November 2013
Volume 54, Issue 12
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Visual Neuroscience  |   November 2013
Myosin 6 Is Required for Iris Development and Normal Function of the Outer Retina
Author Affiliations & Notes
  • Ivy S. Samuels
    Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio
    Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
  • Brent A. Bell
    Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
  • Gwen Sturgill-Short
    Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio
    Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
  • Lindsey A. Ebke
    Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
  • Mary Rayborn
    Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
  • Lanying Shi
    The Jackson Laboratory, Bar Harbor, Maine
  • Patsy M. Nishina
    The Jackson Laboratory, Bar Harbor, Maine
  • Neal S Peachey
    Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio
    Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
    Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, Cleveland, Ohio
  • Correspondence: Ivy S. Samuels, Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH 44106;SAMUELI@ccf.org
Investigative Ophthalmology & Visual Science November 2013, Vol.54, 7223-7233. doi:10.1167/iovs.13-12887
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      Ivy S. Samuels, Brent A. Bell, Gwen Sturgill-Short, Lindsey A. Ebke, Mary Rayborn, Lanying Shi, Patsy M. Nishina, Neal S Peachey; Myosin 6 Is Required for Iris Development and Normal Function of the Outer Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(12):7223-7233. doi: 10.1167/iovs.13-12887.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: To determine the molecular basis and the pathologic consequences of a chemically induced mutation in the translational vision research models 89 (tvrm89) mouse model with ERG defects.

Methods.: Mice from a G3 N-ethyl-N-nitrosourea mutagenesis program were screened for behavioral abnormalities and defects in retinal function by ERGs. The chromosomal position for the recessive tvrm89 mutation was determined in a genome-wide linkage analysis. The critical region was refined, and candidate genes were screened by direct sequencing. The tvrm89 phenotype was characterized by circling behavior, in vivo ocular imaging, detailed ERG-based studies of the retina and RPE, and histological analysis of these structures.

Results.: The tvrm89 mutation was localized to a region on chromosome 9 containing Myo6. Sequencing identified a T→C point mutation in the codon for amino acid 480 in Myo6 that converts a leucine to a proline. This mutation does not confer a loss of protein expression levels; however, mice homozygous for the Myo6tvrm89 mutation display an abnormal iris shape and attenuation of both strobe-flash ERGs and direct-current ERGs by 4 age weeks, neither of which is associated with photoreceptor loss.

Conclusions.: The tvrm89 phenotype mimics that reported for Myosin6-null mice, suggesting that the mutation confers a loss of myosin 6 protein function. The observation that homozygous Myo6tvrm89 mice display reduced ERG a-wave and b-wave components, as well as components of the ERG attributed to RPE function, indicates that myosin 6 is necessary for the generation of proper responses of the outer retina to light.

Introduction
Myosins are actin-based motor proteins, coupling adenosine triphosphate (ATP) hydrolysis to mechanical motion along actin filaments. To date, more than 30 myosin proteins have been identified in species ranging from Drosophila to human, and they are known to contribute to essential cellular functions, including secretion, cell division, differentiation, and migration. 1 Myosin 6 is an unconventional myosin motor protein and is the only myosin that moves toward the minus end of the actin filament. 2  
In mice, only one functionally unique isoform of myosin 6 is expressed. The gene was first identified in the Snell's waltzer mouse (sv), which is characterized by circling behavior and head tossing secondary to vestibular dysfunction. 3,4 The presence of cochlear dysfunction in Myo6sv mice identified Myo6 as a deafness gene. 46 MYO6 mutations have subsequently been identified in autosomal recessive nonsyndromic deafness (DFNB37) 7,8 and autosomal dominant nonsyndromic hearing loss (DFNA22) in humans. 6,9,10 Myosin 6 has also been found in the retina and is highly expressed in photoreceptors and RPE cells 1113 and throughout the inner retina. Notably, two of nine patients documented with DFNB37 displayed retinal abnormalities. 7 In photoreceptors, myosins are present in the actin-containing domain within the connecting cilium of the inner segment, where the initiation and regulation of disc membrane morphogenesis occurs. 14 In the mouse photoreceptor, myosin 6 is localized exclusively to the inner segment. Myo6sv and two allelic mutants (Myo6sv-2J and Myo6sv-4J ), which are all effective Myo6-null mutants, display reductions in a-wave and b-wave amplitudes as early as age 6 weeks, 15 with no evidence of photoreceptor degeneration or disruption in disc morphogenesis. 12 In the RPE, myosin 6 is localized to the periphery of the cell and colocalizes with LysoTracker 12 corroborating its role in vesicle trafficking 1619 and providing a potential explanation for the functional abnormalities found despite the absence of anatomical changes to the retina. 
A large number of new mouse models that demonstrate irregular structural and/or functional eye phenotypes have been developed in a mutagenesis program conducted at The Jackson Laboratory. 20 The translational vision research models (TVRM) program uses N-ethyl-N-nitrosourea (ENU) to induce random mutations, and neurological and ocular screens are used to identify mutants of interest. As described herein, the tvrm89 mutant was identified by its circling behavior, and the mutation involved was subsequently identified as a leucine to proline substitution in myosin 6. Myo6tvrm89 mutants phenocopy, in many respects, the sensory abnormalities identified in Myo6sv , Myo6sv-2J , and Myo6sv-4J mice. Unlike these well-studied mutants, myosin 6 protein expression is retained in the Myo6tvrm89 -mutant retina. The Myo6tvrm89 mouse provides a novel mutant for Myo6 that confers a loss of function but does not affect expression of the protein. 
Methods
Mice: Mutagenesis, Mapping, and Genotyping
All animal procedures were approved by the institutional animal care and use committees of the institutions involved and are in agreement with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Homozygous tvrm89 mice were identified from a mutagenesis program 20 in which male C57BL/6J (B6) mice were mutagenized with ENU administered in intraperitoneal injections of 80 mg/kg for 3 weeks. 21 The G3 offspring, generated using a three-generation backcross mating scheme to identify recessive mutations, 22 were screened by a series of neurological protocols. The tvrm89 mutant was identified based on its circling behavior and head bobbing, indicative of inner ear dysfunction. 
To map the gene involved, which is inherited as an autosomal recessive trait, B6 tvrm89 homozygous female mice were mated to male DBA/2J mice to generate F1 progeny, which were subsequently intercrossed. The F2 progeny were assessed at age 12 weeks, and DNA was isolated from tail snips using a modified version of published methods. 23 A genome-wide scan to determine the chromosomal location of tvrm89 was performed with simple sequence-length polymorphic markers. Products of PCR were separated by electrophoresis on a 4% agarose gel (MetaPhor; FMC, Rockland, ME), stained with ethidium bromide, and visualized by UV light. 
For sequencing of candidate genes, RNA and cDNA were prepared from three mutant mice and three control B6 mice. The RNA was isolated from snap-frozen eyes (TRIzol; Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The cDNA was generated with a reverse transcription kit (Retroscript; Ambion, Austin, TX). The Myo6 coding region was amplified from cDNA using PCR amplification, and purified products were sequenced. 
The tvrm89 mouse colony at The Jackson Laboratory is maintained by heterozygous matings. Mice were shipped to the Cleveland Clinic to establish a satellite colony that is maintained on a 14-hour light–10-hour dark cycle. 
An allele-specific PCR assay for Myo6tvrm89 was established, and PCR amplification of the region was carried out as follows: (1) 94°C for 2 minutes, (2) 94°C for 20 seconds, (3) annealing temperature of 60°C for 10 seconds, and (4) 65°C for 50 seconds; steps two through four were repeated for 40 cycles, followed by one cycle at 65°C for 7 minutes. The following oligonucleotides were used for PCR amplification: 
  •  
    F1: 5′-AGCCCAGACTATTAACGTACATT-3′
  •  
    R1: 5′-CCTTTCATTAAAAAACTGTTGTG-3′
  •  
    R2: 5′-CCTTCAGGATGGTTTCATTAAAAAACTGTAGGA-3′
Scanning Laser Ophthalmoscopy and Spectral-Domain Optical Coherence Tomography
Mice were anesthetized with 64 mg/kg of sodium pentobarbital. Mydriasis was induced by administration of 1 μL of 0.5% Mydrin-P (tropicamide-phenylephrine combination) drops (Santen Pharmaceutical Co., Ltd., Osaka, Japan). The drops were gently massaged into the eye by artificially blinking the eyelids. Eyes were then immediately covered with Systane Ultra artificial tears (Alcon Laboratories, Inc., Ft. Worth, TX). Mice were then placed in a warmed, humidified, oxygenated acrylic plastic sheet chamber for a minimum of 5 minutes to permit time for pupil dilation. Mice were then removed for imaging by scanning laser ophthalmoscopy (SLO) (model HRA2; Heidelberg Engineering, Inc., Vista, CA) and spectral-domain optical coherence tomography (SDOCT) (model Envisu SDOIS; Bioptigen, Inc., Research Triangle Park, NC). The SLO imaging involved collection of different imaging modalities, including dark-field reflectance and autofluorescent images with both blue (488 nm) and infrared (795 nm and 830 nm) illumination wavelengths. Using a wide-field objective lens with a 55° field of view (FOV), retinal images were collected with the optic nerve centrally positioned. Additional views of the peripheral regions were obtained to further investigate the nasal, temporal, superior, and inferior quadrants. Eyes were occasionally rehydrated with balanced salt solution or Systane Ultra artificial ears (Alcon Laboratories) and mechanically massaged to simulate blinking as needed. After SLO imaging, the mouse was transferred to the Envisu SDOIS system (Bioptigen, Inc.) for SDOCT imaging. The SDOCT volumetric scans (250 a-scans per b-scan × 250 b-scans per volume) were obtained with the optic nerve centrally located within the FOV. Using a 50° objective lens, the SDOIS system afforded a retinal FOV of approximately 1.5 mm, with an axial, in-depth resolution of approximately 6 μm. After imaging, both eyes received bacitracin zinc and polymyxin B sulfate ophthalmic ointment (Bausch & Lomb, Inc., Tampa, FL) to prevent corneal dehydration. During recovery, mice were placed in a bottom-warmed (33–36°C), oxygenated (21%–60%) acrylic plastic sheet chamber until they fully recovered from general anesthesia. 
Electroretinography
After overnight dark adaptation, mice were anesthetized (80 mg/kg of ketamine and 16 mg/kg of xylazine), the cornea was anesthetized (1% proparacaine hydrochloride), and the pupils were dilated (1% tropicamide, 2.5% phenylephrine hydrochloride, and 1% cyclopentolate). Mice were placed on a temperature-regulated heating pad throughout each recording session. 
The protocols used to record ERG components generated by the outer neural retina or the RPE have been described. 24 In brief, responses of the outer retina were recorded with a stainless steel electrode referenced to a needle electrode placed in the cheek in response to strobe-flash stimuli presented in the dark or superimposed on a steady 20 candela (cd)/m2 rod-desensitizing adapting field. The amplitude of the a-wave was measured 8 milliseconds after flash onset from the prestimulus baseline. The amplitude of the b-wave was measured from the a-wave to the peak of the b-wave or, if no a-wave was present, from the prestimulus baseline. Implicit times were measured from the time of flash onset to the a-wave trough or the b-wave peak. 
Components of the direct-current coupled (dc)–ERG generated by the RPE were recorded with a silver/silver chloride electrode bridged to the corneal surface with Hanks' balanced salt solution in response to stimuli presented for 7 minutes. The amplitude of the c-wave was measured from the prestimulus baseline to the peak of the c-wave. The amplitude of the fast oscillation (FO) was measured from the c-wave peak to the trough of the FO. The amplitude of the light peak (LP) was measured from the FO trough to the asymptotic value. The amplitude of the off-response was measured from the LP asymptote to the peak of the off-response. 
Histology and Immunohistochemistry
After mice were killed, the superior cornea was marked before enucleation. After removal of the cornea and lens, eyes were fixed in 0.1 M sodium phosphate buffer (pH 7.4) containing 4% paraformaldehyde for 4 hours. The posterior pole was then immersed through a graded series of sucrose solutions as follows: 10% for 1 hour, 20% for 1 hour, and 30% overnight. Eyes were embedded in optimum temperature cutting compound freezing medium, flash frozen on dry ice, and immediately transferred to −80°C. Tissue was sectioned at 10-μm thickness with a cryostat (Leica, Wetzlar, Germany) at −30°C, mounted on Superfrost slides (Fisherbrand, Pittsburgh, PA), and stored at −80°C until processed. Sections were incubated in 0.1% Triton X-100 and 10% normal goat serum in PBS for 1 hour (PBS-T) at room temperature and then washed three times with PBS for 5 minutes each. The sections were incubated overnight at 4°C with the primary antibody. Sections were rinsed with PBS-T three times for 10 minutes each and incubated with secondary antibody (AlexaFluor 594, 1:500; Molecular Probes, Eugene, OR) for 1 hour at RT. After rinsing sections three times for 5 minutes each with PBS-T, sections were mounted with 4′,6-diamidino-2-phenylindole (Vectashield; Vector Laboratories, Burlingame, CA) and coverslipped. Primary antibody was rabbit anti–Myosin-VI (1:200; Proteus BioSciences, Inc., Ramona, CA). 
For light microscopy, eyes were fixed in 0.1 M sodium cacodylate buffer (pH 7.4) containing 2% formaldehyde and 2.5% glutaraldehyde. The tissues were then osmicated, dehydrated though a graded ethanol series, and embedded in epoxy resin (Epon/Araldite; Polysciences, Inc., Washington, PA). Semithin sections (1 μm) were cut approximately along the horizontal meridian and through the optic nerve and stained with toluidine blue O for evaluation. 
Western Blot
Retinal and RPE tissues were dissected from enucleated eyes and frozen in lysis buffer (50 mM Tris [pH 8.0], 150 mM sodium chloride, 10% glycerol, 0.5% Triton X-100, and 0.1% NP40) supplemented with protease inhibitors (Roche Applied Science, Indianapolis, IN). Lysates were homogenized by manual grinding with a disposable pestle within a microcentrifuge tube three times, followed by five brief sonication pulses. Lysates were digested at 4°C for 1 hour, followed by centrifugation at 6600g for 10 minutes at 4°C. The supernatants were collected, and the total protein in each sample was determined by bicinchronic acid assay (Thermo Scientific, Rockford, IL) according to the manufacturer's instructions. Samples containing 25 μg of total protein for retina or 10 μg of protein for RPE with Lane Marker Reducing Sample Buffer (Thermo Scientific) were heated to 95°C for 5 minutes to denature the samples. Proteins were separated on 4% to 20% Tris-glycine SDS-PAGE gels and transferred to polyvinylidene difluoride membranes using 1× running buffer and transfer buffer (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked for 1 hour in a 5% milk solution in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). The primary antibodies were applied in blocking solution overnight at 4°C. After washes with TBS-T, the peroxidase-conjugated secondary antibodies (Jackson Immuno-Research Laboratories, Inc., West Grove, PA) were applied at 1:5000 in blocking buffer for 1 hour at room temperature, and proteins were detected by enhanced chemiluminescence (Western Lightning kit; Perkin-Elmer, Waltham, MA). Primary antibodies used were Myosin VI (1:200; Proteus BioSciences, Inc.) and β-actin (1:1000; Abcam, Cambridge, MA). 
Results
A Myo6 Missense Mutation Confers Pathology in tvrm89 Homozygous Mice
Linkage analysis localized the gene mutated in tvrm89 mice to a region of chromosome 9 containing Myo6. Because the phenotype of affected tvrm89 homozygotes matched that of Myo6sv/sv mice, 3 including elevated auditory brainstem response threshold (data not shown), the coding region of Myo6 was sequenced. We detected a T→C nucleotide transition that converts a leucine (CTC) to a proline (CCC) at amino acid 480 (Fig. 1A). The first 759 amino acids of mouse myosin 6 represent the head-motor region of the protein containing both the ATP-binding domain and the actin-binding sites (http://www.uniprot.org/uniprot/Q9UM54 [in the public domain]). Conversion of amino acid 480 from leucine to proline is likely to affect actin and/or ATP binding and to destabilize protein structure because of the substitution of proline's unique 5-membered ring into the main-chain conformation. 
Figure 1
 
The tvrm89 mutants bear a point mutation in Myosin6 but retain myosin 6 protein levels. (A) Positional cloning and direct sequencing demonstrate a T→C transition mutation found in homozygous tvrm89 mutants. (B) Allele-specific genotyping was accomplished using PCR amplification of DNA extracted from tail snips. (C) Western blot analysis of retinal (left) and RPE (right) protein lysates demonstrates myosin 6 protein levels in wild-type (+/+), heterozygous (tvrm89/+), and homozygous (tvrm89/tvrm89) mice. Actin serves as a loading control. (D) Cryosections of eyecups from +/+, tvrm89/+, and tvrm89/tvrm89 mice were immunostained with Myosin-VI antibody (red) and counterstained with 4′,6-diamidino-2-phenylindole (blue). Myosin 6 is present in all three genotypes and is highest in rod inner segments and the inner retina. At the right is the no primary antibody control. Low levels of autofluorescence are found in the rod outer segments; however, no specific immunostaining was apparent. Scale bar: 20 μm.
Figure 1
 
The tvrm89 mutants bear a point mutation in Myosin6 but retain myosin 6 protein levels. (A) Positional cloning and direct sequencing demonstrate a T→C transition mutation found in homozygous tvrm89 mutants. (B) Allele-specific genotyping was accomplished using PCR amplification of DNA extracted from tail snips. (C) Western blot analysis of retinal (left) and RPE (right) protein lysates demonstrates myosin 6 protein levels in wild-type (+/+), heterozygous (tvrm89/+), and homozygous (tvrm89/tvrm89) mice. Actin serves as a loading control. (D) Cryosections of eyecups from +/+, tvrm89/+, and tvrm89/tvrm89 mice were immunostained with Myosin-VI antibody (red) and counterstained with 4′,6-diamidino-2-phenylindole (blue). Myosin 6 is present in all three genotypes and is highest in rod inner segments and the inner retina. At the right is the no primary antibody control. Low levels of autofluorescence are found in the rod outer segments; however, no specific immunostaining was apparent. Scale bar: 20 μm.
The PCR analysis of cDNA encompassing the tvrm89 mutation confirmed the change in both the F2 intercross and tvrm89 maintenance colony and was used for genotyping Myo6 mutants because the products demonstrate a size difference (wild type is 135 base pair [bp] and mutant is 108 bp) (Fig. 1B). From this point forward, the tvrm89 allele will be referred to as Myo6tvrm89
Western blot analyses of lysates generated from retina or RPE were probed with an antibody that recognizes an epitope containing amino acids 1049 through 1254 within the tail region of myosin 6. The immunoblot (Fig. 1C) demonstrated that myosin 6 is retained in homozygous Myo6tvrm89 mutants. Localization of myosin 6 was investigated by fluorescent immunohistochemistry on frozen cryosections from adult mice. No differences in myosin 6 expression or localization were apparent between homozygous Myo6tvrm89 mutants and control littermates (Fig. 1D). General histological analysis of mutant mice was further performed using semithin epoxy resin sections of adult eyecups, and no overt changes were identified in any retinal layer (Fig. 2, top). High-magnification micrographs of the RPE–outer segment interface demonstrated normal morphology (Fig. 2, bottom). 
Figure 2
 
Normal retinal anatomy in Myo6tvrm89 mutants. Retinal cross sections obtained from control Myo6+/+ (A), Myo6tvrm89/+ (B), and mutant Myo6tvrm89/tvrm89 (C) mice. Retinal histology was normal in all genotypes. Top: Representative light micrographs of the retina from ultrathin sections of adult eyecups. Scale bar: 20 μm. Bottom: Representative light micrographs displaying RPE morphology from semithin sections of adult eyecups. Scale bar: 10 μm. INL, inner nuclear layer; ONL, outer nuclear layer; RIS, rod inner segment; ROS, rod outer segment.
Figure 2
 
Normal retinal anatomy in Myo6tvrm89 mutants. Retinal cross sections obtained from control Myo6+/+ (A), Myo6tvrm89/+ (B), and mutant Myo6tvrm89/tvrm89 (C) mice. Retinal histology was normal in all genotypes. Top: Representative light micrographs of the retina from ultrathin sections of adult eyecups. Scale bar: 20 μm. Bottom: Representative light micrographs displaying RPE morphology from semithin sections of adult eyecups. Scale bar: 10 μm. INL, inner nuclear layer; ONL, outer nuclear layer; RIS, rod inner segment; ROS, rod outer segment.
Iris Abnormalities in Homozygous Myo6tvrm89 Mice
At age 4 weeks, in vivo imaging demonstrated a bilateral, abnormal, scalloped pupil shape in homozygous Myo6tvrm89 mutants (Fig. 3, left, middle). This phenotype was fully penetrant and generally caused by formation of synechiae, including adhesions of the iris to the cornea (anterior synechiae) or to the lens (posterior synechiae). Subsequent imaging at 8 weeks and at 1 year or older showed similar but less pronounced findings (Fig. 3). The SDOCT imaging of mutants with bilateral synechiae revealed multiple abnormalities within the anterior segment at both 4 weeks and 8 weeks (Fig. 4). At 4 weeks, a lenticular opacity (arrowhead) can be observed immediately adjacent to a keratolenticular adhesion (arrow) involving the posterior cornea and the lens. At 8 weeks, the misshapen iris and keratolenticular adhesion persist, but the lenticular opacity is less apparent and somewhat resolved. A corneal ulceration (arrowhead) can also be observed on the posterior cornea at 8 weeks. Anterior chamber cavities are also abnormally small at both time points (asterisks). The SLO of mice at 4 weeks, 8 weeks, or 1 year or older did not identify any clinical RPE or retinal abnormalities (Fig. 3, right). 
Figure 3
 
Iris but not fundus abnormalities in homozygous Myo6tvrm89 mice. Myo6tvrm89 mutants display iris abnormalities (synechiae) that resolve with age. Left, middle: Low-magnification photomicrographs obtained by SLO displaying scalloped pupil shape in both left and right eyes at 4 weeks (top), 8 weeks (middle), and 1 year or older (bottom). Right: Infrared (dark field) SLO images of the retina taken at 4 weeks (top), 8 weeks (middle), and 1 year (bottom). No abnormalities were observed in the retina or the RPE at any age.
Figure 3
 
Iris but not fundus abnormalities in homozygous Myo6tvrm89 mice. Myo6tvrm89 mutants display iris abnormalities (synechiae) that resolve with age. Left, middle: Low-magnification photomicrographs obtained by SLO displaying scalloped pupil shape in both left and right eyes at 4 weeks (top), 8 weeks (middle), and 1 year or older (bottom). Right: Infrared (dark field) SLO images of the retina taken at 4 weeks (top), 8 weeks (middle), and 1 year (bottom). No abnormalities were observed in the retina or the RPE at any age.
Figure 4
 
Abnormal iris shape and anterior segment morphology in homozygous Myo6tvrm89 mice. The SDOCT imaging of homozygous Myo6tvrm89 mice found with bilateral synechiae revealed abnormal anterior segment morphology in approximately 50% (4/7) of animals. The horizontal line through the en face fundus image (left) indicates the location of a single SDOCT B-scan frame (right). Asterisks denote anterior chamber. Arrowhead at the top marks the lenticular opacity. Arrowhead at the bottom marks the corneal ulceration. Arrows indicate keratolenticular adhesions.
Figure 4
 
Abnormal iris shape and anterior segment morphology in homozygous Myo6tvrm89 mice. The SDOCT imaging of homozygous Myo6tvrm89 mice found with bilateral synechiae revealed abnormal anterior segment morphology in approximately 50% (4/7) of animals. The horizontal line through the en face fundus image (left) indicates the location of a single SDOCT B-scan frame (right). Asterisks denote anterior chamber. Arrowhead at the top marks the lenticular opacity. Arrowhead at the bottom marks the corneal ulceration. Arrows indicate keratolenticular adhesions.
Homozygous Myo6tvrm89 Mutants Display Reduced Light-Evoked Responses of the Retina and RPE
The ERG is reduced in amplitude in Myo6sv/sv mice. 12 To determine whether this feature is shared in the Myo6tvrm89 mutants (Fig. 1C), we used ERGs to examine the function of the outer retina and of the RPE. The strobe-flash ERG depicts both photoreceptor activity in the form of the a-wave and bipolar cell function as revealed by the b-wave. Figure 5 shows averaged ERG tracings at 4 weeks (A), 8 weeks (D), and 1 year or older (G) in response to a subset of the strobe-flash stimuli (−2.4, −0.6, and 1.4 log cd s/m2, respectively). At each age, the overall amplitude of ERGs obtained from homozygous Myo6tvrm89 mutants was reduced compared with that of control (+/+ and tvrm89/+) littermates. Luminance-response functions for the major components of the ERG are shown in Figure 5 at 4 weeks (B, C), 8 weeks (E, F), and 1 year or older (H, I). Compared with control, response functions of homozygous Myo6tvrm89 mutants are reduced by a consistent factor across flash luminance. The amplitude of the a-wave was reduced on average by 40% at 4 weeks (B), 53% at 8 weeks (E), and 31% at 1 year or older (H), and the amplitude of the b-wave was reduced by 35% at 4 weeks (C), 46% at 8 weeks (F), and 34% at 1 year or older (I). These reductions are comparable to the 25% (a-wave) and 30% (b-wave) reductions reported in Myo6sv/sv mutants aged 6 to 7 weeks. 12 We found no change in b-wave latency at any age (data not shown). 
Figure 5
 
Homozygous Myo6tvrm89 mice display attenuated ERGs. Averaged strobe-flash ERG responses at 4 weeks (AC), 8 weeks (DF), and 1 year or older (GI). (A, D, G) Averaged tracings from control (+/+ and tvrm89/+) and mutant (tvrm89/tvrm89) mice in response to stimuli of −2.4, −0.6, and 2.4 log cd s/m2. (B, E, H) The a-wave luminance-response functions (points indicate the average ± SEM). (C, F, I) The b-wave luminance-response functions (points indicate the average ± SEM). Data summarize results from 22 control and 10 mutants at 4 weeks, from 24 controls and 7 mutants at 8 weeks, and from 4 controls and 2 mutants at 1 year or older. ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.01 by Student's t-test.
Figure 5
 
Homozygous Myo6tvrm89 mice display attenuated ERGs. Averaged strobe-flash ERG responses at 4 weeks (AC), 8 weeks (DF), and 1 year or older (GI). (A, D, G) Averaged tracings from control (+/+ and tvrm89/+) and mutant (tvrm89/tvrm89) mice in response to stimuli of −2.4, −0.6, and 2.4 log cd s/m2. (B, E, H) The a-wave luminance-response functions (points indicate the average ± SEM). (C, F, I) The b-wave luminance-response functions (points indicate the average ± SEM). Data summarize results from 22 control and 10 mutants at 4 weeks, from 24 controls and 7 mutants at 8 weeks, and from 4 controls and 2 mutants at 1 year or older. ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.01 by Student's t-test.
We next assessed the light-evoked responses of the RPE. Figure 6A shows representative dc-ERG tracings from homozygous mutant Myo6tvrm89 and control mice at 4 weeks and 8 weeks. The four main components of the dc-ERG (Fig. 6A) are generated because of changes in ion conductance across the basal and apical RPE membranes in response to light stimuli. 24 Compared with control, the overall amplitude of the dc-ERG was reduced in homozygous Myo6tvrm89 mutants at both age 4 weeks and 8 weeks; as shown in Figure 6, the individual components at these two ages were reduced by 36% and 30% (respectively) for the c-wave (B), 45% and 27% for the FO (C), and 31% and 25% for the LP (D), as well as by 18% at both ages for the off-response (E). Collectively, these results demonstrate that ERG components generated by the RPE response were diminished in homozygous Myo6tvrm89 mutants but that the magnitude of this reduction was less pronounced at age 8 weeks. 
Figure 6
 
The RPE function is reduced in homozygous Myo6tvrm89 mice. (A) Representative dc-ERGs of control (+/+ or tvrm89/+) mice (black) and mutant (tvrm89/tvrm89) mice (gray) at 4 weeks and 8 weeks. Baseline measurements are recorded for 30 seconds before initiation of a 7-minute light stimulus of 2.4 log cd/m2. The major dc-ERG components are labeled. (BE) Amplitude of c-wave (B), FO (C), LP (D), and off-response (E) obtained from mice aged 4 weeks or 8 weeks. Bars indicate the average ± SEM of 20 control and 7 mutant mice at 4 weeks and of 15 control and 7 mutant mice at 8 weeks. **P ≤ 0.001 by Student's t-test.
Figure 6
 
The RPE function is reduced in homozygous Myo6tvrm89 mice. (A) Representative dc-ERGs of control (+/+ or tvrm89/+) mice (black) and mutant (tvrm89/tvrm89) mice (gray) at 4 weeks and 8 weeks. Baseline measurements are recorded for 30 seconds before initiation of a 7-minute light stimulus of 2.4 log cd/m2. The major dc-ERG components are labeled. (BE) Amplitude of c-wave (B), FO (C), LP (D), and off-response (E) obtained from mice aged 4 weeks or 8 weeks. Bars indicate the average ± SEM of 20 control and 7 mutant mice at 4 weeks and of 15 control and 7 mutant mice at 8 weeks. **P ≤ 0.001 by Student's t-test.
RPE Function in Homozygous Myo6tvrm89 Mice Is Reduced but Is Spared Relative to Photoreceptor Activity
The dc-ERG is generated secondary to rod photoreceptor activity. 24,25 As a consequence, a reduced dc-ERG could reflect RPE dysfunction or a reduced effective stimulus from rod photoreceptors. In view of the reduced a-wave of homozygous Myo6tvrm89 mutants (Fig. 5), we examined the relation between rod photoreceptor activity and the individual dc-ERG components of homozygous Myo6tvrm89 -mutant mice. Each panel of Figure 7 shows the amplitude of a dc-ERG component (c-wave [A], FO [B], LP [C], and off-response [D]) plotted against the amplitude of the a-wave elicited by a high-luminance stimulus after each response measure was normalized to the control average. When the dc-ERG is reduced beyond the a-wave, the plotted points fall below the diagonal line; points fall along the diagonal line when the response measures are reduced by equal amounts. In general, at 4 weeks points fall close to the diagonal, indicating that dc-ERG and ERG a-waves are reduced by equivalent amounts. At 8 weeks, points fall above the diagonal, demonstrating that RPE function is spared despite the decrease in photoreceptor activity. 
Figure 7
 
The RPE function is equivalent to or better than photoreceptor activity in homozygous Myo6tvrm89 mice. Relative changes in amplitude of each major dc-ERG component (c-wave [A], FO [B], LP [C], and off-response [D]) as a function of a-wave amplitude in response to a light stimulus of 1.4 log cd s/m2. Each filled point indicates data obtained from an individual homozygous Myo6tvrm89 -mutant mouse plotted relative to the average control response. Data sets from 4 weeks are denoted by squares, and data sets from 8 weeks are denoted by triangles. The average ± SEM for each age is represented by an open square (4 weeks) or open triangle (8 weeks). The average ± SEM for control mice is represented by an open circle. The diagonal line indicates an equivalent reduction in the a-wave and each major component of the dc-ERG.
Figure 7
 
The RPE function is equivalent to or better than photoreceptor activity in homozygous Myo6tvrm89 mice. Relative changes in amplitude of each major dc-ERG component (c-wave [A], FO [B], LP [C], and off-response [D]) as a function of a-wave amplitude in response to a light stimulus of 1.4 log cd s/m2. Each filled point indicates data obtained from an individual homozygous Myo6tvrm89 -mutant mouse plotted relative to the average control response. Data sets from 4 weeks are denoted by squares, and data sets from 8 weeks are denoted by triangles. The average ± SEM for each age is represented by an open square (4 weeks) or open triangle (8 weeks). The average ± SEM for control mice is represented by an open circle. The diagonal line indicates an equivalent reduction in the a-wave and each major component of the dc-ERG.
Reduced Cone ERGs in Homozygous Myo6tvrm89 Mice
Myosin 6 localization has been reported in cone ellipsoids of various fish species, 11,13 and it is highly expressed at the outer limiting membrane and inner segment of mouse photoreceptors 12 (Fig. 1). Therefore, we assessed the cone ERGs of homozygous Myo6tvrm89 mice. Figure 8 shows representative responses of homozygous mutant Myo6tvrm89 and control mice elicited by a 1.9 log cd s/m2 flash stimulus for mice aged 4 weeks (A), 8 weeks (C), or 1 year or older (E). As shown in Figure 8, the cone ERG is reduced by a comparable degree across flash luminance, averaging 21% at 4 weeks (B), 38% at 8 weeks (D), and 34% at 1 year or older (F). 
Figure 8
 
Cone ERG is reduced in homozygous Myo6tvrm89 mice. Averaged light-adapted strobe-flash ERG responses at 4 weeks (A, B), 8 weeks (C, D), and 1 year or older (E, F). Mice were light adapted for 7 minutes following dark-adapted testing and underwent strobe-flash ERGs under a steady rod-desensitizing adapting field (20 cd/m2). (A, C, E) Representative tracings from control (+/+ and tvrm89/+) and mutant (tvrm89/tvrm89) mice in response to a light stimulus of 1.9 log cd s/m2. (B, D, F) Cone ERG amplitude measured from the peak of the b-wave to the amplitude of the a-wave at 8 milliseconds. Data points indicate the average ± SEM of 22 control and 10 homozygous Myo6tvrm89 mice at age 4 weeks, of 24 control and 7 homozygous Myo6tvrm89 mice at age 8 weeks, and of 4 control and 2 homozygous Myo6tvrm89 mice at age 1 year or older. ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.01 by Student's t-test.
Figure 8
 
Cone ERG is reduced in homozygous Myo6tvrm89 mice. Averaged light-adapted strobe-flash ERG responses at 4 weeks (A, B), 8 weeks (C, D), and 1 year or older (E, F). Mice were light adapted for 7 minutes following dark-adapted testing and underwent strobe-flash ERGs under a steady rod-desensitizing adapting field (20 cd/m2). (A, C, E) Representative tracings from control (+/+ and tvrm89/+) and mutant (tvrm89/tvrm89) mice in response to a light stimulus of 1.9 log cd s/m2. (B, D, F) Cone ERG amplitude measured from the peak of the b-wave to the amplitude of the a-wave at 8 milliseconds. Data points indicate the average ± SEM of 22 control and 10 homozygous Myo6tvrm89 mice at age 4 weeks, of 24 control and 7 homozygous Myo6tvrm89 mice at age 8 weeks, and of 4 control and 2 homozygous Myo6tvrm89 mice at age 1 year or older. ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.01 by Student's t-test.
Discussion
Myosin 6 is involved in many cellular processes important to sensory function. 26,27 Herein, we describe the retinal phenotype of the Myo6tvrm89 mutant that carries a novel missense mutation in the motor domain of Myosin6. Our results add to the literature noting critical roles for myosin 6 at the inner and outer ear stereocilia in mice and in human deafness. 4,15,2833 Moreover, our analysis of the functional and structural effects of the tvrm89 mutation provides a detailed report of the retinal phenotype in Myo6 mutants. 
Unlike previously described Myo6 mutants, which involve null mutations that result in a loss of myosin 6 in the actin-rich domains within the retina/RPE, including rod photoreceptor inner segments, horizontal cells, and Müller glia, 11,13,34 myosin 6 protein expression is retained in the homozygous Myo6tvrm89 retina (Fig. 2). This feature indicates that the Myo6tvrm89 mouse model presented herein may provide insight into phenotypic features attributed to loss of myosin 6 function. 
Iris Abnormalities in Myo6tvrm89 -Mutant Mice
A novel finding in the homozygous Myo6tvrm89 mutant is the scalloped shape of the iris margin and abnormal morphology of the anterior segment (Figs. 3, 4). A similar feature was not reported for any of the Myo6sv strains; however, it is unknown if this was because the iris abnormalities were not present or were missed. Bilateral synechiae were present in all homozygous Myo6tvrm89 mutants, and approximately 50% of these animals also displayed lesions within the anterior chamber (Figs. 3, 4). We noted that these phenotypic features typically became less pronounced with age in the majority of animals imaged, which may be attributable to the use of mydriatics for ERG studies. The mydriatics and/or cyclopegics utilized for routine ERG studies are known to break synechiae and are used clinically in the treatment of iritis and iridocyclitis. 35 Consistent with this idea, posterior and anterior adhesions were visualized at 4 weeks and were maintained at the 8-week time point in the subset of animals that only underwent anesthesia by isofluorane inhalation and which were never treated with xylazine or topical mydriatics (Fig. 4). 
Nonprogressive Reduction in Retinal Function of Myo6tvrm89 Mice Without an Anatomical Correlate
Our detailed ERG analysis demonstrated that retinal function was reduced in 4-week-old homozygous Myo6tvrm89 mutants, the earliest age examined. The Myo6tvrm89 -mutant phenotype, which involves reduced ERG amplitude in the face of normal retinal structure, is unusual but has been reported in other models and could reflect a reduced photoreceptor dark current, altered regulation of ions and pH in the subretinal space, a change in resistance of the retinal circuit through which the ERG is recorded, or some other mechanism. 3638  
The dark current is the steady influx of a primarily sodium ion current that occurs in photoreceptors in the dark, maintaining them in a depolarized state. Upon presentation of light stimuli, the cation channels close, and photoreceptors hyperpolarize, releasing neurotransmitters to second-order neurons. Changes in the distribution or expression of the sodium ion channels or sodium-calcium exchanger in the rod outer segment could thus lead to reductions in both photoreceptor activity and responses of RPE and bipolar cells. 
The general decrease in retinal function that we observed in homozygous Myo6tvrm89 mutants could alternatively relate to a change in ion conductance across the RPE and/or a change in pH homeostasis that can occur in response to altered function of the endolysosomal pathway. Myosin 6 is associated with both the autophagosome 39 and lysosome, 1619 where the mutant form of myosin 6 could prevent either proper phagocytosis of shed outer segments, exchange of visual cycle components, or disk turnover and morphogenesis. Notably, we found that myosin 6 localization is unchanged in homozygous Myo6tvrm89 mice (Fig. 1D) but that its protein expression is higher within the RPE compared with wild-type and heterozygous mice (Fig. 1C). Within polarized epithelial cells such as the RPE, myosin 6 internalizes and transports receptors away from apical microvilli via clathrin-mediated endocytosis. 2,11,12,17,26,31,4046 Myosin 6 is known to mediate endocytosis of the cystic fibrosis transmembrane receptor (CFTR) in polarized epithelial cells. 47,48 Cystic fibrosis transmembrane receptor underlies a chloride ion conductance that is partially responsible for generation of the FO. In Myosin6-null mice, CFTR expression is maintained on the apical membrane of intestinal epithelium. 49 We have previously reported that dc-ERG is abnormal in CFTR-mutant mice. 25 Our data therefore suggest that the Myo6tvrm89 mutation may confer loss of endocytosis of CFTR at the apical RPE and contribute to the reduction in the ERG via altered chloride ion conductances. 
Other mouse mutants that demonstrate reduced ERG amplitude in the absence of anatomical changes to the retina include the Mct3−/− mouse, in which the ERG reductions were attributed to altered ion and pH homeostasis in the subretinal space due to the absence of normal lactate transport by monocarboxylate transporter 3 (MCT3). 36 The sodium-driven bicarbonate exchanger (NCBE)–knockout mouse also displays a reduction in b-wave amplitude, with no change in retinal morphology. 37 The altered ERG in this mouse is also thought to result at least partially from impaired intracellular pH regulation and chloride ion concentration. Lysosomal dysfunction can also lead to changes in pH via dysfunction of the ATP-driven proton pump in the RPE membrane. 38  
Loss of Myo6 could cause a change in resistance across the retinal circuit through which the ERG is recorded. Such a change could account for an overall reduction in ERG components despite preservation of retinal structure. While it is possible that the adhesions present in the mutant could potentially attenuate the amount of light reaching the retina because of this phenotype, a change in pupil dilation would lead to decreased sensitivity and prolonged latency, which were not observed. Whether any of these potential mechanisms underlie the Myo6-mutant phenotype of reduced ERG amplitudes in the face of normal retinal structure will require further analysis. 
We have presented a mouse model that can be used to better understand the role of myosin 6 and how it may interact with and compensate for other myosin isoforms. Despite the addition of two small amino acid inserts (amino acids 9 and 13) within the motor domain and a unique 53–amino acid insert between the converter domain and light chain–binding helix, which confers its minus-end directionality, 50 myosin 6 maintains a high degree of homology to other myosin isoforms, most notably in the head-motor domain. 5154 The phenotypes of Myo6-null and Myo6tvrm89 mice are similar to that of Myo7a (shaker)–null mice, which is a known Usher IB gene. This mouse mutant also presents with deafness and circling/head tossing behaviors associated with vestibular defects 55,56 and demonstrates reductions in ERG component amplitudes, without photoreceptor degeneration (unless combined with the loss of Cadherin-23, Cdh23). 5761 Most important, Myo7a is also expressed in the RPE and at the actin-rich domain of the inner segment. 62,63 It is interesting to speculate on the potential functional redundancy that may exist between these two myosin isoforms and determine how each acts independently. The generation of mice lacking both Myo6 and Myo7a would provide insight into this possibility. 
Acknowledgments
The authors acknowledge Charles Kaul for his assistance with in vivo imaging experiments, William Dupps for helpful analysis of iris and lens abnormalities, and Joe Hollyfield and Brian Perkins for critical reading of the manuscript. 
Supported by National Institutes of Health Grant R01 EY16501 (PMN), The Jackson Laboratory National Cancer Institute Cancer Center Support Grant CA34196, the Department of Veterans Affairs, the Foundation Fighting Blindness, and an unrestricted grant from Research to Prevent Blindness to the Cleveland Clinic Lerner College of Medicine of Case Western Reserve University. 
Disclosure: I.S. Samuels, None; B.A. Bell, None; G. Sturgill-Short, None; L.A. Ebke, None; M. Rayborn, None; L. Shi, None; P.M. Nishina, None; N.S. Peachey, None 
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Figure 1
 
The tvrm89 mutants bear a point mutation in Myosin6 but retain myosin 6 protein levels. (A) Positional cloning and direct sequencing demonstrate a T→C transition mutation found in homozygous tvrm89 mutants. (B) Allele-specific genotyping was accomplished using PCR amplification of DNA extracted from tail snips. (C) Western blot analysis of retinal (left) and RPE (right) protein lysates demonstrates myosin 6 protein levels in wild-type (+/+), heterozygous (tvrm89/+), and homozygous (tvrm89/tvrm89) mice. Actin serves as a loading control. (D) Cryosections of eyecups from +/+, tvrm89/+, and tvrm89/tvrm89 mice were immunostained with Myosin-VI antibody (red) and counterstained with 4′,6-diamidino-2-phenylindole (blue). Myosin 6 is present in all three genotypes and is highest in rod inner segments and the inner retina. At the right is the no primary antibody control. Low levels of autofluorescence are found in the rod outer segments; however, no specific immunostaining was apparent. Scale bar: 20 μm.
Figure 1
 
The tvrm89 mutants bear a point mutation in Myosin6 but retain myosin 6 protein levels. (A) Positional cloning and direct sequencing demonstrate a T→C transition mutation found in homozygous tvrm89 mutants. (B) Allele-specific genotyping was accomplished using PCR amplification of DNA extracted from tail snips. (C) Western blot analysis of retinal (left) and RPE (right) protein lysates demonstrates myosin 6 protein levels in wild-type (+/+), heterozygous (tvrm89/+), and homozygous (tvrm89/tvrm89) mice. Actin serves as a loading control. (D) Cryosections of eyecups from +/+, tvrm89/+, and tvrm89/tvrm89 mice were immunostained with Myosin-VI antibody (red) and counterstained with 4′,6-diamidino-2-phenylindole (blue). Myosin 6 is present in all three genotypes and is highest in rod inner segments and the inner retina. At the right is the no primary antibody control. Low levels of autofluorescence are found in the rod outer segments; however, no specific immunostaining was apparent. Scale bar: 20 μm.
Figure 2
 
Normal retinal anatomy in Myo6tvrm89 mutants. Retinal cross sections obtained from control Myo6+/+ (A), Myo6tvrm89/+ (B), and mutant Myo6tvrm89/tvrm89 (C) mice. Retinal histology was normal in all genotypes. Top: Representative light micrographs of the retina from ultrathin sections of adult eyecups. Scale bar: 20 μm. Bottom: Representative light micrographs displaying RPE morphology from semithin sections of adult eyecups. Scale bar: 10 μm. INL, inner nuclear layer; ONL, outer nuclear layer; RIS, rod inner segment; ROS, rod outer segment.
Figure 2
 
Normal retinal anatomy in Myo6tvrm89 mutants. Retinal cross sections obtained from control Myo6+/+ (A), Myo6tvrm89/+ (B), and mutant Myo6tvrm89/tvrm89 (C) mice. Retinal histology was normal in all genotypes. Top: Representative light micrographs of the retina from ultrathin sections of adult eyecups. Scale bar: 20 μm. Bottom: Representative light micrographs displaying RPE morphology from semithin sections of adult eyecups. Scale bar: 10 μm. INL, inner nuclear layer; ONL, outer nuclear layer; RIS, rod inner segment; ROS, rod outer segment.
Figure 3
 
Iris but not fundus abnormalities in homozygous Myo6tvrm89 mice. Myo6tvrm89 mutants display iris abnormalities (synechiae) that resolve with age. Left, middle: Low-magnification photomicrographs obtained by SLO displaying scalloped pupil shape in both left and right eyes at 4 weeks (top), 8 weeks (middle), and 1 year or older (bottom). Right: Infrared (dark field) SLO images of the retina taken at 4 weeks (top), 8 weeks (middle), and 1 year (bottom). No abnormalities were observed in the retina or the RPE at any age.
Figure 3
 
Iris but not fundus abnormalities in homozygous Myo6tvrm89 mice. Myo6tvrm89 mutants display iris abnormalities (synechiae) that resolve with age. Left, middle: Low-magnification photomicrographs obtained by SLO displaying scalloped pupil shape in both left and right eyes at 4 weeks (top), 8 weeks (middle), and 1 year or older (bottom). Right: Infrared (dark field) SLO images of the retina taken at 4 weeks (top), 8 weeks (middle), and 1 year (bottom). No abnormalities were observed in the retina or the RPE at any age.
Figure 4
 
Abnormal iris shape and anterior segment morphology in homozygous Myo6tvrm89 mice. The SDOCT imaging of homozygous Myo6tvrm89 mice found with bilateral synechiae revealed abnormal anterior segment morphology in approximately 50% (4/7) of animals. The horizontal line through the en face fundus image (left) indicates the location of a single SDOCT B-scan frame (right). Asterisks denote anterior chamber. Arrowhead at the top marks the lenticular opacity. Arrowhead at the bottom marks the corneal ulceration. Arrows indicate keratolenticular adhesions.
Figure 4
 
Abnormal iris shape and anterior segment morphology in homozygous Myo6tvrm89 mice. The SDOCT imaging of homozygous Myo6tvrm89 mice found with bilateral synechiae revealed abnormal anterior segment morphology in approximately 50% (4/7) of animals. The horizontal line through the en face fundus image (left) indicates the location of a single SDOCT B-scan frame (right). Asterisks denote anterior chamber. Arrowhead at the top marks the lenticular opacity. Arrowhead at the bottom marks the corneal ulceration. Arrows indicate keratolenticular adhesions.
Figure 5
 
Homozygous Myo6tvrm89 mice display attenuated ERGs. Averaged strobe-flash ERG responses at 4 weeks (AC), 8 weeks (DF), and 1 year or older (GI). (A, D, G) Averaged tracings from control (+/+ and tvrm89/+) and mutant (tvrm89/tvrm89) mice in response to stimuli of −2.4, −0.6, and 2.4 log cd s/m2. (B, E, H) The a-wave luminance-response functions (points indicate the average ± SEM). (C, F, I) The b-wave luminance-response functions (points indicate the average ± SEM). Data summarize results from 22 control and 10 mutants at 4 weeks, from 24 controls and 7 mutants at 8 weeks, and from 4 controls and 2 mutants at 1 year or older. ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.01 by Student's t-test.
Figure 5
 
Homozygous Myo6tvrm89 mice display attenuated ERGs. Averaged strobe-flash ERG responses at 4 weeks (AC), 8 weeks (DF), and 1 year or older (GI). (A, D, G) Averaged tracings from control (+/+ and tvrm89/+) and mutant (tvrm89/tvrm89) mice in response to stimuli of −2.4, −0.6, and 2.4 log cd s/m2. (B, E, H) The a-wave luminance-response functions (points indicate the average ± SEM). (C, F, I) The b-wave luminance-response functions (points indicate the average ± SEM). Data summarize results from 22 control and 10 mutants at 4 weeks, from 24 controls and 7 mutants at 8 weeks, and from 4 controls and 2 mutants at 1 year or older. ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.01 by Student's t-test.
Figure 6
 
The RPE function is reduced in homozygous Myo6tvrm89 mice. (A) Representative dc-ERGs of control (+/+ or tvrm89/+) mice (black) and mutant (tvrm89/tvrm89) mice (gray) at 4 weeks and 8 weeks. Baseline measurements are recorded for 30 seconds before initiation of a 7-minute light stimulus of 2.4 log cd/m2. The major dc-ERG components are labeled. (BE) Amplitude of c-wave (B), FO (C), LP (D), and off-response (E) obtained from mice aged 4 weeks or 8 weeks. Bars indicate the average ± SEM of 20 control and 7 mutant mice at 4 weeks and of 15 control and 7 mutant mice at 8 weeks. **P ≤ 0.001 by Student's t-test.
Figure 6
 
The RPE function is reduced in homozygous Myo6tvrm89 mice. (A) Representative dc-ERGs of control (+/+ or tvrm89/+) mice (black) and mutant (tvrm89/tvrm89) mice (gray) at 4 weeks and 8 weeks. Baseline measurements are recorded for 30 seconds before initiation of a 7-minute light stimulus of 2.4 log cd/m2. The major dc-ERG components are labeled. (BE) Amplitude of c-wave (B), FO (C), LP (D), and off-response (E) obtained from mice aged 4 weeks or 8 weeks. Bars indicate the average ± SEM of 20 control and 7 mutant mice at 4 weeks and of 15 control and 7 mutant mice at 8 weeks. **P ≤ 0.001 by Student's t-test.
Figure 7
 
The RPE function is equivalent to or better than photoreceptor activity in homozygous Myo6tvrm89 mice. Relative changes in amplitude of each major dc-ERG component (c-wave [A], FO [B], LP [C], and off-response [D]) as a function of a-wave amplitude in response to a light stimulus of 1.4 log cd s/m2. Each filled point indicates data obtained from an individual homozygous Myo6tvrm89 -mutant mouse plotted relative to the average control response. Data sets from 4 weeks are denoted by squares, and data sets from 8 weeks are denoted by triangles. The average ± SEM for each age is represented by an open square (4 weeks) or open triangle (8 weeks). The average ± SEM for control mice is represented by an open circle. The diagonal line indicates an equivalent reduction in the a-wave and each major component of the dc-ERG.
Figure 7
 
The RPE function is equivalent to or better than photoreceptor activity in homozygous Myo6tvrm89 mice. Relative changes in amplitude of each major dc-ERG component (c-wave [A], FO [B], LP [C], and off-response [D]) as a function of a-wave amplitude in response to a light stimulus of 1.4 log cd s/m2. Each filled point indicates data obtained from an individual homozygous Myo6tvrm89 -mutant mouse plotted relative to the average control response. Data sets from 4 weeks are denoted by squares, and data sets from 8 weeks are denoted by triangles. The average ± SEM for each age is represented by an open square (4 weeks) or open triangle (8 weeks). The average ± SEM for control mice is represented by an open circle. The diagonal line indicates an equivalent reduction in the a-wave and each major component of the dc-ERG.
Figure 8
 
Cone ERG is reduced in homozygous Myo6tvrm89 mice. Averaged light-adapted strobe-flash ERG responses at 4 weeks (A, B), 8 weeks (C, D), and 1 year or older (E, F). Mice were light adapted for 7 minutes following dark-adapted testing and underwent strobe-flash ERGs under a steady rod-desensitizing adapting field (20 cd/m2). (A, C, E) Representative tracings from control (+/+ and tvrm89/+) and mutant (tvrm89/tvrm89) mice in response to a light stimulus of 1.9 log cd s/m2. (B, D, F) Cone ERG amplitude measured from the peak of the b-wave to the amplitude of the a-wave at 8 milliseconds. Data points indicate the average ± SEM of 22 control and 10 homozygous Myo6tvrm89 mice at age 4 weeks, of 24 control and 7 homozygous Myo6tvrm89 mice at age 8 weeks, and of 4 control and 2 homozygous Myo6tvrm89 mice at age 1 year or older. ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.01 by Student's t-test.
Figure 8
 
Cone ERG is reduced in homozygous Myo6tvrm89 mice. Averaged light-adapted strobe-flash ERG responses at 4 weeks (A, B), 8 weeks (C, D), and 1 year or older (E, F). Mice were light adapted for 7 minutes following dark-adapted testing and underwent strobe-flash ERGs under a steady rod-desensitizing adapting field (20 cd/m2). (A, C, E) Representative tracings from control (+/+ and tvrm89/+) and mutant (tvrm89/tvrm89) mice in response to a light stimulus of 1.9 log cd s/m2. (B, D, F) Cone ERG amplitude measured from the peak of the b-wave to the amplitude of the a-wave at 8 milliseconds. Data points indicate the average ± SEM of 22 control and 10 homozygous Myo6tvrm89 mice at age 4 weeks, of 24 control and 7 homozygous Myo6tvrm89 mice at age 8 weeks, and of 4 control and 2 homozygous Myo6tvrm89 mice at age 1 year or older. ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.01 by Student's t-test.
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