Retinal Degeneration 6 (rd6): A New Mouse Model for Human Retinitis Punctata Albescens

Abstract

purpose. To characterize the genetics and phenotype of a new mouse mutant with retinal degeneration, rd6, that is associated with extensive, scattered, small white retinal dots seen ophthalmoscopically.

methods. The phenotype was characterized using ophthalmoscopy, fundus photography, electroretinography, light microscopy, immunocytochemistry, and electron microscopy. Genetic characterization and linkage analysis studies were performed using standard methods.

results. The inheritance pattern of rd6 is autosomal recessive. Linkage analysis mapped rd6 to mouse Chromosome 9 approximately 24 cM from the centromere, suggesting that the human homolog may be on chromosome 11q23. Ophthalmoscopic examination of mice homozygous for rd6 revealed discrete subretinal spots oriented in a regular pattern across the retina. The retinal spots appeared by 8 to 10 weeks of age and persisted through advanced stages of retinal degeneration. Histologic examination revealed large cells in the subretinal space, typically juxtaposed to the retinal pigment epithelium. The white dots seen on fundus examination corresponded both in distribution and size to these large cells. By 3 months of age, the cells were filled with membranous profiles, lipofuscin-like material, and pigment. These cells reacted strongly with an antibody directed against a mouse macrophage-associated antigen. Photoreceptor cells progressively degenerated with age, and an abnormal electroretinogram was initially detected between 1 and 2 months of age.

conclusions. The fundi of mice homozygous for rd6 exhibit phenotypic similarities to the human flecked retinal disorder retinitis punctata albescens. Thus, rd6/rd6 mice may be a model for understanding the etiology of this or similar disorders. The relationship between the aberrant subretinal cells and the concomitant photoreceptor degeneration remains to be established.

Several human retinal disorders are characterized by scattered white retinal dots or lesions. Such inherited retinal disorders were originally classified by Krill ¹ as “flecked retina diseases.” The main disorders that he and others have described within this category are retinitis punctata albescens (RPA), fundus albipunctatus (FA), Doyne’s familial maculopathy, and fundus flavimaculatus, now more commonly referred to as Stargardt’s disease. ^{1 2} Our understanding of the fundus appearance of Stargardt’s disease was substantially advanced by a histopathologic study showing that the flecks are aggregates of swollen retinal pigment epithelial (RPE) cells. ³ The retinal dots seen in human RPA and FA are similar funduscopically; however, the human retinal histopathology of these two conditions is unknown. RPA is a progressive disorder that causes visual loss similar to that observed in patients with retinitis pigmentosa, whereas FA is a form of congenital stationary night blindness, which tends to be more stable over time. ⁴  

Animal models with spontaneous retinal degeneration have been used for many years to provide potential insight into the etiologies of retinal degenerations and tissue to study disease progression and pathology. Many of these animal models have come from screening mice from genetically independent mouse strains and stocks at The Jackson Laboratory (TJL) by indirect ophthalmoscopy and electroretinography (ERG). ^{5 6 7 8} We have recently identified a new retinal degeneration mutation, retinal degeneration 6 (rd6), which is associated with distinctive white dots or flecking on the retina. This article presents the genetic analysis of rd6, showing that it is a new autosomal recessive mutation that maps to mouse Chromosome 9, and phenotypic characterization of the clinical and morphologic features of the rd6/rd6 mutant mouse. 

Methods

Animals

The mice in this study were bred and maintained in standardized conditions in the Research Animal Facility at TJL. They were maintained on NIH31 6% fat chow and acidified water, with a 14-hour light/10-hour dark cycle in conventional facilities that are monitored regularly to maintain a pathogen-free environment. All experiments were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Origin

rd6 was discovered in the inbred mouse strain C3HfB/Ga Cas1 ^b , hereafter referred to as C3HfB. This strain was derived from offspring of irradiated (C3Hx101) F1 male mice crossed to the Oak Ridge National Laboratory “multiple recessive test stock”; the Cas1 ^b mutation was subsequently backcrossed to C3HfB, a C3H subline fostered (f) on C57BL/6 (B). Although all C3H strains are fixed for Pdeb ^rd1 , a mutation in the beta subunit of cGMP phosphodiesterase, ⁹ this particular strain exhibited an unusual granular retinal appearance compared with other strains homozygous for Pdeb ^rd1 . To test for the possibility of a new mutation different from Pdeb ^rd1 , mice from this strain were mated to C57BL/6J mice, which have a normal retina. The F1 progeny, which did not show ocular abnormalities, were backcrossed to the parental strain C3HfB. The backcross offspring could be separated into 3 groups: Group 1, normal; group 2, Pdeb ^rd1 homozygotes with characteristic patches of pigment deposits and large diffuse depigmented regions observable by 3 to 4 weeks of age; and group 3, mice with small, discrete dots present throughout the fundus (Fig. 1) . On examination by indirect ophthalmoscopy, the dots gave the retina a granular appearance, a phenotype first observed between 8 and 10 weeks of age. Mice from group 3 were intercrossed to fix the mutation in a stock, free of Pdeb ^rd1 but exhibiting the new retinal phenotype. Subsequently, the rd6 stock has been maintained by repeated backcrossing to C57BL/6J to make a congenic inbred strain. Phenotypic characterization was performed on mice from the mixed background stock and the incipient B6 congenic. Major aspects of the phenotype do not appear to differ on the two genetic backgrounds. 

Clinical Retinal Evaluation

All mice in the characterization studies and linkage crosses had pupils dilated with 1% atropine ophthalmic drops and were evaluated by indirect ophthalmoscopy with a 78 diopter lens. Signs of retinal degeneration, such as vessel attenuation, alterations in the RPE, and presence or absence of retinal dots were noted. Fundus photographs were taken with a Kowa Genesis small animal fundus camera (Torrance, CA). ¹⁰  

Electroretinography

After at least 2 hours of dark-adaptation, mice were anesthetized with an intraperitoneal injection of normal saline solution containing ketamine (15 mg/gm) and xylazine (7 mg/gm body wt). Electroretinograms (ERGs) were recorded from the corneal surface of one eye after pupil dilation (1% atropine sulfate) using a gold loop electrode referenced to a gold wire in the mouth. A needle electrode placed in the tail served as ground. A drop of methylcellulose (2.5%) was placed on the corneal surface to ensure electrical contract and to maintain corneal integrity. Body temperature was maintained at a constant temperature of 38°C using a heated water pad. 

All stimuli were presented in a Ganzfeld dome (LKC Technologies, Gaithersburg, MD) whose interior surface was painted with a highly reflective white matte paint (No. 6080; Eastman Kodak, Rochester, NY). Stimuli were generated with a Grass Photic Stimulator (model PS33 Plus; Grass Instruments, Worcester, MA) affixed to the outside of the dome at 90° to the viewing porthole. Rod-dominated responses were recorded to short-wavelength (λ_max = 470 nm; Wratten 47A filter) flashes of light over a 4.0-log unit range of intensities (0.3 log unit steps) up to the maximum allowable by the photic stimulator. Cone-dominated responses were obtained with white flashes (0.3 steps) on the rod-saturating background after 10 minutes of exposure to the background light to allow complete light adaptation. 

Responses were amplified (Grass CP511 AC amplifier, ×10,000; 3 dB down at 2 and 10,000 Hz) and digitized using an I/O board (model PCI-1200; National Instruments, Austin, TX) in a personal computer. Signal processing was performed with custom software (LabWindows/CVI; National Instruments). Signals were sampled every 0.8 msec over a response window of 200 msec. For each stimulus condition, responses were computer-averaged with up to 50 records averaged for the weakest signals. A signal rejection window could be adjusted online to eliminate electrical artifacts. 

Histology and Electron Microscopy

A total of 40 mice (2 at each time point), ranging in age from 1 week to 29 months, were studied histologically. The eyes for light microscopy were immediately removed and immersed in cold fixative (1% paraformaldehyde, 2% glutaraldehyde, and 0.1 M cacodylate buffer). Eyes were left in fixative for 24 hours, after which time they were transferred to cold 0.1 M cacodylate buffer solution for an additional 24 hours. Samples were embedded in methacrylate historesin, and sections were cut and stained with hematoxylin and eosin (H&E). The eyes for ultrastructural studies were fixed in one-half strength Karnovsky fixative (2% formaldehyde and 2.5% glutaraldehyde in 100 mM cacodylate buffer, pH 7.4, containing 0.025% CaCl₂) for at least 24 hours. Eyes were cut into small wedges (∼1 × 2 mm), rinsed (3 × 10 min) in 100 mM cacodylate buffer (pH 7.4), and post-fixed with 1% osmium tetroxide for 2 hours. Tissues from all four quadrants of each eye were subsequently processed for transmission electron microscopy. ¹¹  

Immunocytochemistry

Eyecups were fixed in 4.0% paraformaldehyde in 100 mM cacodylate buffer, pH 7.4, for 2 to 4 hours. Eyes were rinsed in 100 mM sodium cacodylate buffer (3 × 10 minutes), infiltrated, and embedded in acrylamide (Boehringer–Mannheim, Indianapolis, IN) and optimal cutting temperature (OCT) compound (Miles, Elkhart, NY), ¹² snap-frozen in liquid nitrogen, sectioned to a thickness of 6 to 8 μm on a cryostat, and mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Unfixed eyecups were embedded directly in OCT, without acrylamide infiltration or embedment, and sectioned. Sections that bisected the optic nerve in temporonasal orientation were prepared from each eyecup. The sections were blocked in 10 mM sodium phosphate, pH 7.4, containing 0.85% NaCl, 1 mM each of calcium chloride and magnesium chloride, and 1 mg/ml globulin-free bovine serum albumin (PBS/M/C/BSA). Sections were then rinsed and incubated in MOMA-2 (Chemicon, Temecula, CA) antibody for 1 hour. ¹³ Slides were rinsed in PBS/M/C (2 × 10 minutes) and subsequently incubated for 30 minutes in fluorescein isothiocyanate (FITC)–conjugated secondary antibody. Sections were then washed in PBS/M/C (2 × 10 minutes), and placed on coverslips. For negative controls, sections were exposed to PBS/M/C/BSA containing no primary antibody or 1% (vol/vol) normal mouse serum, followed by secondary antibody. Unlabeled, adjacent sections were examined at 450 to 550 nm excitation (Olympus BP545 filter set; Melville, NY) to detect the presence of autofluorescence that is characteristic of RPE cells at this wavelength. 

Gene Mapping

To determine the chromosomal location of the rd6 gene we mated C3HfB/Ga-Cas1 ^b mice to C57BL/6J mice. The F1 mice, which exhibited no retinal abnormalities, were intercrossed for the initial linkage study. In a second cross, similar F1 mice were backcrossed to C3HfB/Ga-Cas1 ^b mice. In both crosses, all the Pdeb ^rd1 /Pdeb ^rd1 mice were discarded at 1 month of age. Tail DNA was isolated as previously reported. ¹⁴ For PCR (polymerase chain reaction) amplification, 25 ng DNA was used in a 10-μl volume containing 50 mM KCl, 10 mM Tris–Cl, pH 8.3, 2.5 mM MgCl₂, 0.2 mM oligonucleotides, 200 μM dNTP, and 0.02 U AmpliTaq DNA polymerase. The reactions, which were initially denatured for 2 minutes at 95°C, were subjected to 49 cycles of 20 seconds at 94°C, 20 seconds at 50°C, 30 seconds at 72°C, and then a final 7-minute extension at 72°C. PCR products were separated by electrophoresis on 4% MetaPhor (FMC, Rockland, ME) agarose gels and visualized under UV light after staining with ethidium bromide. Initially a genome scan of microsatellite (Mit) DNA markers was carried out on pooled DNA samples. ¹⁵ After detection of linkage on Chromosome 9, the microsatellite markers D9Mit2, D9Mit54, D9Mit171, D9Mit191, D9Mit197, D9Mit227, D9Mit247, and D9Mit286 were scored on individual DNA samples. 

Results

Clinical Phenotype

Mice homozygous for rd6 have small, evenly spaced white dots throughout their retinas (Fig. 1B) . These small white dots become apparent on ophthalmoscopic examination by 8 weeks of age in male and female mice. They are easily visible through about 7 months of age after which they persist but are more difficult to distinguish. Homozygous rd6 mice begin to show clinical signs of retinal degeneration at 7 months of age. The fundus develops a mildly pigmented granular and mottled appearance by 15 months; individual spots can still be seen, although less frequently, in mice up to about 2 years of age. Retinal vessels are pale and attenuated by 7 months of age and are not detectable by ophthalmoscopy by 22 months. 

ERG Phenotype

ERGs of eyes from homozygous rd6 mice show a slow progressive retinal dysfunction of both rods and cones over a 70-week period, beginning at about 1 month of age (data not shown) and being extinguished by 70 weeks of age (Fig. 2) . The rod and cone systems are affected by the degenerative process and diminish at a similar rate. 

Histologic Phenotype

Histologic examination revealed progressive changes in the retina and RPE (Fig. 3) . By 1 month of age, the distal portions of the outer segments were mildly distorted, a finding confirmed by electron microscopy (see below). In addition, by 3 weeks of age, a few small cells characterized by an eosinophilic cytoplasm lacking pigment granules were identified within the subretinal space, juxtaposed to the RPE. From 1 to 2 months of age, there was a progressive increase in the number of aberrant cells in the subretinal space, and many were characterized by the presence of pigment granules similar to those in the RPE. The photoreceptor cell outer segments were reduced slightly in length, with a decrease in the number of photoreceptor cells. Between 3 to 5 and 6 to 11 months of age, there was progressive loss of photoreceptor cells in the outer nuclear layer (ONL), shortening and disorientation of the outer segments, and persistence of the abnormal pigmented cells in the subretinal space. Overall degeneration appeared to stabilize temporarily between 3 and 6 months (Fig. 3) . 

From 15 months of age, the photoreceptor outer segments were totally absent, and in some areas, the ONL could not be positively identified. The large pigmented cells in the subretinal space persisted through 22 months of age but could no longer be found after that time (up to 29 months). Between 4 and 11 months, many of these subretinal aberrant cells doubled or tripled in size compared with their size at 2 months of age. The other characteristic feature of progressive retinal degeneration was the decrease in thickness of the ONL. In the nearly normal 1-week-old mice, the ONL was 12 to 14 nuclei thick. By 1 month, the ONL decreased to a 7- to 8-nuclei thickness; by 1 year of age to 2 to 4 nuclei layers; and by 2 years was only 0 to 1 nuclei (Fig. 3 , last panel). Higher magnifications of atypical large aberrant cells in the subretinal space are shown in Figure 3 , bottom panels. 

Immunocytochemical Analyses

To better characterize the nature of the unusual cells within the subretinal space, sections from eyes of 6-month-old rd6/rd6 mice were exposed to various antibodies and lectins. Only one monoclonal antibody, MOMA-2, directed against an intracellular antigen associated with mouse macrophages and monocytes, ¹⁶ reacted strongly with these cells (Fig. 4) . The MOMA-2 antibody did not react with the RPE or retina. Reactive choroidal cells, assumed to be monocytes/macrophages, confirmed the specificity of the reaction. The aberrant subretinal cells are autofluorescent under 450 to 550 nm excitation. 

Electron Microscopy

Ultrastructural studies were performed on eyes derived from mice at 2, 4, and 8 months of age (Figs. 5 6 and 7) . Pyknotic photoreceptor cell bodies increase in number over the first year of life. Approximately four rows of photoreceptor nuclei were observed at 8 months of age (Fig. 5) . Outer segments become disoriented and disrupted, but the depth of the subretinal space does not decrease appreciably over the first 8 months (Figs. 5A 5B 5C) . In the subretinal space, distinct aberrant cells are seen that are regularly spaced at intervals that correspond to the dots observed funduscopically (Figs. 5A 5C 6A 6B 6C and 7B) . Furthermore, their diameters average 35 μm (range, 22–42 μm) in mice between 2 and 8 months of age. This size is similar to that of the dots observed ophthalmologically; the average size of the white dots is slightly greater than 50% of the diameter of the large retinal vessels, which is approximately 60 to 80 μm. Higher magnifications of these subretinal space–associated cells in 2-, 4-, and 8-month-old rd6/rd6 mice show that they possess cytoplasmic inclusions that are indistinguishable from the pigment granules and lipofuscin in adjacent RPE cells and phagosome-like vesicles (Figs. 6A 6B 6C 6D) . In addition, distinct regions of membranous material and vacuoles can be observed in both RPE cells and the cells within the subretinal space (Figs. 7A and 7B) . This material is not typically observed in the RPE of control mice. 

Genetic Analysis

rd6 segregated as an autosomal recessive mutation in the genetic crosses carried out to isolate it on the C57BL/6J genetic background. These data were confirmed by the (C3HfB-rd6/rd6 x C57BL/6J[B6])F1 x C3HfB-rd6/rd6 backcross that produced 22 rd6/rd6 and 23 wild-type progeny. Linkage to Chromosome 9 was initially detected by genome analysis of pooled DNA samples from 21 progeny (42 meioses) from the C3HfB-rd6/rd6 x B6 intercross described in the Methods section. These 42 meioses showed 1/42 recombinant chromosome with D9Mit227 and 2/42 with D9Mit171. An additional 45 mice from the backcross gave order and recombination percentages of D9Mit227 − 2/87 = 2.3 ± 1.6 − rd6 − 2/87 = 2.3 ± 1.6 − D9Mit171 (Fig. 8) . The allele sizes of markers surrounding the mutation are like C3H, suggesting that the mutation arose in a stretch of C3H DNA either from the originally irradiated C3HX101 F1 hybrid mouse or from the C3HfB strain to which Cas1 ^b was backcrossed. C3HfB alleles in this region of Chromosome 9 are identical with strain C3H. The homologous region in humans is chromosome 11q22-23. 

Discussion

Histologic and physiological evidence indicate that homozygous rd6 mice develop an early onset, but slow, retinal degeneration. Alterations in the photoreceptor layer can be seen shortly after the retina develops; the outer segments shorten and soon show significant disorganization. Photoreceptor cells degenerate from the normal 10 to 12 cell–layer thickness to 1 to 3 cell layers by 12 months. Functional changes, as measured by ERG, occur concomitantly with the histologic changes; early rod and cone dysfunction is coincident with initial photoreceptor and ONL cell loss, and the ERG is extinguished by 70 weeks. 

Unique funduscopic features are observed in rd6/rd6 mice in association with the retinal degeneration. By 2 months of age there are discrete dots distributed in a regular fashion throughout the retina; these are easily observed by indirect ophthalmoscopy. Histologic analysis showed a correlation of the retinal spots with large, aberrant cells that lie within the subretinal space. These cells are relatively small in the first month of life and then enlarge and become pigmented by the second month. Many of these cells reach a size of approximately 35 to 40 μm in diameter (or about one half the diameter of the retinal vessels, see the Results section) by 8 to 10 weeks of age when the retinal dots are first observed. The appearance of pigment similar to that seen in the RPE, the presence of vesicles and membranous profiles at the electron microscopic level, and the strong reaction to the monocyte/macrophage-specific MOMA-2 monoclonal antibody suggest that these may be phagocytic cells that have invaded the subretinal space. We have observed histologically similar cells in other mouse mutants with retinal degenerations (authors’ unpublished data). The large aberrant cells in the subretinal space are consistent with the small white dots seen by ophthalmoscopy based on their relative size and distribution and on the lack of any other histologic finding. 

It is likely that a number of genes will be found to be associated with disorders in which retinal spots and flecks are associated with retinal degeneration. For example, FA, which gives rise to retinal spots, has been found to be associated with 11-cis retinol dehydrogenase mutations. ¹⁷ The 11-cis retinol dehydrogenase (Rdh1) gene has not yet been mapped in the mouse; its location in human chromosome 12q13-q14 predicts its mouse homolog would map to mouse Chromosomes 10 or 15. ¹⁸ In addition, genes have been identified for other flecked retinal diseases, for example, Stargardt’s disease, ¹⁹ and Doyne’s familial drusenosis. ²⁰ The homologs of the genes mutated in Stargardt’s disease (ABCR) and Doyne’s (EFEMP1) also have not yet been mapped in the mouse. Their locations in human chromosomes 1p13 and 2p16, respectively, predict that their homologs will map to mouse Chromosomes 3 and 17. All three genes can be examined as potential candidates for rd6; however, the Chromosome 9 location of rd6 suggests that it may identify yet another mutant gene associated with flecked retina disorders. However, the retinal phenotype in homozygous rd6 mice resembles most the changes seen clinically in human RPA. RPA is a rare, progressive, recessively inherited disease that is characterized by whitish-yellow spots radiating out from the posterior pole with no macular involvement. ERG responses are reduced in early stages of RPA and eventually become nonrecordable as the disease progresses. ⁴ The RPA phenotype in humans is genetically heterogeneous because it has recently been shown to be caused by mutations in both cellular retinaldehyde-binding protein ²¹ and peripherin. ²² Because the mouse cellular retinaldehyde-binding protein gene maps to mouse Chromosome 1 and because the retinal-related peripherin gene maps to mouse Chromosome 17, ¹⁸ the rd6 mutation is likely to identify yet another gene involved in flecked retina diseases. Further study of this new retinal degeneration model mouse may give valuable clues about these human disorders characterized by white dots in the retina. 

Mouse mutants with retinal disorders provide excellent models for human retinal diseases. From a clinical point of view, the mouse retina has an ophthalmoscopic and histologic appearance very similar to those of the human retina. The main funduscopic difference is that mice have no fovea. However, the mouse retina has a cone system that can be easily measured by ERG and detected histologically. Genetically, it is estimated that there is at least 95% conservation of sequence between essential coding regions in the human and mouse genomes. This means that human homologs of the mutant genes that cause inherited disorders in the mouse are likely to be involved in similar human disorders. Homology maps that allow comparison of mouse chromosomes to their corresponding human segments are well developed; and when a mutation is found in mouse, the human gene location can frequently be predicted. ²³  

A number of ocular disorders, vitelliform macular degeneration 2 (VMD2), neovascular inflammatory vitreoretinopathy (ADNIV), Usher Syndrome 1B, and familial exudative vitreoretinopathy (FEVR), have been mapped to human chromosome 11q, which has homology to mouse Chromosome 9. ²² However, none of these ocular diseases appears to map unambiguously to the region of human chromosome 11q, which is directly homologous with the rd6 region of mouse Chromosome 9, neither is their phenotype similar. Whether rd6 is an ortholog of a previously described human ocular disease or whether it identifies a new retinal degenerative disorder locus in this region, which contains a cluster of genes essential for normal eye function, will be answered by the identification of the gene. 

 Supported by National Eye Institute Grants EY07758 (JRH, MTD), EY06463 (GSH), EY11515 (GSH), and EY11996 (PMN); Cancer Center Core Grant CA34196 from the National Cancer Institute (The Jackson Laboratory); a grant from The Foundation Fighting Blindness; a Senior Scientist Award (JRH); a Lew R. Wasserman Merit Award (GSH) from Research to Prevent Blindness; and an unrestricted grant to The University of Iowa from Research to Prevent Blindness.

 Submitted for publication February 7, 2000; revised April 14, 2000; accepted April 26, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

(A, B) Fundus photographs of normal control C57BL/6J mouse (A) and a 4-month-old homozygous rd6 mouse (B) demonstrating multiple subretinal retinal spots. The white spots are first detectable ophthalmoscopically at approximately 8 to 10 weeks. They are uniform in size, being slightly larger in diameter than 50% of the largest retinal vessels, and evenly distributed throughout the retina.

Figure 2.

 

Stardardized ERGs showing rod- and cone-mediated ERGs across different ages. (A) Representative cone-mediated ERGs for a range of intensities from −0.19 to 1.13 log photopic trolands in a normal adult C57BL/6J mouse (left) and homozygous rd6 mice ranging in age from 10 to 70 weeks (left to right). (B) Representative rod-mediated responses obtained in the dark-adapted state, using a range of intensity settings from −3.0 to 1.42 log scotopic trolands, and short-wavelength flashes of light.

Figure 3.

 

Composite showing histologic progression of rd6 retinal degeneration. The upper half left panel depicts a normal retina, RPE, and choroid from a 2-year-old control, followed by histologic retinal sections from rd6/rd6 mutants at 1, 3, 5, 7, 11, and 29 months (magnification, ×400). The lower half shows the aberrant RPE-like cells in the subretinal space at 3 weeks and at 1, 3, 5, 7, and 11 months, and their absence at 29 months (magnification, ×900). Eyes from two to four mice were examined at each time point. There is progressive loss of the photoreceptors over approximately 16 months. The aberrant cells are initially unpigmented at 3 weeks as seen in the left panel and become pigmented and enlarged over time. Vacuoles and open spaces in the subretinal space are observed surrounding these aberrant cells.

Figure 4.

 

Fluorescence light micrographs depicting immunoreactivity of MOMA-2 antibody to the neural retina (A) and RPE (B) of a 4-month-old rd6/rd6 mouse. The two retinal layers became separated during tissue processing. Distinct cells located within the subretinal space react strongly with this antibody. Reactivity is positive based on negative controls (not depicted). MOMA-2 antibody is not detected in any other region of the RPE or retina, but some reactive cells were observed in the choroidal stroma (not shown).

Figure 5.

 

Transmission electron micrographs depicting the outer retina of the homozygous rd6 mouse. (A through C) RPE (labeled PE) and choroid (CH) shown in the top panel (magnification, ×2110); (D through F) the corresponding ONL (magnification, ×2400) shown in the bottom panel, from 2-month-old (A, D), 4-month-old (B, E), and 8-month-old (C, F) rd6 homozygous mice. Photoreceptor degeneration, as evidenced by pyknotic photoreceptor cell bodies (asterisks) in the ONL, occurs slowly over the first year of life. Cells throughout this layer degenerate such that approximately 4 rows remain by 8 months. Concomitantly, the outer segments become disoriented, although the depth of the subretinal space, between the RPE and outer limiting membrane (OLM), does not decrease appreciably between 2 and 8 months. Most significantly, distinct cells (arrows; A, C) are observed within the subretinal space at these ages. The cells average 35 μm in diameter at these ages. They are regularly spaced at intervals that correspond to the whitish-yellow dots observed ophthalmoscopically. Note that no other morphologically detectable structures are observed that might account for the spots.

Figure 6.

 

Higher magnification electron micrographs of the RPE (PE) and photoreceptor outer segment (OS) interface from 2-month-old (A; magnification, ×6415), 4-month-old (B; magnification, ×4187), and 8-month-old (C, magnification,× 4260; and D, magnification, ×3580) rd6/rd6 mice. The cells located within the subretinal space are round to somewhat flattened and are characterized by cytoplasmic inclusions that are indistinguishable from pigment granules and lipofuscin in the PE. In addition, they often contain electron-lucent vacuoles and whorls of membranous material. Note that the PE from the 8-month-old mouse shown in (C) and (D), but not in the younger mice, contains material that is indistinguishable from that observed in the subretinal cells. This is shown clearly in Figure 7 .

Figure 7.

 

Electron micrographs depicting membranous whorls and vacuoles in the cells within the subretinal space (asterisk; A, magnification, ×4200) and associated RPE cells (asterisk; B, magnification, ×5260) of an 8-month-old rd6/rd6 mouse.

Figure 8.

 

(A) Eighty-seven meioses from an intercross and a backcross between C3HfB-rd6/rd6 and C57BL/6J were phenotyped and genotyped. Linkage to several markers on mouse Chromosome 9 was observed. The columns of squares represent haplotypes (open boxes, C57BL/6J allele; filled boxes, C3HfB allele). The numbers of chromosomes with each haplotype are indicated below each column. (B) Genetic map of Chromosome 9 in the rd6 region showing the closest markers and the region of human homology.

The authors thank Cindy S. Avery, Ron E. Hurd, Priscilla Jewett, Heidi Hoopes Nienhaus, and Lisa Thayer for technical assistance; Jennifer Smith for graphic services; and Robert Mullins for assistance with the immunocytochemical analyses.