Journal of Neuroscience Research 27:400-407 (1990)

Rapid Communication Type 1 Astrocytes and Oligodendrocyte-Type 2 Astrocyte Glial Progenitors Migrate Toward Distinct Molecules R. C. Armstrong, L. Harvath, and M. E. Dubois-Dalcq Laboratory of Viral and Molecular Pathogenesis, National Institute of Neurological Disorders and Stroke, Bethehda, Maryland ( R . C . A . , M.E.D.-D.); Division of Blood and Blood Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland (L.H.)

During central nervous system (CNS) development, glial precursors proliferate in subventricular zones and then migrate throughout the CNS to adopt their final destinations and differentiate into various types of mature glial cells. Although several growth factors promoting the proliferation and/or differentiation of glial precursors have been identified, very little is known about the nature of signals that guide glial cell migration in the CNS. Therefore, we have investigated whether polypeptide growth factors and/or extracellular matrix molecules may mediate the migration of two major glial cell types, type 1 astrocytes and oligodendrocyte-type 2 astrocyte (0-2A) progenitor cells. We show that, in a microchemotaxis chamber assay, type 1astrocytes move toward laminin and complement-derived C5a. Astrocyte migration toward laminin is inhibited by a laminin-specific pentapeptide, YIGSR-NH2. In contrast, 0-2A progenitors migrate toward platelet-derived growth factor (PDGF), which also functions as a mitogen for these cells. Using a new method to simultaneously assay migration and DNA synthesis, we also demonstrate that 0-2A progenitors can migrate toward PDGF even when DNA replication is inhibited with an antimitotic agent. Thus, migration of different types of glial cells can be induced in vitro by specific signaling molecules, which are present in the developing brain and may stimulate migration of glial cells prior to CNS myelination. Key words: growth factors, extracellular matrix, myelination, laminin, platelet-derived growth factor

from the inner layer of the neural tube to the cortical plate (reviewed in Hatten, 1990). Later during development, radial glia give rise to astrocytes which then move to cortical and subcortical regions (Rakic, 1984; LeVine and Goldman, 1988). Precursors of oligodendrocytes are generated in subventricular zones and migrate into developing white matter regions (Paterson et al., 1973; LeVinc and Goldman, 1988; Reynolds and Wilkin, 1988). Neuronal migration appears to involve one or more cell adhesion molecules associated with radial glial cells (reviewed in Hatten, 1990). The nature of the molecules mediating glial cell migration has not been identified. Therefore, we have used an in vitro assay to determine which molecules can stimulate directed glial cell migration. We studied the migration of two major glial cell types, type 1 astrocytes and 0-2A progenitors. Type 1 astrocytes emerge during early CNS development and synthesize growth factors that can influence the development of 0-2A progenitor cells (reviewed in Raff, 1989). 0-2A progenitors give rise to oligodendrocytes, which form myelin sheaths that facilitate rapid impulse conduction along CNS axons. In vitro migration can be evaluated quantitatively with a microchemotaxis chamber (Falk et al., 1980) in which chemoattractants are added to the lower wells and cell suspensions are placed in the upper wells. Since the upper and lower compartments of the microchemotaxis chamber are separated by a filter with 8 Fm pores, cells adhere to the upper surface of the filter and then move through the pores in response to a gradient of attractant. The number of cells which migrated to the lower surface

INTRODUCTION

Received June 20, 1990; revised July 20, 1990; accepted July 30. 1990.

Cell migration is an essential feature of CNS development. Immaturc neurons migrate along radial glia

Address reprint requests to Dr. Regina Annstrong, NINDSiNIH, Bldg. 36 Rm. 5DW. Bethesda, MD 20892.

0 1990 Wiley-Liss, Inc.

Astrocyte and 0 - 2 A Progenitor Chemotaxis

of the filter can then be counted with the aide of an image analysis system. This quantitation requires that purified or enriched cell suspensions are used in the chemotaxis assay. Therefore, we prepared purified type 1 astrocytes and enriched populations of 0-2A progenitor cells from neonatal rat brain. For comparison, we also prepared enriched suspensions of microglia from neonatal rat brain, which have been shown to respond to specific chemoattractants (Yao et al., 1990). We then selected candidate molecules which might stimulate directed migration of these glial populations. The following factors were tested as potential chemoattractants: fibronectin and laminin, which are both synthesized in vitro by astrocytes and Schwann cells and promote directed Schwann cell migration (McCarthy et al., 1983; Price and Hynes, 1985; Selak et al., 1985; Baron-Van Evercooren et al., 1986); C5a, which attracts phagocytic leukocytes and microglia (Shin et al., 1968; Yao et al, 1990); PDGF, which is synthesized by cultured astrocytes (Richardson et al., 1988), stimulates proliferation and motility of 0-2A progenitors in vitro (Noble et al., 1988; Raff, 1989), and is a chemoattractant for fibroblasts (Seppa et al., 1982); fibroblast growth factor (FGF), which is another mitogen for 0-2A progenitors (Noble et al., 1988; Besnard et al., 1989b) and induces endothelial cell migration (Presta et al., 1986); transforming growth factor p (TGFP), which may regulate the effects of PDGF and FGF on 0-2A progenitor cells (Besnard et al., 1989a) and is a chemoattractant for monocytes (Wahl et al., 1987); and insulin-like growth factor 1 (IGFl), which enhances proliferation of oligodendrocyte precursors (McMorris and Dubois-Dalcq, 1968). Our in vitro analysis shows that astrocytes and 02A progenitors migrate toward distinct molecules, which are known to be present in the developing CNS. We propose a scenario by which these molecules could guide different types of glial cells to their final destinations.

MATERIALS AND METHODS Preparation of cell suspensions Mixed glial cell cultures were prepared from cerebral hemispheres of 2 day old rat pups (Behar et al., 1988). After 8 days in vitro, these cultures became stratified as microglia and 0-2A progenitors grew on top of a monolayer of astrocytes (McCarthy and de Vellis, 1980; Giulian and Baker, 1986). Microglia, 0-2A progenitors, and astrocytes were separated in three successive steps, and the purity of each cell population was confirmed by staining with cell-type-specific markers. (1) Shaking the cultures for 1 hr on a rotary shaker (180 rpm, 37°C) dislodged mainly microglial cells which were then passed through a 60 k m Nytex filter, spun down,

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and diluted to 40,000 cells per 54 p1 of defined medium (see below). The majority of these cells (90.7 2 5.6%) were stained by Dil-labeled acetylated low density lipoprotein (Dil-LDL; 10 pgiml for 2 hrs at 37°C; Biomedical Technologies, Stoughton, MA), which binds to LDL receptors on microglia (Giulian and Baker, 1986). (2) The cultures were subsequently shaken overnight to yield 0-2A progenitor cells (McCarthy and de Vellis, 1980; Behar et a]., 1988), which were also filtered and diluted to 40,000 cells per 54 pl of defined medium. Sixteen hrs after seeding, 71.2 +- 6.4% of these cells were 0-2A progenitors (Behar et al., 1988; Raff, 1989) that expressed cell-surface gangliosides recognized by the A2B5 monoclonal antibody (Eisenbarth et al., 1979) but not galactocerebroside (GC), an oligodendrocyte marker (Raff et al., 1979; Ranscht et al., 1982), or glial fi brillary acidic protein (GFAP), an astrocyte marker (Pruss, 1979; Raff et al., 1979), as determined by threefluorochrome immunocytochemistry (Armstrong et al., 1990). The other cell types present in these suspensions were mainly oligodendrocytes (10.0 +- 2.9%) and microglia (12.3 ? 2.5%). (3) The remaining monolayer of adherent astrocytes was split (1 :2) and treated for 24 hrs with 10- ’M cytosine arabinoside (ara C). Residual 0-2A progenitors and oligodendrocytes were eliminated by A2B5 and anti-GC complement-mediated cytotoxicity (Noble and Murray, 1984). Subconfluent cultures of purified astrocytes were dissociated and diluted to 20,000 cells per 54 p,l of defined medium. In these suspensions, 96.6 L 0.25% of the cells were GFAP-positive type 1 astrocytes. [These “type 1 astrocytes” will be referred to as simply “astrocytes” since “type 2 astrocytes” are not considered in this study (see Raff, 1989)].

Chemotaxis assay Migration of each population of glial cells in response to a series of potential chemoattractants was assayed in a microchemotaxis chamber (Neuro Probe, Cabin John, MD) (Falk et al., 1980; Harvath et al., 1980). Attractants and cells were diluted in defined medium containing 0.5% fetal bovine serum (Armstrong et al., 1990; 50 ng/ml insulin was used in the present study). Laminin, purified from mouse EHS sarcoma cell line (Grafet al., 1987), was tested at I , 5 , 10, and 20 pg/ml. C5a, produced by activating normal rat serum with 1 mgiml of zymosan (Sigma, St. Louis, MO) for 45 min. at 37°C (Harvath et al., 1980), was assayed at dilutions of l:lO, 1:20, 1:40, and 1:80. All other attractants were prepared commercially and tested at the indicated concentrations (human PDGF, predominantly AB heterodimer form, [ I , 5, 10, 20,40 ngiml], R & D Systems, Minneapolis, MN; bovine basic FGF [5, 10, 20 ngiml], R & D Systems; human fibronectin L25,50 pg/ml] , Boehringer Mannheim, Indianapolis, IN; human TGFPl (0.5,

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I , 5 , 10 ngiml], R & D Systems; human IGFl [50, 100 ngiml] , Amgen Biologicals, Thousand Oaks, CA j. The 48 lower wells of the chemotaxis chamber were each filled with 29 p1 of attractant. The attractants were covered with a polycarbonate filter containing 8 pm pores. [Filtcrs were precoated with poly-D-lysine (10 pgiml; Sigma, St. Louis, MO), unless indicated to have been precoated with laminin (10 Fgirnl), fibronectin (50 pgi ml), or collagen types I and 111 (100 pgiml; Vitrogen 100, Collagen Corp., Palo Alto, CA)]. A plate of 48 upper wells was then secured over the filter and each well was filled with 54 ~1 of cell suspension. After 4 hrs (astrocytes) or !6 hrs (microglia and 0-2A progenitors) at 37°C in humidified air, migrated cells on the lower side of the filter were fixed in methanol, visualized with DiffQuik histological stain (American Scientific Products, McGaw Park, IL), and quantitated with an Optomax 4010 Image Analysis System (Optomax lnc., Hollis, NH). This assay measured directed cell movement toward a gradient of chemoattractant, or “chemotaxis.” Yet a specific molecule could also stimulate random motility, or “chemokinesis.” Chemotaxis was distinguished from chemokinesis by adding an equal concentration of attractant to both the upper and lower wells of the chemotaxis chamber. which abolished chemotaxis but not chemokinesis. Fluorescence af migrated cells Migrated cells on the lower surface of the filter were labeled with fluorescent markers to identify each specific glial cell typc. Astrocytes were fixed with 5%’ glacial acetic acid in ethanol for 10 rnin at -20°C and then imniunolabeled with a rabbit polyclonal antiserum to GFAP (1:1000; Pruss, 1979), which was visualized with biotinylatcd donkey anti-rabbit IgG (10 pgiml; Amersham, Arlington Heights, IL) followed by streptavidin conjugated 7-amino-4-methyl-coumarin-3-aceticacid (25 pg/ml; Molecular Probes, Eugene, OR). Astrocytc nuclei were stained with propidiutn iodide (50 pgiml; Sigma; St. Louis, MO). Microglia were labeled with Dil-LDL (2.5 pg/ml in the lower well; Biomedical Technologies, Stoughton, MA) during the 16 hr migration period and then fixed with 2% paraformaldehyde for IS min. 0-2A progenitors were fixed with 2% paraformaldehyde for 15 min and then immunostaincd with A2BS (1:100 ascites; Eisenbarth et al., 1979) followed by rhodamine-conjugated goat anti-mouse IgM (Jackson Immunoresearch, West Grove, PA).

BrdU assay Bromodeoxyuridine (BrdU; Sigma, St. Louis, MO) was diluted into the attractants to a concentration of 2 p M in the lower wells, and thus was present during the entire 16 hr migration period. Migrated cells on the

lower filter surface were immersed in methanol for 10 min, 0.2% paraformaldehyde for 1 min, 0.07M NaOH for S min, and 2.0% paraformaldehyde for 5 min. The cells were immunostained with a monoclonal antibody to BrdU (Becton Dickinson, San Jose, CA) followed by fluorescein-conjugated goat anti-mouse IgG (35 Fg/rnl; Jackson Immunoresearch, West Grove, PA). The nuclei of all migrated cells were then stained with propidium iodide (50 pgiml; Sigma, St. Louis, MO). Using optics to visualize fluorescein and allow bleed through from the rhodamine channel, nuclei labeled with only propidium iodide appear orange-red while nuclei double-labeled with propidium iodide and BrdU appear yellow. While focusing on each nucleus, only those with a homogeneous yellow stain were counted as having incorporated BrdU.

RESULTS Type 1 astrocytes, microglia, and 0-2A progenitors each responded differently to the set of chemoattractants (Figures 1 and 2). In each case, the phenotype of the migrated cells was confirmed with cell-specific markers. Type 1 astrocytes, stained with antibody to GFAP, exhibited significant migration only in response to laminin and CSa. Microglia, identified by LDL receptor labeling, migrated to C5a as described previously (Yao ct al., 1990). 0-2A progenitors, which expressed gangliosides recognized by the A2B5 antibody, responded strongly to PDGF. The migration of astrocytes and 0-2A progenitors toward their specific attractant(s) was then examined in greater detail. Astrocytes migrated in a dose-dependent manner toward both CSa and laminin within a 4 hr period. Migration loward CSa was maximal when a 1:20 dilution of zyniosan-activated rat serum was present in the lower well (not shown). C5a stimulation of directed migration, or chemotaxis, was distinguished from stimulation of random movement, or chemokinesis, by comparing astrocyte migration in the presence and the absence of a gradient of C5a. When zymosan-activated serum (1:20) was present in both the upper and lower wells, the number of migrated cells was diminished by 44.2 2 22.0% ( n = 4 ) . Thus, C5a induced astrocytc chemotaxis (approximately half of the response) as well as chemokinesis. Astrocytes also exhibited significant migration toward laminin (Figures 1A and 3A), contrary to a previous report (Bressler et al., 1985). This migration was a specific response to laminin since it was substantially inhibited by the pentapeptide YIGSR-NH,, which corresponds to a unique sequence in the B 1 chain of laminin and binds to the 67 kD laminin receptor of epithelial cells (Graf et al., 1987). YIGSR-NH, (100 pgiml) added to the lower well, instead of laminin, did

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Fig. 1 . Different types of glial cells migrate toward distinct chemoattractants in a microchemotaxis chamber assay. Astrocytes migrate to CSa and laminin (A), microglia to CSa (B), and 0 - 2 A progenitors to PDGF (C). A range ol: concentrations of each potential attractant was assayed and only the maximal positive rcsponses are shown for comparison between cell types. Attractants were tested in triplicate during each experiment and each value shown represents the average (2sem) of 3-5 experiments. "CSa" indicates rat serum that was stimulated with zyrnosan to pencratc C5a (Harvath et al., 1980).

not stimulate astrocyte migration, which may require interaction with additional sites within the laminin molecule. However, increasing concentrations of YIGSRNH, in the upper wells reduced astrocyte migration toward laminin in a dose-dependent manner (Figure 3B), probably by competing with laminin for receptor binding. A similar pentapeptide. YICSK, which binds only weakly to the laminin receptor (Graf et al., 1987), did not inhibit migration toward laminin (not shown). Addi-

tion of laminin at 10 pgiml to the upper and lower wells decreased migration by 47.0 rfi 7.2% ( n = 4 ) . Thus, directed and random movement contributed almost equally to the astrocyte response to laminin, as was the case with C5a. Since laminin can act as a soluble or a substratebound molecule (McCarthy et al., 1983; Aznavoorian et al., 1990) and may bind to the filter during the incubation period, this directed astrocyte migration could involve cheniotaxis and/or haptotaxis, which is dirccted movement stimulated by a substrate-bound attractant. When filters were precoated to bind laminin (10 Fgirnl), fibronectin (50 pgjml), or collagen (100 pglml), instead of poly-D-lysine, the astrocytes still migratcd toward only CSa and laminin (not shown). 0-2A progenitors migrated toward PDGF in a dose-dependent manner (Figure 4A). Unexpectedly, this response was not observed after 4 hrs or 8 hrs (not shown) but was significant at 16 hrs (Figure 1C). Since 0-2A progenitors required an extended incubation period for migration, the concentration of PDCF in the upper and lower wells was determined by ['251]PDGFradioimmunoassay (Atnersham, Arlington Heights, IL) at 0.5, 4, 8, 12, and 16 hrs after addition of 40 ngiml of PDGF to the lower wells. The lower to upper well gradient of PDGF concentration was approximately 10:1 at 0.5 hrs and decreased to 211 at 16 hrs, when measured in the absencc of the cells. Therefore, a gradient of PDGF was present throughout the 16 hr assay. 0-2A progenitors responded to PDGF by directed migration since the response was reduced by 85.8 ? 12.7% (n=S) when 20 ngiml of PDGF was added to both thc upper and lower wells. Simultaneous analysis of 0-2A progenitor migration and DNA synthesis indicated that in a given cell PDGF can act as a chemoattractant and as a mitogen. Mitotic activity was assayed during migration by adding bromodeoxyuridine (BrdU) to the attractants in the lower wells. When an attractant was not added to the medium in the lower wells (Figure 4B). a small fraction of the migrated cells were labeled with BrdU ( 1 1.2 4.1%: n = 3). which is incorporated during DNA replication (Gratzner, 1982). In response to 20 ng/ml of PDGF (Figure 4C), a larger proportion of' migrated cells were labled with BrdU (32.6 2 6.4%; n = 3). which is incorporatedTreatInent with ara C , an antimitotic agent, niarkedly blocked BrdU incorporation in response to PDGF (1.2 2 0.6%; n = 3 ) but only slightly inhibited migration (Figure 4A and D). Therefore, 0-2A progenitors probably migrated before undergoing DNA replication. In contrast to PDGF, FGF was not a potent chemoattractant for 0-2A progenitors (Figure 1C) although it stimulated a high level of BrdU incorporation (36.5 .i_ 0.9%; n = 3 ) . These experiments demonstrate that DNA replication is not required for 0 - 2 A progenitor migration toward

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Fig. 2. Migrated cells on the lower side of the filters visualized with Diff-Quik histological stain (A,C,E) or phenotypic markers (B,D,F). Arrows indicate cells passing through filter pores. A,B) Astrocytes which migrated toward laminin (10 pg/ml) extended long flat processes and were immunostained with polyclonal antisera to GFAP that was visualized with 7-amino4-methyl-coumarin-3-acetic acid [blue fluorescence]; nuclei

are stained with propidium iodide [red fluorescence]. C,D) Microglia which migrated to C5a (1 :20 dilution of zymosanactivated rat serum) were labeled with Dil-LDL. E,F) 0-2A progenitors which migrated to PDGF (10 ngiml) were bipolar or multipolar cells that expressed antigens recognized by A2B5 and visualized with rhodamine. Bars = SO p.m.

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Fig. 3. Astrocyte migration toward laminin is inhibited by a laminin-specific peptide. A) Increasing the concentration of laminin in the lower wells induced a corresponding increase in the number of migrated cells. B) Adding thc pentapeptidc YIGSR-NH, to the cell suspension in the upper wells inhibited migration toward 10 pg/ml laminin in the lower wells in a dose-dependent manner. Each condition was tested in triplicate during each chemotaxis assay and the values shown represent the average (k sem) of 4 experiments.

PDGF. Yet this migration occurs slowly perhaps because of the time required to stimulate the intracellular signalling pathway involved in chemotaxis or because 0-2A progenitors need to be in specific cell cycle stage to respond to PDGF by movement.

DISCUSSION Our in vitro analysis of glial cell migration reveals that distinct molecules induce the migration of different glial cell types. The migratory responses demonstrated in these in vitro studies may be indicative of mechanisms utilized in vivo during CNS development. In the immature rat CNS, astrocytes migrate from the optic nerve head into the retina in close association with blood vessels (Watanabe and Raff, 1988). Similarly, fetal and neonatal cortical astrocytes transplanted into adult rat CNS

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enter the Virchow-Robin space, migrate along parenchymal blood vessels close to the basement membranes and are found in the perivascular space (Emmett et al., 1988; Goldberg and Bernstein, 1988). Thus, laminin present in developing CNS tissue (Licsi, 1985) and the basement membrane of perivascular regions could be an important mediator of astrocyte migration. In CNS lesions, CSa in perivascular regions might recruit astrocytes and microglia through a specific chemotactic effect, which could be partially responsible for the inflanimatory and astrocytic responses. oligodendrocyte precursor cells show extensive migration in the stages preceding myelination. From embryonic day 16 through the postnatal period, glial precursors migrate from subventricular regions to populate axonal tracts, while acquiring oligodendrocyte differentiation markers (LeVine and Goldman, 1988; Reynolds and Wilkins, 1988). Similarly, transplanted immature oligodendrocytes and/or their precursors often migrate long distances prior to myelinating CNS axons (Lachapelle ct a1 ., I983/84). In addition, 0-2A progenitors progressively populate the developing rat optic nerve from the chiasma toward the retina (Small et al., 1987). 0-2A progenitors possess PDGF a-receptors (Hart et al., 1989b) and PDGF A chain mRNA is expressed in the CNS during development (Richardson et al., 1988; Pringle et a]., 1989). If expression and/or secretion of PDGF is developmentally regulated in different CNS regions and results in the formation of a gradient, then PDGF could direct 0-2A progenitor migration in vivo. Our study presents evidence that each type of glial cell migrates in response to very specific signals. Our current working hypothesis is that laminin may guide the positioning of astrocytes in the developing CNS. Astrocyte-derived PDGF may then stimulate 0-2A progenitor proliferation and migration into formative white matter regions. As 0-2A progenitors mature into oligodendrocytes, they no longer divide in response to PDGF but maintain functional PDGF receptors (Hart et al., 1989a). Thus, PDGF might continue to stimulate chemotaxis but not mitosis in differentiating 0-2A progenitors. In addition, ncuronal surface molecules may modulate the final migration and axonal adhesion of the oligodendrocyte lineage cells just before myelination (Trotter et al., I 989).

ACKNOWLEDGMENTS We thank Hynda Kleinman for advice in this study and for the gift of purified laminin and the pentapeptides, Suresh Shelat for technical assistance, Brynmor Watkins for advice on the BrdU immunostaining, and Randall McKinnon for comments on the manuscript.

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Fig. 4. PDGF stimulates 0 - 2 A progenitor migration and mitosis. A) Increasing the concentration of PDGF in the lower wclls induced a corresponding increase in the number of migrated cells. Addition of 10-5M ara C to each PDGF concentration slightly inhibited migration. Each condition was tested in triplicate during each chemotaxis assay and the values shown represent the average (% sem) of 5 experiments. B,C,D) Migrated cells on the lower surface of the filter are labeled with propidium iodide (orange-red nuclei). Cell nuclei that incorporated BrdU during the migration period are double-

labeled for propidium iodide and BrdU (yellow nuclei). B) With only defined medium in the lower well, very few cells migrated and incorporated BrdU. C) Many cells migrated toward PDGF (20 ngiml) and a substantial proportion of these cells incorporated BrdU. D) In the presence of 10psM ara C, PDGF (20 ngiml) induced migration but BrdU incorporation was blocked, as demonstrated by the decreased number of nuclei that arc double-labeled relative to panel (C). Bar = 50 )-Lm.

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