help with immunofluorescence analy-ning and developmental stage-specific primary culture of oligoden-ses, and our colleagues for valuable discussions (T. Kim, D. Madison,drocyte progenitors (O4/GalC0) directly from postnatal rodent cere-W. Kruger, E. Joly, J. P. Vos, and M. Hurley).brum. NeuroProtocols 2: 209– 218.

Gogate, N., Verma, L., Zhou, J. M., Milward, E., Rusten, R., O’Connor,M., Kufta, C., Kim, J., Hudson, L., and Dubois-Dalcq, M. (1994). Plas-ticity in the adult human oligodendrocyte lineage. J. Neurosci. 14:REFERENCES4571– 4587.

Grinspan, J. B., Stern, J. L., Franceschini, B., and Pleasure, D. (1993).Asai, T., Wanaka, A., Kato, H., Masana, Y., Seo, M., and Tohyama, M.Trophic effects of basic fibroblast growth factor (bFGF) on differenti-(1993). Differential expression of two members of FGF receptor geneated oligodendroglia: A mechanism for regeneration of the oligoden-family, FGFR-1 and FGFR-2 mRNA, in the adult rat central nervousdroglial lineage. J. Neurosci. Res. 36: 672–680.system. Mol. Brain Res. 17: 174–178.

Hart, I. K., Richardson, W. D., Heldin, C. H., Westermark, B., and Raff,Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G.,M. C. (1989). PDGF receptors on cells of the oligodendrocyte-type-2Smith, J. A., and Struhl, K., Eds. (1991). Current Protocols in Molecularastrocyte (O-2A) cell lineage. Development 105: 595–603.Biology. Wiley, New York.

Iwamoto, M., Shimadzu, A., Nakashima, K., Susuki, F., and Kato, Y.Bansal, R., and Pfeiffer, S. E. (1994). Inhibition of protein and lipid(1991). Reduction in basic fibroblast growth factor receptor is coupledsulfation in oligodendrocytes blocks biological responses to FGF-2with terminal differentiation of chondrocytes. J. Biol. Chem. 266: 461–and retards cytoarchitectual maturation but not developmental lin-467.eage progression. Dev. Biol. 162: 511–524.

Jaye, M., Schlessinger, J., and Dionne, C. A. (1992). Fibroblast growthBansal, R., Kumar, M., Murray, K., and Pfeiffer, S. E. (1996). Develop-factor receptor tyrosine kinases: Molecular analysis and signal trans-mental and FGF-2-mediated regulation of syndecans (1 –4) and glypi-duction. Biochim. Biophys. Acta 1135: 185–199.can in oligodendrocytes. Mol. Cell. Neurosci. 7: 276– 288.

Johnson, D. E., and Williams, L. T. (1993). Structural and functionalBogler, O., Wren, D., Barnett, S. C., Land, H., and Noble, M. (1990).diversity in the FGF receptor multigene family. Adv. Cancer Res. 60:Cooperation between two growth factors promotes extended self-1–41.renewal and inhibits differentiation of oligodendrocyte-type-2-

Johnson, D. E., Lee, P. L., Lu, J., and Williams, L. T. (1990). Diverseastrocyte (O-2A) progenitor cells. Proc. Natl. Acad. Sci. USA 87: 6368–forms of a receptor for acidic and basic fibroblast growth factors.6372.Mol. Cell. Biol. 10: 4728– 4736.Chellaiah, A. T., McEwen, D. G., Werner, S., Xu, J., and Ornitz, D. M.

Johnson, D. E., Lu, J., Chen, H., Werner, S., and Williams, L. T. (1991).(1994). Fibroblast growth factor receptor (FGFR) 3: Alternative splic-The human fibroblast growth factor receptor genes: A common struc-ing in immunoglobin-like domain III creates a receptor highly specifictural arrangement underlies the mechanisms for generating receptorfor FGF/FGF-1. J. Biol. Chem. 269: 11620 –11627.forms that differ in their third immunoglobulin domain. Mol. Cell.Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNABiol. 11: 4627– 4634.isolation by acid guanidium thiocyanate–phenol –chloroform extrac-

Keegan, K., Johnson, D. E., Williams, L. T., and Hayman, M. J. (1991).tion. Anal. Biochem. 162: 156–159.Isolation of an additional member of the fibroblast growth factorDeloulme, J. C., Janet, T., Pettmann, B., Laeng, P., Knoetgen, M-F.,receptor family, FGFR-3. Proc. Natl. Acad. Sci. USA 88: 1095 –1099.Sensenbrenner, M., and Baudier, J. (1992). Phosphorylation of the

Lai, C., and Lemke, G. (1991). An extended family of protein-tyrosineMARCKS protein (P87), a major protein kinase C substrate, is not ankinase genes differentially expressed in the vertebrate nervous sys-obligatory step in the mitogenic signaling pathway of basic fibroblasttem. Neuron 6: 691– 704.growth factor in rat oligodendrocytes. J. Neurochem. 58: 567– 578.

Mansukhani, A., Dell’Era, P., Moscatelli, D., Kornbluth, S., Hanfusa, H.,Dionne, C. A., Crumley, G., Bellot, F., Kaplow, J. M., Searfoss, G., Ruta,and Basilico, C. (1992). Characterization of the murine BEK fibroblastM., Burgess, W. H., Jaye, M., and Schlessinger, J. (1990). Cloning andgrowth factor (FGF) receptor: Activation by three members of theexpression of two distinct high-affinity receptors cross-reacting withFGF family and requirement for heparin. Proc. Natl. Acad. Sci. USAacidic and basic fibroblast growth factors. EMBO J. 9: 2685 –2692.89: 3305–3309.Earp, H. S., Hepler, J. R., Petch, L. A., Miller, A., Berry, A. R., Harris,

Mansukhani, A., Moscatelli, Talarico, D., Levytska, V., and Basilico, C.J., Raymond, V. W., McCune, B. K., Lee, L. W., Grisham, J. W., and(1990). A murine fibroblast growth factor (FGF) receptor expressedHarden, T. K. (1988). Epidermal growth factor (EGF) and hormonesin CHO cells is activated by basic FGF and Kaposi FGF. Proc. Natl.stimulate phosphoinositide hydrolysis and increase EGF receptorAcad. Sci. USA 87: 4378 –4382.protein synthesis and mRNA levels in rat epithelial cells: Evidence

Mayer, M., Bogler, O., and Noble, M. (1993). The inhibition of oligoden-for protein-kinase C-dependent and -independent pathways. J. Biol.drocytic differentiation of O-2A progenitors caused by basic fibro-Chem. 263: 13868 –13874.blast growth factor is overridden by astrocytes. Glia 8: 12–19.Eckenstein, F. P. (1994). Fibroblast growth factors in the nervous system.

McCarthy, K. D., and DeVellis, J. (1980). Preparation of separate astro-J. Neurobiol. 11: 1467 –1480.glial and oligodendroglial cell cultures from rat cerebral tissue. J. CellFressinaud, C., Laeng, P., Labourdette, G., Durand, J., and Vallat,

J. M. (1993). The proliferation of mature oligodendrocytes in vitro is Biol. 85: 890– 902.



McKinnon, R. D., Matsui, T., Dubois-Dalcq, M., and Aaronson, S. A. skeletal muscle growth and differentiation are controlled by a highaffinity receptor, FGFR-1. Dev. Biol. 154: 169– 181.(1990). FGF modulates the PDGF-driven pathway of oligodendrocyte

Wanaka, A., Milbrandt, J., and Johnson, E. M. (1991). Expression of FGFdevelopment. Neuron 5: 603– 614.receptor gene in rat development. Development 111: 455– 468.Miki, T., Bottaro, D. P., Fleming, T. P., Smith, C. L., Burgess, W. H.,

Wang, F., Kan, M., Jianming, X., Yan, G., and McKeehan, W. L. (1995a).Chan, A. M., and Aaronson, S. A. (1992). Determination of ligand-Ligand-specific structural domains in the fibroblast growth factorbinding specificity by alternative splicing: Two distinct growth factorreceptor. J. Biol. Chem. 270: 10222– 10230.receptors encoded by a single gene. Proc. Natl. Acad. Sci. USA 89:

Wang, F., Kan, M., Yan, G., Xu, J., and McKeehan, W. L. (1995b). Alter-246– 250.nately spliced NH2-terminal immunoglobulin-like loop I in the ecto-Ono, K., Bansal, R., Payne, J., Rutishauser, U., and Miller, R. H. (1995).domain of the fibroblast growth factor (FGF) receptor 1 lowers affin-Early development and dispersal of oligodendrocyte precursors inity for both heparin and FGF-1. J. Biol. Chem. 270: 10231 –10235.embryonic chick spinal cord. Development 121: 1743– 1754.

Wang, J., Gao, G., and Goldfarb, M. (1994). Fibroblast growth factorOrnitz, D. M., and Leder, P. (1992). Ligand specificity and heparinreceptors have different signaling and mitogenic potentials. Mol. Cell.

dependence of fibroblast growth factor receptors 1 and 3. J. Biol.Biol. 1: 181–188.

Chem. 23: 16305–16311. Warrington, A. E., and Pfeiffer, S. E. (1992). Proliferation and differenti-Orr-Urtreger, A., Givol, D., Yayon, A., Yarden, Y., and Lonai, P. (1991). ation of O4/ oligodendrocytes in postnatal rat cerebellum: Analysis

Developmental expression of two fibroblast growth factor receptors, in unfixed tissue slices using anti-glycolipid antibodies. J. Neurosci.flg and bek. Development 113: 1419 –1434. Res. 33: 338–353.

Partanen, J., Makela, T. P., Eerola, E., Korhonen, J., Hirvonen, H., Claes- Werner, S., Duan, D-S., DeVries, C., Peters, K. G., Johnson, D. E., andson-Welsh, L., and Alitalo, K. (1991). FGFR-4, a novel acidic fibroblast Williams, L. T. (1992). Differential splicing in the extracellular regiongrowth factor receptor with a distinct expression pattern. EMBO J. of fibroblast growth factor receptor 1 generates receptor variants with6: 1347 –1354. different ligand-binding specificities. Mol. Cell. Biol. 12: 82– 88.

Peters, K., Werner, S., Chen, G., and Williams, L. T. (1992). Two FGF Williams, E. J., Furness, J., Walsh, F. S., and Doherty, P. (1994). Activa-receptor genes are differentially expressed in epithelial and mesen- tion of the FGF receptor underlies neurite outgrowth stimulated by

L1, N-CAM, and N-cadherin. Neuron 13: 583–594.chymal tissues during limb formation and organogenesis in theYamaguchi, F., Saya, H., Bruner, J. M., and Morrison, R. S. (1994). Differ-mouse. Development 114: 233–243.

ential expression of two fibroblast growth factor-receptor genes isPeters, K., Ornitz, D., Werner, S., and Williams, L. (1993). Unique ex-associated with malignant progression in human astrocytomas. Proc.pression pattern of the FGF receptor 3 gene during mouse organogen-Natl. Acad. Sci. USA 91: 484– 488.esis. Dev. Biol. 155: 423–430.

Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M.Pfeiffer, S. E., Warrington, A. E., and Bansal, R. (1993). The oligodendro-(1991). Cell surface, heparin-like molecules are required for bindingcyte and its many cellular processes. Trends Cell Biol. 3: 191–197.of basic fibroblast growth factor to its high affinity receptor. Cell 64:Rapraeger, A. C., Krufka, A., and Olwin, B. B. (1991). Requirement of841–848.heparan sulfate for bFGF-mediated fibroblast growth and myoblast

Yazaki, N., Hosoi, Y., Kawabata, K., Miyake, A., Minami, M., Satoh, M.,differentiation. Science 252: 1705 –1708.Ohta, M., Kawasaki, T., and Itoh, N. (1994). Differential expression

Rutishauser, U., and Jessell, T. M. (1988). Cell adhesion molecules inpatterns of mRNAs for members of the fibroblast growth factor recep-

vertebrate neural development. Physiol. Rev. 68: 819–857. tor family, FGFR-1-FGFR-4, in rat brain. J. Neurosci. Res. 37: 445–452.Saito, H., Kasayama, S., Kouhara, H., Matsumoto, K., and Sato, B. (1991). Yong, V. W., Dooley, N. P., and Noble, P. G. (1994). Protein kinase C

Up-regulation of fibroblast growth factor (FGF) receptor mRNA lev- in cultured adult human oligodendrocytes: Characteristics, lack ofels by basic FGF or testosterone in androgen-sensitive mouse mam- down-regulation and isoform a as a mediator of fiber outgrowth. J.mary tumor cells. Biochem. Biophys. Res. Commun. 174: 136– 141. Neurosci. Res. 39: 83– 96.

Shaoul, E., Reich-Slotky, R., Berman, B., and Ron, D. (1995). Fibroblast Yuguchi, T., Kohmura, E., Yamada, K., Wanaka, A., Otsuki, H., Saka-growth factor receptors display both common and distinct signaling guchi, T., Yamashita, T., Tohyama, M., and Hayakawa, T. (1994).pathways. Oncogene 10: 1553 –1561. Messenger RNA and protein expression of basic fibroblast growth

factor receptor after cortical ablation. Mol. Brain Res. 25: 50–56.Templeton, T. J., and Hauschka, S. D. (1992). FGF-mediated aspects of

Received February 16, 1996Revised April 1, 1996

Accepted April 11, 1996


Regulation of FGF receptors in the oligodendrocyte lineage

Documents

Transcript of Regulation of FGF receptors in the oligodendrocyte lineage