DEVELOPMENTAL

1.52, 293-303 (19%)

BIOLOGY

Multiple Molecular Weight Forms of Basic Fibroblast Growth Factor Are Developmentally Regulated in the Central Nervous System SUZANNE GIORDANO, LARRY SHERMAN,* WILLIAM LYMAN,? AND RICHARD MORRISON’

Basic hbroblast growth factor (bFGF) is a heparin-binding protein implicated in the differentiation, proliferation, and maintenance of cells in the central nervous spstcm (CNS). It is not clear how bFGF achieves this multiplicity of effects. Multiple molecular weight forms of bFGF have recently been identified, ho\\tver, and each form may have distinct activities during CNS development. We have examined the pattern of bFGF expression during CNS develop)merit using protein immunoblot and RNA blot analyses. RNA blot analysis detected a major bFGF transcript of 3.7 kb in embryonic and adult rat brain; however, this message decreased in abundance during development. Three bFGF protein forms were identified on immunoblots of adult rat brain extract with approximate molecular weights of 18, 21, and 22 kDa. Embryonic rat brain extracts also contained the 1% and 21-kDa bFGF protein forms, but lacked the 22-kDa form. Expression of the 22kDa form was first detected in the neonate and then steadily increased to adult levels by 1 month of age. Immunoblots of adult human brain extracts also showed the presence of three bFGF protein forms with approximate molecular weights of 18, 22, and 24 kDa. In human second trimester fetal brain extracts, only the 1%kDa bFGF protein was detected. Comparison of bFGF proteins in developing rat spinal cord, cerebellum, and cortex demonstrated that distinct patterns of bFGF protein forms exist in different regions of the CNS. Therefore, the expression of indivitlual bFGF protein forms is regulated in the CNS with regard to both developmental stage and location. These data support the idea that different forms of bFGF may be associated with specific developmental events during the matura‘c‘ 1992 Academic Press. Inc. tion and organization of the nervous system.

proliferation of cells in the CNS (Walicke, 1988). High concentrations of bFGF are found, for example, in both embryonic (Gonzalez ef (xl., 1990; Seed ef al., 1988) and adult brain (Esch et (II., 1985; Baird et crl., 1986; GospobFGF has been darowicz et ul., 1986). Furthermore, shown to stimulate the proliferation of 02A cells (McKinnon ef al., 1990; Bogler et (AZ.,1990), astrocytes (Morrison and de Vellis, 1981; Pettmann ef (II., 1985), and oligodendrocytes (Eccelston and Silberberg, 1985; Saneto and de Vellis, 1985) and can support the survival of postmitotic neurons (Morrison et ah, 1986,1988; Walicke et al., 1986; Unsicker ef (xl., 198’7; Hatten et t/l., 1988). Thus, bFGF appears to have a wide variety of effects on the major cell types comprising the CNS. What is not clear, however, is how one growth factor can mediate such a multitude of effects on these varied populations of cells. The recent identification of multiple forms of bFGF may explain some of its diverse actions. Different molecular weight forms of bFGF corresponding to approximately l&22.5,23, and 24 kDa can be translated from a single human bFGF mRNA transcript (Florkiewicz and Sommer, 1989; Prats ef trl., 1989). The higher molecular

INTRODUCTION

Most cells in the mammalian central nervous system (CNS) are derived from a precursor cell population within the germinal neuroepithelium (GN). These precursor cells differentiate in the developing brain and spinal cord and give rise to CNS neurons and glia. Little is known, however, about the mechanisms controlling the establishment of diversity and maintenance of these cells or their derivatives (Skoff 1975; Turner and Cepko 1987; Wetts and Fraser 1988; Frederiksen et al., 1988). There is increasing evidence that various peptide growth factors expressed in the CNS are involved in the differentiation and maintenance of GN cells and their progeny (Hughes et al., 1988; Noble et al., 1988; Raff et nl., 1988; Richardson et (xl., 1988; Lillien et ab, 1988). Basic fibroblast growth factor (bFGF), a heparin-binding protein originally identified in pituitary and brain extracts (Gospodarowicz ef al., 1984; Risau, 1986), is one such growth factor implicated in the differentiation and

i To whom correspondence

should be addressed 293

0012-1606/92 $5.00 Copgright All rights

o 1992 by Academic Press. Inc. of reproduction in any form reserved

294

DEVELOPMENTALBIOLOGYVOLUME152,1992

weight proteins represent amino terminal extensions of the 18-kDa bFGF and appear to be initiated at leucine codons upstream of the first methionine. Potential upstream CTG start sites are also present in the bFGF message of other species, including rat (Kurokawa et al., 1988). Moreover, the different molecular weight forms have been shown to have distinct intracellular localizations, indicating that they may have unique activities (Renko et (xl., 1990; Bugler et al., 1991). To address the possibility that each molecular weight form of bFGF might have distinct activities that are important for the differentiation and maintenance of cells in the CNS, we have examined the pattern of bFGF expression in the developing and adult rat and human CNS. In this report we demonstrate that three forms of bFGF are expressed in the adult rat and adult human CNS and that each form has a unique developmentally regulated pattern of expression. MATERIALS AND METHODS

(Boehringer-Mannheim) and centrifuged at 14,OOOgfor 30 min. The supernatant was removed and stored at -80°C. Aliquots were taken for protein determinations by the method of Lowry (Lowry et al., 1951). Supernatants were either analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) or first incubated with heparin-Affigel (BioRad) to concentrate heparin-binding proteins. Extracts to be enriched for heparin-binding proteins were diluted 1:5 in phosphate-buffered saline (PBS) and incubated overnight with 300 ~1 of heparin-Affigel at 4°C. This volume of heparin-Affigel binds at least 1 pg of purified human recombinant bFGF (hr-bFGF, Synergen, Boulder Co.). Heparin-Affigel was then centrifuged at 14,OOO.ofor 10 min and the supernatant was removed. The heparin-Affigel was rinsed twice in PBS and proteins were eluted by boiling for 5 min in sample buffer containing 5% 2-mercaptoethanol and 2.5% SDS.

Gel Electrophoresis and Western Blot Analysis

Fetal tissues were obtained for this study as part of an ongoing research protocol that has been approved by the Albert Einstein College of Medicine Committee on Clinical Investigation and the Health Corporation of the City of New York. Informed consent was obtained from the participants. Fetal tissues were obtained at the time of elective pregnancy termination from normal females. The gestational ages of the abortuses ranged from 14 to 21 weeks. In order to be confident in the consistency of different samples, only tissues from regions immediately anterior to the middle cerebral artery were used and the abortus tissue was dissected in the operating room under sterile conditions. Adult brain tissues were obtained from temporal lobectomies performed for epilepsy at Good Samaritan Hospital and Medical Center in Portland, Oregon.

Tissue extracts or heparin-binding proteins were resolved by SDS-PAGE using a 15% gel. Proteins were then transferred to nitrocellulose (Bio-Rad) by electroblotting at 100 V for 2 hr at 4°C. Nonspecific sites were blocked by incubating nitrocellulose in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 1% bovine serum albumin (Sigma). Blots were either stained with Coomassie blue or incubated overnight with the anti-bFGF monoclonal antibody DE6 at a 1:lOOO dilution (Reilly et al., 1989; provided by Dr. J. Gross, E. I. DuPont) or the bFGF monoclonal antibody 148.6.1.1 at 1:lOOO dilution (provided by C. Hart, Zymogenetics Seattle, WA). In control experiments blots were incubated in TBST in the absence of primary antibody or with protein A-purified mouse IgG. Blots were washed three times for 5 min each in TBST and subsequently incubated with an alkaline phosphataseconjugated goat anti-mouse secondary antibody (Promega) (1:7500) for 30 min at room temperature. Immunoreactive bands were visualized by developing the blot with nitroblue tetrazolium (NBT) and 5-bromo-4chloro-3-indoyl phosphate (BCIP) according to the manufacturer’s specifications (Promega). The molecular weights of bFGF proteins were determined by comparison with prestained markers (Diversified Biotech).

Preparation qf Tissue and Cell Extracts

Densit@metry

Tissues and cultured cells were homogenized in a buffer of 10 mM Tris-HCl, pH 7.0, 2 M NaCl, and 0.1% Chaps detergent containing the protease inhibitors leupeptin 10 Fg/ml, 0.2 mM PMSF, and 100 pg/ml of pepstatin, bestatin, and aprotinin (Boehringer-Mannheim). The homogenate was incubated with 1 U/ml of DNase

Semiquantitative determinations of bFGF protein levels were determined with a scanning densitometer (Hoefer Scientific). Values were normalized to a standard of 20 ng hr-bFGF loaded onto each polyacrylamide gel. A standard hr-bFGF curve was linear with respect to the optical density of the scanned bands between 5

Sprague-Dawley rats (Simonson Labs; Gilroy, CA) ranging in age from 15 days of gestation (E15) to adult animals were studied in these experiments. Rats were anesthetized with CO, prior to decapitation, and tissues were excised and immediately frozen in dry ice/ethanol.

Human Brain Sawzples

and 50 ng (Gross et nZ., 1990). Data from three separate Western blots were compiled and graphed to analyze changes in the expression of the three bFGF protein forms during the observed stages of development. Cell Culture The SNB19 cell line was derived from a left parietooccipital high-grade human glioblastoma (Gross et al., 1988). This cell line is derived from the astrocyte lineage and is positive for glial fibrillary acidic protein. This cell line has previously been shown to express bFGF protein (Morrison, 1991). The preparation and addition of sense and antisense oligodeoxynucleotides to cell cultures were performed as previously described (Morrison, 1991). One week after the addition of oligodeoxynucleotides cells were washed in PBS and cell extracts were prepared as described above.

Total RNA was prepared by the LiCl/urea method of Auffry and Rougeon (1980). Poly(A)+ RNA was isolated on oligo(dT) cellulose. Twenty micrograms of RNA was separated on a 1.5% agarose formaldehyde gel and transferred to nitrocellulose (Thomas, 1980). Prehybridization was carried out at 42°C in a solution containing 20 mM KPO,, 5~ SSC (0.1 A4 NaCl, 0.15 mM sodium citrate, pH 7.0), 5~ Denhardt’s solution (Denhardt, 1976), 50 pg/ml calf thymus DNA (Sigma), and 50% formamide. Hybridizations were performed at 42°C in prehybridization solution containing 1 X 10’ cpm labeled riboprobe. Three consecutive washes were performed at 75°C in 0.1X SSC and 0.2% SDS followed by one wash in 0.1X SSC. The RNA probe was transcribed from the T7 promoter of plasmid pGb270 containing 0.284 kb of the human bFGF cDNA (bases 659-943) (Abraham, 1986) inserted into the pGem4Z vector (Promega). Blots were exposed to X-ray film (Kodak) for 1 week and then stripped and reprobed with a cyclophillin cDNA probe to demonstrate the amount of RNA loaded in each lane. RESULTS

Rnt Brain To determine the pattern of bFGF expression during CNS development, tissue extracts were made from rats ranging in age from El5 to adult and assayed for bFGF on Western blots. Several distinct changes in the expression of bFGF protein forms were observed during this time course (Figs. 1 and 2). Embryonic rat brain (El%PO; PO is Postnatal Day 0) expressed only two immunoreactive forms of the bFGF protein with molecu-

la4

-

FIG. 1. Western blot analysis of bFGF expression in developing rat brain. Proteins were extracted from the entire rat brain as described under Materials and Methods. Equal amounts of total protein (600 pgl were subsequently incubated with heparin-AfIigel overnight at 4°C. Heparin-binding proteins were eluted with sample buffer, loaded onto a 15V* SDS-polgacrglamide gel, and then transferred to nitroccllulose. Blots were incubated in DE6 monoclonal antibody and bFCF immunoreactivity was visualized as described under Materials and Methods. Proteins were extracted from rat brain at ages El.5 E18, PO, P7, P14, and adult (AD) and from rat liver at age El8 (LV ElX). In addition, 20 ng of human recombinant bFGF (18 kDa) and molecular weight markers were run as standards.

lar weights corresponding to 18 and 21 kDa (Fig. 1). These forms were also present in embryonic liver. Adult brain (P7-1 year), in contrast, contained three immunoreactive bFGF forms, including the 1% and 21-kDa forms seen in embryonic brain and an additional form at 22 kDa (Fig. 1). The staining of immunoreactive bands observed at 34 kDa on immunoblots was also detected in the absence of any primary antibody (Fig. 68). These proteins appear to bind nonspecifically to the secondary antibody and therefore are not likely to be related to bFGF (see below). Expression of the 22-kDa form, which was prominent in adult brain, was not observed until after birth in whole brain extracts. The expression of this form steadily increased after birth, reaching maximum levels in the adult. The expression of the 18-kDa form peaked at PO and the expression of this protein form was significantly reduced in adult brain (Figs. 1 and 2). Expression of the 21-kDa form was also maximal in embryonic brain (Fig. 1). The predominant bFGF proteins in adult brain were the 21- and 22-kDa forms. In addition, when the relative areas of all the forms were examined by densitometric scanning, an overall decrease in bFGF protein levels relative to the total protein was observed during this time course. The same results were obtained for three separate immunoblots for which different extractions were performed at each developmental time point. The immunoreactive

296

DEVELOPMENTAL BIOLOGY

VOLUME 152, 1992

Developmental

El5

El8

PO

P7

P14

AD

El5

El8

PO

P7

P14

AD

1

Expression

of bFGF in Human Brain

Human fetal and adult brains were examined to determine if the regulation of bFGF molecular forms may be conserved between species. Extracts were prepared from human fetal CNS tissue, ranging in age from 13 to 21 weeks of gestation, and adult brain, ranging in age from 30 to 45 years. Human brain tissue exhibited the same basic pattern of bFGF regulation as that found in the rat. Species-specific molecular weight differences, however, were observed for the bFGF forms as previously reported (Brigstock et ah, 1990). Both human and rat brain contained the 18-kDa form but adult human tissue contained slightly larger molecular weight forms, of approximately 22 and 24 kDa, than the adult rat brain (21 and 22 kDa). Interestingly the higher molecular weight forms were associated with later stages of human brain development just as we observed in the rat. In human fetal brain (13-21 weeks), however, only the 18-kDa form of bFGF was detected (Fig. 3). The absence of the 22-kDa form in human fetal brain (13-21 weeks) in contrast to embryonic rat brain (E18), which contained both the 18- and the 21-kDa forms, may reflect differences in the developmental stage of the samples. The above results indicate that similar mechanisms regulate bFGF expression in the CNS of these two species.

0 El5

El8

PO

P7

PI4

AD

FIG. 2. Densitometric analysis of individual rat bFGF protein forms. The relative values of absorbance for each bFGF protein form were determined by measuring the optical density of each band on a scanning densitometer. Three Western blots representative of three separate protein extractions were subjected to densitometric scanning. Data from three separate Western blots (including Fig. 1) were compiled and the mean values are shown in the graph. The relative absorbance of each band is shown for all developmental time points examined. (A) 18 kDa. (B) 21 kDa. (C) 22 kDa.

proteins at approximately 16 kDa, seen in Fig. 1, have been observed in other studies and appear to represent specific proteolysis of the bFGF protein (Moscatelli et al., 1987). To control for the possibility that heparin incubations altered the observed pattern of bFGF expression during development, cell extracts were examined directly on SDS-polyacrylamide gels. The pattern of expression of each bFGF form was the same as that observed following heparin-Affigel incubation (data not shown).

r--- 1 36

-

29

-

18.4

-

FIG. 3. Western blot analysis of bFGF in human adult and fetal brain. Proteins were extracted from human adult (30-45 years) and fetal brain (1X and 20 weeks) and equal amounts of total protein (1 mg) were incubated with heparin-Affigel overnight at 4°C. Samples were resolved by SDS-PAGE, and immunoblotting was performed with the DE6 bFGF antibody as described under Materials and Methods. The band at 34 kDa, marked with an arrow, is seen in the absence of primary antibody and is not related to bFGF (see Fig. 6B).

HUMAN LGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTM 4 24.2

c23.1

+ 22.5

; 17.8 -RAT LAARGRAALGGRGRGRGRGAPRAAAAGSRGRGGAM f 21 8

+ 20.5

+ 17.5

FIG. 4. Predict& amino acid sequence for the amino terminus of the t)E’(;F protein initiating at alternatil-e CUG codons (Florkiewicz and Sommvr, 19X9; Kurakawa rt (I/., 1988). The amino terminus of the l)FGF molecular forms predicted from the human and rat cDNA sequences, respcctivclg. Potential initiating leucincs arc in boldface. The predicted molecular weights of hFGF proteins initiating at these leucines and the first methionine are indicated. The sequence (:GR(:R(; present in the rat and human sequences is underlined.

The specificity of the immunoreactive bands observed on Western blots was verified by several different criteria. Immunoblots with the DE6 antibody recognized four immunoreactive proteins. Three of these proteins (18, 21, and 22 kDa in rat and 18, 22, and 24 kDa in humans) have been previously reported (Brigstock c,t ul.. 1990) and the molecular weights of these forms are in agreement with those predicted for bFGF proteins initiating at alternative initiation sites on the bFGF message (Fig. 4). An additional triplet of immunoreactive bands at approximately 34 kDa was also observed. The staining of this protein, however, varied on different gels and in different preparations of extracts. Therefore, to demonstrate that the three smaller proteins were related to bFGF and to evaluate further the identity of the 34-kDa protein, the following control experiments were performed. Adult rat brain extracts and 20 ng of hr-bFGF were resolved by SDS-polyacrylamide gel electrophorcsis (Fig. 5). The proteins were transferred to nitrocellulose and probed with native DE6 (Fig. 5A) or DE6 depleted of bFGF IgG by passing the antibody over a heparin-Afigel-bFGF complex (Fig. 5C). Blots probed with native DE6 resulted in four immunoreactive forms (Fig. 5A). In blots incubated with depleted DE6 antibody no staining of the 1%, 21-, or 2%kDa forms or hr-bFGF was observed. Some staining, however, of the 34-kDa protein was retained (Fig. 5C). The DE6 antibody was also reacted with heparin-Affigel alone and this antibody gave identical results to native DE6 (Fig. 5B). Immunoreactive bands identical to those observed with DE6 were also recognized on immunoblots using an additional bFGF monoclonal antibody (Fig. 6A). Subsequent experiments, however, in which primary antibody was omitted, still resulted in the staining of the 34-kDa triplet, but not the 1%, 21/22-, or

22/24-kDa proteins (Fig. 6B). These data suggest that the 34-kDa protein is not related to bFGF. 1~ vitro studies have indicated that the 1%, 22-, and 24-kDa bFGF proteins can be translated from a single human mRNA (Prats rt ul., 1989; Florkiewicz and Sommer, 1989). We utilized this information to develop another method for evaluating the specificity of bFGF immunoreactivity observed on immunoblots. The human glioma cell line, SNB19, used in this study was previously shown to express bFGF (Morrison et (xl., 1990). Furthermore, the addition of bFGF-specific antisense oligodeoxynucleotides to these cells was shown to suppress bFGF expression and dramatically inhibit cell growth (Morrison, 1991). Therefore, we asked whether antisense oligodeoxynucleotides complementary to the bFGF iZTG initiation site would reduce all three forms of bFGF expressed in SNB19 cells. As shown in Fig. 6C treatment with antisense primers results in a significant reduction in the intensity of all three bFGF protein forms. Oligonucleotides in the sense orientation did not alter the expression of bFGF proteins (Morrison, 1991). These results suggest that all three immunoreactive bands are related to bFGF.

Distinct regions of the rat CNS were examined to determine if the expression of different forms of bFGF varied throughout the central nervous system. Proteins were extracted from rat spinal cord, cortex, and cerebellum at ages E18, PO, and adult. Immunoblot analysis demonstrated different patterns of bFGF proteins in these regions of the CNS. In spinal cord and cortex, the predominant bFGF forms in adult and embryonic tissues were the same as those observed in whole brain extracts. Embryonic spinal cord and cortex expressed predominantly the 1%, and 21-kDa forms. Adult spinal cord and cortex expressed high levels of the 21- and 22kDa forms and lower levels of the 1%kDa form (Fig. 7A). Although the general pattern of expression was similar in spinal cord and cortex, the temporal expression of the 22-kDa form was variable. For example, the 22-kDa form appeared earlier in the development of the spinal cord (PO) than in cortex or in whole brain extracts (Fig. 7A). In contrast, the cerebellum exhibited a significantly different pattern of bFGF expression (Fig. 7). In Fig. 6B an excess of total protein was applied to heparin-Affigel to maximize detection of the different bFGF forms. As seen in this figure the pattern of bFGF forms in El8 cerebellum was similar to that observed in other regions of the embryonic CNS. The predominant bFGF proteins

298

DEVELOPMENTALBIOLOGYV0~~~~152.1992

184

-

124

-

FIG. 5. Specificity of the DE6 antibody for bFGF protein forms. Equal amounts of protein (400 fig) extracted from adult rat brain were first reacted with heparin-Affigel and then resolved on a 15% reducing SDS-polyacrylamide gel. Twenty nanograms of hr bFGF was loaded next to each brain sample. Following protein transfer to nitrocellulose and blocking of nonspecific sites, the nitrocellulose was incubated with (A) DE6 antibody, (B) DE6 antibody reacted with heparin-Affigel, or (C) DE6 depleted of bFGF-IgG. Prestained molecular weight markers were resolved on each blot.

in embryonic cerebellum correspond to the 18- and 21kDa forms. However, during postnatal maturation of the cerebellum there was an increase in the level of the 18-kDa form and a significant reduction in the 21-kDa form, which became barely detectable in the adult. This is in marked contrast to what was observed in the developing spinal cord and cortex. In addition, the 22-kDa form was weakly expressed following birth so that in adult cerebellum the 18-kDa form predominates. This differs from the pattern observed in adult spinal cord and cortex which clearly contain high levels of the 21and 22-kDa forms. To address the possibility that the absence of larger bFGF proteins in the cerebellum was due to degradation of these proteins by specific proteases, extracts of adult whole brain and adult cerebellum were incubated together. Immunoblot analysis of this mixed extract identified all three bFGF proteins, suggesting that the absence of larger bFGF proteins in the cerebellum is not due to proteolysis. Analysis

of bFGF mRNA in Developing

Rat Brain

Northern blot analysis was performed on poly(A)+ RNA isolated from El8 and adult rat brain to determine if there were changes in bFGF mRNA expression during CNS development. Under high stringency conditions a major band of 3.7 kb was observed in both embryonic

and adult whole brain. Embryonic brain (E18) contained significantly higher levels of the 3.7-kb transcript than adult brain (Fig. 8A). In addition to the 3.‘7kb transcript, an additional band of 3.0 kb was observed in the adult. This transcript was not detected in embryonic brain. As controls, blots were stripped and rehybridized with a cyclophilin probe to control for differences in the amount of RNA loaded in each lane (Fig. 8B). DISCUSSION

Although bFGF was first identified in pituitary and brain extracts, its function in neural tissue has not been completely defined. A variety of in vitro and in vivo assays illustrate that bFGF can influence all major cell types in brain, including astrocytes, oligodendrocytes, and multiple neuronal populations. Basic FGF has been localized by immunohistochemical methods mainly to glial cells in the adult CNS and is detected in only a few neurons (Woodward et al, 1992; Gomez-Pinilla et al., 1992). In agreement with these findings, we observe all three bFGF forms in rat astroglial cell cultures but do not detect bFGF in neuronal cultures (unpublished observations). These studies indicate that bFGF has multiple roles in nervous system development, maintenance, and response to injury (for review see Gospodarowicz et al., 1987; Haynes, 1988). It is not clear, however, how one factor can be responsible for such a multitude of biologi-

Regulation

GIORDANOETAL.

of bFGF

C

B

299

Forms during CNS Development 2 !A

55

-

43

-

36

-

29

-

0

a +

.

FIG. 6. Evaluation of the specificity of bFGF (Lane 1) and hr-bFGF (Lane 2) immunoreactivity. (A) Adult rat brain extract (600 pg total protein) was incubated with heparin-Affigel and resolved by SDS-PAGE. Immunoblotting was performed using the monoclonal bFGF antibody 148.6.1.1 as described under Materials and Methods. (B) Adult rat brain extract (600 pg) stained with the omission of primary antibody. (C) Equal amounts of total protein (200 pg) extracted from SNB19 cells were incubated with heparin-Affigel and resolved by SDS-PAGE. Immunoblots were performed with the DE6 antibody. Lane C, extract from untreated cells. Lane ASl, extract from cells incubated for ‘7 days with bFGF-specific antisense oligonucleotide AS1 (35 @cM)as described (Morrison, 1991). Molecular weight markers are indicated in A, B, and C.

cal effects in a tissue with as many diverse cell types as the brain. In the present study, we investigated the possibility that there are temporal and regional variations in bFGF expression which might correlate with diverse activities in the nervous system. Multiple Protein Forms of bFGF Are Expressed in the CNS The results of this study demonstrate that the rat and human CNS contain multiple molecular weight forms of bFGF. The bFGF forms we identified correspond to molecular weights predicted for proteins initiating at the first AUG and two upstream CUG start sites present in the rat and human bFGF message (Florkiewicz and Sommer, 1989; Kurokawa et al., 1988). An additional start site in the human mRNA coding for a protein of 23 kDa is not clearly resolved on our gels and appears to be migrating with the 22-kDa form. Initiation of translation at alternate start sites has been reported for other proteins including other members of the FGF family (Acland et al., 1990). Basic FGF proteins corresponding to the same approximate molecular weights we observed have been identified previously in neural and nonneural tissues (Moscatelli et al., 1987; Presta et al., 1988; Caday et al., 1990; Westermann et ab, 1990; Grothe et al., 1990, Brigstock et al., 1990; Sherman et ab, 1991). Our results show

specific staining of these proteins on immunoblots using two different bFGF monoclonal antibodies. Furthermore, experiments with the human glioma cell line SNB19 demonstrate that bFGF-specific antisense oligodeoxynucleotides selectively suppress the expression of all three bFGF proteins. Some previous studies of bFGF levels in adult rat brain did not detect all three bFGF molecular weight forms that were observed in this and other studies (Caday et al., 1990; Eckenstein et al, 1991). The absence of the higher molecular weight forms may reflect differences in the method of protein extraction. The present study employed 2 M NaCl and Chaps detergent in the extraction buffer whereas previous studies used only 1 MNaCl. A concentration of 1 MNaCl may not be sufficient to remove bFGF from low affinity binding sites (Moscatelli, 1987). Therefore, it is conceivable that these forms are preferentially bound to low affinity sites and were not extracted with lower salt concentrations. The extended amino terminal domains of the higher molecular weight forms, which are very rich in arginine and glycine residues, may confer functional differences to these growth factors or alter their affinities for intracellular or extracellular targets. This concept is strengthened by the recent finding that the 18-kDa bFGF form has a cytoplasmic localization whereas the higher molecular weight forms are predominantly localized in the nucleus (Renko et ab, 1990; Bugler et ab, 1991).

300

DEVELOPMENTALBIOLOGY

V0~~ME152,1992

LB--29

-

FIG. ‘7. Regional expression of bFGF in the developing rat CNS. Proteins were extracted, enriched, resolved by SDSPAGE, and visualized on immunoblots using the DE6 antibody as explained under Materials and Methods. (A) Protein extracts were prepared from rat spinal cord (SC), cerebellum (Cb), and cortex (Cx) at ages El8, PO, and adult (AD). 600 fig of total protein was incubated with heparinAHige1. (B) Western analysis of proteins extracted from developing rat cerebellum. Adult whole hrain extract (AD BRAIN) and hr-bFGF are also shown for comparison. 1.2 mg of total cerebellar protein was incubated with heparinAHigel. Molecular weight markers are indicated.

Multiple Protein Forms of bFGF Are Developmentally

The present results demonstrate that the expression of multiple molecular weight forms of bFGF is regulated during the development of the rat and human CNS. In the rat, the 22-kDa form, for example, is not present in embryonic CNS tissues, but is expressed in neonates and adults. In contrast, the 18-kDa form peaks during embryonic development and is minimally expressed in the adult. Similarly, adult human brain extracts contain three immunoreactive bFGF protein forms of 18, 22, and 24 kDa whereas only the 18-kDa form was detected in fetal brain at the time points we examined. At least part of this regulation probably occurs at the level of translation. Several potential stem loop structures can be identified in the upstream region of the bFGF message (Florkiewicz and Sommer, 1989). The efficiency of translation at upstream start sites may be effected by proteins that bind to these sites or other regions of the mRNA. Basic FGF expression also appears to be regulated at the level of transcription. As shown here and in a previous study, the major bFGF mRNA detected in rat brain is a 3.7-kb transcript (Ernfors et ul., 1990). Basic FGF mRNA expression was significantly greater in embryonic rat brain than in adult brain. This finding is in agreement with previous investigations of bFGF mRNA expression in the rat CNS (Ernfors et al., 1990). The decrease in bFGF mRNA levels observed during CNS de-

velopment appeared to correlate with the overall decreased levels of immunoreactive bFGF protein in adult rat brain compared with those in embryonic (E18) brain. Although we observed an overall decrease, the levels of bFGF protein fluctuated during the time course examined. For example, we observed a slight increase in total immunorcactive bFGF protein expression in rat brain between ages P7 and adulthood. This is in agreement with a previous study demonstrating an increase in bFGF levels in postnatal rat brain (Caday et al., 1990).

Basic FGF Expression Exhibits Re.yiowal Specificity Specific regions of the rat CNS show distinct patterns of bFGF molecular weight forms. The observation that the 22-kDa form is found in adult cortex and spinal cord but not in cerebellum implies that individual forms of bFGF may have distinct functions in different parts of the nervous system. Basic FGF has been shown to stimulate granule cell precursor proliferation (Gao et al., 1991) and cell neurite extension (Hatten et al., 1988). Therefore, the changes in bFGF expression in the neonate may have a role in granule cell differentiation. During development of the CNS the proliferative region of the neural tube contains precursor cells, which in some cases give rise to both neurons and glia (Turner and Cepko, 1987; Galileo et uZ., 1990). Environmental cues, such as growth factors, may influence the developmental fate of these precursor cells. Basic FGF repre-

301

2 I

E I

FIG. S. Analysis of bFGF mRNA expression in developing rat brain. Polg (A)’ RNA was isolated from El8 and adult (AD) rat brain. Twenty micrograms of Poly(A)+ RNA was separated on a 1.5%) formaldehyde gel, transferred to nitrocellulose, and prehgbridized. (A) Hybridization was carried out for 8 hr in prehybridization solution containing 1 X 108 cpm of the [32P]UTP-labeled RNA probe transcribed from the T7 promotor of plasmid pGb270 containing a human bFGF cDNA insert. Washes were done at high stringency (75”C, 0.1X SSC) and the blot was exposed to X-ray film for 1 week. The major 3.7-kb bFGF message present in embryonic and adult brain is indicated. An arrow marks the 3.0-kb transcript expressed in adult brain. (B) The top blot was stripped and reprobed with a cyclophillin clone to account for the atnounts of RNA loaded in each lane.

sents a strong candidate for this type of factor since it is expressed very early in development and is widely dispersed throughout the embryonic nervous system (Gonzalez et (xl., 1990; Kalcheim and Neufeld, 1990). Moreover, bFGF stimulates the proliferation and differentiation of glial cells and glial cell progenitors (McKinnon et al., 1990) and enhances neuronal survival and neurite outgrowth (Morrison et ul., 1986; Walicke, 1988). Basic FGF also has potent angiogenic properties (Folkman and Klagsburn, 1987) and may influence vascularization of the nervous system (Gospodarowicz ef ul., 1986; Risau et al., 1988). It is not yet clear if bFGF influences all these cell types during the course of normal brain development. Variations in bFGF receptor expression have, however, been observed during CNS development (Wanaka et al., 1990, 1991; Heuer ef al., 1990). The temporal and regional variations we observed

for bFGF protein forms may reflect functional differences for these proteins. These results suggest that subpopulations of CNS cell types may exhibit differential responses to bFGF. It is also conceivable that the response of a single cell type to bFGF changes as the nervous system matures. It remains to be determined if the multiple bFGF proteins exhibit different affinities for the various FGF receptors. In summary, we show here that there is species-specific expression of bFGF forms. Moreover, the multiple forms of bFGF that are present in both the rat and the human CNS display specific changes in expression during the course of CNS development. Furthermore, the presence of individual forms of bFGF appears to be regulated independently in different brain regions. The expression of these multiple bFGF protein forms may account for some of the varied reported effects of bFGF in the CNS. We thank M. Tang for technical assistance and Dr. J. De Toledo for providing the adult human brain samples. In addition we thank C. Bush, J. Gregory, and 8. Smith for help in preparing the manuscript. We also thank Drs. G. Ciment, P. Copenhaver, and K. Stocker for critically reading the manuscript. This work is supported by Grant NS26125 from the NIH, a Grant from the M. J. Murdock Charitable Trust, and a grant from the Medical Research Foundation of Oregon to R.M. REFERENCES Abraham, J. il., Whang, J. L., Tumolo, A., Mergia, A., Friedman, J., Gospodarowicz, D., and Fiddes, J. C. (1986). Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO J. 5( lo), 2523-2528. Aeland, P., Dixon, M., Peters, G., and Dickson, C. (1990). Subcellular fate of the Int-2 oncoprotein is determined by choice of initiation codon. h’oture 343, 662665. Auffrey, and Rougcon (1980). Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J Biocl~em. 107, 303-314. Baird, A., Esch, F., Mormede, P., Ueno, N., Ling, N., Bohlen, P., Ying, S. Y., Wehrenberg, W. B., and Guillemin, R. (1986). Molecular characterization of fibroblast growth factor: Distribution and biological activities in various tissues. Recent Ploy. Harm. Res. 42, 143-205. Bogler, O., Wren, D., Barnett, S. C., Land, H., and Noble, M. (1990). Cooperation between two growth factors promotes extended self renewal and inhibits differentiation of oligodendrocyte type-2 astrocyte (O-2A) progenitor cells. Proc. Nntl. Acad. Sci. USA 87,63686372 Brigstock, D. R., Klagsbrun, M., Sasse, J., Farber, P. A., and Iberg, N. (1990). Species-specific high molecular weight forms of basic fibroblast growth factor. Growth Fuctors 4, 45-52. Bugler, B., Amalric, F., and Prats, H. (1991). Alternative initiation of translation determines cytoplasmic or nuclear localization if basic fibroblast growth factor. ilfol. Cell. Riol. 11, 573-577. Caday, C. G., Klagsbrun, M., Fanning, P. J., Mirzabegian, A., and Finklestein, S. P. (1990). Fibroblast growth factor (FGF) levels in the developing rat brain. I)ci,. Bruin Res. 52, 241-246. Denhardt, D. T. (1976). A membrane-filter technique for the detection of complementary DNA. Biochcnl. Biophys. Rex Corn mun. 23, 641646.

302

DEVELOPMENTAL BIOLOGY

Eccleston, P. A., and Silberberg, D. H. (1985). Fibroblast growth factor is a mitogen for oligodendrocytes in vitro. Dev. Bruin Res. 21,315318. Eckenstein, F. P., Shipley, G. D., and Nishi, R. (1991). Acidic and basic fibroblast growth factors in the nervous system: Distribution and differential alteration of levels after injury of central versus peripheral nerve. J. Neurosci. 11, 412-419. Ernfors, P., Lnnerberg, P., Ayer-LeLievre, C., and Persson, H. (1990). Developmental and regional expression of basic fibroblast growth factor mRNA in the rat central nervous system. J. Neurosci. Res. 27, 10-15. Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen, P., and Guillenia, R. (1985). Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. hoc. Nutl. Acad. Sci. USA 82,6507-6511. Finkelstein, S. P., Apostolides, P. J., Caday, C. G., Prosser, J., Philips, M. F., and Klagsbrun, M. (1988). Increased basic fibroblast growth factor (bFGF) immunoreactivity at the site of focal brain wounds. Bruin Res. 460, 253-259. Florkiewicz, R. Z., and Sommer, A. (1989). Human basic fibroblast growth factor gene encodes four polypeptides: Three initiate translation from non-AUG codons. Proc. N&l. Acad. Sci. USA 86, 39% 3981. Folkman, J., and Klagsbrun, M. (1987). Angiogenic factors. Sciertce 235,442-447. Frederiksen, K., Jat, P. S., Valtz, N., Levy, D., and McKay, R. (1988). Immortalization of precursor cells from the mammalian CNS. Neuror, 1, 439-448. Galileo, D. S., Gray, G. E., Owens, G. C., Majors, J., and Sanes, J. R. (1990). Neurons and glia arise from a common progenitor in chicken optic tectum: Demonstration with two retroviruses and cell-type specific antibodies. proc. N&l. Acud. Sci. USA 87, 458-462. Gao, W., Heintz, N., and Hatten, M. (1991). Cerebellar granule cell neurogenesis is regulated by cell-cell interactions in vitro. Neuron 6,705715. Gomez-Pinilla, F., Lee, J. W-K. and Cotman, C. W. (1992). Basic fibroblast growth factor in adult rat brain: Cellular distribution and response to entorhinal lesion and fimbria-fornix transection. J. Lveurosci. 12, 345-355. Gonzalez, A. M., Buscaglia, M., Ong, M., and Baird, A. (1990). Gospodarowicz, D., Chang, J., Lui, G. M., Baird, A., and Bohlen, P. (1984). Isolation of brain fibroblast growth factor by heparinsepharose affinity chromatography: Identity with pituitary fibroblast growth factor. Proc. Nutl. Acad. Sci. USA 81,6963-6967. Gospodarowicz, D., Massoglia, S., Cheng, J., and Fujii, D. K. (1986). Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex and corpus luteum capillaries. J. Ce:ellPhysiol. 136, 121-136. Gospodarowicz, D., Ferrara, N., Schweigerer, L., and Neufeld, G. (1987). Structural characterization and biological functions of fibroblast growth factor. Endocr. Rex: 8, 95-114. Gross, J. L., Behrens, D. L., Mullins, D. E., Kornblith, P. L., and Dexter, D. L. (1988). Plasminogen activator and inhibitor activity in human glioma cells modulation by sodium butyrate. Cancer Res. 48, 291-296. Gross, J. L., Morrison, R. S., Eidsvoog, K., Herblin, W. F., Kornblith, P. L., and Dexter, D. L. (1990). Basic libroblast growth factor: A potential autocrine regulator of human glioma cell growth. J. Neurosci. Rrs. 27, 689-697. Grothe, C., Zachmann, K., Unsicker, K., and Westermann, R. (1990). High molecular weight forms of basic fibroblast growth factor recognized by a new anti-bFGF antibody. FEBS Lett. 260,35-38. Hatten, M. E., Lynch, M., Rydel, R. E., Sanchez, J., Joseph-Silverstein,

VOLUME 152,1992

J., Moscatelli, D., and Rifkin, D. B. (1988). 17~vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor. Dev. Biol. 125, 280-289. Haynes, L. W. (1988). Fibroblast (heparin-binding) growth factors in neuronal development. Mol. Neurobiol. 2,263-289. Heuer, J. G., Von Bartheld, C. S., Kinoshita, Y., Evers, P. C., and Bothwell, M. (1990). Alternating phases of FGF receptor and NGF receptor expression in the developing chicken nervous system. Neuron 5, 283-296. Hughes, S. M., Lillien, L. E., Raff, M. C., Rohrer, H., and Sendtner, M. (1988). Ciliary neurotrophic factor induces type-2 astrocyte differentiation in culture. Nature 335,70-73. Kalcheim, C. and Neufeld, G. (1990). Expression of basic fibroblast growth factor in the nervous system of early avian embryos. Development 109, 203-215. Kimelman, D., and Kirschner, M. (1987). Synergistic induction of mesoderm by FGF and TGF-beta and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 51,860-877. Kurokawa, T., Seno, M., and Igarashi, K. (1988). Nucleotide sequence of rat basic fibroblast growth factor cDNA. Nucleic Acids. Res. 16, 5201. Lillien, L. E., Sendtner, M., Rohrer, H., Hughes, S. M., and Raff, M. C. (1988). Type-2 astrocyte development in rat brain cultures is initiated by a CNTF-like protein produced by type-l astrocytes. Neuron 1,485-494. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin-phenol reagent. J. Biol. Cl/em. 193, 265-275. McKinnon, R. D., Matsui, T., Dubois-Dalcq, M., and Aaronson, S. (1990). FGF modulates the PDGF-driven pathway of oligodendrocyte development. Neurolc 5, 603-614. Morrison, R., and de Vellis, J. (1981). Growth of purified astrocytes in a chemically defined medium. Pmt. Natl. Aca,d. Sci. USA 78, 72057209. Morrison, R. S. (1991). Suppression of basic fibroblast growth factor expression by antisense oligonucleotides inhibits the growth of transformed human astrocytes. J. Biol. Chem. 266(2), 728-734. Morrison, R. S., Gross, J. L., Herblin, W. F., Reilly, T. M., LaSala, P. A., Alterman, R. L., Moskal, J. R., Kornblith, P. L., and Dexter, D. L. (1990) Basic fibroblast growth factor-like activity and receptors are expressed in a human glioma cell line. Cuncer Rex 50,2524-2529. Morrison, R. S., Keating, R. F., and Moskal, J. R. (1988). Basic fibroblast growth factor and epidermal growth factor exert differential trophic effects on CNS neurons. J. Neurosci Res. 21, 71-79. Morrison, R. S., Sharma, A., de Vellis, J., and Bradshaw, R. (1986). Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture. Proc. N&l. Acud. Sci. l!SA 83, 7537-7541. Moscatelli, D. (1987). High and low affinity binding sites for basic fihroblast growth factors on cultured cells: Absence of a role for low affinity binding sites in the stimulation of plasminogen activator production by bovine capillary endothelial cells. J. Cell Physiol. 131, 123-130. Moscatelli, D., Joseph-Silverstein, J., Manejias, R., and Rifkin, D. B. (1987). M, 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc. Nutl. Acad. Sci. USA 84, 5778-5782. Noble, M. D., Murray, K., Stroobant, P., Waterfield, M. D., and Riddle, P. (1988). Platelet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell. Nature 333, 560-562. Park, C. M., and Hollenberg, M. J. (1989). Basic fibroblast growth factor induces retinal regeneration in oir:o. Den Biol. 134, 201-205. Pettmann, B., Weibel, M., Sensenbrenner, M., and Labourdette, G.

(1985). Purification of two astroglial growth factors from bovine brain. FEES’ Lcff. 189, 102-108. Prats, H., Kaghad, M., Prats, A. C., Klagsbrun, M., Lelias, J. M., Liauzun, P., Chalon, P., Tauber, J. P., Amalric, F., Smith, J. A., and Caput, D. (1989). High molecular mass forms of basis fibroblast growth factor are initiated by alternative CIJG codons. Ptw Not!. Actrtl. Sci. USA 86, 18:36-1840. Presta, M., Rusnati, M., Maier, J. A. M., and Ragnotti, G. (1988). Purification of basic fibroblast growth factor from rat brain: Identification of a .t-I, 22,000 immunoreactive form. Biockew. Biol/h!/s. Rrs. CO))/,,(,(li. 155, 1161-1172. Ratf. M. C., Lillien, L. E., Richardson, IV. D., Burne, J. E., and Noble, M. D. (1988). Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture. IVutrcw 333, 562-565. Reilly, T. M., Taylor, D. S., Herblin, W. F., Thoolcn, M. J., Chiu, A. T., Ratson. D. W., and Timmermans, P. 8. M. W. M. (1989). Monoclonal antibodies directed against basic fibroblast growth factor which inhibit its biological activity in vitro and in viva. Kiockerrc. Bioph!/s. Rf%. (‘/,I,/ ,,I I, ,1. 164, 736-743. Rrnko, M., Quarto, N., Morimoto, T., and Rifkin, D. B. (1990). Nuclear and cytoplasmic localization of different basic fibroblast growth factor species. .I. CfdI. I’!/qsiol. 144, 108-114. Richardson, TV’. D., Pringle, N., Mosley, M. J., Westermark, B., and Dubois-Dalcq, M. (1988). A role for platelet-derived growth factor in normal gliogenesis in the central nervous system. &II 53, 309-319. Rifkin. D. B., and Moscatelli, D. (1989). Recent developments in the cell biology of basic fibroblast growth factor. J. Ceil Rid. 109, l-6. Risau, W. (1986). Developing brain produces an angiogenic factor, I’tYK: .Vnll. Acud. SC;. K&1 83, 3855-31859. Risau. \V.. Gautschi-S ova, P., and Bohlcn, P. (1988). Endothelial cell growth factors in embryonic and adult chick brain are related to human acidic fibroblast growth factor. EilfBO ,I 7, 959-962. Saneto. R. I’., and de Veilis, J. (1985). Effect of mitogens in various organs and cell culture conditioned media on rat oligotlcndroc~tes. Jk/,. A~f’/~,YJ.SC~i. 7, 340-350. Seed, J., Oln-in, B. B.. and Hauschka, S. D. (1988). Fibroblast growth factor levels in the whole embryo and limb bud during chick developmen1. Ikl! l?id. 128, X-57.

Sherman, L., Stocker, K., Rees, S., Morrison, R., and Ciment, G. (1991). Expression of multiple forms of bFGF in early avian embryos and their possible role in neural crest cell commitment. AUK IVY Accctl. sci. 638, 470-473. Skoff, R. P., Price, D. L., and Stocks, A. (1975). Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. J. Cow/~. Nmrd. 169, 291X334. Thomas, P. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. P/w. hictl. Actrtl. Sri. I!%1 77 5201~5205. Turier, D. L., and Cepko, C. L. (1987). A common progenitor for ntrurons and glia persists in rat retina late in development. ,Vo:trt/cw328, 131-136. Unsicker, K., Rr,ichcrt-Preihsch, H., Schmidt, R., Pettmann, B., Labourdettc, G., and Sensenbrenner, M. (1987). Astroglial and fibroblast growth factors have neurotrophic functions and cultured peripheral and central nervous system neurons. E’roc. i\‘etl. Acctrl. S’ci. Us/l 84, 5459~463. Walicke, P., Cowan, 1%“.M., Lena, N., Baird, A., and Guillemin, R. (1986). Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. P~oc. ;V~tl. AU/Cl. sci. 1:s.~ 83, 3012-3016. Walickc, P. A. (1988). Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions. .J. X~~urowi. 8(7), 2618-2(X7. Wanaka, A., Jr., Johnson, E. M., and Milbrandt, J. (1990). Localization of FGF receptor mRN.4 in the adult rat central nervous system b3 in situ hybridization. ~‘v’e/cro~l5, 267~281. Wanaka, A., Milbrandt, J., and Johnson, E. M. (1991). Expression of FGF receptor gene in rat development. IIri&/~r,rc,,/t 111, 455+t68. Wcstermann, R.. Johannscn, M., IJnsicker, K., and Grothe, C. (1990). Basic fibroblast growth factor (bFGF) immunoreactivity is present in chromafhn granules. ,/. A:c,rcwc~llc,w. 55, 28%%!)2. Wetts, R., ant1 Fraser, S. E. (1988). Multipotent precursors can give rise to all major cell tj-pes in the frog retina. S’cierccc,312, 1142-l 14;,. IVoodwarti. \I’. R., Nishi, R., Meshul, C. K., Williams, T. E., Coulombe. M., and Eckenstein, F. P. (1992). Nuclear and cytoplasmic localization of basic fibroblast growth factor in astrocytes and (‘A2 hippocampal neurons. .J. ~V~u~~osci.12, 142-1Z.