Neuroscience Letters, 116 (1990) 23-28
23
Elsevier Scientific Publishers Ireland Ltd. NSL 07034
Localization of acidic fibroblast growth factor (aFGF) mRNA in mouse and bovine retina by in situ hybridization E. Jacquemin, C. Halley, J. Alterio, M. Laurent, Y. Courtois and J.C. Jeanny Unitk de Recherches G~rontologiques, U.118-1NSERM, Unitk affiliOe CNBS, Association Claude Bernard, Paris (France)
(Received 15 September 1989; Revisedversion received 12 April 1990; Accepted 12 April 1990) Key words." Fibroblast growth factor; Retina; Mouse; Bovine;In situ hybridization
Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current viewsabout the role of FGF in retinal cell biology.
Fibroblast growth factors (FGFs) of mol.wt. 15,000--18,000 Da are mitogenic polypeptides existing in two forms: acidic (aFGF, pI 5.6; 6.0) and basic (bFGF, pI 9.6). They have received various names depending on the tissue of origin (e.g. eyederived growth factor), on the process of purification (e.g. heparin-binding growth factor) or on their target cells (e.g. F G F ) (see ref. 15 for review). They have been extracted from various tissues, including brain and retina [3, 4, 7, 22]. An a F G F c D N A has been isolated from a bovine retinal c D N A library [2, 9]. The c D N A clone, 4004 bp in size, encodes the entire a F G F (155 amino acids). The coding region shows 87% homology with human a F G F [11] and 63% with bovine b F G F [1]. Additional homologies were observed with the oncogenes hst (58%) [21] and int2 (66%) [17]. The open reading frame is preceded by 917 bp at the 5' end and is followed by an untranslated region of about 3000 bp. Northern blot analysis of bovine retina poly A ÷ m R N A s showed the existence of 4 sizes of m R N A , 3 of them (9.9; 6.0; 4.2 Kb) coding for the a F G F [2]. The retina is a complex nervous structure made of several layers: the photoreceptors (rods and cones), the inner nuclear layer (cell bodies of bipolar, horizontal and Correspondence: J.C. Jeanny, Unit6 de Recherches G~rontologiques, U. I18-INSERM, Unit6 affili6e CNRS, Association Claude Bernard, 29 rue Wilhem, 75016 Paris, France.
0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
24 amacrine cells) and the ganglion layer (cell bodies of ganglion cells). Besides the nerve cells there are numerous neuroglial and vessel associated cells. Despite the recent characterization of a F G F in rod outer segment from bovine retina [1619] it is not known which retinal cells express aFGF. We have used a F G F cDNA probes to detect by in situ hybridization the m R N A coding for a F G F in ocular tissues from adult mouse and bovine. The results show that all the nuclear layers of the neural retina contain the a F G F mRNA. Adult mice (Swiss OF1, Iffa Cr6do, Domaine des Oncins, France) were sacrified by neck dislocation. Biological samples from adult bovine were directly collected at the Slaughterhouse. The probes were the a F G F cDNA clone (4004 bp) [2, 9]) and the a F G F cDNA clone Bam HI-Sph I which contains 438 bp of the coding region. Both do not cross hybridize with b F G F mRNA. As a control for the hybridization specificity, DNA of plasmid pBR 322 and f12 crystallin cDNA clone were used as probes under identical conditions. Mouse eyes and posterior part of the bovine ocular globes were dissected out and immediately mounted in OCT, frozen in liquid nitrogen and conserved at -80°C. Frozen sections 10/~m thick were thawed on sterile slides. The conditions for fixation, in situ hybridization and autoradiography were performed essentially as described previously [ 12]. The sections were fixed 5 min in methanol/acetic acid (3/1) and post fixed 20 min into 0.1% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.2), freshly prepared and maintained on ice. The DNA probes were labelled with [32p]dCTP (3000 Ci/mmol) by Nick translation or Random priming to a specific radioactivity of approximately 3-8 × l0 s cpm./~g -l. The probes were denatured by heating and mixed with the hybridization cocktail immediately before use. Some slides were pretreated with HCI (0.2 N, 10 min, at room temperature) and proteinase K (10/tg/ml, 15 min, 37°C) and preincubated with hybridization medium. The sections were then hybridized to the probes as were unpretreated sections. The final hybridization cocktail contained 50% formamide, 0.6 M NaC1, 10 mM Tris pH 7.0, 1 mM EDTA, 1 × Denhart's solution, 250/tg-ml-l of single stranded salmon sperm DNA, 500/lg.ml- 1 of yeast tRNA and 1% dextran sulphate. Hybridization cocktail (10/A) containing 5 ng of labelled probe (approx. 2-4 × 106 cpm) was applied to each group of 2 sections. Hybridizations were carried out overnight at 42°C. After hybridization, the slides were rinsed 3 times 10 min with 2 x SSC at room temperature (1 × SSC =0.15 M NaC1, 0.015 M sodium citrate) and washed successively twice 30 min in 0.1 x SSC at 55°C and three times 30 min in 2 x SSC at 4°C. The slides were then dehydrated in graded ethanols and air dried. They were dipped in 50% Ilford K2 nuclear emulsion. After exposure for 1-2 weeks at 4°C the slides were developed in Kodak D 19, and fixed in Hypam Ilford fix. Photographs were taken using PAN-F film (Ilford) on a Zeiss photomicroscope. 32p-labelled a F G F cDNA clone (4004 bp) was hybridized to mouse eye sections.
25
b
Fig. 1. Mouse eye sections respectively hybridized to 32p-labelled aFGF cDNA clone (4004 bp) (a,b) or to 32p-labelled PBR322 plasmid DNA (control probe) (c,d). a,c: phase contrast; b,d: bright-field. The labelling with the aFGF probe is mainly localized on the ganglion cells (gc), inner nuclear layer (inl) and photoreceptors (p). e, epithelium; ch, choroid; m, muscles; s, sclera; on, optic nerve, x 120.
After 2 weeks exposure, nuclei of 3 neuronal layers (ganglion cell layer, inner nuclear layer and photoreceptors) were all uniformly intensely labelled (Fig. la, b). A slight labelling on the pigmented epithelium was also visible. In contrast, all non-retinal tissues (optic nerve, choroid and sclera) were persistently negative. Sections of all tissue types hybridized with control probes contained no labelled cells (Fig. lc, d). Pretreatment with hydrochloric acid or proteinase K and preincubation with hybridization medium did not give a better signal (data not shown). Specific localization of aFGF mRNA has been performed by similar experiments on mouse eye (Fig. 2) and bovine retina (Fig. 3) with 32p-labelled cDNA clone Bam HI-SphI (438 bp) specific of the coding region. As shown in Fig. 1, this probe hybridized to aFGF mRNA localized on the 3 nuclear layers. We could also observe few silver grains at the border of the pigmented epithelium and outer segments of the photoreceptors specially in the case of the bovine. In the same conditions f12crystallin probe only hybridized on the lens (data not shown) and not on the nuclear layers of the retina (Figs. 2b and 3b). In the posterior part of the eye aFGF is mainly localized in neural retina. The two probes, the complete cDNA or the coding sequence for aFGF, display the same pattern but more intense for the first one which can be correlated with the size of the probe. We cannot precise which mRNA is revealed in these experiments since northern blot analysis with different fragments of the cDNA does not show a specific probe
26
Fig. 2. Mouse eye sections respectively hybridized to 32p-labelled aFGF cDNA clone BamHI SphI (438 bp) (a) or to ~2p-labelledfl crystallin cDNA clone (control probe) (b). The labelling pattern in (a) is similar to Fig. la,b on the 3 neuronal layers: photoreceptors (p) (al), inner nuclear layer (inl) (a2) and ganglion cell layer (gc) (a3). Hybridization with control probe (b) gave negative results on the same layers. (bl, b2, b3). e, epithelium. × 465. Higher magnification 1-3: × 1000.
for each species o f m R N A coding for a F G F (unpublished results). We do not know also at this level o f resolution if there is a differential expression between the different types o f neurons or if glial cells can express it, since the latter represents a minor population in the neural retina. This problem is currently under investigation. O u r results suggest that a F G F isolated from the retina [3, 4, 7] and more recently from photoreceptors [16, 19], as well as F G F localized by antibodies against a and b F G F [5, 6] are synthesized by retinal cells. Thus despite that F G F is expressed and isolated-from neural retina, its putative role(s) in retinal cell biology is still poorly understood. F G F s exhibit angiogenic activities and a role in retinal vascularization
27
Fig. 3. Bovineretina sectionsrespectivelyhybridizedto 32P-labelledaFGF cDNA clone BamHI-Sphl (438 bp) (a) or to 3zp-labelledp crystallincDNA clone (control probe) (b). The labellingseen in (a) is still localized in the 3 nuclear layers of the neural retina. × 370. Higher magnification1, 2, 3, 3': × 795. can be postulated [8]. The presence of a F G F in photoreceptors and the release of the peptide from rod outer segment (ROS) membranes also suggest that it plays a role in photoreceptor metabolism and phototransduction [18, 19]. Addition of a F G F or b F G F to newborn rat retina photoreceptor cells in monolayer culture stimulates a 5- to 10-fold increase in the levels of opsin and prolongs cell survival by up to 6 days [10]. a F G F also has a potent influence on processes such as regeneration by rat retinal ganglion cells (RGCs) in vitro [14] and promotes RGCs survival after transection of the optic nerve [20]. Recent experiments also demonstrate the presence of F G F receptor on retina sections and in purified preparations of ROS respectively by autoradiography or radioreceptor competition and cross linking [13, 16]. The presence and effects of a F G F in highly specialized retinal cell types suggest that F G F may play a very important role in the biology and physiology of the eye. We thank J. Piatigorsky for the P2 crystallin clone, L. Jonet for his technical assistance, H. Co6t for the photographs, N. Breugnot and Y. Maville for typing. This work was supported by grants from D R E T to C.H., Ligue Nationale pour le Cancer and Association pour la Recherche sur le Cancer.
28 1 Abraham, J.A., Mergia, A., Whang, J.L., Tumolo, A., Friedman, J., Hjerrild, K.A., Gospodarowicz, D. and Fiddes, J.C., Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor, Science, 233 (1986) 545-548. 2 Alterio, J., Halley, C., Brou, C., Soussi, T., Courtois, Y. and Laurent, M., Characterization of a bovine acidic FGF cDNA clone and its expression in brain and retina, FEBS Lett., 242 (1988) 41~J,6. 3 Arruti, C. and Courtois, Y., Morphological changes and growth stimulation of bovine epithelial lens cells by a retinal extract in vitro, Exp. Cell Res., 117 (1978) 283-292. 4 Baird, A., Esch, F., Gospodarowicz, D. and Guillemin, R., Retina- and eye-derived endothelial cell growth factors: partial molecular characterization and identity with acidic and basic fibroblast growth factors, Biochemistry, 24 (1985) 7855 7860. 5 Caruelle, D., Groux-Muscatelli, B., Gaudric, A., Sestier, C., Coscas, G., Caruelle, J.P. and Barritault, D., Immunological study of acidic fibroblast growth factor (aFGF) distribution in the eye, J. Cell. Biochem., 39 (1989) 117 128. 6 Courtois, Y., Fayein, N.A., Soubrane, G., Raulais, D., Diry, M., Vigny, M. and Jeanny, J.C., Specific interaction between eye derived growth factors and ocular basement membranes, Invest. Ophthalmol. Vis. Sci., 28 (1987) 80. 7 Courty, J., Loret, C., Moenner, M., Chevallier, B., Lagente, O., Courtois, Y. and Barritault, D., Bovine retina contains three growth factor activities with different affinity to heparin: eye derived growth factor 1, II, III, Biochimie, 67 (1985) 265-269. 8 Glaser, B.M., D'Amore, P.A., Michels, R.G., Patz, A. and Fenselau, A., Demonstration of vasoproliferative activity from mammalian retina, J. Cell Biol., 84 (1980) 298-304. 9 Halley, C., Courtois, Y. and Laurent, M., Nucleotide sequence of bovine acidic fibroblast growth factor cDNA, Nucl. Acid Res., 16 (1988) 10913. 10 Hicks, D. and Courtois, Y., Acidic fibroblast growth factor stimulates opsin levels in retinal photoreceptor cells in vitro, FEBS Lett., 234 (1988) 475M79. I I Jaye, M., Howk, R., Burgess, W., Ricca, G,A., Chiu, I.M., Ravera, M.W., O'Brien, S.J., Modi, W.S., Maciag, T. and Drohan, W.N., Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization, Science, 233 (1986) 541 545. 12 Jeanny, J.C., Bower, D.J., Errington, L.H., Morris, S. and Clayton, R.M., Cellular heterogeneity in the expression of the S-crystallin gene in non-lens tissues, Dev. Biol., 112 (1985) 94- 99. 13 Jeanny, J.C., Fayein, N., Moenner, M., Chevallier, B., Barritault, D. and Courtois, Y., Specific fixation of bovine brain and retinal acidic and basic fibroblast growth factors to mouse embryonic eye basement membranes, Exp, Cell Res., 171 (1987) 63 75. 14 Lipton, S.A., Wagner, J.A., Madison, R.D. and D'Amore, P.A., Acidic fibroblast growth factor enhances regeneration of processes by postnatal mammalian retinal ganglion cells in culture, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 2388 2392. 15 Lobb, R.R., Harper, J.W. and Fett, J.W., Purification of heparin-binding growth factors, Anal. Biochem., 154(1986) 1 14. 16 Mascarelli, F., Raulais, D. and Courtois, Y., Fibroblast growth factor phosphorylation and receptors in rod outer segments, EMBO J., 8 (1989) 2265--2273. 17 Moore, R., Casey, G., Brookes, S., Dixon, M., Peters, G. and Dickson, C., Sequence, topography and protein coding potential of mouse int-2: a putative oncogene activated by mouse mammary tumour virus, EMBO J., 5 (1986) 919~24. 18 Plouat, J., Mascarelli, F., Lagente, O., Dorey, C., Lorans, G., Faure, J.P. and Courtois, Y., Eye derived growth factor: a component of rod outer segment implicated in phototransduction. In E. Agardh and B. Ehinger (Eds.), Retinal Signal Systems, degenerations and transplants, Elsevier/North Holland, Amsterdam, 1986, pp. 311 320. 19 Plou~t, J., Mascarelli, F., Loret, M.D., Faure, J.P. and Courtois, Y., Regulation of eye derived growth I;actor binding to membranes by light, ATP or GTP in photoreceptor outer segments, EMBO J., 7 (1988) 373 376. 20 Sievers, J., Hausmann, B., Unsicker, K. and Berry, M., Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve, Neurosci. Lett., 76 (1987) 157-162. 21 Taira, M., Yoshida, T., Miyagawa, K., Sakamoto, H., Terada, M. and Sugimura, T., cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 2981L2984. 22 Thomas, K.A., Rios-Candelore, M. and Fitzpatrick, S., Purification and characterization of acidic fibroblast ~rowth factor from bovine brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 357 361.
23
Elsevier Scientific Publishers Ireland Ltd. NSL 07034
Localization of acidic fibroblast growth factor (aFGF) mRNA in mouse and bovine retina by in situ hybridization E. Jacquemin, C. Halley, J. Alterio, M. Laurent, Y. Courtois and J.C. Jeanny Unitk de Recherches G~rontologiques, U.118-1NSERM, Unitk affiliOe CNBS, Association Claude Bernard, Paris (France)
(Received 15 September 1989; Revisedversion received 12 April 1990; Accepted 12 April 1990) Key words." Fibroblast growth factor; Retina; Mouse; Bovine;In situ hybridization
Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current viewsabout the role of FGF in retinal cell biology.
Fibroblast growth factors (FGFs) of mol.wt. 15,000--18,000 Da are mitogenic polypeptides existing in two forms: acidic (aFGF, pI 5.6; 6.0) and basic (bFGF, pI 9.6). They have received various names depending on the tissue of origin (e.g. eyederived growth factor), on the process of purification (e.g. heparin-binding growth factor) or on their target cells (e.g. F G F ) (see ref. 15 for review). They have been extracted from various tissues, including brain and retina [3, 4, 7, 22]. An a F G F c D N A has been isolated from a bovine retinal c D N A library [2, 9]. The c D N A clone, 4004 bp in size, encodes the entire a F G F (155 amino acids). The coding region shows 87% homology with human a F G F [11] and 63% with bovine b F G F [1]. Additional homologies were observed with the oncogenes hst (58%) [21] and int2 (66%) [17]. The open reading frame is preceded by 917 bp at the 5' end and is followed by an untranslated region of about 3000 bp. Northern blot analysis of bovine retina poly A ÷ m R N A s showed the existence of 4 sizes of m R N A , 3 of them (9.9; 6.0; 4.2 Kb) coding for the a F G F [2]. The retina is a complex nervous structure made of several layers: the photoreceptors (rods and cones), the inner nuclear layer (cell bodies of bipolar, horizontal and Correspondence: J.C. Jeanny, Unit6 de Recherches G~rontologiques, U. I18-INSERM, Unit6 affili6e CNRS, Association Claude Bernard, 29 rue Wilhem, 75016 Paris, France.
0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
24 amacrine cells) and the ganglion layer (cell bodies of ganglion cells). Besides the nerve cells there are numerous neuroglial and vessel associated cells. Despite the recent characterization of a F G F in rod outer segment from bovine retina [1619] it is not known which retinal cells express aFGF. We have used a F G F cDNA probes to detect by in situ hybridization the m R N A coding for a F G F in ocular tissues from adult mouse and bovine. The results show that all the nuclear layers of the neural retina contain the a F G F mRNA. Adult mice (Swiss OF1, Iffa Cr6do, Domaine des Oncins, France) were sacrified by neck dislocation. Biological samples from adult bovine were directly collected at the Slaughterhouse. The probes were the a F G F cDNA clone (4004 bp) [2, 9]) and the a F G F cDNA clone Bam HI-Sph I which contains 438 bp of the coding region. Both do not cross hybridize with b F G F mRNA. As a control for the hybridization specificity, DNA of plasmid pBR 322 and f12 crystallin cDNA clone were used as probes under identical conditions. Mouse eyes and posterior part of the bovine ocular globes were dissected out and immediately mounted in OCT, frozen in liquid nitrogen and conserved at -80°C. Frozen sections 10/~m thick were thawed on sterile slides. The conditions for fixation, in situ hybridization and autoradiography were performed essentially as described previously [ 12]. The sections were fixed 5 min in methanol/acetic acid (3/1) and post fixed 20 min into 0.1% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.2), freshly prepared and maintained on ice. The DNA probes were labelled with [32p]dCTP (3000 Ci/mmol) by Nick translation or Random priming to a specific radioactivity of approximately 3-8 × l0 s cpm./~g -l. The probes were denatured by heating and mixed with the hybridization cocktail immediately before use. Some slides were pretreated with HCI (0.2 N, 10 min, at room temperature) and proteinase K (10/tg/ml, 15 min, 37°C) and preincubated with hybridization medium. The sections were then hybridized to the probes as were unpretreated sections. The final hybridization cocktail contained 50% formamide, 0.6 M NaC1, 10 mM Tris pH 7.0, 1 mM EDTA, 1 × Denhart's solution, 250/tg-ml-l of single stranded salmon sperm DNA, 500/lg.ml- 1 of yeast tRNA and 1% dextran sulphate. Hybridization cocktail (10/A) containing 5 ng of labelled probe (approx. 2-4 × 106 cpm) was applied to each group of 2 sections. Hybridizations were carried out overnight at 42°C. After hybridization, the slides were rinsed 3 times 10 min with 2 x SSC at room temperature (1 × SSC =0.15 M NaC1, 0.015 M sodium citrate) and washed successively twice 30 min in 0.1 x SSC at 55°C and three times 30 min in 2 x SSC at 4°C. The slides were then dehydrated in graded ethanols and air dried. They were dipped in 50% Ilford K2 nuclear emulsion. After exposure for 1-2 weeks at 4°C the slides were developed in Kodak D 19, and fixed in Hypam Ilford fix. Photographs were taken using PAN-F film (Ilford) on a Zeiss photomicroscope. 32p-labelled a F G F cDNA clone (4004 bp) was hybridized to mouse eye sections.
25
b
Fig. 1. Mouse eye sections respectively hybridized to 32p-labelled aFGF cDNA clone (4004 bp) (a,b) or to 32p-labelled PBR322 plasmid DNA (control probe) (c,d). a,c: phase contrast; b,d: bright-field. The labelling with the aFGF probe is mainly localized on the ganglion cells (gc), inner nuclear layer (inl) and photoreceptors (p). e, epithelium; ch, choroid; m, muscles; s, sclera; on, optic nerve, x 120.
After 2 weeks exposure, nuclei of 3 neuronal layers (ganglion cell layer, inner nuclear layer and photoreceptors) were all uniformly intensely labelled (Fig. la, b). A slight labelling on the pigmented epithelium was also visible. In contrast, all non-retinal tissues (optic nerve, choroid and sclera) were persistently negative. Sections of all tissue types hybridized with control probes contained no labelled cells (Fig. lc, d). Pretreatment with hydrochloric acid or proteinase K and preincubation with hybridization medium did not give a better signal (data not shown). Specific localization of aFGF mRNA has been performed by similar experiments on mouse eye (Fig. 2) and bovine retina (Fig. 3) with 32p-labelled cDNA clone Bam HI-SphI (438 bp) specific of the coding region. As shown in Fig. 1, this probe hybridized to aFGF mRNA localized on the 3 nuclear layers. We could also observe few silver grains at the border of the pigmented epithelium and outer segments of the photoreceptors specially in the case of the bovine. In the same conditions f12crystallin probe only hybridized on the lens (data not shown) and not on the nuclear layers of the retina (Figs. 2b and 3b). In the posterior part of the eye aFGF is mainly localized in neural retina. The two probes, the complete cDNA or the coding sequence for aFGF, display the same pattern but more intense for the first one which can be correlated with the size of the probe. We cannot precise which mRNA is revealed in these experiments since northern blot analysis with different fragments of the cDNA does not show a specific probe
26
Fig. 2. Mouse eye sections respectively hybridized to 32p-labelled aFGF cDNA clone BamHI SphI (438 bp) (a) or to ~2p-labelledfl crystallin cDNA clone (control probe) (b). The labelling pattern in (a) is similar to Fig. la,b on the 3 neuronal layers: photoreceptors (p) (al), inner nuclear layer (inl) (a2) and ganglion cell layer (gc) (a3). Hybridization with control probe (b) gave negative results on the same layers. (bl, b2, b3). e, epithelium. × 465. Higher magnification 1-3: × 1000.
for each species o f m R N A coding for a F G F (unpublished results). We do not know also at this level o f resolution if there is a differential expression between the different types o f neurons or if glial cells can express it, since the latter represents a minor population in the neural retina. This problem is currently under investigation. O u r results suggest that a F G F isolated from the retina [3, 4, 7] and more recently from photoreceptors [16, 19], as well as F G F localized by antibodies against a and b F G F [5, 6] are synthesized by retinal cells. Thus despite that F G F is expressed and isolated-from neural retina, its putative role(s) in retinal cell biology is still poorly understood. F G F s exhibit angiogenic activities and a role in retinal vascularization
27
Fig. 3. Bovineretina sectionsrespectivelyhybridizedto 32P-labelledaFGF cDNA clone BamHI-Sphl (438 bp) (a) or to 3zp-labelledp crystallincDNA clone (control probe) (b). The labellingseen in (a) is still localized in the 3 nuclear layers of the neural retina. × 370. Higher magnification1, 2, 3, 3': × 795. can be postulated [8]. The presence of a F G F in photoreceptors and the release of the peptide from rod outer segment (ROS) membranes also suggest that it plays a role in photoreceptor metabolism and phototransduction [18, 19]. Addition of a F G F or b F G F to newborn rat retina photoreceptor cells in monolayer culture stimulates a 5- to 10-fold increase in the levels of opsin and prolongs cell survival by up to 6 days [10]. a F G F also has a potent influence on processes such as regeneration by rat retinal ganglion cells (RGCs) in vitro [14] and promotes RGCs survival after transection of the optic nerve [20]. Recent experiments also demonstrate the presence of F G F receptor on retina sections and in purified preparations of ROS respectively by autoradiography or radioreceptor competition and cross linking [13, 16]. The presence and effects of a F G F in highly specialized retinal cell types suggest that F G F may play a very important role in the biology and physiology of the eye. We thank J. Piatigorsky for the P2 crystallin clone, L. Jonet for his technical assistance, H. Co6t for the photographs, N. Breugnot and Y. Maville for typing. This work was supported by grants from D R E T to C.H., Ligue Nationale pour le Cancer and Association pour la Recherche sur le Cancer.
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