A missense mutation in the previously undescribed
gene Tmhs underlies deafness in hurry-scurry
Chantal M. Longo-Guess, Leona H. Gagnon, Susan A. Cook, Jian Wu*, Qing Y. Zheng, and Kenneth R. Johnson†
The Jackson Laboratory, Bar Harbor, ME 04609
Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 14, 2005 (received for review January 31, 2005)
Mouse deafness mutations provide valuable models of human
hearing disorders and entry points into molecular pathways im-
portant to the hearing process. A newly discovered mouse muta-
tion named hurry-scurry (hscy) causes deafness and vestibular
dysfunction. Scanning electron microscopy of cochleae from 8-day-
old mutants revealed disorganized hair bundles, and by 50 days of
age, many hair cells are missing. To positionally clone hscy, 1,160
F2 mice were produced from an intercross of (C57BL?6-hscy ?
CAST?EiJ) F1hybrids, and the mutation was localized to a 182-kb
region of chromosome 17. A missense mutation causing a critical
cysteine to phenylalanine codon change was discovered in a
previously undescribed gene within this candidate interval. The
gene is predicted to encode an integral membrane protein with
four transmembrane helices. A synthetic peptide designed from
the predicted protein was used to produce specific polyclonal
antibodies, and strong immunoreactivity was observed on hair
bundles of both inner and outer hair cells in cochleae of newborn
??? controls and ??hscy heterozygotes but was absent in hscy?
hscy mutants. Accordingly, the gene was given the name ‘‘tetra-
span membrane protein of hair cell stereocilia,’’ symbol Tmhs. Two
related proteins (>60% amino acid identity) are encoded by genes
on mouse chromosomes 5 and 6 and, together with the Tmhs-
encoded protein (TMHS), comprise a distinct tetraspan subfamily.
Our localization of TMHS to the apical membrane of inner ear hair
cells during the period of stereocilia formation suggests a function
in hair bundle morphogenesis.
mouse ? hair cell ? stereocilia ? tetraspan
than half of these childhood cases are thought to be genetic (1).
The development and maintenance of the intricate structures
and complex mechanisms of the mammalian inner ear require
the proper functioning and concerted interactions of hundreds
of genes and their products. To date, ?100 forms of human
nonsyndromic deafness have been genetically mapped (Hered-
itary Hearing Loss Homepage (http:??dnalab-www.uia.ac.be?
dnalab?hhh), and many of the genes responsible have been
identified and characterized (2). More than 200 mouse muta-
have been developed for ?50 human hearing disorders (Hearing
Impairment in Mice, www.jax.org?hmr?index.html). Because of
the complex nature of the ear, it is likely that many more
deafness-related genes remain to be discovered.
The inner ear is comprised of the vestibular region, which
controls balance, and the cochlea, which is important in detect-
ing, amplifying, and transmitting auditory information to the
brain. In the organ of Corti of the cochlea, two distinct types of
sensory cells, inner and outer hair cells, are essential for the
transduction of sound into nerve impulses. Stereocilia are mod-
ified microvilli that project from the apical membranes of inner
ear hair cells. The actin-filled stereocilia contain mechanically
gated ion channels that open or close in response to sound-
induced deflections and thus are crucial to the hearing process
earing loss is the most prevalent sensory disorder in human
populations, occurring in ?0.2–0.3% of all live births. More
(3). Many mouse mutations have been valuable in identifying
and characterizing genes that are important in the development
and maintenance of hair cells and their stereocilia, including
Myo7a, Myo6, Myo15, Cdh23, Pcdh15, Ush1c, Ush1g, Whrn,
Actg1, and Espn (4).
Here, we describe a mouse mutation in a gene that encodes a
protein we believe to be involved in the formation of hair cell
stereocilia. We named the spontaneous mutation hurry-scurry
(hscy) because of the characteristic rapid circling behavior of
homozygous mutant mice. Mutant mice are also congenitally
deaf. Using a positional cloning approach, we mapped hscy to
chromosome (Chr) 17 and identified the underlying gene, which
is predicted to encode an integral membrane protein with four
stereocilia,’’ gene symbol Tmhs. Its spatial and temporal expres-
sion pattern indicates a likely role in hair bundle morphogenesis.
Materials and Methods
Mice. The hscy mutation arose spontaneously at The Jackson
Laboratory in a B6.MOR-Gusbaline. Mutants were crossed to
C57BL?6 mice for three generations followed by sibling matings
to maintain the line. All mice were obtained from the Mouse
Mutant Resource at The Jackson Laboratory, and all procedures
involving their use were approved by the Institutional Animal
Care and Use Committee.
Genetic Mapping. A pooled DNA strategy using microsatellite
markers (5) was used to initially localize the mutation to Chr 17.
DNAs from individual mice then were typed to refine the map
position with the aid of the MAP MANAGER computer program
(6). PCR conditions for typing microsatellite markers were as
described (7). Mutant mice (hscy?hscy) from the linkage cross
were easily identified by their overt circling and head-shaking
behavior. To distinguish ??? and ??hscy genotypes of nonmu-
tant recombinant mice, progeny tests with hscy?hscy mice were
Auditory-Evoked Brainstem Response (ABR). Hearing in mice was
assessed by ABR thresholds as described (8).
Histopathology and Scanning Electron Microscopy (SEM). Cross sec-
tions of the inner ear were obtained in the following manner.
Inner ears were dissected out of the skull, decalcified in Bouin’s
for ?2 weeks, and embedded in paraffin. Tissue sections were
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ABR, auditory-evoked brainstem response; SEM, scanning electron micros-
copy; En, embryonic day n; Pn, postnatal day n.
*Present address: Veterans Affairs Medical Center, University of Tennessee Health Science
Center, Memphis, TN 38104.
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
May 31, 2005 ?
vol. 102 ?
no. 22 www.pnas.org?cgi?doi?10.1073?pnas.0500760102
cut 4 ?m thick and stained with hematoxylin?eosin. Tissues for
SEM analysis were dissected and fixed in 2.5% glutaraldehyde in
0.1 M phosphate buffer (pH 7.2) for 3–4 h at 4°C followed by
several washes in 0.1 M phosphate buffer. Bone and stria
surrounding the cochlea were dissected away and the tectorial
membrane removed to expose the organ of Corti. Tissues were
processed in 2% osmium tetroxide, dehydrated, and dried. The
organ of Corti was sputter-coated with gold and examined at 15
kV under a Hitachi (Tokyo) 3000N scanning electron micro-
scope. For SEM analysis, the following numbers of mice of each
genotype and developmental stage were examined: hscy?hscy
[two postnatal day (P)0, one P8, three P15, one P50), ??hscy
(two P0, one P8, one P15), and ??? (two P15, one P50)].
Genomic DNA and RNA Isolation and cDNA Synthesis.GenomicDNA
for PCR was prepared from mouse tail tips using the Hot Shot
method (9). Total RNA from inner ear, whole brain, and kidney
tissues was isolated with TRIzol reagent following the manu-
facturer’s protocol (Invitrogen). Poly(A)?mRNA for Northern
blot analysis was isolated by using the PolyATract mRNA
Isolation System (Promega). Mouse cDNA was synthesized by
using SuperScript II reverse transcriptase according to the
manufacturer’s instructions (Invitrogen).
Northern Blot Hybridization. Northern blots were prepared and
hybridized as described (10). Commercially prepared Northern
blots from adult mouse tissues and mouse embryos (MTN blots,
to nucleotides 22–875 of the XM?283418 cDNA sequence.
Production of Antibodies and Immunohistochemistry. A synthetic
16-aa peptide corresponding to the C-terminal end of the
predicted mouse Tmhs-encoded protein (TMHS) was injected
into rabbits, and high-titer antipeptide antiserum was collected
and affinity purified by a commercial vendor (Alpha Diagnos-
tics, San Antonio, TX). For immunofluorescence, tissues were
dissected and fixed overnight in 4% paraformaldehyde, embed-
ded in paraffin, and cut 4–6 ?m. Embryonic stages were
determined by checking vaginal plugs. Noon of the day the
vaginal plugs were detected was considered embryonic day
(E)0.5. Inner ears from mice older than P5 were decalcified in
7% EDTA?PBS for 1 week before embedding. Tissues were
treated with 0.1% trypsin for 15 min at 37°C. After several PBS
washes, the tissue sections were incubated overnight at 4°C with
the anti-TMHS antibody (1:50) or anti-Myosin VIIa (1:500)
(Affinity BioReagents, Golden, CO). Slides incubated without
primary antibody were used as negative controls. Primary anti-
bodies were detected with goat anti-rabbit Alexa Fluor 488
(1:500) (Molecular Probes). Images were visualized by using a
Leitz (Wetzlar, Germany) DMRXE microscope and a Leica
The following numbers of mice of each genotype and devel-
opmental stage were examined by immunohistochemistry with
the anti-TMHS antibody: hscy?hscy (one E14.5, one E15.5, one
E16.5, one E17.5, two P0, one P9, one P30, and one P60), ??hscy
(one E14.5, one E17.5, one P0, one P9, one P30, and one P60),
and ??? (one E14.5, one E15.5, one E16.5, one E17.5, and
DNA Sequencing and Mutation Genotyping.Primersandsequencing
methods are described in Supporting Text, which is published as
supporting information on the PNAS web site.
The hscy Phenotype. The overt phenotype of hscy homozygotes
consists of circling behavior, frequent head shaking from side to
side, and an inability to swim. The hearing ability of both mutant
and control mice was measured by using ABR thresholds. We
tested 15 mutant and 9 control mice between 26 and 131 days of
age. None of the hscy?hscy mutant mice tested showed any
response to auditory stimuli up to 110 dB sound pressure level.
The ABR thresholds of control mice were in the range of normal
hearing. The deafness and circling behavior are recessive and
fully penetrant in mice on the C57BL?6 background and also in
F2hybrid linkage cross mice.
Examination of cochlear cross sections revealed severe de-
generation of the organ of Corti in mutant but not control mice
decreased spiral ganglia (Fig. 1A). Degeneration was more
pronounced in the basal region of the cochlea than in the apex.
Hair cell morphology was investigated further by SEM. At P0,
mutant hair cells appear normal but begin to degenerate by P8
(Fig. 1B). At P8, stereocilia of both outer and inner hair cells in
mutants are disorganized compared with those of controls. The
outer hair cells have lost their rigid V-shaped pattern, and the
inner hair cells have a more splayed appearance than those of
controls. By P50, there are patches in the basal portion of the
mutant cochlea in which the outer hair cells have completely
degenerated, and inner hair cell stereocilia are severely splayed.
sections through basal turns of cochleae from control and mutant mice at 4
months of age. Mutant cochleae exhibit severe degeneration of the organ of
Corti (OC) and secondary degeneration of spiral ganglion cells (SG). (B) SEM
mice at postnatal ages P0 and P8, and from mutant mice at P15 and P50. In P8
mutants, hair bundle disorganization and some hair cell loss is apparent; by
P50, few stereocilia remain, and most outer hair cells have degenerated in
basal portions of the cochlea.
Cochlear pathology associated with the hscy mutation. (A) Cross
Longo-Guess et al. PNAS ?
May 31, 2005 ?
vol. 102 ?
no. 22 ?
Positional Cloning and Mutant Gene Identification. To fine map the
hscy mutation, we produced 1,160 F2mice from an intercross
between (C57BL?6-hscy ? CAST?Ei) F1 hybrid mice. All F2
progeny produced from the linkage cross were genotyped at
weaning for flanking markers, and only mice with informative
recombinant chromosomes were further analyzed with addi-
tional markers. Crossover analysis with known markers limited
the candidate interval to a 289-kb region between the genes
Fkbp5 and Mapk14 (Fig. 2A). Using the available mouse genome
sequence for the 289-kb candidate region, we designed custom
primers to generate seven additional genomic CA repeat mark-
ers, which further narrowed the genetic interval to 182 kb (Fig.
2B). The narrowed region contained only two known genes, Clps
and Srpk1, and two uncharacterized genes, LOC67645 and
LOC328789. Of these four genes, we considered LOC328789 the
most likely candidate because of its expression primarily in
neural tissue, as represented by two full-length cDNAs,
AK020389 (from diencephalon) and AK020670 (from neonate
cerebellum), as well as 15 neurospecific ESTs (Unigene
To evaluate the LOC328789 gene, we designed PCR primers
to amplify overlapping regions of the 1,237-bp National Center
for Biotechnology Information reference cDNA sequence
XM?283418. We detected a substitution from G to T in DNA
from hscy?hscy mutant mice at nucleotide 482 of the protein-
coding portion of the cDNA (where nucleotide ? 1 is the A of
the ATG initiation codon). The mutation in cDNA is hence
in the cDNA of mutant mice. To verify the mutation detected in
cDNA, we sequenced PCR fragments obtained from genomic
DNA of three additional mutants as well as two additional
C57BL?6J animals and a mouse of the genetically divergent
To confirm the correspondence of Tmhs genotypes with hscy
phenotypes, we sequenced DNA from 99 mice produced from
intercrosses of ??hscy heterozygotes and identified 22 with ???
genotypes, 56 with ??hscy genotypes, and 21 with hscy?hscy
genotypes. (DNA chromatographs are shown in Fig. 6, which is
published as supporting information on the PNAS web site.)
Mice with two copies of the c.482G?T mutation (hscy?hscy)
always had a mutant phenotype, whereas mice with two copies
of the G nucleotide (???) and mice with both G and T (??hscy)
always had normal phenotypes. The symbol Tmhs has been
assigned to designate the previously unnamed and uncharacter-
ized mouse gene (LOC328789) that underlies the hscy mutation.
Molecular Characterization of the Tmhs Gene and hscy Mutation.
Comparison of the mouse cDNA sequence (XM?283418) with
genomic DNA sequence (NT?039649) revealed that the Tmhs
gene spans ?7.9 kb and is composed of four exons (Fig. 2C). The
protein coding sequence of XM?283418 (nucleotides 285–944)
occurs in exon 2 of Tmhs, which corresponds to nucleotide 766
of XM?283418. Exon 4 includes a B1 sine repeat (nucleotides
1058–1164 of XM?283418) that includes a potential polyA signal
(ATTAAA). This signal appears to have been used to form most
of the mRNAs and ESTs comprising UniGene Mm.284760 (such
as AK020670, AW492878, BB125404, BB132044, BB258096,
BB250980, and others), but a few transcripts apparently recog-
nize a more 3? signal (such as AK020389 and BB196812). The
FIRSTEF computer program (11) analysis of genomic DNA
strongly predicts a CpG-related promoter sequence and a first
exon 5? boundary near the 5? end of the XM?283418 sequence,
to be within 100 bp of the 5? end of this cDNA.
The G to T transversion causes a nonconservative amino acid
change from cysteine to phenylalanine at amino acid position
161 (codon UGU to UUU). The amino acid mutation is thus
designated C161F. The Tmhs gene is predicted by the TMHMM
computer program (12) to encode a protein with four trans-
membrane helices and two extracellular loops, as shown sche-
matically in Fig. 2D. The C161F mutation occurs in the second
extracellular loop. Cysteine is a weakly polar hydrophilic amino
acid with a thiol side chain, whereas phenylalanine is a nonpolar
highly hydrophobic amino acid with an aromatic side chain. The
large difference between these two amino acids would likely
cause the mutant protein to be targeted for degradation early in
development, consistent with the absence of protein expression
that we observed in the mutant inner ear. Thiol side chains are
essential for the formation of disulfide bonds between cysteine
residues, which help to stabilize the tertiary structure of the
protein. The cysteine mutated in hscy mice (C161F) is highly
custom-designed markers (arrowheads) enabled further refinement of the candidate interval to an ?182-kb region containing two known and two
uncharacterized genes. (C) The uncharacterized gene (LOC328789) identified by the XM?283418 cDNA sequence is predicted to have four exons (shown as
rectangles connected by lines representing introns); a G?T transversion was found in exon 2, within the protein coding sequence of this gene (black regions of
exons). (D) Schematic representation of the predicted protein structure showing the four transmembrane domains and the two cysteines within each
extracellular loop. The Cys?Phe change caused by the hscy mutation is indicated.
www.pnas.org?cgi?doi?10.1073?pnas.0500760102 Longo-Guess et al.
conserved in the orthologous proteins of other species, including
rat, human, chicken, pufferfish, Drosophila, and Caenorhabditis
elegans (Fig. 7, which is published as supporting information on
the PNAS web site).
Tmhs Gene and Protein Expression. Gene expression was examined
at the RNA level by performing Northern blot analysis and
RT-PCR. A multiple tissue Northern blot from a commercial
vendor (Clontech, BD Biosciences) consisting of poly(A)?RNA
testes of adult mice was hybridized with a Tmhs DNA probe. Of
the eight tissues analyzed, a transcript was detected only in brain
RNA (Fig. 3A), consistent with the neurospecific sources of
database ESTs. The estimated size of the transcript (1.3 kb)
agrees with the length of the XM?283418 cDNA sequence. A
Northern blot of poly(A)?RNA extracted from brains of ???
control mice and hscy?hscy mutant mice and hybridized with the
same probe revealed no differences in transcript levels between
mutant and control mice (Fig. 3B). This result was anticipated,
not likely disrupt transcription.
We used RT-PCR to examine Tmhs gene expression in the
inner ear. Total RNA was extracted from the entire inner ear
(cochlea ? vestibule), cochlea, vestibule, brain, and kidney of an
adult ??? control mouse and used to make cDNA. Tmhs-
specific PCR primers amplified the expected 480-bp product
from each of the tissue-specific cDNAs, although a lower level
of expression was seen in the kidney (Fig. 3C). Although not
quantitative, these results verify inner ear expression of the gene.
To examine the localization of the protein in the brain and
inner ear, we used peptide antisera specific to the carboxyl
terminal end of the predicted protein. We examined sagittal
sections of brains from adult and newborn mice but did not
detect any TMHS-specific immunofluorescence that was signif-
icantly above background (data not shown). In the inner ear,
however, intense fluorescence clearly indicated a concentrated
presence of TMHS on stereocilia of both inner and outer hair
cells in newborn ??? and ??hscy mice (Fig. 4 A and C). We saw
no TMHS expression in cochlear hair cells of P0 hscy?hscy
mutants (Fig. 4 B and D), confirming the specificity of the
antibody and the severe consequence of the hscy missense
mutation. The same TMHS expression pattern observed in P0
mice was seen in the inner ears of wild-type but not mutant mice
at E16.5 and E17.5. We did not detect TMHS immunofluores-
cence in the inner ears of wild-type or mutant embryos or
postnatal mice when examined at E14.5, E15.5, P9, P30, and P60.
To verify that hair cells were intact in newborn hscy mutant
mice, we examined the expression of myosin VIIa with a
commercially available antibody. Myosin VIIa is expressed
throughout the cell body in both inner and outer hair cells (13),
and its expression appeared normal in cochleae of P0 hscy?hscy
mice (Fig. 8, which is published as supporting information on the
PNAS web site).
Because hscy?hscy mice exhibit circling and head-tossing
behaviors characteristic of vestibular dysfunction, we also exam-
ined TMHS expression in the vestibular neuroepithelia of the
inner ear. As in the cochlea, TMHS immunofluorescence was
pronounced in the stereocilia of hair cells in the vestibular
maculae and cristae of P0 wild-type mice (Fig. 9, which is
published as supporting information on the PNAS web site).
Related Sequences. A search of the Pfam, InterPro, Prosite,
CDART, and Smart protein databases with the TMHS amino
acid sequence resulted in no matches to any known protein
domains. An Ensembl BLAST search of the public mouse genome
database with the TMHS amino acid sequence revealed two
closely related proteins (62–66% amino acid identity) that are
encoded by genes located on mouse Chrs 5 and 6. These two
related proteins were provisionally given the symbols LHFPL3
and LHFPL4 by database curators because of their ?25% amino
acid sequence similarity to the lipoma HMGIC fusion partner
protein, LHFP (14). We used the CLUSTALW computer program
(15) to create a phylogram of amino acid sequence similarities to
illustrate the orthologous relationships of the mouse and human
genes encoding TMHS and LHFP-like proteins (Fig. 5A). The
similarities of TMHS, LHFPL3, and LHFPL4 with one another
(62–71% amino acid identities) are much higher than their
similarities with LHFP, LHFPL1, or LHFPL2 (21–26% amino
The Tmhs gene is predicted to encode a four transmembrane
from multiple adult mouse tissues (H, heart; B, brain; S, spleen; Lu, lung; Li,
probe. (B) Northern blot of polyA? RNA extracted from brains of adult
cDNA probe (Upper) and subsequently with a ? actin control probe (Actb,
from total RNA extracted from adult C57BL?6J tissues (I, inner ear; C, cochlea;
(corresponding to exon 2 sequence) and 1212R (corresponding to exon 4
sequence) produced ?480 bp of product from cDNA but failed to amplify a
product from genomic DNA.
Tmhs gene expression. (A) Commercial Northern blot of polyA? RNA
a hscy?hscy mutant (B) showing hair cell-specific localization. (Bar, 50 ?m.) (C
and D) Higher magnification of boxed regions from A and B. (Bar, 10 ?m.)
Intense immunofluorescence was observed on stereocilia (indicated by 2) of
both inner hair cells (IHC) and outer hair cells (OHC) in the ??hscy heterozy-
gote (C) but not in the hscy?hscy mutant (D).
Subcellular localization of TMHS protein. (A and B) Cross sections
Longo-Guess et al. PNAS ?
May 31, 2005 ?
vol. 102 ?
no. 22 ?
domain protein, making it a member of the large tetraspan
superfamily. This family includes the claudin tight junction
proteins, the connexin gap junction proteins, clarins, proteolipid
proteins, peripheral myelin, and epithelial membrane proteins,
calcium channel ?-subunit-like proteins, and members of the
tetraspanin family. We used CLUSTALW multiple sequence align-
ments to compare the mouse TMHS protein sequence with
representative members of these other tetraspan families in the
mouse and with related sequences in other species (Fig. 6B).
TMHS, LHFPL3, and LHFPL4 form a separate subfamily that
is clearly distinct from all other tetraspan proteins.
We present several lines of evidence that support Tmhs as the
gene underlying the deafness and balance dysfunction of hscy
mutant mice: (i) The hscy candidate gene region is genetically
limited to only 182 kb, which includes the Tmhs gene. (ii) Tmhs
genotypes (determined by DNA sequence analysis of the mu-
tated 482G?T region) agree with hscy phenotypes in all ?100
mice examined. (iii) The coisogenic nature of the hscy mutation
(its spontaneous occurrence in a genetically homogeneous in-
bred strain) eliminates the possibility that the Tmhs 482G?T
missense mutation is a population polymorphism. (iv) The
cysteine residue of TMHS that is altered in hscy mutant mice is
highly conserved in the orthologous proteins of other species
from rats to C. elegans and is predicted to be essential for proper
protein structure and function. (v) The absence of the TMHS
protein in the inner ears of hscy?hscy mice demonstrates a
causative connection between the hscy phenotype and the Tmhs
The human ortholog of the mouse Tmhs gene (GeneID
222662), which we designate TMHS, is located on chromosome
6p21.3. The hscy mutation of Tmhs thus may provide a model for
human hearing disorders that map to this region, such as the
dominant nonsyndromic disorders DFNA21 (16) or DFNA31
(17). According to the genetic and physical map positions of
flanking markers (17), the TMHS gene is outside of the candi-
possibility of mapping errors, it may still be worthy of consid-
a recessive nonsyndromic hearing disorder, DFNB53, has been
mapped to the 6p21.3 region (Wenjie Chen, Hereditary Hearing
Loss Homepage, http:??webhost.ua.ac.be?hhh), and TMHS
might also be considered a candidate gene for this newly mapped
In mammals, TMHS, LHFPL3, and LHFPL4 comprise a
distinct subfamily within the large superfamily of tetraspan
proteins (Fig. 5). In insects and worms, there appears to be a
single orthologous protein that corresponds to the three-
ESTs found in the UniGene databases indicate that all three
as brain, spinal cord, and retina. Although the majority of
expression database entries for Tmhs are from brain tissue, and
our Northern blot results detected expression in the brain (Fig.
primary site of TMHS function. By immunohistochemistry with
specifically in stereocilia of inner ear hair cells but not in brain
between gene and protein expression in the brain is that the
TMHS protein may be distributed more diffusely among cells of
in hair cell stereocilia. Alternatively, Tmhs mRNA in brain may
not be translated, or the protein may be unstable because of
improper modification or localization.
Mutations of tetraspan proteins have been shown to underlie
both human and mouse deafness disorders. Mutations of the gap
junction proteins GJA1 (18), GJB2 (19), GJB3 (20), and GJB6
(21) are responsible for several human nonsyndromic deafness
disorders, including DFNB1 and DFNA3. Mutations of the tight
junction protein CLDN14 underlie DFNB29 (22), and mutations
of clarin-1 underlie Usher syndrome type 3A (23). Targeted
mutations of Cldn11 (24), Cldn14 (25), Gjb2 (26), and Gjb6 (27)
cause hearing impairment in mice. The gap junction and tight
junction tetraspan proteins that underlie hearing disorders are
crucial for maintaining proper ion concentrations in the various
subcompartments of the inner ear (2). Within the mammalian
are found in supporting cells and in fibroblasts of the spiral
ligament and limbus (18, 28, 29), the tight junction protein
CLDN11 is expressed in the basal cell layer of the stria vascularis
(24), and CLDN14 is expressed in hair cells and supporting cells
(22). Clarin-1, the tetraspan protein underlying USH3A, is
expressed only in hair cells and is thought to play a role in the
formation or structure of the hair cell synapse (23). Although
CLDN14 and clarin-1 are expressed in hair cells, they are
ments. (A) Phylogram of orthologous and paralogous relationships of mouse
and human TMHS and LHFP-like proteins. GenBank reference nos. for these
proteins and the chromosome positions of their respective genes are also
shown. Numbers shown were calculated from pairwise distance scores. (B)
Unrooted tree of tetraspan protein relationships. Representative members of
all tetraspan subgroups are included. Only mouse sequences were used for
these comparisons, except for Anopheles, Drosophila, and C. elegans.
Protein relationships analyzed by CLUSTALW multiple sequence align-
www.pnas.org?cgi?doi?10.1073?pnas.0500760102 Longo-Guess et al.
localized to the basolateral membranes of these cells. TMHS is
the only tetraspan protein known to localize to the apical
membrane and stereocilia of hair cells, although other tetraspan
proteins have been localized to the apical membranes of polar-
ized epithelial cells in other tissues (30, 31).
The period of peak TMHS expression corresponds with the
developmental period of stereocilia formation in inner ear hair
cells of the mouse, between E15 and P8. These temporal and
spatial patterns of Tmhs expression coupled with the early onset
of stereocilia disorganization observed in hscy?hscy mutant mice
indicate a likely role for this gene in hair bundle morphogenesis.
Cdh23 (32, 33) and Pcdh15 (34, 35) encode cadherin proteins
thought to be involved in hair bundle morphogenesis and whose
dysfunction results in stereocilia phenotypes similar to Tmhs
mutants. Cadherins are transmembrane proteins of adherens
junctions that mediate cell–cell adhesion and connections to the
cytoskeleton. Some tetraspan proteins recently have been shown
to localize to adherens junctions and are thought to play a role
in regulating cellular adhesion and epidermal morphology
through F-actin attachments (36, 37). In a like manner, it is
possible that TMHS may associate specifically with inner ear
cadherins and help direct development and morphogenesis of
the hair bundle.
contributions: Amy Kiernan and Verity Letts for critical review of this
manuscript, Heping Yu for ABR technical assistance, Peter Finger for
EM imaging assistance, Priscilla Jewett for histology assistance, and
Kenneth Bosom and Sandra Gray for mouse colony management. We
also thank Dawn Young for the initial discovery of the mutant mice and
Gordon Watson (Children’s Hospital Research Center, Oakland, CA)
for providing them to us. This work was supported by National Institutes
of Health (NIH) Grants DC04301 and RR01183. The Jackson Labora-
tory institutional shared services are supported by NIH Grant CA34196.
1. Morton, N. E. (1991) Ann. N.Y. Acad. Sci. 630, 16–31.
2. Friedman, T. B. & Griffith, A. J. (2003) Annu. Rev. Genomics Hum. Genet. 4,
3. Muller, U. & Littlewood-Evans, A. (2001) Trends Cell Biol. 11, 334–342.
Rev. Genet. 5, 489–498.
5. Taylor, B. A., Navin, A. & Phillips, S. J. (1994) Genomics 21, 626–632.
6. Manly, K. F., Cudmore, R. H., Jr., & Meer, J. M. (2001) Mamm. Genome 12,
7. Johnson, K. R., Gagnon, L. H., Webb, L. S., Peters, L. L., Hawes, N. L., Chang,
B. & Zheng, Q. Y. (2003) Hum. Mol. Genet. 12, 3075–3086.
8. Zheng, Q. Y., Johnson, K. R. & Erway, L. C. (1999) Hear. Res. 130, 94–107.
M. L. (2000) BioTechniques 29, 52, 54.
10. Johnson, K. R., Cook, S. A., Erway, L. C., Matthews, A. N., Sanford, L. P.,
Paradies, N. E. & Friedman, R. A. (1999) Hum. Mol. Genet. 8, 645–653.
11. Davuluri, R. V., Grosse, I. & Zhang, M. Q. (2001) Nat. Genet. 29, 412–417.
12. Sonnhammer, E. L., von Heijne, G. & Krogh, A. (1998) Proc. Int. Conf. Intell.
Syst. Mol. Biol. 6, 175–182.
13. Self, T., Mahony, M., Fleming, J., Walsh, J., Brown, S. D. & Steel, K. P. (1998)
Development (Cambridge, U.K.) 125, 557–566.
14. Petit, M. M., Schoenmakers, E. F., Huysmans, C., Geurts, J. M., Mandahl, N.
& Van de Ven, W. J. (1999) Genomics 57, 438–441.
15. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G.
& Thompson, J. D. (2003) Nucleic Acids Res. 31, 3497–3500.
16. Kunst, H., Marres, H., Huygen, P., van Duijnhoven, G., Krebsova, A., van der
Velde, S., Reis, A., Cremers, F. & Cremers, C. (2000) Clin. Otolaryngol. 25,
17. Snoeckx, R. L., Kremer, H., Ensink, R. J., Flothmann, K., de Brouwer, A.,
Smith, R. J., Cremers, C. W. & Van Camp, G. (2004) J. Med. Genet. 41, 11–13.
18. Liu, X. Z., Xia, X. J., Adams, J., Chen, Z. Y., Welch, K. O., Tekin, M., Ouyang,
X. M., Kristiansen, A., Pandya, A., Balkany, T., et al. (2001) Hum. Mol. Genet.
19. Kelsell, D. P., Dunlop, J., Stevens, H. P., Lench, N. J., Liang, J. N., Parry, G.,
Mueller, R. F. & Leigh, I. M. (1997) Nature 387, 80–83.
20. Xia, J. H., Liu, C. Y., Tang, B. S., Pan, Q., Huang, L., Dai, H. P., Zhang, B. R.,
Xie, W., Hu, D. X., Zheng, D., et al. (1998) Nat. Genet. 20, 370–373.
21. Grifa, A., Wagner, C. A., D’Ambrosio, L., Melchionda, S., Bernardi, F.,
Lopez-Bigas, N., Rabionet, R., Arbones, M., Monica, M. D., Estivill, X., et al.
(1999) Nat. Genet. 23, 16–18.
22. Wilcox, E. R., Burton, Q. L., Naz, S., Riazuddin, S., Smith, T. N., Ploplis, B.,
23. Adato, A., Vreugde, S., Joensuu, T., Avidan, N., Hamalainen, R., Belenkiy, O.,
Olender, T., Bonne-Tamir, B., Ben-Asher, E., Espinos, C., et al. (2002) Eur. J.
Hum. Genet. 10, 339–350.
24. Gow, A., Davies, C., Southwood, C. M., Frolenkov, G., Chrustowski, M., Ng,
25. Ben-Yosef, T., Belyantseva, I. A., Saunders, T. L., Hughes, E. D., Kawamoto,
K., Van Itallie, C. M., Beyer, L. A., Halsey, K., Gardner, D. J., Wilcox, E. R.,
et al. (2003) Hum. Mol. Genet. 12, 2049–2061.
26. Cohen-Salmon, M., Ott, T., Michel, V., Hardelin, J. P., Perfettini, I., Eybalin,
M., Wu, T., Marcus, D. C., Wangemann, P., Willecke, K., et al. (2002) Curr.
Biol. 12, 1106–1111.
27. Teubner, B., Michel, V., Pesch, J., Lautermann, J., Cohen-Salmon, M., Sohl,
G., Jahnke, K., Winterhager, E., Herberhold, C., Hardelin, J. P., et al. (2003)
Hum. Mol. Genet. 12, 13–21.
28. Lopez-Bigas, N., Olive, M., Rabionet, R., Ben-David, O., Martinez-Matos,
J. A., Bravo, O., Banchs, I., Volpini, V., Gasparini, P., Avraham, K. B., et al.
(2001) Hum. Mol. Genet. 10, 947–952.
29. Lautermann, J., ten Cate, W. J., Altenhoff, P., Grummer, R., Traub, O., Frank,
H., Jahnke, K. & Winterhager, E. (1998) Cell Tissue Res. 294, 415–420.
30. Bosse, F., Hasse, B., Pippirs, U., Greiner-Petter, R. & Muller, H. W. (2003)
J. Neurochem. 86, 508–518.
31. Frank, M., van der Haar, M. E., Schaeren-Wiemers, N. & Schwab, M. E. (1998)
J. Neurosci. 18, 4901–4913.
32. Siemens, J., Lillo, C., Dumont, R. A., Reynolds, A., Williams, D. S., Gillespie,
P. G. & Muller, U. (2004) Nature 428, 950–955.
33. Di Palma, F., Holme, R. H., Bryda, E. C., Belyantseva, I. A., Pellegrino, R.,
Kachar, B., Steel, K. P. & Noben-Trauth, K. (2001) Nat. Genet. 27, 103–107.
34. Alagramam, K. N., Murcia, C. L., Kwon, H. Y., Pawlowski, K. S., Wright, C. G.
& Woychik, R. P. (2001) Nat. Genet. 27, 99–102.
35. Ahmed, Z. M., Riazuddin, S., Ahmad, J., Bernstein, S. L., Guo, Y., Sabir, M. F.,
Mol. Genet. 12, 3215–3223.
Nat. Cell Biol. 5, 619–625.
37. Kearsey, J., Petit, S., De Oliveira, C. & Schweighoffer, F. (2004) Eur.
J. Biochem. 271, 2584–2592.
Longo-Guess et al. PNAS ?
May 31, 2005 ?
vol. 102 ?
no. 22 ?