Download Impairment of Vision in a Mouse Model of Usher Syndrome Type III

Retina
Impairment of Vision in a Mouse Model of Usher
Syndrome Type III
Guilian Tian, Richard Lee, Philip Ropelewski, and Yoshikazu Imanishi
Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States
Correspondence: Yoshikazu Imanishi, Department of Pharmacology,
Case Western Reserve University,
2123 Adelbert Road, Cleveland, OH
44106, USA;
[email protected].
GT and RL contributed equally to the
work presented here and should
therefore be regarded as equivalent
authors.
Submitted: March 24, 2015
Accepted: January 20, 2016
Citation: Tian G, Lee R, Ropelewski P,
Imanishi Y. Impairment of vision in a
mouse model of Usher syndrome type
III. Invest Ophthalmol Vis Sci.
2016;57:866–875. DOI:10.1167/
iovs.15-16946
PURPOSE. The purpose of this study was to obtain an Usher syndrome type III mouse model
with retinal phenotype.
METHODS. Speed congenic method was used to obtain Clrn1 exon 1 knockout (Clrn1/) and
Clrn1N48K knockin (Clrn1N48K/N48K) mice under A/J background. To study the retinal
functions of these mice, we measured scotopic and photopic ERG responses. To observe if
there are any structural abnormalities, we conducted light and transmission electron
microscopy of fixed retinal specimens.
RESULTS. In 3-month-old Clrn1/ mice, scotopic b-wave amplitude was reduced by more than
25% at the light intensities from 2.2 to 0.38 log cds/m2, but scotopic a-wave amplitudes
were comparable to those of age-matched wild type mice at all the light intensities tested. In
9-month-old Clrn1/ mice, scotopic b-wave amplitudes were further reduced by more than
35%, and scotopic a-wave amplitude also showed a small decline as compared with wild type
mice. Photopic ERG responses were comparable between Clrn1/ and wild type mice.
Those electrophysiological defects were not associated with a loss of rods. In Clrn1N48K/N48K
mice, both a- and b-wave amplitudes were not discernable from those of wild type mice aged
up to 10 months.
CONCLUSIONS. Mutations that are Clrn1/ biallelic cause visual defects when placed under A/J
background. The absence of apparent rod degeneration suggests that the observed phenotype
is due to functional defects, and not due to loss of rods. Biallelic Clrn1N48K/N48K mutations did
not cause discernible visual defects, suggesting that Clrn1 allele is more severely
dysfunctional than ClrnN48K allele.
sher syndrome type III (USHIII) is characterized by
progressive loss of hearing and vision as well as by variable
degrees of balance disorder.1,2 Onsets of both hearing and
vision loss are highly variable. For example, vision in the
peripheral field is lost in the first 2 to 3 decades of life, and
thereafter central vision can last for 1 or 2 more decades.2–4
Clarin-1 (Clrn1) is currently the only gene associated with
USHIII.5,6 The gene is predicted to encode a four transmembrane domains protein (CLRN1) with unknown function.
Proteomics analysis of CLRN1-interacting proteins suggests
that CLRN1 is associated with the regulation of actin
homeostasis. 7 Consistently, studies of CLRN1-deficient
mouse7–9 and zebrafish10 indicate structural and functional
defects in the stereocilia: actin-filled membrane structures
responsible for the mechanosensation of auditory hair cells.
Furthermore, recent study using CLRN1-deficient zebrafish
suggests that CLRN1 is also potentially involved in synaptic
transmission by hair cells.10 Currently, the role of CLRN1 in the
retina is unknown, largely because there are no model animals
that recapitulate the visual defects of USHIII.
Severities of Mendelian diseases, including Usher syndrome
(USH), are affected by other genes collectively called modifiers.11–14 Accordingly, interpretations of phenotypic outcomes
tend to be complicated by the genetic interactions between
USH causative genes and their modifiers. Such genetic
interaction is potentially one of the causes for variable onsets
of hearing or vision loss observed in human USH pa-
U
tients.1,2,15,16 In mouse, studies of different genetic backgrounds were instructive to understand the effect of modifiers
on the age-dependent deterioration of vision. For example,
effects of different genetic backgrounds were clearly illustrated
by the study of retinitis pigmentosa 1 homolog (RP1h) mutant
allele, RP1htm1Eap.17 When RP1htm1Eap allele was placed in
C57BL/6J or DBA1/J backgrounds, it did not induce apparent
retinal degeneration phenotype. When placed in the A/J
background, however, the RP1htm1Eap allele induced severe
age-dependent retinal degeneration. Unlike other genetic
backgrounds, the A/J background predisposes mice to agerelated retinal degeneration18,19 which is a hallmark of many
inherited blinding disorders.20 In human, age-related deterioration of vision is a natural process,21 and may affect the visual
performance of USH patients. Thus, the A/J mouse is potentially
a useful tool to exacerbate age-related retinal phenotypes that
are observed in human USH patients but difficult to detect in
most murine USH models.16
In the past, we and others generated and characterized
murine models of USHIII.8,9,22 There are two major mutations1,5,6,23 causative to USHIII: Y176X mutation, which is
commonly observed in Finnish USHIII patients and causes a
premature termination of the translation, and N48K mutation,
which is commonly observed in USHIII patients of Ashkenazi
Jewish population and causes defects in the N-linked glycosylation of CLRN1.7 Mutation Y176X is considered functionally
null, and thus the Clrn1 knockout would recapitulate this
iovs.arvojournals.org j ISSN: 1552-5783
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017
866
IOVS j March 2016 j Vol. 57 j No. 3 j 867
USHIII Model Mouse With Reduced ERG Responses
genetic condition. Mutation N48K may be a hypomorphic
allele as the progression of hearing impairment is slower in
Clrn1N48K knockin mouse than in Clrn1 knockout mouse.
While these models demonstrate hearing impairments, they
did not exhibit visual phenotype. Thus, it has been difficult to
quantify and compare the effects of different Clrn1 mutations
to the retina. In this study, we backcrossed Clrn1 exon 1
knockout (Clrn1) and N48K knockin (Clrn1N48K) alleles to A/
J strain, and obtained Clrn1-deficient and mutant mice with
more than 96% of the genes derived from A/J background.
These newly generated USHIII models allowed us to investigate
the role of CLRN1 in the mouse retina and to compare the
properties of Clrn1 and Clrn1N48K alleles. Thus, in conjunction with the previous characterizations of hearing phenotypes,8,9 this characterization of retinal phenotype is relevant
to understanding the etiologies of human USHIII caused by
Y176X and N48K mutations.
METHODS
Animals
All animal experiments employed procedures approved by
Case Western Reserve University Animal Care Committee and
conformed to both the recommendations of the American
Veterinary Medical Association Panel on Euthanasia and the
Associate of Research for Vision and Ophthalmology. Mice
were maintained in a 12-hour light/12-hour dark (6 AM/6 PM)
cycle and fed standard mouse chow. All electroretinogram
experiments were performed under dim red light produced
using a medium red filter (Roscolux 27, transmittance >580
nm; Rosco Laboratories, Inc., Stamford, CT, USA).
The Generation of Clrn1 Mutant Mice Under A/J
Background
The Clrn1/ and Clrn1N48K/N48K mice under C57BL/6J
background8,9,22 were backcrossed to A/J background using
Jackson Laboratory’s Speed Congenic Service. This method
identifies the progenies which carried the highest percentage
of targeted background by testing the cross-strain differences
in the single nucleotide polymorphisms (SNPs; also called
microsatellite polymorphisms). At Jackson, SNPs were scanned
throughout the genome24,25 to confirm that Clrn1þ/ and
Clrn1þ/N48K mice carried more than 96% of the genome from
the A/J strain. Those mice were again backcrossed with A/J
mice to obtain Clrn1þ/ and Clrn1þ/N48K offspring, which were
then expanded into colonies at Case Western Reserve
University. The presence of Clrn1 and Clrn1N48K alleles were
confirmed by PCR amplification of genomic DNA (Supplementary Figs. S1A–C). The mice used for this study were negative
to rd8 allele as confirmed using the PCR-based method,26 and
thus our analyses were not affected by this mutation.26 We
found that the A/J strain was difficult to breed. Accordingly, the
number of offspring we could obtain was lower than that for
mice in C57BL/6J background.
Genomic DNA Isolation From Mouse Tail
Genomic DNA was isolated from mouse tail snips by
incubating in tail lysis buffer (0.1 M Tris [pH 7.5], 5 lM EDTA,
0.2% SDS [wt/vol], 0.2 M NaCl, and 700 lg/lL Proteinase K)
either overnight at 558C or for 2 hours at 558C in a
Thermomixer (Eppendorf) set to 1200 RPM. After digestion,
samples were spun down and the supernatant was mixed with
an equal volume of isopropanol to precipitate the genomic
DNA. Solutions were spun down again, and the precipitated
genomic DNA was washed with 70% ethanol to remove salts.
Genomic DNA was then dissolved in 200 lL water and 10 lL
was used for subsequent PCR analysis.
Genotyping PCR
The genotypes of the A/J Clrn1/ mice were confirmed by
performing PCR using two sets of primer pairs: P3/P4 and
C1/P4. P3 (5 0 -GGAGTAAGAAGTAGTCAACGG-3 0 , forward
primer) is located upstream of exon 1 of Clrn1 and P4 (5 0 GCATTTCTCAGCAGATCAC-3 0 , reverse primer) is located
downstream of exon 1. Together, P3/P4 amplifies a 2066
bp product for wild-type (WT) allele and a 782 bp product
for Clrn1 allele. C1 (5 0 -TTTACCGAAGCCTTTTCTCG-3 0 ,
forward primer) is located within exon 1, and when paired
with P4, a 1035 bp product is produced for WT allele, while
no product results from Clrn1 allele. Template DNA was
mixed with dH2O, 0.5 U HotStarTaq (Qiagen, Hilden,
Germany), the manufacturer-provided reaction buffer, 200
lM deoxyribose–nucleotide triphosphates (dNTPs), and 0.5
lM of each primer. Polymerase chain reaction cycler
conditions consisted of an initial denature/enzyme heat
activation step of 15 minutes at 958C followed by 35 cycles
of 30 seconds at 948C, 30 seconds at 538C for P3/P4 or 558C
for C1/P4, and 165 seconds at 728C, followed by a final
elongation step of 7 minutes at 728C. Allele Clrn1N48K was
identified using PCR primers 2L (5 0 -GACAAGATGACTACCAT
TAAGCTATGTG-3 0 ) and 2R (5 0 -CTTTCGTTTTTGAAAATTCA
CACACACAC-3 0 ) which flank the loxP site present in the
Clrn1N48K knock-in allele but not in the WT allele. This set
of primers produced a 275 bp PCR product for the knock-in
allele and a 230 bp product for WT allele. The reaction
composition is identical to that given above for Clrn1/
genotyping. Products were amplified using the following
cycler conditions: 15 minutes at 958C, followed by 35 cycles
of 30 seconds at 948C, 30 seconds at 558C, and 150 seconds
at 728C, concluded by a final elongation step of 10 minutes
at 728C. Brightness and contrast of the gel images were
adjusted using photo editing software (Adobe Photoshop
CS6; Adobe Systems, Inc., San Jose, CA, USA). Only linear
adjustments were made.
ERGs
We analyzed Clrn1/ and WT mice by ERG at the ages of 3, 9,
and 12 months. ClrnN48K/N48K and WT mice were analyzed by
ERG at the ages of 4 and 10 months. Mice were dark-adapted at
least overnight before analysis. Under dim red light, mice were
anesthetized with intraperitoneal injection using 10 lL/g body
weight of 12.5 mg/mL ketamine and 0.5 mg/mL xylazine
diluted with 13 PBS (Hyclone cat# SH30256.01). The pupils
were dilated with 0.01% tropicamide. A contact lens electrode
was placed on the eye, and a reference electrode and ground
electrode were placed under the skin of the tail and between
the ears. We recorded ERGs with a universal electrophysiological system (UTAS E-3000 MF; LKC Technologies, Inc.,
Gaithersburg, MD, USA). The light intensity was calibrated by
the manufacturer and was computer-controlled. The mice
were placed in a Ganzfeld dome, and scotopic and photopic
responses to flash stimuli were each obtained from both eyes
simultaneously. We noted variability in A/J mouse’s response to
the anesthetics, which might have affected the ERG responses.
Thus, at the end of the procedure, each eye was inspected for
the presence of cataracts. If the mouse formed severe cataracts,
indicated any signs of morbidity, or did not survive the
procedure, data from these mice were excluded from the
analysis.
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017
IOVS j March 2016 j Vol. 57 j No. 3 j 868
USHIII Model Mouse With Reduced ERG Responses
Immunofluorescence Staining
Eye cups were fixed in 4% paraformaldehyde for 6 hours at
room temperature, followed by incubation in a succession of
phosphate buffer (0.02 M NaH2PO4, 0.08 M Na2HPO4) with
increasing concentrations of sucrose (5%–20% wt/vol in
phosphate buffer) at room temperature, and overnight incubation in a mixture of one part OCT (Sakura) to two parts SPB
(20% sucrose wt/vol in phosphate buffer). at 48C. Eye cups were
then frozen and sectioned on a cryostat (CM1850; Leica
Microsystems, Wetzlar, Germany) at 208C. Fixed eye sections
were blocked in 1.5% normal goat serum diluted in PBS with
vol/vol 0.1% Triton X-100 (PBST) for 1 hour, incubated with
primary antibody overnight at room temperature, washed, and
then incubated in secondary antibody for 1 hour at room
temperature, followed by a final wash and mounting. The
following primary antibodies were used: mouse mAb anti-b
peripherin/rds (a gift from Brian Kevany, Department of
Pharmacology, Case Western Reserve University, Cleveland,
OH, USA); mouse mAb anti-rhodopsin 1D427 (an antibody
originally generated by Robert S. Molday et al., and a gift from
Vera Moiseenkova-Bell, Department of Pharmacology, Case
Western Reserve University, Cleveland, OH, USA); rabbit pAb
anti-glial fibrillary acidic protein (catalog no. Z0334; Dako, Santa
Clara, CA, USA); rabbit pAb anti PKCa (catalog no. SC-208; Santa
Cruz Biotechonology, Inc., Dallas, TX, USA); mouse mAb anti
PSD-95 (catalog no. 05-494; Upstate Biotechnology, Cambridge,
MA, USA); mouse IgG anti-RIBEYE (catalog no. 612044; BD
Transduction Laboratories, San Jose, CA, USA); mouse mAb antiGo alpha (catalog no. MAB3073; Chemicon, Billerica, MA, USA);
mouse mAb anti-Bassoon (Stressgen, Farmingdale, NY, USA);
mouse mAb anti-Calbindin-D-28K (clone CB-955, catalog no.
C9848; Sigma-Aldrich Corp., St. Louis, MO, USA); mouse mAb
anti-Calretinin (catalog no. MAB1568; Chemicon); and mouse
mAb anti-myosin VIIA (catalog no. MYO7A 138-1; Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Goat antimouse Cy3, donkey anti-rabbit Alexa Fluor 488, or donkey antimouse Alexa Fluor 488 (Jackson ImmunoResearch) were used as
secondary antibodies. Samples were visualized by confocal
microscopy.
Histology and Light Microscopy
For light microscopy, mouse eyecups were fixed and processed
as described above. The sections of fixed eye cups were
stained with Hoechst 33342 nuclear staining and mouse mAb
anti-rhodopsin 1D4 also as described above. After washing
with PBST and mounting, the sections were imaged using a
Leica DM6000B microscope. Brightness and contrast of
differential interference contrast (DIC) images were adjusted
using photo editing software (Adobe Photoshop CS6; Adobe
Systems, Inc.); only linear adjustments were made. Composite
images were made using image analysis software (Image-Pro
MDA, ver. 5.1.0.20; Media Cybernetics, Rockville, MD, USA).
Immunoblotting
Whole retinal lysate was prepared by sonicating excised retina in
radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4],
150 mM NaCl, 1% IGEPAL CA-630, and 0.5% sodium deoxycholate) containing complete protease inhibitor (Roche Pharmaceuticals, Basel, Switzerland), mixing for 1 hour at 48C, and then
clearing by centrifugation. Proteins were resolved using SDSPAGE, transferred to polyvinylidene fluoride membrane, blocked
in PBS containing 0.1% Tween-20 and 5% dry milk, and probed
with primary antibodies against rhodopsin, peripherin, or glial
fibrillary acidic protein (GFAP; see section Immunofluorescence
Staining for detailed antibody information). Membranes were
washed in PBS containing 0.1% Tween-20 and incubated in
secondary antibody conjugated with horseradish peroxidase.
Tubulin probed by mAb anti-beta-tubulin (catalog no. E7;
Developmental Studies Hybridoma Bank) was used for a loading
control for each lane. Immuno-positive bands were quantified
using ImageJ (http://imagej.nih.gov/ij/; provided in the public
domain by the National Institutes of Health, Bethesda, MD, USA).
Electron Microscopy
Eye cups were prepared using 4.5-month-old AJ Clrn/ mice
along with age-matched WT AJ mice obtained from Charles
River Laboratories, Inc. (Wilmington, MA, USA). Tissue
sections were prepared as described previously28,29 and
examined by a FEI Tecnai Spirit (T12) transmission electron
microscope with a charge-coupled device camera (US 4000 4k
3 4k; Gatan, Inc., Pleasanton, CA, USA).
RESULTS
Characterization of Clrn1/ Mice Under A/J
Background
In the past, we have analyzed the phenotypes of Clrn1 knockout
(Clrn1/) and Clrn1N48K knockin (Clrn1N48K/N48K) mice under
C57BL/6J background. These Clrn1/ and Clrn1N48K/N48K mice
did not demonstrate any detectable defects in the retina.8,9,22
Compared with C57BL/6J mice, A/J mice demonstrate a
pronounced age-related deterioration of vision, which is also a
characteristic of the human visual system.17,18 To study the
function of CLRN1 in context to age-related deterioration of
vision, we generated Clrn1/ and Clrn1N48K/N48K under A/J
background. To investigate whether the loss of Clrn1 gene
causes retinal abnormalities, visual functions of Clrn1/ and WT
control mice were compared by ERG analyses at different ages
under scotopic conditions (Figs. 1, 2). At age 3 months, the
Clrn1/ mice displayed b-wave amplitudes significantly lower
than WT counterparts (Figs. 1A, 2A). With the light stimuli at the
intensities of 2.2 to 0.38 log cds/m2, the responses of Clrn1/
mice were 25% to 31% lower than those of WT mice (P < 0.013
by t-test). At the same age, there was no significant difference in
a-wave amplitude between Clrn1/ and WT mice (Figs. 1A,
right panel, 2B). B-wave originates from the secondary
neurons,30 and thus the reduction in b-wave amplitudes
observed in Clrn1/ is likely caused by deficit in the synaptic
communication between photoreceptors and secondary neurons, or deficits in the inner retina.
At age 9 months, b-wave amplitudes became further
separated (Fig. 2A, 9 months), and a-wave amplitudes became
significantly different between Clrn1/ and WT mice (Fig. 2B).
B-wave amplitudes of Clrn1/ mice was 43% to 51% lower
than those of WT mice (P < 0.001 by t-test) at the light
intensities of 2.2 to 0.38 log cds/m2 (Fig. 1B, left and middle
panel). A-wave amplitudes of Clrn1/ mice were also
significantly lower than those of WT mice by 30% to 40% for
the three highest light intensities (0.6, 0.38, and 1.6 log cds/
m2). As a-wave originates from photoreceptor cells,31 this agedependent decline suggests that photoreceptor function is
compromised in Clrn1/ mice. The above ERG responses
were measured under scotopic conditions, and were primarily
contributed by rod photoreceptors. To test the contribution of
cone photoreceptors, we measured the ERG responses under
photopic conditions. Under photopic conditions, Clrn1/ and
WT mice gave similar b-wave amplitudes under different
intensities of light, suggesting that it was not the cone
photoreceptor function, but the rod photoreceptor function
which was primarily affected in Clrn1/ (Fig. 3A).
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017
IOVS j March 2016 j Vol. 57 j No. 3 j 869
USHIII Model Mouse With Reduced ERG Responses
FIGURE 1. Electroretinogram responses of Clrn1/ and WT mice in A/J background at age 3 to 12 months. Electroretinogram responses (Left) were
measured under scotopic conditions. Amplitudes of b- (Middle) and a-wave (Right) were plotted as a function of light intensity. (A) At age 3 months,
b-wave amplitudes were significantly lower in Clrn1/ than in WT mice at the light intensities of 2.2, 0.6, and 0.38 log cds/m2. (B) At age 9
months, b-wave amplitudes of Clrn1/ were significantly lower than those of WT mice at all the intensities tested. A-wave amplitudes of Clrn1/
mice were also significantly lower than those of WT mice at the light intensities of 0.6, 0.38, and 1.6 log cds/m2. (C) At age 12 months, there was
no significant difference in a- and b-wave amplitudes between Clrn1/ and WT mice at all light intensities tested (P > 0.11 by t-test). ***P < 0.001.
**P < 0.01. *P < 0.05 by t-test.
In analyzing the phenotype caused by Clrn1/, it is crucial
to note that WT A/J mice demonstrated age-dependent decline
in both a- and b-wave amplitudes independent of the Clrn1
defect. For example, WT A/J mice demonstrated 52% and 59%
decline in a- and b- wave amplitudes from age 3 to 12 months at
the light intensity of 0.38 log cds/m2 (WT, Figs. 1A, 1C). Thus,
this age-dependent decline of ERG amplitudes in A/J background eventually masked the decline caused by Clrn1
deficiency (Fig. 2). At age 12 months, neither the a- nor bwave amplitude was significantly different between Clrn1/
FIGURE 2. Age-dependent decline in ERG a- and b-wave amplitudes. Amplitudes of a- and b-waves (light stimuli: log cds/m2) were measured and
compared between WT and Clrn1/ mice at ages 3, 9, and 12 months. Both WT and Clrn1/ demonstrated age-dependent decline of a- and b-wave
amplitudes from 3 to 9 months. At age 9 months, a- and b-wave amplitudes of Clrn1/ were lower than those of WT. From 9 to 12 months,
additional age-dependent decline was observed in WT mice, and as a result a- and b-wave amplitudes became similar between Clrn1/ and WT
mice. The data are represented by mean 6 SD. For these analyses, the numbers and ages of animals used were as follows: eight WT and seven
Clrn1/ mice at age 3 months, seven WT and seven Clrn1/ at age 9 months, and eight WT and eight Clrn1/ at age 12 months. ***P < 0.001. **P
< 0.01. *P < 0.05. NS, not significantly different (P > 0.05) by t-test.
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017
IOVS j March 2016 j Vol. 57 j No. 3 j 870
USHIII Model Mouse With Reduced ERG Responses
FIGURE 3. Electroretinogram responses of 9-month-old Clrn1/ and WT mice under photopic conditions (A) and recovery of a-wave amplitude
during dark adaptation (B). (A) Representative ERG responses (left) and a plot of b-wave amplitudes as a function of light intensity (right). B-wave
amplitudes were similar between Clrn1/ and WT mice (P > 0.14 by t-test). (B) Dark-adapted mice were exposed to intense illumination (>445 cd/
m2) for 3 minutes, and then dark-adapted again. The recovery of a-wave amplitudes was monitored with single-flash ERG (0.2 log cds/m2) every 5
minutes for 60 minutes. There was no significant difference in recovery rate between Clrn1/ and WT mice (P > 0.29 by t-test). Nine-month-old
Clrn1/ and WT mice were used for these two tests.
and WT mice (Figs. 1C, 2). In general, a- and b-wave amplitudes
observed for 12-month-old Clrn1/ and WT mice were similar
to those observed for 9-month-old Clrn1/ mice. Thus, from 9
to 12 months, the decline of ERG amplitudes caused inherently
by A/J genetic background masked the decline caused by Clrn1
defect.
Dark adaptation is mediated by biochemical processes such
as 11-cis-retinal regeneration32 and translocation of proteins
associated with rhodopsin-mediated GPCR cascade.33,34 To
understand the contribution of these processes, the rate of
dark adaptation was compared between Clrn1/ and WT
mice. Initially, the photoreceptor cells were photobleached
with strong light, which diminished the a-wave amplitude by
100%. Then, the recovery of the a-wave amplitude was
monitored every 5 minutes, up to 1 hour. Recovery rates of
a-wave amplitude were similar between Clrn1/ and WT (Fig.
3B). Therefore, this study suggests that CLRN1 protein is not
involved in the 11-cis-retinal regeneration or other biochemical
processes associated with dark adaptation.
apparent difference in the extent of aberrant innervation
between WT and Clrn1/ mice (Fig. 5A, Clrn1/, arrow). The
majority of synapses were formed appropriately in the outer
plexiform layer as demonstrated by localization of PSD95positive presynapse structures and PKCa positive dendrites in
the outer plexiform layer (Fig. 5A, OPL). A similar result was
obtained for another on-bipolar cell marker Go a (Supplementary Fig. S3A). Likewise, dendrites of horizontal cells, labelled
by anti-calbindin antibody,42 demonstrated similar innervation
patterns in both WT and Clrn1/, mostly connecting
appropriately at the outer plexiform layer (Supplementary
Fig. S3B). Anti-calretinin, which is known to label amacrine and
ganglion cells,42 did not show discernible differences in WT
Absence of Global Morphologic Abnormalities in
the Ciliary and Synaptic Compartments of Clrn1/
Retina
Given assumptions that Usher syndrome is a cilia- or synapserelated disorder,15,35 and considering a possible role of CLRN1
in the organization of actin7 that often mediates protein
trafficking or synaptic transmission,36 we tested the localization of two major ciliary trafficked proteins, rhodopsin,37,38
and peripherin/rds,39 in Clrn1/ mice. Despite these assumptions, both rhodopsin and peripherin/rds localized properly to
the rod outer segments of Clrn1/ mice (Fig. 4), and protein
expression levels were not significantly different between WT
and Clrn1/ mice as assessed by immunoblotting (Supplementary Fig. S2, P > 0.42). Thus, Clrn1/ is not involved in
the trafficking of those primary proteins in the outer segment.
The b-wave defect found in Clrn1/ is possibly a sign that
the photoreceptor synapse is affected. Ectopic synapse
formation has been reported to be one of age-dependent
phenotypes in A/J strain of mice.40 To examine if ectopic
synapse formation is exacerbated in Clrn1/ mice, we labelled
the eye sections of 9-month-old Clrn1/ and WT mice using an
antibody against PSD95 to visualize the photoreceptor synaptic
termini41 and another antibody against PKCa to visualize the
dendritic regions from rod on-bipolar cells.42 As anticipated,
ectopic synapses formed in WT A/J mice as evidenced by
aberrant innervation of PKCa positive dendrites into the outer
nuclear layer (Fig. 5A, WT, arrow), however there was no
FIGURE 4. Deficiency of CLRN1 does not affect the localization of
outer segment proteins rhodopsin and peripherin/rds. Sections of
fixed eye cups of 9-month-old WT (top panel) and Clrn1/ (bottom
panel) mice were probed with Hoechst 33342 nuclear staining dye
(blue), antibody against GFAP (green), and antibody against either the
1D4 epitope of rhodopsin (red, left panels) or peripherin/rds (red,
right panels). Rhodopsin and peripherin/rds localized properly in rod
outer segments of WT and Clrn1/ mice. Arrows indicate vascular–
IgG labeled with the secondary antibody employed in the study. Images
are maximum projections of a series of optical sections. Scale bar: 40
lm.
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017
IOVS j March 2016 j Vol. 57 j No. 3 j 871
USHIII Model Mouse With Reduced ERG Responses
FIGURE 5. Deficiency of CLRN1 did not promote the formation of ectopic synapse. (A) Sections of fixed eye cups of 9-month-old WT (top row) and
Clrn1/ (bottom row) mice were probed with antibody against PSD95 (red), and PKCa (green). In WT and Clrn1/, the majority of synapses are
formed appropriately in the outer plexiform layer (OPL). Images are single confocal optical sections. Scale bar: 5 lm. (B) Ultrastructure of rod
spherule in WT (left) and Clrn1/ (right) mice at age 4.5 months. Triad structures, characterized by association of a synaptic ribbon and two
horizontal cell processes (H), are intact in the retinas of both WT and Clrn1/. Innervations of bipolar dendrites (Bi) were observed both in WT and
Clrn1/. Scale bar: 100 nm.
and Clrn1/ mice (Supplementary Fig. S3C). Calretininpositive neurons stratified in three major layers of inner
plexiform layers in WT and Clrn1/ mice (Supplementary Fig.
S3C). Photoreceptors are characterized by ribbon synapse.36
Ribbon synapses contain the ribbon structures, which play
critical roles in regulating the synaptic vesicle cycle.36 AntiBassoon and RIBEYE labeled individual puncta, characteristic
of photoreceptor ribbons. The distribution, size and number of
ribbons were not significantly different between WT and
Clrn1/ mice (Supplementary Figs. S3D, S3E).
To further investigate the ultrastructural organization of
photoreceptor synapse, we conducted transmission electron
microscopy. In a photoreceptor ribbon synapse, a pair of
horizontal processes associate with a rod synaptic terminus in
a structure termed the triad. Photoreceptor ribbon synapse is
further characterized by innervation of dendrite processes
from bipolar cells. Those triads and bipolar processes were
observed in rod spherules of WT and Clrn1/ mice (Fig. 5B),
further supporting the lack of structural abnormality in rod
synapses.
To more globally test if Clrn1 defect is causing structural
abnormality of retina, we conducted morphometric analysis of
9-month-old mice, when the most significant decline in the
ERG responses was documented. Thicknesses of different
retinal layers were measured using retinal sections transecting
optic nerve heads, and compared between WT and Clrn1/
retinas (Fig. 6A). We found no significant difference in either
the outer segment length or outer nuclear layer thicknesses,
suggesting that Clrn1 deficiency does not cause rod photoreceptor degeneration (Figs. 6B, 6C). Likewise, thicknesses of the
outer plexiform layer were not significantly different between
WT and Clrn1/ mice (Fig. 6D). This is in contrast to CaBP4/
mice, which demonstrated dramatic attenuation of b-wave and
thinning of outer plexiform layer.43 We also did not observe
significant changes in the thicknesses of inner nuclear and
plexiform layers that consist of secondary neurons (Figs. 6E,
6F). Consistent with the results above, we found no significant
difference in GFAP localization in the process of Müller cells
between Clrn1/ and WT mice (Fig. 4). Using immunoblotting, protein expression levels of GFAP were also assessed
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017
IOVS j March 2016 j Vol. 57 j No. 3 j 872
USHIII Model Mouse With Reduced ERG Responses
FIGURE 6. Deficiency of CLRN1 did not affect the morphology of retina. (A) Sections of fixed eye cups of 9-month-old WT (left) and Clrn1/ (right)
mice were probed with nuclear staining dye (blue) and antibody against rhodopsin (red), and then imaged by light microscopy. Differential
interference contrast images (gray scale) were merged with fluorescence images (blue and red). INL: inner nuclear layer; IPL: inner plexiform layer;
IS: inner segment; ONL: outer nuclear layer; OS: outer segment. From optic nerve (ON), the thicknesses of OS (B), ONL (C), OPL (D), INL (E), and
IPL (F) were measured every 250 lm on superior and inferior sides. There was no significant difference in the thicknesses of any of the layers
measured between WT and Clrn1/ mice (n ¼ 4 retinas from 4 animals for each group, P > 0.095 by t-test). Scale bar: 20 lm.
quantitatively on retinas collected at age 4.5 months, when
progressive functional changes are anticipated to be dramatic
in Clrn1/ mice based on the time course of decay in ERG
amplitudes (Fig. 2). This analysis also failed to detect significant
difference between Clrn1/ and WT (Supplementary Fig. S2,
GFAP), suggesting that Clrn1 deficiency does not accelerate
retinal degeneration. Taken together, these results suggest that
the reduction in the a-wave amplitude is not a result of rod
photoreceptor degeneration or compromised rod photoreceptor-bipolar connectivity. Instead, the observed deficiency is
likely associated with the function of CLRN1.
Characterization of ClrnN48K Mice in A/J
Background
Previous comparison of Clrn1N48K/N48K and Clrn1/ mice
indicate that the onset of hearing loss is delayed for
Clrn1N48K/N48K, although both Clrn1/ and Clrn1N48K/N48K
mice lose hearing eventually.8 Thus, this past study suggests
that, unlike Clrn1 allele which is null, the function of
Clrn1N48K product is dramatically attenuated but still present.
Such attenuated function of Clrn1N48K is not sufficient to
sustain hair cells, thus leading eventually to deafness.8 Taking
advantage of the visual testing of A/J mice, we tested the
hypothesis that Clrn1N48K is more functional than Clrn1 and
thus constitutes a hypomorphic allele. To test this hypothesis,
we investigated if Clrn1N48K/N48K exhibit declined a- and bwave amplitudes as observed for Clrn1/ mice. The visual
performances of Clrn1N48K/N48K mice were compared with
WT mice at age 4 and 10 months. Those two time points (4
and 10 months) were offset by 1 month compared with those
selected for Clrn1/ mice, because of an anticipation that
onset of degenerative events are delayed in Clrn1N48K/N48K
mice over Clrn1/ mice based on hearing phenotype.8 At
both age 4 and 10 months, the a-wave amplitudes were similar
between WT and Clrn1N48K/N48K mice at all light intensities
tested (Figs. 7A, 7B, right panel), suggesting that Clrn1N48K/N48K
mutation did not cause an appreciable decline in photoreceptor
function. Likewise, there were no detectable defects of the inner
retinal response, as b-wave amplitudes were also similar for both
WT and Clrn1N48K/N48K at both ages under all tested light
intensities (Figs. 7A, 7B, left panel). The a- and b-wave
amplitudes of these 10-month-old mice were similar to those
of the 9-month-old WT mice (Figs. 1B, 7B, WT). Thus, visual
performance of the A/J mouse strain does not decline
significantly from age 9 to 10 months. These comparisons
indicate that Clrn1N48K/N48K alleles did not exacerbate agerelated decline in the a- and b-wave amplitudes, as Clrn1/
alleles did.
DISCUSSION
To the best of our knowledge, the A/J Clrn1/ mouse is the
first USHIII model that exhibits retinal phenotype. Contrary to
Clrn1/, the lack of observable visual phenotype in A/J
Clrn1N48K/N48K mouse suggests that Clrn1N48K is a hypomorphic allele compared with Clrn1/ which is functionally null.
This assumption is consistent with the delayed onset of hearing
loss in Clrn1N48K/N48K mouse as compared to Clrn1/ mouse
in the previous study.8 In the past, it has been challenging to
obtain a visual phenotype in USHIII model animals. This study
demonstrates that the A/J strain of mouse is a useful tool to
obtain statistically and biologically significant attenuation of
visual performance caused by deficiency in the Clrn1 gene
which is causative to USHIII. A similar approach has been
successful for obtaining a visual phenotype for the Rp1h
mutant allele, suggesting the A/J strain is useful to analyze the
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017
IOVS j March 2016 j Vol. 57 j No. 3 j 873
USHIII Model Mouse With Reduced ERG Responses
FIGURE 7. Electroretinogram responses of Clrn1N48K/N48K and WT mice in A/J background. Amplitudes of a- (right) and b- (left) waves were
measured under scotopic conditons at age 4 (A) and 10 (B) months, and plotted as a function of light intensity. At both ages, there were no
significant differences in a- and b-wave amplitudes between Clrn1N48K/N48K and WT mice at all the light intensities tested (P > 0.41 by t-test).
functions of human blindness causative genes whose phenotypic outcomes have been obscured in other strains of mice.
The observed defects in scotopic ERG responses are
suggestive of rod photoreceptor dysfunction in Clrn1/ mice.
Absence of differences in photopic ERG responses between
Clrn1/ and WT further suggests that defects are originating
primarily from rods, and not from cones. This is consistent
with clinical studies of USHIII in which rod photoreceptor cells
are initially affected.2 In A/J Clrn1/, decline of ERG
amplitude was progressive, characterized initially by b-wave
defect followed by combined a- and b-wave defects. This bwave defect is a characteristic of attenuated synaptic transmission from rod photoreceptors to secondary neurons. A similar
progression was observed for CaBP4/ mice which are
defective in a calcium binding protein localized to photoreceptor synapses. CaBP4 deficiency dramatically reduced bwave responses due to compromised calcium signaling in the
photoreceptor synaptic termini.43 This decline in the b-wave is
then followed by eventual a-wave defects.43 Thus the defects
originating at photoreceptor synapses can lead to depressed awave function as observed in this study of A/J Clrn1/ mice.
While the molecular mechanism causing compromised a- and
b-wave amplitudes is unclear, we believe that the reduced ERG
amplitudes is not due to a loss of photoreceptor cells, as no
global morphologic differences were discernable between
Clrn1/ and WT mice. Instead, it is likely correlated with the
unique function of CLRN1 in photoreceptor synapse.
Our interactive proteomics study suggests the binding of
CLRN1 with other protein components associated with the
actin cytoskeleton.7 It is crucial to note that we did not observe
significant differences in the distribution and localization of Factin between Clrn1/ and WT mice (Supplementary Fig. S3G,
green), but it remains possible that the CLRN1 deficit is leading
to functional defects of F-actin-associated biochemical processes. For example, F-actin is used by the USHIB gene product
myosin VIIA, which is a motor protein carrying cargo along Factin. The genetic interaction between Clrn1 and Myo7a is
also suggestive of a functional association between CLRN1 and
myosin VIIA mediated by actin.44 Intriguingly, myosin VIIA
expression is attenuated in A/J mice,19 and thus the absence of
Clrn1 in conjunction with reduced myosin VIIA may exacerbate the visual phenotype in A/J mice, as observed for human
USH patients.44 We confirmed that the expression of Myosin
VIIA is very low both in Clrn1/ and WT A/J mice; only
marginal signal was detected in RPE microvilli by immunofluorescence microscopy (Supplementary Fig. S3F, green). Nevertheless, the observed localization pattern is consistent with
the previous study of myosin VIIA in rodents.45 Other genes
may also act as modifiers for USHIII or other USH subtypes, and
require further investigation. This is a promising area of
research given that the severity of vision and hearing
impairments are highly variable among Usher syndrome
patients.
Usher syndrome is a devastating disorder that drastically
compromises the quality of life of those afflicted. Recent
studies suggest that CLRN1 is possibly involved in the function
of synapses in auditory hair cells of mouse and zebrafish.10
Other studies, however, failed to detect such synapse
defects,8,9,46 and instead demonstrated roles of CLRN1 in
stereocilia of hair cells that are not present in photoreceptor
neurons. For the studies of Usher syndromes, the major
challenges are that sensory deficits are observed in retina and
cochlea which have no apparent functional similarities, and
that animal models which demonstrate retinal deficits are often
missing. In this regard, retinal ERG phenotype observed in A/J
Clrn1/ is a viable starting point to understand the difference
and similarity of CLRN1 roles in the retina and cochlea.
Acknowledgments
We would like to thank the Elden family for their support; Ina
Nemet for her technical assistance; and Hisashi Fujioka, director of
the Electron Microscopy Core Facility at CWRU, for his support on
transmission electron microscopy. Confocal microscopy was
conducted at the light microscopy imaging core facility at CWRU.
The mouse mAb anti-myosin VIIA (developed by Dana Jo Orten)
and mouse mAb anti-beta-tubulin E7 (developed by Michael
Klymkowsky) were obtained from the Developmental Studies
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017
IOVS j March 2016 j Vol. 57 j No. 3 j 874
USHIII Model Mouse With Reduced ERG Responses
Hybridoma Bank, created by the National Institute of Child Health
and Human Development and maintained at the University of
Iowa, Department of Biology, Iowa.
Supported by Usher III Initiative (YI), Hope for Vision (YI), and
startup funding from Case Western Reserve University (YI).
18.
Disclosure: G. Tian, None; R. Lee, None; P. Ropelewski, None;
Y. Imanishi, None
19.
References
20.
1. Ness SL, Ben-Yosef T, Bar-Lev A, et al. Genetic homogeneity
and phenotypic variability among Ashkenazi Jews with Usher
syndrome type III. J Med Genet. 2003;40:767–772.
2. Herrera W, Aleman TS, Cideciyan AV, et al. Retinal disease in
Usher syndrome III caused by mutations in the clarin-1 gene.
Invest Ophthalmol Vis Sci. 2008;49:2651–2660.
3. Pakarinen L, Tuppurainen K, Laippala P, Mantyjarvi M,
Puhakka H. The ophthalmological course of Usher syndrome
type III. Int Ophthalmol. 1995;19:307–311.
4. Plantinga RF, Pennings RJ, Huygen PL, et al. Visual impairment
in Finnish Usher syndrome type III. Acta Ophthalmol Scand.
2006;84:36–41.
5. Adato A, Vreugde S, Joensuu T, et al. USH3A transcripts
encode clarin-1, a four-transmembrane-domain protein with a
possible role in sensory synapses. Eur J Hum Genet. 2002;10:
339–350.
6. Fields RR, Zhou G, Huang D, et al. Usher syndrome type III:
revised genomic structure of the USH3 gene and identification
of novel mutations. Am J Hum Genet. 2002;71:607–617.
7. Tian G, Zhou Y, Hajkova D, et al. Clarin-1, encoded by the
Usher Syndrome III causative gene, forms a membranous
microdomain: possible role of clarin-1 in organizing the actin
cytoskeleton. J Biol Chem. 2009;284:18980–18993.
8. Geng R, Melki S, Chen DH, et al. The mechanosensory
structure of the hair cell requires clarin-1, a protein encoded
by Usher syndrome III causative gene. J Neurosci. 2012;32:
9485–9498.
9. Geng R, Geller SF, Hayashi T, et al. Usher syndrome IIIA gene
clarin-1 is essential for hair cell function and associated neural
activation. Hum Mol Genet. 2009;18:2748–2760.
10. Ogun O, Zallocchi M. Clarin-1 acts as a modulator of
mechanotransduction activity and presynaptic ribbon assembly. J Cell Biol. 2014;207:375–391.
11. Nadeau JH. Modifier genes in mice and humans. Nat Rev
Genet. 2001;2:165–174.
12. Badano JL, Katsanis N. Beyond Mendel: an evolving view of
human genetic disease transmission. Nat Rev Genet. 2002;3:
779–789.
13. Ebermann I, Phillips JB, Liebau MC, et al. PDZD7 is a modifier
of retinal disease and a contributor to digenic Usher
syndrome. J Clin Invest. 2010;120:1812–1823.
14. Louie CM, Caridi G, Lopes VS, et al. AHI1 is required for
photoreceptor outer segment development and is a modifier
for retinal degeneration in nephronophthisis. Nat Genet.
2010;42:175–180.
15. Reiners J, Nagel-Wolfrum K, Jurgens K, Marker T, Wolfrum U.
Molecular basis of human Usher syndrome: deciphering the
meshes of the Usher protein network provides insights into
the pathomechanisms of the Usher disease. Exp Eye Res. 2006;
83:97–119.
16. Williams DS. Usher syndrome: animal models, retinal function
of Usher proteins, and prospects for gene therapy. Vision Res.
2008;48:433–441.
17. Liu Q, Saveliev A, Pierce EA. The severity of retinal
degeneration in Rp1h gene-targeted mice is dependent on
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
genetic background. Invest Ophthalmol Vis Sci. 2009;50:
1566–1574.
Danciger M, Yang H, Ralston R, et al. Quantitative genetics of
age-related retinal degeneration: a second F1 intercross
between the A/J and C57BL/6 strains. Mol Vis. 2007;13:79–85.
Mustafi D, Maeda T, Kohno H, Nadeau JH, Palczewski K.
Inflammatory priming predisposes mice to age-related retinal
degeneration. J Clin Invest. 2012;122:2989–3001.
Wright AF, Chakarova CF, Abd El-Aziz MM, Bhattacharya SS.
Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait. Nat Rev Genet. 2010;11:273–284.
Jager RD, Mieler WF, Miller JW. Age-related macular degeneration. N Engl J Med. 2008;358:2606–2617.
Geller SF, Guerin KI, Visel M, et al. CLRN1 is nonessential in
the mouse retina but is required for cochlear hair cell
development. PLoS Genet. 2009;5:e1000607.
Joensuu T, Hamalainen R, Yuan B, et al. Mutations in a novel
gene with transmembrane domains underlie Usher syndrome
type 3. Am J Hum Genet. 2001;69:673–684.
Petkov PM, Ding Y, Cassell MA, et al. An efficient SNP system
for mouse genome scanning and elucidating strain relationships. Genome Res. 2004;14:1806–1811.
Yang H, Ding Y, Hutchins LN, et al. A customized and versatile
high-density genotyping array for the mouse. Nat Methods.
2009;6:663–666.
Mattapallil MJ, Wawrousek EF, Chan CC, et al. The Rd8
mutation of the Crb1 gene is present in vendor lines of C57BL/
6N mice and embryonic stem cells, and confounds ocular
induced mutant phenotypes. Invest Ophthalmol Vis Sci. 2012;
53:2921–2927.
MacKenzie D, Arendt A, Hargrave P, McDowell JH, Molday RS.
Localization of binding sites for carboxyl terminal specific antirhodopsin monoclonal antibodies using synthetic peptides.
Biochemistry. 1984;23:6544–6549.
Imanishi Y, Sun W, Maeda T, Maeda A, Palczewski K. Retinyl
ester homeostasis in the adipose differentiation-related protein-deficient retina. J Biol Chem. 2008;283:25091–25102.
Nemet I, Tian G, Imanishi Y. Submembrane assembly and
renewal of rod photoreceptor cGMP-gated channel: insight
into the actin-dependent process of outer segment morphogenesis. J Neurosci. 2014;34:8164–8174.
Stockton RA, Slaughter MM. B-wave of the electroretinogram.
A reflection of ON bipolar cell activity. J Gen Physiol. 1989;93:
101–122.
Breton ME, Schueller AW, Lamb TD, Pugh EN Jr. Analysis of
ERG a-wave amplification and kinetics in terms of the Gprotein cascade of phototransduction. Invest Ophthalmol Vis
Sci. 1994;35:295–309.
Wang JS, Nymark S, Frederiksen R, et al. Chromophore supply
rate-limits mammalian photoreceptor dark adaptation. J
Neurosci. 2014;34:11212–11221.
Sokolov M, Lyubarsky AL, Strissel KJ, et al. Massive light-driven
translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation.
Neuron. 2002;34:95–106.
Strissel KJ, Sokolov M, Trieu LH, Arshavsky VY. Arrestin
translocation is induced at a critical threshold of visual
signaling and is superstoichiometric to bleached rhodopsin. J
Neurosci. 2006;26:1146–1153.
Maerker T, van Wijk E, Overlack N, et al. A novel Usher protein
network at the periciliary reloading point between molecular
transport machineries in vertebrate photoreceptor cells. Hum
Mol Genet. 2008;17:71–86.
Sterling P, Matthews G. Structure and function of ribbon
synapses. Trends Neurosci. 2005;28:20–29.
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017
IOVS j March 2016 j Vol. 57 j No. 3 j 875
USHIII Model Mouse With Reduced ERG Responses
37. Lodowski KH, Lee R, Ropelewski P, Nemet I, Tian G, Imanishi
Y. Signals governing the trafficking and mistrafficking of a
ciliary GPCR, rhodopsin. J Neurosci. 2013;33:13621–13638.
38. Nemet I, Ropelewski P, Imanishi Y. Rhodopsin trafficking and
mistrafficking: signals, molecular components, and mechanisms. Prog Mol Biol Transl Sci. 2015;132:39–71.
39. Tian G, Ropelewski P, Nemet I, Lee R, Lodowski KH, Imanishi
Y. An unconventional secretory pathway mediates the cilia
targeting of peripherin/rds. J Neurosci. 2014;34:992–1006.
40. Higuchi H, Macke EL, Lee WH, et al. Genetic basis of agedependent synaptic abnormalities in the retina. Mamm
Genome. 2015;26:21–32.
41. Koulen P, Fletcher EL, Craven SE, Bredt DS, Wassle H.
Immunocytochemical localization of the postsynaptic density
protein PSD-95 in the mammalian retina. J Neurosci. 1998;18:
10136–10149.
42. Haverkamp S, Wassle H. Immunocytochemical analysis of the
mouse retina. J Comp Neurol. 2000;424:1–23.
43. Haeseleer F, Imanishi Y, Maeda T, et al. Essential role of Ca2þbinding protein 4, a Cav1.4 channel regulator, in photoreceptor synaptic function. Nat Neurosci. 2004;7:1079–1087.
44. Adato A, Kalinski H, Weil D, Chaib H, Korostishevsky M,
Bonne-Tamir B. Possible interaction between USH1B and
USH3 gene products as implied by apparent digenic deafness
inheritance. Am J Hum Genet. 1999;65:261–265.
45. Hasson T, Heintzelman MB, Santos-Sacchi J, Corey DP, Mooseker MS. Expression in cochlea and retina of myosin VIIa, the
gene product defective in Usher syndrome type 1B. Proc Natl
Acad Sci U S A. 1995;92:9815–9819.
46. Gopal SR, Chen DH, Chou SW, et al. Zebrafish models for the
mechanosensory hair cell dysfunction in usher syndrome 3
reveal that clarin-1 is an essential hair bundle protein. J
Neurosci. 2015;35:10188–10201.
Downloaded From: http://www.journalofvision.com/pdfaccess.ashx?url=/data/journals/iovs/935065/ on 05/06/2017