Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) mice

Neuron,

Vol. 9, 113-119, July, 1992, Copyright

0 1992 by Cell Press

Complete Rescue of Photoreceptor Dysplasia and Degeneration in Transgenic retinal degeneration slow (rd.s) Mice Gabriel H. Travis,* Karen R. Groshan,* Marcia Lloyd,+ and Dean Bok+ *Department of Psychiatry University of Texas Southwestern Medical Center Dallas, Texas 75235 +Jules Stein Eye Institute and Department of Anatomy and Cell Biology University of California Los Angeles School of Medicine Los Angeles California 90024

Summary retinal degeneration slow (rds) is a semidominant mutation of mice with the phenotype of abnormal development of rod and cone photoreceptors, followed by their slow degeneration. The rds gene has been putatively cloned and its novel protein product initially characterized biochemically. In the present study we undertook to correct in vivo the retinal phenotype of mice with the rds mutation. We assembled a transgene containing a regulatory segment of the opsin gene positioned upstream of the wild-type rds coding region. Mice from three transgenic lines, homozygous for the rds mutation, were analyzed for expression of the transgene and for their retinal phenotypes. In two high expressing lines, we observed complete reversion to wild-type retinal morphology. In a third, low expressing line, we observed a retinal phenotype intermediate between wild type and rdslrds, suggesting partial rescue of the mutation. These results constitute formal proof that we have cloned fhe rds gene. Introduction The vertebrate photoreceptor is a specialized neuron consisting of a cell body and an outer segment attached by a connecting cilium. The outer segment is a membrane-rich structure, densely packed with flattened discs containing rhodopsin visual pigment and other proteins involved .in visual transduction. Outer segments appear developmentally in mouse retina late during the first postnatal week. In mice homozygous for the retinal degeneration slow (rds) mutation, no outersegments are formed (Van Nie et al., 1978; Cohen, 1983; Sanyal, 1987). Instead, rhodopsin-containing vesicular structures are present in the subretinal space and have been observed budding from the ciliary processes of the otherwise normal inner segments (Nir and Papermaster, 1986; Usukura and Bok, 1987; jansen et al., 1987). The second phase of the rds phenotype is degeneration of the photoreceptor cell bodies, a process that requires up to 1 year to reach completion. At this time the retina is totally devoid of photoreceptors, with all other retinal cell types present and morphologically intact. In rdsl+ het-

erozygotes, a milder phenotype is seen. Outer segments are present, but are shortened and disorganized, with swelling and vacuolation of the discs (Hawkins et al., 1985). The degeneration of photoreceptors is much slower in rdsl+ heterozygotes. The rds gene was cloned in a subtractive screen for photoreceptor-specific mRNAs, without prior knowledge of its encoded protein (Travis et al., 1989). The rds mutation was found to result from a10 kb insertion of a mouse genomic repetitive element into a proteincoding exon. The product of the wild-type rds gene is a 39 kd membrane-associated glycoprotein that is present exclusively in photoreceptor outer segment discs (Travis et al., 1991a; Connell et al., 1991). The function of the normal rds protein is unknown. Its distribution and primary structure, in conjunction with the above-described ultrastructural features of rds mutants, suggest that it may play a role in the folding or stabilization of outer segment discs. In the present study we undertook to correct the rds mutation in vivo, to confirm that we have cloned the rds gene and as a first step toward a genetic analysis of the function of the rds protein. Results Generation of Transgenic Mice We assembled a transgene containing an opsin regulatory fragment positioned upstream of the wild-type rds coding region (Figure 1). In other studies, opsin promoter segments fused to /acZ reporter genes have been shown to drive rod-specific expression of B-galactosidase with a developmental onset that shortly follows the appearance of outer segment structures (Lem et al., 1991; Zack et al., 1991). The construct was microinjected into fertilized eggs from rds/+ heterozygous mutant mice. A total of six transgenic lines were established. Three werechosen for study based upon their breeding productivity. To move the transgene onto an rdslrds homozygous mutant background, FI mice from lines 96,113, and 80 were mated with nontransgenic rdslrds homozygotes. The genotype of each Fz mouse at rds was determined by following a Bglll restriction fragment length polymorphism that results from an insertional mutation within the rds gene on Southern blots (Figure 2; Travis et al., 1989). Only mice homozygous for the rds mutation were analyzed further. Expression of the Transgene To quantitate expression of the transgene in the three lines, RNAwas prepared from the retinas of individual F2 mice for Northern blot analysis, using cloned SV40 and a mouse rds cDNA as probes. The SV40 probe hybridized to a 2.6 kb band in lanes containing retinal RNA from the transgenic mice, but not the nontransgenic control mice (Figure 3a). This was the pre-

NeU”3” 114

opsin promoter segment // N 6.5 kb

Barn HI (60)

Bgl II

Map of the Opsin-rds-SV40

(1.527)

SV40 terminator I

f--------------Figure 1. Schematic

Bgl II

Pst I (963)

(451)

2.6 kb transcript

Fusion Transgene

A segment of the mouse opsin gene from nucleotide -6500 to f80 is followed by a central fragment of cDNA clone B9A (Travis et al., 1989) containing the complete wild-type rds coding block, followed by the SV40 t-intron and polyadenylation signal. Restriction sites with nucleotide positions of rds clone B9A are indicated. The opsin transcription initiation site (Baehr et al., 1988) is indicated by the arrow, the rds coding region by the open box, and the polyadenylation site by (A),.

transgenic lines was estimated by immunoblotting, using polyvalent antisera raised against a p-galactosidase-rds fusion protein (Travis et al., 1991a). These antisera reacted with a protein species of approximately 39 kd in retinal extracts from wild-type mice, but not with those from retinal degeneration (rdlrd) mutant mice, which virtually lack photoreceptors (Figure4; Carter-Dawson et al., 1978). No immunoreactivity was observed in retinal extracts from predegenerate rdslrds mutant mice. Given that the gene is transcribed in rds mutants (Figure 3b), the absence of immunoreactivity in extracts from these animals may be due to either instability of the mutant translation product, or exclusive reactivity of the anti-fusion protein seraagainst epitopeswithin thecarboxy-terminal third of the normal protein, which is deleted in rds mutants (Travis et al., 1989). An immunoreactive band of 39 kd was present in lanes containing retinal extracts from line 96 and 113 transgenic mice on an rdsl r& mutant background (Figure 4). This band was shifted downward by approximately 2 kd after endoglycosidase F digestion (data not shown), similar to the shift seen with the wild-type protein (Travis et al., 1991a). Thus, an rds-immunoreactive glycoprotein of normal size is produced in the retinas of high expressing transgenic lines 113 and 96. There was no detectable immunoreactivity in the lanes containing extracts

dieted size for the transcript from the opsin-r&-W40 transgene (Figure 1). The 2.6 kb transcript was abundant in RNAfrom lines96and 113, butwas rare in RNA from line 80. The rds probe detected two mRNAs of 2.7 and 1.6 kb, previously shown to arise from alternative polyadenylation (Travis et al., 1989) in wild-type retina, and a pair of mRNAs of 12-14 kb in rdslrds mutant retina (Figure 3b). As predicted, all four bands were detected in retinal RNAfrom rdsl+ heterozygotes.The 2.6 kb mRNA from the transgene was present in retina from lines 113 (data not shown), 96,.and 80, but not in their nontransgenic littermate controls. The abundance of the 2.6 kb transgene transcript in line 96 was approximately equal to the 2.7 kb transcript of the endogenous rds gene in wild-type retina. In line 80, the 2.6 kb transgene transcript was only about-onetenth as abundant as the 2.7 kb wild-type mRNA, based upon autoradiograph signal intensity (Figure 3b). There was a rough correlation between the number of copies of the transgene in each line detected by Southern blot analysis and the levels of transgene expression. Lines 96 and 113 each had IO-20 copies per haploid genome, and line 80 had 2-4 copies (data not shown). All transgenic mice studied were hemizygous for the transgene. The level of expression oftherds protein in thethree

e $

8.16.15.14.1-

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Figure2. Southern Blot Analysis of Tail DNA Samples Showing the Genotype at rds

p

-

8.1 6.1 5.1 4.1

- 3.1

Each sample was digested with Bglll, and the blot was probed with DNA from clone B9A. Wild-type (w/t), rdsl+ heterozygote, and rdsirds homozygote DNAs are ineluded as genotype controls. The Bglll polymorphic pattern for each of the test animals is identical to that of the rdslrds homozygote control. The region of the gel showing the signal from the transgene was deleted for clarity. Positions of DNA size standards are indicated on the sides of both gels.

Rescue of rds in Transgenic

Mice

115

a

llO-

84-

2.6 Mr

G3PDH

47-

3324-

b Figure 4. Protein

Expression

in rds Transgenic

Mice

lmmunoblot analysis of retinal proteins showing expression of the transgene. Retinal extracts from transgenic lines 96 (animal 96d), 113 (pooled animals 113b and 113e), and 80 (pooled animals 80mm and 8Oqq), each on an rdslrds genetic background, were reacted with an anti+galactosidase-rds fusion protein antiserum. Retinal extracts from nontransgenic C57BU6 wild-type (w/t), fully degenerate,rd (rdlrd), and predegenerate rds (rdslrds) miceare included as controls. Positions of molecularweight size standards are shown on the left in kilodaltons.

2.7 1.6-

G3PDH Figure 3. Northern Blot Analysis sion of the Transgene

of Retinal RNA Showing

Expres-

(a) RNA from line 96, 113, and 80 transgenic (Xl and nontransgenic (nTG) mice probed with the transcription terminator fragment of SV40. RNAs from nontransgenic C57BU6 wild-type (w/t) and rdslrds homozygous mutant control retina are included as controls. (b) RNA from line 96 and 80 transgenic and nontransgenic mice probed with clone B9A. Samples of nontransgenic wild-type (w/t), rdsl+ heterozygote, and rdslrds homozygote retinal RNA are included as controls. Sizes of the major RNA bands in kilobases are indicated to the left. The insets i,, -led G3PDH shows the autoradiographic signal after the blots were stripped and reprobed with a cDNA of glyceraldehyde-3-phosphate dehydrogenase CTso et al., 1985) as a control for equal loading of lanes.

from transgenic line 80. This was probably because the low level of transgene expression in line 80. Phenotype in Transgenic rdslrds Homozygous Mutants Retinas from line 96, 113, and 80 transgenic

of

mice on

an rdslrds mutant background at 2 and 5 months of age were studied by light and electron microscopy. Nontransgenic littermates were used as controls. All features of the rds phenotype were present in these mutant controls (Figure 5a), including absence of outer segments, partial degeneration of photoreceptor cell bodies, and vesicular debris in the subretinal space (Jansen and Sanyal, 1984). In contrast, retinas from transgenic lines 96 and 113 were indistinguishable from wild type, with normal outer segment morphology and no evidence of photoreceptor degeneration by both light (Figures 6a and 6b) and electron microscopy (Figure 5b and 5~). A complete rescue of the rds phenotype was thus effected in rod cells from line 96 and 113 mice. Interestingly, retina from transgenic line 80 had an appearancethat was intermediate between that of retinas from wild-type mice and rdslrds mutants (Figure 5d). There were fewer extracellular vesicles. In their place, whorls of disorganized discs were present in the subretinal space, likely representing dysplastic outer segments. This pattern is similar to that seen in rds/+ heterozygotes (Hawkins et al., 1985), except that in animals from line 80, the outer segment dysplasia was more severe. This difference in severity may be duetoadosageeffect;inline80thelevelofthemRNA encoding the normal rds protein is only about 10% of wild type, whereas in heterozygotes it is approximately 50% (Figure 3b).

NE!UWl 116

Figure 5. Electron Microscopic Analysis of Transgenic Lines 96, 113, and 80 on an rdsirds ground

Retinas Mutant

from Back-

In all cases, the retinal pigment epithelium (RPE) is at the top of the figure. (a) Nontransgenic control mouse 96j (2 months old). Rod inner segments (IS) and connecting cilia (arrows) are present, but outer segments are absent. Magnification, 3300x. (b) Superior nasal retinal quadrant of transgenic mouse 96q (littermate of 96j; 2 months old). Fully developed outer segments (OS) containing regularly arranged disc structures are visible between the retinal pigment epithelium (RPE) and inner segments (IS). Magnification, 2900x. (c) Superior temporal quadrant of transgenic mouse 113h (5 months i old). As with 96q, the outer segments (OS) appear morphologically normal and there is no evidence of photoreceptor degeneration. Magnification, 3800x (d) Inferior temporal quadrant of low expressing transgenic mouse * 80q (2 months old). Partial development of outer segment discs is evident from the membranous whorls (arrows) between the retina I pigment epithelium (RPE) and inner segments (IS). Magnification, 3800 x

Rescue 117

of rds in Transgenic

Figure 6. Light Micrographs

Mice

of Retina from

the Inferior

Nasal and Inferior

Temporal

Quadrants

of Transgenic

Line 96

(a) Inferior nasal quadrant. (b) Inferior temporal quadrant. The retinal pigment epithelium (RPE), rod outer segments (OS), and inner segments (IS) appear normal, as does the thickness of the outer nuclear layer, which contains the photoreceptor cell bodies. Magnification, 560x.

Discussion Here we have shown a correlation between correction of the retinal phenotype in rdslrds mutant mice and the level of expression of a transgene encoding the wild-type rds protein. In lines 96 and 113, which both had morphologically normal retina, the transgenewas expressed at levels similar to those of the endogenous rds gene in wild-type mice. The intermediate phenotype in low expressing line 80 probably represents a partial titration of the mutation. In normal mice, the rds gene is expressed in both rods and cones (Arikawa et al., 1992). If we assume that the r&-containing transgene, transcriptionally regulated bytheopsin promoter, hasthesame pattern of expression as the endogenous opsin gene, then we would expect the rds phenotype to have been rescued in rod but not cone photoreceptors. W e were unable to confirm rod-specific correction, however, since only 3% of photoreceptors in mice are cones (CarterDawson et al., 1978) and since the retinas of 5-monthold rdslrds mice, the oldest animals examined in this study, are only partially degenerate (Sanyal, 1987). The rds mutation has been shown to result from the insertion of a IO kb mousegenomic repetitiveelement into a protein-coding exon of the rds gene (Travis et al., 1989). This gene is transcribed in rds mutants, giving rise to a pair of mRNAs of approximately 12 kb, each bearing the entire inserted element (Figure 3b). It is not known whether these mRNAs are translated. If so, the last 116 residues of their putative mutant protein products would be truncated, including the

glycosylated region of the D2 loop, the fourth membrane-spanning segment, and thecytoplasmic C3 segment (Travis et al., 1989). In their place is an aberrant stretch of amino acids encoded by the inserted element, before the first in-frame stop codon. There are two possible mechanisms for the dysplastic phenotype in rdsl+ heterozygotes consistent with these observations. One is a dominant-negative effect of the putative anomalous protein in outer segment discs, perhaps through displacement of the normal protein from its sites of interaction with other proteins (genetic antimorph). The second mechanism is haploinsufficiency, by which a 50% reduction in wild-type gene dosage results in a critical loss of the normal rds protein (genetic hypomorph). The results presented in this paper are more consistent with the second mechanism. In lines 96 and 113, both the mRNA and the protein products of the transgene were at levels similar to the levels in normal mice of the endogenous, wild-type rds mRNA and protein. Since the transgenics were on a background with two mutant alleles of rds, if a dominant-negative mechanism were operative, we would not have expected a complete rescue, but rather a phenotype similar to that of the rdsl+ heterozygote. Similarly, in the case of low expressing line 80, if a dominant-negative mechanism were operative, the toxic product of two mutant rds alleles would have overwhelmed the small amount of normal rds protein encoded by the transgene, and no rescue of the phenotype would have been expected. Two previous reports describe the use of smaller upstream genomic segments of opsin driving expres-

sion of a /acZ reporter gene (Lem et al., 1991; Zack et al., 1991). Nonuniform expression patterns in retinaof the /acZ product were observed in several of these transgenic lines. In the present study, rescue of the rds phenotype in photoreceptors was uniform across the retina in the three fines studied, with no patchy or gradient changes in the morphology of outer segments (Figures 5 and 6). The absence of apparent retinal sector gradients in outer segment morphology may be due to a threshold effect, whereby photoreceptors situated in regions of the retina showing low expression still make sufficient transgene product to complement the rds phenotype fully. Alternatively, expression of the transgene may actually be uniform throughout the retina in these lines, as the result of our useof a largeropsin promoter segment.Thisquestion should be resolved by quantitative in situ hybridization. Recently it has been shown that the human homologoftherdsgene(RDS) (Traviset al., 1991b) isaffected inasubsetof patientswithautosomaldominant retinitis pigmentosa (ADRP; Kajiwara et al., 1991; Farrar et al., 1991). The mutations in RDS that have been observed in ADRP all result in single residue deletions or nonconservative amino acid substitutions and hence are likelyto be less severealleles than rds in mice. This is in agreement with the much slower progression of blindness in humans with ADRP than in rds/+ mice, although species differences may also be playing a role. The photoreceptor outer segments are completely absent from rdslrds mice. Despite this absence, the electroretinogram is detectable in predegenerate rds mutants (Reuter and Sanyal, 1984), suggesting that visual transduction can occur in the plasma membrane of photoreceptor cell bodies and that the progressive blindness observed in rds mice and humans with mutations in RDS is primarily due to photoreceptor degeneration. In summary, we have rescued the morphologic phenotype in rds mice by transgenic complementation. This represents formal confirmation that rds has been cloned. The relationship between the levels of transgene expression and the retinal phenotype in the three lines studied suggests that rds is a simple lossof-function mutation. This has ramifications for the eventual development of somatic gene therapy, using nonpathogenic herpes simplex virus vectors, for the subset of ADRP patients with mutations in the RD.5 gene. Experimental

Procedures

Generation of Transgenic Mice The mouse opsin gene was cloned in phage h by probing a BALBlc genomic library with a mouse opsin cDNA (Travis et al., 1989). The full-length clone !39A of the wild-type rds cDNA was isolated as previously described (Travis et al., 1989). A 0.9 kb Bglll-BamHI fragment containing the SV40 t-intron and polyadenylation signal was prepared from plasmid pSV2bg (Subramani and Southern, 1983). The transgenic rescue construct (pOBSK) wasassembled in the plasmid pSK(Stratagene, La Jolla, CA) using standard techniques for the manipulation of recombinant DNA.

Insert DNA from pOBSKwas purified on a sucrose gradient and injected into the pronuclei of fertilized eggs from BALBic x C57BU6 hybrid mice that were heterozygous for rds. Eggs surviwing micromanipu!ation were transferred to the oviducts of pseudopregnant foster mothers, following described methods (Hogan et al., 1986). All mice were maintained on 12 hr light/dark cycles at low levels of illumination to minimize photic injury. Southern Analysis of Transgenic Mice Mice were analyzed for the presence of the transgene by Southern blot analysis of tail DNA (data not shown). Ten microgram samples of DNA were digested with EcoRI. These were separated by electrophoresis in 0.8% agarose and transferred to Biotrans Plus filters (ICN Biomedicals, Irvine, CA) in alkaline conditions. An SV40-containing restriction fragment of pSV2& was radiolabeled with aP by random priming (Feinberg and Vogelstein, 1983) for use as a probe. To determine the genotype at the rds locus of each transgenic mouse, Southern blot analysis was performed on tail DNA digested with Bglll and probed with the 513 bp Bglll-Pstl fragment of rds clone B9A. Final stringency wash conditions fo: all Southern blots were 0.2x SSC at 65’C. Northern Blot Analysis of Retinal RNA RNA was prepared from the retinas of individual animals as previously described (Travis et al., 1989). Five microgram samples of total RNA were separated by electrophoresis on 1.2% agarose formaldehyde gels and transferred to nitrocellulose (Thomas, 1980). Probes were made from SV40 and B9A DNA as described above. Final stringency wash conditions were 0.2x SSC at 65OC. Preparation of Anti-rds Antisera The 1076 bp Bglll fragment of rds clone B9A, encoding residues 81-346 (Travis et al., 1989), was cloned into the BamHl site of the prokaryotic expression plasmid pUR292 (Rtither and Miiller-Hill, 1983). Upon induction with isopropyl-P-n-thiogaiaclopyranoside, a bacterial culture transformed with this plasmid expresses a P-galactosidase-rds fusion protein at high levels (Travis et al., cultures were separated 1991a). Extracts from these transformed by electrophoresis in 12% polyacrylamide gels containing 0.1% SDS. After destaining in 10% acetic acid, a major band of M. 150,000, corresponding to the induced fusion protein, was dissected from the gel. This was pulverized in liquid Nz and suspended in a 50% emulsion of Freund’s incomplete adjuvani for subcutaneous immunization of rabbits, following procedures approved by the Animal Resources Center at UT Southwestern. lmmunoblot Analysis of Retinal Protein Extracts Frozen mouse retinas from each line were homogenized on ice in 300 PI of phosphate-buffered saline (PBS) and centrifuged at 27,000 x g far 15 min. The pellets were washed twice with 200 kl of PBS and then extracted with 100 hi of PBS containing 2% SDS.Thesupernatantswerecollectedafteral5minspinat13,400 x g. These sampies were incubated in 5% P-mercaptoethanol at room temperature. Samples containing 5 pg of protein were electrophoresed in 12% polyacrylamide gels containing 0.1% SDS. Proteins in the gels were transferred to nitrocellulose, and the immunoreactive bands were visualized using a l/100 dilution of the rabbit anti+galactosidase-rds fusion protein antiserum #X640-5/17 and the ABC detection procedure supplied by Pierce Chemical Co., Rockford IL. Preparation of Samples for Light and Electron Microscopy Animals were fixed by vascular perfusion of formaldehyde and glutaraldehyde (1.0%:2.0%, except mouse 113h, which was 1.0%: 1.0%) in 100 mM sodium phosphate buffer (pH 7.2), followed by fixation in 1.0% osmium tetroxide. The tissue was dehydrated and embedded in Araldite 502 (Ciba Geigy Corp.). Staining was with toluidine blueafter sectioning for light microscopy and with uranium and lead salts after sectioning for electron microscopy. Acknowledgments We gratefully

acknowledge

Jenny Price for her skillful

manipula-

Rescue of rds in Transgenic 119

Mice

tion of mouse oocytes, Orna Yaron for her assistance with the Immunoblots, and Prakash Bhatia, Jessie E. Hepler, John Mercer, and James Norton for their useful comments on the manuscript. This work was supported by grants from the National Eye Institute and the National Retinitis Pigmentosa Foundation. G. H. T. is a John Merck Fund scholar. D. B. is the Dolly Green Professor of Ophthalmology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

11, 1992; revised

April

U., and Miiller-Hill, B. (1983). Easy identification EMBO J. 2, 1791-1794.

of cDNA

Sanyai, S. (1987). Cellular site of expression and genetic interaction of therdand rds loci in the retinaof the mouse. In Degenerative Retinal Disorders: Clinical and Laboratory Investigations (New York: Alan R. Liss, Inc.), pp. 175-194. Subramani S., and Southern P. J. (1983). Analysis of gene expression using simian virus 40 vectors. Anal. Biochem. 135, 1-15. Thomas, P. S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201-5205. Travis, G. H., Brennan, M. B., Danielson, P. E., Kozak, C. A., and Sutcliffe, J. G. (1989). Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds). Nature 338, 70-73.

27, 1992.

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Travis, C. H., Sutcliffe, J. C., and Bok, D. (1991a). The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated glycoprotein. Neuron 6, 61-70. Travis, G. H., Christerson, L., Danielson, P. E., Klisak I., Sparkes, R. S., Hahn, L. B., Dryja, T. P., and Sutcliffe, 1. G. (1991b). The human retinal degeneration slow (rds) gene: chromosome assignment and structure of the mRNA. Genomics 70, 733-739. Tso, J. Y., Sun, X. H., Kao, T. H., Reece, K. S., and Wu, R. (1985). Isolation and characterization of rat and human glyceraldehyde3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucl. Acids Res. 73,2485-2502. Usukura, J., and Bok, D. (1987). Changes in the localization and content of opsin during retinal development in the rds mutant mouse: immunocytochemistry and immunoassay. Exp. Eye Res. 45, 501-515. Van Nie, R., Ivanyi, D., and Demant, P. (1978). A new H-2 linked mutation, rds, causing retinal degeneration in the mouse. Tissue Antigens 12, 106-108. Zack, D. J., Bennett, J., Wang, Y., Davenport, C., Klaunberg, B., Gearhart, J., and Nathans, J. (1991). Unusual topography of bovine rhodopsin promoter-/acZ fusion gene expression in transgenie mouse retinas. Neuron 6, 187-199.