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Xenopus laevis as a Model for Understanding Retinal DiseasesO L Moritz and D C Lee, University of British Columbia, Vancouver, BC, Canada

ã 2010 Elsevier Ltd. All rights reserved.

Glossary

AP20187 – A small molecule modeled on a dimer of

the immunosuppressive drug FK506. One molecule

of AP20187 can bind with high affinity to two FK506-

binding protein (Fv) domains. Thus, Fv domains can

be used to produce fusion proteins that will dimerize

in the presence of AP20187. This in turn can be used

to control the activity of proteins or enzymes whose

activities are influenced by dimerization, such as

caspases.

Bardet–Biedl syndrome – An autosomal recessive

disorder characterized by obesity, retinal

degeneration, polydactyly, hypogonadism,

developmental delay, and mental retardation. Genes

implicated in this syndrome are involved in ciliary

transport processes.

Caspase-9 – An initiator caspase in the apoptotic

cascade. Caspases are a family of cysteine

proteases, which play essential roles in programmed

cell death. Once activated, caspase-9 triggers

activation of other caspases, precipitating the

apoptotic process.

Optical coherence tomography (OCT) –

A technique that permits three-dimensional imaging

within tissues that scatter light. The technique is

frequently used in ophthalmology to noninvasively

image the cell layers of the retina.

Peripherin/RDS – A transmembrane glycoprotein

found in the outer segment of both rod and cone

photoreceptor cells. It is thought to be a structural

protein important for disk morphogenesis. Mutations

in the gene encoding peripherin/RDS are associated

with a variety of autosomal dominant retinal

dystrophies; also referred to in publications as rds,

peripherin, or peripherin-2.

Rab proteins and Arf4 – The members of the Ras

superfamily of monomeric guanine-nucleotide-binding

proteins (G proteins), which are involved in the

regulation of membrane trafficking.

Retinal degeneration – A phenotype associated

with many different retinal disorders, involving

progressive death of retinal cells, usually of a specific

cell type.

Retinitis pigmentosa – A hereditary retinal

dystrophy characterized by defective dark

adaptation, progressive loss of peripheral vision that

may eventually extend to loss of central vision, and

the appearance of black pigment in the fundus.

RNA helicase Ddx39 – A member of the DEAD box

protein family of putative RNA helicases. Family

members are characterized by the conserved motif

Asp-Glu-Ala-Asp (D-E-A-D), and are involved in RNA

metabolism.

Stargardt’s disease – An autosomal recessive

juvenile-onset form of macular dystrophy arising from

mutations in the ABCA4 gene. The ABCA4 gene

product is expressed in photoreceptor cells and is

thought to be an ATP-dependent transporter for

N-retinylidene-PE.

Introduction

The amphibian retina has many unique properties thathave intrigued visual scientists for decades. Many studieshave been conducted on the retina of Xenopus laevis, a frogspecies commonly used as a laboratory animal. These frogswere introduced to the research community in the 1930sand soon became widely available, after the discovery thatthey can be induced to lay eggs by injection of humanchorionic gonadotropin (known as the Hogben test forpregnancy). As a research subject,X. laevis have advantagesover other amphibians in that they have low maintenance

317

ONL

ONL

INL

INL

RPE

RPE

OS

OS

ONL

ONL

INL

INL

RPE

RPE

Figure 1 Expression of a bovine rhodopsin P23H mutant

transgene causes retinal degeneration in Xenopus laevis.

Combined confocal/DIC (upper panels) or confocal micrographs(lower panels) of developmental stage 47/48 wild-type retinas

(left panels) or transgenic retinas expressing bovine-rhoP23H

(right panels). Animals were raised in bright cyclic light for

2 weeks, beginning at fertilization. The 12-mm retinalcyrosections were stained with wheat germ agglutinin (red),

B630N anti-rhodopsin (green), and Hoechst 33342 nuclear stain

(blue). The central retinas of animals expressing bovine P23H

rhodopsin (right panels) have almost no remaining rods, withthose that remain having short and irregular outer segments.

Note the relatively large diameter (7 mm) of the rod

photoreceptors relative to those of the mammalian retina. Due tothe relatively large diameter of the photoreceptors, only a single

row of nuclei is required in the ONL. RPE, retinal pigment

epithelium; OS, outer segment; INL, inner nuclear layer; ONL,

outer nuclear layer. Scale = 10 mm.

318 Xenopus laevis as a Model for Understanding Retinal Diseases

requirements (they are entirely aquatic and do not requirelive food), and they can easily be induced to lay eggs. Thismade them a desirable model organism for developmentalbiologists studying fertilization and embryonic develop-ment; however, they have also been adopted by visionresearchers as a model amphibian retina. The large sizeof the principle rod photoreceptors makes them highlyamenable to biochemical, morphological, and electro-physiological studies (Figure 1). Early studies on theproperties of the X. laevis visual system laid the ground-work for the use of this animal as a model organism for thestudy of visual disorders, while more recently developedtechniques for genetic manipulation have resulted inX. laevismodels of inherited retinal disease that are partic-ularly amenable to certain forms of analysis.

Early Work on X. laevis – Biochemistry,Electrophysiology, and Microscopy

Modern research on the amphibian retina dates backto the extraordinary anatomical studies of Cajal andothers in the 1800s that defined many of the principalcell types of the retina. Most early studies dealt with Ranaand Bufo species. However, X. laevis have been used as asubject of retinal research dating back more than 50 years.In the 1950s, Wald and co-workers used X. laevis forinvestigations of visual pigment biochemistry. From themost abundant rod photoreceptors, it was found thatthe principal pigment exhibited maximal absorbanceat 523 nm.

The amphibian retina is also of interest to electrophy-siologists. Although the first electroretinogram (ERG)was recorded by Holmgren in the 1860s from a frog eye,X. laevis frogs were not used extensively for electrophysi-ological studies until the 1970s, with initial studies ofX. laevis retinal physiology performed by Ripps and co-workers, correlating visual pigment content and photo-receptor threshold.

In the 1950s and 1960s, detailed analysis of the ultra-structure of photoreceptors was obtained by electronmicroscopy, leading to the current understanding of pho-toreceptor disk membrane structure, and the mechanismsof disk membrane synthesis and renewal. Many of thesestudies utilized amphibian retina, again typically Ranaspecies, although Lanzavecchia examined the ultrastruc-ture of X. laevis rods and cones in 1960. Further studies byKinney and Fisher further characterized the morphogen-esis and ultrastucture of the principal rod photoreceptorin X. laevis.

In the 1940s and 1950s, work by Sperry and co-workersdemonstrated regeneration of the optic nerve after tran-section in various amphibians. Beginning in the late 1960s,Jacobson and co-workers conducted investigations of ret-inal development in X. laevis, with particular emphasis onthe development of retinotectal projections. These studiesemployed embryonic surgery (e.g., inversion of the eye) toidentify the origin of signals for optic nerve axon guid-ance, and demonstrated that the location of synapses ofganglion cell axons in the optic tectum are specified bythe location (dorsal, ventral, nasal, or temporal) of the cellbodies in the retina.

These early investigations established the X. laevisretina as a viable subject for retinal research that is stillin use, including ongoing studies of X. laevis disk sheddingand renewal, electrophysiology, biochemistry, and retinaldevelopment, that are further improving our understand-ing of retinal function. Additionally, several studies dis-cussed below have directly modeled human retinaldisorders in X. laevis, in order to better understand retinaldysfunction.

Xenopus laevis as a Model for Understanding Retinal Diseases 319

X. laevis as a Model for Vitamin ADeprivation

One of the first instances of modeling retinal disease inX. laevis was a study by Witkovsky and co-workers onvitamin A deprivation in tadpoles. This model repro-duced features of night blindness (i.e., decreased rod sen-sitivity) seen in vitamin-A-deprived patients. Amongother novel findings, the authors demonstrated thatbleaching of photopigment causes a greater reduction inphotoreceptor sensitivity than can be accounted for byreduction in pigment quantity alone. This was accountedfor in later studies, which demonstrated that opsin has agreater tendency to activate the visual transduction cas-cade than rhodopsin. More recently, studies on vitamin Adeprivation in X. laevis have been continued by Solessioand co-workers, who have also pioneered the use of psy-chophysical measurements of X. laevis visual sensitivity.

X. laevis as a Model for Glaucoma

Early studies by Jacobson and Keating involving opticnerve transection in X. laevis are similar to paradigmscurrently used in research of optic nerve injury or glau-coma, although more typically the research subject is amammal. However, unlike a mammalian optic nerve, asevered X. laevis optic nerve regenerates (a property thatwould be highly desirable in glaucoma patients). Researchfocused on X. laevis models of retinal regeneration of thistype is currently being continued by Belecky-Adams andco-workers, who recently identified a possible role of theRNA helicase Ddx39 in the regulation of stem cells in theretina.

X. laevis and Studies of the Transport ofRhodopsin

The first investigations of the mechanism of outer seg-ment renewal utilized amphibians injected with radioac-tive amino acids in a pulse–chase paradigm that allowednewly synthesized membranes to be visualized by autora-diography. The original experiments by Young and co-workers demonstrated that new disks were formed at thebase of the rod outer segment, and subsequently becamediscontinuous with the outer segment plasma membrane.These studies were further extended by Besharse andHollyfield, who examined the influence of light on disksynthesis in X. laevis photoreceptors, demonstrating diur-nal regulation of disk membrane synthesis. Collectively,these studies demonstrated the extremely high rate of rodphotoreceptor disk membrane synthesis in X. laevis retina,

estimated at roughly 50-fold higher than in mammalianphotoreceptors.

These findings suggested that, due to the enormousrate of outer segment membrane synthesis, the amphibianretina would be an excellent choice for studying thebiosynthesis of rhodopsin. This work was pioneered byPapermaster and Dreyer, and subsequently continuedby Papermaster and co-workers. These studies largelyused Rana and Xenopus species, and resulted in identifica-tion of molecular and ultrastructural details of the rho-dopsin transport pathway. Components of the biosyntheticmachinery and transport mechanisms first identified inamphibia included associated features of the connectingcilium (the pericilliary ridge complex), and vesicles trans-porting rhodopsin (RTCs or rhodopsin transport carriers).Deretic was able to reconstitute rhodopsin transport inamphibian retinal extracts, and using this assay demon-strated that the rhodopsin transport signal was located inthe cytoplasmic C-terminal domain. This in vitro assay andassociated methodologies subsequently led to identifica-tion of a number of molecules associated with rhodopsintransport, including the small G proteins, rab6, rab8,rab11, and arf4.

However, due to the lack of an effective system forculturing photoreceptors, it was not possible to incorpo-rate molecular biology approaches into the study ofamphibian rhodopsin transport pathways until the 1990s,when techniques for the production of transgenic X. laevisdeveloped by Kroll and Amaya, and cloning of promot-ers suitable for driving expression in X. laevis rods, allow-ed the study of rhodopsin transport in a geneticallymanipulated amphibian retina. Tam and colleaguesfound that rhodopsin-green fluorescent protein (GFP)fusion proteins expressed in X. laevis retina were trans-ported correctly to rod outer segments. This allowedfurther identification and in-vivo demonstration of thefunction of the outer segment localization signal QVAPA,located at the extreme C-terminus of rhodopsin. Severalmutations affecting this region cause retinitis pigmentosa(RP). These studies also demonstrated the extraordinaryutility of transgenic X. laevis for conducting compari-sons between transgenic animals, as numerous primarytransgenic animals carrying different transgenes can begenerated in a relatively short amount of time. For ex-ample, the rhodopsin outer segment localization signalwas identified and refined using 15 distinct transgeneconstructs.

Using the same transgenic X. laevis system, the func-tion of rab8 in rhodopsin transport was explored usingdominant-negative and constitutively active mutants.Expression of these mutant rab8-GFP fusion proteins inX. laevis rods generated the first X. laevis models with aninherited retinal degeneration (RD) phenotype, as thesefusion proteins proved to be quite toxic to retinal rods,

320 Xenopus laevis as a Model for Understanding Retinal Diseases

and the phenotype was passed to F1 offspring. Theexpression of dominant-negative GFP-rab8T22N causeda particularly rapid death of photoreceptors associatedwith accumulation of rhodopsin-containing intracellularvesicles in the vicinity of the base of the connectingcilium. Although it was not clear at the time, subsequentstudies indicate that the resulting phenotype may beclosely related to the RD associated with Bardet–Biedelsyndrome (BBS). Similar investigations of the smallG-protein Arf4, which binds the rhodopsin outer segmentlocalization signal, are ongoing.

The RD observed in this study demonstrated a uniquecentral-to-peripheral distribution subsequently seen in allother X. laevis models of RD. This is associated with therapid growth of the eye in young X. laevis, which results incontinuous addition of new photoreceptors to the periph-eral retina. Thus, a single cryosection can demonstrate allstages of photoreceptor degeneration moving from centralretina to periphery.

Modeling RP in Transgenic X. laevis

Subsequently, genetically modified transgenic X. laeviswas used in a number of studies that directly examinedmutations associated with the human disorder RP, aninherited form of RD. The pioneering study involvedtransgenic expression of mutant forms of peripherin/rds,a protein found at the periphery of rod outer segmentdisks. Peripherin/rds and its mutants were expressed asGFP fusion proteins using the rod opsin promoter todrive expression in retinal rods, at levels sufficientlyhigh to cause RD in some cases. Confocal microscopy ofthe intrinsic GFP fluorescence showed that several fusionproteins had unique localization patterns distinct fromwild type that respectively suggested either specific dis-ruptions in normal function, or misfolding and endoplas-mic reticulum (ER) retention. Furthermore, electronmicroscopy revealed unique abnormalities in disk organi-zation, possibly associated with disruption of the normalfunctions of the peripherin/rds C-terminus.

Previous attempts to use rhodopsin-GFP fusion pro-teins to develop similar models of RP were unsuccessful,most likely due to low expression levels. In order to adaptthe system for the study of RD induced by rhodopsinmutants, a system was devised for detection of non-fluorescent transgene products based on nonconservedrhodopsin antibody epitopes, such that epitope tagsinvolving minimal (or no) sequence changes could beintroduced. Initially, this system was applied to the studyof an RP-causing mutation (Q348ter) that disrupts thepreviously identified rhodopsin outer segment localiza-tion signal. In these studies, the power of the X. laevissystem for drawing comparisons between transgenes wasfurther expanded. Rhodopsin mutants defective in signal

transduction properties were combined with rhodopsinmutants responsible for RP to dissect the role of rhodop-sin signal transduction in rod cell death pathways. Theresults demonstrated rhodopsin mislocalization was asso-ciated with axonal sprouting and cell death, regardlessof whether rhodopsin signal transduction propertieswere inhibited.

The same system was subsequently applied to thestudy of the rhodopsin mutation P23H, the most commoncause of autosomal dominant RP in North America.Despite numerous studies of this rhodopsin mutant incultured cells and transgenic rodents, there was no clearconsensus as to the effects of this mutation on rhodopsinfunction; in cultured cells, it was classified as a mutantdefective in folding and ER exit, while transgenic animalstudies suggested it was transported correctly to rod outersegments, where it caused RD that was exacerbated bylight.

Studies in transgenic X. laevis compared several differ-ent forms of P23H rhodopsin, including P23H rhodopsinsbased on different species, and P23H rhodopsins defec-tive in signal transduction and chromophore binding.Interestingly, all forms of P23H rhodopsin caused RD,but varied in terms of ER retention, expression level,and light sensitivity. P23H rhodopsins that exhibited dra-matic ER retention (X. laevis P23H rhodopsin) caused RDunder all circumstances, while P23H rhodopsins that weretransported in small quantities to the OS (bovine P23Hrhodopsin) caused RD only on light exposure (Figure 1).Furthermore, for bovine P23H rhodopsin, disruptionof the chromophore-binding site was associated withreduced expression levels and RD, regardless of lightexposure. This result reconciles the differences seenbetween previous studies, suggesting that in some formsof P23H-induced RD, chromophore binding promotesER exit of newly synthesized rhodopsin. The dramaticsensitivity of these phenotypes to the underlying rhodop-sin sequence was confirmed by Zhang and colleagues, whodemonstrated light-sensitive RD in an X. laevis rhodopsinthat differed from that used previously only in thesequences of the epitope tags. In addition to providinginsight into the mechanisms underlying RD, these studiesdramatically emphasize the difficulties in extrapolatingresults reported from a single disease model to humandisease states.

A unique finding in this system was the presence ofconsiderable quantities of truncated P23H rhodopsin, inwhich a significant portion of the N-terminal domain(including the mutated H23 residue) was removed; infact, this was the dominant species observed in retinasexpressing bovine P23H rhodopsin. This truncated spe-cies was also previously observed in cultured cells,although in smaller quantities. Identification of this spe-cies in other transgenic models would be difficult due tolack of a suitable reagent for detection, but was readily

Xenopus laevis as a Model for Understanding Retinal Diseases 321

achieved in X. laevis due to the availability of both N- andC-terminal specific antibodies that did not cross-reactwith endogenous rhodopsin.

Subsequent studies of the same X. laevis models ofP23H-rhodopsin-induced RD probed the association ofchromophore binding and ER exit. In order to address thequestion of whether the causative factor in light-inducedRD was a reduction in the supply of free 11-cis retinal, orisomerization of 11-cis retinal bound to P23H rhodopsinas chromophore, the sensitivity of RD to different wave-lengths of light was examined. It was determined that theprofile of light sensitivity was consistent with photoi-somerization of rhodopsin (which maximally absorbsgreen light) rather than free chromophore (with maximalabsorbance in the UV). This also brings to mind similarstudies of the constitutively active rhodopsin mutantK296E, classified as misfolding by some studies incultured cells, suggesting that the active conformation ofrhodopsin and/or loss of chromophore can be associatedwith altered kinetics of ER exit.

Studies of additional RP-causing rhodopsin mutationsin transgenic X. laevis are ongoing, and have been reportedat international meetings, including K296E rhodopsin andthe glycosylation-defective mutants T4K and T17M.Glycosylation-defective rhodopsin mutants are alsoreported to be associated with light-exacerbated RD inX. laevis.

Inducible RD

In an alternate approach to modeling RP in the transgenicX. laevis retina, Hamm and co-workers designed a drug-inducible model of RD driven by a modified form ofcaspase-9. Dimerization and subsequent activation ofthis caspase-9 transgene is driven by AP20187, a smallmolecule based on a dimer of FK506. Administration ofAP20187 to these transgenic X. laevis induces rapid RDthat is not dependent on any particular environmentalcondition (such as special lighting). The system isdesigned to examine the effects of rod degeneration onother cell types, including cone photoreceptors and cellsof the inner nuclear layer, and to examine the capacity forregeneration of rods in the X. laevis retina. This study wasalso the first to provide functional (i.e., electrophysiologi-cal) data for an X. laevis model of RD.

Interestingly, this model demonstrated a dramaticreduction in electrophysiological responses to stimulidesigned to isolate cone function (e.g., a rapid flickerstimulus), despite the fact that no associated cone deathwas detected. This reduction in cone sensitivity maybe associated with the cones themselves, or with othercells associated with the cone pathway and the ERGB-wave (e.g., cone bipolar cells and/or Muller glia). Therestoration of responses to flicker stimuli was associated

with a thickening of the inner nuclear layer, and a similarthickening can be observed by optical coherence tomog-raphy (OCT) in human RP patients.

Other Transgenic X. laevis Models ofRetinal Disease

In addition to rhodopsin and peripherin/rds mutationsresponsible for RP, gene products related to Stargardt’sdisease have been expressed in X. laevis retina as GFPfusion proteins in order to examine their localizationproperties, although this has not yet resulted in a replica-tion of a RD phenotype.

In studies by Kefalov and colleagues, cone opsins wereexpressed in X. laevis rods. As cone photoreceptors areconsiderably noisier than rod photoreceptors, this alloweda determination of the proportion of cone dark noise(activation of transduction in the absence of photons)that is purely due to the cone pigment sequence, andnot other aspects of the transduction cascade or photore-ceptor environment. Although generating a model of dis-ease was not a goal of this study, the resulting animalscould be considered a model for congenital stationarynight blindness, which is due to abnormally high activityof the visual transduction cascade in the absence ofphotons.

X. laevis as a Model for Eye Development/Developmental Disorders

The rapid development of the X. laevis embryos makesthese animals of particular interest to developmentalbiologists, including those concerned with eye devel-opment. The first studies of the development of theX. laevis eye were conducted by Hollyfield throughradioactive monitoring of the growth of the developingretina. Chung and colleagues histologically monitoredthe structural changes in the developing larval X. laevisretina and correlated these changes with electrophysio-logical changes, notably that while the receptive field ofa ganglion cell remains constant in the developinglarvae through metamorphosis, the inhibitory periph-eral region expands to the entire retina of the adultX. laevis.

The development of the X. laevis eye has been manipu-lated by both the overexpression and by the knockdownof transcription factors. El-Hodiri and colleagues haveextensively studied the transcriptional regulation of pho-toreceptor development. More recently, they identified aretinal homeobox gene family member, Rx-L, which reg-ulates photoreceptor-specific gene expression. Expressed indeveloping embryos, the knockdown of Rx-L expression

322 Xenopus laevis as a Model for Understanding Retinal Diseases

adversely affected photoreceptor development, causingsubtle phenotypes of altered photoreceptor morphology.

Using a similar embryonic transfection paradigm,Knox and colleagues found that overexpression of thetranscription factors, Nrl and Nr2e3, in X. laevis retinaresulted in an increase in numbers of rods, with concomi-tant reduction in cone photoreceptors, indicative of theroles of these factors in determining the developing pho-toreceptor cell fate. This system may prove extremelyuseful in modeling developmental disorders of the retinawith similar underlying mechanisms.

X. laevis Models of Retinal Regeneration

Some recent studies have investigated the fascinatingcapacity of the X. laevis retina to repair itself after severetraumatic injury. In these studies, the entire retina isexcised from an X. laevis tadpole eye. The retina subse-quently demonstrates a dramatic capacity to completelyregenerate by transdifferentiation of the remaining cellsof the retinal pigment epithelium (RPE). Certain aspectsof this transdifferentiation can be reproduced in culture,and it appears to be dependent on diffusible factors (pos-sibly fibroblast growth factor 2 (FGF2)) released from thechoroid. These results could have implications for trau-matic eye injuries such as retinal detachment, retinaldegenerative disorders, and glaucoma.

Summary

As an unconventional system for modeling retinal disease,X.laevis presents a number of advantages. As it is quite easy togenerate transgenic X. laevis, they are an excellent systemfor comparing the effects of multiple transgenes. Otheradvantages include the relative ease of microscopic andelectrophysiological studies due to the large size of thephotoreceptor cells, regenerative capacity of the retina, andnon-cross-reactivity of mammalian antibodies. However,there are also significant disadvantages, such as the currentlack of knock-out or gene-replacement capabilities, longgeneration time (1 year), pseudotetraploid genome, and rel-atively small eyes, such that it is clearly not an ideal systemappropriate for all experiments. Rather, X. laevis models ofretinal disease are a very useful addition to the library ofsystems and models available to vision researchers.

See also: The Photoreceptor Outer Segment as a Sensory

Cilium; Primary Photoreceptor Degenerations: Retinitis

Pigmentosa; Primary Photoreceptor Degenerations: Ter-

minology; Retinal Degeneration through the Eye of the Fly;

Secondary Photoreceptor Degenerations: Age-Related

Macular Degeneration; Secondary Photoreceptor Degen-

erations; Zebrafish as a Model for Understanding Retinal

Diseases: Pde6, Apoptosis, and the Bystander Effects;

Zebrafish: Retinal Development and Regeneration.

Further Reading

Araki, M. (2007). Regeneration of the amphibian retina: Role of tissueinteraction and related signaling molecules on RPEtransdifferentiation. Development, Growth and Differentiation 49:109–120.

Besharse, J. C., Hollyfield, J. G., and Rayborn, M. E. (1977). Turnover ofrod photoreceptor outer segments. II. Membrane addition and loss inrelationship to light. Journal of Cell Biology 75: 507–527.

Deretic, D., Williams, A. H., Ransom, N., et al. (2005). RhodopsinC terminus, the site of mutations causing retinal disease, regulatestrafficking by binding to ADP-ribosylation factor 4 (ARF4).Proceedings of the National Academy of Sciences of the UnitedStates of America 102: 3301–3306.

Hamm, L. M., Tam, B. M., and Moritz, O. L. (2009). Controlled rod cellablation in transgenic Xenopus laevis. Investigative Ophthalmologyand Visual Science 50(2): 885–892.

Hollyfield, J. G. (1971). Differential growth of the neural retina in Xenopuslaevis larvae. Developmental Biology 24: 264–286.

Pan, Y., Nekkalapudi, S., Kelly, L. E., and El-Hodiri, H. M. (2006). TheRx-like homeobox gene (Rx-L) is necessary for normal photoreceptordevelopment. Investigative Ophthalmology and Visual Science 47:4245–4253.

Papermaster, D. S., Schneider, B. G., Zorn, M. A., and Kraehenbuhl, J. P.(1978). Immunocytochemical localization of opsin in outer segmentsand Golgi zones of frog photoreceptor cells. An electron microscopeanalysis of cross-linked albumin-embedded retinas. Journal of CellBiology 77: 196–210.

Sperry, R. W. (1944). Optic nerve regeneration with return of vision inAnurans. Journal of Neurophysiology 7: 57–69.

Tam, B. M. and Moritz, O. L. (2007). Dark rearing rescues P23Hrhodopsin-induced retinal degeneration in a transgenic Xenopuslaevis model of retinitis pigmentosa: A chromophore-dependentmechanism characterized by production of N-terminally truncatedmutant rhodopsin. Journal of Neuroscience 27: 9043–9053.

Tam, B. M., Moritz, O. L., Hurd, L. B., and Papermaster, D. S. (2000).Identification of an outer segment targeting signal in the COOHterminus of rhodopsin using transgenic Xenopus laevis. Journal ofCell Biology 151: 1369–1380.

Tam, B. M., Xie, G., Oprian, D. D., and Moritz, O. L. (2006). Mislocalizedrhodopsin does not require activation to cause retinal degenerationand neurite outgrowth in Xenopus laevis. Journal of Neuroscience26: 203–209.

Witkovsky, P., Gallin, E., Hollyfield, J. G., Ripps, H., and Bridges, C. D.(1976). Photoreceptor thresholds and visual pigment levels in normaland vitamin A-deprived Xenopus tadpoles. Journal ofNeurophysiology 39: 1272–1287.

Young, R. W. and Droz, B. (1968). The renewal of protein in retinal rodsand cones. Journal of Cell Biology 39: 169–184.

Zhang, R., Oglesby, E., and Marsh-Armstrong, N. (2008). Xenopuslaevis P23H rhodopsin transgene causes rod photoreceptordegeneration that is more severe in the ventral retina and ismodulated by light. Experimental Eye Research 86: 612–621.