|Molecular Vision 2004;
Received 28 May 2003 | Accepted 7 November 2003 | Published 8 January 2004
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: email@example.com
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.
Cone photoreceptors are dominant in the fovea centralis of primates where they reach a peak density of up to 300 K/mm2 . 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) . 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 ) with small developing outer segments evident from fetal day (Fd) 75 in macaques  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  also proposed. FGFs have a diverse range of functions in the central nervous system (reviewed in ), 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.
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.
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).
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.
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; ). 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).
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.
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).
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 ) 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 . 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 . 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).
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  and immunoreactivity for FGFR1 through FGFR4 in developing and adult rat retina, as well as in adult human retina . 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.
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 . No specific analysis has been made of the FGF receptors stimulated in those studies. Our findings and those of Kinkl et al.  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 , 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 ( and present study).
Our finding of low to absent FGFR2-IR on photoreceptors contrasts with the findings of Kinkl et al. . They showed strong immunoreactivity for FGFR2 in the outer segments of cone photoreceptors in frozen sections of adult human retina . 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 . 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).
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 . Cone opsins are detectable by immunolabeling in macaque fovea from around Fd 75, or about 5 days earlier by in situ hybridization , while other cone-specific markers including peripherin and α-transducin are detected either simultaneously with or shortly after opsin expression . Rhodopsin-IR is detected on the membranes of rods near the incipient fovea from around Fd 65 . 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 . 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  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  and inner segments , 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  but that over time cones will degenerate in the absence of a substantial rod population . Indeed it has been suggested that cones derive a diffusible "survival factor" from rods  or Müller cells , 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  (present study), and the identification of signalling cascades that mediate these effects is an important initial phase in developing this understanding.
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).
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