Expression of CNTF Receptor-α in Chick Violet-Sensitive Cones with Unique Morphologic Properties

Abstract

purpose. Application of ciliary neurotrophic factor (CNTF) can rescue mature photoreceptors from lesion-induced and hereditary degeneration. In the chick retina, expression of the CNTF receptor is present in a subpopulation of photoreceptor cells. The present study was undertaken to identify the CNTF receptor-expressing photoreceptors and to describe the subcellular localization of the receptor protein.

methods. The localization of the CNTF receptor was analyzed by light and electron microscopic immunocytochemistry in chick retinal wholemount preparations, with an antibody for CNTF receptor α (CNTFRα). Immunoreactive cells were identified by double labeling with immunocytochemical markers for photoreceptor subpopulations.

results. The CNTFRα antibody labeled evenly distributed outer segments (OS) of a photoreceptor subpopulation. CNTFRα-positive OS were associated with oil droplets of uniform size. Receptor immunoreactivity did not colocalize with markers for rods and red-green cones. Complete overlap was found after double labeling with the antibody CERN 933, which recognizes violet-sensitive cones in the chick retina. Ultrastructurally, the CNTFRα-immunoreactive OS showed rodlike properties: an elongated shape and stacks of membrane discs separated from the plasma membrane. Immunoreactivity was completely restricted to the plasma membrane of the OS and the inner membrane sheet of the photoreceptor calices present in avian retinas.

conclusions. CNTFRα expression identifies a unique type of photoreceptors in the avian retina which does not fit into the classic morphologic definition of rods and cones. The specific expression in violet-sensitive photoreceptors suggests that CNTF may have a neuroprotective role related to the specific function of these cells.

Ciliary neurotrophic factor (CNTF) represents a pleiotropic protein with a broad spectrum of target cells in the peripheral and central nervous system. It has been implicated in the regulation of neuronal development, ^{1 2} but evidence is accumulating that it is of particular importance in the mature nervous system for the prevention of neuronal degeneration and injury-induced cell death. ^{3 4 5}  

In cultures from developing vertebrate retinas, CNTF has been shown to promote neuronal development ^{6 7} and, in particular, to regulate the differentiation of photoreceptors. ^{8 9 10 11 12 13 14} In the adult rodent retina, application of CNTF in vivo improves rod survival after lesions ^{15 16} or in hereditary retinal degeneration, ^{17 18 19 20} indicating that it plays a role in the maintenance of photoreceptor function. 

Effects of CNTF are mediated by a tripartite cytokine receptor complex consisting of two transmembrane signal-transducing components (LIF receptor-β, gp130) and a ligand-binding α-subunit (CNTFRα) that is specific for CNTF and thus defines cells that are responsive to this cytokine. CNTFRα is expressed in a variety of distinct neuronal cell types of the peripheral and central nervous systems, including retinal ganglion cells and inner nuclear layer neurons, as shown in the rat and chick retina. ^{21 22 23 24} Expression of the receptor protein in the outer nuclear layer of rat retina is prominent during early postnatal development but decreases with maturation, as shown by immunoblot analysis. ¹⁴ In the adult chick retina, prominent immunoreactivity for CNTFRα has been described in outer segments (OS) of a photoreceptor subpopulation. ²⁴  

This study describes the identification of CNTFRα-expressing photoreceptors as representing violet-sensitive cones with the protein being exclusively located in the plasmalemma of the OS. Electron microscopic analysis further shows that these CNTF-responsive cones exhibit a rodlike membrane morphology in their OS. 

Materials and Methods

Tissue Preparation

All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Fertilized white leghorn eggs were incubated in a humidified egg incubator at 37°C. Chicks were killed by decapitation at embryonic day 19 or at 5 to 17 days after hatching, as indicated in figure legends. Eyecups were opened by removing the cornea, lens, and vitreous body and then were fixed for light microscopy for 1 hour in phosphate-buffered fixative (0.1 M, pH 7.4) containing 4% paraformaldehyde. For electron microscopic immunocytochemistry, the fixative contained 0.1% glutaraldehyde and 4% paraformaldehyde, and for conventional electron microscopy, the glutaraldehyde concentration was increased to 2.5%. Eyecups were postfixed in 4% paraformaldehyde at 4°C overnight. Retinal tissue was carefully dissected, and the neural retina was separated from the pigmented epithelium. For light microscopic staining, pieces 2 × 4 mm were cut from the central retina and processed for immunocytochemistry. In one experiment, the fixed retina was cryoprotected, and vertical sections were cut at a thickness of 12 μm. For pre-embedding immunoelectron microscopy, central parts of the retina with adhering pigmented epithelium were embedded in 5% agar and sectioned (70 μm) on a vibratome for immunostaining. 

Immunocytochemistry

Retinal pieces and cryostat sections were pretreated for 1 hour with 10% normal goat serum, 1% bovine serum albumin, and 0.1% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4). The detergent was omitted for electron microscopic immunolabeling. Incubation with primary antibodies in PBS containing 3% normal goat serum and 1% bovine serum albumin was performed overnight at 4°C. The rabbit anti-CNTFRα antibody used in this study (at a dilution of 1:3000–1:10,000) was kindly provided by Hermann Rohrer and Stefan Heller (Max-Planck-Institute for Brain Research, Frankfurt, Germany). Its preparation and specificity have been described previously. ^{24 25} In immunofluorescence double-labeling experiments, the monoclonal mouse antibody rho-4D2 (dilution 1:300; kindly provided by Robert S. Molday, University of British Columbia, Vancouver, British Columbia, Canada ⁸ ) was used to identify rods. The antibody has been raised against bovine rhodopsin ²⁶ and could be shown by immunoelectron microscopy to label the OS membranes of rods also in the chick retina. ⁸ In addition, rho-4D2 labels a subpopulation of cones in the chick retina that, as judged on the basis of close homologies between the rod epitope recognized by the antibody ²⁶ and a corresponding sequence in the green-sensitive visual pigment of the chick, most likely represent the green cone subpopulation. ⁸ The rabbit antibody CERN 933 (dilution 1:2000), raised against human blue visual pigment, ²⁷ and FITC-conjugated lectin were used in double-labeling experiments to identify cone subpopulations. Antibody binding was visualized by appropriate Cy3- or Cy2-conjugated secondary antibodies (Rockland, Gilbertsville, PA) applied at a dilution of 1:300 for 1 to 3 hours. Alternatively, biotinylated goat anti-rabbit antibodies (1:200; Vector Laboratories, Burlingame, CA) and biotin avidin-peroxidase complex (1:100; ABC Elite, Vector Laboratories) with diaminobenzidine (0.05%) and H₂O₂ (0.01%) as substrates were used for CNTFRα immunolabeling. ²⁴ FITC-conjugated lectin (Vector Laboratories) was applied for 30 minutes at a concentration of 20 μg/mL in PBS containing 0.1% bovine serum albumin. Appropriate immunocytochemical control experiments were performed in which the primary antibody was omitted or replaced by a corresponding normal serum. For all staining protocols, including those for electron microscopy, the reported specific signals were absent in these experiments. Labeled retinal tissue was placed on gelatin-coated coverslips, air dried, and mounted (Moviol; Hoechst, Darmstadt, Germany). Sections were coverslipped in Kaiser gelatin (Merck, Darmstadt, Germany). Flatmounts and sections were viewed and photographed with a microscope (BX60; Olympus, Tokyo, Japan) equipped with Nomarski optics and appropriate fluorescence filter combinations. Photographs were digitally contrast enhanced and color adjusted with image-management software (Photoshop; Adobe, Mountain View, CA). Confocal images of CNTFRα immunofluorescence (Cy3)/FITC-peanut agglutinin double-labeled retinas were obtained with a confocal laser microscope (TCS NT; Leica, Heidelberg, Germany) using filter combinations for tetrarhodamine isothiocyanate (TRITC) and FITC, respectively. Images were acquired with a z-scan of 0.02 μm. 

In Situ Hybridization

To prepare digoxigenin-labeled riboprobes, we used pCRII vectors from Invitrogen (Karlsruhe, Germany) encoding a 504-bp PCR fragment of the chicken violet opsin (GenBank accession no. P28684, position 187-691; available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD) or a 497-bp fragment of the chick blue opsin (GenBank accession no. P28682). Probes were synthesized (DIG RNA labeling kit; Roche Diagnostics, Mannheim, Germany) from the BamHI-linearized template, using the T7 RNA polymerase (antisense probe), and the XhoI-linearized template, using Sp6 RNA polymerase (sense probe). Cryosections were thawed, washed twice in PBS and treated with proteinase K (10 μg/mL PBS) for 20 minutes at 37°C. Sections were washed with glycerin solution (2 mg/mL), postfixed in 4% paraformaldehyde (in PBS) for 20 minutes, and rinsed twice in PBS. Prehybridization was performed for 4 hours at 70°C in hybridization solution (50% formamide, 2× SSC, 500 μg/mL sheared herring sperm DNA, 250 μg/mL yeast tRNA, 1× Denhardt’s solution, 10% dextran sulfate, and 0.1% Tween). Each section was incubated with 200 μL hybridization solution (containing 100 ng of the riboprobe) overnight at 70°C in a humidified chamber. After hybridization, slides were incubated as follows: four times for 15 minutes each in 2× SSC at 70°C; two times for 30 minutes each in 0.2× SSC at 70°C; two times for 15 minutes each in 0.1× SSC at room temperature; and two times for 10 minutes each in PBST (PBS containing 0.1% Tween). For visualization of the digoxygenin-labeled probes, sections were treated with 300 μL blocking solution (10% goat serum in PBST) for 2 hours at room temperature. Anti-digoxygenin Fab fragments conjugated to alkaline phosphatase (Roche Diagnostics) were diluted 1:2000, and sections were incubated for 3 hours at room temperature. Finally, retinas were stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate (NBT/BCIP; Roche Diagnostics) and then washed four times in PBST. For double labeling, sections were subsequently processed for immunocytochemistry, as described earlier. 

Electron Microscopic Immunocytochemistry

Free-floating vibratome sections of agar-embedded chick retinas from central regions were treated for CNTFRα immunocytochemistry, as described earlier. For intensification of peroxidase reaction product, a modified version of previously described procedures ^{28 29} was applied. Briefly, sections were rinsed in 0.1 M cacodylate buffer (pH 7.4), postfixed in 2.5% glutaraldehyde in cacodylate buffer for 2 hours at 4°C, kept in cacodylate buffer at 4°C overnight, and then carefully washed in water. Sections were then sequentially treated with silver nitrate and gold chloride. ³⁰ Postfixation was performed in 0.5% OsO₄ for 30 minutes at 4°C and sections were dehydrated in a graded series of ethanol (30%–100%) and propylene oxide and flat embedded (Durcupan, ACM; Fluka Chemie, Buchs, Switzerland). Selected sections were cut out and re-embedded in blocks for sectioning (50 nm). Ultrathin sections collected on single-slot polyvinyl formal–coated copper grids were examined and photographed in an electron microscope (CM 100; Philips, Eindhoven, The Netherlands). For conventional electron microscopy, retinas fixed in 2.5% glutaraldehyde were postfixed in 1% OsO₄ for 2 hours at 4°C, dehydrated in ethanol, stained with 1% uranyl acetate, and further processed as described. 

Results

The antibody used in this study has been demonstrated to label the OS of a subpopulation of photoreceptors heavily in sections of chick retinas. ¹² Immunoblot analysis of isolated OS confirmed that the labeling is due to the presence of high levels of CNTFRα protein in this cellular compartment. Expression of CNTFRα is observed at the earliest stages of OS formation, which starts at approximately embryonic day (E)18. In flatmounted pieces from E19 retinas, CNTFRα-positive OS appeared as intense fluorescent tips arranged in a semiregular mosaic, indicating that the corresponding photoreceptors represent a distinct subpopulation (Fig. 1A) . At 8 days after hatching (P8), immunolabeled OS had a more mature, elongated shape (Fig. 1B) . With further development the labeling pattern did not change except that the OS still increased in length (data not shown; see Ref. ²⁴ ). Inspection of labeled OS at higher magnification indicated that the receptor protein is enriched in the plasma membrane and less abundant in the interior of the OS (Fig. 1C) . Vertical sections of the chick retina were also immunolabeled for CNTFRα with the peroxidase method, to confirm that the protein is restricted to the OS compartment of the photoreceptor layer (Fig. 1D) . 

In a previous study, CNTFRα-expressing photoreceptors in the adult chick retina were tentatively identified as rods on the basis of the long, relatively thin shape of their OS and the failure to demonstrate oil droplets characteristic of avian cones in the inner segments (IS) of these cells. ¹² To determine the identity of CNTFRα-positive photoreceptors, retinas were double labeled using the anti-CNTFRα antibody and the monoclonal antibody rho-4D2 raised against bovine rhodopsin, which has been shown to recognize rod OS in the chick retina. ^{8 26} The OS labeled by the two antibodies were very similar in shape, but the density of rho-4D2–positive OS was substantially higher (Figs. 2A 2B) and there was no colocalization of the two signals (Fig. 2C) , indicating that CNTFRα is not expressed in rods. 

Consequently, we assumed that the cytokine receptor is present in cone OS. In the avian retina, cones contain characteristic oil droplets in their IS that differ in color and size, depending on the cone subtype. Therefore, to confirm that CNTFRα is expressed in a subpopulation of cones, we examined the presence of an oil droplet in immunoreactive photoreceptors. By viewing immunofluorescence-labeled retinal flatmounts stained for CNTFRα by Nomarski optics, we were able to demonstrate that the immunoreactive OS belong to oil droplet-containing cells. As shown in Figure 3A , all the CNTFRα-immunoreactive OS were associated with oil droplets at their vitreous end, corresponding to the location of the IS. The color of the oil droplets was no longer discernible in the immunostained specimen, but their uniform size and optical density (Fig. 3B) indicates that the receptor is expressed in a specific subtype of cones. 

The chick retina contains four types of single cones and one type of double cones, with distinct spectral sensitivities due to the presence of specific visual pigments. ^{31 32} To identify the CNTFRα-expressing cone type, we performed double-labeling experiments with two fluorescent markers. The lectin peanut agglutinin (PNA) has been demonstrated to label specifically the IS and OS membranes of red and green-sensitive cone photoreceptors, ³³ which represent the most abundant cone population in the chick retina. ^{32 34 35} Inspection of double-labeled retinas by confocal laser microscopy revealed that CNTFRα immunoreactivity did not colocalize with PNA fluorescence labeling (Figs. 4A 4B) . In the flatmount shown, the photoreceptor IS and OS are oriented more horizontally, which more clearly demonstrates the labeling of both segments by PNA fluorescence. Although a partial overlap with the CNTFRα signal was occasionally observed due to the high density of PNA-binding cones, the CNTFRα-positive OS heavily labeled in red were never delineated by the green PNA fluorescence. 

As a second marker we used an antibody (CERN 933) that was raised against human blue-sensitive pigment. ²⁷ Immunofluorescence double-labeling revealed that this antibody and the anti-CNTFRα antibody recognize the same subpopulation of chick cones (Figs. 4C 4D 4E) . Quantitative evaluation confirmed that there was a 100% colocalization between the two markers. Inspection of 400 to 500 CNTFRα-positive OS in each of three flatmounts from three different retinas showed that all these OS were also positive for CERN 933. Vice versa, all OS labeled by CERN 933 also exhibited CNTFRα immunofluorescence. 

The antibody CERN 933 had not been tested in the chick retina, so far. However, the human blue pigment used as an antigen to produce the CERN 933 antibody shows considerable sequence identity with the chick violet pigment but not with other cone pigments of the chick. ³¹ To confirm further that the CNTFRα-expressing cone population identified by CERN 933 represents the violet-sensitive cones, CNTFRα immunocytochemistry was combined with in situ hybridization for violet pigment mRNA in retinal sections (Figs. 4F 4G 4H) . Strong mRNA signals were observed proximal to the layer of the oil droplets in the IS of a subpopulation of photoreceptors (Fig. 4F) . When the adjacent section was immunolabeled for CNTFRα (Fig. 4G) , the digital overlay of the two images revealed that each of the IS expressing violet cone opsin mRNA was associated with an immunofluorescent OS (Fig. 4H) . establishing that the CNTFRα-expressing photoreceptors were violet-sensitive cones. 

Because the results did not completely rule out the possibility that CERN 933 (and antibodies to CNTFRα) in addition to violet-sensitive cones also recognize the visual pigment of blue cones, we double labeled retinal sections by CERN 933 immunocytochemistry and in situ hybridization for blue cone opsin mRNA. No colocalization was observed in these experiments (data not shown) demonstrating that CERN 933 specifically labels violet cones. 

For ultrastructural localization of CNTFRα in photoreceptor OS, a pre-embedding immunocytochemical technique employing conversion of the primary DAB signal to an electron dense silver-gold precipitate ²⁸ was used. This method resulted in strong labeling of a subpopulation of OS which, in agreement with the light microscopic observations, had an elongated rod-like appearance when compared with typical cone OS, but clearly were part of photoreceptor cells with an oil droplet (Fig. 5A) . CNTFRα immunoreactivity was observed over the OS plasma membrane that continuously envelopes the inner membrane stacks, which were not labeled. In addition, membranes of calices opposing the OS were heavily labeled, whereas the outer leaf of the caliceal membrane was not stained. The calices, which are found in avian retinas, represent cellular protrusions emanating from the IS and surrounding the OS. ³⁶ The limited spatial resolution of the pre-embedding 3,3′-diaminobenzidine (DAB) staining technique used in our experiments does not allow a precise localization of the antigen with respect to cellular compartments. However, the CNTFRα is most likely located in the membranes delineated by the immunostaining, although labeling also extended into the adjacent cytoplasmic and extracellular compartments (compare Figs. 5A 5B 5E ). 

The ultrastructure of photoreceptor OS has been studied in many different species (see Cohen ³⁷ and Dowling ³⁸ ), including the avian retina. ³⁹ Both rods and cones contain stacks of transversely oriented membranes. However, whereas these membranes were described in cones to form lamellar invaginations which are continuous with the plasma membrane, the membranes of rod OS show no such continuity but form separate membrane discs (see schematic representation in Fig. 5C ). The labeled membrane of the OS forming a continuous sheath appeared to be difficult to reconcile with the typical membrane configuration of cone OS. In fact, at higher magnification CNTFRα-labeled OS always exhibited a rod-like membrane configuration as no continuity between the plasma membrane and the nonlabeled inner membranes could be observed (Fig. 5B) . Because the preservation of the ultrastructure was not optimal in the immuno-stained specimens, ultrathin sections were prepared at optimized conditions and without prior immunocytochemical processing. With careful inspection of these sections, photoreceptors with the characteristic elongated shape and an oil droplet could be identified (Fig. 5D) . The configuration with membrane discs and a separated plasma membrane in these OS was confirmed in these sections (Fig. 5E) . 

Discussion

Results of the present study show that the CNTFRα protein is specifically expressed in a cone subpopulation of the chick retina that could be identified as violet-sensitive cones. Four types of cones can be distinguished in the chick retina according to their spectral sensitivity which is based on the presence of specific red-, green-, blue- or violet-sensitive opsin proteins. ³² Based on sequence homology and phylogenetic identity the violet pigment of the chick is grouped in one class together with blue pigments of mammalian retinas, whereas the blue-sensitive pigments of birds and fish form a separate class. ^{31 40} In line with this classification, the polyclonal antibody CERN 933, which was raised against the human blue-sensitive pigment protein recognizes the violet cone population in the chick retina. Each of the cone pigments is associated with an oil droplet of distinct color located in the distal part of the inner cone segment. ^{41 42} By counting the number of the different oil droplets, we determined that violet-sensitive cones make up approximately 10% of all cones in the avian retina. ³¹ We did not systematically analyze the number and distribution of CNTFR-positive OS, but by counting CNTFRα-immunoreactive OS and the total number of oil droplets in randomly selected flatmounts of embryonic (E19) and hatched (P8, P14) retinas we obtained a very similar ratio of 8% to 12% (data not shown). It will be interesting to examine, if expression of the CNTF receptor is a common feature of mammalian blue cones and violet/UV-sensitive cones in nonmammalian retinas. 

The most intriguing observation of this study was the untypical OS morphology of the CNTFRα-positive photoreceptors which by their immunocytochemical properties, their pigment protein and their oil droplet were identified as cones. Cone OS are generally described to consist of lamellae which are continuous with the plasma membrane ³⁸ (see Fig. 4C ). The CNTFRα-expressing violet cones, however, contained discs that have become separated from the plasmalemma after invagination, the typical configuration of rod OS. To our knowledge, such a combination of cone and rod features has not been described so far. This type of photoreceptor may have escaped detection in previous electron microscopic studies due to its low abundance but became noticeable here because of its immunoreactivity for CNTFRα. It would be of interest to know, whether this type of photoreceptors can also be found in other species with violet- or UV-sensitive cones. 

The subcellular distribution of the CNTFRα has not been studied, so far. On the light microscopic level, CNTFRα immunoreactivity in neurons of the mammalian peripheral and central nervous systems did not appear to be confined to specific cellular compartments, ^{21 43} whereas the chick receptor protein was preferentially found in fiber tracts and neuropil of central and peripheral structures. ⁴⁴ Immunocytochemistry in the retina of the chick indicated a distinct subcellular localization in different cell classes. ²⁴ Here, the subcellular distribution of the CNTFRα protein could be shown to be highly specific in photoreceptors. According to the intensity of the immunocytochemical signals, very high levels of CNTFRα are present in OS plasmalemma but expression was not detectable in the disc membranes, as would be expected for the receptor of an intercellular signal molecule. At the cilium, the plasma membrane of the OS is continuous with that of the distal IS and forms villous protrusions, the calices, ⁴⁵ that surround the proximal part of the OS. CNTFRα immunoreactivity extended to the inner sheet of these calices. With this localization, the receptor is in close proximity to the interdigitating processes of the pigmented epithelium as a potential source of the ligand. Actually, a protein with CNTF-like activity has been first purified from chick eye tissue encasing the neural retina. ⁴⁶ Later, chick CNTF, originally termed growth promoting activity (GPA), was cloned ⁴⁷ and shown by immunocytochemistry to be expressed in eye tissue. ⁴⁸ In the rat, CNTF could be demonstrated to be produced by pigmented epithelium cells. ⁴⁹ A second CNTF source for retinal neurons is the Müller glia, ^{22 50} but processes of these cells do not reach the OS layer from which they are separated by the outer limiting membrane. Thus, it is more likely that the ligand for CNTFRα in OS is supplied by the pigmented epithelium, although this remains to be demonstrated in the chick retina. The restriction of CNTFRα expression to OS further raises the question whether CNTF signals are processed in the photoreceptor cell. Effects mediated by the CNTF receptor complex are known to be relayed to the nucleus, primarily through the JAK/STAT signal transduction pathway, where they influence the transcription of target genes. ⁵¹ An interesting question is whether and how activation of the CNTF receptor in the OS membrane is signaled to the photoreceptor nucleus. 

The finding that CNTFRα is specifically expressed in one class of cones in the adult chick adds an new aspect to existing data on the role of CNTF in photoreceptor development and function. In the rod-dominated rat retina, CNTFRα is expressed in the outer nuclear layer during the period of cell differentiation and was shown to mediate inhibitory effects on the differentiation of rods in vitro. ^{10 11 14 52} During the corresponding developmental period of the cone-dominated chick retina, the receptor is also present in progenitor cells of the photoreceptor layer and CNTF was found to promote the differentiation of rods and/or green cones in vitro. ^{12 13 24} With retinal maturation, CNTFRα was shown to be strongly downregulated in the outer nuclear layer of the rat retina. ¹⁴ Together with the present observation, this indicates that the receptor is not expressed by mature rods. This suggests that the protective effects of exogenous CNTF on injured or degenerating rods in adult rodent retinas described in many studies ^{15 16 17 18 19 20} is not due to direct action of the factor on these cells. This conclusion is in agreement with previous studies in rodent retinas showing that application of CNTF or experimental damage induces STAT3 and MAP kinase activation in ganglion and glial cells but not in rods suggesting that CNTF induces indirect rod-protecting influences in glial cells. ^{53 54} The prominent expression of CNTFRα in chick violet-sensitive cones suggests a direct action of CNTF on this cell type, although there are no experimental data, so far, showing CNTF effects on cone photoreceptors. However, it is puzzling why only one class of cones would be under neuroprotective influence. There are two possible explanations: Either the other cone populations are supported by other neurotrophic proteins or violet cones are particularly dependent on neuroprotective signals. One may argue that by absorbing short wavelength photons violet/UV-sensitive cones are most susceptible to light damage. Although light exposure has been shown to upregulate CNTF expression in the pigmented epithelium, ⁴⁹ this explanation remains purely speculative. 

In summary, this study leads to the following conclusions: CNTF can directly act on mature cones of the chick retina via its receptor located in the OS plasma membrane. The factor specifically acts on violet-sensitive cones representing a cone subpopulation. The violet-sensitive cones have unique morphologic characteristics that distinguish them from other retinal photoreceptors. 

 Supported by Deutsche Forschungsgemeinschaft Grant SFB 505/A4 (VS, H-DH) and by an unrestricted grant from Research to Prevent Blindness, Inc., to the Department of Ophthalmology and Visual Sciences, University of Utah (SF).

 Submitted for publication February 21, 2003; revised April 30 and September 11, 2003; accepted September 13, 2003.

 Disclosure: V. Seydewitz, None; A. Rothermel, None; S. Fuhrmann, None; A. Schneider, None; W.J. DeGrip, None; P.G. Layer, None; H.-D. Hofmann, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Immunolabeling for CNTFRα in flatmounts and sections of the chick retina. (A) Immunofluorescence staining with Cy3-conjugated secondary antibodies of an E19 retina. Photoreceptors have just started to form OS. Labeled OS appear as small tips forming a semiregular mosaic. (B) Retina stained at P8 showing CNTFRα expression in more mature, elongated OS. (C) Higher magnification of a P8 retina indicating that CNTFRα protein is largely confined to the outer cell membrane. (D) Vertical section from a P8 retina stained for CNTFRα with the peroxidase method showing that receptor expression is entirely confined to OS in the photoreceptor layer. Scale bars: (A, B, D) 20 μm; (C) 10 μm.

Figure 2.

 

CNTFRα immunoreactivity was not localized to rod OS. (A) Flatmount of a P8 chick retina immunofluorescence labeled with antibodies to rhodopsin and Cy2-conjugated secondary antibodies. (B) Same visual field as in (A) stained for CNTFRα with Cy3-conjugated secondary antibodies. Note that labeled OS were very similar in size and shape segments to those of rods (see A) but were present at lower density. (C) Double exposure for Cy2 and Cy3 fluorescence demonstrates that the two signals did not colocalize. Scale bar, 10 μm.

Figure 3.

 

Association of CNTFRα-immunoreactive OS with oil droplets. Retinal flatmount (P8) immunofluorescence labeled for CNTFRα was photographed sequentially with fluorescence and Nomarski optics, and the two images were digitally overlaid. (A) Lower magnification showing that each of the labeled OS was associated with an oil droplet at its proximal tip. (B) Higher magnification demonstrating the reproducible localization of the oil droplets with respect to the labeled OS and their uniform appearance. Scale bars: 10 μm.

Figure 4.

 

Identification of CNTFRα-immunoreactive photoreceptors as violet-sensitive cones. (A) Binding of FITC-conjugated peanut agglutinin in a P8 chick retina viewed by confocal microscopy. (B) Same visual field as in (A) double labeled for CNTFRα immunoreactivity; digital overlay of confocal images. Binding of PNA specific for red- and green-sensitive cones did not colocalize with CNTFRα expression. (C–E) Double labeling of a retinal flatmount (P17) with the antibody CERN933 raised against human blue-sensitive pigment (green Cy2 signal in C) and antibodies to CNTFRα (red Cy3 signal in D). Double exposure for both fluorescence markers in (E) shows the colocalization of the two antigens in the same set of OS. Note that the yellowish-red CNTFRα staining is prominent over the plasma membrane. (F–H) In situ hybridization for mRNA of the chick violet-sensitive chick pigment (F) and CNTFRα immunocytochemistry (G) in adjacent retinal sections of an E19 chick retina. The digital overlay of the images in (H) demonstrates that the mRNA signal in the IS was exactly associated with immunoreactive OS at the same position. Scale bars: (A, B, F–H) 10 μm; (C–E) 20 μm.

Figure 5.

 

Ultrastructural properties of CNTFRα-immunoreactive photoreceptors. (A) Electron micrograph showing an immunolabeled photoreceptor with strong staining of the OS plasma membrane (arrowheads) and the inner sheet of the caliceal membranes (arrows). Note the elongated shape of the immunoreactive OS compared with neighboring unlabeled cone OS. OD, oil droplets partially destroyed by immunocytochemical processing; ( ) black granula of the pigment epithelium. (B) At higher magnification the inner membranes of the OS could be seen to form discs that are closed on both ends and separated from the labeled plasma membrane (arrowheads). (C) Diagrammatic representation of membrane configurations classically assigned to OS of rods (left) and cones (right; with oil droplet). (D) Photoreceptor in a section processed for electron microscopy without prior immunostaining (at P5). The cell contained a well-preserved oil droplet and had a slender elongated OS with a rodlike (see C) membrane configuration visible at higher magnification in (E). Arrowheads: closed tips of membrane discs that are separated from the plasma membrane. Scale bars: (A, B, D) 20 μm; (C) 10 μm.

The authors thank Gabriele Kaiser and Jutta Hofmann for excellent technical assistance.