Molecular Vision 2002; 8:149-160 <http://www.molvis.org/molvis/v8/a21/> Received 13 March 2002 | Accepted 11 June 2002 | Published 14 June 2002

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Fibroblast growth factor receptor (FGFR) and candidate signaling molecule distribution within rat and human retina

Norbert Kinkl,¹ Gregory S. Hageman,² José A. Sahel,³ David Hicks³
 
 

¹Institut für Humangenetik, GSF Forschungszentrum, Ingoldstaedter Landstrasse 1, Neuherberg, D-85764, Germany; ²Department of Ophthalmology and Visual Sciences, The University of Iowa Center for Macular Degeneration, 11190 PFP, 200 Hawkins Drive, Iowa City, IA, 52242, USA; ³Laboratoire de Physiopathologie Cellulaire et Moléculaire de la Rétine, INSERM Université Louis Pasteur E9918, Clinique Médicale A, CHUR, 1 Place de l'Hôpital, 67091 Strasbourg, France

Correspondence to: D. Hicks; Phone: 33 390 243423; FAX: 33 390 243417; email: hicks@neurochem.u-strasbg.fr

Abstract

Purpose: To map the expression and distribution of FGFR and potential FGFR-related signaling molecules within rat and human retina.

Methods: Sections of postnatal 5 day old and adult rat, and aged human retina, and cell cultures prepared from selected cell populations of young rat retina, were immunolabeled with specific antisera to FGFR (FGFR-1, -2, -3, and -4) or candidate signaling molecules [phospholipase Cg1 (PLCg1), son of sevenless 1 and 2 (SOS1, SOS2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein tyrosine phosphatase (SH-PTP2) and SH2-containing protein (Shc)], and with multiple retinal cell-type specific antibodies. Controls were conducted using primary antisera pre-adsorbed with the corresponding immunizing peptide.

Results: All FGFR antisera showed strong labeling of inner retina [inner nuclear layer, inner plexiform layer and ganglion cell layer (INL, IPL and GCL respectively)] in rat and human retina, although there were distinct differences in individual patterns. FGFR-3 was particularly intense in ganglion cell bodies and dendrites, and was absent from photoreceptors and bipolar cells in vitro. FGFR-1 and FGFR-4 also labeled the outer nuclear layer (ONL), more intensely in adult than in young tissue, and FGFR-4 was especially prominent within inner segments. FGFR-2 and -3 were only weakly expressed in the ONL, but FGFR-2 showed specific labeling of cone outer segments in human retina. Candidate FGFR-signaling molecules also showed generally higher expression in the inner than outer retina in the different samples. Shc immunolabeling was apparent in the GCL and nascent photoreceptor outer segments in young and adult retina. SOS1 expression was much more intense than SOS2 in the ONL, although the latter showed selective intense staining of a sub-population in the INL and GCL. These ex vivo data were confirmed in cultures prepared from young rat retina. Pure photoreceptor cultures exhibited strong expression of FGFR-1 and -4, and faint expression of FGFR-2 and -3. In mixed inner retinal cultures, anti-FGFR-1 labeled neurons and Müller glia with equal intensity, while the other FGFR antisera showed preferential staining of neurons. FGFR-3 was strongly expressed by ganglion and amacrine cells but not by other types. Signaling molecules showed widespread expression, but of variable intensity, in all cells. All control experiments using corresponding peptide pre-adsorption led to complete removal of immunostaining.

Conclusions: Rat and human retinal cells showed a largely similar, widespread expression of multiple FGFR and candidate FGFR-related signaling molecules. Distinct differences in development, species, cell- and sub-cell type distribution were apparent, suggesting that specific FGFR/FGF ligands and transduction pathways may operate in different cells.

Introduction

The potential use of neurotrophic factors to reduce or arrest incurable hereditary neurodegenerations is an area of intense investigation. The pioneering work of Faktorovich and collegues clearly demonstrated the feasibility of such an approach: intraocular injections of fibroblast growth factors (FGFs) were able to slow down photoreceptor loss in the Royal College of Surgeons rat displaying retinal dystrophy [1]. These original studies paved the way for numerous studies of retinal neuroprotection, involving screening of a wide variety of neurotrophic factors, in a number of experimental animal models, and using a range of drug delivery strategies [2-4]. The last fifteen years have revealed that the FGF family contains at least 23 separate gene members, many of which exist in multiple isoforms owing to differential splicing or initiation from alternative start codons [5]. These diffusible proteins exert their biological effects through four distinct high affinity FGF receptors (FGFR), which in order to be fully activated operate in conjunction with low affinity heparan sulfate proteoglycans [6,7]. An additional FGFR, termed the cysteine-rich FGFR or CFR, has been cloned in the chicken [8]. Given the potential clinical interest of these investigations in neurology [9,10], one area of research that has received relatively little attention are the physiological role(s) and mode of action of, as well as the signaling pathways used by these molecules within the central nervous system (CNS). FGFs and FGFRs have been extensively studied using immortalized or transfected cell lines (especially a large body of work using the rat pheochromocytoma line PC12 [11,12]) and transgenic mouse models, but the inherent complexity of the CNS has rendered comparable analyses very difficult. Classical FGFR1 and FGFR2 knockout mice are lethal during the pre-implantation embryonic period and offer little information on the role of these receptors in later development or adulthood [13,14]. FGFR3 and FGFR4 homozygous recombinant mice are viable and have relatively little obvious phenotype, although perturbations in lung development [15] and cholesterol biosynthesis [16] have been observed. FGF2 knockout mice exhibit selective deficits in the CNS [17,18], but the large number and widespread distribution of other FGFs make functional redundance likely.

The retina offers an attractive tissue for investigating FGF function within the CNS: the organisation is relatively simple and highly structured, numerous antibody and oligonucleotide probes exist against the various cell types, development and differentiation are largely postnatal in rodents, and there is an ever increasing body of genetic, molecular and cell biological data ([19,20] and the databases at RetNet). FGF exerts neuroprotective effects on photoreceptors in vivo [1] and in vitro [21]. Our laboratory has been interested in defining signaling pathways involved in such processes, and has developed well characterized cell culture systems facilitating biochemical, immunological and molecular biological analyses [22-24]. Nevertheless, much basic information is still lacking concerning the cell-type specific distribution of the different FGFR and candidate signaling molecules, especially within humans. FGFR1 and FGFR2 have both been detected in developing and adult chick and cow retina [25-30], and FGFR4 has been described in adult rat and human retina [31]. Nothing is known concerning the retinal distribution of FGFR3. In the present study we have made a comprehensive survey of all four FGFR and multiple candidate intracellular substrates, at two different stages (early postnatal and adult), in two different species (rat and human), and under in vivo and in vitro conditions. Comparison of immunostaining with cell-type markers reveals that there is broad expression of all the proteins examined, with notable specific differences in cellular and sub-cellular distribution.

Methods

Tissue Collection

All animal experimentation adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Postnatal (PN) 5 day and adult (3 month) Wistar rats were killed by CO₂ inhalation and decapitation, the eyes dissected and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 4 h. In order to facilitate entry of fixative, for PN5 eyes a small needle hole was made near to the limbus, while for adult eyes the cornea and lens were removed. Tissue was rinsed in 0.1 M phosphate buffer, pH 7.4 and then passaged through PBS containing 10%, 15%, and 20% sucrose respectively (each for 1 h), frozen in OCT and sectioned at 8 μm by cryostat. Human donor eyes from individuals over 50 years of age were acquired from the University of Iowa Center for Macular Degeneration, and processed within four hours of death. Institutional Review Board committee approval for the use of human donor tissues was obtained from the Human Subjects Committee of the University of Iowa. Retinas were dissected free from the posterior eyecup and fixed in 4% paraformaldehyde for 2 h, cut into 1 cm² fragments and then passaged through PBS containing 10%, 15% and 20% sucrose respectively, each for 1 h. Retinal specimens were frozen in OCT as above and sectioned at 8 μm by cryostat. Rat and human retinal sections were stored in sealed humidity-free boxes at -20 °C until ready for staining. A total of four separate eyes from each of PN5 and adult rat, and three human retinas, were used in the present study, and sections were taken in the central posterior region in close proximity to the optic nerve head.

Cell Culture

Retinal cultures from PN5 rats were prepared exactly as previously described [18,21], and maintained in chemically defined medium in the absence of any added growth factors. Both pure photoreceptor (PR) and inner retinal (IR) cultures were used for the present studies, and cells were examined after 3 d in vitro, subsequent to fixation in 4% paraformaldehyde for 15 min. Three independent experiments were performed for the present study.

Immunofluorescence labeling

Cryosections were blocked with PBS containing 0.1% Triton X-100, 0.5% bovine serum albumin and 0.1% NaN₃ for 20 min and labeled overnight at 4 °C with the different combinations of polyclonal anti-FGFR/candidate signaling molecules and monoclonal antibody cell-specific markers. The samples were rinsed six times in PBS and labeled for 2 h with Alexa-594-conjugated goat anti-rabbit IgG and Alexa-488-conjugated goat anti-mouse IgG (both used at 10 μg/ml final concentration; Molecular Probes, Eugene, OR). The solution also contained 1 μg/ml 4,6-diaminodiphenyl-2-phenylindole (DAPI; Sigma-Aldrich, Poncy-Cergoise, France). The different antibodies used are given in Table 1. Controls were performed for each FGFR and signaling molecule by pre-incubating a five fold excess of the immunizing peptide together with the primary antibody solution for 2 h prior to addition to sections and cells. Labeled tissues and cells were examined under a Nikon Optiphot 2 fluorescent microscope equipped with the Visiolab digital image analysis system (Biocom, Lyon, France) and images prepared using Adobe Photoshop 5.0 Limited Edition. All photographs were taken close to the optic nerve head, and although we did not control for orientation with respect to nasal, temporal, superior or inferior quadrants, similar observations were made in every case examined.

Results

Distribution of FGFR in rat retina ex vivo

Labeling of PN5 rat retina (Figure 1A) with antisera to FGFR-1 through -4 revealed largely similar patterns with slight differences in intensity between different layers. All four FGFR were more heavily expressed within the inner retina, FGFR1-3 being especially prominent in cells along the vitreal border of the inner nuclear layer (INL) and within the ganglion cell layer (GCL), as well as throughout the inner plexiform layer (IPL) and nerve fiber layer (NFL; Figure 1B,C,E). FGFR-4 was only weakly expressed by cell bodies but was present in the IPL (Figure 1F). FGFR-1 and -4 also showed faint to moderate staining of the outer nuclear layer (ONL; Figure 1B,F), while FGFR-2 and -3 were more prominent in the INL, particularly the horizontal cells (Figure 1C,E). All four FGFR were strongly expressed in the retinal pigmented epithelium (RPE). Pre-incubation of all four antisera with the corresponding peptide completely abolished labeling (Figure 1D).

Similar studies within the adult rat retina (Figure 2A) showed largely the same patterns, with some alterations. FGFR-1 was strongly labeled in all nuclear layers (Figure 2B), FGFR-2 and -3 were intensely stained in the INL and GCL but only very faint in the ONL (Figure 2C,E), and FGFR-4 was now strongly expressed in the INL including GCL, and also very intense within the inner segments (IS) of photoreceptors (Figure 2F). As previously, all four FGFR were abundantly expressed in RPE, and pre-absorption of antisera with corresponding peptides abolished all staining (Figure 2D).

Greater attention was paid to the GCL staining of FGFR-3, which was especially prominent. Distinct from the other FGFR, this member could be detected on the cell bodies and large dendrites running into the IPL, as well as in axons of the NFL (Figure 3).

Distribution of candidate FGF-related signaling molecules in rat retina ex vivo

The different candidate intracellular messenger molecules followed similar overall patterns to those observed for the FGFR, being more abundant within the inner retina than the outer half. In PN5 rat retina (Figure 4A) there was moderate to heavy labeling across the retina with antisera to PLCg1, SOS1 and SOS2 (Figure 4B-D), while ERK1/2 and SH-PTP2 were mainly concentrated within the INL, IPL and GCL (Figure 4F,G). Shc was only faintly expressed, mostly within the GCL and IPL, but also was seen as punctate labeling in the newly emerging IS and outer segments (OS; Figure 4H). Each of these proteins was also observed localized within the RPE. Pre-adsorption of each antibody with its respective immunizing peptide led to disappearance of staining (Figure 4E). Adult rat retina (Figure 5A) again showed a largely similar profile with some interesting changes. PLCg1 and ERK1/2 were detected across the retina, appearing still only faintly in the ONL but moderately intense in the INL and GCL (Figure 5B,F). SOS1 and SOS2 showed quite distinct patterns, SOS1 being strongly expressed within the photoreceptor IS, through the ONL and especially intense in the outer plexiform layer (OPL) and/or horizontal cells (Figure 5C), while SOS2 was virtually absent from the IS, ONL and OPL but very robust in a sub-set of cell bodies within the INL and GCL (Figure 5D). SH-PTP2 was present across the retina but seemingly absent from the GCL (Figure 5G). Shc was seen in a similar pattern but with a notable expression in the IS and OS (Figure 5H). All intracellular messengers were seen in the RPE, and labeling was blocked by pre-incubation with each corresponding immunizing peptide (Figure 5E).

Distribution of FGFR in rat retina in vitro

We further examined distribution of each FGFR in dissociated cultures prepared from PN5 rat retina. Labeling of dissociated photoreceptor cultures with antibodies for each FGFR has been previously published [24], and confirmed the observations made here in vivo, namely strong expression of FGFR-1 and -4 but only weak expression of FGFR-2 and -3. Inner retinal cultures revealed that FGFR-1 was equally expressed by all neurons and glia while the other three were preferentially present in neurons [24]. In agreement with this generalised staining pattern, double immunolabeling with selected neuronal markers (115A10 monoclonal antibody for rod and cone on bipolar cells, HPC1 monoclonal antibody for amacrine cells, neurofilament 68 kDa sub-unit monoclonal antibody for horizontal and ganglion cells) and FGFR revealed that these receptors were present on each sub-population (data not shown).

Distribution of candidate FGF-related signaling molecules in rat retina in vitro

Staining of such cultures with antibodies to the various intracellular messenger proteins also echoed the data obtained in vivo. All six were detected in photoreceptors, albeit at different levels of intensity and with slight differences in sub-cellular localization. PLCg1, SOS1 and ERK1/2 were visible throughout the cytoplasm in cell bodies and neurites (Figure 6A,B,D,E,G,J), whereas SOS2, SH-PTP2, and Shc were only detected in the cell bodies (Figure 6C,F,H,I,K,L). Within IR cultures, PLCg1 and ERK1/2 showed the most generalised labeling, being equally expressed in both neurons and glia (Figure 7A,D,G,J), while SOS1, SOS2, and SH-PTP2 showed greater labeling of neurons than glia (Figure 7B,C,E,F,H,K). Shc was only faintly expressed in IR cells (Figure 7I,K). Pre-adsorption with each corresponding peptide abolished all immunolabeling (data not shown).

As for FGFR, double immunolabeling with signaling molecule and cell-type specific markers revealed a generalised distribution: candidate signaling molecules were detected in horizontal, bipolar, amacrine and ganglion cells (Figure 8).

Distribution of FGFR in human retina ex vivo

FGFR-1 immunoreactivity was relatively uniform across the human retina (Figure 9A), visible within the rod and cone IS, ONL, INL and GCL and fiber layers (Figure 9B). Anti-FGFR-2 showed only faint immunolabeling of the ONL, and more pronounced staining of cells in the INL (especially horizontal cells) and GCL (Figure 9C), as observed for rat retina. In contrast, intense labeling of cone OS was observed with this antibody. Anti-FGFR-3 was also similar to data from the rat, in that the strongest label was apparent at the level of the GCL, in cell bodies and dendrites as well as the NFL. There was also noticeable staining of the horizontal cells (Figure 9E). FGFR-4 immunoreactivity was present at the level of photoreceptor IS, as seen for rat, but was also detected throughout the ONL, IPL and GCL (Figure 9F). Identical staining patterns were observed in all three human retinas examined, and staining was completely abolished by pre-incubation with the appropriate peptide (Figure 9D).

We performed double immunolabeling studies to see whether FGFR-2 immunreactivity within the cone OS co-localized with any particular spectral sub-population. Staining with FGFR-2 antisera and the primate cone monoclonal antibody 7G6 or peanut agglutinin (PNA) showed that all cone cells were double labeled (Figure 10A-F). Furthermore, the band of FGFR-2 immunolabeling visible along the margin of the OPL co-localized with parvalbumin-immunoreactive horizontal cells (Figure 10G-I).

Immunolabeling of human post-mortem retina (Figure 11A) with the different candidate signaling molecules gave generally similar patterns to those observed for adult rat retina. PLCg1, SOS1 and ERK1/2 were expressed throughout the retina, ganglion cell bodies showing notably strong staining with anti-PLCg1 (Figure 11B), and IS being heavily stained for all three (Figure 11B,C,F). SOS2 was restricted to a sub-set of cell bodies in the INL and GCL (Figure 11D), whereas SH-PTP2 and Shc showed a faint generalised distribution, with Shc showing higher staining of the IS (Figure 11G,H). Pre-adsorption of the different antibodies with their corresponding immunogen abolished all staining (Figure 11E).

Discussion

These data represent the first comprehensive description of the presence and distribution of all FGFR and some candidate second messenger molecules within the rat and human retina. Cross comparisons were made at several levels, between different stages of maturation and between in vivo and in vitro conditions in the rat, and between adult rat and human retina. Furthermore, within the limits of resolution at the light microscope level, distinct changes in sub-cellular distribution of certain proteins could be observed.

Investigations of FGFR within the retina have concerned almost exclusively FGFR-1 and -2. In the chick retina, both receptor types are strongly expressed during early embryogenesis in the undifferentiated, proliferating neuroepithelium [25-27]. FGFR-1 and -2 expression have been detected in adult rat and bovine retina, principally in the OPL, the outer limiting membrane and the GCL with weaker labeling in the IPL [28-30,32]. Retinal detachment leads to rapid upregulation of FGFR-1 expression in photoreceptors [33], and phosphorylation of FGFR-1 in Müller glia and RPE [34]. Truncated forms of FGFRs have been found in blood, vitreous and basement membranes of retinal vascular endothelial cells [35-37]. Soluble truncated forms of FGFR-1 are also present in rat retina where it has been hypothesized they are involved in regulating FGF activity in normal and degenerating retina [38]. A single publication refers to the presence of FGFR-4 within rat and human retina using RT-PCR and in situ hybridisation, where it appears to be especially prominent within photoreceptors [31]. In functional studies, antisense oligonucleotide perturbation of FGFR-1 expression leads to cell degeneration in cultured chick retina [39]. Dominant-negative constructions of FGFR-1 and -2 have also been used to examine functional roles of these proteins in retinal development and survival. Transfection of truncated FGFR-1 into Xenopus embryos leads to a 50% loss in photoreceptors and amacrine cells, and a parallel increase in Müller glia, suggesting a role of FGFR in cell fate determination [40]. Additional studies by this group suggested a role for FGFR in axon guidance of retinal ganglion cells [41]. Transgenic mouse strains in which truncated FGFR-1 were targeted to RPE show defects in both choroidal angiogenesis and photoreceptor survival [42]. Finally, targeting of truncated FGFR-1 and -2 to photoreceptors induces progressive retinal degeneration [43]. Thus considerable experimental data point to important roles of FGFR in regulating retinal development and survival.

Several molecules which are known to be involved in FGF (and other growth factor) signal transduction have been detected in retinal cells by immunohistochemical techniques or by western blotting. PLCg1 was localized in photoreceptor IS, ONL, OPL, IPL and GCL of bovine retina [44], whereas in another study using the same tissue significant PLCg1 staining was observed only in the choriocapillaris region [30]. Expression of three Shc-A isoforms (p66, p52, p46), C-Src, PI3-K, PLCg1, and ERK1/2 was found in adult rat and cat retina [45,46]. ERK expression has also been found in chick retinal cells in vitro [39,47], but in chick only one ERK is expressed that seems to be the homologue of mammalian ERK2 [47]. There are few reports of SOS expression or function within the nervous system, and none within the retina. They constitute guanine nucleotide exchange factors linking tyrosine kinase receptor activation to downstream Ras activation [48], and SOS1 null mice die in mid gestation [49]. There is widespread distribution of RNA transcripts for both forms [48], but detailed cellular localizations have not often been described [50]. There was almost mutually exclusive retinal localization of SOS1 and SOS2 in both adult rat and human retina, the former being abundant in photoreceptors and faint in ganglion cells, the latter showing the opposite pattern. Western blot analysis of SOS1 and SOS2 in purified rat photoreceptors also demonstrated much higher levels of the former [24]. Hence the histological data suggest that use of one or the other in trophic factor signaling is cell type-dependent. The Shc (SH2 containing protein) adaptor proteins are important mediators of growth factor and cytokine receptor signaling [51] by virtue of their association with phosphotyrosine residues of the activated receptors [52,53]. Shc immunolabeling in rat retina revealed distinct localization to the photoreceptor OS (in addition to the inner retina), indicating participation in cell signaling within this organelle. Further work will be necessary to understand its function in this sub-cellular compartment, since it normally associates with SOS and Grb2 to mediate receptor-mediated signaling, and OS staining of SOS1 and SOS2 was below detection.

A compelling motive for investigating neurotrophic factor function is their potential use as pharmacological tools to limit neuronal loss in pathologies such as inherited retinal degeneration. Such effects have currently only been demonstrated in animals [1], and it is hence necessary to examine whether the receptors and signaling molecules mediating such effects are present in humans. Furthermore, exploration of growth factor effects has often been modeled in vitro using embryonic tissue, and hence it is also important to compare expression in these different experimental scenarios. There was general correspondence between labeling in PN5 rat retina in vivo and in vitro, and between adult rat and human retina, with a few notable exceptions. FGFR and signaling molecule distributions were generally more widespread in young than in adult rat retina, becoming increasingly restricted and qualitatively more intense with maturation. For example, SOS1 and SOS2 were uniformly distributed at PN5 but became differentially localised in adult tissue, suggesting that different signaling pathways could operate for a given cell type according to its differentiation state. Expression was higher for all FGFR and intracellular messengers within the inner than the outer retinal layers, and a similar preponderance of signaling molecules within the inner retina and their activation following neurotrophic factor application has been observed by others [54,55]. Although we have no data to indicate functional roles of the different proteins examined in this study, their presence in distinct cellular and sub-cellular locations suggests they may subserve particular processes. In both rat and human retinas, FGFR-1 and -4 are the predominant forms present within the ONL and photoreceptor IS, where they may regulate autocrine or paracrine FGF signaling. This localization places them at the level of the interphotoreceptor matrix where they may be exposed to FGF released from any of the neighbouring cell types, photoreceptors, RPE and Müller glia. FGF1 and FGF2 have both been detected in the retina and/or RPE [56-58]. A newly discovered member, FGF19, exhibits selective affinity for FGFR-4 and has been detected in embryonic human retina [59]. The intense immunolabeling of human cone OS by FGFR-2 is especially interesting, raising the possibility that FGFs may represent therapeutically important trophic factors. Cone degeneration is the most devastating lesion in many inherited retinal degenerations, and occurs even when the primary gene mutation is present in other cell types such as rods [60]. Since we have demonstrated that diffusible molecules originating from rods are capable of stimulating cone survival in animal models of retinal degeneration both in vitro [61] and in vivo [62], neurotrophic factor-based therapy may constitute a viable clinical approach.

FGFR-3 has not been previously reported within the vertebrate neural retina, but is reported to be mostly expressed by glia within the CNS [63]. The abundant presence of this receptor around ganglion cell bodies and dendrites indicates it may play a role in their development and/or survival. Some studies have demonstrated that FGF1 and FGF2 stimulate ganglion cell survival or neurite regeneration in vivo [64] and in vitro [65,66]. Although the FGFR(s) activated in any of these studies was/were not identified, both FGF1 and FGF2 can activate FGFR-3 as well as other FGFR [67]. FGF members that show selective stimulation of FGFR-3, such as FGF9 [68], may be particularly interesting candidates to examine for potential neurotrophism or neuroprotection.

In conclusion, these immunohistochemical data provide a catalogue of the expression of FGFR and several putative FGF signaling molecules within rat and human retina. The existence of distinct binding patterns (e.g., preferential expression of FGFR-1 and -4 within photoreceptors, FGFR-2 expression in human cone OS, FGFR-3 expression in ganglion cells, differential expression of SOS1 and SOS2 genes) predicates further examination of these proteins in cell type-dependent signaling cascades, and underlines the potential interest of such neurotrophic factor complexes in treatment of retinal degenerations.

Acknowledgements

The authors wish to thank the following sources for generous financial assistance: Foundation Fighting Blindness, British Retinitis Pigmentosa Society (DH), and ProRetina Deutschland (NK).

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