Biomedical subjects

Y Courtois

Publications and source records attributed to Y Courtois.

At least 91 records · Page 5Linked to original sources

PubMed1994

Expression of inducible nitric oxide synthase in cytomegalovirus-infected glial cells of retinas from AIDS patients.

The inducible isoform of nitric oxide synthase has been detected in cytomegalovirus (CMV)-infected retinas from acquired immunodeficiency syndrome (AIDS) patients by immunohistochemistry and NADPH-diaphorase staining. Subsequent immunohistochemistry using antibodies against CMV antigens and glial fibrillary acidic protein (GFAP) demonstrated that inducible NOS was localized in CMV-infected glial cells, particularly Müller cells. These findings indicate that inducible NOS is expressed in vivo in the human retina as a result of viral infection, and suggest that high levels of NO production might be involved in CMV-induced retinitis.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Human retinal pigmented epithelial cells produce nitric oxide in response to cytokines.

The present study demonstrates that human retinal pigmented epithelial cells produce nitric oxide (NO) upon co-treatment with interferon gamma (IFN gamma) and interleukin-1 beta (IL-1 beta). The biosynthesis of NO, which was measured by the accumulation of the stable end-product nitrite, requires an induction period of approximately 12 hours and continues for at least three days. The synthesis was abolished by the stereoselective inhibitors of NO synthase (NOS), NG-monomethyl-L-arginine, N omega-nitro-L-arginine and N omega-nitro-L-arginine methyl ester and by cycloheximide. Transforming growth factor beta suppressed cytokine-induced NOS. The results indicate that cytokines such as IFN gamma and IL-1 beta are capable of inducing NOS, while TGF beta prevents this induction, in subcultured pigmented epithelial cells from the human retina.

Amino Acid Oxidoreductases↗

PubMed1994

DNA strand breakage during physiological apoptosis of the embryonic chick lens: free 3' OH end single strand breaks do not accumulate even in the presence of a cation-independent deoxyribonuclease.

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosomes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation.

Adenosine Diphosphate Ribose↗

PubMed1994

Up-regulation of aFGF expression in quiescent cells is related to cell survival.

Exogenously administrated acidic FGF modulates the proliferation of several cell types, controls cell differentiation, and promotes cell survival. Most cells that are sensitive to exogenous aFGF are also capable of expressing it at very low levels. Thus in order to establish the role of endogenous aFGF as a mitogenic, differentiation, or survival factor, we studied the regulation of aFGF expression by evaluating the level of mRNA by PCR amplification and the concentration of protein by Enzyme Immuno Assay (EIA). In the lens, the amount of aFGF transcripts in nondividing cells of the central epithelium and in the differentiated fiber cells located at the periphery of the lens is similar, suggesting that endogenous aFGF is not involved with lens differentiation. In cultures, depending on the growth conditions, the endogenous aFGF expressed by Bovine Epithelial Lens (BEL) cells is subject to modulation. Cells arrested either by contact inhibition or by serum deprivation express more aFGF transcripts and protein than in exponentially growing cells, implying that endogenous aFGF has no mitogenic role under these conditions. In serum-deprived cells, the addition of specific aFGF antisense primers inhibits endogenous aFGF expression and leads to the death of these cells. These results associated with the higher expression of aFGF in nondividing BEL cells, suggesting that, contrary to exogenous aFGF, endogenous aFGF is not a mitogenic factor but a survival factor.

Animals↗

PubMed1994

Nitric oxide decreases in vitro phagocytosis of photoreceptor outer segments by bovine retinal pigmented epithelial cells.

The presence of nitric oxide synthase (NOS) in the retina, the constitutive isoform in photoreceptor outer segments and the inducible form in retinal pigmented epithelial (RPE) cells, has been demonstrated, but the role of the free radical NO produced, remains unknown. We have investigated the effect of NO on the process of rod outer segment (ROS) phagocytosis. Using an in vitro assay for phagocytosis in primary cultures of bovine RPE cells, we demonstrate that NO released by SIN-1 (3-morpholinosydnonimine) in the culture medium inhibits the phagocytosis of ROS. Furthermore, endogenous NO, produced by RPE cells cotreated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), is also able to decrease RPE cell phagocytic activity. This effect depends upon the continuous presence of NO during the assay and is abolished by the scavenging of NO by hemoglobin or by the inhibition of NO synthase activity by L-arginine analog, NG-monomethyl-L-arginine. Pretreatment of ROS with SIN-1 failed to impair subsequent phagocytosis, demonstrating that NO directly affects the RPE cells ability to phagocytose ROS. The inhibitory effect of NO is cGMP independent, since 8-bromo-cGMP does not modify this process. This decrease of ROS phagocytosis by RPE cells caused by NO may occur as a result of retinal inflammation, and could lead to photoreceptor degeneration.

Animals↗

PubMed1994

Effects of exogenous FGFs on growth, differentiation, and survival of chick neural retina cells.

Fibroblast growth factors (FGFs) are known to play important roles in various processes including development and differentiation. Chick embryo neural retina cells, capable of transdifferentiation into lentoid bodies and pigmented cells, were used in vitro to examine the effects of exogenous acidic (aFGF) and basic FGF (bFGF) on proliferation and protein accumulation. We demonstrate that both factors increase the proliferation of glial cells and modulate the survival of neurons without affecting protein accumulation within these cells. Moreover, FGFs stimulate the differentiation of the photoreceptors. The rate of proliferation varies over the period of culture, with a maximum occurring after 2 weeks, followed by a decrease concommitant with the appearance of lentoid bodies. The concentration of aFGF was measured using an enzyme immuno assay and showed an accumulation of this protein only in bFGF-treated cultures, suggesting that bFGF positively modulates aFGF synthesis in neural retina cell cultures.

Animals↗

PubMed1994

Superoxide inhibits proliferation and phagocytic internalization of photoreceptor outer segments by bovine retinal pigment epithelium in vitro.

Experiments were performed to investigate the effect of free radical damage on two aspects of retinal pigmented epithelium (RPE) metabolism, namely, proliferation and phagocytosis. Bovine RPE cells were maintained in monolayer cultures, either as passaged (for proliferation and lysosomal activity assays) or primary cultures (for phagocytosis measurements). Free radicals (superoxide anions) were generated by a xanthine oxidase (XO)-hypoxanthine (HX) reaction. Total phagocytosis (binding plus ingestion of rod outer segments (ROS)) was quantitated by radioimmunoassay using a specific anti-opsin antibody and iodinated secondary antibody. In some cases, agents with known or possible protective influences against oxidative damage, i.e., superoxide dismutase (SOD), vitamin E, and basic fibroblast growth factor (bFGF), were tested for their activity in this model system. RPE cell proliferation was inhibited in a HX-XO dose-dependent manner, in the absence of cell toxicity. Modifications of cell morphology were also noticed. Either simultaneous exposure of RPE cells to ROS membranes and HX-XO or pretreatment of ROS membranes with HX-XO prior to their addition to RPE monolayers led to a statistically significant 20-30% decrease in phagocytosis relative to control values. This decrease was essentially observed in the binding phase of phagocytosis, indicating damage to ROS surface molecules as the primary event. Addition of SOD or vitamin E prevented this loss of phagocytic activity, whereas bFGF had no effect. Superoxide radicals did not, however, affect phagocytosis when RPE cells were exposed to them alone, prior to incubation with ROS; nor did they alter a later stage in the phagocytic process, acid phosphatase activity. This tissue culture model represents a convenient system for analyzing free radical damage in different aspects of RPE-photoreceptor behavior and may be useful in studying this phenomenon in several retinal disorders.

Acid Phosphatase↗

PubMed1994

Spatial and temporal expression patterns of FGF receptor genes type 1 and type 2 in the developing chick retina.

Fibroblast growth factors are known to influence the growth and differentiation of cultured cells derived from the chick retina. The fibroblast growth factors can interact with a family of at least four closely related receptor kinases. To find a correlation between the presence of fibroblast growth factor receptors and eye development, the patterns of expression of transcripts encoding the type 1 (FGF-R1) and type 2 (FGF-R2) receptors in the developing chick retina have been studied. Northern blot analysis of RNA of the whole retina was used to observe that FGF-R1 transcripts are abundant at embryonic day 4 and then decrease until day 11. After this stage, the level of expression of FGF-R1 increases and its peak of expression at embryonic day 18 is concomitant with the detection of the opsin transcript. FGF-R2 transcript is also detected by Northern blots of RNA of the whole retina until embryonic day 6. However, the re-expression after embryonic day 11 of FGF-R2 could only be demonstrated by PCR studies. The same pattern of expression is observed with in situ hybridization of sagittal sections. The two genes are coexpressed in the pigmented epithelium and in the neural retina during embryonic development. The expression follows the retinal layering according to a gradient from the vitreous humor to the choroid. Quantification of the in situ hybridization signals demonstrates that the pattern of expression of both receptors diverges after embryonic day 6 between the pigmented epithelium and the neuronal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Induction and regulation of nitric oxide synthase in retinal Müller glial cells.

Müller glial cells from the rat retina were examined for their capacity to produce nitric oxide (NO). Treatment of retinal Müller glial (RMG) cells with lipopolysaccharide (LPS), interferon-gamma, and tumor necrosis factor-alpha induced NO synthesis as determined by nitrite release in media. Simultaneous addition of LPS, interferon-gamma, and tumor necrosis factor-alpha caused the largest increase in NO synthesis. NO biosynthesis was detected after 12 h and was dependent on the dose of LPS, interferon-gamma, and tumor necrosis factor-alpha. Stereoselective inhibitors of NO synthase (NOS), cycloheximide and transforming growth factor-beta, blocked cytokine-induced NO production. Cytosol from LPS/cytokine-treated RMG cultures, but not from unstimulated cultures, produced a calcium/calmodulin-independent conversion of L-arginine to L-citrulline that was completely blocked by NOS inhibitor. The expression of NOS in RMG cells was confirmed by northern blot analysis, in which stimulation of these cells led to an increase in NOS mRNA levels. We conclude that RMG cells can express an inducible form of NOS similar to the macrophage isoform. High NO release from activated RMG cells might represent a protection from infection but may also contribute to the development of retinal pathologies.

Amino Acid Oxidoreductases↗

PubMed1994

Basic fibroblast growth factor experimentally induced choroidal angiogenesis in the minipig.

Basic fibroblast growth factor (bFGF), a soluble mitogen, has been isolated and purified from various organs, including the retina. In vivo angiogenic activity of bFGF has been demonstrated with several assays. An experimental model of choroidal neovascularization was developed in the mini pig by perfusion of recombinant human bFGF through an osmotic minipump. Endogenous bFGF and bFGF receptors were localized in the normal pig retina by immunohistochemistry and autoradiography after binding. The perfusion of exogenous bFGF induced well-organized new vessels along the last 3 mm of the catheter in the suprachoroidal space. This neovascularization did not penetrate the normal Bruch's membrane. Vascular cells (identified by von Willebrand factor antibody staining) increased in number and in surface from the proximal part to the end of the intraocular catheter in all bFGF perfused eyes. In eyes perfused with phosphate buffered saline (controls), but not in the bFGF perfused eyes, an inflammatory response occurred (identified by a macrophage specific antibody). These results demonstrate that choroidal angiogenesis can be achieved without an inflammatory response by perfusing an excess of bFGF in the suprachoroidal space.

Animals↗

PubMed1994

Basic fibroblast growth factor modulates corneal wound healing after excimer laser keratomileusis in rabbits.

We have previously demonstrated beneficial effects of basic fibroblast growth factor (bFGF) on corneal epithelial healing in different in vivo models. In the present study, we investigated the influence on epithelial and stromal healing of topical bFGF after excimer laser keratomileusis in a rabbit model. The corneas of 24 New Zealand white rabbits were deepithelialized (7-mm diameter) and subsequently received identical deep stromal laser ablations (depth, 50 microns; 6 D; diameter, 5 mm). The animals were randomly assigned to one of four treatment groups: PBS (control); bFGF at 500 ng/application; dexamethasone 0.1%, and bFGF+dexamethasone (n = 12 eyes/group). All treatments were given four times daily; with bFGF being given until complete epithelial healing and dexamethasone and PBS, until 3 months postsurgery. Wound surface regression on time was determined by means of computer-assisted image analysis of fluorescein-stained corneas. Corneal opacity was scored using slit-lamp observation and graded following a previously established scoring system. As compared with the other three treatment groups, a highly significant acceleration in the rate of epithelial wound healing was found after bFGF application for 2-3 days only (P < 0.001). A combined therapy (bFGF+steroid) had no effect on the healing rate. As compared with control values, the peak scores for subepithelial haze were significantly lower in eyes that had received bFGF; the regression on time of the haze in the growth factor group paralleled that in eyes receiving continuous steroid treatment. These results demonstrate for the first time the efficiency of a growth factor in modulating the wound-healing response after excimer laser photoablation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Protection against light-induced retinal degeneration by an inhibitor of NO synthase.

The existence of nitric oxide synthase (NOS) in retinal rod outer segments and pigmented epithelial cells suggests that NO in excess could impair the interaction between these cells, resulting in photoreceptor degeneration. To test this hypothesis, NG-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, was intraperitoneally injected daily into rats subjected to constant illumination for 7 days in order to destroy their photoreceptors. By measuring photoreceptor nuclear layer thicknesses, we found that L-NAME partially protects (by up to 35%) against the degeneration of photoreceptors and acts to maintain their organization. Thus NO may be involved in the process by which photoreceptor degeneration results from constant illumination of the retina.

Amino Acid Oxidoreductases↗

PubMed1993

Acidic fibroblast growth factor is expressed abundantly by photoreceptors within the developing and mature rat retina.

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.

Animals↗

PubMed1993

[Influence of the running conditions of a diesel engine on the mutagenic effects of its emissions].

A direct exposure method to detect mutagenicity of car exhausts was applied to study the influence of the running conditions of a diesel engine functioning on an assay platform. Biological and physico-chemical approaches were carried out together to appreciate the quality of diesel emissions. Exhausts, emitted by a diesel engine (Renault, 2068 cm3) were sampled from a dilution tube connected to a constant volume sampler. Regulated pollutants (CO, NOx, HC and particles) and some non-regulated pollutants (monoaromatic hydrocarbons (MAH) and polyaromatic hydrocarbons (PAH), aldehydes) were analysed when the engine ran at different operating conditions of load (full, 3/4, 2/4 and 1/4) and of engine speed (from 1000 to 4000 rev./min). The mutagenicity (Ames test) of diesel exhaust was assessed by a direct exposure method which does not need extraction of the particles. Bacteria are pre-seeded onto the agar plate and exposed directly to the diesel exhausts parallelly to the measurements of pollutants. The engine speed and the load influence the level, the composition and the induced mutagenicity of the diesel emissions and the results show that: when engine speed increases all the gaz phase indicators increase independently of the load, those of the particulate phase and mutagenicity increase at low load but decrease at full load; when load increase, all the indicators of both phases decrease except NOx which increases.

Carbon Monoxide↗

PubMed1993

Differential regulation of inducible nitric oxide synthase by fibroblast growth factors and transforming growth factor beta in bovine retinal pigmented epithelial cells: inverse correlation with cellular proliferation.

Bovine retinal pigmented epithelial (RPE) cells express, after activation with interferon gamma (IFN-gamma) and lipopolysaccharide (LPS), an inducible nitric oxide synthase (NOS). Experiments were done to investigate the effects of the transforming growth factor beta 1, epidermal growth factor, and fibroblast growth factors (FGFs), which are abundant in the retina, on NOS activity. Transforming growth factor beta 1 slightly increases the production of nitrite, an oxidation product of NO, induced by LPS plus IFN-gamma, whereas acidic and basic FGFs markedly inhibit the nitrite release due to LPS/IFN-gamma in a concentration-dependent manner, and epidermal growth factor did not modify LPS/IFN-gamma-induced NOS activity. The growth factors alone did not stimulate nitrite release. We have attempted to elucidate the mechanism of FGF inhibition. Results with heparin, suramin, and tyrphostin suggest involvement of the high-affinity receptor for FGF in its inhibition of LPS/IFN-gamma-stimulated NOS activity. Continued stimulation of RPE cells with LPS/IFN-gamma was essential for the induction of NO synthesis, and maximal inhibition was obtained when FGF was present during stimulation with LPS/IFN-gamma, suggesting that FGF inhibits NOS induction. Furthermore, an antiproliferative action of NO was demonstrated by an inverse correlation between the amounts of nitrite or citrulline produced in response to different stimuli (LPS/IFN-gamma or LPS/IFN-gamma with growth factors) and the level of cellular proliferation. Similar inhibition of growth was obtained when RPE cells were incubated with an NO donor, sydnonimide. Because NO acts as a cytotoxic compound in the retina, FGF, by inhibiting the induction of NOS in RPE cells, may have beneficial effects in protecting the retina from cytokine and endotoxin-mediated tissue damage.

Amino Acid Oxidoreductases↗

PubMed1993

Fibroblast growth factor receptor deficiency in dystrophic retinal pigmented epithelium.

The retinal pigmented epithelium (RPE) is known to be the site of the primary lesion in inherited retinal dystrophy in the Royal College of Surgeons (RCS) rat, a model for retinitis pigmentosa. Although the only functional defect so far detected in these cells is their failure to efficiently phagocytose shed photoreceptor outer segment debris, the actual cause of photoreceptor cell death is still unknown. Recently the possibility of "trophic factors" important in photoreceptor survival produced by normal RPE but not by dystrophic RPE has been suggested. Hence we decided to investigate the presence and abundance of two candidate diffusible factors, the acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), as well as their high affinity cell surface receptors (FGF-R). mRNA was isolated from primary cultures of purified normal and dystrophic RPE and analyzed by PCR amplification using specific oligonucleotide primers for aFGF and bFGF: the size and abundance of amplified fragments was similar for both cell types. Also, aFGF protein, detected by immunocytochemistry using specific antisera, appeared to be present in approximately equal amounts and distributed in a similar pattern. However, scatchard analysis of radio-labelled bFGF binding to primary cultures of normal and dystrophic rat RPE revealed that dystrophic RPE possess only 29% the number of surface receptors compared to congenic normal cells. Furthermore, the level of expression of FGF-R2 mRNA, but not that of FGF-R1, was significantly different. Other parameters measured (receptor affinity, profile of ligand internalization and degradation, receptor molecular weight and mitogenic activity) did not show any significant differences between normal and dystrophic RPE. The precise role of FGF-R deficiency in the etiology of the disease hence remains to be determined, but it indicates the importance of trophic factors in the normal functioning of the retina.

Animals↗

PubMed1993

aFGF binding to low and high affinity receptors induces both aFGF and aFGF receptors dimerization.

Acidic Fibroblast Growth Factor (aFGF) binds on two classes of fibroblast growth factor receptors, the high affinity receptors (HAR) a family of four known transmembrane tyrosine kinases and the low affinity receptors (LAR), related to cell surface heparan sulfate proteoglycan (HSPG). We analysed the relationship between the binding of aFGF on the HAR and on the LAR in bovine lens epithelial (BEL) cells in the presence of heparin or suramin. Through Northern blotting analysis we demonstrated that the three immunoglobulin-like transcript of FGF receptor type 1 (FGF-R1) is the major expressed high affinity receptor in BEL cells. On the contrary, HAR-aFGF complexes are present in two forms (150 kDa and 135 kDa) revealed by cross-linking experiments with 125I aFGF. Moreover 125I aFGF binding to BEL cell surface induces the spontaneous formation of a 125I aFGF dimer (31 kDa) which is then internalized and degraded in the cells as the 15.5 kDa aFGF native form is. It has been observed that heparin at 10 micrograms/ml (1) in cross-linking experiments, reduces by half the total number of HAR complexes by preventing the formation of the 150 kDa complex but does not affect the 135 kDa complex, (2) in binding experiments, suppress the spontaneous formation of the 125I aFGF dimer bound to LAR, and then its internalization and degradation in the cells. Moreover, we demonstrate that (1) only HAR contributes specifically and directly to the aFGF internalization process, (2) HAR internalization is ligand concentration and time saturable, (3) there is no desensitization of aFGF internalization induced by ligand binding to HAR, (4) a FGF dimerization process is highly dependent on the apparent affinity of FGF for heparin, since aFGF mutant with a reduced affinity for heparin does not promote the dimerization. These data strongly suggest that a heteroreceptor-aFGF complex (150 kDa) is formed by one molecule of HAR (FGF-R1) associated to one molecule of LAR through their respective interactions with a very stable aFGF homodimer. Such a three component receptor induced by FGF dimerization may be a process involved in the mechanism of action of FGFs which could explain the diversity of the biological response of FGF depending on the presence of the HSPG on the extra cellular matrix. In addition prebinding of unlabelled aFGF to the cells induces a 4 fold increase in the affinity of HAR to 125IaFGF concomitant with its down regulation by 80% and initiates the formation of the HAR homodimer.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗

PubMed1993

Developmental regulation of acidic fibroblast growth factor (aFGF) expression in bovine retina.

Acidic fibroblast growth factor (aFGF) is a signalling molecule implicated in a wide variety of biological processes such as cell growth, differentiation and survival. It has been purified from bovine retina. The present study was carried out to detect which cells in the bovine retina expressed aFGF at the different stages of embryonic and post-natal development. The specific aFGF mRNA and protein were detected by in situ hybridization employing riboprobes and immunocytochemistry using affinity purified polyclonal human recombinant aFGF antibodies respectively. No signal was detected by either technique until 4-5 months and then there was progressive expression of aFGF with terminal morphogenesis of the retina. By 8-9 months of embryonic development, nuclei of the 3 neuronal layers (ganglion cell layer, inner and outer nuclear layers) were all uniformly and intensely labeled. A slight labeling of the pigmented epithelium of the retina was also visible throughout development and maturation. These results showed a good correlation between message and protein expression in these cell types. In contrast, glial cells in the nerve fiber layer and vascular endothelial cells displayed a nuclear immunostaining for the protein in the absence of message. These data suggest that aFGF plays a role in the late steps of retinal differentiation by autocrine and paracrine mechanisms.

Animals↗