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RP1 Is Required for the Correct Stacking of Outer Segment Discs

From the F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania.

	Abstract

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PURPOSE. Mutations in RP1 are a common cause of dominant retinitis pigmentosa (RP), but the mechanism by which the identified mutations lead to photoreceptor cell death and blindness has not been determined. To investigate the function of the RP1 protein in photoreceptors and gain insight into the mechanism of disease, gene-targeting techniques were used to produce mice with a mutant Rp1 allele that mimics the truncation alleles found to cause disease.

METHODS. RT-PCR was used to amplify illegitimate RP1 transcripts from lymphoblasts. Gene targeting was used to create mice with a mutant Rp1-myc allele. Confocal immunofluorescence microscopy was used to identify the location of the mutant Rp1-myc protein in photoreceptors. The structure of the photoreceptors in the resultant Rp1-myc mice was studied by light and electron microscopy. The retinal function of the mutant mice was investigated using analysis of full-field ERGs.

RESULTS. Wild-type and mutant RP1 mRNA were both detected in lymphoblasts from patients with RP1 disease. Rp1-myc mice produced a truncated version of the Rp1 protein, containing the N-terminal 662 amino acids, which localized correctly to the axoneme of the photoreceptor outer segments. Mice homozygous for the mutant Rp1-myc allele underwent a rapid-onset retinal degeneration characterized by incorrectly oriented outer segment discs that failed to stack properly into outer segments. In contrast, the photoreceptors of heterozygous mice remained relatively healthy.

CONCLUSIONS. The presence of mutant RP1 mRNA in lymphoblasts from patients with RP1 disease implies that the mutant message can escape nonsense-mediated mRNA decay and that a truncated RP1 protein may be produced in the retina. The truncated Rp1-myc protein appears to be nonfunctional, and not to exert a dominant negative effect in the photoreceptors of heterozygous mice. Results from homozygous Rp1-myc mice indicate that RP1 is required for the correct orientation and higher order stacking of outer segment discs.

The junction between the connecting cilium and the outer segment is also the location of disc morphogenesis. Thus, proteins found in this region may also participate in this process. New outer segment discs are formed by a process of membrane evagination and fusion at the base of the outer segment, adjacent to the axoneme of the outer segment.^{7 13 14 15} Actin is concentrated at the base of the outer segment and has been implicated in disc formation, based on microscopy studies and use of inhibitors of actin polymerization.^{16 17} RPGRIP is also a component of connecting cilia, and RPGRIP knockout mice demonstrate defects in outer segment disc size and orientation, leading to the hypothesis that RPGRIP is required for disc morphogenesis.¹⁸ The roles of other proteins shown to be located in or around the cilium, such as -actinin, cadherin, centrin, and myosin II are not as clearly defined.^{19 20 21 22}

Data from mice with targeted removal of exons 2 and 3 of the Rp1 gene suggest roles for Rp1 in both protein transport and disc formation.²³ These mice produce an Rp1 mRNA resulting from abnormal splicing of exons 1 and 4 of the Rp1 gene. This abnormal mRNA is translated, and an abnormal Rp1 protein that is lacking the N-terminal 269 amino acids is produced in the retinas of heterozygous and homozygous Rp1-exon 2/3 deletion mice.²³ It is thus not clear whether the defect in outer segment formation observed in these mice is due to the lack of the Rp1 protein or to a dominant effect of the abnormal protein. In addition, the mislocalization of opsin is minimal compared with that observed with other mutations that alter protein trafficking.^{10 11}

All the 20 pathologic mutations in RP1 found to date are nonsense or frameshift mutations that are located between codons 500 and 1053; 18 are found between codons 658 and 872.^{3 24} These mutations all cluster at the beginning of exon 4 (codons 263-2156) and are thought to generate a truncated RP1 protein that lacks the C-terminal one half to two thirds of the protein. Because these premature termination mutations are all located after the final intron–exon boundary of the RP1 gene, it is predicted that the mutant mRNAs could escape nonsense mediated decay.²⁵ To study the function of the RP1 protein and gain insight into disease pathogenesis, we investigated the expression of mutant RP1 mRNA in lymphoblasts from patients with RP1 disease. We then used gene-targeting techniques to produce mice with a mutant Rp1 allele that mimics the truncation alleles found to cause disease. Our data show that RP1 has a role in controlling the orientation of outer segment discs and in the organization of discs into outer segments.

	Materials and Methods

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Preparation of DNA and Mutation Screening
This research adhered to the tenets of the Declaration of Helsinki and was approved by the institutional review board of the University of Pennsylvania. Samples from the Foundation Fighting Blindness (FFB) Histopathology Collection (Scheie Eye Institute, Philadelphia, PA) were selected for genetic screening based on history and histology. Only those specimens with a history of autosomal dominant RP and retinal tissue containing some residual photoreceptors were chosen to be screened. Genomic DNA was isolated from frozen eye tissue or blood samples (QIAamp DNA Mini Kit; Qiagen, Valencia, CA), and the region of RP1 (codon 603-891) that contains 18 of 20 known mutations was amplified by PCR. The primers used were forward 5'-AGGTTCAGTCCTATTTCAGCAGATG-3' and reverse 5'-CCCTGGTTGTAGCATGTTGTTTA-3'. The PCR products (865 bp) were electrophoresed, eluted from the gel and purified by extraction with phenol-chloroform followed by precipitation with ethanol. Automated sequencing using both the forward and reverse primers was performed using 100 ng of purified DNA as a template.

Detection of RP1 mRNA in Lymphoblasts
Lymphoblasts from members of the RP01 pedigree with the Arg677Ter mutation in the RP1 gene were obtained from the Coriell Cell Repository (http:/locus.umdnj.edu). The cells were grown in RPMI 1640 with 2 mM L-glutamine plus 15% fetal bovine serum at 37°C with 5% CO₂. Total RNA (50–100 µg) was extracted from approximately 10 million cells (TRIzol; Invitrogen, Carlsbad, CA), and mRNA was prepared from this total RNA (Oligotex mRNA Minikit; Qiagen). Poly(A)⁺ RNA (1–4 µg) was treated with DNase I (Ambion, Austin, TX), and reverse transcribed (Superscript II; Invitrogen). The first PCR reaction was then performed using 1 µL of first-strand cDNA to amplify the region of RP1 transcript containing codon 677. Two microliters of the first PCR reaction were used as the template for the second nested PCR reaction. Thirty cycles of amplification were used for both PCR reactions. The primers used for the first PCR reaction were: forward, 5'-CAATAACAATGATCAAATGGAGGAGTC-3'; and reverse, (5'-CCTTGAACCTTGGAATTTTGAGTA-3'. The internal primers for the second PCR reaction were: forward, 5'-AGGTTCAGTCCTATTTCAGCAGATG-3'; and reverse, 5'-ATTCTACCTTTTGTGTTTATTCTCTCA-3'. The final PCR products were electrophoresed on agarose gels, extracted from the gel, and purified by extraction with phenol-chloroform, followed by precipitation with ethanol. The purified PCR products were then digested with Taq1 to detect the Arg667Ter mutation, or sequenced. To control for genomic DNA contamination, parallel reactions without reverse transcriptase were included for each sample. These no–reverse-transcriptase control experiments were carried through the nested PCR steps with the experimental samples.

Generation of Rp1-myc Mice
This research followed the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the guidelines of the University of Pennsylvania for Animal Care and Use. The Rp1-myc targeting vector was constructed using segments of genomic DNA amplified by high-fidelity PCR from a mouse bacterial artificial chromosome (BAC) clone that contains the entire mouse Rp1 gene. This BAC clone, 314A, was identified by screening high-density filters from the RPCI-22 (129S6/SvEvTac) Mouse BAC Library (BACPAC web site: http://www.chori.org/bacpac/libraryres.htm/ provided in the public domain by the National Center of Excellence in Genomics at Children’s Hospital Oakland Research Center, Oakland, CA). The 3' arm of the targeting vector, including bases 16,110 to 16,879 of the Rp1 gene (based on GenBank AF291754; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), plus an additional 1,174 bases 3' to the gene, was amplified with PCR primers containing XhoI and KpnI restriction sites. The purified, digested PCR product was then cloned into loxpneoBS, which contains a floxed pgk-neo cassette,²⁶ creating plasmid pSP2. The 5' arm of the targeting vector including bases 6,822 to 12,003 of the Rp1 gene was amplified and cloned into the NotI and SalI sites of the pCMV-tag5 vector (Stratagene) adjacent to the myc tag to create plasmid pSP6. The 5'-myc arm, including the myc tag, stop codon and polyadenylation signal from pCMV-tag5, was then excised from pSP6, using the enzymes NotI and MluI, and cloned into the NotI-MluI sites of pSP2, upstream of the floxed pgk-neo cassette, creating the final Rp1-myc targeting vector (see Fig. 2 ).

View larger version (48K): [in this window] [in a new window]   FIGURE 2. Targeted disruption of the Rp1 gene in mice. (A) Maps of the wild-type Rp1 locus, the targeting vector, and the targeted Rp1 locus are shown. The exons of the Rp1 gene are shown as open boxes. The DCX domain encoded by exons 2 and 3 is indicated, as is the region that contains all pathologic mutations at the beginning of exon 4. The map of the targeting vector shows the portion of exon 4 after Arg662 of the mouse Rp1 coding sequence that is replaced with a neomycin resistance gene cassette (pgk-Neo-pA). A 10-amino-acid myc tag coding sequence (shaded area) was placed at the C-terminal end of the truncated Rp1 gene. The 5' and 3' probes used for Southern blot analyses are shown. The locations of EcoRI (E) and BamHI (B) are indicated, as are the expected sizes of the restriction fragments for the wild-type and targeted loci. (B) Southern blot analysis of EcoRI and BamHI digested genomic DNA isolated from control (left) and targeted (right) ES cells. The 7.3-kb band detected with the 5' probe, and the 5.7-kb band detected with the 3' probe confirm correct recombination at the Rp1 locus.

The single ES cell clone with correct recombination was microinjected into C57BL/6 blastocysts to generate chimeric mice by the Chimeric and Transgenic Mouse Core Facility at the University of Pennsylvania School of Medicine (Philadelphia, PA). Highly chimeric founders were crossed with C57BL/6 mice to generate lines of heterozygous (Rp1^+/myc) Rp1-myc mutant mice. Mice homozygous (Rp1^myc/myc) for the targeted allele were generated by sibling crosses. Genotyping was performed by PCR analysis of tail DNA using two pairs of primers: 5'-GTGCCACAACAATGCTTTACCGTCAAC-3' and 5'-GGACTGAGGGGCCTGAAATGA-3' to amplify the mutant allele, and 5'-CTCAGCAGCGAATGATAACTTC-3' and 5'-CACTTGGAACGGTGGTCTGT-3' to amplify the wild-type allele. The sizes of the PCR products amplified from wild-type and mutant Rp1 alleles are 488 and 612 bp, respectively. Sequence analysis of the mutant PCR product verified correct gene targeting.

Western Blot Analysis
Retinas from animals of each genotype (Rp1^+/myc, Rp1^myc/myc, and Rp1^+/+) at the desired ages were collected and homogenized in sample buffer (NuPAGE; Invitrogen). Solubilized retinal protein samples were then processed for Western blot analysis as described.⁴ Briefly, equivalent amounts of protein from each genotype were separated on 4% to 12% Tris-acetate gel (NuPage; Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA).^{30 31} The membranes were blocked with 10% nonfat dry milk in TBS-T (50 mM Tris-C1 [pH 8.0], 150 mM NaCl, 0.1% Tween-20) and incubated with primary antibodies. Chicken polyclonal anti-C'-Rp1 antibody (1:2000) raised against codons 1708 to 1928 from the C-terminal region of mouse Rp1 was used to detect the normal Rp1 protein.⁴ Monoclonal anti-myc antibody (1:1000, Cell Signaling Technology; Beverly, MA) was used to detect the mutant Rp1-myc protein. Antibody binding was detected with alkaline phosphatase conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) and enhanced chemifluorescence (ECF) substrate (Amersham Biosciences, Piscataway, NJ). Positive signals were visualized and quantified by fluorometry using a Storm 860 PhosphorImager (Amersham Biosciences-Molecular Dynamics, Sunnyvale, CA).

Immunofluorescence Microscopy
Eyes from postnatal day (P)14, P30, and 10-month-old mice were enucleated after cardiac perfusion with 4% PFA in 0.1 M phosphate buffer and fixed in 4% PFA for 3 hours. Fixed eyecups were infiltrated with 30% sucrose, frozen in OCT freezing medium, and cryosectioned at 10 µm. For immunostaining, retinal sections were blocked in PBS containing 1% normal goat serum, 1% bovine serum albumin, and 0.5% Triton X-100 for 1 hour and then incubated overnight with primary antibodies at 4°C. After they were rinsed with PBS, the sections were treated with fluorochrome-conjugated secondary antibodies for 1 hour, washed in PBS, and mounted (Fluoromount-G; Southern Biotechnology Associates, Birmingham, AL). The primary antibodies used were anti-C'-Rp1,⁴ anti-myc monoclonal antibody, mouse monoclonal anti-rhodopsin 4D2, anti-rds/peripherin (Per 5H2), anti-Rom1 (Rom 1D5), and anti-rod cyclic nucleotide gated channel -subunit (PMc 1D1), anti-ABCR (rim 3F4) (Per 5H2, Rom 1D5, PMc 1D1 and rim 3F4 were obtained from Robert Molday, University of British Columbia, Vancouver, Canada) anti-arrestin,³² anti-blue cone (JH455), and red/green cone (JH492).³³ Cy2- and Cy3-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. Alexa 468– and Alexa 584–conjugated secondary antibodies were from Molecular Probes (Eugene, OR). Control sections were treated with preimmune anti-C'-Rp1 or without primary antibodies. Stained sections were viewed with a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Thornwood, NY). Frozen retinal sections from the same animals were also dried and stained with Richardson stain to evaluate the thickness of the outer nuclear layer. Bright-field digital images were captured using a microscope (TE300; Nikon USA, Melville, NY) equipped with a digital camera (Spot RT; Diagnostic Instruments, Sterling Heights, MI).

Light and Electron Microscopy
Anesthetized mice were perfused through the heart with freshly prepared 2% PFA+2% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.4). Eyes were removed and eyecups were fixed in the same buffer for 4 hours at room temperature. After they were rinsed with 0.2 M cold sodium cacodylate buffer (pH 7.4), eyecups were trimmed into 1 x 2-mm pieces and post fixed in 1% osmium tetroxide. Specimens were then stained with 1% uranyl acetate, dehydrated and embedded in Epon resin (EMbed812; Electron Microscopy Sciences) according to the manufacturer’s instructions. Sections of 1-µm thickness were cut and stained with alkaline toluidine blue for light microscopy. Ultrathin sections of 60- to 80-nm thickness were cut, stained with lead citrate and uranyl acetate, and examined using a transmission electron microscope (CX100; JEOL, Tokyo, Japan). Electron micrographs were captured using film or a digital-image capture system.

ERG Measurements
The methods for electrical recording of light stimulation have been described in detail previously.³⁴ In brief, full-field ERGs were recorded from both eyes of anesthetized mice with differential amplifiers having a bandwidth of 0.1 Hz to 1 kHz. The filtered traces were digitized at 5 kHz and stored on a computer for further analysis. The corneal electrodes were platinum wires embedded in the contact lenses, placed on the eye on a layer of hydroxypropyl methylcellulose (Goniosol; Ciba Vision, Duluth, GA). The reference electrode was a tungsten needle inserted subcutaneously in the forehead. The recording chamber served dually as a Faraday cage and a Ganzfeld, with appropriate ports and baffles to ensure uniform illumination. Intensities were calibrated as previously described.³⁴ Mice were dark adapted for a minimum of 12 hours before the ERG experiments. Preparations of the animals for recordings were made under dim red light. The mice were anesthetized with an intraperitoneal injection containing (in micrograms per gram body weight) 25 ketamine, 10 xylazine, and 1000 urethane and their pupils dilated with 1% tropicamide solution (Alconox, New York, NY). Before recording commenced, animals were maintained in complete darkness for 15 minutes.

	Results

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Expression of Mutant RP1 mRNA in Human Lymphoblasts
The 20 pathologic mutations found in RP1 to date all cluster toward the beginning of exon 4 and are all truncation mutations.^{3 24} Because all the pathologic mutations in RP1 occur within the final exon, it is possible that the mutant RP1 mRNA escapes nonsense-mediated mRNA decay to produce a truncated RP1 protein in the retina.²⁵ To ask if the mutant RP1 mRNA escapes nonsense-mediated mRNA decay, we attempted to find retinal tissue samples of patients with RP1 disease in the FFB Histopathology Collection, which was then housed at the Scheie Eye Institute. One sample in this collection was from a patient known to have the Arg677Ter mutation in RP1. However, histopathologic evaluation of this sample showed end-stage retinal degeneration, with no photoreceptors remaining in the retina. Thus, this sample could not be used for detection of mutant RP1 mRNA or protein in the retina. Tissue samples from 40 patients with less than end-stage retinal degeneration were identified by review of the pathologic descriptions for all retinal degeneration samples in the FFB collection. Genomic DNA was isolated from ocular or blood samples for each of these 40 patients, and the region of RP1 that contains the known cluster of mutations was amplified and sequenced. Unfortunately, no mutations in the RP1 gene were detected in these patients with early-stage RP (data not shown).

As a proxy for retinal tissue, we screened lymphoblasts isolated from patients with the Arg677Ter mutation, the most common RP1 mutation, and related control subjects, for illegitimate RP1 transcripts. Lymphoblasts from patients of the RP01 pedigree were obtained from the Coriell Cell Repository (http://locus.umdnj.edu/ Coriell Institute for Medical Research, Camden, NJ).³⁵ mRNA was extracted from cultured lymphoblasts and nested RT-PCR was performed to amplify the region of the RP1 transcript containing codon 677. This codon, and the primers used to amplify it, are within exon 4 of the RP1 gene. As shown in Figure 1 , RT-PCR successfully amplified RP1 mRNA of patients homozygous and heterozygous for the Arg677Ter mutation, as well as unaffected control subject. Control PCR reactions using RNA that was not reverse transcribed (no RT) showed that the mRNA samples were not contaminated with genomic DNA (Fig. 1A) . Restriction digests and DNA sequencing confirmed that the mutant mRNA was amplified in both heterozygous and homozygous patients (Figs. 1B 1C) .

View larger version (57K): [in this window] [in a new window]   FIGURE 1. RP1 mRNA is produced as an illegitimate transcript in lymphoblasts. (A) RT-PCR analysis of RNA from lymphoblasts of unaffected (+/+), heterozygous (+/-), and homozygous (-/-) members of the RP01 pedigree. Nested RT-PCR was performed with mRNA that had been isolated from cultured lymphoblasts. The 314-bp product from the second PCR reaction was detected in patients heterozygous and homozygous for the Arg677Ter mutation, as well as in unaffected control members of the RP01 pedigree. There were no products in no-reverse-transcriptase (NRT) control experiments, indicating no contamination with genomic DNA. (B) Restriction enzyme digestion of the PCR products from (A). The final PCR products from three genotypes were gel purified and digested with Taq1. The undigested 314-bp fragment from the mutant RP1 allele was present in both heterozygous and homozygous patients. Bands of 219 and 95 bp from the digested wild-type PCR product were detected in heterozygous and unaffected control subjects. (C) Sequence analysis of the nested RT-PCR products. The CT (Arg667Ter) mutation was detected in the patients with Arg667Ter RP1 allele.

Localization of the Truncated Rp1-myc Protein
To determine whether the mutant Rp1-myc allele produces a truncated Rp1-myc protein in mouse retina, equal amounts of total retinal protein from 4-week-old Rp1^+/+, Rp1^+/myc, and Rp1^myc/myc mice were subjected to Western blot analysis. Probing with anti-myc and anti-C'-Rp1 antibodies revealed that a truncated Rp1-myc protein with a predicted molecular mass of 70 kDa is present in mice with a mutant Rp1 allele, but not in the wild-type mice (Fig. 3) . In contrast, the normal Rp1 protein was detected in wild-type and heterozygous mice, but no signal could be detected in the homozygous mice (Fig. 3) . The level of the normal Rp1 protein in Rp1^+/myc heterozygous mice was approximately half of that detected in the wild-type. Similarly, the level of truncated Rp1-myc protein in Rp1^+/myc heterozygous mice was approximately half that in Rp1^myc/myc homozygous mice.

View larger version (46K): [in this window] [in a new window]   FIGURE 3. Western-blot analysis of wild-type Rp1 and mutant Rp1-myc protein expression. Total retinal proteins were extracted from 4-week-old Rp1+/+, Rp1+/myc, and Rp1myc/myc mice. Equal amounts of total protein from each genotype were electrophoresed and transferred to polyvinylidene difluoride (PVDF) membrane and then probed with either antibodies to the C terminus of Rp1 (top: anti-C'-Rp1 antibody) or anti-myc (bottom). The normal mouse Rp1 protein (240 kDa) was detected in Rp1+/+ and Rp1+/myc mice, but not in Rp1myc/myc mice. An 70-kDa protein corresponding to the predicted size of the mutant Rp1-myc protein was present in Rp1+/myc and Rp1myc/myc mice, but absent in Rp1+/+ mice.

View larger version (101K): [in this window] [in a new window]   FIGURE 4. The Rp1-myc protein localizes to the axoneme of photoreceptor outer segments. (A) Immunofluorescence and Nomarski confocal images of retinal sections from 2-week-old mice after double labeling with antibodies against Rp1 (red) and myc (green). Left: wild-type retina. Strong Rp1 immunoreactivity was observed along the photoreceptor axonemes; no myc immunoreactivity was detected. Middle: Rp1+/myc retina. Both Rp1 and myc signals were detected, and the distribution of the myc signal matched that of wild-type Rp1 signal (yellow). Right: Rp1myc/myc. The mutant Rp1-myc protein signal was located correctly in photoreceptor axonemes; no normal Rp1 was detected. (B) Close examination of the immunofluorescence signal revealed a subtle difference between the wild-type Rp1 and mutant Rp1-myc signals. The wild-type protein demonstrated a concentration at the proximal end of the signal, at the junction of the outer segment and connecting cilium (arrow, left). This distinct, intense signal is not observed for the mutant Rp1-myc protein (arrowheads, right). (C) Images of single photoreceptors from (B) at higher magnification show more clearly that the concentration of signal at the proximal end was present in wild-type Rp1 staining (arrow; left), but not in the mutant Rp1-myc staining (arrowhead; right).

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Outer segment proteins were correctly localized in Rp1-myc mice. Frozen retinal sections from 2-week-old wild-type (Rp1+/+) and homozygous mutant (Rp1myc/myc) mice were labeled with antibodies to rhodopsin, arrestin, and -CNGC. No mislocation of rhodopsin was detected in the Rp1myc/myc mice. The distribution of arrestin and -CNGC were also identical in the wild-type and mutant retinas.

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Electroretinograms of mice of the three genotypes at 4 to 5 weeks of age. (A–C) Scotopic b-waves obtained in response to a series of 510-nm flashes with luminances of 3.2 x 10-4, 9.2 x 10-4, 1.6 x 10-3, 3.4 x 10-3, and 1.8 x 10-2 scot cd-s · m-2 estimated to produce 0.16, 0.46, 0.79, 1.7, and 9.0 photoisomerizations/rod, respectively, in a normal mouse. Each trace is the average of nine individual records (the traces of the two eyes were also averaged during analysis). The saturating amplitude of the b-wave was extracted by fitting a hyperbolic saturation function to the intensity versus amplitude function.36 (D–F) ERG a-waves, obtained in response to flashes having luminances of 0.74, 1.9, 4.0, 87.0, and 220 scot cd-s · m-2, estimated to produce 370, 960, 2,000, 4,000, and 110,000 photoisomerizations/rod, respectively in a wild-type mouse. The saturating amplitude of the a-wave was obtained directly from the response to the most intense flash. (G–I) The a-wave data of panels (D–F) are presented normalized by the saturating a-wave amplitude and fitted with a model of the activation phase of the phototransduction cascade to extract the amplification constant.37 With flash intensities expressed in luminance units, the amplification parameters are KA 2000, 1800, and 1000 seconds-2 (scot cd-s · m-2)-1 for the data of the Rp1+/+, Rp1+/myc, and Rp1myc/myc mice, respectively. For further details see Table 1 .

View larger version (24K): [in this window] [in a new window]   FIGURE 7. Saturating amplitudes of the principal components of the electroretinograms. Each panel presents the mean saturating amplitudes (±SEM) of the a-wave (rod amax), the scotopic b-wave (rod bmax), and the cone-driven b-wave (cone bmax) obtained from populations of animals of the indicated genotypes of ages 4 to 5 weeks (A) and 4 to 8 months (B). Differences between the components of the wild-type (Rp1+/+) mice and mice of the other two genotypes that are statistically significant are indicated by *P < 0.05, **P < 0.01, and ***P < 0.001. See also Table 1 .

View larger version (113K): [in this window] [in a new window]   FIGURE 8. Progressive degeneration of photoreceptors in the Rp1myc/myc mice. (A) The extent of photoreceptor loss in Rp1myc/myc mice was estimated by observing the thickness of the outer nuclear layer (ONL) in sections of frozen retina from mutant and control mice at different ages. Lens-optic nerve sections were stained with Richardson stain. At age P14, the thickness of the ONL was comparable between Rp1myc/myc and wild-type littermates (left). By 30 days of age, retinas in Rp1myc/myc mice showed significant degenerative changes. The thickness of the ONL was reduced by approximately two to three rows compared with wild-type littermate control animals (middle). By 10-months of age, only two rows of nuclei remained in the retinas of Rp1myc/myc mice (right). (B) Toluidine blue staining of Epon-embedded retinal sections from 8-month-old Rp1+/+ and Rp1+/myc mice shows that the thickness of ONL and the length of outer segments did not differ significantly between Rp1+/+ and Rp1+/myc mice. (C) Immunofluorescence and Nomarski confocal images of retinas in 10-month-old Rp1+/+ and Rp1myc/myc mice. Antibodies against rhodopsin (red, top) and red/green cone opsin (green, bottom) show that both rods and cones remain in the degenerated Rp1myc/myc retinas. Mislocalization of both proteins to the inner segment and ONL was detected in the mutant mice.

View larger version (138K): [in this window] [in a new window]   FIGURE 9. Ultrastructure of photoreceptors in Rp1-myc mice. Eyes of mice with indicated ages and genotypes were embedded in Epon, sectioned at 60- to 80-nm thickness and evaluated by electron microscopy. (A) Outer segments in the P14 wild-type retina appeared as straight, parallel cylinders from inner segments to the RPE. The stacks of discs were organized and perpendicular to the long axis of the cell (left). In contrast, outer segments in P14 Rp1myc/myc mice were disorganized. Small packets of discs were scattered in the space between the inner segments and RPE (middle). Close examination of the disorganized discs (right) showed that these small packets contained approximately 20 to 40 discs with diameters two to three times normal (2–4 µm), oriented obliquely or parallel to the long axis of the cell. Although abnormally oriented, the discs in Rp1myc/myc retinas were intact, with identifiable rims. (B) The morphology of the photoreceptor axoneme and newly forming discs at the base of the outer segments were examined at P10. The nascent discs were tightly aligned along the axoneme (arrowhead) at the base of the outer segment in the wild-type mice (left). In the Rp1myc/myc mice, even the early lip (L) of new membrane evagination at the base of the outer segment was misoriented, and nascent discs were parallel to the axoneme (arrowhead, middle). In addition, the disc rims were no longer associated with the axoneme. A cross section showed a normal axoneme structure, but also demonstrated discs oriented parallel to the axis of the outer segment (right). (C) At age P30 the outer segment dysplasia was more severe, with shortening and disorganization of outer segments structures in the photoreceptors of Rp1myc/myc mice (middle), compared with the wild-type control (left). Misoriented disc stacks (arrowhead) and short packets of discs () were still observed (right). (D) The ultrastructure of photoreceptors in 8-month-old Rp1+/myc mice (right) was grossly normal compared with that of wild-type mice (left). CC, connecting cilium; IS, inner segment; OS, outer segment.

By 1 month of age, retinas from Rp1^myc/myc mice exhibited severely shortened and disorganized outer segments compared with wild-type or Rp1^+/myc mice (Fig. 9C) . Again, the predominant feature of the abnormal discs in the Rp1^myc/myc mice were small packets of intact but incorrectly oriented discs. For evaluation of the ultrastructure of photoreceptors in heterozygous Rp1^+/myc mice, retinas from seven Rp1^+/myc mice and seven littermate control animals at ages 6 to 8 months were examined extensively by electron microscopy. In contrast to the Rp1^myc/myc mice, heterozygous Rp1^+/myc mice had essentially normal photoreceptor ultrastructure up to 8 months of age, including normal cell bodies, as well as outer segments (Fig. 9D) .

	Discussion

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Rp1 Required for Correct OS Disc Orientation
The data presented above show that Rp1^myc/myc homozygous mice have a profound defect in outer segment formation. Stacks of membrane discs in the Rp1^myc/myc mice were oriented obliquely or parallel to the long axis of the outer segments, instead of perpendicularly. The discs were also large and were grouped in small packets of 20 to 40 discs, instead of the long arrays of mature discs found in healthy photoreceptors. This defect in disc orientation appears to occur during disc formation. Even the early evaginations of membrane at the base of the outer segment were misdirected in the homozygous mutant mice (Fig. 9B) . In control animals, the new discs always formed perpendicular to the axoneme, and discs retained their alignment with the axoneme after formation.³⁹ In homozygous mutant animals, the discs lost this association with the axoneme. Thus, it appears that Rp1 is required for the correct orientation and higher order stacking of outer segment discs.

Given the location of Rp1 in the axoneme and the abnormal disc orientation observed in the Rp1^myc/myc mice, it is attractive to hypothesize that Rp1 functions as a connection between newly formed discs and the axoneme and that this interaction helps discs form in the correct orientation and stack up into outer segments. Proteins known to be located in the disc rims, such as rds/peripherin, Rom1, and ABCR are potential candidates for such an interaction.^{40 41 42} Mutations in RP1, all of which cause loss of the C-terminal one half to two thirds of the protein, would then cause photoreceptor cell dysfunction and ultimately death by disrupting the interaction between the axoneme and the discs. We are currently searching for proteins that interact with RP1 and can mediate this interaction.

The importance of the connecting cilium and axoneme in disc formation has been recognized for many years. Indeed, the photoreceptor outer segment is considered to be a modified cilium adapted to optimize light absorption.⁴³ As a testament to the importance of the connecting cilium and axoneme in disc formation, targeted deletion of other protein components of the cilium can also lead to defects in disc formation. For example, mice deficient in RPGRIP exhibit defective outer segment orientation, in a fashion similar to that observed in the Rp1-myc mice.¹⁸

Localization of the Truncated Rp1-myc Protein
It was interesting to find that the truncated Rp1-myc protein localized correctly to the photoreceptor axoneme. This indicates that at least part of the information required for association of Rp1 with the axoneme is contained in the N-terminal third of the protein. It is likely that the two tandem doublecortin domains (DCX) at the N terminus of RP1 participate in the interaction of RP1 with the axoneme (Fig. 2) . These domains (codons 35-236 of Rp1) are homologous to the microtubule binding domain of the doublecortin protein.⁴⁴ Doublecortin is a microtubule-associated protein (MAP) that is essential in the proper development of the layers of the cerebral cortex, and mutations in doublecortin cause X-linked lissencephaly and double cortex syndrome, which are inherited defects of the cerebral cortex associated with arrest of migrating cerebral cortical neurons.^{45 46 47} The two tandem DCX domains in doublecortin are thought to stabilize microtubules by tethering tubulin dimers together or by cross-linking adjacent microtubules.⁴⁸ Additional data show that lack of the DCX domains from the Rp1 protein in vitro or in vivo leads to altered localization of the Rp1 protein (Liu Q, et al. IOVS 2003;44:ARVO E-Abstract 4528). These data imply that the DCX domains in the N-terminal region of RP1 are active and raise the possibility that RP1 is a photoreceptor-specific MAP.

Aside from tubulin, axonemes are known to contain MAPs, and the components of the protein transport system, including dynein, kinesin, and IFT proteins.¹² It is likely that many other proteins are also required for ciliary axoneme structure and function, as a recent proteomic analysis detected more than 240 protein components in ciliary axonemes from respiratory epithelia.⁴⁹ It is reasonable to assume that photoreceptor axonemes contain unique proteins, given the specialized functions required to produce and maintain outer segments. Although RP1 was detected in the proteomic analysis of cultured respiratory epithelial cells mentioned earlier, we have not been able to detect the Rp1 protein in tissues, or cultured cells, with motile or primary cilia by using immunofluorescence assays (Pierce, et al. IOVS 2003;44:ARVO E-Abstract 1529). RP1 thus appears to be one of the photoreceptor-specific, or at least photoreceptor-enriched, axoneme components.

One subtle difference between the location of the Rp1-myc protein and the wild-type Rp1 protein is detected at the junction of the outer segment and connecting cilium. The immunofluorescence signal for the wild-type protein demonstrates a concentration in this area, whereas such a concentration is not observed with antibodies against the myc-tagged mutant protein (Figs. 4B 4C) . This finding suggests that the C-terminal two thirds of Rp1 interacts with a protein or proteins found at this junction. Data from Rp1-exon 2/3 deletion mice support this concept. In these mice, abnormal splicing between exons 1 and 4 occurs, and a mutant Rp1 protein without the DCX domains is produced. The mutant exon 2/3 deletion protein is found at the junction between inner segments and outer segments, but only as a punctate signal.²³ Additional experiments show that the punctate signal is located at the base of the axoneme in outer segments (Liu Q, et al. IOVS 2003;44:ARVO E-Abstract 4528). Actin and myosin II are known to be concentrated in this region at the base of the OS, and are thought to be important in disc formation, although their exact roles remain unclear.^{17 19 21} A photoreceptor-specific form of cadherin (prCAD) has been identified recently and found to be localized in the superficial aspects of newly evaginated disc membranes at the base of the outer segment. Targeted deletion of prCAD also results in fragmentation and disorganization of outer segments.²² It is possible, therefore, that interactions between the C-terminal portion of RP1 and actin, myosin, cadherin, or another protein located at the base of the outer segment, are part of the mechanism by which Rp1 helps discs form in the correct orientation and stack up into outer segments.

The 10-amino-acid myc tag was chosen for these studies because it is commonly used as an epitope tag and is recognized with high affinity by the well-characterized antibody 9E10.⁵⁰ Although we cannot rule out definitively that the myc tag did not alter the function of the truncated Rp1-myc protein, the myc tag itself does not generally affect protein function.⁵¹ For example, introduction of the myc tag into vascular endothelial growth factor (VEGF) did not alter its biological activity.⁵²

Retinal Degeneration in Rp1^myc/myc Homozygous Mice
The Rp1^myc/myc homozygous mice undergo a rapid retinal degeneration. The amplitude of their rod a-waves is decreased by 4 weeks of age and is almost undetectable by 4 to 8 months of age. Rod and cone b-waves were also decreased in homozygous animals, although not to the same extent as the rod a-waves. Because the a-wave is the field potential produced by light suppression of the rod-circulating current,^{36 53} the loss of the saturating a-wave amplitude in the Rp1^myc/myc mice can be understood as a consequence of both loss of rods and disorder of the residual outer segments. Nonetheless, the proteins underlying the activation phase of rod phototransduction appear to be present in their normal ratios, because the amplification of the a-wave appears largely unchanged in the 4- to 5-week-old mice (Fig. 6 ; Table 1 ).^{54 55} These observations are consistent with the conclusion that the primary defect in the rods is the misorientation of the outer segment discs and loss of large-scale outer segment structure, but not in the molecular mechanisms that deliver the lipid and protein components required for outer segment synthesis and for phototransduction. A similar phenotype was observed in the prCAD null mice.²²

The finding that the components of the phototransduction cascade are present in normal ratios, even though outer segments are abnormal in the Rp1^myc/myc mice, is consistent with the immunofluorescence data showing the correct location of outer segment proteins in the retinas of mutant mice (Fig. 5) . No rhodopsin mislocalization was noted in the retinas of 2-week-old Rp1^myc/myc mice, in contrast to findings with Rp1-exon 2/3 deletion mice.²³ The reason for this difference between the two animal models is not clear. It is possible that the differently truncated Rp1 proteins lead to different defects in protein function. It is also possible that protein transport is not a major function of Rp1, because the amount of rhodopsin mislocalization observed in the Rp1-exon 2/3 deletion mice was quite small compared with that observed in the retinas of mice with mutations in transport proteins, such as kinesin II or myosin VIIa.^{10 11}

Mutant RP1 mRNA and Nonsense-Mediated Decay
We chose to make the Rp1-myc allele after detecting the mutant RP1 mRNA as an illegitimate transcript in lymphoblasts derived from patients with RP1 disease. We used lymphoblasts as a proxy for retina because a suitable sample of retina from a patient with RP1 was not available. Lymphoblasts of members of the RP01 pedigree were deposited in the Coriell Repository when this family was originally ascertained.^{35 56} These lymphoblasts are a valuable resource, and can be used as an alternative to retinal tissue because there is a low level of illegitimate transcription of nonexpressed genes in many cell types, including lymphocytes.⁵⁷ RT-PCR data show that mutant RP1 mRNA is present in lymphoblasts of patients heterozygous or homozygous for the Arg677Ter mutation in RP1. These data indicate that the mutant mRNA escapes nonsense-mediated mRNA decay, as predicted by the location of the truncation mutation in the final exon.²⁵ Although only indirect, the data also imply that truncated RP1 protein is made in the retina, because, when present, the mutant mRNA should be translated.

RP1 Disease Mechanism: Haploinsufficiency Versus Dominant Negative?
With the finding that mutant RP1 mRNA can be detected in lymphoblasts, we hypothesized that mutations in RP1 could lead to production of a truncated protein in the retina and may cause photoreceptor cell death through a dominant negative mechanism mediated by the truncated protein. We created the Rp1-myc allele in part to test this hypothesis. Our results indicate that the truncated Rp1-myc protein is nonfunctional, as animals homozygous for the mutant allele experience a rapid retinal degeneration. The lack of a major phenotype in the heterozygous animals, however, suggests that the truncated protein does not have a dominant negative effect. It is thus more likely that RP1 disease in humans is caused by decreased expression of functional RP1 protein, or haploinsufficiency. In this case, the haploinsufficiency probably occurs because the Rp1-myc protein complement is nonfunctional. It still remains possible, however, that the mutant RP1 mRNA is degraded in the retina. Additional evaluation of this question by using human tissue or point mutation knockin mice is warranted.

If mutations in RP1 cause disease by haploinsufficiency, then the Rp1^+/myc mice would still be expected to undergo retinal degeneration, because they have only one normal Rp1 allele. However, the photoreceptors of the heterozygous mice remained relatively healthy. The lack of a major phenotype in the Rp1^+/myc mice may be due to differences between mouse and human photoreceptors or to the genetic background of the Rp1-myc mice. It is known that the phenotype of patients with RP can vary dramatically, even among family members with the same mutation. This is true of patients with mutations in RP1.⁵⁸ The difference in disease severity has been suggested to be due to variations in disease-modifying genes. Effects of genetic background have also been observed in mice. A dramatic example involves the so called "rumpshaker" mutation of the proteolipid protein (Plp) gene, which causes dysmyelination in humans and mice. The phenotype of mice with this mutation depends critically on the genetic background in which the mutation is expressed. On the C3H background, there is normal longevity, whereas changing to a C57BL/6 strain results in seizures and death by postnatal day 30.⁵⁹ Similarly, the extent of neovascularization observed in response to an identical experimental stimulus varies up to 10-fold in different strains of mice.⁶⁰ We are currently outcrossing the Rp1-myc allele onto several different mouse strains to assess the effect of genetic background on the retinal phenotype.

Although photoreceptor structure appeared to be normal in the Rp1^+/myc mice at 8 months of age, the Rp1-myc protein appeared to cause some alterations in photoreceptor function, because rod b-waves were significantly decreased at 4 to 8 months of age in the heterozygous animals. This finding suggests alterations in the signaling between rods and their bipolar cells.³⁶ However, given the apparently normal ultrastructure of photoreceptor cells in the retinas of these mice, it is unclear why the b-wave is diminished. At present, however, it is not certain whether these changes are the early signs of a slow retinal degeneration. The ERGs of a small number of 1-year-old F1 Rp1^+/myc mice were not different from those of control subjects, implying that Rp1^+/myc mice do not have a progressive retinal degeneration phenotype (data not shown). Additional investigations of the retinal function of older Rp1^+/myc mice are in progress to address these questions.

To determine definitively whether haploinsufficiency of RP1 can cause disease, we are preparing mice with a complete knockout of the Rp1 coding region. We are hopeful that comparison of Rp1-myc mice and Rp1 null mice will help clarify the mechanism of RP1 disease. This is an important issue, because the answer will dictate which potential therapeutic approaches are applicable to RP1 disease. Members of the RP1 Consortium are also preparing Rp1 knockin mice, with a single nonsense mutation at Arg662 of the Rp1 gene, but no other alterations, to further investigate the mechanism by which mutations in RP1 cause photoreceptor cell death. These knockin mice will be especially useful for determining whether the mutant Rp1 mRNA survives to produce a truncated protein. We are also hopeful that use of these different animal models will help elucidate the pathway(s) by which disorganized outer segments lead to photoreceptor cell death.

	Acknowledgements

 
The authors thank Romaica Omarrudin, Suzanne Pavluk, David Pugh, Andrei Savchenko, and Xinyu Zhao for their technical assistance; Kathy Battaglia and Ann Milam for their comments on the manuscript; the other members of the RP1 Consortium for their advice; and Peter Sterling for use of his electron microscope.

	Footnotes

 
Supported by National Eye Institute Grants EY12910 and EY02660, Bethesda, Maryland; Research to Prevent Blindness, New York, New York; the Foundation Fighting Blindness, Owings Mills, Maryland; the Rosanne Silbermann Foundation, Livingston, New Jersey; and the F. M. Kirby Foundation, Morristown, New Jersey.

Submitted for publication April 25, 2003; revised June 6, 2003; accepted June 10, 2003.

Disclosure: Q. Liu, None; A. Lyubarsky, None; J.H. Skalet, None; E.N. Pugh, Jr, None; E.A. Pierce, 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.

Corresponding author: Eric A. Pierce, F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, 305 Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, PA 19104; epierce{at}mail.med.upenn.edu.

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