Human Molecular Genetics, 2002, Vol. 11, No. 16 1899-1907
© 2002 Oxford University Press
Species-specific subcellular localization of RPGR and RPGRIP isoforms: implications for the phenotypic variability of congenital retinopathies among species
Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
Received April 26, 2002; Accepted June 5, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The retinitis pigmentosa GTPase regulator (RPGR) is encoded by the X-linked RP3 locus, which upon genetic lesions leads to neurodegeneration of photoreceptors and blindness. The findings that RPGR specifically and directly interacts in vivo and in vitro with retina-specific RPGR-interacting protein 1 (RPGRIP) and that human mutations in RPGR uncouple its interaction with RPGRIP provided the first clue for the retina-specific pathogenesis of X-linked RP3. Recently, mutations in RPGRIP were found to lead to the retinal dystrophy, Leber congenital amaurosis. However, mouse models null for RPGR had, surprisingly, a very mild phenotype compared with those observed in XlRP3-affected humans and dogs. Moreover, recent reports are seemingly in disagreement on the localization of RPGR and RPGRIP in photoreceptors. These discrepancies were compounded with the finding of RPGR mutations leading exclusively to X-linked cone dystrophy. To resolve these discrepancies and to gain further insight into the pathology associated with RPGR- and RPGRIP-allied retinopathies, we now show, using several isoform-specific antibodies, that RPGR and RPGRIP isoforms are distributed and co-localized at restricted foci throughout the outer segments of human and bovine, but not mice rod photoreceptors. In humans, they also localize in cone outer segments. RPGRIP is also expressed in other neurons such as amacrine cells. Thus, the data lend support to the existence of species-specific subcellular processes governing the function and/or organization of the photoreceptor outer segment as reflected by the species-specific localization of RPGR and RPGRIP protein isoforms in this compartment, and provide a rationale for the disparity of phenotypes among species and in the human.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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X-linked RP3 is a progressive and very severe neurodegenerative disease ultimately leading to the death of photoreceptors and to rapid and complete loss of vision, often with an onset in the first decade of life (1–3). The X-linked RP3 locus encodes the retinitis pigmentosa GTPase regulator (RPGR) (4,5). About 70% of the mutations in RPGR lie in the cryptic exon ORF15, which upon alternative splicing leads to the production of a transcript encoding a larger protein, RPGRORF15, without an isoprenylation motif (6). RPGR is ubiquitously expressed (4,5), but a retina-specific splice variant apparently exists (7). The N-terminal half of RPGR has homology to the RanGTPase nucleotide exchange factor, RCC1 (8,9), leading to the proposal that RPGR may play a role in photoreceptor biology through a RanGTPase-dependent mechanism (10). However, this does not seem to be the case. Instead, RPGR was found to interact directly and specifically with a retina-specific RPGR-interacting protein (RPGRIP) in vitro and in vivo, and through its C-terminal domain, RPGR-interacting domain (RID) (11,12). Others have also found that RPGR interacts with RPGRIP (13,14). In addition, XlRP3-associated mutations in RPGR disrupted the interaction of RPGR with RPGRIP (12,13). Antibodies against conserved domains of these proteins localized them to outer segments of photoreceptors in bovine retinas (12). Altogether, these data provided the first insight into the retina-specific effects of RPGR mutations via a mechanism dependent on RPGRIP and localized in the outer segment of photoreceptors (12). More recently, mutations in the RPGRIP gene were found to lead to the autosomal recessive neuroretinopathy, Leber congenital amaurosis (LCA), in the human (15,16), thus underscoring the essential function of RPGRIP in human photoreceptor biology.
However, three significant findings have been reported that seem to be at odds with the observations described above and with those from other studies. First, RPGR knockout mice have extremely mild electrophysiological and morphological phenotypes (10) compared to those observed in humans (1–3,17) and the dog disease model (18,19). Second, an RPGR isoform and RPGRIP were localized only to the connecting cilium, but not outer segments, of mouse (and squirrel) photoreceptors (10,14). Third, a recent report implicated specific mutations in RPGRORF15 in X-linked cone dystrophy in the human (20), thus sparing rod photoreceptor function. The localization of these proteins to the connecting cilium of mouse photoreceptors led the authors to speculate that differences in handling techniques of fixation of retinas may account for the discrepant results obtained with the bovine (and human) retinas (14). In light of the numerous sections analyzed by various fixation methods that were not reported, such an explanation became very implausible. Still, the possibility of the presence of RPGR and RPGRIP in discrepant subcellular compartments of photoreceptors would have profound implications for the interpretation of the molecular pathogenesis of the diseases. For example, localization of these proteins at the connecting cilium would imply a potential role for these in the maintenance of this structure and/or transport of cargoes between the inner and outer segments. In contrast, the presence of these proteins in the outer segments would imply a direct role for RPGR/RPGRIP in the structural and/or functional maintenance of this compartment. Thus, it became critical to investigate further the distribution of these proteins in photoreceptors. In addition, the localization of RPGRORF15 has never been reported. To address these discrepancies and the localization of the major RPGR and RPGRIP isoforms in the retina across species, we generated several RPGR- and RPGRIP-isotype specific antibodies and determined the localization of the cognate isoforms in the retina by using high-resolution epifluorescense microscopy. Among other locales, we found that RPGR and RPGRIP isoforms localize at distinct and spaced foci in rod and cone outer segments of photoreceptors of humans and bovine, while in the mouse they are localized to the connecting cilium. These results imply the existence of species-specific molecular processes involved in the function and/or structure of the outer segment compartment of photoreceptors and with profound consequences for the phenotypic expression and variability of congenital neurodegenerative retinal dystrophies among species.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation and characterization of RPGR, RPGRORF15 and RPGRIP antibodies
To investigate the localization of RPGR and RPGRIP isoforms in the retina, we generated, in duplicate and independently, RPGR and RPGRIP isotype-specific antibodies. To this end, four independent antibodies, MCW21 and MCW22, and MCW27 and MCW28, respectively, were generated against linear peptides comprising the unique C-terminal ends of RPGR and RPGRORF15 (Fig. 1A). Likewise, two antibodies, MCW3 and MCW4, were generated against a linear peptide covering an intermediate spliced domain of RPGRIP (Fig. 1A). As expected, the anti-RPGR and anti-RPGRORF15 antibodies recognized single protein species in human retinal homogenates with the predicted and apparent molecular masses of
97 and 145 kDa (Fig. 1B). The anti-RPGRIP antibody rec-ognized 175 kDa protein and some other lower-molecular-weight degradation (or processed) species (Fig. 1B), consistent with results reported with other well-characterized antibodies against the RID (12) and N-terminal coiled-coil domains (data not shown) of RPGRIP.
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Immunocytolocalization of RPGR and RPGRIP isoforms in bovine and murine retinas
We performed high-resolution epifluorescence microscopy on bovine, mouse and human retinal cryosections with the antibodies described to determine the localization of RPGR- and RPGRIP-specific isoforms in the retina among species and resolve the discrepant results reported of these isoforms in different subcellular compartments of photoreceptors (10,12, 14). To this effect, immunolocalization of RPGR with MCW21 and MCW22 antibodies detected RPGR strongly in rod outer segments (ROS) of bovine retinas (Fig. 2A and B). In this compartment, RPGR was highly localized at distinct foci spaced throughout the ROS (insets in Fig. 2B). Likewise, MCW4 against an RPGRIP isoform localized this protein strongly to ROS of bovine photoreceptors (Fig. 2C). These RPGR and RPGRIP isoforms co-localized largely throughout the ROS (Fig. 2D). Then, we determined the localization of these proteins in mouse retinas with the same antibodies. In striking contrast to what was observed in bovine retinas, these antibodies localized these RPGR (Fig. 2E and F) and RPGRIP (Fig. 2G and H) isoforms in a highly punctate fashion and strongly at the interface region between the outer and inner segments of photoreceptors, consistent with the localization of these proteins to the connecting cilium of photoreceptors. The outer segments of photoreceptors were conspicuously free of any staining for these proteins.
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Immunocytolocalization of RPGRORF15 and RPGRIP isoforms in the bovine neuroretina
The RPGRORF15 isoform contains the translation of cryptic exon 15 leading to the production of a larger protein of
140 kDa (Fig. 1C) without an isoprenylation motif at its C terminus. The absence of this membrane-anchoring motif from RPGRORF15 and the preponderance of mutations found in this isoform (6) prompted us to find its localization in the retina. As shown in Figure 3A, the MCW27 antibody against RPGRORF15 showed that this isoform was present strongly in the outer segments of rod photoreceptors. Like its splice variant counterpart, RPGR (Fig. 2B), RPGRORF15 was present at distinct foci throughout the rod outer segments (inset in Fig. 3A). A similar distribution and localization pattern was also observed for RPGRIP (Fig. 3A and inset). RPGRORF15 and RPGRIP co-localized partially in the outer segments of rod photoreceptors. However, as shown in the inset in Figure 3, there seems to be a higher number of RPGRORF15-positive foci than that observed for RPGRIP, and not all RPGRORF15-positive foci co-localized with RPGRIP.
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RPGRORF15, RPGR and RPGRIP isoforms are localized to rod and cone outer segments of human retinas
We then employed the same antibodies to localize RPGRORF15, RPGR and RPGRIP isoforms in the human retina. RPGRORF15 (Fig. 4B), RPGR (Fig. 4E) and RPGRIP (Fig. 4H) were present strongly in the outer segments of rod photoreceptors. The distribution pattern of these proteins in this subcellular compartment was highly punctate and spatially restricted throughout the ROS. Likewise, these proteins were present in outer segments of cone photoreceptors (arrows in Fig. 4B, C, E and H), where the distribution was also highly punctate, albeit seemingly less intense than that observed in ROS.
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All RPGRIP isoforms are localized to the rod outer segments of bovine but not mouse retinas
We have previously reported the identification of several RPGRIP isoforms (12). All of these shared a conserved RPGR-interacting domain (RID) (12). A previous report by Hong et al. (14) described the localization of RPGRIP to the ciliary axoneme of the connecting cilium of mouse photoreceptors with an antibody against the conserved RID of RPGRIP. These results were in apparent disagreement with those reported in bovine retinas with an independent set of antibodies against the same domain of RPGRIP (12). Therefore, we used one of these well-characterized antibodies, Ab39 (12), to revisit the immunolocalization of all RPGRIP isoforms in bovine and mouse retinas, but now applying high-resolution epifluorescence microscopy. As shown in Figure 5(A and B), bovine retinal sections immunostained with Ab39 showed that all RPGRIP isoforms were present in ROS and distributed in spatially confined foci throughout the ROS. Likewise, a similar distribution pattern was observed in cone outer segments (although cone photoreceptors required Triton X-100 permeabilization of the photoreceptors with this particular antibody). In addition, a restricted number of ganglion cells were also strongly immunoreactive towards RPGRIP (Fig. 5A), where RPGRIP distribution was conspicuously punctate and disperse throughout the perikarya. However, the distribution of RPGRIPs in the mouse retina was strikingly different from that observed in bovine (and human) retinas. To this end, all RPGRIP isoforms were confined to the junction region between the inner and outer segments of photoreceptors in a highly punctate fashion, and some weaker immunoreactivity was detected in the synaptic layer of photoreceptors.
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RPGRORF15, RPGR and RPGRIP isoforms are expressed and localized in selective neuroretinal cells other than photoreceptors
Previous observations with less sensitive immunohistochemistry detection techniques (12) (data not shown) and Figure 5A suggested that RPGR and RPGRIP isoforms might be expressed in other neuroretinal cells in addition to the photoreceptors. To this end, we generated independent antibodies (e.g. MCW3 and MCW28) against the same peptide domains of RPGRIP and RPGR previously described (Fig. 1A). We used them to confirm the results obtained and to overcome potential antibody-dependent recognition of partially masked epitopes due to potential cell-specific/dependent effects. To this end, immunohistochemistry with an antibody (MCW3) against the same epitope used for the production of MCW4 antibody confirmed the localization of RPGRIP in the outer segment of photoreceptors. However, this antibody also led to strong staining of amacrine cells located at the proximal edge of the inner nuclear layer, indicating expression of RPGRIP in these cells (Fig. 6A and B; arrow). Likewise, the MCW28 antibody against RPGRORF15 showed that this isoform was localized in the outer segment compartment of photoreceptors and that its presence could also be seen in amacrine cells, albeit with much less intensity (Fig. 6C and D; arrowheads). The localization of this isoform was observed also in a restricted set of ganglion cells, where its distribution was highly spotty (Fig. 6E and F). Finally, longer exposures of retinal sections with MCW22 detected the RPGR isoform also in amacrine cells, in particular, in punctate spots at the periphery of these cells (Fig. 6Gand H). Thus, the localization of RPGR isoforms in amacrine cells paralleled that seen for RPGRIP, albeit with much less preponderance.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We employed a battery of independently raised antibodies to show that RPGR and RPGRIP isoforms are consistently localized in photoreceptors of bovine, human and mouse retinas. However, the subcellular localization of these isoforms is strikingly different among species. In mouse photoreceptors, all RPGR and RPGRIP isoforms are localized between the junction of inner and outer segments, consistent with their reported localization to the connecting cilium of photoreceptors (10,14). In contrast, all RPGR and RPGRIP isoforms were distributed with a similar pattern throughout the outer segment subcellular compartment of bovine and human photoreceptors. RPGRORF15 co-localized partially with an immunoreactive RPGRIP isoform, supporting previous reports of RPGRIP as a natural photoreceptor partner of RPGR, but indicating that RPGRORF15 and RPGRIP are also likely to play independent roles in photoreceptor function. In addition, the expression of RPGR and RPGRIP isoforms is not restricted to ROS of photoreceptors, where the localization of these was prevalent. RPGR, RPGRORF15 and, in particular, RPGRIP were expressed and localized in amacrine cells and often throughout the perikarya of some ganglion cells. Thus, the differential cellular and subcellular distribution of these proteins among retinal neurons, in particular photoreceptors, of different species reflect most likely fundamental differences in the organization of the outer segment subcompartment between species and the degree of participation of these proteins in its maintenance and/or function. Thus, we propose that these factors account for the large variability of phenotypic penetrance of the XlRP3 locus observed between the mouse (10) and the human (1–3,17) and dog model of XlRP3 (18,19). Moreover, these results indicate that RPGR- and RPGRIP-associated mutations do not lead to human photoreceptor dystrophy due to ciliary axoneme dysfunction, as previously proposed (10,14). Instead, a dysfunction of the outer segment compartment of photoreceptors remains the determinant locale for the pathogenesis of RPGR- and RPGRIP-associated neuroretinopathies.
The presence of RPGRIP in postreceptoral neurons may also significantly contribute to the extreme severity of the pathologies associated with genetic lesions in RPGRIP1. For example, the strong expression of RPGRIP in amacrine cells may contribute significantly to abolishing the full-field rod-plus-cone ERGs (16) in light of the significant input of these cells into the neural circuitry modulating ganglion cell responses from rods and cone polar cells. Moreover, cholinergic amacrine cells have been reported to play a role in the transient targeting of the synaptic terminals of rods and cones to the inner plexiform layer during retinal development (21). Thus, the panretinal distribution of RPGRIP may explain why mutations in RPGRIP lead to LCA instead of RP. This also suggests that RPGRIP acts upstream of RPGR and may play wider roles in the retina. LCA is genetically heterogeneous and the most severe form of congenital retinal dystrophy, often leading to complete blindness from the first months of life and extinguished ERGs (22). To this end, mutations in RPGRIP were reported to lead to severe impaired vision since early in childhood (15,16). Finally, in light of the striking differences in the localization of RPGR/RPGRIP and the wide differences in phenotypic penetrance caused by genetic lesions in RPGR (and possibly RPGRIP) among species, RPGR/RPGRIP-allied retinopathies may join other human-specific retinopathies, such as the human Usher 1B (23–25), which retinal phenotype has no equivalent counterpart in the shaker-1 mouse (26,27). This is also supported by the absence of myosin VIIa in rat and guinea-pig photoreceptors (28). Thus, it is tempting to postulate that the roles of myosin VIIa, RPGR and RPGRIP may be linked in humans.
Another central question raised by these data pertains to the reasons behind the striking differences in subcellular distribution of RPGR and RPGRIP isoforms in photoreceptors between the mouse and other species, such as bovine and human. In addition to key differences in the cellular spatial organization of the retina between the mouse and other species, it is possible that the variable subcellular distribution of RPGR/RPGRIP among species reflects differences in the subcellular and molecular organization of the outer segments of photoreceptors, which are known to vary significantly among species. For example, the disk of the outer segments exhibits petal-like invaginations, incisures, and the number of these is species-specific (29–31). In contrast to other species with multilobulated disks, the disks of murine rod photoreceptors contain only one such incisure (30). In addition, species-specific filaments lengths have been reported to ‘crosslink’ the rims of the disks with the plasma membrane and to bridge the lobes of each disk (32,33). To this effect, it will be interesting to determine the degree of participation of RPGR and RPGRIP in orchestrating the species-specific architectural organization of ROS of photoreceptors and the implications of these for human retinopathies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tissue sources and primary antibodies
Fresh bovine (Bos taurus) eyes with a postmortem time of less than 30 minutes were obtained from a local slaughterhouse. Human eyes with a postmortem time of 6–12 hours were provided by the Eye Bank at the Eye Institute of the Medical College of Wisconsin. Male C57BL/6 mice approximately 6 months old were obtained from Charles River Laboratories. Enucleation and processing of bovine and mouse retinas were performed between 11 a.m. and 1 p.m. All tissue manipulation procedures complied with institutional and federal guidelines.
Several rabbit polyclonal antibodies were generated in duplicate against linear peptide sequences comprising unique domains in RPGRORF15, RPGR and RPGRIP. MCW3 and MCW4 antibodies were produced against the amino acid sequence, ARFPVLVTSDLD, of RPGRIP. MCW21 and MCW22 antibodies were produced against the amino acid sequence, SDNKDADQNHMSQN, of human RPGR. MCW27 and MCW28 antibodies were produced against the amino acid sequence, KNGPSGSKKFWNNILPHYLELK, of human RPGR. The Ab39 antibody against the RID of bovine RPGRIP has been previously described (12). Each peptide was conjugated to KLH, mixed with complete Freund's adjuvant and inoculated into two rabbits (Cocalico Biological, Inc., Reamstown, PA). Up to six booster injections of the peptides in incomplete Freund's adjuvant were followed and spaced 4–6 weeks apart. Antibody titers were determined by ELISA assay, and antibodies were affinity-purified against the cognate peptides.
Preparation of retinal homogenates and western blot analysis
Homogenates of human retinas were prepared as previously described (12). Briefly, human retinas were homogenized in 3x SDS-sample buffer (5% w/v SDS, 0.15 M Tris–HCl, pH 6.7, and 30% glycerol) by several passes through an 18G11/2 needle and then a 25G5/8 needle, followed by 1 : 3 dilution in RIPA buffer (25 mM Tris, pH 8.2, 50 mM NaCl, 0.5% Nonidet P40, 0.5% deoxycholate, 0.1% SDS and 0.1% azide) containing 10 mM iodoacetamide and complete protease inhibitor cocktail (Boehringer Mannheim). Protein concentration was measured by the BCA method. All protein samples were boiled and then resolved on SDS–PAGE, and Western blots with antibodies against RPGR, RPGRORF15 and RPGRIP1 were carried out as previously described (34). Blots were developed with a SuperSignal chemiluminescence substrate (Pierce).
Immunohistochemistry
Immunohistochemistry on retinal sections was carried out as previously described (35). Posterior eyecups were fixed in 2% paraformaldehyde in phosphate buffer, pH 7.4, at 4°C for about 1 hour and infused sequentially for 12 hours with 15% and 30% sucrose. Retinal sections not fixed and fixed in more stringent conditions (4% paraformaldehyde, 0.1% gluteraldehyde) produced identical results. Bovine and human retinas and mouse eyecups were embedded in OCT medium, frozen in liquid nitrogen and sectioned radially at 3 µm in a Reichert–Jung cryostat. Retinal cryosections were mounted onto gelatin-coated slides, incubated with 10% normal goat serum (Jackson Immunoresearch Laboratories, West Grove, PA) in incubation buffer (0.1% Triton X-100 or 0.1% Saponin in PBS, pH 7.4) for 15 minutes, followed by another incubation for about 2 hours at room temperature in the same buffer containing the primary antibody at the concentrations described in the figure legends. For single-label immunofluorescence microscopy, sections were then rinsed trice for 10 minutes with incubation buffer, followed by incubation in the same buffer with goat anti-rabbit antibodies (2.5 µg/ml) conjugated with the fluorescent dyes Alexa488 or Alexa594 (Molecular Probes, Eugene, OR) for 1 hour at room temperature. Finally, sections were washed twice with incubation buffer for about 5 minutes, mounted with Antifade mounting medium (Molecular Probes, Eugene, OR) and coversliped. For double-label immunofluorescence microscopy, affinity-purified MCW4 was conjugated directly with Alexa594 at 1 : 3 molar ratio (Molecular Probes, Eugene, OR). Then, non-labeled primary antibody incubation and detection was carried out as described previously for single-label immunofluorescence, followed by intermediate washing steps, incubation with Alexa594-conjugated MCW4 antibody and two final washing steps. Control experiments were carried out whenever appropriate with cognate preimmune serums and/or omitting the primary antibodies. Visualization of retinal sections and localization of proteins were carried out by Nomarski and wide-field epifluorescence microscopy at various focal planes with an E600 Nikon research light microscope. This was equipped with fluorescence, appropriate filters, a 100 W mercury light source, Nomarski DIC and Plan Apochromat optics (100x and 60x oil objectives with NA of 1.4, and 40x, 20x and 10x objectives). Crossover of fluorescent probes, background and autofluorescence were found to be negligible. Images were acquired with a SPOT-RT digital camera coupled to the microscope and driven by SPOT Imaging v3.4 software (Diagnostic Instruments, Sterling Heights, MI, USA). All images were captured at non-saturating integration levels, 12-bit mono black/white (except for DAPI staining), and then pseudocolored, and, whenever applicable, fluorescent images were merged with the same software. Overall arrangements and cropping of images were performed by importing these to Adobe Photoshop v5.5 (Adobe, Mountain View, CA, USA).
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ACKNOWLEDGEMENTS |
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This work was supported by NIH Grants EY11993 and EY012665 to P.A.F. T.A.M. was supported in part by a postdoctoral fellowship from the Fight for Sight research division of Prevent Blindness America.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 4144568877; Fax: +1 4144566545; Email: ferreira{at}mcw.edu
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