Human Molecular Genetics, 2000, Vol. 9, No. 12 1729-1738
© 2000 Oxford University Press
The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells
1Laboratory of Molecular Genetics and 2Laboratory of Cell Biology, NIDCD, Rockville, MD 20850, USA, 3Department of Human Genetics, 4Department of Otolaryngology and 5Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI 48109-0638, USA and 6Laboratory of Cellular Biology, NIDCD, Bethesda, MD 20892, USA
Received 20 March 2000; Revised and Accepted 22 May 2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Recessive mutations in myosin 15, a class XV unconventional myosin, cause profound congenital deafness in humans and both deafness and vestibular dysfunction in mice homozygous for the shaker 2 and shaker 2J alleles. The shaker 2 allele is a previously described missense mutation of a highly conserved residue in the motor domain of myosin XV. The shaker 2J lesion, in contrast, is a 14.7 kb deletion that removes the last six exons from the 3"-terminus of the Myo15 transcript. These exons encode a FERM (F, ezrin, radixin and moesin) domain that may interact with integral membrane proteins. Despite the deletion of six exons, Myo15 mRNA transcripts and protein are present in the post-natal day 1 shaker 2J inner ear, which suggests that the FERM domain is critical for the development of normal hearing and balance. Myo15 transcripts are first detectable at embryonic day 13.5 in wild-type mice. Myo15 transcripts in the mouse inner ear are restricted to the sensory epithelium of the developing cristae ampularis, macula utriculi and macula sacculi of the vestibular system as well as to the developing organ of Corti. Both the shaker 2 and shaker 2J alleles result in abnormally short hair cell stereocilia in the cochlear and vestibular systems. This suggests that Myo15 may be important for both the structure and function of these sensory epithelia.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The human hereditary deafness locus DFNB3 was mapped to chromosome 17p11.2 in a large kindred from the village of Bengkala, Indonesia (1,2). Genes in this region of human chromosome 17p were conserved along a portion of mouse chromosome 11 to which shaker 2 (sh2) had been mapped (2,3). Shaker 2 homozygous mice are congenitally deaf and have vestibular defects that cause circling behavior (4,5). Myo15, a novel unconventional myosin, was discovered to be the gene responsible for the sh2 phenotype by genetic complementation with a bacterial artificial chromosome (BAC) transgene and DNA sequence analysis (3). The Myo15 gene is ~60 kb in length and contains 66 exons (GenBank accession no. AF144093) (6). The predicted full-length transcript is >12 kb and encodes a 3511 amino acid protein.
Myosins are proteins that bind to actin filaments and function as molecular motors. They produce force by cycling between two conformation states controlled by the binding and hydrolysis of ATP (7). Myosins form a superfamily of proteins which includes the conventional class II myosins and 14 classes of unconventional myosins which are distinguished based on homology of the motor domains. All myosins share a common structural organization. The motor domain contains the actin- and ATP-binding sites followed by a flexible neck region that contains myosin light chain-binding sites. The C-terminal portion of a myosin protein forms a tail structure that is divergent between the different classes of myosins (7).
The myosin XV motor domain is predicted to contain ATP- and actin-binding sites, which are followed by two myosin light chain-binding sites (IQ domains). Preceding the myosin XV motor domain is a large (1223 amino acids) proline-rich N-terminal extension, most of which is encoded by exon 2 (6). Another myosin with an N-terminal extension of 266 amino acids is the class III myosin NinaC, which is found in the photoreceptor cells of Drosophila retinas (8). The motor domain of NinaC is required for the structural integrity of the photoreceptor cells, whereas the N-terminal extension, a protein kinase domain, is involved in signal transduction. The myosin XV N-terminal extension, in contrast, is proline rich with no other predicted sequence similarity to reported proteins.
The myosin XV tail region contains five domains which are, in order from N- to C-terminal: a MyTH4 (myosin tail homology four) domain, a FERM-like domain, an SH3 (src homology 3) domain, a second MyTH4 domain and, finally, a FERM domain (6). SH3 domains are protein modules that recognize and bind proline-rich sequences (9) and have been described in proteins that are involved in such functions as synaptic vesicle endocytosis (10) and proper subcellular localization of proteins (11). FERM domains are found in a group of homologous proteins that include the band 4.1 superfamily (F, ezrin, radixin and moesin) (12). Proteins with FERM domains interact with the cytoplasmic domains of integral membrane proteins such as CD44, CD43 and ICAM-2 and may function as cross-links between the cell membrane and the actin cytoskeleton (13). MyTH4 domains are found in the tails of myosins IV, VIIA, X and XII (14) and at present their functions are unknown. Myosin VIIA contains two FERM domains, two MyTH4 domains and an SH3 domain (14,15) organized in the same order as in myosin XV (6). The similarity between the myosin XV and myosin VIIA tails is interesting because these unconventional myosins are both involved in hereditary hearing loss in humans and required for the proper structure and organization of the stereocilia in the organ of Corti (3,16,17).
Mutations in Myo15 were discovered in sh2 mice and in three human kindreds with DFNB3 (18), implicating several regions of the protein as critical for proper function. Of the three mutations described in humans, two cause amino acid substitutions in the first MyTH4 domain and the third creates a premature stop codon that truncates the tail region (6). The sh2 mutation is a substitution of a tyrosine residue for a cysteine in a highly conserved position of the myosin XV motor domain (3). Another spontaneous allele, shaker 2J (sh2J) (19), also results in congenital deafness and vestibular defects. We report the characterization of the sh2J mutation at the genomic and cellular level. This allele demonstrates the critical importance of the FERM domain in myosin XV function in both the auditory and vestibular systems. We also describe the developmental profile of Myo15 expression and show that Myo15 is expressed in the inner hair cells (IHCs) and outer hair cells (OHCs) of the post-natal cochlea.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Developmental expression of Myo15 mRNA
To determine the temporal and spatial expression of Myo15 during normal development of the mouse inner ear, a Myo15 riboprobe specific for exons 48–51 (Tail 1) was hybridized in situ to paraffin-embedded sections from C57BL/6J mouse embryos of different developmental stages (Fig. 1). Myo15 transcripts were first detected at 13.5 days post-coitum (d.p.c.). No Myo15 transcripts were observed in the developing inner ear at either 11.5 or 12.5 d.p.c. (data not shown). Myo15 transcripts were restricted to discrete regions of the three cristae ampularis, macula utriculi and macula sacculi and the cochlea from 15.5 d.p.c. onward. The hybridization signal in 18.5 d.p.c. cochlea is restricted to the sensory epithelium of the organ of Corti. In situ hybridization of adjacent sections from these same embryos with a riboprobe specific for exon 2, the exon that encodes most of the N-terminal extension, demonstrated the same age of onset and restricted pattern of Myo15 expression as observed with the Tail 1 riboprobe (data not shown). Hybridization of post-natal day 8 (P8) mouse inner ears with the Tail 1 probe demonstrates that Myo15 mRNA is expressed in the IHCs and OHCs of the organ of Corti and is not detected in the surrounding supporting cells (Fig. 1).
|
Abnormal stereocilia on hair cells in Myo15sh2-J/sh2-J mice
Scanning electron microscopy of cochleae from 1-month-old wild-type, Myo15sh2-J/sh2-J and Myo15sh2/sh2-J compound heterozygote mice shows that Myo15sh2-J/sh2-J mice have very short stereocilia on both their IHCs and OHCs (Fig. 2), much like those of the Myo15sh2/sh2 mouse (3). The normal organizational pattern of the stereocilia is largely preserved although there are some ectopic stereocilia. A similar mutant phenotype is observed in Myo15sh2/sh2-J compound heterozygotes, which indicates that the two alleles do not complement each other.
|
Laser scanning confocal microscopy of the rhodamine-conjugated phalloidin-stained cochleae of 1-month-old Myo15sh2-J/+ and Myo15sh2-J/sh2-J mice detected long, actin-containing structures (referred to as ‘cytocauds’ in ref. 20) in the IHCs of Myo15sh2-J/sh2-J animals but not in Myo15sh2-J/+ animals (Fig. 3C and D). These structures, which can be as long as 50 µm, originate below the cuticular plate within each IHC of the cochlea and extend through the cell and outward from the base. Such structures were never observed in the OHCs. Hair cells in the vestibular epithelium were similarly abnormal. Epifluorescence analysis of whole-mount utricles labeled with rhodamine-conjugated phalloidin revealed long, well-organized stereocilia on wild-type utricular hair cells (Fig. 3E). In contrast, very short stereocilia bundles were observed in the Myo15sh2-J/sh2-J macula utriculi (Fig. 3F). Elongated actin-containing structures, similar to those in the IHCs of the organ of Corti, were seen beneath the reticular lamina in most of the hair cells of the macula utriculi. Stereocilia in the sensory epithelium of the saccule and the three ampulae were short, similar to those in the utricle (data not shown). Based on the similarity between the sh2 and sh2J phenotypes and their failure to complement, the defects in the sh2J homozygous mice were presumed to be due to a mutation in Myo15.
|
The sh2J allele is a deletion of part of Myo15
To identify the molecular defect in the sh2J allele, Southern blots of HindIII-digested Myo15sh2-J/sh2-J and Myo15sh2-J/+ genomic DNA were hybridized with two probes located near the 3"-terminus of the gene (Fig. 4A and B). Probe 1 (nucleotides 65 098–65 346, 248 bp, GenBank accession no. AF144093) hybridized to the same 6.2 kb restriction fragment from both Myo15sh2-J/sh2-J and Myo15sh2-J/+ genomic DNA. Probe 2 (nucleotides 74 210–74 491, 282 bp) failed to hybridize to Myo15sh2-J homozygote DNA, indicating a deletion of part of the Myo15 gene in Myo15sh2-J/sh2-J mice. PCR primers were designed to amplify across the sh2J deletion (Fig. 4A). In wild-type DNA, primers 4 and 5 amplify a 231 bp product across the 5" break point and primers 6 and 7 amplify a 242 bp product across the 3" break point. Primers 4 and 7 amplify a 185 bp product from Myo15sh2-J/sh2-J DNA. Amplification of DNA from mice heterozygous for the Myo15sh2-J deletion yields all three bands. Sequence analysis of the PCR products demonstrates that the deletion spans 14.7 kb from nucleotide 67 056 (GenBank accession no. AF144093) at the 5"-end to nucleotide 81 783 at the 3"-end. This deletion removes the sequences for exons 61–66, the 3"-UTR and ~5.6 kb of 3"-flanking DNA. The sh2J mutation results in loss of the coding region for the C-terminal FERM domain and the presumed 3" cleavage and polyadenylation signals from the mRNA. A 9 bp fragment was detected between the deletion break points (Fig. 4D). Inspection of the sequence revealed that a 13 bp inverted repeat was created during the deletion process. Exons 2–59 of Myo15 from an sh2J homozygote were also sequenced and no other mutations in the coding region were detected.
|
Myo15 transcripts can be detected in Myo15sh2-J/sh2-J mice
In order to determine the effect of the deletion on Myo15 mRNA expression, antisense riboprobes specific for exons 48–51 (Tail 1) and exons 61–65 (Tail 2) were hybridized in situ to sections of P1 Myo15sh2-J/sh2-J and Myo15sh2-J/+ mice (Fig. 5). Sections from Myo15sh2-J/+ mice demonstrate the expected expression pattern with both antisense probes. No Myo15 transcripts were detected in sections from Myo15sh2-J/sh2-J mutants hybridized with the Tail 2 riboprobe, which is within the region of the sh2J deletion. Myo15 transcripts were, however, detected in the Myo15sh2-J/sh2-J homozygous mutants with the Tail 1 riboprobe. It appears that deletion of the last six exons from the mRNA does not affect the accumulation of Myo15sh2-J transcript or alter its tissue distribution.
|
Myosin XV protein is present in inner ears of Myo15sh2-J/sh2-J mice
To determine whether the abnormal Myo15 mRNA transcripts were translated, myosin XV protein expression was analyzed by immunofluorescence with anti-mouse myosin XV antibody (TF1) (6) directed against a region of the tail encoded 5" to the sh2J deletion (Fig. 6). Whole-mount cochlear tissues from wild-type, Myo15sh2/sh2, Myo15sh2-J/+ and Myo15sh2-J/sh2-J adult animals were treated with TF1 antibody and a fluorescein-conjugated secondary antibody and then examined with a Fluorescence Microscopy Imaging System (CELLscan; Scanalitics, Fairfax, VA). Myosin XV expression was detected in the OHCs and IHCs. Labeling of the typical V-shaped groups of stereocilia on the three rows of OHCs is evident in the tissues of both wild-type mice and Myo15sh2-J/+ heterozygotes (Fig. 6, column 1). This labeling is absent from the Myo15sh2/sh2 and Myo15sh2-J/sh2-J tissues, which is consistent with the developmental defect in the length of the stereocilia (Fig. 6, column 1).
|
In the wild-type, immunofluorescence was also detected at the level of the actin-rich cuticular plate (Fig. 6, column 2) and below the level of the apical cell body (Fig. 6, column 3). Myosin XV staining at the cuticular plate appears as a dispersed fluorescence in wild-type and Myo15sh2-J/+ mice with some localized increase in staining corresponding to the rootlet of the kinocilium (Fig. 6, column 2) in 4 of the 12 cochleae examined. This localized increase was not observed in the other 8 cochleae. The staining in both Myo15sh2/sh2 and Myo15sh2-J/sh2-J mice appears punctate and is more defined than the pattern observed in wild-type mice, which is suggestive of an altered intracellular distribution (Fig. 6, column 2). This punctate staining was observed in all 12 homozygous mutant cochleae examined (6 Myo15sh2/sh2 and 6 Myo15sh2-J/sh2-J ). Despite the deletion of 14.7 kb of genomic DNA, which includes the exons for the second FERM domain, the Myo15sh2-J/sh2-J animals accumulate a presumably truncated form of myosin XV.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The shaker 2 and shaker 2J mutant mice provide a model system for studying the molecular pathology of human hereditary non-syndromic deafness due to mutations at the DFNB3 locus (6). In wild-type mice, Myo15 transcripts are first detectable at ~13.5 d.p.c. during the initial stages of hair cell maturation. The temporal and spatial expression of Myo15 appears to parallel the expression of Myo6 and Myo7A (21), two other unconventional myosins that are mutated in mice with hearing loss and vestibular defects (22,23). Mutant alleles of the human ortholog MYO7A are associated with non-syndromic deafness DFNB2 and Usher syndrome type 1B (24). Transcripts from Myo6 and Myo7A are first detected in the sensory epithelium of 13.5 d.p.c. otocysts and can be detected at high levels in the vestibular and cochlear sensory hair cells at 15.5 d.p.c. onward (21). The similar temporal profiles of Myo6, Myo7A and Myo15 expression in hair cells suggests that they may be co-regulated.
The shaker 2J mutation is a deletion of 14.7 kb of Myo15 genomic sequence that results in loss of the last six exons and a portion of the 3" flanking region. Despite the loss of the putative polyadenylation signal at position 76 103 (GenBank accession no. AF144093), stable Myo15 transcripts are synthesized and present in the correct spatial expression pattern in shaker 2J mice. This indicates that essential transciptional regulatory elements are probably not located within the deletion region. Moreover, the abnormal Myo15 transcripts must utilize an alternative polyadenylation signal upstream of the deletion or a downstream sequence that was brought into proximity by the deletion. The abnormal Myo15 transcripts are translated since myosin XV protein is present in Myo15sh2-J/sh2-J mice. In spite of the presence of the myosin XV protein the mice have profound congenital deafness and circling behavior. Thus, the mutant protein is not functional for either hearing or proper balance.
Humans homozygous for DFNB3 mutations have not been rigorously tested for vestibular function (18); however, deaf individuals from Bengkala, Indonesia, indicate that they feel ‘inebriated’ in the absence of visual cues (25). This suggests that Myo15 may perform a similar role in the vestibular system in humans and mice and that it has a common role in the development and function of the sensory epithelia of both the vestibular system and the organ of Corti.
The effects of myosin XV defects on the cells of the inner ear were evaluated by microscopy of the organ of Corti and the vestibular sensory epithelia in shaker 2 and shaker 2J mutants. Abnormally short stereocilia are present on the IHCs and OHCs of the organ of Corti and on hair cells of the utricular, saccular and ampullary epithelia in both mutants. In addition, the IHCs and vestibular hair cells of both mutants contain abnormal elongated structures with an actin core. Similar actin structures were first observed in the shaking-waltzing guinea pig (26,27).
The cellular phenotypes of the shaker 2 and shaker 2J mice suggest that myosin XV has an important structural role in actin cytoskeleton organization. The stereocilia normally have a highly enriched actin content. In the presence of abnormal myosin XV, the stereocilia are very short and abnormal actin structures form, suggesting a failure in organization of the intracellular actin cytoskeleton. During development microvilli initially cover the apical surface of hair cells. The stereocilia are thought to develop by elongation of some microvilli whereas the remaining microvilli are eventually lost (28,29). The presence of short and ectopic stereocilia on the surface of the IHCs in shaker 2 and shaker 2J mutants demonstrates that the normal development of stereocilia is disrupted when myosin XV function is altered by mutations. The characteristic three rows of V-shaped stereocilia groups develop on the OHCs of the mutants, which indicates that this aspect of OHC development is still present.
The shaker 2 and shaker 2J mutant alleles have very different molecular lesions, yet they appear to produce the same phenotype. Myo15sh2/sh2, Myo15sh2-J/sh2-J and Myo15sh2/sh2-J animals display head tossing, circling, hyperactivity and deafness with no apparent differences in the cellular phenotypes in the vestibular system or the cochlea. The shaker 2 mutation affects the motor domain of myosin XV, whereas the shaker 2J allele lacks the six exons that encode the C-terminal FERM domain of the myosin XV tail. FERM domains are found in membrane-associated proteins like moesin, radixin and talin that are involved in linking the cytoskeleton to the membrane (12). The myosin XV C-terminal FERM domain may also play a role in anchoring the protein to the cell membrane. Deletion of the FERM domain may result in an inability to exert force on the actin cytoskeleton because the truncated protein is not anchored properly, causing a failure to form the scaffolding required for normal stereocilia structure. At this time it is unknown what proteins, other than actin, can bind to myosin XV or influence its function.
The importance of a C-terminal FERM domain was suggested by a shaker 1 allele of Myo7A. The Myo7A3336SB allele is a chemically induced mutation that introduces a premature stop codon, resulting in truncation of the C-terminal FERM domain and a severe deafness phenotype (15). This mutation does not appear to affect the steady-state levels of mutant mRNA, much like the deletion of the FERM domain in Myo15sh2-J/sh2-J mice. However, the protein levels in the Myo7A3336SB mutants were only 13% that of wild-type. Results from these studies of Myo7A and our comparison of Myo15sh2-J and Myo15sh2 mutants support the hypothesis that the FERM domain is as critical for myosin function in the auditory system as the motor domain. Moreover, the protein partners that interact with the FERM domains of Myo7A and Myo15 are good candidates for genes involved in the development of normal hearing in mice and humans.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Mice
Mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were cared for according to the National Institutes of Health Guidelines. Experiments were approved by the University of Michigan Committee on the Use and Care of Animals (protocol no. A3314-01), Laboratory of Molecular Genetics, NIH, NIDCD IUCAC (protocol no. 791-97) and Laboratory of Cellular Biology, NIH, NIDCD IUCAC (protocol no. 923-99).
In situ hybridization
In situ hybridization was performed as described previously (30,31). Embryos from C57BL/6J mice were collected from timed pregnancies and staged according to Thieler (32) at 13.5, 15.5, 17.5 and 18.5 d.p.c. Embryos up to 15.5 d.p.c. were fixed intact in 4% paraformaldehyde in 1x phosphate-buffered saline (PBS). Animals at 17.5 d.p.c. and older were decapitated and only the heads were fixed. The skin and brain were removed from 18.5 d.p.c. and older heads prior to fixation. The tissues were embedded in paraffin and sectioned in the transverse orientation at a thickness of 10 µm.
Heads from a single litter produced by a Myo15sh2-J/sh2-J x Myo15sh2-J/+ mating were collected at P1, the skin and brains removed, the heads fixed in 4% paraformaldehyde in 1x PBS, embedded in paraffin and sectioned in the transverse orientation at a thickness of 12 µm. Genotyping was performed on tissue obtained at the time of dissection in order to identify animals heterozygous for the Myo15sh2-J allele and the homozygous mutants. Riboprobes were designated Tail 1 (exons 48–51, nucleotides 8726–9199) and Tail 2 (exons 61–65, nucleotides 10 212–10 723) (GenBank accession no. AF144095). These cDNA fragments were subcloned into pGemT-easy (Promega, Madison, WI) and oriented so that both the sense and antisense riboprobes could be produced from the T7 RNA polymerase promoter. The riboprobes were double labeled with [
-35S]UTP and [-35S]ATP using a Stratagene RNA transcription kit (catalog no. 200340; Stratagene, La Jolla, CA). Non-radioactive in situ hybridization of P8 mouse inner ears was performed on frozen sectioned tissue with digoxigenin-labeled Tail 1 probe as previously described (33).
Southern blotting
Genomic DNA from the mouse strain on which shaker 2J arose, C57BL/Ks-db m, was obtained from the Jackson Laboratory. DNA from all other strains was prepared from lung, liver, kidney and spleen tissue and Southern blotting was performed as described (34). Probe 1 (nucleotides 65 098–65 346, 248 bp) and Probe 2 (nucleotides 74 210–74 491, 282 bp) (GenBank accession no. AF144093) were amplified from genomic DNA by PCR (see Table 1 for primer sequences) and were used to serially hybridize a single Southern blot, which was stripped between hybridizations. The blot was washed in 0.1x SSC, 0.1% SDS at 57°C and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) for 1–2 days.
|
Genotyping
Four different oligonucleotide primers (primer 4, nucleotides 66 972–66 992; primer 5, nucleotides 67 193–67 213; primer 6, nucleotides 81 653–81 674; primer 7, nucleotides 81 866–81 883; GenBank accession no. AF144093) were designed for genotyping (Table 1). The primers were used in separate reactions in three combinations. Primers 4 and 5 amplify a 242 bp product which spans the 5" break point and primers 6 and 7 amplify a 231 bp product which spans the 3" break point. Primers 4 and 7 amplify a 185 bp product from the sh2J allele, but do not result in a product from the wild-type allele because 14.7 kb is significantly longer than the PCR conditions allow. The PCR was carried out with 0.2 U of Taq polymerase (Roche Molecular Biochemicals, Indianapolis, IN) in a 25 µl reaction with the buffer supplied by the manufacturer, 0.2 mM dNTPs and 6 pmol each primer. Cycling conditions were: 92°C for 2 min; 30 cycles of 92°C for 30 s, 55°C for 30 s and 72°C for 30 s; 72°C for 10 min. Ten microliters of the reaction from each primer pair were pooled and electrophoresed on a 2% agarose, 1% NuSieve (FMC BioProducts, Rockland, ME), 0.5x TBE gel.
Sequencing
DNA from Myo15sh2-J/sh2-J and C57BL6/J animals was amplified by PCR using primers designed within intron sequences 10–200 nucleotides from the intron–exon boundaries. The amplification products were separated by agarose gel electrophoresis and the appropriate bands were extracted, reamplified and purified by the Qiagen gel extraction method (Qiagen, Santa Clarita, CA). The purified products were sequenced on ABI 377 sequencer using one of the two PCR primers and analyzed using Sequencher software (Gene Codes, Ann Arbor, MI). Myo15 sequencing primers are available on request.
Scanning electron microscopy
Cochleae from 1-month-old Myo15sh2-J/+, Myo15sh2-J/sh2-J and Myo15sh2/sh2-J mice (n 
2 each) were locally perfused with 2.5% glutaraldehyde in phosphate buffer (0.1 M) and dissected to reveal the surface of the organ of Corti epithelium. Samples were treated with 1% osmium tetroxide for 1 h, dehydrated in ethanol, critical point dried (Samdri 790; Tousimis, Rockville, MD), sputter coated with gold and viewed in an Amray 1000B scanning electron microscope operated at 10–15 kV.
Histochemistry and light microscopy
Cochleae from 1-month-old Myo15sh2-J/+, Myo15sh2-J/sh2-J and Myo15sh2/sh2-J mice were locally perfused with 4% paraform-aldehyde (n 2 each). The overlying otic capsule was removed to allow penetration of reagents into the organ of Corti. The bone above the utricle and the ampulae was removed to expose the vestibular organs. Tissues were permeabilized in 0.3% Triton X-100 for 10 min, then incubated with rhodamine–phalloidin (1:100 dilution; Molecular Probes, Eugene, OR) as previously described (35). Isolated segments of the organ of Corti were dissected free of surrounding tissues and mounted on glass microscope slides with Crystal Mount (Biomeda, Foster City, CA). Vestibular organs were removed from the bone as described previously (36) and mounted similarly to the cochlear samples. Preparations were examined and photographed using a Leica DMRB epifluorescence microscope using a 100x 1.3 numerical aperture objective. Samples analyzed with a confocal fluorescence microscope were prepared in a similar way. Analysis was performed using a Bio-Rad 600 confocal microscope (Bio-Rad, Hercules, CA) and a Z series of 1.0 µm thick optical sections was obtained, as described previously (37).
Immunolocalization studies
Cochleae from 1- and 1.5-month-old B6C3F1/J, Myo15sh2/sh2, Myo15sh2-J/sh2-J and Myo15sh2-J/+ mice were perfused through the round and oval windows and a small fenestra in the apical cochlear bony capsule with 4% paraformaldehyde in PBS, pH 7.4, followed by incubation in this fixative for 1 h at room temperature (38). Then cochleae were washed in 1x PBS and opened, and organs of Corti were dissected out of the modiolus using a fine needle. Samples were permeabilized with 0.5% Triton-X100 in 1x PBS for 30 min and washed in 1x PBS, followed by overnight incubation at 4°C in blocking solution (2% bovine serum albumin and 2% donkey serum in 1x PBS). Affinity-purified Myo15 antiserum TF1 (1.15 µg/µl) (6) was diluted 1:100 v/v in blocking solution and used in a 1 h room temperature incubation. After washing in 1x PBS, the tissue was incubated for 40 min using fluorescein-conjugated donkey anti-rabbit secondary antibody (Amersham, Arlington Heights, IL), diluted 1:200 v/v in blocking solution. After several washes in PBS, samples were mounted for microscopy with ProLong Antifade kit (Molecular Probes). Images were obtained using a Zeiss Axiophot microscope equipped with a 63x 1.4 numerical aperture objective, CCD camera (Photometric, Tuscon, AZ) and fluorescence microscopy imaging system (CELLscan). Control samples were treated as above but with omission of primary antibody or using preincubation of affinity-purified Myo15 antiserum (1.15 µg/µl) with a GST–Myo15 (amino acids 2157–2347; GenBank accession no. AF144094) fusion peptide (0.75 µg/µl) (6) in the proportions 1:1.6 w/w for 1 h at room temperature (data not shown).
|
ACKNOWLEDGEMENTS |
|---|
We thank Jill Karolyi for maintaining the mouse colonies at the University of Michigan and Raquel Cantos and Quianna Burton for their help with the frozen section and non-isotopic in situ hybridization. This work was funded by a University of Michigan Rackham Predoctoral Fellowship (F.J.P.), extramural grants from programs of the National Institutes of Health HD30428 (S.A.C.) and DC01634 (Y.R.) and NIDCD intramural funds 1 Z01 DC00048-02 LMG (T.B.F.).
|
FOOTNOTES |
|---|
+ These authors contributed equally to this work
§ Present address: Virology, Department 106, Bristol-Myers Squibb, 5 Research Parkway, Wallingford, CT 06492, USA
¶ To whom correspondence should be addressed at: Department of Human Genetics, 4301 MSRB III, 1150 West Medical Center Drive, University of Michigan, Ann Arbor, MI 48109-0638, USA. Tel: +1 734 763 0682; Fax: +1 734 763 7672; Email: scamper@umich.edu
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Friedman, T.B., Liang, Y., Weber, J.L., Hinnant, J.T., Barber, T.D., Winata, S., Arhya, I.N. and Asher, J.H.J. (1995) A gene for congenital, recessive deafness DFNB3 maps to the pericentromeric region of chromosome 17. Nature Genet., 9, 86–91.[ISI][Medline]
2 Liang, Y., Wang, A., Probst, F.J., Arhya, I.N., Barber, T.D., Chen, K.S., Deshmukh, D., Dolan, D.F., Hinnant, J.T., Carter, L.E. et al. (1998) Genetic mapping refines DFNB3 to 17p11.2, suggests multiple alleles of DFNB3, and supports homology to the mouse model shaker-2. Am. J. Hum. Genet., 62, 904–915.[ISI][Medline]
3 Probst, F.J., Fridell, R.A., Raphael, Y., Saunders, T.L., Wang, A., Liang, Y., Morell, R.J., Touchman, J.W., Lyons, R.H., Noben-Trauth, K. et al. (1998) Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene. Science, 280, 1444–1447.
4 Gates, W.H. (1934) Linkage tests of the new shaker mutation with other factors in the house mouse (Mus musculus). Am. Nature, 68, 173.
5 Clark, F.H. (1935) Linkage relations of Zavaskaia Shaker in the house mouse (Mus musculus). Proc. Natl Acad. Sci. USA, 21, 247–251.
6 Liang, Y., Wang, A., Belyantseva, I.A., Anderson, D.W., Probst, F.J., Barber, T.D., Miller, W., Touchman, J.W., Jin, L., Sullivan, S.L. et al. (1999) Characterization of the human and mouse unconventional myosin XV genes responsible for hereditary deafness DFNB3 and shaker 2. Genomics, 61, 243–258.[ISI][Medline]
7 Mooseker, M.S. and Cheney, R.E. (1995) Unconventional myosins. Annu. Rev. Cell Dev. Biol., 11, 633–675.[ISI][Medline]
8 Montell, C. and Rubin, G.M. (1988) The Drosophila ninaC locus encodes two photoreceptor cell specific proteins with domains homologous to protein kinases and the myosin heavy chain head. Cell, 52, 757–772.[ISI][Medline]
9 Yu, H., Chen, J.K., Feng, S., Dalgarno, D.C., Brauer, A.W. and Schreiber, S.L. (1994) Structural basis for the binding of proline-rich peptides to SH3 domains. Cell, 76, 933–945.[ISI][Medline]
10 Shupliakov, O., Löw, P., Grabs, D., Gad, H., Chen, H., David, C., Takei, K., De Camilli, P. and Brodin, L. (1997) Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions. Science, 276, 259–263.
11 Anderson, B.L., Boldogh, I., Evangelista, M., Boone, C., Greene, L.A. and Pon, L.A. (1998) The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization. J. Cell Biol., 141, 1357–1370.
12 Chishti, A.H., Kim, A.C., Marfatia, S.M., Lutchman, M., Hanspal, M., Jindal, H., Liu, S.C., Low, P.S., Rouleau, G.A., Mohandas, N. et al. (1998) The FERM domain: a unique module involved in the linkage of cytoplasmic proteins to the membrane [letter]. Trends Biochem. Sci., 23, 281–282.[ISI][Medline]
13 Yonemura, S., Hirao, M., Doi, Y., Takahashi, N., Kondo, T. and Tsukita, S. (1998) Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J. Cell Biol., 140, 885–895.
14 Chen, Z.Y., Hasson, T., Kelley, P.M., Schwender, B.J., Schwartz, M.F., Ramakrishnan, M., Kimberling, W.J., Mooseker, M.S. and Corey, D.P. (1996) Molecular cloning and domain structure of human myosin-VIIa, the gene product defective in Usher syndrome 1B. Genomics, 36, 440–448.[ISI][Medline]
15 Mburu, P., Liu, X.Z., Walsh, J., Saw, D.J., Cope, M.J., Gibson, F., Kendrick-Jones, J., Steel, K.P. and Brown, S.D. (1997) Mutation analysis of the mouse myosin VIIA deafness gene. Genes Funct., 1, 191–203.[Medline]
16 Self, T., Mahony, M., Fleming, J., Walsh, J., Brown, S.D. and Steel, K.P. (1998) Shaker-1 mutations reveal roles for myosin VIIA in both development and function of cochlear hair cells. Development, 125, 557–566.[Abstract]
17 Friedman, T.B., Sellers, J.R. and Avraham, K.B. (1999) Unconventional myosins and the genetics of hearing loss. Am. J. Hum. Genet. (Semin. Med. Genet.), 89, 147–157.
18 Wang, A., Liang, Y., Fridell, R.A., Probst, F.J., Wilcox, E.R., Touchman, J.W., Morton, C.C., Morell, R.J., Noben-Trauth, K., Camper, S.A. and Friedman, T.B. (1998) Association of unconventional myosin MYO15 mutations with human nonsyndromic deafness DFNB3. Science, 280, 1447–1451.
19 Cook, S.A. and Davisson, M.T. (1993) Re-mutation to shaker-2 (sh-2). Mouse Genome, 91, 312.
20 Beyer, L.A., Odeh, H., Probst, F.J., Lambert, E.H., Dolan, D.F., Camper, S.A., Kohrman, D.C. and Raphael, Y. (2000) Hair cells in the inner ear of the pirouette and shaker 2 mutant mice. J. Neurocytol., 29, in press.
21 Xiang, M., Gao, W.Q., Hasson, T. and Shin, J.J. (1998) Requirement for Brn-3c in maturation and survival, but not in fate determination of inner ear hair cells. Development, 125, 3935–3946.[Abstract]
22 Avraham, K.B., Hasson, T., Steel, K.P., Kingsley, D.M., Russell, L.B., Mooseker, M.S., Copeland, N.G. and Jenkins, N.A. (1995) The mouse Snell’s waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells. Nature Genet., 11, 369–375.[ISI][Medline]
23 Gibson, F., Walsh, J., Mburu, P., Varela, A., Brown, K.A., Antonio, M., Beisel, K.W., Steel, K.P. and Brown, S.D. (1995) A type VII myosin encoded by the mouse deafness gene shaker-1. Nature, 374, 62–64.[Medline]
24 Keats, B.J. and Corey, D.P. (1999) The Usher syndromes. Am. J. Med. Genet., 89, 158–166.[ISI][Medline]
25 Friedman, T.B., Hinnant, J.T., Fridell, R.A., Wilcox, E.R., Raphael, Y. and Camper, S. (2000) DFNB3 families and shaker-2 mice: mutations in an uncoventional myosin, Myo15. Adv. Otorhinolaryngol., 56, 131–144.[Medline]
26 Ernstson, S., Lundquist, P.G., Wedenberg, E. and Wersall, J. (1969) Morphologic changes in vestibular hair cells in a strain of the waltzing guinea pig. Acta Otolaryngol. (Stockh.), 67, 521–534.[Medline]
27 Flock, A., Cheung, H. and Wersall, J. (1979) Pathological actin in vestibular hair cells of the waltzing guinea pig. Adv. Otorhinolaryngol., 25, 12–16.[Medline]
28 Zine, A. and Romand, R. (1996) Development of the auditory receptors of the rat: a SEM study. Brain Res., 721, 49–58.[ISI][Medline]
29 Anniko, M. (1983) Cytodifferentiation of cochlear hair cells. Am. J. Otolaryngol., 4, 375–388.[ISI][Medline]
30 Ressler, K.J., Sullivan, S.L. and Buck, L.B. (1994) Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell, 79, 1245–1255.[ISI][Medline]
31 Sassoon, D.A., Garner, I. and Buckingham, M. (1988) Transcripts of alpha-cardiac and alpha-skeletal actins are early markers for myogenesis in the mouse embryo. Development, 104, 155–164.[Abstract]
32 Theiler, K. (1989) The House Mouse: Atlas of Embryonic Development. Springer-Verlag, New York, NY.
33 Morsli, H., Choo, D., Ryan, A., Johnson, R. and Wu, D.K. (1998) Development of the mouse inner ear and origin of its sensory organs. J. Neurosci., 18, 3327–3335.
34 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
35 Raphael, Y. and Altschuler, R.A. (1991) Reorganization of cytoskeletal and junctional proteins during cochlear hair cell degeneration. Cell Motil. Cytoskeleton, 18, 215–227.[ISI][Medline]
36 Meiteles, L.Z. and Raphael, Y. (1994) Scar formation in the vestibular sensory epithelium after aminoglycoside toxicity. Hear. Res., 79, 26–38.[ISI][Medline]
37 Raphael, Y., Athey, B.D., Wang, Y., Lee, M.K. and Altschuler, R.A. (1994) F-actin, tubulin and spectrin in the organ of Corti: comparative distribution in different cell types and mammalian species. Hear. Res., 76, 173–187.[ISI][Medline]
38 Kurc, M., Dodane, V., Pinto, D.S. and Kachar, B. (1998) Presynaptic localization of G protein isoforms in the efferent nerve terminals of the mammalian cochlea. Hear. Res., 116, 1–9.[ISI][Medline]
39 Deol, M.S. (1954) The anomalies of the labyrinth of the mutants varitint-waddler, shaker-2 and jerker in the mouse. J. Genet., 52, 562–588.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Liu, S. Woolner, J. E. Johndrow, D. Metzger, A. Flores, and S. M. Parkhurst Sisyphus, the Drosophila myosin XV homolog, traffics within filopodia transporting key sensory and adhesion cargos Development, January 1, 2008; 135(1): 53 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Schneider, A. C. Dose, F. T. Salles, W. Chang, F. L. Erickson, B. Burnside, and B. Kachar A New Compartment at Stereocilia Tips Defined by Spatial and Temporal Patterns of Myosin IIIa Expression J. Neurosci., October 4, 2006; 26(40): 10243 - 10252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Lin, R. Cantos, M. Patente, and D. K. Wu Gbx2 is required for the morphogenesis of the mouse inner ear: a downstream candidate of hindbrain signaling Development, May 15, 2005; 132(10): 2309 - 2318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Rzadzinska, M. E. Schneider, C. Davies, G. P. Riordan, and B. Kachar An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal J. Cell Biol., March 15, 2004; 164(6): 887 - 897. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. A. Belyantseva, E. T. Boger, and T. B. Friedman Myosin XVa localizes to the tips of inner ear sensory cell stereocilia and is essential for staircase formation of the hair bundle PNAS, November 25, 2003; 100(24): 13958 - 13963. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. J. Karolyi, F. J. Probst, L. Beyer, H. Odeh, G. Dootz, K. B. Cha, D. M. Martin, K. B. Avraham, D. Kohrman, D. F. Dolan, et al. Myo15 function is distinct from Myo6, Myo7a and pirouette genes in development of cochlear stereocilia Hum. Mol. Genet., November 1, 2003; 12(21): 2797 - 2805. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Hurle, E. Ignatova, S. M. Massironi, T. Mashimo, X. Rios, I. Thalmann, R. Thalmann, and D. M. Ornitz Non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice caused by mutations in otopetrin 1 Hum. Mol. Genet., April 1, 2003; 12(7): 777 - 789. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ekelund, D. Lichtermann, I. Hovatta, P. Ellonen, J. Suvisaari, J. D. Terwilliger, H. Juvonen, T. Varilo, R. Arajarvi, M.-L. Kokko-Sahin, et al. Genome-wide scan for schizophrenia in the Finnish population: evidence for a locus on chromosome 7q22 Hum. Mol. Genet., April 12, 2000; 9(7): 1049 - 1057. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||