acidic fibroblast growth factor is expressed abundantly by photoreceptors within the developing and...

European Journal of Neuroscience, Vol. 5, pp. 1586-1595 @ I993 European Neuroscience Association

Acidic Fibroblast Growth Factor is Expressed Abundantly by Photoreceptors Within 'the Developing and Mature Rat Retina

K. Bugra', L. Oliver, E. Jacquemin, M. Laurent, Y . Courtois and D. Hicks2 INSERM U. 118, affilibe CNRS, Association Claude-Bernard, 29 rue Wilhem, 75016, Paris, France 'Present address: INSERM U. 29, HBpital Cochin, 124 Blvd Port Royal, 75014, Paris, France 2Present address: Laboratoire Laveran, Clinique Ophthalmologique, HBpital Civil, 1 place de I'HBpital, BP 426, 67091 Strasbourg Cedex, France

Key words: differentiation, mRNA, PCR analysis, immunohistochemistry, immunoblotting

Abstract

In order to further understand the role(s) of fibroblast growth factors (FGFs) in the development, differentiation and function of the central nervous system, we analysed the expression of the mRNA, and the presence and tissue distribution of the translated product, of one member of the FGF family, acidic FGF (aFGF), within the mammalian retina. Firstly, the relative abundance of aFGF mRNA was assayed in embryonic (between 14 and 17 days of gestation), postnatal (between 1 and 17 days after birth) and adult rat retina by quantitative reverse transcription-coupled polymerase chain reaction amplification using specific aFGF oligonucleotides. The level of expression remained uniformly low throughout the embryonic period and until postnatal day 7. Therefore the quantity of aFGF mRNA increased rapidly, reaching 80% of adult levels by eye opening (postnatal day 13). Adult levels were three-fold higher than at early developmental times. In situ hybridization of adult rat retina using specific antisense aFGF riboprobes revealed labelling in all cellular layers. Antisera raised against recombinant human aFGF revealed very little labelling of 4-day postnatal retina, but by postnatal days 8 and 17 immunoreactive aFGF was localized mainly within the photoreceptor cell bodies. Western blots of retinal extracts derived from 17-day embryonic, 4-day postnatal and adult retina probed with the same antibody revealed a single immunoreactive band of the expected molecular weight (18 kDa) in all extracts. Thus aFGF is mostly transcribed and translated within the retina subsequent to the major steps of cell birth, migration and differentiation, and seems to be abundantly expressed by maturing photoreceptor cells.

Introduction

In recent years, a great deal of interest has been shown in the roles played by neurotrophic factors in the development, differentiation and survival of cells within the central nervous system (CNS). The fibroblast growth factors (FGFs), a family of seven currently identified low molecular weight polypeptide growth factors and oncogenes (for review see Burgess and Maciag, 1989), have been especially highlighted as molecules playing essential roles in CNS metabolism (for review see Wagner, 1991). Much evidence now points to the synthesis (Alterio et al., 1988; Wilcox and Unnerstall, 1991), distribution (Fu et al., 1991; Ishikawa et al., 1991), modulation (Eckenstein et al . , 1991) and effects (Otto er al., 1987; Anderson er al . , 1988) of the two prototype members, acidic FGF (aFGF) and basic FGF (bFGF), within the CNS in vivo. In addition, another family member, int-2, is transiently expressed during CNS development (Wilkinson et al., 1989). There are also abundant data available demonstrating the survival, differentiation and neurite outgrowth-promoting effects of aFGF and bFGF on different populations of primary cultures of brain neurons and glia,

and cell lines, in vitro (e.g. Walicke et al., 1986; Momson et al., 1988; Walicke and Baird, 1991).

The vertebrate retina develops initially as an evagination of the diencephalon, rapidly folding and flattening to form a cup-shaped neuroepithelium (Barnstable, 1990). Following extensive cell proliferation and migration, at maturity it consists of an orderly layered arrangement of relatively few neuronal and glial cell types, many of which have been well characterized as to their biochemical, physiological and pharmacological properties (Blanks, 1982). The processes underlying the control of cell type differentiation and layer formation within the retina are poorly understood, but it seems clear that the timing of cell birth alone is insufficient to confer a particular phenotype, as retroviral clonal analysis has revealed that at any one time a pluripotential precursor can give rise to several cell types (Turner and Cepko, 1987; Wetts and Fraser, 1988). Recently Harris and Messersmith (1992) have proposed that two inductive steps are necessary for the determination of the rod photoreceptor phenotype, and that these inductions must

Correspondence to: D. Hicks, as above

Received 21 January 1993, revised 24 May 1993, accepted 19 July 1993

aFGF expression in developing and adult retina 1587

be at short distance. It is hence possible that local environmental cues, such as diffusible factors operating over short range, are involved in the development of the retina and other regions of the CNS.

Molecules displaying mitogenic activity, subsequently identified as the retinal forms of FGFs, were first isolated 15 years ago (Arruti and Courtois, 1978). Since that time, much evidence has accumulated concerning the presence (Plouet et al., 1988), synthesis (Jacqemin et al., 1990; Noji et al., 1990), distribution (Caruelle et af., 1989; Hanneken et al., 1989; De h n g h and McAvoy, 1992), binding (Jeanny et al., 1987; Fayein et al., 1990), and possible functions of aFGF and bFGF (Mascarelli et al., 1989, 1991; reviewed in Hicks et al., 1991), within the retina. The light-sensitive photoreceptor cells have been particularly well studied in this respect; FGF has been purified and characterized from the outer segments of these cells, where its binding has been shown to be dependent on cyclic nucleotides and phosphorylation (Plouet et al., 1988; Mascarelli et al., 1989). In addition, an important role for aFGF and bFGF in photoreceptor differentiation and survival has been demonstrated in v i m (Hicks and Courtois, 1988, 1992) and in vivo (Faktorovich et al., 1990). Lastly, implantation of bFGF-impregnated foam into retinotectomized chick embryo eyes stimulates regeneration of a new neural retina from the overlying retinal pigmented epithelium (RPE) (Park and Hollenberg, 1989).

Much of this data are qualitative and have been obtained from relatively mature tissue, and we still know very little about the role of endogenous FGFs in retinal development and differentiation. As a first step to answering this question, we have analysed both the relative abundance and distribution of aFGF messenger RNA (mRNA) transcripts and the appearance and distribution of aFGF protein within the developing and mature rat retina, and show that aFGF expression occurs mostly subsequently to precursor proliferation and migration, and that it is present in abundance within photoreceptors. Parts of this work have been communicated previously in abstract form (Hicks et al., 1992).

Materials and methods

Tissue collection Female Long -Evans rats with dated pregnancies were maintained on a normal light/dark cycle (12 h light:12 h dark) until ready for use. For embryonic samples, mothers were killed by C 0 2 anaesthesia and cervical dislocation, and the embryos rapidly dissected free using aseptic techniques and placed in warmed tissue culture medium. For embryonic (E) day 14, the entire optic lobe was harvested, while for subsequent stages (E17) the neural retina was carefully dissected free of surrounding tissue such as the RPE. Tissue was instantly frozen in liquid nitrogen and stored at -70°C until ready for use. Newborn [postnatal (PN) day 11 and PN4, PN7, PNlO, PN13, PN17 and PN23, as well as adult retinas were also collected. Entire litters (about ten rats) were used for data points at E l l to PN4, and half-litters for all older times.

mRNA analysis Total RNA was isolated from the frozen tissues and cells by the acid guanidinium thiocyanate - phenol - chloroform method (Chomczynski and Sacchi, 1987). The RNA samples were treated with RNase-free DNase to eliminate contaminating DNA according to the manufacturer's instructions (Promega). The quality and the relative concentrations of the RNA preparations were verified by agarose gel electrophoresis and ethidium bromide staining. The exogenous RNA added for co- amplification was extracted from light-induced tobacco leaves by the method of Chirgwin et al. (1979).

The oligonucleotide primers for aFGF amplification were selected from different exons: the sequence for the 3'-complementary primer is 5'-AAGCCCGTCGGTGTCCATGG-3', and that for the 5'-identical primer is 5'-GATGGCACAGTGGATGGGAC-3' (Abraham et al., 1986; Alterio et ul., 1988; Goodrich et al., 1989). The 3'complementary primer sequence for nitrate reductase is 5'-AGGAGCTGATGT- GTTGCCCGG-3' (Vaucheret et al., 1991). The primers were synthesized with an Applied Biosystems automated DNA synthesizer at the Pasteur Institute.

For amplification, to each 1 pg retinal total RNA under investigation 40 ng tobacco leaf RNA was added as a source of internal control. Reverse transcription of aFGF and nitrate reductase RNAs was carried out simultaneously using the 3'-complementary primers at 42°C by Moloney monkey leukemia virus reverse transcriptase (BRL), and the reactions were stopped by heating to 95°C for 10 min. One-tenth of the cDNA product was amplified in a Perkin - Elmer Cetus Thermal Cycler in a final volume of 100 pl buffer containing 50 mM Tris-HC1, pH 9, 15 mM (NH4)S04, 7 mM MgC12, 50 mM KCl, 1 mh4 deoxyribonucleotide triphosphate, 30 pmoles each of aFGF and nitrate reductase primer pairs and 1 unit Tuq polymerase. Each amplification cycleconsisted of 92°C for 1 min; 62°C for 1 min; 72°C for 1 min. The exponential phase of the reaction was experimentally determined, and for comparative analysis the samples were accordingly amplified for 24 cycles, the extent of the amplification reflecting the initial concentration of transcripts (Chelly et al., 1988).

To analyse the amplified products, 20 pl of amplified DNA was separated on a 10% acrylamide gel and transferred to nylon membranes (Hybond N+, Amersham), hybridized with specific probes. Bovine aFGF probe corresponding to the first two exons, a full-length nitrate reductase cDNA (gift from Dr C. Meyers, INRA), and bFGF cDNA (gift from Drs Abraham and Fiddes, California Institutes of Technology) were labelled with [32P]deoxyribocytidine triphosphate by random priming (Feinberg and Vogelstein, 1984). Hybridizations were carried out at 45°C overnight, and final washing of filters was done at 55°C in 0.1 xSSC, 1 % sodium dodecyl sulphate (SDS) for 30 min. The filters were exposed to X-OMAT AR5 (Kodak) film at -80°C for variable times. The densitometric scannings were done using a Biocom image analyser, and the aFGF readings were normalized using the nitrate reductase scannings as reference.

aFGF in situ hybridization Sections (10 pm) of adult rat eyes mounted on sterile gelatine-coated slides were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated and stored at -20°C until used. Pretreatment of the slides was performed according to Chariaut-Marlangue et al. (1992): prehybridizations were done at 37"C, 2 h in 50% formamide, 750 mM NaCl, 25 mM PIPES, pH 6.8,25 mM EDTA, 1 XDenhardt's solution, 100 mM dithiothreitol, 0.2% SDS, 250 pg/ml Escherichia coli DNA, 250 p g / d poly(A).

The PstI-XbaI fragment of the bovine cDNA was cloned into Bluescript KS+ transcription vector (KB5-15). The plasmid was linearized with HindIII to generate antisense transcript using T7 RNA polymerase, while for sense transcript KB5-15 was linearized with Not1 and T3 RNA polymerase. We used [35S]UTP, loo0 Cilmmol, at a final concentration of 25 pM of UTP, 500 pM for each of the other three nucleotides, and followed the protocol provided by Stratagene. Subsequently the template was removed by DNase digestion, and the transcripts were subjected to limited alkaline hydrolysis to obtain an average fragment size of 200 nucleotides. Hybridizations were performed at 42°C overnight with lo7 c.p.m./d of RNA probe. Subsequent to the RNase digestions (50 pg/ml for 30 min at 37"C),

1588 aFGF expression in developing and adult retina

slides were washed twice at 42°C with 2 XSSC, and once at 50°C with 0.1 XSSC. Slides were then dehydrated, dipped in Amersham LM1 emulsion and exposed for 2 weeks.

Antibody preparation Adult New Zealand White rabbits were injected subcutaneously with 200 pg recombinant human aFGF supplemented with 400 pg heparin suspended in Freund's complete adjuvant. Animals received a single booster, and were bled according to standard ethical procedures. Titre and specificity of antibody preparations were determined by ELISA and Western blots using purified aFGF and bFGF as standards (Oliver et al., 1992). In these studies, antibodies obtained from two rabbits, and from different bleeds, were used.

Western blotting Retinas isolated from El7 (one litter), PN4 (n = 10) and adult (n = 6) rats were lysed in 1 ml PBS containing 2 M NaCl, 0.1 % 3-[(cholamido- propy1)-dimethylammonio]-1-propane sulphonate and protease inhibitors in a hand-held tissue homogenizer on crushed ice. Cold PBS (5 ml) was added and an aliquot taken to determine protein. concentration (Bio-Rad). 200 pl heparin-ultrogen (IBF Labs, France) was added and incubated overnight at 4"C, the suspension centrifuged and the supernatant discarded. The pellet was washed once in 40 ml cold PBS, and ten times in 1 ml cold PBS. Finally, 100 pl Laemmli buffer was added to each sample and then electrophoresed on an 18 % polyacrylamide gel. Representative lanes were silver-stained, while duplicates were transferred onto nitrocellulose and soaked in PBS containing 1 % fat-free milk powder and 0.05 % Tween 20 (buffer A). The blots were then incubated in anti-aFGF antibody (diluted 1:5OOO in buffer A for 2 h at 22"C), washed and incubated in biotinylated donkey anti-rabbit IgG (dar-Bio, Amersham; diluted 1: lo00 in buffer B for 1 h at 22"C), and finally washed and incubated in streptavidin - horseradish peroxidase (Amersham; diluted 1: lo00 in buffer B for 1 h at 22°C). Bands were detected using the ECL system (Amersham) with 1 s exposure on Kodak X-ray film.

lmmunohistochemistry Retinal setions (10 pm) were used either unfixed or fixed rapidly in cold (-20°C) acetone for 2 min, or in 4% paraformaldehyde in PBS at 4°C for 10 min. Sections were preincubated in PBS containing 5 % fat-free milk powder and 0.1 % Tween 20 (buffer B) for 30 min at 22"C, and then incubated in anti-aFGF antibody diluted 1 : lo00 in buffer B for 3 - 15 h at 4°C. Control studies included the use of preimmune sera, sera obtained by injecting rabbits wtih 400 pg heparin only, anti-aFGF antisera preadsorbed with 10 pg purified aFGF and omission of the primary antibody. All sections were washed thoroughly in PBS, then incubated sequentially in dar-Bio (10 pglml in buffer A, 60 min at 22 "C) followed by ExtrAvidin - Tetrarhodamine Isothiocyanate (Sigma) (10 pglml in buffer A, 60 min at 22°C). For double labelling, rho4D2 anti-rhodopsin monoclonal antibcdy (Hicks and Molday, 1986) was included in the primary antibody solution, and binding revealed using goat anti-mouse IgG - fluorescein isothiocyanate (Biosys, France). Slides were examined using a Leitz Axioplan microscope equipped with fluorescence and Nomarski optics, and photographs taken using the Leitz Multiphot automated exposure system on Ilford HP5 film.

Results aFGF mRNA transcription The amplified aFGF target fragment was 135 bp, and its identity was verified by restriction digestions. The PvuII digestion produced 89 and

FIG. 1. The 135 bp fragment (lane 1) was digested with Pvun (lane 2) or with PsrI (lane 3) prior to Southern blotting and hybridization. The 135 bp fragment (arrow) was amplified from 1 pg retinal total RNA for 24 cycles, and 1/5 of the reaction mix was used per lane in the Southern blot preparation. The blot was hybridized with the specific probe for aFGF (lane 4) or bFGF (lane 5). Molecular weight markers were obtained from Ekehringer Mannheitn (marker 5).

FIG. 2. Developmental expression of aFGF in the rat retina. One microgram of total RNA from dissected embryonic (El4 and E17) and postnatal retinas (PNl -adult) was added to 40 pg tobacco leaf RNA and cDNA synthesis was carried out in the presence of 3'-complementary primers of aFGF and nitrate reductase. Amplifications were performed as described in Materials and methods for 24 cycles.


FIG. 3. In situ hybridization of adult rat retina using antisense (a) and sense (b) aFGF riboprobes. The antisense probe reveals large numbers of silver grains overlying the outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) (a), while the sense control reveals only sparse grains scattered over the retina @).

46 bp fragments, and the PsrI digestion resulted in 86 and 49 bp fragments, as predicted from the restriction map of the rat aFGF sequence (Goodrich el al . , 1989) (Fig. 1). The 135 bp fragment specifically hybridized with aFGF but not with bFGF cDNA probes (Fig. 1).

Quantitation of the relative abundance of aFGF mRNA during retinal development revealed a uniform steady-state level of - 30% of adult levels throughout embryogenesis and up to PN7 (Fig. 2). Thereafter there was a rapid linear increase in the relative abundance of message, with 80% maximal levels attained by PN13. Maximal expression of aFGF mRNA was observed by early adulthood (PN23), and was maintained at this level until the oldest time point examined (12 months).

aFGF in situ hybridization Sections of adult rat retina incubated with antisense aFGF riboprobes revealed the presence of silver grains overlying the three cellular layers: the outer and inner nuclear layers and ganglion cell layer (ONL, INL and GCL respectively) (Fig. 3a). The use of the corresponding (control) sense aFGF riboprobe revealed only background levels of silver grains overlying these cellular compartments (Fig. 3b). Similar incubations of developing (PN1, PN4 and PN8) retinal sections failed to reveal any deposition of silver grains above background levels (data not shown).

Western blotting The aFGF antibodies used in the present study showed strong binding to samples of purified human recombinant aFGF, but no cross-reactivity with purified human recombinant bFGF (Fig. 4). Retinal extracts

isolated from E17, PN4 and adult rats and purified through heparin- ultrogel affinity columns each contained a single immunoreactive band at 18 kDa when incubated in anti-aFGF antibody (Fig. 4). Although the heparin affinity chromatography made it impossible to quantitate the relative amounts of growth factor in the different preparations, it was indispensable to remove the large excess of soluble proteins which otherwise masked the signal.

aFGF and opsin immunohistochemistry Labelling of sections of PN4 normal rat retina (in which layer formation is still fairly rudimentary, and photoreceptor differentiation is only just beginning; Fig. 5a) with anti-opsin antibody revealed faint but positive immunoreactivity within the posterior retina in the area of the developing ONL (Fig. 5b). Use of anti-aFGF antiserum revealed very little binding (Fig. 5c). By PN8, most layer formation is complete in the rat retina, with a central-to-peripheral gradient of maturation (Fig. 6a). Labelling of such sections with anti-opsin antibody revealed a strong signal in the central ONL (Fig. 6b), decreasing in intensity towards the peripheral ONL. Double labelling of these same sections with anti-aFGF antiserum also revealed a central-to-peripheral gradient of binding intensity. Labelling was primarily associated with the photoreceptor cells in the central ONL, with some faint immunoreadon product present in the IPL (Fig. 6c). As with the opsin immunolabelling, the intensity of aFGF immunoreactivity decreased toward the periphery. By PN17 all cellular proliferation and migration had finished, and the retina was essentially in its adult form, the photoreceptors having largely elaborated their outer segments (Fig. 7a). Opsin immunoreactivity was


FIG. 4. Silver-stained polyacrylamide and immunoblotted E17, PN4 and adult retinal samples. Retinal homogenates from rat retinas were prepared as described in Materials and methods, and fractionated on heparin-ultrogel. The eluants were subsequently run on SDS-PAGE, and silver-stained (PN4, lane F; adult, lane G). Following transfer to nitrocellulose, the samples were incubated with anti-aFGF IgG (E17, lane C; PN4, lane D; adult, lane E). In all three cases, a single band of 18 kDa is visible. The bands comigrate with purified aFGF (lane A), whereas purified bFGF does not bind the antibody (lane B). Molecular weight markers are indicated on the right.

observed throughout the ONL, from the posterior (Fig. 7b) to peripheral retina. aFGF-immunoreactive product was now present throughout the entire ONL, from the centre (Fig. 7c) to the periphery, and as at PN8 was restricted to the perinuclear cytoplasm of the photoreceptors with very little binding to the nuclear regions. Outer segments were devoid of aFGF-like immunoreactive product, contrary to the opsin labelling pattern (compare Fig. 7b and c). Some faint binding was observed in the lower retina (arrow, Fig. 7c), although this was variable. At all ages examined, control sections showed reduced levels of labelling as compared to test sections (Figs 5-7d), except for weak non-specific labelling of the sclera and inner segments. These labelling patterns were obtained with aFGF antisera obtained from two rabbits, and from separate bleeds.

Discussion We have shown in the present study that aFGF transcription, as measured by PCR amplification using oligonucleotide primers, remains at a steady, low level throughout embryonic and early postnatal retinal development, then rises quickly through the following week to attain near (80%) maximal levels by PN13. This time course coincides with the expression of the translated protein as detected by immunohistochemical means, present primarily within the newly forming and differentiating photoreceptors.

Specificity of aFGF mRNA and protein analysis We have set up a PCR amplification system where the extent of coamplified exogenous nitrate reductase levels was used as means of

normalization. It has been shown that reverse transcription-coupled PCR amplification reactions at the exponential phase reflect the initial RNA levels (Chelly et al. , 1988; Wang er al. , 1989).

Concerning previously published data on the immunohistochemical localization of aFGF within the retina, Caruelle er al. (1989) reported widespread immunolabelling of retinal cell bodies and fibre layers, Elde er al. (1991) only observed retinal ganglion cell staining, and De Longh and McAvoy (1992) detected ganglion cell and some neuroblastic zone labelling. Our results differ from those of Elde er al. (1991) in that we did not observe strong binding to retinal ganglion cells, while they did not detect photoreceptor labelling. Curiously, neither Elde er al. (1991) nor we detected aFGF immunoreactivity within the photoreceptor outer segment, while in fact these organelles are a relatively rich source of this factor (Plouet et al., 1988). We agree with Elde er al.’s findings that Miiller glial cells (Hicks and Courtois, 1990) appear to contain very little aFGF, either mRNA or protein (Hicks et al. , 1991; Malecaze et al . , 1991). Elde er al. (1991) also localized aFGF expression by in situ hybridization, and showed it to be expressed mainly within the GCL, while we (Jacquemin er al., 1990) and others (Noji et al., 1990) have revealed message throughout the adult retina. Western blots of retinal preparations showed that the antibody used here recognized a single band of 18 kDa, and that no higher molecular weight forms of aFGF were detected, contrary to the results of Fu et al. (1991). In apparent discrepancy with the data on aFGF immunolocalization, we did observe aFGF in immunoblots of immature retina. And although levels of expression of aFGF mRNA during early development were too low to be detected by in siru hybridization, aFGF was expressed outside the ONL in adult retina.


FIG. 5. Immunohistochemistxy of PN4 rat retina. (a) PN4 retina is relatively immature, with a broad neuroblastic zone (NZ) lying above the granular cell layer (GCL). (b) Anti-opsin antibody labels a few elongated immature cells at the scleral surface (arrow). (c) Anti-aFGF antiserum does not bind significantly to retina at this age. (d) Control sections incubated in preimmune serum, same dilution as test sera. Scale bar = 25 pm.

FIG. 6. Immunohistochemistry of PN8 rat retina. (a) The neuroblastic zone has now split into the outer nuclear layer (ONL) and inner nuclear layer (INL), and the inner plexiform layer (IPL) and granular cell layer (GCL) are clearly seen (Nomarski image). (b) Anti-opsin labelling is strong in the central ONL, restricted to the perinuclear cytoplasm of the photoreceptor cell bodies. (c) Anti-aFGF IgG primarily labels the ONL as well, and the immunoreactive product is localized to the perinuclear cytoplasm. Some faint labelling of a few scattered cells in the INL, IPL and GLC is also evident. (d) Control sections using IgG from the non-retained fraction obtained from the aFGF-Sepharose affinity column. Scale bar = 25 pn.


FIG. 7. Immunohistochemistry of PN17 retina. (a) By this age the retina is almost fully developed, with the three cellular layers (outer and inner nuclear layers and granular cell layer; ONL, INL and GLC) separated by the outer plexifonn layer (OPL) and inner plexifonn layer (IPL) respectively. In addition, the photoreceptors now possess both an inner segment (IS) and an outer segment (0s) at their apical surface (Nomarski image). (b) Anti-opsin antibody labels the ONL, and especially heavily the outer segment region. (c) Anti-aFGF IgG labelling of the same section reveals heavy immunoreaction within the ONL, but complete absence of label from the outer segment. There is also strong labelling of the OPL, and some faint staining of the upper INL (arrow), IPL and GCL. (d) Control sections using antisera raised against heparin only. Notice there is weak non-specific labelling of the inner segment. Scale bar = 25 pm.

Taken together, the data presented here indicate that the immunohistochemical detection of aFGF within the photoreceptors correlates with the late rising phase of aFGF mRNA expression. If this is true, it indicates that these cells contain -70% of total adult retinal aFGF. The remaining 30% present in other retinal compartments and at earlier times, although detectable by in situ hybridization and Western blotting, seems to be immunologically masked in tissue sections. In particular the abrupt cut-off in labelling observed at the level of the outer segment indicates that a change in aFGF conformation may be occurring between the site of synthesis and transport to its (functional?) site.

The antibodies used in the present study were raised by the co-injection of recombinant aFGF and heparin (Oliver er al., 1992). Previous attempts at raising anti-aFGF antisera in our laboratory, in which recombinant aFGF alone was injected, produced a generally weak immune response with specific antibodies of low titre. As heparin has been shown to stabilize the structure of aFGF (Schreiber er al., 1985) and to protect both aFGF and bFGF against thermal, acidic and enzymatic degradation (Gospodarowicz and Cheng, 1986; S o w e r and Rifkin, 1989), it may favour an immune response directed against a folded configuration. Heparin alone did not induce generation of anti-aFGF antibodies (see Results). Hence an interesting possibility is that heightened aFGF-like immunoreactivity within the photoreceptors

reflects the presence of an immunologically distinct form of the molecule, perhaps coupled with heparin sulphate proteoglycan. It is clear from the differing immunolocalization patterns for aFGF cited above, and those reported for bFGF (e.g. Hanneken etal . , 1989; Hageman et al., 1991; Connolly et al., 1992) and for transforming growth factor P (TGF-P) (Kardami et al., 1990) that caution must be observed in interpreting such data. Factors such as tissue fixation, species and regional differences, antibody titre and methods of antibody preparation can all affect the particular signal observed.

aFGF expression and retinal function With the methodological approaches used here, the majority of aFGF expression is of relatively late onset and is predominantly localized to the photoreceptors. As cell birth in the rodent retina extends from early in embryonic development ( - 12 days of gestation) to approximately PN7 (Young, 1985; Turner and Cepko, 1987), and layer formation is for the most part also complete by PN7 (Blanks, 1982), the low, non-varying levels of endogenous aFGF mRNA expression at these ages suggests it is less likely to be involved in these processes. It is clear that the photoreceptors have commenced terminal differentiation, as witnessed by the expression of the visual pigment opsin prior to immunohistochemically detected aFGF expression. They


are not, however, fully mature at the onset of aFGF synthesis as outer segment formation and synaptogenesis are ongoing at this time (Weidman and Kuwabara, 1968). aFGF expression in these cells hence correlates with the onset of their ‘functionality’, as opsin can be stimulated by light from around PN7 (Hicks and Barnstable, 1987), an electroretinogram can be recorded at PN12 (Weidman and Kuwabara, 1968) and phagocytosis of shed outer segments by the RPE commences around PN12 (Tamai and Chader, 1979). Considering that RPE cells possess high-affinity FGF receptors at their cell surface (Sternfeld et al., 1989; Malecaze er al., 1993), that the interphotoreceptor matrix contains bFGF (Hageman et al., 1991), that both aFGF and bFGF are synthesized by photoreceptor cells (Jacquemin et al., 1990; Noji et a/. , 1990) and RPE cells (Schweiger et al., 1987; Hicks et al., 1991), and that RPE cells derived from RCS rats with hereditary retinal dystrophy are deficient in FGF receptors (Malecaze et al., 19!93), it is clear that FGFs are somehow involved in photoreceptor/RPE interactions.

Exogenous aFGF can stimulate embryonic and early postnatal differentiation of rat retinal neurons in vitro (Hicks and Courtois, 1988; Guillemot and Cepko, 1992). As in vivo, little retinal aFGF is synthesized at this time, two possibilities exist that may explain the discrepancies. Firstly, the in vivo source of this factor may be from the RPE cells, which are capable of producing many growth factors and cytokines (e.g. Campochiaro et al., 1989; Tombran-Tink et al., 1991; Waldbillig et al., 1992; Malecaze et al., 1993). We are currently examining the developmental expression of aFGF and bFGF by the RPE cells to resolve this issue. Secondly, alternative members of the FGF family may be synthesized at earlier times (see below) and activate FGF receptors present in the retina at this time (Heuer et al., 1990), as in certain cases individual receptor types can respond to multiple ligands (Johnson et al., 1990). The Miiller glial cells themselves seem to make very little aFGF, either in vitro (Hicks et al., 1991) or in vivo (Malecaze et al., 1991), so unless their own aFGF transcription is up-regulated in certain conditions they do not represent a major source of this protein.

Temporal expression of aFGF Although the evidence is still fragmentary and somewhat contradictory, a general picture of successive appearance of FGF family members during CNS development and maturation appears to be emerging. FGF-5 is present very early, at early postimplantation (Hkbert et al., 1990); int-2 is expressed transiently during mid gestation (Wilkinson et al., 1989); and bFGF seems to be synthesized towards mid or late gestation (HCbert et al., 1990), and analysis of brain extracts reveals very low levels prior to birth (Caday et al., 1990). The developmental expression of aFGF seems to be more controversial, as some groups using molecular biological techniques detect significant quantities of this molecule early in development (Hkbert et al., 1990; Wilcox and Unnerstall, 1991), whilst biochemical analyses reveal only trace levels of aFGF protein up to birth, followed by large postnatal increases in CNS expression (Caday et al., 1990; Ishikawa et al., 1991). These same two groups report that expression of both bFGF and aFGF continues at high levels in the adult CNS, whilst Wilcox and Unnerstall (1991) signal a decline in aFGF expression with age. Schnurch and Risau (1991) demonstrate a developmental increase in aFGF expression in chick CNS, and Fu et al. (1991) detect an increasing intensity of aFGF immunoreactivity during embryonic development. It is difficult at the present time to resolve the differences in these results for aFGF, but the use of whole embryos in certain cases may obscure tissue- specific variations such as those signalled here. Developmental variations are also observed for other heparin-binding growth factors such as

retinoic acid-induced heparin binding growth factor (Urios et al., 1991), as well as certain forms of FGF receptors and nerve growth factor receptors (Heuer et al., 1990). Thus it is possible that each particular member plays a specific role in the establishment and maintenance of a given differentiating neural tissue.

To extend our knowledge of the synthesis and possible functions of the FGF family during development and maturation of the retina, we are currently using similar technical approaches to quantitate the developmental expression and distribution of bFGF, int-2, F G F J and several high-affinity FGF receptors (flg, bek, etc.) to examine their role in retinal cell function.

Acknowledgements The authors wish to thank D. Raulais for assistance in preparing the anti-aFGF antibodies, H. Cob for photographic work and V. Kirschenbaum for typing the manuscript.

Abbreviations aFGF bFGF CNS E EDTA FGF GCL INL ONL PN PBS PIPES W E

acidic fibroblast growth factor basic fibroblast growth factor central nervous system embryonic day ethylene diamhe tetraacetic acid fibroblast growth factor ganglion cell layer inner nuclear layer outer nuclear layer postnatal day phosphate-buffered saline piperazine-N,N’-bis[2-ethanesulphonic acid] retinal pigmented epithelium

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acidic fibroblast growth factor is expressed abundantly by photoreceptors within the developing and...

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