specific staining was obtained with CLRN1-B2 antibody in human, monkey, rabbit, cat, and rat retinas (data not shown). These data suggest that CLRN1 might be expressed at very low levels in the retina. Similarly, other published data using CLRN1 antibodies that work well on western blots and on expressed CLRN1 protein in vitro do not provide specific localization of the CLRN1 protein on retinal tissue sections by immunocytochemistry [23]. Taken together, these data suggest that antibody access to protein epitopes exposed on the short extracellular and intracellular loops of CLRN1 may be limited. It is known that tetraspanins protrude only 4–5 nm from the plasma membrane [61,62]. Additionally, tetraspanins are known to function as organizers of multi- molecular membrane complexes [56], similar to USH protein complexes [3,16,57,58]. One explanation for the lack of results by immunocytochemistry on intact tissue (using the antibodies that do stain cultured cells) may be that additional proteins simply block access of antibodies to the CLRN1 epitopes. Clrn1 Knockout Retinas Appear Normal and Cochleas Degenerate It has been shown that in humans, CLRN1 mutation results in a phenotype consistent with rod photoreceptor death and RP [10,59]. However, in mice, histological (Figure 6) and immuno- histochemical (Figure 7) analyses in WT (+/+) and Clrn1 KO (2/ 2) animals revealed no adverse consequences of Clrn1 gene deletion. Moreover, ERG analysis of the aged Clrn1 KO mouse failed to demonstrate either photoreceptor (A-wave) or inner retinal (B-wave) dysfunction (Figure S6) even at 2 years of age. Staining of second and third order neurons (Figure 7A–7H) and the matrix sheaths around cone photoreceptors (Figure 7I and 7J) suggests that these cells are similarly unaffected by Clrn1 deletion. Thus, structural and functional analyses of Clrn1 KO retinas reveal that their retinas and their vision is normal. In contrast, yet similar to other mouse USH KO mouse models, the lack of CLRN1 dramatically affected cochlear structure and function (Figure 8 and Figure S7, respectively). Using ABR in WT C57BL/6J animals as a measure of hearing function, and similar to other data from the Clrn1 KO mouse [22], we found Clrn1 KO mice to be essentially deaf as early as postnatal P18 (Figure S7). Whole mount immunostaining of WT (+/+) and Clrn1 KO (2/2) cochlea show a severe disruption in the stereocilia of 4 month old animals (Figure 8), with some early signs of pathology as early as P15. Thus, in the Clrn1 KO mouse we have established another model of USH where deafness is apparent, but blindness is absent. The mechanism underlying the cochlear degeneration remains an open question, and in fact, the role of clarin 1 in the inner ear is still a mystery. Based on homology to Stargazin and its proposed four transmembrane protein structure, CLRN1 was suggested previously to have a role in the excitatory ribbon synapse junctions between hair cells and cochlear ganglion cells [12]. It is interesting to note that Clrn1 deletion results in a broader distribution of longer stereocilia in cochlear IHCs. While degeneration of hair cells and supporting cells is a common phenomenon in hearing impaired mouse mutants, elongation of stereocilia is usually specific to defects in proteins involved in actin polymerization. The elongated stereocilia present in the Clrn1 mutant mice suggest a similar role. Stereocilia contain a rigid paracrystalline array of parallel and polarized cross-linked actin filaments, whose length is tightly regulated, with continuous actin turnover [63]. Many factors influence the structure of the actin core of stereocilia, including for example, myosin activity and membrane tension. Defects in actin polymerization and/or depolymerization may be caused by a mutation in any one of the factors that this dynamic process is dependent on, including any of actin’s known Clarin 1 Is Necessary for Hearing but Not Vision interacting proteins. For example, myosin VIIa is involved in the dynamic regulation of elongation of stereocilia, as demonstrated by stereociliar defects in Myo7a mutant mice [64,65]. Other genes with mutations leading to Usher syndrome encode proteins that are expressed and are functional in the stereocilia. For example, whirlin, associated with mutations in USH2A [18], is expressed in the stereocilia and indeed Whrn mouse mutants have shorter stereocilia [66]. Precise localization of clarin1 in the cochlear hair cells and further identification of its interacting proteins [23] will be vital to elucidating the cause for the stereociliar pathology. Our data suggests a new, previously unexplored function for clarin1 in hair cells. Conclusions Similar to most other USH mouse models, our Clrn1 KO animal exhibits a non-syndromic phenotype. In contrast to our original hypothesis, Clrn1 is not expressed in photoreceptor cells; but rather, Clrn1 transcripts are produced by M u¨ller cells, the retinas primary glial cell. These data cannot exclude the possibility of another developmental phase of Clrn1 expression or that Clrn1’s expression is absolutely restricted to Mu¨ ller glia. Additional studies will be required to ascertain how CLRN1 may be participating in the unique relationship between Mu¨ ller and photoreceptor cells. Photoreceptors in the Clrn1 knockout mice remained anatom- ically and physiologically normal, without exhibiting any pheno- typic characteristics of retinal degeneration. In contrast, the Clrn1 KO mouse is physiologically deaf within 2–3 weeks of birth, although IHCs and OHCs appear largely anatomically intact at this time point. These data suggest that loss of hearing function precedes cell loss in the cochlea: within several months of birth, the absence of CLRN1 protein(s) cause damage evidenced by progressive morphological disruption, proceeding to eventual complete loss of the primary sensory hair cells. Taken together, these data support the notion that an Usher protein complex [3] is required for inner ear structure and function, but dispensable for retinal structure and function in mice. Moreover, the presence of abnormal hair cell stereocilia in the Clrn1 KO mouse suggests that CLRN1 may function at the apical part of the cell in addition to (or in place of) serving a role in synapse structure and function, as previously predicted [12]. Our data also suggest that the cellular site of the defect in human sensory tissues must be ascertained to design a rational therapy for these debilitating conditions, and early diagnosis and intervention will be critical to the success of human therapies for USH3-related hearing and vision losses. Materials and Methods Knockout Mouse Production A Clrn1 knockout mouse was generated by IngenKO, Pty. Ltd. (Clayton, Victoria, Australia). Briefly, the targeting construct was produced using the ET cloning system [67] with C57BL/6J genomic DNA. The construct included loxP sites that flanked part of the Clrn1 upstream promoter, the first exon (59 UTR and protein coding sequence), 269 bp of the first intron, and a neomycin resistance gene (NeoR). The targeting construct was electroporated into C57BL/6J embryonic stem (ES) cells, and recombinant clones were selected using G418. Selected clones were transfected with Cre recombinase and screened by PCR for removal of the Clrn1 gene fragment noted above. Recombined ES cells were microinjected into BALB/c blastocysts and implanted into pseudo-pregnant mothers. Chimeric progeny were obtained, and highly chimeric animals were subsequently mated with C57BL/6J mice to identify germ-line transmission of the Clrn1 PLoS Genetics | www.plosgenetics.org 12 August 2009 | Volume 5 | Issue 8 | e1000607