Cone Degeneration Following Rod Ablation in a
Reversible Model of Retinal Degeneration
Rene Y. Choi,1,2Gustav A. Engbretson,1,3Eduardo C. Solessio,1Georgette A. Jones,1
Adam Coughlin,1Ilija Aleksic,1and Michael E. Zuber1,2
PURPOSE. Amphibian retinas regenerate after injury, making
them ideal for studying the mechanisms of retinal regeneration,
but this leaves their value as models of retinal degeneration in
question. The authors asked whether the initial cellular
changes after rod loss in the regenerative model Xenopus
laevis mimic those observed in nonregenerative models. They
also asked whether rod loss was reversible.
METHODS. The authors generated transgenic X. laevis express-
ing the Escherichia coli enzyme nitroreductase (NTR) under
the control of the rod-specific rhodopsin (XOP) promoter. NTR
converts the antibiotic metronidazole (Mtz) into an interstrand
DNA cross-linker. A visually mediated behavioral assay and
immunohistochemistry were used to determine the effects of
Mtz on the vision and retinas of XOPNTR F1tadpoles.
RESULTS. NTR expression was detected only in the rods of
XOPNTR tadpoles. Mtz treatment resulted in rapid vision loss
and near complete ablation of rod photoreceptors by day 12.
Mu ¨ller glial cell hypertrophy and progressive cone degenera-
tion followed rod cell ablation. When animals were allowed to
recover, new rods were born and formed outer segments.
CONCLUSIONS. The initial secondary cellular changes detected in
the rodless tadpole retina mimic those observed in other mod-
els of retinal degeneration. The rapid and synchronous rod loss
in XOPNTR animals suggested this model may prove useful in
the study of retinal degeneration. Moreover, the regenerative
capacity of the Xenopus retina makes these animals a valuable
tool for identifying the cellular and molecular mechanisms at
work in lower vertebrates with the remarkable capacity of
retinal regeneration. (Invest Ophthalmol Vis Sci. 2011;52:
photoreceptors.1Rod photoreceptor death is followed by sec-
etinitis pigmentosa (RP) is a heterogeneous group of in-
herited disorders characterized by the initial loss of rod
ondary degenerative changes, the most notable and debilitating
of which is the subsequent death of cone photoreceptors.2
Other cellular changes include Mu ¨ller glial cell hypertrophy,
synaptic layer loss, and retinal interneuron death.2
Mammalian animal models that mimic aspects of RP have
been critical for investigating the mechanisms of rod cell death
and the secondary changes that follow.3,4In contrast, amphib-
ian, fish, and, more recently, chicken model systems have been
predominantly used to study retinal regeneration because of
the remarkable ability of these species to partially or even fully
recover from retinal damage.5–7The retinal damage/regenera-
tion paradigms used in lower vertebrates have ranged from
complete and partial retinectomy to the ablation of individual
retinal cell classes using chemical and genetic approac-
hes.5,6,8–14Given their ability to replace dead or dying retinal
cells, we asked whether a regenerative animal model (Xenopus
laevis) would also exhibit the secondary cellular changes ob-
served in nonregenerative animal models.
To address this question, we generated a transgenic line of
X. laevis driving the expression of Escherichia coli nitroreduc-
tase (NTR) under the control of the rod-specific rhodopsin
promoter (XOP).15,16Nitroreductase converts nitroimidazole
prodrugs, such as metronidazole (Mtz), into a cytotoxic DNA
cross-linker.17,18Taking advantage of a low-light, visually based
behavioral assay, we show Mtz-treated XOPNTR transgenic
tadpoles rapidly lose their vision.19,20Rod cell death is swift,
specific, and parallels the loss in sight. Apoptotic rods were
detected at 24 hours, and rod outer segment degeneration was
extensive by 5 days. Mu ¨ller cell hypertrophy and cone photo-
receptor degeneration and death followed rod degeneration.
Furthermore, new rod photoreceptors were generated after
selective rod ablation. These results suggest that retinal degen-
eration after rod loss in Mtz-treated XOPNTR animals mimics
retinal degeneration in other animal models, despite the ability
of Xenopus to regenerate all retinal cell classes. Consequently,
Xenopus may serve not only as a model system for retinal
development and regeneration but also for understanding the
cellular and molecular mechanisms of retinal degeneration.
Generation of XOPNTR Transgene
and Transgenic Animals
The transgene construct pXOP(?508/?41)-NTR was generated by
replacing eGFP of pXOP(?508/?41)GFP with the E. coli NTR gene
from plasmid F116.16XOPNTR F0transgenic X. laevis were generated
using restriction enzyme-mediated integration, and four XOPNTR
founders, two males and two females, were grown to adulthood.21
Progeny from the founder females, XOPNTR1 and XOPNTR2, were
generated, and only XOPNTR2 tadpoles responded to metronidazole
From the Departments of1Ophthalmology and2Biochemistry and
Molecular Biology, Center for Vision Research, SUNY Eye Institute,
Upstate Medical University, Syracuse, New York; and the3Department
of Biomedical and Chemical Engineering, Syracuse University, Syra-
cuse, New York.
Supported by National Institutes of Health Grants EY015748 and
EY017964 (MEZ); Research to Prevent Blindness Career Development
Award (MEZ) and unrestricted grant to the Upstate Medical University
Department of Ophthalmology; the E. Matilda Zeigler Foundation for
the Blind (MEZ); and the Lions of Central New York.
Submitted for publication February 8, 2010; revised June 26 and
July 17, 2010; accepted August 2, 2010.
Disclosure: R.Y. Choi, None; G.A. Engbretson, None; E.C. So-
lessio, None; G.A. Jones, None; A. Coughlin, None; I. Aleksic,
None; M.E. Zuber, None
Corresponding author: Michael E. Zuber, Departments of Ophthal-
mology and Biochemistry and Molecular Biology, SUNY Upstate Med-
ical University, 750 East Adams Street, Syracuse, NY 13210;
Investigative Ophthalmology & Visual Science, January 2011, Vol. 52, No. 1
Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
Generation of F1Tadpoles and Genotyping
The transgenic female was injected 1 week and 1 day before egg
collection with pregnant mare’s serum gonadotropin (Sigma Aldrich,
St. Louis, MO) and human chorionic gonadotropin (Intervet, Millsboro,
DE), respectively, to induce egg laying, and the eggs were fertilized in
vitro using wild-type sperm. DNA from tail snips of F1tadpoles was
isolated (DNeasy Blood and Tissue Kit; Qiagen Inc., Valencia, CA).
Primers specific for the XOPNTR transgene (5? XOPNTR, 5?-CGCTA-
AATCCTTTGTTGCTGACGC-3?; 3? XOPNTR, 5?-GTTGAACACGTAAT-
TACCGGCAGC-3?) were used to identify transgenic tadpoles and their
nontransgenic siblings, which were used as wild-type controls. The
Committee for the Humane Use of Animals at SUNY Upstate Medical
University approved all procedures, and all procedures adhered to the
ARVO Statement for the Use of Animals in Ophthalmic and Vision
Metronidazole Preparation and Use
Metronidazole (Sigma Aldrich; product no. M-1547) was dissolved in
0.1? MMR containing 0.4% dimethyl sulfoxide (DMSO; Sigma-Aldrich;
product no. D8418) to a final concentration of 10 mM immediately
before use.22Control animals were cultured in the same solution
without Mtz. Preliminary experiments demonstrated that higher con-
centrations of Mtz were toxic. No more than 30 stage 50 to stage 53
(stages are noted in figure legends) transgenic or nontransgenic sibling
tadpoles were cultured in 600 mL Mtz solution and raised at 22°C in
complete darkness (Mtz is light sensitive) for the indicated time.
Preliminary experiments demonstrated that there were no differences
in the response to Mtz for tadpoles between stages 50 and 53. For
regeneration experiments, animals were Mtz-treated then allowed to
recover for periods of up to 30 days in 0.1? MMR in ambient labora-
Cryosectioning, Immunohistochemistry, and In
Tadpoles were euthanatized in 1% methanesulfonate (Tricaine; Sigma-
Aldrich), fixed in 4% paraformaldehyde for 1 hour, immersed in 20%
sucrose, mounted in OCT, and cryostat sectioned (12 ?m). When
staining for rod transducin, animals were incubated for 2 hours in
room light to ensure labeling of rod somata. The following primary
antibodies were used for immunostaining: anti–transducin polyclonal
(1:100; product no. sc-389; Santa Cruz Biotechnology, Santa Cruz, CA),
anti–XAP2 monoclonal (1:10; clone 5B9; Developmental Studies Hy-
bridoma Bank [DSHB], Iowa City, IA), anti–calbindin polyclonal (1:
500; product no. 80001-624; VWR, West Chester, PA), anti–islet-1
monoclonal (1:100; clone 39.4D5; DSHB), anti–calretinin polyclonal
(1:100; product no. NB200-618; Novus Biologicals, Littleton, CO),
anti–GABA polyclonal (1:500; product no. 20094; Immunostar, Hud-
son, WI), mouse R5 monoclonal (1:5; kindly provided by W.A. Harris,
Cambridge University), and anti–vimentin monoclonal (1:50; clone
14h7; DSHB). The following secondary antibodies tagged with fluores-
cent molecules were used for immunostaining: goat anti–mouse IgM
Alexa 555 (1:500; product no. A-21426), donkey anti–rabbit IgG Alexa
488 (1:500; product no. A-21206), goat anti–mouse IgG Alexa 488
(1:500; product no. A-11001), goat anti–mouse IgG3 Alexa 594 (1:750;
product no. A-21155), goat anti–mouse IgG2b Alexa 555 (1:500; prod-
uct no. A-21147), and goat anti–mouse IgG1 Alexa 488 (1:500; product
no. A-21121). All secondary antibodies were purchased from Invitro-
gen (Carlsbad, CA). In situ hybridizations were performed as previ-
ously described.23Substrate (Fast Red; Roche Applied Science, India-
napolis, IN) was used for the in situ hybridization procedure. The slides
were mounted in a solution of reagent (FluorSave; VWR), 2% 1,4-
diazabicyclo[2.2.2]octane (DABCO; Sigma-Aldrich), and 10 mg/mL 4,6-
diamidino-2-phenyindole, dilactate (DAPI; Sigma-Aldrich).
EdU (5-ethynyl-2?-deoxyuridine; 10 mM) was injected intra-abdomi-
nally on days 3 and 10 of Mtz treatment and on days 3, 10, and 21
during recovery. On day 30 of recovery, animals were processed for
immunohistochemistry, as described, and then for EdU using an Alexa-
Fluor 488 imaging kit according to the manufacturer’s instructions
(Click-iT EdU; product no. C10337; Invitrogen).
TUNEL Cell Apoptosis Assay
Dying cells were detected with an in situ apoptosis detection kit in
accordance with the manufacturer’s instructions (ApopTag Red; prod-
uct no. S7165; Millipore, Billerica, MA). A rhodamine-conjugated pri-
mary antibody was used to detect labeled cells.
Imaging and Cell Counts
Stained sections were visualized using an upright fluorescence light
microscope with motorized Z-focusing (DM6000 B; Leica Microsys-
tems, Bannockburn, IL) fitted with a camera (Retiga-SRV; Q-Imaging,
Surrey, BC, Canada) for image capture. Images were processed (Vo-
locity software, version 5.0.3; Improvision Inc., a PerkinElmer Com-
pany, Waltham, MA). For each treatment group, no fewer than three
animals were used. A single central retinal section from each animal
was scored. In Figure 6, a region between the ventro-temporal and the
central retina was selected for scoring. Cells were counted within a
region spanning 100 ?m. Unless otherwise noted, n equals the number
of animals analyzed (*P ? 0.05; **P ? 0.01). Statistical analysis was
performed using a Student’s t-test, paired two-tailed distribution.
Dim-Light Visually Mediated Behavioral Assay
Behavioral assays were performed as previously described with several
changes.19Stage 50 tadpoles were housed individually during drug
treatment and behavioral testing. Twelve hours before testing, tanks
containing transgenic or control animals were wrapped in aluminum
foil (Mtz-treated tadpoles and their respective controls were exposed
to room light during feeding and, therefore, had to be dark adapted).
The behavioral response was recorded with a digital camcorder using
infrared illumination for off-line analysis. (Preliminary experiments
demonstrated the infrared light source on the camcorder did not elicit
a behavioral response.) Stimulus light was provided from a 70-W
tungsten-halide lamp collimated and attenuated in 0.5-log increments
with neutral density filters. The beam was focused onto the entrance
aperture of a fiberoptics light pipe. The light pipe terminated in a ring
illuminator (2-inch inside diameter, 3.5-inch outside diameter; product
no. NT54-173; Edmund Optics, Barrington, NJ) centered 9 inches
above the tank housing the tadpoles. Maximal luminance at the tank
was 120 photometric cd/m2measured with a photometer (model 370;
Graseby Optronics, Orlando, FL). See Supplementary Movie S1, http://
for further details and an example of the response observed before and after
Mtz treatment. Calculations used to determine the light threshold needed to
elicit the observed response can be found in the Supplementary Text, http://
Results in figures are shown as the mean percentage of time spent on the
white side of the tank ? SE mean. A two-tailed Student’s t-test with P ? 0.05
was considered significant.
Expression of Nitroreductase in Rod
Photoreceptors of F1XOPNTR Tadpoles
We generated F0transgenic X. laevis expressing NTR under
the control of the ?508 to ?41 region of the X. laevis rho-
dopsin promoter (XOP).16,24Transgenics were identified by
genotyping using XOPNTR-specific PCR primers, then grown
to adulthood. Tadpoles generated from one founder female
(XOPNTR) were used for all experiments in this study.
In situ hybridization was used to determine the expression
pattern of NTR in stage 50 F1tadpoles. Consistent with the
known activity of the ?508 to ?41 region of the rhodopsin
IOVS, January 2011, Vol. 52, No. 1
Cone Degeneration Follows Rod Ablation in Xenopus
gene, NTR expression was detected only in the outer nuclear
layer (ONL) of transgenic tadpoles (Fig. 1A; n ? 4).24In
contrast, NTR was undetectable in wild-type (nontransgenic
sibling) retinas (Fig. 1B; n ? 4). The intensity and distribution
of NTR expression in the ONL varied from animal to animal,
consistent with previous reports attributing these differences
to position-effect variegation, which can result in altered trans-
gene expression on the order of days or even hours in genet-
ically nonmosaic animals.25To determine whether NTR ex-
pression was detectable in cone photoreceptors, retinas were
costained for the NTR transcript and calbindin, which specif-
ically labels cone photoreceptors in Xenopus.26NTR expres-
sion was never detected in cones (156 cells from four retinas;
Figs. 1C, 1C?, and 1C?), suggesting that NTR expression was
rod photoreceptor specific.
Rapid Vision Loss and Rod Photoreceptor
Ablation in Mtz-Treated XOPNTR Tadpoles
Nitroreductase reduces the antibiotic Mtz to a cytotoxic DNA
cross-linker.17Therefore, Mtz treatment of transgenic tadpoles
should result in rod photoreceptor ablation. We used both a
visually based behavioral assay and immunohistochemistry to
determine the effect of Mtz on rod cell function and viability in
control and XOPNTR transgenic tadpoles (Fig. 2). The behavior
and retinal histology of nontransgenic, sibling tadpoles gener-
ated from XOPNTR adults and wild-type tadpoles were indis-
tinguishable (not shown). Therefore, nontransgenic tadpoles
generated from the same clutch of XOPNTR eggs were used as
controls and are labeled here as wild-type.
Transgenic and control tadpoles were cultured in 10 mM Mtz
for 17 days. At 6-day intervals, the effect of Mtz treatment on the
dim light vision of tadpoles was determined using a modified
visually mediated behavioral assay.19,20The behavioral responses
of untreated XOPNTR tadpoles and controls were similar (Fig. 2A,
day 0). In contrast, we observed a significant drop in the behav-
ioral response of Mtz-treated XOPNTR transgenic tadpoles in as
little as 6 days (Fig. 2A; n ? 17). By day 12, Mtz-treated XOPNTR
animals were unable to distinguish between the white and black
sides of the test tank (n ? 14). In contrast, the responses of
DMSO-only treated XOPNTR transgenic (n ? 5) and Mtz-treated
wild-type tadpoles (n ? 6) were unchanged.
Immunohistochemistry using the rod-specific XAP2 antibody
was used to determine the effect of Mtz on rod cells.27The
morphology of rods in Mtz-treated wild-type (n ? 14) and un-
treated XOPNTR tadpoles (n ? 16) was normal (Figs. 2B, 2C,
2C?). In contrast, the retinas of all Mtz-treated, XOPNTR tadpoles
were severely altered. At 5 days, rod outer segments were re-
duced in number, shorter, and fragmented (Figs. 2D, 2D?; n ? 8).
After 10 days of exposure there was a near complete lack of rod
outer segments (Figs. 2E, 2E?; n ? 15). Intact rod outer segments
were sometimes, but not consistently, observed in the most pe-
ripheral region of the retina (Fig. 2E; n ? 15). However, no
consistent pattern of rod loss was observed. Together, these
results suggest Mtz treatment results in vision loss because of
rapid rod outer segment degeneration and possibly rod photore-
ceptor ablation in XOPNTR animals.
Apoptotic Cell Death Is Initially Photoreceptor
Specific in Mtz-Treated XOPNTR Tadpoles
The restricted expression pattern of NTR predicts rod photo-
receptors would be primarily affected by Mtz treatment. To
determine which retinal cell classes were initially affected,
stage 53 tadpoles were cultured in Mtz for 1 day and apoptotic
cells were identified using terminal deoxynucleotidyl trans-
ferase–mediated deoxyuridine triphosphate nick end-labeling
(TUNEL) detection. TUNEL-positive cells were seldom ob-
served in either Mtz-treated wild-type or untreated XOPNTR
tadpoles (Figs. 3A, 3B). In the retinas of untreated XOPNTR
tadpoles, TUNEL-positive cells were infrequently observed in
the ganglion cell layer (GCL; 0.5% of cells; n ? 7), whereas the
XOPNTR tadpoles. (A, B) In situ hy-
bridization was used to detect NTR
expression (purple) in transgenic
and control tadpoles. NTR expres-
sion was detected in the outer nu-
clear layer of XOPNTR tadpoles (A)
but was undetectable in wild-type
sibling embryos (B). (C–C?) Retinas
were triple stained to label NTR-ex-
pressing cells (Fast red; red), cones
(calbindin; green), and nuclei (DAPI;
blue). NTR-expressing cells (C, ar-
rowheads) do not express calbindin
(C?, C?). Scale bars: 100 ?m (A, B);
25 ?m (C–C?).
NTR expression in rod
of stage 50F1
366 Choi et al.
IOVS, January 2011, Vol. 52, No. 1
only TUNEL-positive cells detected in Mtz-treated wild-type
animals were located in the inner nuclear layer (INL; 0.4% of
cells; n ? 10). Although a similarly small number of INL cells
were apoptotic in the retinas of Mtz-treated XOPNTR animals
(0.4% of INL cells), we observed dramatic cell death in the
outer nuclear layer (Fig. 3C; n ? 8).
To determine the extent to which rods and cones were
affected, we costained retinas for TUNEL and cone calbindin.
No TUNEL-positive cells were detected in the central retina
ONL of either Mtz-treated wild-type or untreated XOPNTR
tadpoles. In contrast, in Mtz-treated XOPNTR tadpoles, 50.3%
of rods but only 3.4% of cone nuclei were TUNEL-positive
(Figs. 3D, 3D?, 3D?).
The Xenopus retina contains approximately equivalent
numbers of rod and cone photoreceptors. Therefore, these
results suggest nearly 15-fold more rods than cones are TUNEL-
outer segment loss in Mtz-treated
stage 50 XOPNTR tadpoles. (A) A be-
havioral assay was used to determine
the effect of metronidazole on the
dim-light vision of XOPNTR trans-
genic tadpoles and their wild-type
(WT) siblings. Histogram bars indi-
cate the average percentage of time
spent on the white side of the test
tank. Blue, red, and green bars de-
note wild-type Mtz-treated, XOPNTR
DMSO-treated (referred to as un-
treated), and XOPNTR Mtz-treated
tadpoles, respectively. The mean ?
SEM is indicated. The behavioral
responses of WT Mtz-treated and
XOPNTR untreated animals were not
statistically different at any time
point, whereas the Mtz-treated ani-
mals were significantly different from
both control groups on treatment
days 6, 12, and 17. Immunohisto-
chemistry was used to determine the
effect of Mtz treatment on rod pho-
toreceptors. Retinal sections were
stained to detect rod photoreceptor
outer segments (XAP2; red) and cell
nuclei (DAPI; blue). Wild-type (B)
and transgenic (C–E) tadpoles were
treated for 5 days with DMSO only
(C) or for 5 or 10 days with Mtz
(B, D, E). Higher magnification
views of the boxed regions in C–E
are shown in C?–E?. Scale bars: 100
?m (B–E); 25 ?m (C?–E?). **P ?
Rapid vision loss and rod
apoptotic cells (TUNEL; red) and all cell nuclei (DAPI; blue). Wild-type (A) and transgenic (B, C) tadpoles were left untreated (B) or were treated
with Mtz (A, C) for 1 day. (D–D?) To identify the class of apoptotic cells in the ONL, retinal sections were stained for TUNEL (D; red) and the cone
marker calbindin (D?; green). D and D? are merged in D?. Scale bars: 200 ?m (A–C); 25 ?m (D–D?). Arrowheads: TUNEL-positive cells.
Rapid rod cell death in stage 53 transgenic XOPNTR tadpoles treated with metronidazole. (A–C) Retinal sections were labeled to detect
IOVS, January 2011, Vol. 52, No. 1
Cone Degeneration Follows Rod Ablation in Xenopus
positive after 1 day of treatment. This is to be expected be-
cause NTR should only be expressed in rods given that it is
under the control of the rhodopsin promoter. Another expla-
nation for the relatively low number of TUNEL-positive cones
is that cones might have been rapidly killed, cleared from the
retina, and therefore not detected. To test this hypothesis, we
compared the number of cones in treated and control trans-
genic animals. We found no statistically significant difference
in the number of cones after 1 day of Mtz-treatment (XOPNTR
untreated 20.1 ? 0.8, XOPNTR Mtz-treated 20.5 ? 1.2; P ?
0.79). Taken together, these results suggest rod photorecep-
tors are the initial and predominant cell type ablated in Mtz-
treated XOPNTR tadpoles.
Progressive Cone Outer Segment Degeneration
and Death Follow Rod Ablation
In mammals, cone degeneration follows rod cell loss. We
wondered whether a similar dynamic was detectable in the
Xenopus retina. XOPNTR tadpoles were treated with Mtz for 1,
3, 5, 10, and 17 days. The total number and the percentage of
TUNEL-positive rods and cones were determined at each time
point. Rod transducin and cone calbindin were used to label
photoreceptors because these markers are detected in both the
outer segments and the somata of these respective cell types.
No change in the number of rods or cones was observed in
XOPNTR tadpoles during the first 3 days of Mtz treatment
(rods: day 0, 21.6 ? 1.2 [n ? 7] vs. day 3, 21.0 ? 0.6 [n ? 3];
cones: day 0, 15.4 ? 0.9 [n ? 7] vs. day 3, 15.0 ? 1.5 [n ? 3]).
Similar rod (20.6 ? 0.9; n ? 7) and cone (15.9 ? 0.6; n ?
7) numbers were detected in age-matched nontransgenic
siblings. However, 93.7% of rods and only 6.7% of cones
were TUNEL-positive on day 3. By day 5, rod cell loss was
obvious, and by day 10, the few remaining rods (0.7 ? 0.7;
n ? 3) were all TUNEL-positive (Fig. 4A). Rod transducin
was undetectable on day 17 of treatment. By comparison,
the number of cones was unchanged through day 10 of
rod ablation. Stage 52 XOPNTR tad-
days. Retinal sections were stained for
rods (transducin), cones (calbindin),
apoptotic cells (TUNEL), and nuclei
(DAPI). The blue, green, and orange
lines indicate the number of rods,
cones, and cones with outer segments
over time, respectively. The percent-
point is shown. The numbers of rods
(blue) and cones (green) in control
wild-type animals treated with Mtz for
17 days are shown for comparison (all
cones had outer segments). Similar re-
sults were observed in transgenic
control animals treated with DMSO
for 17 days (18.3 ? 0.9 rods; 16.3 ?
0.9 cones). Retinal sections of wild-
type Mtz-treated (B, C), XOPNTR
untreated (B?, C?) and XOPNTR
Mtz-treated (B?, C?) tadpoles were
stained for calbindin. Animals were
treated for either 17 (B, B?) or 35
(C, C?) days. All sections were
counterstained for nuclei (DAPI).
Asterisks: region lacking an outer
plexiform layer (C?). Arrowheads:
cones with outer segments. ND,
not determined. Scale bar, 20 ?m.
368Choi et al.
IOVS, January 2011, Vol. 52, No. 1
treatment (compare day 0 [15.4 ? 0.9] with day 10 [16.3 ?
0.9]), and the percentage of TUNEL-positive cones remained
relatively constant (Fig. 4A). No cone cell loss was detected
until day 17 of treatment (Fig. 4A).
In mammalian model systems, cone outer segment loss
precedes and is an indicator of pending cone cell death.2,28,29
Therefore, we also monitored cone outer segment loss in
Mtz-treated XOPNTR tadpoles. A progressive reduction in the
number of cones with outer segments was observed from day
0 (15.4 ? 0.9; n ? 7) through day 10 (8.7 ? 4.3; n ? 3),
despite no apparent change in the total number of calbindin-
positive cells (Fig. 4A). By day 17, approximately one-third of
surviving cones had outer segments (n ? 8; Fig. 4A). To
determine whether more prolonged exposure to Mtz would
result in more dramatic cone outer segment degeneration, we
treated XOPNTR transgenics for 17 and 35 days and stained
central retinal sections for calbindin. In contrast to retinas from
35-day controls (100% of the cones had outer segments) and
17-day Mtz-treated XOPNTR animals (38% of the cones had
outer segments), the retinas of transgenics treated for 35 days
lacked cone outer segments (Figs. 4B? ? 4C?; compare with
calbindin-positive cells with outer segments: wild-type Mtz-
treated, 20.5 ? 1.2 [n ? 4]; XOPNTR untreated, 19.8 ? 0.6
[n ? 5]; XOPNTR Mtz-treated, 0 [n ? 4]). In addition, the
retinas of Mtz-treated XOPNTR tadpoles lacked a distinct outer
plexiform layer (OPL) compared with control retinas. Small
patches of calbindin-positive cone somata were separated by
acellular gaps in the ONL in the retinas of Mtz-treated XOPNTR
tadpoles (Figs. 4C?, asterisks).
Together, these results suggest the rapid loss of rod photo-
receptors followed by more gradual cone outer segment de-
generation, and eventually cone cell death, in Mtz-treated
Secondary Cellular Changes in the Rodless
In addition to cone loss, other secondary retinal changes follow
rod photoreceptor death in all nonregenerative animal models.
To determine whether this was also true in the rodless tadpole
retina, stage 52 XOPNTR transgenics were cultured in Mtz for
17 days, and immunostaining for neuronal and glial markers
was used to identify retinal cell classes. Markers used included
islet-1 (retinal ganglion and a subset of inner nuclear layer
cells), ?-aminobutyric acid (GABA; horizontal and GABAergic
amacrine cells), calretinin (bipolar, GABAergic and serotoner-
gic amacrine and a subset of retinal ganglion cells), R5 and
vimentin (Mu ¨ller glia), and calbindin.30–34The number of cells
stained for each marker was determined and compared with
results from control untreated XOPNTR and Mtz-treated wild-
type animals. Only the number of calbindin-positive cells
(cones) differed significantly when the retinas from control
and Mtz-treated XOPNTR animals were compared (Fig. 5A and
Supplementary Fig. S1, http://www.iovs.org/lookup/suppl/
The number of Mu ¨ller glia was unchanged in rodless retinas
(Fig. 5A; wild-type Mtz-treated, 8.0 ? 0.2 [n ? 7]; XOPNTR-
untreated, 8.9 ? 0.4 [n ? 7]; XOPNTR Mtz-treated, 8.5 ? 0.3
[n ? 8]). However, the morphology of Mu ¨ller cells was dra-
matically altered in Mtz-treated transgenic animals. We ob-
served a time-dependent increase in R5 immunoreactivity in
the retinas of Mtz-treated XOPNTR transgenic animals (Figs.
5D–G). The increase in R5 staining was most notable in the
outer nuclear (Figs. 5D–G, asterisks) and ganglion cell (Figs.
5D–G, double asterisks) layers. An increase in R5 immunore-
activity with no apparent change in Mu ¨ller cell number could
result from either cell hypertrophy or an increase in the ex-
pression of the protein recognized by the R5 monoclonal
antibody (the R5 epitope is unknown). To distinguish between
these two possibilities, we stained control and day 17 treated
retinas with a second Mu ¨ller cell marker, vimentin.34We ob-
served no change in the number of vimentin-positive cells in
rodless day 17 tadpoles (wild-type Mtz-treated, 9.2 ? 0.4 [n ?
6]; XOPNTR untreated, 9.1 ? 0.4 [n ? 8]; XOPNTR Mtz-
treated, 9.0 ? 0.2 [n ? 7]). As with R5, vimentin staining was
unchanged in controls but was dramatically increased in de-
generating retinas (not shown), suggesting hypertrophy of
Mu ¨ller cells is also a secondary effect of rod cell loss in Mtz-
treated XOPNTR transgenic tadpoles.
Regeneration of Rod Photoreceptors after
The retinas of X. laevis frogs can completely regenerate after
partial or complete retinectomy.6,11,14To determine whether
rod loss was reversible in the intact retinas of transgenic
XOPNTR tadpoles, stage 53 tadpoles were treated with Mtz for
12 days and allowed to recover for 30 days.
XOPNTR transgenic animals treated with Mtz showed no
central retina XAP2 staining (Figs. 6A, A?; n ? 4). The only rod
outer segments detected were located in the most peripheral
region of the outer nuclear layer immediately adjacent to the
ciliary marginal zone (CMZ) and consisted, on average, of
fewer than five cells per section (Fig. 6A; asterisk). As ex-
pected, rod outer segments of both control groups were unaf-
fected (not shown; n ? 4 for both wild-type treated and
XOPNTR untreated groups). After 30 days of recovery, XAP2-
positive rod outer segments were once again observed in
Mtz-treated XOPNTR transgenic animals (compare Figs. 6A, A?
with 6B, B?). Rods were detected throughout the outer nuclear
layer, but their regeneration was incomplete, with some rod-
less regions persisting (Fig. 6B, arrowheads; n ? 3). Compared
with the long finger-like outer segments of control retinas
(Figs. 6C?, 6D?), the regenerated rod outer segments were
shorter and wider in appearance, resembling the immature
rods of younger retinas (Fig. 6B?). We compared the number of
ONL nuclei in Mtz-treated XOPNTR tadpoles with those of
controls. Metronidazole treatment reduced the number of ONL
nuclei by approximately 50% (XOPNTR untreated 12 days,
38.7 ? 1.7 [n ? 3]; XOPNTR Mtz-treated 12 days, 19.5 ? 1.6
[n ? 3]). After recovery, the density of nuclei in ONL regions
containing rod outer segments was similar to that of untreated
transgenics, suggesting the new outer segments are generated
from newly born rod photoreceptors (XOPNTR untreated 12
days, recovery 30 days, 39.7 ? 0.7 [n ? 3]; XOPNTR Mtz-
treated 12 days, recovery 30 days, 40.7 ? 0.3 [n ? 3]).
The marker XAP2 labels only rod outer segments. There-
fore, regenerated outer segments could originate from either
newly born rods or from undetected rod somata that had lost
their outer segments. To distinguish between these possibili-
ties, we treated XOPNTR tadpoles with Mtz for 17 days, al-
lowed them to recover for 30 days, and periodically injected
the animals with the thymidine analog EdU, which is incorpo-
rated into the DNA of replicating cells during S phase. Trans-
ducin-expressing cells were not detected in the central retinas
of Mtz-treated XOPNTR tadpoles (Fig. 6E). Similar to the XAP2
staining, transducin was only detected in the most peripheral
retina, immediately adjacent to the CMZ (two to three cells on
average per section, not shown). By comparison, transducin
was strongly expressed in both the soma and the outer seg-
ments of control rods (compare Figs. 6E and 6G: XOPNTR
Mtz-treated, 0 ? 0 [n ? 3] vs. XOPNTR untreated, 19.3 ? 0.3
[n ? 3]). Transducin-positive rods were once again observed in
Mtz-treated transgenics after a 30-day recovery phase (compare
Figs. 6E and 6F; XOPNTR Mtz-treated 17 days, recovery 30
days, 5.2 ? 1.3 [n ? 5]). Importantly, 65.4% of the rods
IOVS, January 2011, Vol. 52, No. 1
Cone Degeneration Follows Rod Ablation in Xenopus
observed were EdU-positive, demonstrating these outer seg-
ments were generated by newly born rods (Fig. 6F; XOPNTR
Mtz-treated 17 days, recovery 30 days, 65.4% EdU? [n ? 5] vs.
control XOPNTR untreated 17 days, recovery 30 days, 1.5%
EdU? [n ? 4]). Together, these results indicate that rod loss is
reversible in XOPNTR transgenic tadpoles. Within 30 days of
rod ablation, new rods were born and generated outer seg-
We report that in spite of its regenerative capacity, rod pho-
toreceptor loss in X. laevis results in secondary cellular
changes similar to those observed in nonregenerative models.
Cone cell degeneration and death are observed in patients with
RP and in all nonregenerative RP animal models.2,35–43Simi-
larly, ablation of rod photoreceptors using the NTR-metronida-
zole enzyme-prodrug system resulted in outer segment degen-
eration and cone cell death in X. laevis (Fig. 4). In contrast to
these results, a recent study also using X. laevis did not report
cone loss after rod cell ablation.9Activation of a modified
caspase-9 (iCasp9) in rod photoreceptors resulted in rod cell
death in both premetamorphic and postmetamorphic X. lae-
vis. Although cone death was not reported, cone function was
compromised after 3 months in postmetamorphic animals.
Interestingly, photopic ERGs recovered by 5 months, prompt-
ing the speculation that recovery resulted from either func-
tional restoration or regeneration of cones. The differences
observed in these two Xenopus models might have been due
to the enzyme-prodrug system used, the time at which it was
rod photoreceptor ablation. The reti-
nas of control and XOPNTR stage 52
tadpoles treated with Mtz for 17 days
were immunostained with retinal cell
markers. (A) Blue, red, and green bars
indicate the average number of cells
XOPNTR untreated, and XOPNTR Mtz-
treated tadpoles for each respective
marker. (B–G) Retinal sections of wild-
type Mtz-treated (B), XOPNTR un-
treated (C), and XOPNTR Mtz-treated
(D–G) tadpoles stained for R5 (Mu ¨ller
glia). Animals were treated for 3 (D), 5
(E), 10 (F), and 17 (G) days. All sec-
tions were counterstained for nuclei
(DAPI). Single asterisks: Mu ¨ller cell gli-
osis spreading to the subretinal layer.
Double asterisks: Mu ¨ller cell gliosis
spreading to the GCL. RGC, retinal
ganglion cells; sRGC, subset of retinal
ganglion cells; BPC, bipolar cells; sAM,
subset of amacrine cells; sHC, subset
of horizontal cells; sINL, subset of in-
ner nuclear layer cells; MGC, Mu ¨ller
glial cells. Mean ? SEM is indicated.
**P ? 0.0001. Scale bar, 20 ?m. See
Secondary changes follow
370 Choi et al.
IOVS, January 2011, Vol. 52, No. 1
activated, or both. For instance, XOPNTR tadpoles were con-
tinuously treated with metronidazole, whereas post-metamor-
phic frogs received periodic subcutaneous injections of the
iCasp9 activator AP20187 (the effect of AP20187 on cone
survival in tadpoles was not addressed). The discontinuous
delivery method that must be used in older animals may not
result in cone cell degeneration and death. Alternatively, the
extent and rate of secondary degeneration may be age depen-
dent. Frog cones may be more resistant to the effects of rod
ablation than are the cones of the tadpole retina. Examining
the fate of cones in metronidazole-treated XOPNTR frogs and
AP20187-treated iCasp9 tadpoles should distinguish between
Leakage of the cytotoxic-form of the drug into neighboring
cells could also explain the loss of cones in XOPNTR animals.
Several lines of evidence, however, suggest that this mecha-
nism is unlikely to be driving cone loss in rod-ablated Xenopus
retinas. First, cone cell loss was progressive, mimicking the
(A, A?) or were allowed to recover an additional 30 days (B–D?) before immunohistochemistry. Retinal sections were stained for XAP2 to detect
outer segments of rod photoreceptors. In XOPNTR animals treated with Mtz for 12 days, rod outer segments were only detected in the most
peripheral retina (A, asterisk). After a 30-day recovery, rod outer segments were again detected in the central retina (B, B?), but they were shorter
than rods of wild-type Mtz-treated (C, C?) and XOPNTR untreated (D, D?) tadpoles. (A?–D?) Magnified views of the boxed regions in (A–D).
(B, arrowheads) Regions lacking rod outer segments. Regenerated rods are EdU-positive. Mtz-treated (E, F) and untreated (G, H) stage 53
XOPNTR tadpoles were injected intra-abdominally with EdU, cultured for 17 days, and processed immediately (E, G) or allowed to recover
in Mtz-free media for an additional 30 days (F, H) before immunohistochemistry. Retinal sections were stained to detect nuclei (DAPI; blue),
rod photoreceptors (transducin; red), and cells that had passed through S-phase during treatment (EdU; green). Scale bars: 100 ?m (A–D);
25 ?m (A?–D?, E–H).
Rod photoreceptor regeneration in XOPNTR tadpoles. Stage 53 tadpoles were cultured in Mtz for 12 days and processed immediately
IOVS, January 2011, Vol. 52, No. 1
Cone Degeneration Follows Rod Ablation in Xenopus
temporal sequence of morphologic changes observed in other
animal RP models in which outer segments degenerate first,
followed by the loss of cone soma.28,29Second, cones continue
to die in the absence of rods, which suggests cone loss is
independent of the NTR-Mtz system because rods are no longer
present to convert Mtz to its cytotoxic form. Consistent with
this interpretation, cones lacking outer segments were ob-
served in the ONL nearly 3 weeks after the last rods had been
ablated (Fig. 4C?). Third, metronidazole was specifically devel-
oped as a substrate for NTR to avoid the prodrug-related death
of neighboring cells observed with previous substrates. In cell
culture studies, the death of neighboring cells was minimal,
even under conditions in which targeted and nontargeted cells
share gap junctions.44Fourth, a recent study investigating the
regenerative response of the zebrafish retina to rod ablation
found no evidence of cone cell death when using the NTR-Mtz
In addition to cone loss, Mu ¨ller glia hypertrophy was also
observed in the retinas of Mtz-treated XOPNTR tadpoles. Ex-
pression of the Mu ¨ller cell marker R5 (Fig. 5) and the interme-
diate filament protein vimentin (not shown) were dramatically
increased in Mtz-treated animals. Enlarged Mu ¨ller processes
extend throughout the retina, most notably into the subretinal
space (Fig. 5G). These changes mimic those observed in mam-
malian retinal degenerations.3,45Zebrafish Mu ¨ller glia also re-
spond to rod loss by upregulating the expression of interme-
diate filaments such as glial fibrillary acidic protein.46,47In
contrast to Xenopus, however, extensive gliosis in the subreti-
nal space has not been reported in fish. These results are
intriguing given the distinct response of these two regenerative
models to rod ablation. Cone loss is not observed in the rodless
zebrafish retina.13,48Rod ablation driven by misexpression of a
membrane-targeted form of cyan fluorescent protein under the
control of the Xenopus rhodopsin promoter did not result in
cone degeneration.48Similarly, cone loss was not detected in
Mtz-treated, rodless transgenic fish expressing NTR under the
control of the zebrafish rod opsin promoter.13In zebrafish,
retinal damage results in the activation of Mu ¨ller glia, which
reenter the cell cycle to produce neuronal progenitors that
differentiate into retinal neurons and heal the damaged re-
gion.46,49–54In contrast to fish, retinectomy experiments in
both premetamorphic and postmetamorphic Xenopus indicate
transdifferentiating RPE is the source of new retinal neu-
rons.10,11The correlation between the extent of Mu ¨ller cell
hypertrophy and cone cell death may point to a role for gliosis
in cone cell degeneration.
In Xenopus, rod ablation also resulted in a reduction in the
thickness of the outer plexiform layer (Fig. 4C?; asterisks)
observed in other models of photoreceptor degeneration.55,56
In contrast to nonregenerative models, however, we observed
no statistically significant change in the number of INL or GCL
cells in Mtz-treated tadpoles. Previous studies indicate that near
complete cone cell loss is necessary for extensive neuronal
remodeling, including the death of INL and GCL cells.28After
17 days of Mtz exposure, the number of cone cells was re-
duced to approximately 30% of wild-type levels, possibly ex-
plaining the lack of cell death in other retinal layers. In future
experiments, it will be important to determine whether the
late phases of degeneration (INL, GCL cell death, and neuronal
remodeling) are observed in rodless and coneless XOPNTR
When given time to recover, the retinas of rod-ablated
XOPNTR tadpoles generated new rod photoreceptors with
outer segments; however, regeneration was not complete,
possibly because of insufficient recovery time. Alternatively,
secondary cellular or molecular changes in these regions might
have permanently inhibited rod regeneration. Consistent with
this hypothesis, rod regeneration appeared less robust in trans-
genic animals treated with Mtz for 17 days compared with
12-day treated animals (not shown). However, additional ex-
periments will be necessary to distinguish between these two
Two sources of new cells in the amphibian retina are the
adult retinal stem cells of the CMZ and the retinal pigment
epithelium (RPE). Additional experiments will be necessary to
determine whether the CMZ, RPE, or an unidentified cell class
is the source of the newly born rods. Recently, Mu ¨ller glial cells
have been speculated to contribute to the regeneration of the
retina of higher order vertebrates, as occurs in teleost fish.52,57
However, our preliminary evidence shows no statistically sig-
nificant difference in the number of mitotic or EdU-labeled
Mu ¨ller glial cells between Mtz-treated transgenic and control
animals after 3, 5, or 10 days (data not shown). A more exten-
sive study is necessary to conclusively determine whether
Mu ¨ller glial cells play a role in retinal regeneration.
During normal retinal development, cell classes are born in
a stereotypical order. Retinal ganglion, horizontal, and cone
cells are born early, followed by rods, bipolar, amacrine, and
Mu ¨ller cells. Are the mechanisms of rod regeneration distinct
from those of retinal development? Are the early retinal cell
fates skipped to directly generate rods in XOPNTR tadpoles?
How rapidly is rod vision restored during regeneration? If
retinal degeneration is allowed to progress, will regeneration
no longer be possible? Or will rods, cones, INL, and RGCs all
regenerate and reform the complex neural network necessary
for functional vision? The rapid, synchronous degeneration of
rods, their regeneration, and the ability to control the timing of
these events, coupled with the behavioral assay, makes the
XOPNTR model useful for studying the mechanisms regulating
both retinal degeneration and regeneration.
The authors thank Barry Knox (SUNY Upstate Medical University),
Steve Hobbs (Institute of Cancer Research, Sutton, Surrey, UK), and Bill
Harris for providing the pXOP(?508/?41)GFP construct, F116 con-
struct, and R5 antibody, respectively.
1. Cottet S, Schorderet DF. Mechanisms of apoptosis in retinitis
pigmentosa. Curr Mol Med. 2009;9:375–383.
2. Milam AH, Li ZY, Fariss RN. Histopathology of the human retina in
retinitis pigmentosa. Prog Retin Eye Res. 1998;17:175–205.
3. Jones BW, Marc RE. Retinal remodeling during retinal degenera-
tion. Exp Eye Res. 2005;81:123–137.
4. Ripps H. Cell death in retinitis pigmentosa: gap junctions and the
‘bystander’ effect. Exp Eye Res. 2002;74:327–336.
5. Lamba D, Karl M, Reh T. Neural regeneration and cell replacement:
a view from the eye. Cell Stem Cell. 2008;2:538–549.
6. Del Rio-Tsonis K, Tsonis PA. Eye regeneration at the molecular age.
Dev Dyn. 2003;226:211–224.
7. Fischer AJ. Neural regeneration in the chick retina. Prog Retin Eye
8. Reh TA, Tully T. Regulation of tyrosine hydroxylase-containing
amacrine cell number in larval frog retina. Dev Biol. 1986;114:
9. Hamm LM, Tam BM, Moritz OL. Controlled rod cell ablation in
transgenic Xenopus laevis. Invest Ophthalmol Vis Sci. 2009;50:
10. Vergara MN, Del Rio-Tsonis K. Retinal regeneration in the Xeno-
pus laevis tadpole: a new model system. Mol Vis. 2009;15:1000–
11. Yoshii C, Ueda Y, Okamoto M, Araki M. Neural retinal regeneration
in the anuran amphibian Xenopus laevis post-metamorphosis:
transdifferentiation of retinal pigmented epithelium regenerates
the neural retina. Dev Biol. 2007;303:45–56.
372 Choi et al.
IOVS, January 2011, Vol. 52, No. 1
12. Zhao XF, Ellingsen S, Fjose A. Labelling and targeted ablation of
specific bipolar cell types in the zebrafish retina. BMC Neurosci.
13. Montgomery JE, Parsons MJ, Hyde DR. A novel model of retinal
ablation demonstrates that the extent of rod cell death regulates
the origin of the regenerated zebrafish rod photoreceptors.
J Comp Neurol. 2009;518:800–814.
14. Levine RL. La regenerescence de la retine chez Xenopus laevis.
Rev Can Biol. 1981;40:19–27.
15. Anlezark GM, Melton RG, Sherwood RF, Coles B, Friedlos F, Knox
RJ. The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide
(CB1954)—I: purification and properties of a nitroreductase en-
zyme from Escherichia coli—a potential enzyme for antibody-
directed enzyme prodrug therapy (ADEPT). Biochem Pharmacol.
16. Knox BE, Schlueter C, Sanger BM, Green CB, Besharse JC. Trans-
gene expression in Xenopus rods. FEBS Lett. 1998;423:117–121.
17. Edwards DI. Nitroimidazole drugs—action and resistance mecha-
nisms, II: mechanisms of resistance. J Antimicrob Chemother.
18. Roberts JJ, Friedlos F, Knox RJ. CB 1954 (2,4-dinitro-5-aziridinyl
benzamide) becomes a DNA interstrand crosslinking agent in
Walker tumour cells. Biochem Biophys Res Commun. 1986;140:
19. Viczian AS, Solessio EC, Lyou Y, Zuber ME. Generation of func-
tional eyes from pluripotent cells. PLoS Biol. 2009;7:e1000174.
20. Moriya T, Kito K, Miyashita Y, Asami K. Preference for background
color of the Xenopus laevis tadpole. J Exp Zool. 1996;276:335–
21. Kroll KL, Amaya E. Transgenic Xenopus embryos from sperm
nuclear transplantations reveal FGF signaling requirements during
gastrulation. Development. 1996;122:3173–3183.
22. Sive HL, Grainger RM, Harland RM. Early development of Xenopus
laevis: a laboratory manual. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press; 2000;ix:338.
23. Viczian AS, Vignali R, Zuber ME, Barsacchi G, Harris WA. XOtx5b
and XOtx2 regulate photoreceptor and bipolar fates in the Xeno-
pus retina. Development. 2003;130:1281–1294.
24. Mani SS, Batni S, Whitaker L, Chen S, Engbretson G, Knox BE.
Xenopus rhodopsin promoter: identification of immediate up-
stream sequences necessary for high level, rod-specific transcrip-
tion. J Biol Chem. 2001;276:36557–36565.
25. Moritz OL, Tam BM, Papermaster DS, Nakayama T. A functional
rhodopsin-green fluorescent protein fusion protein localizes cor-
rectly in transgenic Xenopus laevis retinal rods and is expressed in
a time-dependent pattern. J Biol Chem. 2001;276:28242–28251.
rod photoreceptors in Xenopus. J Neurobiol. 1998;35:227–244.
27. Harris WA, Messersmith SL. Two cellular inductions involved in
photoreceptor determination in the Xenopus retina. Neuron.
28. Marc RE, Jones BW, Watt CB, Strettoi E. Neural remodeling in
retinal degeneration. Prog Retin Eye Res. 2003;22:607–655.
29. Lin B, Masland RH, Strettoi E. Remodeling of cone photoreceptor cells
after rod degeneration in rd mice. Exp Eye Res. 2009;88:589–599.
30. Gabriel R. Calretinin is present in serotonin- and gamma-aminobu-
tyric acid-positive amacrine cell populations in the retina of Xeno-
pus laevis. Neurosci Lett. 2000;285:9–12.
31. Ohnuma S, Philpott A, Wang K, Holt CE, Harris WA. p27Xic1, a
Cdk inhibitor, promotes the determination of glial cells in Xeno-
pus retina. Cell. 1999;99:499–510.
32. Seufert DW, Prescott NL, El-Hodiri HM. Xenopus aristaless-related
homeobox (xARX) gene product functions as both a transcrip-
tional activator and repressor in forebrain development. Dev Dyn.
33. Zaghloul NA, Moody SA. Changes in Rx1 and Pax6 activity at eye
field stages differentially alter the production of amacrine neuro-
transmitter subtypes in Xenopus. Mol Vis. 2007;13:86–95.
34. Sakaguchi DS, Moeller JF, Coffman CR, Gallenson N, Harris WA.
Growth cone interactions with a glial cell line from embryonic
Xenopus retina. Dev Biol. 1989;134:158–174.
35. Carter-Dawson LD, LaVail MM, Sidman RL. Differential effect of the
rd mutation on rods and cones in the mouse retina. Invest Oph-
thalmol Vis Sci. 1978;17:489–498.
36. Li T, Snyder WK, Olsson JE, Dryja TP. Transgenic mice carrying the
dominant rhodopsin mutation P347S: evidence for defective vec-
torial transport of rhodopsin to the outer segments. Proc Natl
Acad Sci U S A. 1996;93:14176–14181.
37. Gargini C, Terzibasi E, Mazzoni F, Strettoi E. Retinal organization in
the retinal degeneration 10 (rd10) mutant mouse: a morphological
and ERG study. J Comp Neurol. 2007;500:222–238.
38. Petters RM, Alexander CA, Wells KD, et al. Genetically engineered
large animal model for studying cone photoreceptor survival and
degeneration in retinitis pigmentosa. Nat Biotechnol. 1997;15:
39. Chang B, Hawes NL, Pardue MT, et al. Two mouse retinal degen-
erations caused by missense mutations in the beta-subunit of rod
cGMP phosphodiesterase gene. Vis Res. 2007;47:624–633.
40. Kijas JW, Cideciyan AV, Aleman TS, et al. Naturally occurring
rhodopsin mutation in the dog causes retinal dysfunction and
degeneration mimicking human dominant retinitis pigmentosa.
Proc Natl Acad Sci U S A. 2002;99:6328–6333.
41. Humphries MM, Kiang S, McNally N, et al. Comparative structural
and functional analysis of photoreceptor neurons of Rho?/? mice
reveal increased survival on C57BL/6J in comparison to 129Sv
genetic background. Vis Neurosci. 2001;18:437–443.
42. Machida S, Kondo M, Jamison JA, et al. P23H rhodopsin transgenic
rat: correlation of retinal function with histopathology. Invest
Ophthalmol Vis Sci. 2000;41:3200–3209.
43. Krebs MP, White DA, Kaushal S. Biphasic photoreceptor degeneration
induced by light in a T17M rhodopsin mouse model of cone bystander
damage. Invest Ophthalmol Vis Sci. 2009;50:2956–2965.
44. Bridgewater JA, Knox RJ, Pitts JD, Collins MK, Springer CJ. The
bystander effect of the nitroreductase/CB1954 enzyme/prodrug
system is due to a cell-permeable metabolite. Hum Gene Ther.
45. Jones BW, Watt CB, Frederick JM, et al. Retinal remodeling triggered by
photoreceptor degenerations. J Comp Neurol. 2003;464:1–16.
46. Raymond PA, Barthel LK, Bernardos RL, Perkowski JJ. Molecular
characterization of retinal stem cells and their niches in adult
zebrafish. BMC Dev Biol. 2006;6:36.
47. Morris AC, Scholz TL, Brockerhoff SE, Fadool JM. Genetic dissec-
tion reveals two separate pathways for rod and cone regeneration
in the teleost retina. Dev Neurobiol. 2008;68:605–619.
48. Morris AC, Schroeter EH, Bilotta J, Wong RO, Fadool JM. Cone
survival despite rod degeneration in XOPS-mCFP transgenic ze-
brafish. Invest Ophthalmol Vis Sci. 2005;46:4762–4771.
49. Yurco P, Cameron DA. Responses of Muller glia to retinal injury in
adult zebrafish. Vision Res. 2005;45:991–1002.
50. Fausett BV, Goldman D. A role for alpha1 tubulin-expressing Mul-
ler glia in regeneration of the injured zebrafish retina. J Neurosci.
51. Fimbel SM, Montgomery JE, Burket CT, Hyde DR. Regeneration of
inner retinal neurons after intravitreal injection of ouabain in
zebrafish. J Neurosci. 2007;27:1712–1724.
52. Bernardos RL, Barthel LK, Meyers JR, Raymond PA. Late-stage
neuronal progenitors in the retina are radial Muller glia that func-
tion as retinal stem cells. J Neurosci. 2007;27:7028–7040.
53. Thummel R, Kassen SC, Montgomery JE, Enright JM, Hyde DR.
Inhibition of Muller glial cell division blocks regeneration of the
light-damaged zebrafish retina. Dev Neurobiol. 2008;68:392–408.
54. Thummel R, Kassen SC, Enright JM, Nelson CM, Montgomery JE,
Hyde DR. Characterization of Muller glia and neuronal progenitors
during adult zebrafish retinal regeneration. Exp Eye Res. 2008;87:
55. Aleman TS, Cideciyan AV, Sumaroka A, et al. Retinal laminar
architecture in human retinitis pigmentosa caused by rhodopsin
gene mutations. Invest Ophthalmol Vis Sci. 2008;49:1580–1590.
56. Yu DY, Cringle SJ, Su EN, Yu PK. Intraretinal oxygen levels before
and after photoreceptor loss in the RCS rat. Invest Ophthalmol Vis
57. Karl MO, Hayes S, Nelson BR, Tan K, Buckingham B, Reh TA.
Stimulation of neural regeneration in the mouse retina. Proc Natl
Acad Sci U S A. 2008;105:19508–19513.
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