Molecular Vision 2004; 10:1-14 <http://www.molvis.org/molvis/v10/a1/>
Received 28 May 2003 | Accepted 7 November 2003 | Published 8 January 2004		 Download
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Differential distribution of fibroblast growth factor receptors (FGFRs) on foveal cones: FGFR-4 is an early marker of cone photoreceptors

Elisa E. Cornish,1 Riccardo C. Natoli,1 Anita Hendrickson,2 Jan M. Provis1,3
 
 

1Save Sight Institute and 3Department of Anatomy & Histology, University of Sydney, New South Wales, Australia; 2Department of Biological Structure, University of Washington, Seattle, WA

Correspondence to: Jan M. Provis, Department of Anatomy & Histology (F13), University of Sydney, NSW 2006, Australia; Phone: +61 2 9351 4195; FAX: +61 2 9351 2813; email: jprovis@anatomy.usyd.edu.au

Abstract

Purpose: Relatively little is known of the expression and distribution of FGF receptors (FGFR) in the primate retina. We investigated expression of FGFRs in developing and adult Macaca monkey retina, paying particular attention to the cone rich, macular region.

Methods: One fetal human retina was used for diagnostic PCR using primers designed for FGFR1, FGFR2, FGFR3, FGFR4, and FGFR like-protein 1 (FGFrl1) and for probe design to FGFR3, FGFR4, and FGFrl1. Rat cDNA was used to synthesize probes for FGFR1 and FGFR2 with 90% and 93% homology to human, respectively. Paraffin sections of retina from macaque fetuses sacrificed at fetal days (Fd) 64, 73, 85, 105, 115, 120, and 165, and postnatal ages 2.5 and 11 years were used to detect FGF receptors by immunohistochemistry and in situ hybridization.

Results: PCR showed each of the FGF receptors are expressed in fetal human retina. In situ hybridization indicated that mRNA for each receptor is expressed in all retinal cell layers during development, but most intensely in the ganglion cell layer (GCL). FGFR2 mRNA is reduced in the adult inner (INL) and outer (ONL) nuclear layers, while FGFrl1 mRNA is virtually absent from the adult ONL. FGFR4 mRNA is particularly intense in fetal and adult cone photoreceptors. Immunoreactivity to FGFR1-FGFR4 was detected in the interphotoreceptor matrix in what appeared to be RPE microvilli associated with developing photoreceptor outer segments, and generally is high in the GCL and low in the INL. Different patterns of FGFR3 and FGFR4 immunoreactivities in the outer plexiform layer (OPL) suggest localization of FGFR3 to horizontal cell processes, with FGFR4 being expressed by both horizontal and bipolar cell processes. FGFR1, FGFR3, and FGFR4 immunoreactivities are present in the inner segments and somata of adult cones. The pedicles of developing and adult cones are FGFR1 and FGFR3 immunoreactive, and the basal, synaptic region is FGFR4 immunoreactive. FGFR4 labels cones almost in their entirety from early in development and is not detected in rods. The fibers of Henle are intensely FGFR4 immunoreactive in adult cones.

Conclusions: The results show high levels of FGF receptor expression in developing and adult retina. Differential distribution of FGF receptors across developing and adult photoreceptors suggests specific roles for FGF signalling in development and maintenance of photoreceptors, particularly the specialized cones of the fovea.

Introduction

Cone photoreceptors are dominant in the fovea centralis of primates where they reach a peak density of up to 300 K/mm2 [1]. In the fovea and parafovea, cones are the source of neural pathways mediating both high acuity and colour vision [2-4]. During development, the site of the future foveal depression (the "incipient fovea") is the first region to differentiate, and cone photoreceptors are amongst the earliest cells generated (from around fetal day (Fd) 38 in macaques) [5]. Initially, cones are identifiable as cuboidal, epithelial-like cells that subsequently differentiate morphologically over an extensive period. Foveal cones are involved in synaptic structures shortly after differentiation [6,7] (reviewed in [8]) with small developing outer segments evident from fetal day (Fd) 75 in macaques [9] and about 11 weeks gestation in humans. However, foveal cones achieve final, adult-like proportions at about one year postnatal in macaques and several years postnatal in humans [10-12].

Two specialized features of adult foveal cones are (1) their greatly elongated axons (the fibers of Henle), and (2) their narrow, elongated inner and outer segments, when compared with cones outside the fovea. The fibers of Henle enable synaptic contact between cones in the fovea with bipolar and horizontal cells that are displaced onto the foveal rim during formation of the foveal depression, which takes place in macaque retina between Fd 105 and around birth, at Fd 172 [10,11,13-16]. In adult retina, the narrow and elongated morphology of foveal cones is correlated with high cone density, the most narrow and elongated cones occurring at the site of peak density, in the foveola [1,15,17]. During development, progressive increase in foveal cone density is correlated with a decrease in cone diameter [14,15,18,19]. These observations lead to the suggestion that narrowing and elongation of foveal cones is the mechanism by which cone photoreceptors crowd into, or are displaced towards, the fovea. However, the mechanisms effecting the narrowing and elongation of cones have not been explored.

The mammalian fibroblast growth factor (FGF) family comprises at least 23 structurally related polypeptides that interact with low affinity heparan sulfate proteoglycans molecules to activate high affinity, transmembrane, FGF tyrosine kinase receptors (FGFR) [20,21]. There are at least four such receptors [22-24], with two additional receptors, FGFR5 or FGF receptor-like protein-1 (rl-1) [25-27] and FGFR6 [28] also proposed. FGFs have a diverse range of functions in the central nervous system (reviewed in [29]), including roles in proliferation [30-33], differentiation and survival [34-37], and axon guidance [38,39].

While a considerable number of studies have investigated the effects of FGFRs in cell lines, transgenic models and knockouts [40-43], relatively little is understood about the normal distribution and significance of FGFRs in the mammalian nervous system, including the retina. FGFR distributions have been described in chicken [44-47], bovine [48,49], and rat and human retinas [50-53]. However, few studies have made a thorough analysis of the specific cell classes expressing different FGFRs and relatively little attention had been paid to the FGFRs expressed by cone photoreceptors, especially during development (see however [52-54]). In view of the significant neuroprotective effects of FGFs in the retina [55-58] a systematic analysis of expression of FGFRs is of interest. Furthermore, in view of the known morphogenetic effects of FGF2 in particular [51,59-63] we aimed first, to determine if FGFRs are expressed in the developing human and monkey retina using PCR, and second, to investigate by in situ hybridization and immunohistochemistry if they are expressed by cone photoreceptors in patterns that may be consistent with a role for FGFs in foveal cone morphogenesis.

Methods

Diagnostic PCR and primer design

Primer pairs were designed for the four Fibroblast Growth Factor high affinity receptors (FGFR1, FGFR2, FGFR3, FGFR4) and the Fibroblastic Growth Factor Receptor Like-1 (FGFrl1, also know as FGFR5). Due to the presence of highly conserved regions between receptors, particular care was taken to ensure that only one receptor was amplified by each pair of primers (Table 1). PCR products were analysed using both restriction digestion and sequencing.

Owing to difficulties obtaining fresh macaque retinas, we used human fetal retina for RT-PCR and probe design for FGFR3, FGFR4, and FGFrl1. One human fetal eye at 19 weeks gestation was obtained with informed maternal consent and approval of the Human Ethics Committee of the University of Sydney. The retina was dissected free of the choroid and retinal pigmented epithelium and RNA extracted using TriZOL® (GIBCO-BRL, Invitrogen, Sydney, Australia) for expression analysis. Single stranded cDNA was created from the extracted retinal RNA using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) as per Reverse Transciption System Kit (Promega, Sydney, Australia).

Using the human fetal cDNA as a template, target sequences (as given in Table 1) were PCR amplified using the following solutions (final concentrations in brackets) and protocol parameters. PCR Master solution mix (Promega) containing 10.8 μl of RNase free H20, 2 μl of 10X buffer, 2 μl of 10 mM dNTPs (1 mM), 2 μl of 25 mM MgCl2 (1 mM), 0.2 μl of 5 U/μl Taq DNA Polymerase in storage buffer B (Promega), 1 μl of 40 ng/μl cDNA (40 ng), 1 μl of 1.25 mM primer 1 (1.25 mM; Sigma-Genosys, Sydney, Australia), 1 μl of 1.25 mM primer 2 (1.25 mM; Sigma-Genosys) to make a final volume of 20 μl per reaction mix. PCR protocol: DNA was denatured at 94 °C for 3 min, then amplified over 40 cycles at 94 °C for 25 s, 58 °C for 25 s, 72 °C for 25 s, followed by a final extension step at 72 °C for 5 min. PCR products were run on a 3% agarose gel containing 0.2 ng per ml of ethidium bromide (Bio-Rad, Sydney, Australia) with two standard lanes (Hyperladder-Bioline, Astral, Sydney, Australia). The gel was viewed on a UV transilluminator, photographed on Polaroid film and scanned to produce a digital image.

RNA probes

Complementary DNA from rat used in another study (donated by Andrew Baird, Scripps Institute) was used to prepare RNA probes for FGFR1 (flg) and FGFR2 (bek). Sequencing showed these probes to be 90% and 93% homologous with human FGFR1 and FGFR2, respectively (Genbank accession numbers as per Table 1). We have no data on the sequences for the FGF receptors in macaque, but sequence alignments for each receptor across various species show FGFR to be highly conserved [24,64]. Because fresh macaque tissue was not available to us, we used sequenced DNA fragments obtained from fetal human retina to generate RNA probes for FGFR3, FGFR4, and FGFrl1. DNA fragments were amplified from total RNA of human fetal retina by PCR, ligated to pGem®-T DNA vector (Promega catalog number A3610) and cloned in JM109 competent cells, using the Promega pGem® T DNA vector system protocol. Antisense and sense probes for FGFR1, FGFR2, FGFR3, FGFR4, and FGFrl1 were generated by in vitro transcription of linearized plasmid constructs containing the FGFRs using bacteriophage RNA polymerases (Promega). The templates were purified, sequenced then RNA probes prepared using Digoxigenin (DIG)-labelled-11-UTP using DIG RNA labelling kit (Roche, Sydney, Australia).

Monkey tissue

Macaque monkey retinae aged Fd 64, 73, 85 (before development of the fovea), 105, 115, 120, and 164 (during development of the fovea) and postnatal (P) 2.5 and 11 years (after development of the fovea) were obtained with the ethical approval of the University of Washington, Seattle, from Bogor Agricultural University, Indonesia. All animal procedures were in accordance with guidelines established by the NIH. Fetuses were delivered by caesarean section and mothers returned to the breeding colony after recovery. Fetuses were euthenased by an intravascular overdose of barbiturate, eyes enucleated, then injected with methyl Carnoy's fixative and returned to fixative for 2-4 h. Eyes were embedded in paraffin and sectioned in the horizontal plane at 8 μm.

In situ hybridization

Sections were de-waxed in two changes of xylene for 10 min each, then hydrated in graded ethanols and rinsed in PBS (0.9% NaCl in phosphate buffer, pH 7.4). They were then fixed in 10% Neutral Buffered Formalin (NBF) for 20 min, washed in PBS for 5 min, and then placed in a solution containing 20 mg/ml of proteinase K diluted in TE (50 mM Tris-HCl, 5 mM EDTA, pH 8) for 7 min at 37 °C. The sections were then rinsed in PBS, and re-fixed in NBF for 20 min. Slides were placed into a solution containing 0.1 M triethanolamine (pH 8.0) and acetic anhydride (Sigma catalog number A-6404) for 10 min, washed in PBS then 0.9% NaCl for 5 min each. The slides were dehydrated using graded ethanols and air dried.

The pre-hybridization solution was pre-heated to over 65 °C, before being added to the section, and incubated at 60 °C for a minimum of 1 h under a coverslip. Following incubation, the coverslip and pre-hybridization solution were carefully removed and the pre-heated hybridization solution containing the probe at its particular concentration was added under a new coverslip. Sections were hybridized overnight, at temperatures optimized for each probe. Coverslips were removed and sections washed for 5 min at room temperature (RT) in 2X saline sodium citrate (SSC, pH 7.4), 0.5X SSC and 0.1X SSC, followed by 0.1X SSC at the appropriate post-hybridization wash temperature (55 °C to 75 °C) for 2 h. Post-hybridization temperature was determined as that which produced the lowest levels of background anti-sense labelling, and the cleanest sense labelling. The slides were then washed in 0.1X SSC at RT for 5 min.

Slides were rinsed in washing buffer, placed into blocking solution for 30 min, then incubated in 1:2000 anti-DIG antibody for 1 h. Following antibody incubation, slides were rinsed in two changes of washing buffer then rinsed in detection buffer for 5 min. Visualisation of DIG-labelled probes was performed using Roche HNPP "Fast Red" fluorescent label, incubated for no longer than 1 h. Color reaction was stopped in Milli Q-water (MQ-H20) for 30 min. Sections that were not counter labelled, were fixed in NBF for 20 min, washed in PBS and then coverslipped with DABCO (6 g/L, Sigma; 20% PBS/glycerol) and sealed with nail varnish.

For counter immunolabelling, hybridized sections were blocked for 30 min in normal goat serum (NGS) in preparation for immunolabelling. Sections were incubated overnight at 4 °C in anti-vimentin (1:100; mouse anti-swine, DAKO) or anti-CRALBP (1:1000, courtesy Jack Saari, University of Washington, Seattle). After three 10 min washes in PBS, the antibody was detected using Alexa goat anti-mouse 488 (1:1000, Molecular Probes, Invitrogen, Sydney, Australia), then rinsed, coverslipped with DABCO (6 g/L, Sigma) in 20% PBS/glycerol and sealed with nail varnish. Negative control experiments were performed for each antibody on sections from each animal used in the study, either by omitting the primary antibodies or by using a non-immuno isotype control.

Immunohistochemistry

Paraffin sections were de-waxed and rehydrated, as described above, then rinsed in PBS twice for 10 min. Antigen unmasking involved heating sections in 10 mM sodium citrate butter (pH 6.0) at 80 °C for 10 min then cooling to RT before washing in PBS for 10 min. The area around each section was dried and circled with a DAKO pen to contain solutions and to ensure uniform coverage of the sections. To reduce non-specific staining, sections were initially incubated for 30 min on the shaker table at RT, in 10% NGS, 0.04% saponin in PBS. Sections were then incubated in the FGFR antibody (anti-FGFR1, anti-FGFR2, anit-FGFR3, anti-FGFR4; rabbit anti-human; 1:200 except FGFR4 at 1:400; Santa Cruz, Biotechnology, Inc.) with 0.04% saponin and 2% NGS/PBS for 48-60 h in a humidity chamber at 4 °C, then rinsed with 2% fetal bovine serum (FBS) in PBS for 5 min. After rinsing, sections were incubated in goat anti-rabbit IgG-conjugated Alexa 594 (Molecular Probes; 1:1000) in PBS for 40 min, then washed with 2% FBS/PBS for 15 min. From this point, all incubations and washes were performed covered. Second primary antibodies were diluted in 0.04% saponin, 2% NGS/PBS and incubated overnight at 4 °C. Second antibodies were as follows: Anti-rhodopsin (1:100 Rho4D2; monoclonal mouse courtesy Dr. Robert Molday, University of British Columbia, Canada); anti-vimentin (1:100, mouse anti-swine, DAKO); anti-cellular retinaldehyde-binding protein (1:100 CRALBP courtesy Dr. Jack Saari, University of Washington, Seattle); and α-transducin (1:100, mouse anti-human; [65]). Sections were incubated in goat anti-mouse IgG-conjugated Alexa 488 (Molecular Probes; 1:1000) in PBS for 40 min at RT to visualize bound antibody. Antibodies were raised against human sequences and specificity for monkey FGFRs has not been proven conclusively.

Some sections were triple labelled to demonstrate synaptic terminals, after confocal imaging of the initial double label. After 3 washes in PBS for 15 min each these sections were incubated in anti-synaptophysin (rabbit anti-human, 1:200, DAKO) in PBS overnight. Following several washes in PBS, sections were then incubated in goat biotinylated anti-rabbit secondary antibody for 45 min then washed and visualised using streptavidin-conjugated Cy5 (1:100).

Analyses

All retinae were viewed using a scanning Leica Confocal Microscope and TCSNT software, version 1.6.587 and prepared using Adobe Photoshop version 5.5. Images were captured using a 16x (field size 512x512 μm) or 40x objective (field size 250x250 μm) and a selection of images were zoomed at 2 or 4 times.

Results

FGFR PCR and in situ hybridization.

RT-PCR showed expression of each of the five receptors in human fetal retina. Sense RNA probes for each of the FGFRs produced no detectable labelling in control sections under optimal hybridization conditions. In contrast, distinct patterns of mRNA expression were discerned using each of the FGFR antisense probes (Figure 1, Figure 2).

The ganglion cell layer (GCL) was heavily labelled using RNA probes for each of the FGFR at all ages (Figure 1, Figure 2). In most cases labelling was cytoplasmic, with the exception of FGFR3 probe, which showed both cytoplasmic and nuclear localization (Figure 2A,B). In fetal retina, the inner nuclear layer (INL) was labelled using each of the FGFR probes, but labelling for FGFR2, FGFR3, and FGFrl1 in the INL declined with age (Figure 1A,B,E,F) so that little expression of FGFR2 and FGFrl1 receptors was detected in the adult INL (Figure 1G, Figure 2E) and only low levels of FGFR3 were present (Figure 2B). Expression of each of the FGFRs was detected in the outer nuclear layer (ONL) of fetal retinae (Figure 1, Figure 2). In adult retina, FGFR2 expression in the ONL was at very low levels (Figure 1G) and FGFrl1 is virtually absent (Figure 2E).

Double labelling with anti-L/M opsin at Fd 95 showed co-localization with FGFR1 and FGFR2 mRNA (Figure 1A,E) in cone photoreceptors; similar results were found for FGFR3, FGFR4, and FGFrl1 (not shown). From around Fd 120, photoreceptors showed expression of FGFR1, FGFR2, and FGFR4 mRNA in the developing inner segments, along with some cytoplasmic expression (Figure 1). Postnatally, FGFR1 and FGFR4 were the most strongly expressed FGFRs in cone inner segments (Figure 1C,J). Double labelling using anti-vimentin, a useful marker of Müller cells, indicated that both FGFR1 and FGFR4 are expressed by Müller cells, and that this expression is high during formation of the foveal depression (Figure 1B,I, oblique arrowheads).

Immunoreactivity

FGFR1 immunoreactivity (-IR) was detected throughout the retina at Fd 85 but was most intense in axon bundles in the nerve fiber layer (NFL) and in cone photoreceptors at the level of the developing pedicles (Figure 3A,B, asterisks), on the outer aspect of the nucleus, and on membranes in the subretinal space that appear to be RPE microvilli (Figure 2A,B). These membranes did not colocalize with either anti-CRALBP or anti-vimentin. An attempt to label sections with an antibody to interphotoreceptor retinoid binding protein (IRBP; polyclonal antibody courtesy of Dr. G. Chader [66]) was unsuccessful in the methyl Carnoy-paraffin material (data not shown). However, based on the relationship of these membranes to the outer aspect of the developing cones, we tentatively identify them as developing RPE microvilli. A similar pattern of immunoreactivity in outer retina is more pronounced by Fd 120, when there is a distinct band of FGFR1-IR at the level of the developing outer plexiform layer (OPL; Figure 3C, asterisk) although triple labelling with anti-synaptophysin did not show co-localization with FGFR1 in synaptic pedicles at this stage of development (Figure 3D, asterisk). Intense FGFR1-IR was present also in the inner segments of cones and presumed RPE microvilli that invest the developing outer segments (Figure 2D).

Unlike fetal retina, FGFR1-IR was strong in the GCL and inner plexiform layer (IPL) of adult retina (Figure 3E). Immunoreactivity was also strong on the membranes of semicircular structures adjacent to the OPL, identified as cone pedicles (Figure 3E, asterisk; Figure 4F,G), although the fibers of Henle were not FGFR1-IR (Figure 3E). Triple labelling of adult retina shows synaptophysin immunoreactivity (blue) on the base of the pedicle colocalized with FGFR1 (Figure 3G). The RPE, cone somata, and IS were also FGFR1-IR in adult retina (Figure 2E,F).

FGFR2-IR at Fd 85 was weak in both the retina and RPE (Figure 4A,B). In adult retina, FGFR2-IR was moderate in the INL and GCL but weak in the ONL (Figure 4C) and virtually absent from cone pedicles (Figure 4C-E).

A striking feature of the pattern of FGFR3-IR was the presence of labelling throughout both plexiform layers (Figure 5). The GCL was intensely immunoreactive in both developing and adult retina and it is likely, therefore, that IPL-IR includes the dendrites of ganglion cells. Cells that appeared to be "displaced" ganglion cells in the INL, with dendrites entering the IPL, also were identified at both Fd 120 and in adult retina (Figure 5A,C). Weak IR in the presumed RPE microvilli was seen in fetal retina (Figure 5A). There is some evidence of co-localization of FGFR3 with vimentin in adult retina (Figure 5C, oblique arrowhead) but this was more pronounced in the developing fovea, where Müller cells were clearly double labelled (Figure 5A, oblique arrowheads).

Some presumed horizontal cells in the adult retina were FGFR3-IR (not shown) and it is possible that these are the source of the FGFR3-IR processes spreading throughout most of the depth of the OPL. A prominent feature of the OPL labelling in fetal retina is a distinct non-reactive band, about 2 μm wide, adjacent to the cone pedicles (Figure 5A,B, double asterisks). A similar feature, although not quite so clearly defined, was detected in adult retina abutting the bases of the cone pedicles, which are also FGFR3-IR (Figure 5D). Triple labelling showed synaptophysin immunoreactivity colocalized with FGFR3 on the bases of the cone pedicles, but absent from the non-reactive band (Figure 5E, double asterisks), suggesting that the band comprises post-synaptic elements. Cone somata and inner segments wre FGFR3-IR in fetal and adult retina, as were the proximal parts of the fibers of Henle in adult retina (Figure 5C).

FGFR4-IR in fetal retina was intense in cone photoreceptors and in the processes of ganglion cells, which could be identified clearly within the GCL and IPL (Figure 6A). Co-localization of FGFR4 and vimentin in the inner retina indicates presence of FGFR4 on the inner processes of Müller cells, particularly in the developing fovea (Figure 6A, yellow labelling, oblique arrowhead). Cone photoreceptors were labelled in their entirety in fetal retina, including punctate labelling at the level of the cone pedicles (Figure 6B, asterisk). Presumed RPE microvilli also were mildly FGFR4-IR. A similar pattern of immunoreactivity was seen in adult retina, including FGFR4-IR along the full length of the fibers of Henle, but was reduced significantly on the cone pedicles (Figure 6C). The OPL was moderately FGFR4-IR (Figure 6D), where signal colocalized with synaptophysin (Figure 6E).

Because of the potential value of FGFR4 as a specific, early marker of cones, we investigated FGFR4-IR in specimens at Fd 64, 73, 85, and 105, which were double labelled using antibodies to rhodopsin (to label rods) or antibody to α-transducin, which is specific to cones [65]. Cuboidal cells in the ONL at the incipient fovea were FGFR4-IR at Fd 64 (Figure 7A), that is, about 10 days before L/M opsin can be detected by immunohistochemistry or in situ hybridization in central macaque retina [9]. At Fd 73, many cells in the ONL were FGFR4-IR; a small number of rhodopsin positive cells were detected near the incipient fovea, but none co-localized FGFR4-IR (Figure 7B). Foveal cones co-localizing FGFR4-IR and α-transducin-IR were detected at Fd 85 (Figure 7C). At Fd 105, double labelling with anti-rhodopsin showed many photoreceptors immunoreactive to either rhodopsin or FGFR4, but none that co-localized the two markers (Figure 7D).

Discussion

This study provides a detailed description of the expression patterns of FGF receptor mRNA and protein in developing and adult primate retina. Owing to the unavailability of fresh macaque retinal tissue, we used cDNA from rat to synthesize RNA probes for FGFR1 (90% homolous with human) and FGFR2 (93% homologous), and PCR products from fresh human retina to design RNA probes for FGFR3, FGFR4, and FGFrl1. We have assumed specificity of these probes for FGF receptor mRNAs in macaque tissue, even though specificity has not been confirmed for macaque. Considerable attention has been paid previously to the distribution of FGFR1 and FGFR2 in normal developing and adult retina [67-71], in retinal detachment [72,73], and in several other experimental paradigms [58,74-79]. However, until recently, little was known about the expression of FGFR3 and FGFR4 in the retina [52,53]. Those recent studies have reported expression of FGFR4 mRNA in human retina [52] and immunoreactivity for FGFR1 through FGFR4 in developing and adult rat retina, as well as in adult human retina [53]. The present study confirms and extends those findings, showing detailed patterns of FGFR1-IR through FGFR4-IR in central retina of fetal and adult macaque retina, including differential immunoreactivity on distinct components of central cones.

We also show expression of mRNA for FGFR1 through FGFR4, along with FGFrl1. Those results suggest at least low levels of mRNA for each FGF receptor in each cell layer in both fetal and adult retina with the exception of FGFrl1, which is not expressed in either the INL or ONL in adult retina, but is intense in the GCL. Distribution of the receptor proteins is not as widespread as the patterns of mRNA expression might suggest. While high levels of immunoreactivity in the GCL might be expected on the basis of mRNA expression of each for the receptors, the findings suggest significant post-transcriptional regulation of FGFR1 and FGFR4 protein in the INL and discrete distributions for FGFR1, FGFR3, and FGFR4 proteins in outer retina. Most notable of these are the localization of FGFR3 on horizontal cell processes, cone somas, and the proximal parts of the fibers of Henle, along with the distribution of FGFR4 on almost all parts of the cone.

Inner retina

Previous studies indicate significant roles for FGF ligands and receptors in ganglion cell neurite outgrowth and survival of ganglion cell populations [39,80-83]. In chick retina, blocking FGF receptor activation inhibits ganglion cell differentiation, while stimulation with FGF1 promotes precocious differentiation of ganglion cells [37]. No specific analysis has been made of the FGF receptors stimulated in those studies. Our findings and those of Kinkl et al. [53] indicate that each of the FGF receptors and FGFrl1 (present study) are expressed in the GCL during development and in adults and, since all are activated by a number of FGF ligands [24], have potential roles in ganglion cell differentiation and survival.

Expression of FGFRs in the INL is generally lower than in either of the other two cell layers. Despite counter-immunolabelling with anti-vimentin or anti-CRALBP, mRNA expression by and immunoreactivity of Müller cells was not easy to detect; however, the results suggest that during development, both FGFR3 and FGFR4 are expressed by Müller cells more intensely than either FGFR1 or FGFR2. While mRNA for each of the receptors is detected in presumed neurons in the inner and outer parts of the INL in developing and adult retinae, FGFR4-IR is virtually absent and FGFR1-IR is low in the adult INL. The predominant cell types expressing FGFR2 and FGFR3 in the INL appear to be displaced ganglion cells (present study) and horizontal cells ([53] and present study).

Outer retina

Our finding of low to absent FGFR2-IR on photoreceptors contrasts with the findings of Kinkl et al. [53]. They showed strong immunoreactivity for FGFR2 in the outer segments of cone photoreceptors in frozen sections of adult human retina [53]. In this study we detected immunoreactivity to FGFR1 and FGFR3 in a discrete locus on the outer aspect of the developing inner segments, suggestive of an early stage of outer segment formation. However, we did not detect immunoreactivity to any of the FGF receptors in the outer segments of mature photoreceptors. It is not known if this difference is attributable to different approaches to fixation and embedding in the two studies, or if it is due to inter-species variation.

The present results show generally higher levels of immunoreactivity for FGFR1, FGFR3, and FGFR4 compared with FGFR2 in the ONL of fetal and adult retina and indicate only modest changes in the distribution of receptors between the fetal and adult periods. We did not characterize immunoreactivity for FGFrl1. During development, FGFR1, FGFR2, FGFR3, and FGFR4 immunoreactivities were detected on processes that appear to be the RPE microvilli that invest the developing outer segments, with FGFR1-IR the most intense. Unfortunately, in many cases the retinas (along with the presumed microvilli) were detached from the RPE cells and it was not possible to observe FGFR expression in RPE cell cytoplasm. In adult retina, however, the presumed RPE microvilli were not strongly immunoreactive. This may suggest that FGF signalling is important in establishing the relationship between the RPE microvilli and cone outer segments during development. Consistent with other reports [84,85], we detected FGFR-IR in the RPE where FGF signalling appears to have a role in outer segment phagocytosis [84,86]. The results from our in situ hybridization experiments are difficult to interpret in relation to RPE, due to the tendency of our sense probes to bind non-specifically to the RPE, but not neural retina (Figure 2C,F).

FGFR1-IR is more broadly distributed in adult compared with fetal retina, however, the distinct and early detection of FGFR1 at the level of the developing cone pedicles, as well as persistence of FGFR1-IR in the adult pedicles, suggests a specific role in formation and maintenance of synaptic structures. Cone pedicles also show membranous immunoreactivity to FGFR3 (fetal and adult), with FGFR4 being localized only to the basal, synaptic region of the pedicle in adults. Postsynaptically, both FGFR3 and FGFR4 are expressed on OPL processes, FGFR3 more intensely than FGFR4, with different patterns of immunoreactivity suggesting localization to different OPL elements. Given the intense immunoreactivity of what appear to be horizontal cells to anti-FGFR3 antibody, it is tempting to attribute the FGFR3-IR in the OPL to horizontal cell processes, although this cannot be stated with certainty. Comparison with descriptions of the organization of cone synapses, however, are consistent with this interpretation. In the OPL of the macaque retina, bipolar cell processes aggregate in a narrow band adjacent to the basal portion of the cone pedicle, while horizontal cell processes occupy most of the thickness of the OPL, on the inner aspect of the layer of bipolar cell processes [87]. This would suggest that the band of processes that are non-reactive to FGFR3 (Figure 5) comprises bipolar cells that do not express FGFR3. By comparison, since the full thickness of the OPL is FGFR4-IR, it appears that the processes of both horizontal and bipolar cells express FGFR4 (Figure 6).

Significance

Overall, our findings suggest that immunoreactivities for FGFR3 and FGFR4 are more widespread in the primate retina and more intense than either FGFR1 or FGFR2. Experimental studies indicate that the neurotrophic effects of FGFs on photoreceptors are mediated, at least in part, through upregulation of FGFR1 [73,75,88-90]. The present analysis suggests that expression of FGFR3 and FGFR4 by photoreceptors is constitutively higher than for FGFR1, suggesting that an examination of the roles of FGFR3 and FGFR4 in mediation of the neurotrophic effects of FGFs on photoreceptors is required.

A significant finding in the present study is the early and intense immunoreactivity of cones to FGFR4. Foveal cone photoreceptors are amongst the first cells generated in the retina and can be identified as early as Fd 38, while rods in the vicinity of the incipient fovea are generated later, from about Fd 45 [5]. Cone opsins are detectable by immunolabeling in macaque fovea from around Fd 75, or about 5 days earlier by in situ hybridization [9], while other cone-specific markers including peripherin and α-transducin are detected either simultaneously with or shortly after opsin expression [91]. Rhodopsin-IR is detected on the membranes of rods near the incipient fovea from around Fd 65 [92]. In this study, we detected many intensely FGFR4-IR cones in central retina at Fd 65, suggesting an initial expression somewhat earlier, although sections from younger animals were not available to verify this suggestion. We also show co-localization of FGFR4 with α-transducin at Fd 85, confirming the identity of the FGFR4-IR cones. We found no co-localization of rhodopsin and FGFR4 in fetal retina at Fd 73 or later, indicating that FGFR4 is an early, specific marker of cones. We note, however, that FGFR4 is expressed in photoreceptors in Xenopus retina, where presumably it is not confined to cones [41]. Specific labelling of cones for FGFR4 also appears to occur in adult macaque retina, although the localization of rhodopsin to the outer segment of mature photoreceptors, and of FGFR4 to the soma and axon regions means that a precise assessment of cellular co-localization of the two markers is not possible (not shown).

Of particular interest is the role that FGF signalling may have in the morphogenesis of foveal cones. The high acuity function of the foveal region is directly related to a very high local density of cone photoreceptors [1] that is established progressively during fetal life and the first years postnatal [12,14,19]. This increase in density takes place in the absence of cell division [19,93,94] and in association with a decrease in the diameter of cone somata [19] and inner segments [14], suggesting that narrowing and elongation of foveal cones is the mechanism underlying the crowding of cones into central retina. Understanding of the cellular mechanisms that govern the morphogenesis of cone photoreceptors, including elaboration of the fibers of Henle, is fundamental to understanding how the central retina becomes specialised for high acuity vision. Our present data show that cones are FGFR4-IR throughout the axon, soma and inner segment from very early in development. That is, the distribution of FGFR4 is consistent with a possible role in changing cell shape, including elaboration of the fiber of Henle, although these effects remain to be explored. FGFR1 and FGFR3 have more restricted distributions on cones during development, their arrangements being consistent with potential roles in synaptogenesis.

Photoreceptor loss is a feature of normal aging and is pronounced in disorders including macular degeneration and a variety of retinal dystrophies [74,95,96]. Data suggest that rod photoreceptors are more vulnerable to degeneration than cones [97] but that over time cones will degenerate in the absence of a substantial rod population [98]. Indeed it has been suggested that cones derive a diffusible "survival factor" from rods [99] or Müller cells [100], the identity of which is unknown. In this context, understanding the factors that promote cone morphogenesis and have roles in sustaining normal structure and function is crucial to development of preventative or therapeutic measures, particularly in the areas of transplantation. Characterization of FGFRs expressed by cones that have the potential to mediate neurotrophic effects [53] (present study), and the identification of signalling cascades that mediate these effects is an important initial phase in developing this understanding.

Acknowledgements

This work was supported by National Health and Medical Research Council (Australia) grant number 153825, the Claffy Foundation (EC), the Ophthalmic Research Institute of Australia, and the Sydney Eye Hospital Foundation (RN).

References

1. Curcio CA, Sloan KR, Kalina RE, Hendrickson AE. Human photoreceptor topography. J Comp Neurol 1990; 292:497-523.

2. Dacey DM. Circuitry for color coding in the primate retina. Proc Natl Acad Sci U S A 1996; 93:582-8.

3. Dacey DM. Primate retina: cell types, circuits and color opponency. Prog Retin Eye Res 1999; 18:737-63 [published erratum appears in Prog Retin Eye Res 2000; 19:following 646].

4. Dacey DM. Parallel pathways for spectral coding in primate retina. Annu Rev Neurosci 2000; 23:743-75.

5. La Vail MM, Rapaport DH, Rakic P. Cytogenesis in the monkey retina. J Comp Neurol 1991; 309:86-114.

6. Smelser GK, Ozanics V, Rayborn M, Sagun D. Retinal synaptogenesis in the primate. Invest Ophthalmol 1974; 13:340-61.

7. Linberg KA, Fisher SK. A burst of differentiation in the outer posterior retina of the eleven-week human fetus: an ultrastructural study. Vis Neurosci 1990; 5:43-60.

8. Hendrickson AE. Synaptic development in macaque monkey retina and its implications for other developmental sequences. Perspect Dev Neurobiol 1996; 3:195-201.

9. Bumsted K, Jasoni C, Szel A, Hendrickson A. Spatial and temporal expression of cone opsins during monkey retinal development [published erratum appears in J Comp Neurol 1997; 380:291]. J Comp Neurol 1997; 378:117-34.

10. Hendrickson AE. Development of the primate retina. In: Meisami E, Timiras PS, editors. Handbook of human growth and developmental biology. Boca Raton (FL): CRC Press; 1988. p. 165-78.

11. Hendrickson A. A morphological comparison of foveal development in man and monkey. Eye 1992; 6:136-44.

12. Hendrickson A. Primate foveal development: a microcosm of current questions in neurobiology. Invest Ophthalmol Vis Sci 1994; 35:3129-33.

13. Hendrickson AE, Yuodelis C. The morphological development of the human fovea. Ophthalmology 1984; 91:603-12.

14. Yuodelis C, Hendrickson A. A qualitative and quantitative analysis of the human fovea during development. Vision Res 1986; 26:847-55.

15. Provis JM, Diaz CM, Dreher B. Ontogeny of the primate fovea: a central issue in retinal development. Prog Neurobiol 1998; 54:549-80.

16. Provis JM, Sandercoe T, Hendrickson AE. Astrocytes and blood vessels define the foveal rim during primate retinal development. Invest Ophthalmol Vis Sci 2000; 41:2827-36.

17. Packer O, Hendrickson AE, Curcio CA. Photoreceptor topography of the retina in the adult pigtail macaque (Macaca nemestrina). J Comp Neurol 1989; 288:165-83.

18. Packer O, Hendrickson AE, Curcio CA. Development redistribution of photoreceptors across the Macaca nemestrina (pigtail macaque) retina. J Comp Neurol 1990; 298:472-93.

19. Diaz-Araya C, Provis JM. Evidence of photoreceptor migration during early foveal development: a quantitative analysis of human fetal retinae. Vis Neurosci 1992; 8:505-14.

20. Ornitz DM. FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays 2000; 22:108-12.

21. Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biol 2001; 2:REVIEWS3005.

22. Johnson DE, Williams LT. Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res 1993; 60:1-41.

23. Partanen J, Vainikka S, Korhonen J, Armstrong E, Alitalo K. Diverse receptors for fibroblast growth factors. Prog Growth Factor Res 1992; 4:69-83.

24. Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, Goldfarb M. Receptor specificity of the fibroblast growth factor family. J Biol Chem 1996; 271:15292-7.

25. Sleeman M, Fraser J, McDonald M, Yuan S, White D, Grandison P, Kumble K, Watson JD, Murison JG. Identification of a new fibroblast growth factor receptor, FGFR5. Gene 2001; 271:171-82.

26. Kim I, Moon S, Yu K, Kim U, Koh GY. A novel fibroblast growth factor receptor-5 preferentially expressed in the pancreas(1). Biochim Biophys Acta 2001; 1518:152-6.

27. Wiedemann M, Trueb B. The mouse FGFrl1 gene coding for a novel FGF receptor-like protein. Biochim Biophys Acta 2001; 1520:247-50.

28. Leelayuwat C, Abraham LJ, Pinelli M, Townend DC, Wilks AF, Dawkins RL. The primate MHC contains sequences related to the fibroblast growth factor receptor gene family. Tissue Antigens 1996; 48:59-64.

29. Ford-Perriss M, Abud H, Murphy M. Fibroblast growth factors in the developing central nervous system. Clin Exp Pharmacol Physiol 2001; 28:493-503.

30. Murphy M, Drago J, Bartlett PF. Fibroblast growth factor stimulates the proliferation and differentiation of neural precursor cells in vitro. J Neurosci Res 1990; 25:463-75.

31. Lewis GP, Erickson PA, Guerin CJ, Anderson DH, Fisher SK. Basic fibroblast growth factor: a potential regulator of proliferation and intermediate filament expression in the retina. J Neurosci 1992; 12:3968-78.

32. Bartlett PF, Brooker GJ, Faux CH, Dutton R, Murphy M, Turnley A, Kilpatrick TJ. Regulation of neural stem cell differentiation in the forebrain. Immunol Cell Biol 1998; 76:414-8.

33. Bailly K, Soulet F, Leroy D, Amalric F, Bouche G. Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor. FASEB J 2000; 14:333-44.

34. Desire L, Head MW, Fayein NA, Courtois Y, Jeanny JC. Suppression of fibroblast growth factor 2 expression by antisense oligonucleotides inhibits embryonic chick neural retina cell differentiation and survival in vivo. Dev Dyn 1998; 212:63-74.

35. Hajihosseini MK, Dickson C. A subset of fibroblast growth factors (Fgfs) promote survival, but Fgf-8b specifically promotes astroglial differentiation of rat cortical precursor cells. Mol Cell Neurosci 1999; 14:468-85.

36. Desire L, Courtois Y, Jeanny JC. Endogenous and exogenous fibroblast growth factor 2 support survival of chick retinal neurons by control of neuronal neuronal bcl-x(L) and bcl-2 expression through a fibroblast berowth factor receptor 1- and ERK-dependent pathway. J Neurochem 2000; 75:151-63.

37. McCabe KL, Gunther EC, Reh TA. The development of the pattern of retinal ganglion cells in the chick retina: mechanisms that control differentiation. Development 1999; 126:5713-24.

38. Brittis PA, Silver J, Walsh FS, Doherty P. Fibroblast growth factor receptor function is required for the orderly projection of ganglion cell axons in the developing mammalian retina. Mol Cell Neurosci 1996; 8:120-8.

39. Walz A, McFarlane S, Brickman YG, Nurcombe V, Bartlett PF, Holt CE. Essential role of heparan sulfates in axon navigation and targeting in the developing visual system. Development 1997; 124:2421-30.

40. Deng CX, Wynshaw-Boris A, Shen MM, Daugherty C, Ornitz DM, Leder P. Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev 1994; 8:3045-57.

41. McFarlane S, Zuber ME, Holt CE. A role for the fibroblast growth factor receptor in cell fate decisions in the developing vertebrate retina. Development 1998; 125:3967-75.

42. Arman E, Haffner-Krausz R, Chen Y, Heath JK, Lonai P. Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development. Proc Natl Acad Sci U S A 1998; 95:5082-7.

43. Raffioni S, Thomas D, Foehr ED, Thompson LM, Bradshaw RA. Comparison of the intracellular signaling responses by three chimeric fibroblast growth factor receptors in PC12 cells. Proc Natl Acad Sci U S A 1999; 96:7178-83.

44. Heuer JG, von Bartheld CS, Kinoshita Y, Evers PC, Bothwell M. Alternating phases of FGF receptor and NGF receptor expression in the developing chicken nervous system. Neuron 1990; 5:283-96.

45. Ohuchi H, Koyama E, Myokai F, Nohno T, Shiraga F, Matsuo T, Matsuo N, Taniguchi S, Noji S. Expression patterns of two fibroblast growth factor receptor genes during early chick eye development. Exp Eye Res 1994; 58:649-58.

46. Tcheng M, Fuhrmann G, Hartmann MP, Courtois Y, Jeanny JC. Spatial and temporal expression patterns of FGF receptor genes type 1 and type 2 in the developing chick retina. Exp Eye Res 1994; 58:351-8.

47. Fayein NA, Head MW, Jeanny JC, Courtois Y, Fuhrmann G. Expression of the chicken cysteine-rich fibroblast growth factor receptor (CFR) during embryogenesis and retina development. J Neurosci Res 1996; 43:602-12.

48. Torriglia A, Jeanny JC, Blanquet PR. Immunohistochemical analysis of fibroblast growth factor receptor in bovine retina. Neurosci Lett 1994; 172:125-8.

49. Torriglia A, Blanquet PR. Immunochemical evidence for a fibroblast growth factor receptor in adult retinal optic fiber and synaptic layers. Neuroscience 1994; 60:969-81.

50. Blanquet PR, Jonet L. Signal-regulated proteins and fibroblast growth factor receptors: comparative immunolocalization in rat retina. Neurosci Lett 1996; 214:135-8.

51. de Iongh R, Lovicu FJ, Chamberlain CG, McAvoy JW. Differential expression of fibroblast growth factor receptors during rat lens morphogenesis and growth. Invest Ophthalmol Vis Sci 1997; 38:1688-99.

52. Fuhrmann V, Kinkl N, Leveillard T, Sahel J, Hicks D. Fibroblast growth factor receptor 4 (FGFR4) is expressed in adult rat and human retinal photoreceptors and neurons. J Mol Neurosci 1999; 13:187-97.

53. Kinkl N, Hageman GS, Sahel JA, Hicks D. Fibroblast growth factor receptor (FGFR) and candidate signaling molecule distribution within rat and human retina. Mol Vis 2002; 8:149-60 <http://www.molvis.org/molvis/v8/a21/>.

54. Kitaoka T, Aotaki-Keen AE, Hjelmeland LM. Distribution of FGF-5 in the rhesus macaque retina [published erratum appears in Invest Ophthalmol 1995; 36:976]. Invest Ophthalmol Vis Sci 1994; 35:3189-98.

55. Faktorovich EG, Steinberg RH, Yasumura D, Matthes MT, LaVail MM. Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature 1990; 347:83-6.

56. Faktorovich EG, Steinberg RH, Yasumura D, Matthes MT, LaVail MM. Basic fibroblast growth factor and local injury protect photoreceptors from light damage in the rat. J Neurosci 1992; 12:3554-67.

57. LaVail MM, Yasumura D, Matthes MT, Lau-Villacorta C, Unoki K, Sung CH, Steinberg RH. Protection of mouse photoreceptors by survival factors in retinal degenerations. Invest Ophthalmol Vis Sci 1998; 39:592-602.

58. Xiao M, Sastry SM, Li ZY, Possin DE, Chang JH, Klock IB, Milam AH. Effects of retinal laser photocoagulation on photoreceptor basic fibroblast growth factor and survival. Invest Ophthalmol Vis Sci 1998; 39:618-30.

59. McAvoy JW, Chamberlain CG. Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration. Development 1989; 107:221-8.

60. McAvoy JW, Chamberlain CG, de Iongh RU, Richardson NA, Lovicu FJ. The role of fibroblast growth factor in eye lens development. Ann N Y Acad Sci 1991; 638:256-74.

61. Gu X, Seong GJ, Lee YG, Kay EP. Fibroblast growth factor 2 uses distinct signaling pathways for cell proliferation and cell shape changes in corneal endothelial cells. Invest Ophthalmol Vis Sci 1996; 37:2326-34.

62. de Iongh RU, Lovicu FJ, Hanneken A, Baird A, McAvoy JW. FGF receptor-1 (flg) expression is correlated with fibre differentiation during rat lens morphogenesis and growth. Dev Dyn 1996; 206:412-26.

63. Kay EP, Park SY, Ko MK, Lee SC. Fibroblast growth factor 2 uses PLC-gamma1 for cell proliferation and PI3-kinase for alteration of cell shape and cell proliferation in corneal endothelial cells. Mol Vis 1998; 4:22 <http://www.molvis.org/molvis/v4/a22/>.

64. Coulier F, Pontarotti P, Roubin R, Hartung H, Goldfarb M, Birnbaum, D. Of worms and men: An evolutionary perspective on fibroblast growth factor (FGF) and FGF receptor families. J Mol Evol 1997; 44:43-56.

65. Lerea CL, Bunt-Milam AH, Hurley JB. Alpha-transducin is present in blue-, green-, and red-sensitive cone photoreceptors in the human retina. Neuron 1989; 3:367-76.

66. Bumsted-O'Brien K, Schulte, D Hendrickson A. Expression of photoreceptor-associated molecules during human fetal eye development. Mol Vis 2003; 9:401-409.

67. Caruelle D, Groux-Muscatelli B, Gaudric A, Sestier C, Coscas G, Caruelle JP, Barritault D. Immunological study of acidic fibroblast growth factor (aFGF) distribution in the eye. J Cell Biochem 1989; 39:117-28.

68. Baudouin C, Fredj-Reygrobellet D, Caruelle JP, Barritault D, Gastaud P, Lapalus P. Acidic fibroblast growth factor distribution in normal human eye and possible implications in ocular pathogenesis. Ophthalmic Res 1990; 22:73-81.

69. Noji S, Matsuo T, Koyama E, Yamaai T, Nohno T, Matsuo N, Taniguchi S. Expression pattern of acidic and basic fibroblast growth factor genes in adult rat eyes. Biochem Biophys Res Commun 1990; 168:343-9.

70. Jacquemin E, Jonet L, Oliver L, Bugra K, Laurent M, Courtois Y, Jeanny JC. Developmental regulation of acidic fibroblast growth factor (aFGF) expression in bovine retina. Int J Dev Biol 1993; 37:417-23.

71. Bugra K, Oliver L, Jacquemin E, Laurent M, Courtois Y, Hicks D. Acidic fibroblast growth factor is expressed abundantly by photoreceptors within the developing and mature rat retina [published erratum appears in Eur J Neurosci 1994; 6:1062]. Eur J Neurosci 1993; 5:1586-95.

72. Wen R, Song Y, Cheng T, Matthes MT, Yasumura D, LaVail MM, Steinberg RH. Injury-induced upregulation of bFGF and CNTF mRNAS in the rat retina. J Neurosci 1995; 15:7377-85.

73. Ozaki S, Radeke MJ, Anderson DH. Rapid upregulation of fibroblast growth factor receptor 1 (flg) by rat photoreceptor cells after injury. Invest Ophthalmol Vis Sci 2000; 41:568-79.

74. Gao H, Hollyfield JG. Basic fibroblast growth factor in retinal development: differential levels of bFGF expression and content in normal and retinal degeneration (rd) mutant mice. Dev Biol 1995; 169:168-84.

75. Yamamoto C, Ogata N, Matsushima M, Takahashi K, Miyashiro M, Yamada H, Maeda H, Uyama M, Matsuzaki K. Gene expressions of basic fibroblast growth factor and its receptor in healing of rat retina after laser photocoagulation. Jpn J Ophthalmol 1996; 40:480-90.

76. Yamamoto C, Ogata N, Yi X, Takahashi K, Miyashiro M, Yamada H, Uyama M, Matsuzaki K. Immunolocalization of basic fibroblast growth factor during wound repair in rat retina after laser photocoagulation. Graefes Arch Clin Exp Ophthalmol 1996; 234:695-702.

77. Hackett SF, Schoenfeld CL, Freund J, Gottsch JD, Bhargave S, Campochiaro PA. Neurotrophic factors, cytokines and stress increase expression of basic fibroblast growth factor in retinal pigmented epithelial cells. Exp Eye Res 1997; 64:865-73.

78. Valter K, Maslim J, Bowers F, Stone J. Photoreceptor dystrophy in the RCS rat: roles of oxygen, debris, and bFGF. Invest Ophthalmol Vis Sci 1998; 39:2427-42.

79. Xiao M, McLeod D, Cranley J, Williams G, Boulton M. Growth factor staining patterns in the pig retina following retinal laser photocoagulation. Br J Ophthalmol 1999; 83:728-36.

80. Sievers J, Hausmann B, Unsicker K, Berry M. Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve. Neurosci Lett 1987; 76:157-62.

81. Lipton SA, Wagner JA, Madison RD, D'Amore PA. Acidic fibroblast growth factor enhances regeneration of processes by postnatal mammalian retinal ganglion cells in culture. Proc Natl Acad Sci U S A 1988; 85:2388-92.

82. Bahr M, Vanselow J, Thanos S. Ability of adult rat ganglion cells to regrow axons in vitro can be influenced by fibroblast growth factor and gangliosides. Neurosci Lett 1989; 96:197-201.

83. Chai L, Morris JE. Heparan sulfate in the inner limiting membrane of embryonic chicken retina binds basic fibroblast growth factor to promote axonal outgrowth. Exp Neurol 1999; 160:175-85.

84. Malecaze F, Mascarelli F, Bugra K, Fuhrmann G, Courtois Y, Hicks D. Fibroblast growth factor receptor deficiency in dystrophic retinal pigmented epithelium. J Cell Physiol 1993; 154:631-42.

85. Alizadeh M, Miyamura N, Handa JT, Hjelmeland LM. Human RPE cells express the FGFR2IIIc and FGFR3IIIc splice variants and FGF9 as a potential high affinity ligand. Exp Eye Res 2003; 76:249-56.

86. McLaren MJ, Inana G. Inherited retinal degeneration: basic FGF induces phagocytic competence in cultured RPE cells from RCS rats. FEBS Lett 1997; 412:21-9.

87. Haverkamp S, Grunert U, Wassle H. The cone pedicle, a complex synapse in the retina. Neuron 2000; 27:85-95.

88. Fontaine V, Kinkl N, Sahel J, Dreyfus H, Hicks D. Survival of purified rat photoreceptors in vitro is stimulated directly by fibroblast growth factor-2. J Neurosci 1998; 18:9662-72.

89. Guillonneau X, Regnier-Ricard F, Laplace O, Jonet L, Bryckaert M, Courtois Y, Mascarelli F. Fibroblast growth factor (FGF) soluble receptor 1 acts as a natural inhibitor of FGF2 neurotrophic activity during retinal degeneration. Mol Biol Cell 1998; 9:2785-802.

90. Geller SF, Lewis GP, Fisher SK. FGFR1, signaling, and AP-1 expression after retinal detachment: reactive Muller and RPE cells. Invest Ophthalmol Vis Sci 2001; 42:1363-9.

91. Sears S, Erickson A, Hendrickson A. The spatial and temporal expression of outer segment proteins during development of Macaca monkey cones. Invest Ophthalmol Vis Sci 2000; 41:971-9.

92. Dorn EM, Hendrickson L, Hendrickson AE. The appearance of rod opsin during monkey retinal development. Invest Ophthalmol Vis Sci 1995; 36:2634-51.

93. Provis JM, van Driel D. Retinal development in humans: the roles of differential growth rates, cell migration and naturally occurring cell death. Aust N Z J Ophthalmol 1985; 13:125-33.

94. Sandercoe TM, Madigan MC, Billson FA, Penfold PL, Provis JM. Astrocyte proliferation during development of the human retinal vasculature. Exp Eye Res 1999; 69:511-23.

95. Frank RN. Growth factors in age-related macular degeneration: pathogenic and therapeutic implications. Ophthalmic Res 1997; 29:341-53.

96. Li ZY, Chang JH, Milam AH. Distribution of basic fibroblast growth factor in human retinas with retinitis pigmentosa. Exp Eye Res 1997; 65:855-9.

97. Hicks D, Sahel J. The implications of rod-dependent cone survival for basic and clinical research. Invest Ophthalmol Vis Sci 1999; 40:3071-4.

98. Curcio CA, Owsley C, Jackson GR. Spare the rods, save the cones in aging and age-related maculopathy. Invest Ophthalmol Vis Sci 2000; 41:2015-8.

99. Mohand-Said S, Deudon-Combe A, Hicks D, Simonutti M, Forster V, Fintz AC, Leveillard T, Dreyfus H, Sahel JA. Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse. Proc Natl Acad Sci U S A 1998; 95:8357-62.

100. Wahlin KJ, Campochiaro PA, Zack DJ, Adler R. Neurotrophic factors cause activation of intracellular signaling pathways in Muller cells and other cells of the inner retina, but not photoreceptors. Invest Ophthalmol Vis Sci 2000; 41:927-36.

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