Regulation of FGF Receptors in the Oligodendrocyte Lineage

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Molecular and Cellular Neuroscience 7, 263– 275 (1996) Article No. 0020

Regulation of FGF Receptors in the Oligodendrocyte 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 of developmentally regulated cellular responses involved in the control of growth and differentiation. To identify specific FGF receptor forms involved in these responses, we have characterized FGF receptor transcript expression, and its modulation by FGF-2, as enriched populations of oligodendrocyte progenitors differentiate into mature oligodendrocytes. The data demonstrate that the levels of mRNA expression for FGF high-affinity receptors1, -2, and -3 are differentially regulated during lineage progression: FGF receptor-1 expression increases with lineage progression, FGF receptor-2 is predominantly expressed by terminally differentiated oligodendrocytes, and FGF receptor-3 reaches a peak level of expression in late progenitors and then declines upon further differentiation; FGF receptor-4 expression was not detected in oligodendrocytes. Distinct patterns of alternatively spliced variants of FGF receptor-1 and -2 transcripts are expressed: the predominant FGF receptor-1 transcripts contain three Ig-like domains (FGF receptor-1a), whereas the FGF receptor-2 transcripts contain two Ig-like domains (FGF receptor-2b2) and this form is up-regulated as oligodendrocytes differentiate. In addition, the expression of these receptors is differentially regulated by the ligand, FGF-2: FGF receptor-1 mRNA expression is up-regulated in early progenitors, and FGF receptor-2 mRNA expression is down-regulated in mature oligodendrocytes. Finally, astrocytes express FGF receptor-1, -2, and -3 transcripts, but at different levels and with different exon utilization (FGF receptor-1b, FGF receptor-2b1/b2) compared to oligodendrocytes. To our knowledge this is the first report that demonstrates that the mRNA expression of these three FGF receptor types is differentially regulated in primary cells as they differentiate along a lineage from progenitors to terminally differentiated cells. We propose that this pattern of expression provides a molecular basis for the developmentally varying response of cells to a common ligand. For example, according to this hypothe-

sis, in response to FGF-2, FGF receptor-1 transduces signals that stimulate the prolonged proliferation and migration of early progenitors, FGF receptor-3 promotes the proliferation and arrest of differentiation of late progenitors, and FGF receptor-2 transduces signals for terminal differentiation, but not proliferation, in mature oligodendrocytes.

INTRODUCTION The interrelationship between proliferation and differentiation is a cornerstone of developmental regulation. Fibroblast growth factors (FGFs) are a structurally related family of nine peptides acting at this interface. They target a broad spectrum of cell types derived from mesoderm and neuroectoderm, inducing a variety of biological activities that include either inhibition or stimulation of differentiation, proliferation, and chemotaxis (reviewed in Eckenstein, 1994). The corresponding highaffinity FGF receptors (FGFRs) are a diverse family of tyrosine kinases for which four separate genes (FGFR 1 – 4) have been identified (reviewed in Johnson and Williams, 1993). The basic structure of FGFRs consists of an extracellular ligand-binding region with three immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tyrosine kinase domain. Some FGFRs bind, and are activated by, multiple FGFs, whereas others have a higher degree of specificity (Ornitz and Leder, 1992; Partanen et al., 1991; Johnson and Williams, 1993). Multiple-splice variants of FGFR-1, -2, and -3 can have dramatically altered ligand specificities and binding affinities (Miki et al., 1992; Werner et al., 1992; Johnson et al., 1991). FGF receptor expression has differential spatial distribution during embryogenesis (Peters et al., 1992; Orr-Ur-

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

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treger et al., 1991) and in adult tissues (Asai et al., 1993; Yazaki et al., 1994). In addition, a second class of cell surface components essential for FGF signaling have been identified, a family of low-affinity (Kd 2 – 20 nM ), high-capacity heparan sulfate proteoglycans (Yayon et al., 1991; Rapraeger et al., 1991). With at least nine similar but distinct ligands, four receptors, and their multiple variant isoforms, plus an extended family of coreceptors, it is clear that a cell has ample opportunity to modulate its response to FGF(s) by regulating the combinatorial complexity of a diverse set of cell surface receptors. Oligodendrocytes (OLs) are emerging as a prominent model in which to examine key issues of cell biology in a developmental context (Pfeiffer et al., 1993). OLs form myelin in the central nervous system through an elaboration of their plasma membranes which wrap around neuronal axons to form compact, multilamellar sheaths. The progression of OL progenitors along a carefully regulated lineage, characterized by changes in their morphology and proliferative and migratory capacity, and the ordered expression of OL and myelin-specific proteins and lipids, can be formalized as: [early progenitors (O2A)] r [late progenitors (Pro-OLs)] r [mature OLs (OLs)]. Early progenitors are proliferative and migratory cells; late progenitors are proliferative but nonmigratory cells; mature OLs are postmitotic, terminally differentiated cells expressing myelin proteins and lipids. Purified OL progenitors grown in culture follow a developmental pathway consistent with lineage progression in situ (Warrington and Pfeiffer, 1992). OL developmental lineage is regulated by a variety of growth factors, including FGF-2 (basic FGF). However, the nature of the response to FGF-2 varies markedly as a function of the stage of the OL lineage. FGF-2 up-regulates platelet-derived growth factor (PDGF-a) receptors on early progenitors and, in combination with PDGF, supports their long-term proliferation (McKinnon et al., 1990; Bo¨gler et al., 1990); it is mitogenic for late progenitors and reversibly blocks their terminal differentiation (Gard and Pfeiffer, 1993; McKinnon et al., 1990; Mayer et al., 1993; Bansal and Pfeiffer, 1994); it causes an apparent phenotypic reversion of mature OLs (Grinspan et al., 1993; Fressinaud et al., 1995). We hypothesized that multiple FGF receptors and coreceptors are expressed in a stage-specific manner, accounting in part at least for the variety of responses of OL-lineage cells to FGF-2. In order to test this hypothesis, we have investigated the developmental expression of these receptors and its ligand-mediated regulation. The data demonstrate that during lineage progression, multi-

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ple FGF high-affinity receptor transcripts in specific alternatively spliced isoforms are expressed in a developmentally regulated manner. These results, combined with our observations that multiple putative FGF coreceptors (syndecans 1 – 4, glypican; Bansal et al., 1996) are also developmentally regulated in these cells, suggest that there is a changing repertoire of these signaling molecules on the cell surface as they progress through the developmental lineage. In addition, the data show that FGF-2 itself regulates the mRNA levels of these receptors at specific stages during development.

RESULTS Phenotypic Characterization of Enriched Populations of Cells of the Oligodendrocyte Lineage Highly enriched populations of OL-lineage cells were prepared at three stages of differentiation. The transition from O2A to Pro-OL was marked by a change in morphology from bipolar to multipolar cells, followed by further process and membrane elaboration as the cells differentiated into postmitotic, mature OLs (Fig. 1B). The phenotypic compositions are shown (Fig. 1A). In the absence of FGF-2 (0FGF), the ‘‘early progenitor’’ populations were a mixture of Ç70% early progenitors and Ç30% late progenitors (see Fig. 1 legend) and Ç1% OLs; the ‘‘late progenitor’’ populations were a mixture of Ç80% late progenitors and Ç5% OLs; the ‘‘mature OL’’ population was Ç75% O1/ and Ç54% MBP/. In the presence of FGF-2 (/FGF), because of the synchronizing effect of FGF-2 on developmental progression, progenitor populations with enhanced homogeneity were obtained. ‘‘Early progenitor’’ populations were Ç95% early progenitors and only Ç5% late progenitors; ‘‘late progenitor’’ populations were Ç80% late progenitors and Ç1% OLs; on the other hand, ‘‘mature OL’’ populations treated with FGF-2 for 2 days were reduced in homogeneity to Ç40% O1 / and Ç15% MBP/ cells due to an apparent induction of phenotypic reversion (Introduction). Astrocytes constituted 1 – 6% of the cell populations, depending on conditions of growth.

Oligodendrocytes Express Multiple FGF Receptor Types during Differentiation in Culture We evaluated by Northern blot analyses the expression of individual FGF receptor mRNA by OLs as they undergo growth and terminal differentiation (Figs. 2A and 2B, black bars, 0FGF). Since members of the FGFR family are encoded by genes that show an overall amino

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FIG. 1. Phenotypic characterization of enriched populations of OL-lineage cells grown in the absence or presence of FGF-2. (A) Quantitative distribution of phenotypes within cell populations. Cells were characterized by immunostaining with stage-specific markers. The percentage of total cells that were immunostained with antibodies O4 (Pro-OL / OL marker, black columns), O1 (OL marker, striped columns), anti-MBP (more mature OL marker, spotted columns), or anti-GFAP (astrocyte marker) is shown. 97 –98% of total cells were stained with antibody A2B5 at the two 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 OL lineage used to generate the data in A. Bar, 25 mm.

acid sequence homology of 60 – 70%, specific cDNA probes and hybridization conditions were used that prevent cross-hybridization of the different receptors. The specificity of these probes for the specific receptor type has been established previously (Mansukhani et al., 1990, 1992; Partanen et al., 1991; Ornitz and Leder, 1992). Hybridization with an FGFR-1 probe revealed the expected 4.2-kb species (Templeton and Hauschka, 1992). FGFR-1 mRNA was expressed at the highest levels in mature OLs and in early and late progenitors at levels

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of Ç30 and 50%, respectively. Enriched OL populations showed a 2.3-fold increase in message level over that in P2 brain. We conclude that FGFR-1 transcripts are expressed throughout the OL lineage. Hybridization with an FGFR-2 probe yielded the expected major band at 4.4 kb (Dionne et al., 1990). FGFR-2 expression was barely detectable in early and late progenitors, but was up-regulated (ú10-fold) as OLs entered terminal differentiation. Expression of FGFR-2 was detected in P2 and P5 (not shown) brain at a much lower level than

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in OLs. A second RNA band of Ç1 kb was repeatedly detected only in mature OLs at a level of Ç30% of the major band that could in principle represent a message for a soluble form of the receptor (Johnson and Williams, 1993). We conclude that FGFR-2 is primarily a mature OL receptor and that its developmental expression parallels that of the other major myelin-specific proteins and lipids. Hybridization with a probe for FGFR-3 demonstrated the expected 4.2-kb transcript (Keegan et al., 1991). Unlike FGFR-1 and -2 transcripts that were maximally expressed upon terminal differentiation, FGFR-3 reached peak levels of expression at the late progenitor stage, and then declined upon further terminal differentiation. Expression of FGFR-3 was also detected in adult brain at somewhat higher levels than in OLs. We conclude that FGFR-3 transcripts are maximally expressed by late progenitors. Hybridization with a probe for FGFR-4 resulted in the expected band of Ç3.2 kb in lung and liver (Partanen et al., 1991), but not in OL-lineage cells or brain, consistent with in situ hybridization studies showing a restricted spatial expression of FGFR-4 in the brain (Yazaki et al., 1994) (although GAPDH controls were not available for the example shown in Fig. 2, subsequent analysis of this blot with other probes demonstrated that the RNA and blot were of high quality; other independent blots with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) controls produced the same results). The FGFR-4 probe did hybridize to another smaller RNA band, the identity of which is not known, in all lineage stages of OLs and in brain, but not in liver or lung. We conclude that OLlineage cells do not express the 3.2-kb FGFR-4 transcript.

Oligodendrocytes Express Alternatively Spliced Variants of FGF Receptors FGFR-1, -2, and -3 can be expressed in multiple variant forms arising from alternative splicing of mRNA transcripts (Fig. 3). Receptor variants lack Ig-like domain I, exhibit differences in exon III utilization (exons IIIa, b, c) in the carboxy-terminal half of the third Ig-like do-

main, or contain deletions and insertions within the extracellular or intracellular domains (reviewed in Johnson and Williams, 1993). We determined by nonquantitative RT-PCR if OL-lineage cells expressed variant transcripts of FGF receptors not distinguishable on Northern blots. For RT-PCR amplification of FGFR-1 mRNA, we used a 5*-primer located in the signal sequence and a 3*-primer located between Ig-domains I and II, beyond the ‘‘acid box’’ (Fig. 3). This primer set should identify receptor transcripts that encode for isoforms including either three (FGFR-1a) or two (FGFR-1b) Ig-like domains, producing fragments of 450 and 184 bp, respectively. Amplification of cDNA prepared from early or late progenitors, mature OLs, and brain all produced prominent 450-bp fragments (a very faint band under the FGFR-1a isoform is nonspecific) (Fig. 4A). The expression of transcripts for FGFR-2a and -2b isoforms was examined by RT-PCR (Fig. 4B) and Southern blot analysis using radiolabeled internal sequence oligonucleotides (Fig. 4Da, b). The 5*-primer was located in the untranslated region and a 3*-primer in Ig-like domain III (Fig. 3). These primers should amplify three FGFR2-specific fragments of 1100 bp (FGFR-2a, three Ig-like domains), 900 bp (FGFR-2b1, two Ig-like domains), and 800 bp (FGFR-2b2, two Ig-like domains, but lacking the acid box) (Yamaguchi et al., 1994). In the early and late progenitors, we detected low levels of FGFR-2a, -2b1, and -2b2 isoforms in the ratio 1:1:2.5 (this was clear upon longer exposure of the autoradiograms). In contrast, mature OLs expressed transcripts for FGFR-2a and -2b2 in the ratio 1:35, whereas evidence for FGFR-2b1 was absent (confirmed by the absence of FGFR2-b1 in Southern blot hybridizations; Fig. 4Db). These FGFR-2a, -b1, and -b2 fragments were also amplified from adult rat brain cDNA (Fig. 4B) and a positive control cDNA (Fig. 4B, 4Db). In addition, we detected in mature OLs a PCR fragment of Ç950 bp that may represent another variant form of FGFR-2 (Fig. 4B, asterisk) (confirmed by Southern hybridization with an internal sequence oligonucleotide 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 Northern blotting. (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 whole brain (P2 BR) or adult telencephalon (Ad Tel). Total RNA (20 mg) was loaded per lane and the blots were hybridized with cDNA probes specific for each of the receptor types. Blots were then rehybridized with a probe for GAPDH. Note that the order of minus and plus FGF addition is reversed 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 was set at 100%). Since the FGFR cDNA probes have different specific activities, the values can be used only to compare relative mRNA levels among the 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 the OL-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 also recognized a lower band in the OL (0FGF)-stage population; the quantification does not include this band, which is Ç30% of the known, 4.4-kb FGFR-2 band (arrow). Error bars, standard errors of the mean (N Å 3– 6) or ranges of duplicates.

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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, Iglike domains; AB, acid box; TK1 and TK2, tyrosine kinase domains; KI, kinase insert; CT, cytoplasmic tail. The ligand-binding specifications determined by the use of exon IIIb and IIIc are indicated.

tions; Fig. 4Db, asterisk). The Ç950-kb fragment was also detected in rat adult brain (Fig. 4B), albeit more weakly. We therefore compared areas of adult rat brain that were either enriched (brain stem) or relatively low (forebrain gray matter) in oligodendrocytes and myelin. An amplified band was readily detected by Southern blot analysis in the material from brain stem but not from gray matter (Fig. 4Da, asterisk). Since the choice of exon IIIb or IIIc determines ligandbinding specificity (Miki et al., 1992; Werner et al., 1992), we examined the expression of exon IIIc in the FGFR-2 and -3 transcripts. Since the primers chosen for RT-PCR (Fig. 3) will identify exon IIIc utilization, the appearance of amplified products for FGFR-2 (Fig. 4B) and -3 (Fig. 4C; 145 bp) indicates that they did in fact contain exon IIIc sequences. Based on previous analyses of exon IIImediated ligand-binding specificity, these data suggest that FGFR-2 expressed by OLs is capable of binding both FGF-1 and -2 with equally high affinity (Fig. 3), and that FGFR-3 can bind FGF-1 at higher affinity than FGF-2 (Ornitz and Leder, 1992). The presence of FGFR-3 was further confirmed in OL-lineage cells and astrocytes by amplification of its transcripts utilizing a second set of PCR primers spanning a region of Ig-like domain II and interdomain region II/III (data not shown).

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We conclude that multiple isoforms of FGFR mRNA are expressed by OL-lineage cells and that a potentially novel FGFR-2 mRNA transcript is expressed by mature OLs.

The Expression of FGF Receptors Is Regulated by FGF-2 In order to determine whether the expression of the receptors is regulated by FGF-2 we exposed the cells to FGF-2 for 2 days prior to RNA extraction and Northern blot analysis (Figs. 2A and 2B, hatched bars, /FGF). FGF2 induced an increase in the expression of FGFR-1 mRNA in both early and late progenitors to a common level comparable to the level exhibited by mature OLs with or without growth factor. The expression of FGFR-2 mRNA by early and late progenitors was unaffected by FGF-2, but was strongly down-regulated in mature OLs. The expression of FGFR-3 mRNA was not affected by FGF-2. FGFR-4 mRNA expression was not induced by FGF-2. Therefore, although early progenitors expressed both FGFR-1 and -3 transcripts, FGF-2 regulated the expression of only FGFR-1 in these cells; similarly, although mature OLs expressed all three FGF receptor genes, only the level of FGFR-2 message was regulated by FGF-2.

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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 obtained from 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 culture characterization), 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) using radiolabeled 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-PCR reaction was included in which the template was omitted from the reaction (No Templ). Representative experiments are shown. For FGFR-2, note the 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 analyses were 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, the transcripts of individual FGF receptors are selectively modulated by FGF-2.

Astrocytes Express FGF Receptors The expression of FGF receptor mRNA by cultured astrocytes was analyzed, and the relative levels of expression were compared to that of OL-lineage cells. Alternately spliced isoforms of each receptor were determined by RT-PCR. Astrocytes expressed mRNA transcripts of the predicted sizes for FGFR-1, -2, and -3 (Fig. 2A) but not FGFR-4 (data not shown). The relative levels of expression of FGFR-1 and -3 were 1.5- and 1.8-fold higher, respectively, in astrocytes compared to OL-lineage cells, whereas the levels of FGFR-2 were 2.5-fold higher in OLs (Fig. 5). Astrocytes express predominantly the 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 three types of FGF receptors, the levels of mRNA expression and the isoform patterns of each receptor are different between the two cell types. This also shows that the lowlevel astrocyte contamination (1– 6%) in the OL-lineage

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cell cultures did not contribute to the receptor isoform expression observed by RT-PCR in these cells. Whether FGF receptors are differentially expressed in astrocytes during development, as in OLs, and whether a mechanism of autocrine regulation of these receptors exists, will be of interest since astrocytes also synthesize the ligand.

FIG. 5. Comparison of the relative FGF receptor mRNA expression between astrocytes (hatched bars) and the highest levels of expression observed during the OL lineage (black bars).

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DISCUSSION To examine molecular mechanisms governing sequential developmental responses to FGF, we have studied the regulation of FGF receptor expression by cells as they differentiate through a lineage, using the well-defined OL system. The results demonstrate four main points. First, cells of the OL lineage express FGF high-affinity receptor transcripts -1, -2, and -3, but not -4. Second, the expression of these transcripts is developmentally regulated; specifically, the expression of mRNA for FGFR-1 is increased during lineage progression, for FGFR-3 is transiently up-regulated in the late progenitor stage, and for FGFR-2 is dramatically up-regulated as the progenitors differentiate into mature OLs. Third, FGF-2 up-regulates its own receptor gene expression; for example, FGF-2 up-regulates the expression of FGFR-1 mRNA in progenitors, but down-regulates expression of FGFR2 mRNA in mature OLs. Fourth, distinct patterns of alternatively spliced variants of FGFR-1 and -2 are expressed; specifically, the predominant FGFR-1 transcript contains three Ig-like domains, whereas the FGFR-2 transcripts predominantly contain two Ig-like domains, which are up-regulated as OLs differentiate. The principle conclusion that emerges from these results is that OLs differentially express specific high-affinity FGF receptor transcripts as they undergo lineage progression, leading to a developmentally changing repertoire of these signaling molecules, thus providing a molecular basis for the lineage stage-specific responses of OLs to the common ligand, FGF-2. To our knowledge, this is the first study that has demonstrated that the expression of individual FGF receptors is regulated during lineage progression of primary cells, both as a function of the developmental stage and the availability of the ligand. We predict that these principles will prove to be generally used during the differentiation of other lineages as well.

Developmental Patterns of Expression of FGF Receptor Types Individual FGF receptors have distinct patterns of gene expression during early embryonic development and organogenesis and in adult tissue (Partanen et al., 1991; Orr-Urtreger et al., 1991; Peters et al., 1992, 1993; Yazaki et al., 1994). In in situ hybridization studies of brain, the identity of the cells expressing a particular receptor has been based on their spatial location, morphology, and intensity of Nisel staining, but no distinction was made between the two major glial cell types, OLs and astrocytes. In addition, changes in the expres-

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sion of FGF receptors by cells at distinct stages in a single development lineage of primary cells had not been reported. Because these studies are difficult to perform in vivo, we have taken advantage of a culture system that has allowed us to study FGF receptor expression in highly purified and developmentally synchronized populations of primary cells, at specific, well-defined stages of differentiation. Among the three types of high-affinity FGF receptors (FGFR-1, -2, and -3) expressed during the OL-lineage progression, FGFR-1 was the only one that was up-regulated by its ligand. This effect was restricted to the progenitor stages. In early progenitors, FGF-2 also up-regulates PDGFa receptors (McKinnon et al., 1990). Therefore, the enhanced coexpression of these receptors by FGF-2 may be instrumental in the stimulation of mitotic and migratory capacity that these cells experience in response to the combined administration of PDGF plus FGF-2 (Bo¨gler et al., 1990). Ligand-mediated up-regulation of FGFR-1 mRNA expression has previously been observed for FGF-2 in tumor cells (Saito et al., 1991) and for other growth factors (Earp et al., 1988). Based on in situ hybridization studies, it has been suggested that neurons are the main sites of FGFR-1 expression in the brain (Wanaka et al., 1991; Asai et al., 1993; Yazaki et al., 1994; Peters et al., 1992; Lai and Lemke, 1991), although some expression was also detected in adjacent nonneuronal cells upon ablation of the somatosensory cortex (Yuguchi et al., 1994) and in astrocytomas (Yamaguchi et al., 1994). In the present study, we detected FGFR-1 in highly enriched populations of OLs, perhaps leading to an enhancement of the signal. The pattern of FGFR-2 expression during OL development is particularly interesting. In contrast to the downregulation of FGFR-1 transcripts upon the differentiation of developing myoblasts (Templeton and Hauschka, 1992) and chondrocytes (Iwamoto et al., 1991), and of PDGF a-receptor transcripts in OLs (Hart et al., 1989), the present data show that FGFR-2 mRNA expression is dramatically up-regulated upon terminal differentiation (mirroring the expression of OL differentiation markers such as myelin basic protein). This is consistent with the observation that FGFR-2 mRNA is localized in the myelinated fiber tracts of adult rodent brain (Asai et al., 1993). In addition, FGF-2 has little or no mitogenic effect on mature OLs, but stimulates their process outgrowth (Yong et al., 1994; Gogate et al., 1994; Fressinaud et al., 1993; Bansal et al., unpublished), perhaps via its stimulation of protein phosphorylation of the actin-binding MARKS protein and GAP-43 (Deloulme et al., 1992), proteins involved in the organization of the cytoskeleton. However, an apparent paradox exists, for FGF-2 also down-regulates the expression of FGFR-2 mRNA (these

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data) as well as of myelin-specific genes in these cells (Grinspan et al., 1993; Fressinaud et al., 1995; Bansal et al., unpublished). The ‘‘paradox’’ may be resolved by the possibility that instead of FGF-2, the adhesion molecules NCAM, L1, and N-cadherin may be involved. These molecules are expressed in the vertebrate nervous system where they are involved in neural cell –cell adhesion (Rutishauser and Jessell, 1988). Recently they have been shown to bind to FGFR-1 and -2 via specific sequences and stimulate neurite outgrowth (Williams et al., 1994). Therefore, they could in principle be the preferred ligands for OLs at this point of differentiation and a mechanism similar to that in neurons could occur during OL differentiation and myelination. Additionally, the downregulation of myelin genes is of potential clinical interest, since it could in principle provide a mechanism by which FGF-2 can drive differentiated OLs to dedifferentiate in pathological situations by inhibiting the gene expression of its own receptor. In summary, we suggest that FGFR2 has its principle role in the onset and regulation of terminal differentiation of OLs, and/or the maintenance of the structure and function of mature myelin, and that cell adhesion molecules and/or FGF-2 may modulate these processes via regulation of the level of FGFR-2 expression and/or second messenger metabolism induced by activation of this receptor. FGFR-3 is predominantly expressed in cells with glial morphology in the embryonic and postnatal CNS (Peters et al., 1993; Yazaki et al., 1994). The ventral to dorsal developmental expression of FGFR-3 mRNA in rodent spinal cord coincides well with the origin and subsequent migration route suggested for some OL progenitors (discussed in Ono et al., 1995). Here we have shown that FGFR-3 mRNA is maximally expressed in late progenitors, making FGFR-3 a likely candidate for transducing signals from FGF-2 that reversibly block terminal differentiation by late progenitors. FGFR-3 is the only FGF receptor whose expression is not modulated by FGF-2 at any stage of OL development. Since FGFR-3 has a much higher affinity for FGF-1 than for FGF-2 (Ornitz and Leder, 1992), FGF-1 may be the preferred ligand for FGFR-3 in vivo. Studying the effect of FGF-1 on FGFR3 expression will provide further insight. Based on the available data, we propose the following hypothetical model to explain the varying response of OLs as a function of lineage progression (Fig. 6). At the early progenitor stage, FGF-2 up-regulates both FGFR-1 (these data) and PDGFa receptor (McKinnon et al., 1990) mRNA expression, which together transduce mitogenic and migratory signals in these early progenitors; FGFR3 mRNA is maximally expressed by late progenitors, where its activation signals these cells to proliferate and maintain this developmental stage; FGFR-2, unique in its

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selective expression by mature OLs, is involved in the modulation of differentiation rather than proliferation, leading to cytoskeletal alterations. The study of the signaling pathways initiated by each receptor type is currently an emerging field of investigation. However, differences have been observed in the abilities of FGFR-1 and -4 to induce tyrosine phosphorylation (Wang et al., 1994; Shaoul et al., 1995). Our model implies that differences in the responses to FGF-2 are due to the sequential activation of different signaling cascades, depending on the predominating receptor type at each stage of OL differentiation. However, since FGFR-1 and -3 are expressed throughout the lineage (albeit at varying levels), it is possible that a given receptor activates a series of different pathways, depending on their stage-dependent properties.

Several Isoforms of FGF Receptors Are Expressed by Oligodendrocytes As FGF-responsive progenitors migrate through a series of microenvironments en route to their final destinations, they are likely to be sequentially exposed to multiple members of the FGF family, which in the brain include FGF-1, -2, -3, -5, and -9 (Eckenstein, 1994). The four FGF receptors and their isoforms exhibit redundancy as well as significant differences in their binding specificities and affinities for this family of related ligands. Thus, mechanisms that regulate either preferential expression of different receptor genes or alternative splicing could allow a cell to achieve developmentally regulated, selective responsivity to different FGFs. We therefore examined exon utilization as a function of OL development. Variants of FGFRs are generated by alternative splicing of a single exon, resulting in isoforms containing either all three (a form) or only two (b form, missing Loop-I) Ig-like domains in the extracellular region (Johnson et al., 1990), while the inclusion or exclusion of the ‘‘acid box’’ generates isoforms b1 and b2, respectively. Loop-I and the sequences between Loop-I and Loop-II (which include the acid box) are not needed for ligandbinding activity (Johnson et al., 1990), although their exclusion from FGFR-1 (b isoform) significantly increases the binding affinities for FGF and heparin ligands (Wang et al., 1995b). The a and b isoforms of FGFR-1 and -2 are coexpressed in a variety of cell lines in proportions that generally vary in a tissue-specific manner (Johnson et al., 1990; Werner et al., 1992). Only the a isoform of FGFR3 and -4 has been reported to date (Jaye et al., 1992). Here we have shown that in terminally differentiated OLs, FGFR-1 is expressed primarily as the a isoform transcript, whereas FGFR-2 is expressed primarily as the b2 isoform transcript. Since the b isoform of FGFR-1

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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 compares relative 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 the a isoform (see above), and assuming that the pattern of ligand specificity for FGFR-2 is similar, FGFR-2 may be the more active receptor at this stage of OL differentiation. In addition, a third apparent FGFR-2 isoform transcript was detected in mature OLs, the identity of which is under investigation. A second type of variant results from differences in the carboxy-terminal of Ig-like domain III. This domain can be coded by exon IIIa, IIIb, or IIIc, generating alternatively spliced isoforms of FGFR-1, -2, and -3 (reviewed in Johnson and Williams, 1993). The replacement of exon IIIc by IIIb dramatically alters both the FGF binding affinity and its specificity to that of the ‘‘minimal ligandbinding region’’ of the receptor (Wang et al., 1995a). For example, the IIIb and IIIc variants of FGFR-1, -2, and -3 all bind FGF-1 with high affinity; the IIIb variant of FGFR-2 binds FGF-7 with high affinity, but FGF-2 with lower affinity. Conversely, the IIIc variant leads to high-

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affinity binding of FGF-2 to FGFR-1 and -2, but lowaffinity binding to FGFR-3 (Fig. 3) (Werner et al., 1992; Miki et al., 1992; Ornitz and Leder, 1992). In order to determine the ligand-binding specificity of the FGF receptors expressed by OLs, we examined the mRNA expression of exon IIIc isoforms of FGFR-2 and -3. We found that both receptors utilized exon IIIc and predict, therefore, that in OLs FGFR-2 binds both FGF1 and FGF-2 with high affinity, and that FGFR-3 binds FGF-1 with high affinity but FGF-2 with low affinity. Whether OLs express, in addition, transcripts for the IIIa and IIIb isoforms of FGFR-1 and -2 is not known; both of these forms have been reported in the brain in lower proportions than the IIIc isoform (Werner et al., 1992). Further defining the expression of FGFR variant forms during growth and differentiation will lead to additional insights regarding the identity of the preferred ligands and functions in vivo. Knowledge of the functional specificity of the FGF re-

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ceptors and their isoforms is still limited. Nevertheless, our finding that OLs and their progenitors exhibit a variety of FGF receptor types, in various alternatively spliced forms, the expression of which changes with differentiation, indicates that a large variety of ligand– receptor interactions is possible. In addition, the levels of expression of these receptors are selectively regulated by the ligand. Further, we have found that heparan sulfate proteoglycans belonging to the syndecan and GPI-linked families (putative FGF coreceptors) are also differentially regulated as a function of development and by FGF-2 (Bansal et al., 1996). The combinatorial complexity created by this changing pattern of expression of the multiple components of the FGF signaling system during growth and differentiation is reflected in, and may in fact be responsible for, the varied response of cells to FGF-2 as they progress through their lineage.

EXPERIMENTAL METHODS Cell Culture and Phenotypic Characterization OL progenitors obtained from mixed primary cultures from neonatal rat telencephalon by overnight shaking (McCarthy and DeVellis, 1980) were further enriched (§97%) by differential adhesion and complement lysis and were grown in a serum-free, defined medium (mN2) (Gard et al., 1993; Bansal and Pfeiffer, 1994). Enriched populations of cells under six different experimental conditions were obtained by the use of the following growth conditions: O2A (without FGF stimulation), cells were mitotically expanded for 1 day in mN2 supplemented with PDGF-BB (Upstate Biotechnology; 10 ng/ml); early progenitors (with FGF stimulation), cells were expanded and arrested at this stage by growth in PDGF plus FGF2 (Upstate Biotechnology; 10 ng/ml) for 2 days (Bo¨gler et al., 1990); Pro-OLs (without FGF stimulation), following 2 days of expansion in PDGF/FGF-2 (above), cells were grown in mN2 alone for 1 – 2 days to allow them to differentiate to the late progenitor stage; late progenitors (with FGF stimulation), after the initial expansion of the early progenitor population in PDGF/FGF-2 (above), cells were grown for an additional 3 – 4 days in FGF-2, which blocks lineage progression at the late progenitor stage (McKinnon et al., 1990; Bansal and Pfeiffer, 1994); mature OLs (without FGF stimulation), early progenitors were expanded in PDGF/FGF-2 (above), then grown in mN2 without growth factors for 5 – 7 days to allow them to differentiate into mature OLs; mature OLs (with FGF stimulation), mature OLs (above) were grown in 10 ng/ ml FGF-2 for 2 days before harvesting. Cell purity and phenotypic characteristics were determined by immuno-

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labeling cells with a panel of antibodies (Bansal and Pfeiffer, 1994) (see Results). Astrocytes isolated from monolayer cultures left after releasing OL progenitors from mixed primary cultures (above) (Gard et al., 1993) were ú99% positive for the astrocytic marker glial fibrillary acidic protein.

RNA Isolation and Northern Blotting Total cellular RNA was isolated by acid guanidium thiocyanate– phenol–chloroform extraction (Chomczynski and Sacchi, 1987). For Northern blots (Ausubel et al., 1991), radioactive probes were prepared by random priming (Prime-It II; Stratagene), purified on NucTrap push-columns (Stratagene), and hybridized in the presence of salmon sperm DNA. Membranes were washed twice (21 SSC/0.1% SDS, 5 min, room temperature; 21 SSC/1% SDS, 30 min, 627C; 0.11 SSC, 30 min, room temperature). Blots were quantified with the Packard Instant Imager 2024 (Packard Canberra) and exposed (24 – 72 h, 0707C) to Kodak X-Omat film with intensifying screen. Blots were stripped (0.11 SSC, 1% SDS, 1007C, 15 min) and reprobed for GAPDH to normalize for RNA loading. cDNA probes used were mouse FGFR-1 (1.3-kb EcoRI– BamHI fragment; Mansukhani et al., 1990), mouse FGFR2 (1.2-kb PvuII fragment; Mansukhani et al., 1992), mouse FGFR-3 (430-bp EcoRI– HindIII fragment; Ornitz and Leder, 1992), and mouse FGFR-4 (1-kb EcoRI fragment; Partanen et al., 1991).

RNA Polymerase Chain Reaction First-strand cDNA synthesis used 5 mg total RNA (cDNA synthesis kit and oligo(dT) primers from Stratagene). Primers (Fig. 3): FGFR-1, 5* nt 50– 70, 3* nt 480 – 500 (Yazaki et al., 1994); FGFR-2, 5* nt 113– 136, 3* nt 1196 –1217 (Dionne et al., 1990; Yamaguchi et al., 1994); FGFR-3, 5* nt 970– 990, 3* nt 1095 –1115 (Chellaiah et al., 1994; P. Prinos and P. Tsipuroas, University of Connecticut). RT-PCR was performed (GenAmpPCR kit; Perkin Elmer) (Yamaguchi et al., 1994). As a control, PCR amplification of GAPDH cDNA was performed on each sample using the same amount and batch of cDNA and conditions (primers: 5* nt 27– 46, 3* nt 238– 257). RT-PCR products were separated on 1.5% agarose gels and the band positions were aligned with a control cDNA (S45Y cells for FGFR-2) and molecular weight markers. Each sample was assayed at least three times, resulting in the amplification of the same fragments. For FGFR-2, the PCR-amplified products were further analyzed by Southern blot hybridization using a radiolabeled internal sequence oligonucleotide (nt 192– 212). Signal intensity was measured by phosphorimaging, and data were nor-

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malized to similarly analyzed GAPDH levels for FGFR2 (data not shown).

ACKNOWLEDGMENTS Supported by grants from the NIH (NS10861) and the National Multiple Sclerosis Society (RG2182B-6). We thank Janice Seagren for word processing, Ezinna Anosike for help with immunofluorescence analyses, and our colleagues for valuable discussions (T. Kim, D. Madison, W. Kru¨ger, E. Joly, J. P. Vos, and M. Hurley).

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skeletal muscle growth and differentiation are controlled by a high affinity receptor, FGFR-1. Dev. Biol. 154: 169 – 181. Wanaka, A., Milbrandt, J., and Johnson, E. M. (1991). Expression of FGF receptor gene in rat development. Development 111: 455– 468. Wang, F., Kan, M., Jianming, X., Yan, G., and McKeehan, W. L. (1995a). Ligand-specific structural domains in the fibroblast growth factor receptor. J. Biol. Chem. 270: 10222– 10230. Wang, F., Kan, M., Yan, G., Xu, J., and McKeehan, W. L. (1995b). Alternately spliced NH2-terminal immunoglobulin-like loop I in the ectodomain of the fibroblast growth factor (FGF) receptor 1 lowers affinity for both heparin and FGF-1. J. Biol. Chem. 270: 10231 –10235. Wang, J., Gao, G., and Goldfarb, M. (1994). Fibroblast growth factor receptors have different signaling and mitogenic potentials. Mol. Cell. Biol. 1: 181 –188. Warrington, A. E., and Pfeiffer, S. E. (1992). Proliferation and differentiation of O4/ oligodendrocytes in postnatal rat cerebellum: Analysis in unfixed tissue slices using anti-glycolipid antibodies. J. Neurosci. Res. 33: 338 –353. Werner, S., Duan, D-S., DeVries, C., Peters, K. G., Johnson, D. E., and Williams, L. T. (1992). Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities. Mol. Cell. Biol. 12: 82– 88. Williams, E. J., Furness, J., Walsh, F. S., and Doherty, P. (1994). Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. Neuron 13: 583 –594. Yamaguchi, F., Saya, H., Bruner, J. M., and Morrison, R. S. (1994). Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas. Proc. Natl. Acad. Sci. USA 91: 484– 488. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64: 841 –848. Yazaki, N., Hosoi, Y., Kawabata, K., Miyake, A., Minami, M., Satoh, M., Ohta, M., Kawasaki, T., and Itoh, N. (1994). Differential expression patterns of mRNAs for members of the fibroblast growth factor receptor family, FGFR-1-FGFR-4, in rat brain. J. Neurosci. Res. 37: 445 –452. Yong, V. W., Dooley, N. P., and Noble, P. G. (1994). Protein kinase C in cultured adult human oligodendrocytes: Characteristics, lack of down-regulation and isoform a as a mediator of fiber outgrowth. J. Neurosci. Res. 39: 83– 96. Yuguchi, T., Kohmura, E., Yamada, K., Wanaka, A., Otsuki, H., Sakaguchi, T., Yamashita, T., Tohyama, M., and Hayakawa, T. (1994). Messenger RNA and protein expression of basic fibroblast growth factor receptor after cortical ablation. Mol. Brain Res. 25: 50–56.

Received February 16, 1996 Revised April 1, 1996 Accepted April 11, 1996

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