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Jablonski MM
,
Tombran-Tink J
,
Mrazek DA
,
Iannaccone A
.
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Dysfunction of the retinal pigment epithelium (RPE), its loss, or separation from the underlying neural retina results in severe photoreceptor degeneration. Pigment epithelium-derived factor (PEDF) is a glycoprotein with reported neuroprotective and differentiation properties that is secreted in abundance by RPE cells. The "pooling" of PEDF within the interphotoreceptor matrix places this molecule in a prime physical location to affect the underlying neural retina. The purpose of this study was to analyze the morphogenetic activity of PEDF in a model of photoreceptor dysmorphogenesis induced by removal of the RPE. Eyes were dissected from embryonic Xenopus laevis, and the RPE was removed before culturing in medium containing PEDF, PEDF plus anti-PEDF antibodies, or medium alone. Control retinas were maintained with an adherent RPE. Light and electron microscopic analysis was used to examine retinal ultrastructure. Opsin was localized immunocytochemically and quantified as an index of outer segment membranous material and photoreceptor protein expression. Removal of the RPE resulted in an aberrant assembly of photoreceptor outer segments, loss of fine subcellular ultrastructure in photoreceptors, and a reduction in opsin protein levels when compared with control retinas. The addition of PEDF prevented the dysmorphic photoreceptor changes induced by RPE removal. In particular, photoreceptor ultrastructure, outer segment membrane assembly, and steady-state levels of opsin were equivalent to control conditions. Anti-PEDF antibodies completely blocked the morphogenetic activity of PEDF. These results indicate that PEDF is able to mimic the supportive role of the RPE on photoreceptors during the final stages of retinal morphogenesis.
Fig. 1.
Migration of purified PEDF as a 50 kDa molecular weight species on 10% SDS polyacrylamide gel detected by Coomassie blue staining. Molecular weight markers (Promega midrange standards) are shown in lane 1. Lane 2 is intentionally left blank. Lanes 3, 4, and5 represent the purified PEDF protein at concentrations of 505, 252, and 126 ng, respectively. Lane 6 represents the Western blot analysis showing a very specific interaction, at ∼50 kDa, between 100 ng of purified PEDF and the polyclonal PEDF antibody at 1:1000 dilution. Lanes 7 and 8, respectively, represent anti-PEDF (1:1000 dilution) applied toXenopus retinal homogenates and Xenopusretinal homogenates with the addition of 100 ng of purified PEDF.
Fig. 2.
Outer retinal morphology under the various culture conditions used in this study. a, In retinas that were allowed to undergo the final stages of cytomorphogenesis in vitro with a normally apposed RPE, the outer segments are highly organized and tightly stacked, yielding individual profiles that are in line with individual photoreceptor inner segments.b, In retinas that were maintained in the absence of the RPE, photoreceptor outer segment membrane structure was markedly disorganized, with little evidence of normal disc stacking.c, The addition of 50 ng/ml of PEDF stimulated the proper folding of outer segment membranes. Increased concentrations of PEDF [i.e., 100 ng/ml (d) and 500 ng/ml (e)] failed to induce similar levels of outer segment organization in the absence of the RPE. Under these conditions the majority of photoreceptors had many whorls of membranous discs interspersed with areas of organized outer segments. f,Nonimmune serum (1:500 dilution) was unable to block the morphogenetic properties of PEDF, and most outer segments were highly structured. Although the addition of a 1:1000 dilution of anti-PEDF antiserum (g) partially blocked the dramatic improvement in outer segment organization that was promoted by 50 ng/ml of PEDF, a 1:500 dilution of antiserum (h) significantly disrupted the permissive and protective effects of 50 ng/ml of PEDF.RPE, Retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer. Organized outer segments are indicated bywhite arrows, and disorganized outer segments are marked with black arrows. Scale bar, 10 μm.
Fig. 3.
Ultrastructural analysis of Xenopus laevis retinas with and without an adherent RPE.a, In the presence of the RPE the photoreceptor outer segments were composed of an orderly array of stacked discs surrounded by a plasma membrane. The disc margins were in proper alignment, with all discs of a single photoreceptor being the same diameter. Calycal processes (black arrows) that arise from the inner segment were in close proximity to the vitreal aspect of the outer segments. The apical process of the RPE surrounded the tips of the outer segments (white arrows). b, In the absence of the RPE there was some organization of outer segment discs into flattened, stacked saccules; however, the diameter of outer segment discs differed greatly (black arrows), with many discs terminating prematurely. Also, areas of whorl-like membrane with minimally ordered stacked discs were present (asterisk). Calycal processes were not present. OS, Outer segment;IS, inner segment. Scale bar, 1 μm.
Fig. 4.
PEDF prevented photoreceptor degeneration after removal of the RPE. a, Photoreceptor outer segments were highly structured and ordered in retinas exposed to exogenous PEDF (50 ng/ml). Most discs were flattened and stacked in an orderly array and had an equal diameter. Calycal processes were present and were closely adherent to the outer segments. They extended to the tips of the outer segments, beyond their normal position (black arrows).b, Anti-PEDF (1:500 dilution) blocked the protective effect of PEDF. Photoreceptor outer segments no longer were composed of stacked membranous discs of similar diameter. The addition of both PEDF and anti-PEDF to retinas that lacked an adherent RPE resulted in premature termination of disc elongation and led to a staggered border of the membranous discs (black arrows). Calycal processes were not formed. OS, Outer segment;IS, inner segment. Scale bar, 1 μm.
Fig. 5.
When allowed to complete morphogenesis in the presence of an attached RPE, the average organizational grade of individual outer segments was 3.958 ± 0.200 (mean ± SD) on a scale of −1 to 4, indicating that nearly all of the photoreceptor outer segments were 100% organized. In the absence of the RPE the average grade of photoreceptor outer segment organization decreased to 1.042 ± 1.207 (mean ± SD; p = 0.0001), indicating that nearly 75% of the graded outer segment material was disorganized into whorl-like or staggered profiles. In the presence of 50 ng/ml PEDF the average organizational grade was 3.042 ± 1.338 (mean ± SD), which is not significantly different from that obtained with photoreceptors that matured with an adherent RPE (p = 0.0144). This value indicates that ∼75% of the outer segment membranes were highly structured. In the presence of anti-PEDF (1:500 dilution) the organizational grade significantly decreased to 0.708 ± 1.541(mean ± SD;p = 0.0001 compared with both control retinas and those exposed to PEDF), reflecting that <25% of the outer segment material was organized. The grading scale that was used is as follows: 4 = 100% organization of an individual outer segment; 3 = 75% organization of an individual outer segment; 2 = 50% organization of an individual outer segment; 1 = 25% organization of an individual outer segment; 0 = 0% organization of an individual outer segment; −1 = complete absence of outer segment membrane. Qualities of a disorganized outer segment included whorl-like and staggered profiles. p < 0.009 is considered statistically significant. *Significantly different from control retinas with an attached RPE; ♦, significantly different from eyes exposed to PEDF.
Fig. 6.
PEDF prevented the altered opsin immunolabeling patterns induced by the removal of the RPE. a, In retinas that completed morphogenesis with an adherent RPE, the opsin labeling was very heavy over photoreceptor outer segments, which was indicative of organized, stacked outer segment membranes.b, Removal of the RPE resulted in an altered immunolocalization pattern of opsin in which individual outer segment profiles could not be distinguished. Rather, heavy patches of label alternating with a complete lack of immunopositive label were present distal to the inner segments. c, In the absence of the RPE, PEDF promoted the formation of well organized outer segment membranes. Individual outer segment profiles with linear opsin immunolabeling patterns were evident, consistent with the organized outer segment membrane disc lamellae that were observed morphologically. d, The addition of anti-PEDF disrupted the linear and evenly profiled opsin immunolabeling patterns. Rather, an uneven jagged labeling pattern was present. RPE, Retinal pigment epithelium; OS, outer segment;IS, inner segment. Scale bar, 10 μm.
Fig. 7.
PEDF promoted opsin expression. a,Relative amounts of opsin in control, dysmorphic, and PEDF-supported photoreceptors. Values were normalized to the amounts of opsin in control retinas with an attached RPE. In the absence of the RPE the photoreceptors downregulated the expression of opsin to 70% of that synthesized in the presence of the RPE (p = 0.0001). PEDF prevented the downregulation induced by RPE detachment and sustained opsin expression (p = 0.3938 compared with control eyes; p = 0.0003 compared with eyes without an attached RPE). Anti-PEDF partially blocked the permissive effect of PEDF on opsin synthesis (p = 0.0088 compared with control eyes).p < 0.009 is considered statistically significant. *Significantly different from control retinas with an attached RPE; ♦, significantly different from eyes exposed to PEDF; ✣, significantly different from PEDF plus anti-PEDF antibody.b, Representative immunoblot illustrating the differences in opsin expression under the various experimental conditions.
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