Proc. Natl. Acad. Sci. USAVol. 90, pp. 5499-5503, June 1993Neurobiology

Simulation of human autosomal dominant retinitis pigmentosa intransgenic mice expressing a mutated murine opsin geneMUNA I. NAASH*, JOE G. HOLLYFIELD, MUAYYAD R. AL-UBAIDI*, AND WOLFGANG BAEHRtDepartments of Ophthalmology and Biochemistry, Baylor Coliege of Medicine, Houston, TX 77030

Communicated by Salih J. Wakil, March 19, 1993

ABSTRACT Autosomal dominant retinitis pigmentosa(ADRP), slowly progressing over decades, leads to severe visualimpairment and in some cases to complete blindness. Morethan 40 mutations in the human opsin gene have been linked tosome forms of this genetically heterogeneous disease. In pho-toreceptor cells of ADRP patients with mutations in the opsingene, normal rhodopsin is thought to be synthesized concom-itantly with mutated rhodopsin, which, by an unknown mech-anism, causes the slow degeneration of the photoreceptor cells.To establish a transgenic mouse line that carries a mutatedmouse opsin gene in addition to the endogenous opsin gene, weintroduced a mouse opsin gene containing mutations in exon 1into the germ line of a normal mouse. The alterations consistedof three amino acid substitutions near the N terminus ofrhodopsin, Val-20 -- Gly (V20G), Pro-23 -- His (P23H), andPro-27 -* Leu (P27L). The P23H mutation is the most prev-alent mutation in human ADRP patients. During early post-natal development, mice heterozygous for the mutated opsingene appear to develop normal photoreceptors, but theirlight-sensitive outer segments never reach normal length. Withadvancing age, both rod and cone photoreceptors are reducedprogressively in number. The slow degeneration of the trans-genic retina is associated with a gradual decrease of light-evoked electroretinogram responses. Our results show thatsimultaneous expression of mutated and normal opsin genesinduces a slow degeneration of both rod and cone photorecep-tors and that the course of the retinal degeneration of themutant mouse retina mimics the course of human ADRP.

The term retinitis pigmentosa (RP) comprises a large hetero-geneous group of diseases characterized by photoreceptordegeneration (1). In most cases, RP leads to loss ofperipheraland eventually central vision. The earliest symptoms, mea-surable before visible onset of the disease, are abnormallight-evoked electroretinograms (ERGs) (2, 3). The disease isuntreatable, affects 0O.03% of the U.S. population, and isinherited as an autosomal dominant (ADRP), recessive, or Xchromosome-linked trait (1). The ADRP form is less preva-lent and less severe than the recessive form (1, 3) and hasbeen linked to loci on chromosomes 3q (rhodopsin), 6p RDS(retinal degeneration slow)/peripherin, and 8p (unknown)(4). About 25% of all ADRP cases appear to be caused byvarious mutations in the gene encoding rhodopsin (3, 5-12),the light-sensitive pigment in rod photoreceptors, and to amuch lesser extent peripherin (RDS/peripherin) (12, 13), arod-specific protein of unknown function. The most preva-lent mutation in human ADRP patients, in which Pro-23 nearthe N terminus of rhodopsin is replaced by His (P23H), hasbeen identified in =15% of ADRP families (3, 7, 14) in theUnited States but in none of the families in Europe. Directevidence that a mutated opsin gene may cause an ADRP-likeslow degeneration has not been provided.

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

To understand how the expression of a mutated opsin geneaffects photoreceptor metabolism and how the presence ofboth normal and mutant rhodopsins leads to a slowly pro-gressing photoreceptor degeneration, animal models areneeded. Mouse mutants that mimic the course of humanADRP have not been described, to our knowledge. Thenaturally occurring mouse rd and rds photoreceptor dystro-phies are recessively inherited (15) and were shown to havedefects (loss of function) in the cGMP phosphodiesterase(-subunit gene (16, 17) and in the peripherin gene (18, 19),respectively. Transgenic mice that harbor 15.9-17.5 kb ofnormal and mutant P23H human opsin genes, including 5 kbof upstream regulatory sequences, have recently been gen-erated (20). Three lines containing the mutant gene exhibiteda rapid photoreceptor degeneration uncharacteristic of hu-man ADRP (20). Unfortunately, control experiments, inwhich a normal human gene was introduced, also generateda rapid degeneration in one of two transgenic lines (20). Tocreate a transgenic mouse line that expresses both normaland mutant murine rhodopsins under the control of theendogenous murine promoter in rod photoreceptors and thatcould be utilized to follow gene expression on both thetranscriptional and translational levels, we introduced bymicroinjection a mutant mouse opsin gene into the germ lineof a normal mouse. Except for five point mutations [tworestriction fragment length polymorphisms (RFLPs) andthree altered amino acids], the mutant gene is identical to theendogenous murine gene. In this paper we show that theexpression of the mutant opsin transgene occurs simultane-ously with the wild-type opsin gene and that the consequenceof its expression is a slowly progressing rod and conephotoreceptor degeneration.

MATERIALS AND METHODSPreparation of the Transgene. The transgene was con-

structed by oligonucleotide-directed mutagenesis and inser-tion of a mutated fragment into AMO1 containing the com-plete murine opsin gene (21). The following five oligonucle-otide primers were used for amplification of mutated opsingene fragments: W75 (positions -245 to -209), MN1 (posi-tions 177 to 201), MN2 (positions 211 to 142), MN3 (positions446 to 377), and Wll (positions 1082 to 1062). The positionsand sequences are as described in ref. 21. MN2 and MN3 are70-meric antisense oligonucleotides that introduce the de-sired mutations. Amplified products W75/MN2 (446 bp) andMN1/MN3 (270 bp) were annealed, extended with Taqpolymerase, and reamplified with W75/MN3. This product(681 bp) was cut with Xho I, and the resulting 349-bp Xho Ifragment (see Fig. 1B) was cloned into MOPS2 (22), a 5.1-kbEcoRI fragment containing the entire opsin structural gene,

Abbreviations: RFLP, restriction fragment length polymorphism;RP, retinitis pigmentosa; ADRP, autosomal dominant RP; ERG,electroretinogram; P, postnatal day.*Present address: Department of Ophthalmology and Visual Sci-ences, University of Illinois, 1855 West Taylor Street, Chicago, IL60612.tTo whom reprint requests should be addressed.

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from which the corresponding wild-type Xho I fragment hadbeen removed. The desired clones were identified by thepresence of the predicted RFLPs and checked for correctorientation of the insert. Finally, one clone that displayed thedesired mutations and RFLPs was verified by DNA sequenc-ing. A 5.1-kb EcoRI fragment of this clone was ligated intoAMO1 from which the wild-type EcoRI fragment had beenremoved and packaged into viable EMBL3 phage. The re-sulting phage clones were screened for the correct orientationand sequence. The transgene, a 15-kb Sal I fragment, waspurified by agarose gel electrophoresis, diluted to 10 ng/,ul,and injected into 1-day C57BL/6 x SJL mouse embryos(DNX, Princeton).

Identification of Founder Mice. Of the 11 mice born,founder mice were identified by the absence of the Nco I sitein genomic DNA (Southern blot analysis) or after PCRamplification of exon 1 with primers W75/W11, followed bydigestion with Nco I. Wild-type mice exhibit three fragmentsof 689 bp, 431 bp, and 197 bp. Transgenic (heterozygous)mice have an additional 886-bp fragment (689 bp + 197 bp)due to the deletion of one Nco I site. Founder mice (+/rd)were mated to C57BL/6 mice, and transgenic offspring wereidentified by the presence ofthe Nco I RFLP. Transgenic andnontransgenic offspring were shown to be +/rd or +/+ forthe rd locus by analysis of the Dde I RFLP in exon 7 of thecGMP phosphodiesterase P subunit gene (17). The rd locuswas eliminated by outbreeding transgenic to C57BL/6 mice.In Vitro Transcription of Normal and Mutant RNAs. To

synthesize mutant and normal murine opsin mRNAs, pB-MOPSMUT and pB-MOPS were prepared by subcloning a5.1-kb EcoRI fragment [MOPS2 (21)] with or without themutations, respectively, into pBluescript. Before transcrip-tion with T7 and T3 RNA polymerases, both clones werelinearized with Hae II, which cuts at position 1985 (21) inexon 2 of the opsin gene. The calculated size of the producedRNA starting at the T3 or T7 promoter is 2250 nt.RNA Blot. Retinal RNA from normal and transgenic ani-

mals at ages indicated in Fig. 2 was isolated using theFastTrack procedure (Invitrogen, San Diego). "Adult" RNAwas isolated from retinas of mice ages 1.5-4 months. The24-meric antisense oligonucleotides used as probes are N(5'-CGG CTG CTC GAA GGG GCT CCG CAC) and M(5'-CAG CTG CTC GAA GTG ACT CCG ACC). Oligonu-cleotides N and M were gel-purified, end-labeled with T4polynucleotide kinase and [y-32P]ATP, and separated fromunincorporated ATP by a spin column (Bio-Rad) procedure.Hybridizations were performed in 40o (vol/vol) forma-mide/5x Denhardt's solution/5x standard saline citrate(SSC)/10% (wt/vol) dextran sulfate/50 mM sodium phos-phate, pH 6.8/0.1% SDS/denatured salmon sperm DNA (250ng/ml)/100 ng of end-labeled oligonucleotide at 42°C for 24 has described (23). The blot was washed with 2x SSC/0.2%SDS and 0.5x SSC/0.2% SDS at room temperature. The blotwas examined for the amount of RNA loaded by reprobingwith -y-actin cDNA. The amount of RNA blot was identicalin all lanes (data not shown).

Electroretinograms. The mice were dark-adapted for 2-3 h,followed by ERG recordings in dim red light. The ERGs wererecorded with 50-msec flashes of 500-nm light (maximumintensity, 1100 ,AW/cm2) as described (24).

Histology. For light microscopy, enucleated eyes werefixed and processed as described (25).

RESULTSGeneration ofthe Mutant Mouse. Transgenic mice that have

incorporated a mutated mouse opsin gene were expected tosimultaneously express the mutated and the normal opsingenes in rod photoreceptors, possibly at comparable levels.To distinguish between normal and mutant transcripts and tofollow the fate of normal and mutant rhodopsins, we designed

two types of mutations. (i) We introduced RFLPs that do notalter the amino acid sequence of opsin (addition ofaHinfl siteand removal of a Nco I site; Fig. 1B). Since no foreign DNAwas incorporated, the RFLPs were essential for identificationof a founder mouse by PCR amplification of exon 1 anddigestion of the amplified DNA with the correspondingrestriction enzyme or by Southern blot analysis. (ii) Wegenerated an epitope (Fig. 1A) that may be recognized by ananti-mutant peptide antibody by altering three amino acidsnear the N terminus of rhodopsin (V20G, P23H, and P27L).One of the three altered residues is Pro-23, a well-conservedresidue in guanine nucleotide binding protein-linked recep-tors and the predominant point mutation in ADRP patients.The other two altered residues have not been observed inhuman ADRP and are much less conserved. Thus, wegenerated a murine allele that is not absolutely identical to thehuman disease allele, which carries one missense mutationonly. Expression of the transgene, however, by design willpermit the determination of the fate of mutant and normaltranscripts and the mutant and normal gene product,rhodpsin, that would not be possible with a simple duplica-tion of the human disease allele.The final transgene (Fig. 1C) consisted of a 15-kb mouse

opsin genomic fragment that contained 6 kb of upstream and3.5 kb of downstream sequences [AMO1 (21)], and in whicha part of the wild-type exon 1 of the opsin gene was replacedby a fragment containing the mutations. Injection of the

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FIG. 1. Transgene containing mutations introduced into exon 1 ofthe mouse opsin gene. (A) The N-terminal amino acid sequence ofmouse opsin (first 31 residues). Pro-23 (P), the residue most fre-quently mutated in ADRP patients, is boxed. Arrows mark the threeamino acid residues at positions 20, 23, and 27 that have been alteredin the transgene. An inverted Y symbolizes the carbohydrate chainattached to residues 2 and 15 of rhodopsin. (B) Exon 1 of thetransgene with relevant restriction sites and newly introducedRFLPs. E, EcoRI; X, Xho I; N, Nco I; boxed H, newly generatedHinfl site; (N), deleted Nco I site. The nucleotide numbering system

(21) starts with +1 at the transcription start site, depicted by thebeginning of the open box (5' untranslated region). The solid boxsymbolizes the coding region of exon 1. W75, MN1, MN2, and MN3are primers used for PCR amplification and mutagenesis. The barunderneath symbolizes the mutated 349-bp Xho I fragment that wasused to replace the corresponding wild-type fragment of the opsingene fragment AMO1 (21). (C) Map of the transgene containing thefive exons encoding opsin (solid boxes numbered 1-5), the large 3'untranslated region containing multiple polyadenylylation signals

(21), and the Hinfl and Nco I RFLPs in exon 1. The transgene isdelimited by Sal I (S) sites derived from the multiple cloning site ofthe A vector (EMBL3, hatched boxes).

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FIG. 2. Northern blot of retinal poly(A) mRNA isolated fromtransgenic and wild-type animals at P5 and P20 and as an adult (ad).Autoradiographs of the same blot first probed with end-labeledoligonucleotide N (A) and then stripped and reprobed with end-labeled oligonucleotide M (B) are shown. Lanes: 1, 50 ng of mutantRNA (M-RNA) transcribed from pB-MOPSMUT with T7 RNApolymerase; 2, 50 ng of normal RNA transcribed from pB-MOPSwith T3 polymerase; 3, blank; 4, 6, and 8, 1 ,ug of transgenic retinalpoly(A) RNA isolated from P5, P20, and adult animals, respectively;5, 7, and 9, 1 ug of normal retinal poly(A) RNA (same ages astransgenic); 10-12, transgenic RNA isolated from kidney, liver, andbrain, respectively, each at 1 ,ug.

construct produced two founder mice that were identified bythe Nco I RFLP. One founder mouse passed the transgene toits offspring at 50% transmittance (Mendelian inheritance),whereas the other did not. Southern blot analysis of the F1offspring of the first founder indicated incorporation of twoto five transgene copies per haploid genome into a singleintegration site (data not shown).Normal and Mutated Opsin Genes Are Expressed Simulta-

neously. The mutations introduced into exon 1 of the trans-

gene allowed the design of two 24-meric oligonucleotideprobes (termed N and M) that specifically hybridize withnormal and mutant opsin mRNAs, respectively. As shown inFig. 2, the specificity of oligonucleotides N and M was

excellent: neither oligonucleotide N nor oligonucleotide Mrecognized even trace amounts of RNA (lanes 1 and 2)produced from mutant or normal plasmid vectors, respec-tively, nor did N and M hybridize to RNA of other tissues(lanes 10-12). Thus, we used oligonucleotide M to identifythe onset oftranscription ofthe mutant gene. A Northern blotcontaining both normal and transgenic retinal poly(A) RNAsisolated at postnatal day (P) 5, P20, and from an adult (>1month) was first probed with oligonucleotide N (normal) toidentify the five transcripts of the normal opsin gene (Fig.2A). We have shown (21) that the five species differ only inthe lengths of their 3' untranslated region, that they can bedetected with cloned cDNA probes as early as P1, and thatthe three fastest migrating species are predominant during thefirst 50 days of postnatal retinal development. The undis-turbed pattern of the five wild-type opsin mRNA species inthe transgenic animal (Fig. 2, lanes 6 and 8) indicates that thetransgene has not been incorporated into the mouse opsinlocus on chromosome 6. To determine the transcriptionalpattern of the transgene, the same blot was stripped andreprobed with end-labeled oligonucleotide M (mutant). Theresults show that the mutant RNA was processed correctly,as indicated by the presence of five mRNA species, exactlyas observed for the wild-type gene transcripts (Fig. 2B, lanes6 and 8). Moreover, the transgene appeared to be coex-pressed with the normal opsin gene, as shown by the ap-proximately equal levels ofmRNA at day 20 (Fig. 2B, lane 6).The slight decrease in intensity of mutant opsin mRNAs inFig. 3B, lane 6, as compared to the normal mRNAs in Fig.2A, lane 6, may be due to stripping of the blot, which may

lead to loss of 10-20%o of the bound RNA. Lack of a

hybridization signal at day 5 (Fig. 2 A and B, lanes 4 and 5),where the detectable RNA levels in the normal retina were<0.1% ofthose at day 20, is most likely due to the low specificactivity of the single-label oligonucleotide probe.ERGs Indicate a Slowly Progressing Photoreceptor Degen-

eration. The ERG is an electrical response evoked from theretina by a defined flash of light and is the most important testin the early diagnosis of RP in humans. The earliest detect-able defect is diminished rod function (2). As shown in Fig.3, bright-flash ERGs generated from dark-adapted F1 off-spring displayed a significant reduction in the amplitudes ofa and b waves at P30 (Fig. 3), a time when rod photoreceptors

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FIG. 3. ERGs of transgenic and nontransgenic littermates. (A) Top trace, a single-flash ERG recorded from a dark-adapted nontransgenic210-day-old littermate (N-210 days) (a and b denote a- and b-waves, respectively); lower traces, ERGs of transgenic littermates at ages 34, 85,150, and 210 days. Calibration bars designate 500 ,uV (y axis) and 50 msec (x axis). (B) Plot of dark-adapted a-wave amplitude of nontransgeniclittermates (open circles) and transgenic littermates (solid circles) vs. age in days. Values represent a median standard deviation of data fromthree to eight mice.

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Proc. Natl. Acad. Sci. USA 90 (1993)

FIG. 4. Temporal progressive changes in normal (a, c, and e) and transgenic (b, d, andf) littermates. Retinas shown were recovered at 20days (a and b), 32 days (c and d), and 7 months (e and!) postpartum. The horizontal arrowheads are located at the level of the retinal pigmentepithelium. The paired asterisks in each paired figure are located at the level of the outer plexiform layer. Photoreceptor outer segments, innersegments, and nuclei make up all the cellular mass between the retinal pigment epithelium and outer plexiform layer. The photoreceptor outersegments, located just below the level of the pigment epithelium at 20 days, are shorter in the transgenic than in the normal littermate (compareA and B). At 32 days, the outer segments remain shorter than their nontransgenic littermates (compare outer segments in C and D). Additionally,the number of photoreceptor nuclei in the outer nuclear layer is reduced in the transgenic as compared to the nontransgenic littermates.Photoreceptor cells continue to be lost during subsequent months and by 7 months, the transgenic retina contains only two or three rows ofphotoreceptor nuclei and little outer segment material is apparent (compare E with F). (Bar = 25 Am.)

are fully developed in nontransgenic littermates. BetweenP30 and P210, the a wave, characteristic of the photoreceptorresponse, gradually decreased and the b wave was reduced to=30%o of normal. The gradual a-wave reduction indicates aloss of photoreceptor sensitivity in the transgenic retina,consistent with a slow progressive photoreceptor degenera-tion. Thus, we were observing a slow degeneration thatprogressed over >50% of the lifetime of a normal mouse,paralleling the course of human ADRP, which progressesover many decades.Both Rod and Cone Photoreceptors Slowly Degenerate.

Histological analyses of retinas from both normal and trans-genic mice (Fig. 4) indicated that outer segments were formed

at equivalent times during postnatal development. At P12,shortly before eye opening (at P14), nascent outer segmentswere present in both normal and transgenic animals. By P20,when photoreceptor outer segments were near adult length innormal animals (25 ± 2 Am, mean + SD), outer segmentswere '50% normal length in the transgenic animals (15 ± 2,um). Outer segments in transgenic animals continued toshorten (11.8 + 2 Am at P32 and 9.6 ± 1 Am at P106). Thisdecrease in average length of outer segments was accompa-nied by a decrease in the number ofphotoreceptors, as shownby the reduced thickness of the outer nuclear layer (Fig. 4).Throughout the postnatal period, the pigment epithelium intransgenic eyes appeared to be normal, and no pigmentary

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Proc. Natl. Acad. Sci. USA 90 (1993) 5503

changes typical for human RP were recognized. Pyknoticphotoreceptor nuclei, an indication of ensuing cell death,were observed frequently in the transgenic animals but onlyrarely in nontransgenic littermates. In transgenic retinas ofage P250, the oldest transgenic sample examined, extensiveexpanses of the central fundus were free of photoreceptors,indicating that both rods and cones are affected by thedegeneration, and immunocytochemical studies with anti-rhodopsin antibodies that permit distinction of rod and conephotoreceptors also indicated that both cell types degenerate(unpublished data). Photoreceptor survival was more pro-nounced in the retinal periphery with one to three rows ofphotoreceptor nuclei remaining.

DISCUSSIONThe transgenic mouse line presented here incorporated two tofive copies of a transgene containing three mutations near theN terminus of rhodopsin (VPP mutation) into a single site ofthe mouse genome. The transgene is expressed, and thegenerated phenotype displays many features characteristic ofhuman ADRP. (i) The effect ofexpression of the mutant opsingene results in an autosomal dominant phenotype similar tothat inADRP patients. (ii) The reduced light-evoked responses(ERGs) in mutant mice at P30, when no structural damage ofphotoreceptor cells except a shortening of the outer segmentis evident, reflect a relatively early time of onset of thedegeneration. In human ADRP patients, early ERG changesare the first clinical manifestations of the disease, with amedian onset of night blindness in P23H patients at age 13-14years (14). (iii) The photoreceptor degeneration observed inour transgenic animals progresses slowly, as shown by thegradual reduction of the a wave (Fig. 3), shortening of thephotoreceptor outer segments, and loss ofphotoreceptor cells(gradual decrease of the outer nuclear layer thickness). Inhuman patients, ERGs and fundus analyses, the only effectivemeans to follow the progression of the disease, slowly dete-riorate with age, in most cases progressing overmany decades.(iv) The slow rod degeneration in transgenic mice is accom-panied by a cone degeneration. Widespread loss of conephotoreceptors is a characteristic of human ADRP at latestages. Thus, we have generated a transgenic mouse with aretinal (photoreceptor) degeneration that mimics humanADRP in many aspects. This mouse line will permit a varietyof biochemical, molecular, and cell biological studies that arenot possible in human subjects or with donor eye tissues.We have shown (Fig. 2) that the transgene is transcribed

concurrently with the endogenous murine opsin gene, but wehave no evidence that the mutated rhodopsin is produced,transported to the outer segment, and incorporated into diskmembranes. The P23H mutation is thought to perturb the localsecondary structure of rhodopsin to an extent that the mutantrhodopsin is labile or, alternatively, is processed incorrectlyposttranslationally (26) and, thus, causes a degeneration by ayet unknown mechanism. In transgenic mice expressing ahuman rhodopsin gene containing the P23H mutation, thehuman mutant rhodopsin appeared to be produced and incor-porated into the rod outer segment without apparent grossstructural distortions (20). In tissue culture with embryonickidney cell lines, however, P23H mutant rhodopsins werefound to accumulate in the endoplasmic reticulum (26). Pre-liminary electron microscope studies in transgenic retinas withthe VPP mutation using a monoclonal antibody that onlyrecognizes normal rhodopsin show no dilations of inner seg-ments or abnormal structures indicative of accumulation ofmutant rhodopsin or problems with rhodopsin transport.

If the mutant rhodopsin is produced, then one of thechallenging questions remaining to be answered is howmutations in the rhodopsin gene slowly disable and eventu-ally disrupt photoreceptor cell function. To this end, the fateofthe mutated rhodopsin in the living photoreceptor cellmust

be determined at the level of biosynthesis, posttranslationalmodifications, transport, and incorporation into the diskmembrane of outer segments. A first step toward this goalwill be the generation of a peptide-specific antibody for itsimmunocytochemical localization. Eventually, the mecha-nistic link between rhodopsin mutations and retinitis pigmen-tosa should be understood with the hope ofdesigning rationalapproaches to retard or cure this group of diseases.We thank Kathy Myers and Alain B. Quiambao for excellent

technical assistance, and Drs. R. E. Anderson, J. M. Frederick, R. A.Lewis, and S. J. Pittler for discussion and critical reading of thismanuscript. This research was funded in part by the Retinitis Pig-mentosa Foundation Fighting Blindness (Baltimore), the AdvancedTechnology Program of the Texas Higher Education CoordinatingBoard (Austin, TX), the Retina Research Foundation (Houston), andthe National Eye Institute (EY08123 and EY02363). M.I.N. is therecipient ofan individual National Research Service Award fellowship(5F32 EY06330-02). J.G.H. is the recipient of a Senior InvestigatorAward from the Research to Prevent Blindness Foundation. W.B. isa Jules and Doris Stein Research to Prevent Blindness Professor.

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