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1From the Molekulargenetisches Labor, Universitäts-Augenklinik Tübingen, Germany; 2Max-Planck-Institut für Molekulare Genetik, Berlin, Germany; 4Freie Universität, Berlin, Germany; and 5Labor für Neurohistologie und Zellbiologie, Universitäts-Augenklinik Tübingen, Germany.
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Abstract |
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METHODS. Nyx was isolated by conventional DNA library screening and polymerase chain reaction (PCR)–based approaches. Gene expression in different mouse tissues was studied by RT-PCR. Subsequently, the expression pattern of Nyx and its gene product in mouse and rat retinas was investigated by RNA in situ hybridization and immunohistochemistry with Nyx-specific antibodies.
RESULTS. The Nyx gene encodes a protein of 476 amino acids that contain 11 consecutive LRR motifs flanked by amino- and carboxyl-terminal cysteine-rich LRRs. At the amino acid level, Nyx is highly homologous to its human orthologue (86% identity). The gene is expressed in the eye but also, at lower levels, in brain, lung, spleen, and testis. Nyx expression was found during all stages of postnatal retinal development and was confined to cells of the inner nuclear layer and the ganglion cell layer in adult mouse and rat retinas.
CONCLUSIONS. These data suggest an important function of the Nyx protein in the inner retina and provide evidence that CSNB1 is based on a defect in the inner retinal circuitry.
-1-subunit of an L-type calcium channel.3 4 In the complete form (CSNB1), function of the rod system is completely absent, and cone-evoked signals are mostly normal in amplitude on standard ERG recordings. We and others have recently shown that CSNB1 is caused by mutations in a novel gene of unknown function, designated NYX.5 6 The human NYX gene is composed of three exons and spans approximately 28 kb of genomic DNA in Xp11.4. Its full-length transcript of 2713 bp encodes an open reading frame (ORF) for a deduced protein of 481 amino acids (designated nyctalopin) that shares sequence similarities with members of a protein superfamily characterized by tandem arrays of leucine-rich repeat (LRR) motifs.7 8 Additional sequence features suggest that nyctalopin is a glycosylphosphatidylinositol (GPI)-anchored extracellular protein with 11 typical and 2 cysteine-rich LRRs. The LRR motif plays a role in protein–protein interactions, and members of the LRR superfamily are involved in various processes in development, signal transduction, DNA repair and recombination, and RNA processing, but also cell adhesion and axon guidance.9 10 However, until now, the precise function of NYX and particularly its pathophysiological relationship with CSNB1 was unknown.
Herein, we report on the identification and characterization of the mouse orthologous gene Nyx and its expression pattern at the mRNA and protein levels during postnatal retinal development as well as in the adult mouse and rat tissue.
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Material and Methods |
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Synthesis of cDNA and RACE-PCR
cDNA was synthesized from total mouse eye RNA by using a commercial technology (SMART; Clontech, Palo Alto, CA). 3'-RACE experiments were performed with a forward primer derived from the mouse genomic sequence (5'-GCTACAAGGCCACGTTTCTCTTC-3'). The 2-kb RACE product was cloned into a vector (pCR2.1-TOPO; Invitrogen, Groningen, Germany) and sequenced with M13 and a gene-specific primer (5'-GCACTCTGCGTCTTAACTCTG-3'). 5'-RACE experiments were only successful with the implementation of the polymerase provided by the manufacturer (Advantage-GC 2 PCR Polymerase; Clontech) for PCR amplification and the application of a nested PCR strategy with reverse primers in the 3'-untranslated region (UTR) (5'-CATGAGTTACGTGCTGAGCCCGCC-3') and the coding sequence of exon 3 (5'-GGCCATTCCGGTCCAGATCGATG-3').
RNA Isolation and RT-PCR
Total RNA was isolated from various mouse tissues (RNeasy Midi and Mini Kit; Qiagen). Tissues were homogenized (PT3100 Polytron; Brinkman Instruments, Westbury, NY) in lysis buffer. The RNA preparation was treated with DNase I and tested for the absence of DNA contamination by control PCR amplification with two mND gene intron primers in combination with two neo primers.11 Reverse transcription of total RNA was performed by random hexanucleotide priming with reverse transcriptase (Omniscript; Qiagen). cDNA amplifications were performed with a forward primer in exon 2 (5'- GCCAAGGGATGCTGARCCTG-3') and a reverse primer in exon 3 (5'-CATTCCGGTCCAGATCGATG-3'), using Taq DNA polymerase in a kit (HotStar with Q-solution; Qiagen), applying 35 cycles of 45 seconds at 94°C, 45 seconds at 60°C, and 1 minute at 72°C. Primers for the amplification of Gapdh cDNA were 5'-GACCACAGTCCATGCCATCACT-3' (forward) and 5'-TCCACCACCCTGTTGCTGTAG-3' (reverse).
Animals and Tissue Preparation for Histological Studies
All experiments performed in this study were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. To determine cell specificity of Nyx mRNA expression in the rodent retina, in situ hybridization was performed on mouse and rat retina tissue. For the mouse experiments retinal tissue from adult animals at postnatal day (P)76 and from the developmental stages P3, P5, P10, P15, P20, and P30 were used. The rat retina was mature at P44, and the developmental stages were equal to the mouse stages. The day of birth was designated as P0. Nyx protein was localized immunohistochemically in adult rat retina.
C57BL/6 mice and Brown Norway rats were killed by a short CO2 incubation and decapitation. The eyes were removed and dissected along the ora serrata, and the posterior eyecups were fixed in 2% (in situ hybridization) or 4% (immunohistochemistry) paraformaldehyde in phosphate buffer (PB; 0.1 M, pH 7.4) for 13 to 30 minutes at 4°C. After washing in PB, tissues were cryoprotected by immersion in 30% (wt/vol) sucrose in PB overnight at 4°C. Eyecups were then embedded in cryomatrix (TissueTek; Leica, Nussloch, Germany) and frozen, and radial sections (10–12 µm) were cut on a cryostat. For in situ hybridization, sections were mounted in tissue adhesive (Vectabond; Vector Laboratories, Burlingame, CA) for immunohistochemistry on silane-coated glass slides and dried at 60°C for 2 to 3 hours. Slides were stored at -80°C until further use.
Samples processed for either in situ hybridization or immunohistochemistry were examined by microscope (AX70; Olympus, Tokyo, Japan) with Normarski optics. Images were adjusted for brightness and contrast on computer (Photoshop; Adobe Systems, San Jose, CA).
In Situ Hybridization
As the probe template, a 520-bp PCR fragment of the human NYX (nucleotide positions 1887-2406, GenBank accession number AJ278865; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) was subcloned into an SmaI-linearized vector (pBluescript II SK+; Stratagene) by blunt-end ligation, and a 551-bp fragment of the murine Nyx (corresponding to nucleotide positions 557-1107 in the human sequence) was subcloned into a PstI-linearized vector (pBluescript II SK+; Stratagene). The plasmid pRO4 containing the rat rhodopsin cDNA was kindly provided by Armin Huber, Institut für Zoologie, Universität Karlsruhe, Germany.
One microgram of plasmid DNA was linearized by restriction enzyme digestion and purified by phenol-chloroform extraction. Digoxigenin-UTP–labeled sense and antisense riboprobes were generated by in vitro transcription of linearized plasmids with a kit (DIG RNA Labeling Kit; Roche Molecular Biochemicals, Mannheim, Germany).
Before the in situ hybridization, sections were treated with proteinase K buffer (0.1 M Tris-HCl [pH 8] and 0.05 M EDTA) for 5 minutes at 37°C and digested with 0.3 µg/mL proteinase K (Sigma, Diesenhofen, Germany) for 8 minutes at 37°C. Slides were then washed two times for 3 minutes each in diethyl pyrocarbonate (DEPC)-treated water, postfixed for 15 minutes in paraformaldehyde (PFA; 4% PFA in 0.2 M PB), washed again three times with DEPC-treated water, and air dried.
Sixty microliters of hybridization solution (50% deionized formamide [Sigma], 5x SSC, 5x Denhardt’s solution, 0.5 mg/mL tRNA [Fluka, Buchs, Switzerland]) and 0.2 ng/µL of the digoxigenin-labeled riboprobe were denatured for 5 minutes at 80°C and applied to the sections. The slides were then incubated in a humidified chamber for 16 hours at 64°C. Posthybridization washing steps were performed two times for 30 minutes each in 0.1x SSC at 64°C. After a 10-minute wash in Tris-buffered saline (TBS; 0.15 M NaCl and 0.1 M Tris-HCl [pH 7.5]) at room temperature (RT), the slides were incubated for 30 minutes with blocking solution (10% blocking reagent in 0.1 M maleic acid, 0.15 M NaCl [pH 7.5]; Roche Molecular Biochemicals). Drained slides were incubated with alkaline-phosphatase–conjugated anti-digoxigenin antibody (1:500, in 10% blocking solution, 0.15% Triton X-100 in TBS; Roche Molecular Biochemicals) for 45 minutes at 37°C. Sections were then briefly rinsed two times for 15 minutes each in TBS and preincubated for 10 minutes in substrate buffer (0.1 M Tris-HCl [pH 9.5], 1 mM MgCl2, 10% tetramisole-hydrochloride [Fluka]). Four microliters nitroblue tetrazolium salt (NBT; 30 mg/mL; Bio-Rad Laboratories, Munich, Germany) and 4 µL 5-bromo-4-chloro-3-indolyl phosphate (BCIP; 15 mg/mL; Bio-Rad Laboratories) were mixed with 1 mL of substrate buffer, and each section was incubated in 200 µL of this solution, in a humidified chamber in the dark for 24 to 72 hours at RT. Color reaction was stopped with stop buffer (0.1 M Tris-HCl [pH 7.5] and 0.01 M EDTA), and covered with sorbitol (Merck, Darmstadt, Germany).
Immunohistochemistry
To raise antibodies against NYX, two peptides comprising the amino acid sequences LTTSSPGPSPEPAATTV and ASLSDSLSSRGVG were prepared by solid-phase peptide synthesis, using the Fmoc/But-strategy.12 The peptides were purified to a homogeneity of more than 95% by HPLC, and their identity was confirmed by electrospray mass chromatography. The peptides were coupled to keyhole limpet hemocyanin (KLH) by the glutaraldehyde method13 and used as antigens to raise polyclonal antibodies in New Zealand White rabbits, according to standard immunization protocols (Charles River Service Laboratory, Sulzfeld, Germany). The resultant antiserum was purified by affinity chromatography (Protein A Sepharose; Amersham Biosciences, Freiburg, Germany) and subsequent by immunoaffinity chromatography (applying the peptides used for immunization) and finally concentrated by ultrafiltration on a 20-kDa cut-off membrane.
In sections used for immunohistochemistry endogenous peroxidase was blocked with 3% H2O2 in 40% methanol. To reduce background staining, slides were preincubated for 1 hour in 10% normal goat serum (NGS, Sigma) and 0.3% Triton X-100-PBS (PBST). The primary antibody was diluted 1:1000 in PBST containing 10% NGS and incubated for 3 hours at RT or overnight at 4°C. After a wash with phosphate-buffered saline (PBS), the samples were incubated for 1 hour with a biotin-conjugated secondary antibody (dilution 1:200, Vectastain Elite Kit; Vector Laboratories) in PBST with 5% NGS. After a rinse in PB, retinal sections were processed with an avidin-biotin peroxidase complex, and immunoreaction was visualized with a diaminobenzidine-nickel solution as the chromogen (1 mg/mL diaminobenzidine, 0.2% glucose 0.004% NH4Cl, 0.09% (NH4)2Ni(SO4)2, and 1 µL/mL glucose oxidase in PB). After terminating the reaction in PB the slides were coverslipped with glycerol/PBS (9:1). To determine the specificity of the antigen-antibody reaction, negative control experiments were performed, either by omitting the primary antibody or preabsorbing it with the appropriate peptide.
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Results |
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Gene expression in different mouse tissues was explored by RT-PCR with forward and reverse primers in exons 2 and 3, respectively (Fig. 2) . Transcripts were detected in the eye, brain (cerebrum and cerebellum), lung, spleen, and testis, but not in the kidney, heart, and liver.
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Hybridization with a rat rhodopsin cDNA antisense riboprobe was performed to verify the quality and reliability of our in situ hybridization protocol and the specificity of the staining in the different retinal layers (compare also Bech-Hansen et al.5 ). The reaction time of the rhodopsin probe was the same as for the NYX probe in Figures 3 and 4 . Rhodopsin signals were observed solely in the photoreceptor layer (Fig. 3B) —that is, in the cell bodies and the myoid regions of the photoreceptor cells.
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At early postnatal stages (<P5), the retina is not fully developed, and only the GCL and the adjacent inner plexiform layer (IPL) have already separated from the neuroblast layer (NBL). Strong Nyx expression was observed in the GCL early, at P3. At that stage, the GCL is still multilayered. Nyx-positive cell bodies in the GCL, at that time, were found primarily in the innermost row of the GCL, adjacent to the axon fiber layer. In the undifferentiated NBL of P3 animals, distinctly labeled cells were also present near the NBL–IPL border. In addition, more diffuse staining was scattered over those parts of the NBL that later form the INL (Fig. 4) . The same pattern was present at P5. At P10, when the INL had separated from the photoreceptors, staining was clearly confined to the GCL and INL. With eye opening, which occurred around P15 in both species, the expression pattern of Nyx was essentially the same as in the retina of adult animals, with the exception of a few labeled cells in the more proximal part of the INL. Later developmental stages reflected the murine Nyx expression pattern of the mature rat retina.
To examine cell-specific protein localization we performed immunohistochemistry on retinal sections of adult rats, with polyclonal antibodies against the NYX carboxyl terminus. Immunoreaction to the antibodies was found in the GCL, IPL, INL, and OPL. The photoreceptor region—the ONL and the inner and outer segments—were devoid of immunoreactivity, except for weak staining at the level of the ONL–myoid border, similar to the situation found with the mRNA probe (Fig. 3E) .
The immunoreaction in the GCL and in the inner row of the INL resembled the pattern of mRNA expression. Cells of different size and more than 50% of the somata were labeled in the GCL (Fig. 3E , large arrows), and individual cells were distinguished along the INL-IPL border (Fig. 3E , arrowheads). Besides an overall punctuate labeling of the entire IPL two bands of enhanced immunoreaction were present in sublayers 1 and 3, within which horizontally running processes were observable (Fig. 3E , asterisks). Neurons along the outer margin of the INL were also labeled (Fig. 3E , small double arrows) and, in addition, the OPL showed immunoreactivity with more pronounced staining of fibers running through the proximal part of the OPL above the INL somata (Fig. 3E , small arrow).
No labeling was observed when the primary antibody was either omitted (Fig. 3F) or preabsorbed with the peptide used to raise the antibody.
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Discussion |
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In the adult mouse, Nyx is expressed in several tissues (brain, lung, spleen, and testis) with highest expression levels observed in the eye (Fig. 2) . Similarly, expression of NYX in neural and several non-neural tissues was found in humans.5 6 Expression in kidney, consistently shown in humans, is absent or largely reduced in the mouse (Fig. 2) .
On RNA in situ hybridization, we observed Nyx expression in the cells of the GCL and the inner part of the INL in the mouse and rat retinas. This pattern is consistent with expression of Nyx in amacrine and ganglion cells in the rodent retina. Localization of the Nyx protein by immunohistochemical analysis showed predominant staining in the inner retina, from the GCL up to the terminals of the photoreceptors. However, the photoreceptor layer itself was free of anti-Nyx immunoreactivity, except for a very faintly stained band along the border of the ONL toward the inner segments. In addition to the localization of Nyx mRNA in the cells of the GCL and the inner INL, numerous cells in the outer INL along the INL–OPL border were immunoreactive. These Nyx protein-positive cells outnumbered the sporadically detectable mRNA-expressing cells in the outer region of the INL considerably, thus supporting the idea that Nyx may also be vertically transported to cells that are not able to produce it themselves.
Immunostaining in the outer part of the INL raises the question of the identity of these cells. There is evidence from ERG recordings that the function of depolarizing bipolar cells is impaired in CSNB1.15 16 17 18 However, location, size, and shape of these immunoreactive neurons make it likely that they are horizontal rather than bipolar cells, an assumption further supported by single horizontally oriented fibers running in the OPL close to these cells. Even though the identity of these cells still has to be determined, it is obvious from the in situ hybridization data that most of them do not express Nyx and therefore depend on Nyx produced in the proximal retina. Our results clearly exclude a pronounced expression of Nyx in photoreceptors in the rodent retina. Even though there was a very faint signal in both in situ hybridization and immunohistochemistry approximately at the level of the external limiting membrane, we never observed (in any developmental stage examined) staining in the cell bodies of the ONL or the myoid regions of the photoreceptors comparable to the lucid NYX expression in the INL and GCL. In the human retina, NYX expression of similar intensity has been reported in all nuclear layers, including the ONL and the inner segments.5 This discrepancy may reflect species differences in NYX expression. A similar difference was found in the kidney, where NYX expression was consistently shown in humans,5 6 but was absent, or at least largely reduced, in the mouse (Fig. 2) .
It has been argued that, analogous to the Drosophila LRR proteins chaoptin and capricious, NYX may be involved in the formation of synaptic connections between neurons during development and maturation of the retina. Our in situ results at various developmental stages showed early expression of Nyx (at least as early as P3) and no gross differences in the temporal and spatial expression pattern during postnatal retinal development. The early expression in the developing retina may indicate that NYX at this stage plays a role in synaptogenesis and neuronal circuit formation. Continuous expression in the adult retina suggests a functional relevance throughout life—for example, in the maintenance of the extracellular matrix or cell interaction processes.
CSNB1 was initially thought to be caused by a defect in signal transmission from rods to rod bipolar cells,1 but more recent studies have shown that there is a general impairment of the retinal ON-pathway that involves both rod and cone signaling15 16 17 18 19 and is apparently due to a functional defect postsynaptic to the photoreceptors.18
In the mammalian retinal circuitry, the main (sensitive) signaling pathway of rods involves a single type of depolarizing bipolar cell (rod ON-bipolar), which in turn contact AII amacrine cells through a sign-preserving glutamate synapse. Signals from the AII amacrine cells then infiltrate the cone signaling pathways by exciting the cone ON bipolar cells through gap junction electrical contacts and inhibiting OFF cone bipolar cells through glycinergic synapses.20 The available electrophysiological data suggest that function of rod bipolar cells and also the cone ON pathway through the depolarizing bipolar cells are compromised in CSNB1. Our histologic analyses in the rodent retina localize Nyx expression to cells of the inner half of the INL and the GCL, which probably represents amacrine cells and ganglion cells, respectively. This expression pattern contrasts with the principal electrophysiological findings in CSNB1, which propose a main defect in the depolarizing bipolar cells.15 16 17 18
However, there is also evidence for impairment of more distal neuroretinal function in patients with CSNB1. Miyake et al.21 showed that the scotopic threshold response (STR) is not recordable in these patients. Intravitreous aspartate injections indicate that the STR origin is located postsynaptically to the photoreceptors,22 and microelectrode recordings in the cat retina show that the STR is maximal around the IPL and the GCL.23 24 Furthermore, the oscillatory potentials are extremely small or absent in patients with CSNB1.25 Although the exact site of their origin is still unknown, they are probably generated in or near the IPL,26 27 28 possibly by depolarizing amacrine cells.29 In light of our expression data, we think that the analysis of such distal retinal function in CSNB1 patients deserves further attention.
Because there is no evidence that amacrine or ganglion cells contribute to the rod b-wave or the cone ON response, its loss in patients with CSNB1 cannot be readily explained if the function of NYX is restricted to the inner retina. However, it might be assumed that the absence of a functional defect of nyctalopin in amacrine and ganglion cells will impair the formation of regular synaptic contacts with their input bipolar cells and thus indirectly have also an adverse effect on the functional differentiation of the bipolar cells themselves. Further studies on the neuroretinal circuitry in patients with CSNB1 are necessary to solve this question.
Of note, a naturally occurring mouse model nob (no b-wave) has been described that resembles CSNB1 in humans: stationary course, preserved a-wave, and absent b-wave and oscillatory potentials in ERG recordings.30 Moreover the nob trait displays X-linked recessive inheritance, and recent linkage analysis excludes the gene involved in the incomplete form of CSNB (CSNB2) but maps the nob locus to a region syntenic to the human CSNB1 locus.31
Our in silico mapping results localize Nyx to the centromeric region of the murine X chromosome within the nob-critical interval. This localization further supports the idea that a mutation in Nyx gives rise to the nob phenotype. Thus, further studies of the nob mouse model may provide more insight into the complex pathophysiology of CSNB1 in humans and may help to elucidate the relationship between the principal electrophysiological findings and the restricted expression pattern of NYX in the retina.
Note Added in Proof
After final submission of this paper, Gregg and coworkers published an article in which they showed that nob mice do indeed have a mutation in the Nyx gene (Gregg RG, Mukhopadhyay S, Candille SI, et al. Identification of the gene and the mutation responsible for the mouse nob phenotype. Invest Ophthalmol Vis Sci. 2003;44:378–384).
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Acknowledgements |
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Supported by Grant Fö. 01KS9602 from the Federal Ministry of Education, Science, Research and Technology; the Interdisciplinary Center of Clinical Research, Tübingen; and Grant T-GE-0900-0030 from The Foundation Fighting Blindness.
Submitted for publication February 4, 2002; revised August 6 and December 30, 2002; accepted January 30, 2003.
Commercial relationships policy: N.
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.
Corresponding author: Bernd Wissinger, Molekulargenetisches Labor, Universitäts-Augenklinik, Auf der Morgenstelle 15, D-72076 Tübingen, Germany; wissinger{at}uni-tuebingen.de.
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) and (
marks the ONL-PhR transition.