shows subsequent immunostaining with an anti-CLRN1 affinity- purified antibody (CLRN1-B2). The CLRN1-B2 antibody (rabbit) showed precise co-localization (Figure S4E) with the EGFP- CLRN1 fusion protein. In a similar experiment the Clarin160 antibody (chicken), which was generated against a different CLRN1 peptide, showed equally reliable co-localization with EGFP-CLRN1 (Figure S4F, S4G, S4H). No immunoreactivity (red) was detected when the primary antibody was omitted (Figure S4I, S4J, S4K) or in non-transfected cells (data not shown). Several additional anti-CLRN1 antibodies showed various degrees of specific/non-specific labeling (data not shown). Western blot analysis (Figure S4L) of adult human, mouse, and rat retinal homogenates using the CLRN1-B2 antibody detected a band at ,25–26 kDa in all species, and an additional ,35 kDa protein in the human sample only. Detection was dependent upon sodium dodecylsulfate (SDS) inclusion in the sample buffer, as no reactivity was observed in samples processed without SDS (Figure S4L, right side). We immunoprecipitated CLRN1 from mouse retinas using the CLRN1-B2 antibody to further characterize the CLRN1 protein (Figure S4M). After elution and subsequent immunoblotting with the same antibody, a higher molecular weight (,55–80 kDa) protein complex was detected prominently in the first two eluates (lanes 1 and 2). These data indicate that CLRN1 can homodimerize and/or participate in the formation of larger multi-protein complexes under non-reducing conditions. Studies to identify CLRN1 interacting proteins provide additional insight into CLRN1 function [23], and there have been indications that CLRN1 associates with both actin [23] and microtubules [24]. Immunocytochemistry on P7 WT mouse retinal sections (Figure S5), the peak of Clrn1 mRNA expression as detected by ISH (Figure 2), failed to show specific CLRN1 localization using Clarin160 (Figure S5E, S5F) or CLRN1-B2 (Figure S5G, S5H) antibodies. Similar labeling was observed in both WT and Clrn1 KO control retinal sections for each of the antibodies tested. Retinal and Cochlear Function Recent data suggest that Clrn1 KO mice are functionally normal [22]. We independently performed ERG experiments (Figure S6) in order to measure photoreceptors A- and B-wave function in WT and Clrn1 KO animals up to 2 years of age. Photoreceptor function in the Clrn1 KO animal appeared normal, and no significant difference was observed at any light intensity. In contrast to the lack of a retinal phenotype, but similar to most other mouse models of Usher syndrome [20], the Clrn1 KO mouse exhibits significant structural and functional hearing deficits [22]. Similarly, we demonstrate inner ear dysfunction in the Clrn1 KO mouse by measuring the auditory brainstem response (ABR). Figure S7 illustrates representative ABR traces, and corroborates the functional loss in both juvenile and adult animals [22]. Outer and Inner Hair Cells Degenerate in Clrn1 KO Mice and Exhibit Longer Stereocilia Immunofluorescence with a hair cell-specific marker, myosin VI, and filamentous actin were used to evaluate the cellular arrangement and general morphology of the auditory sensory epithelium of WT and Clrn1 KO (2/2) mice (Figure 8). Recently Clrn1 was shown to be directly associated with the cellular actin networks [23], so staining for f-actin is particularly interesting in the Clrn1 KO animals. Figure 8A illustrates the origin of the cochlear tissue depicting the planes of section used in the analysis of the organ of Corti (middle and apical turns; side and top views). At P15, there is no apparent cell loss in the organ of Corti (Figure 8B and 8C), however, the mechanosensory stereocilia, the Clarin 1 Is Necessary for Hearing but Not Vision actin-rich projections on the apical surface of the inner hair cells (IHC) appear disorganized, elongated, and variable in length (6.0360.51 mm, n = 80, P,0.001) when compared to the WT control (4.6660.174 mm, n = 80) (Figure 8D–8G). Similar non- quantitative morphological changes in stereocilia have been noted previously [22]. At P120, the cochlear outer hair cells (OHC) and IHC, as well as the supporting cells, are severely degenerated (Figure 8H–8M). By P120, the number of cell bodies is significantly reduced, and the remaining cells have reduced apical surfaces and display bundles of severely disorganized and elongated stereocilia (Figure 8M). The supporting pillar cells, two closely adjacent rows of epithelial cells that separate the inner and outer rows of hair cells, appear structurally compromised, as indicated by a reduction in rhodamine-Phalloidin staining of the pillar heads (Figure 8I). Some Clrn1 KO mice on a C57BL/6 background exhibited circling behavior by 5–6 months of age (Video S1). Circling behavior was more prevalent when the KO construct was backcrossed to a mixed C57BL/6 and Sv129 genetic background (data not shown). However, the circling phenotype remained incompletely penetrant, irrespective of the genetic background, consistent with the variable finding of vestibular defects among USH3 patients. We analyzed filamentous actin features of utricular cells in vestibule of the randomly selected (non-circling) 4-month WT and Clrn1 KO animals on the C57BL/6 background (Figure S8). Neither low (Figure S8A, S8B) nor high (Figure S8C, S8D) power images indicate any grossly abnormal structural features in the vestibule at P120. Discussion Photoreceptors are known to express over 300 unique (within the retina) transcripts [32], many of which are unlikely to be expressed outside of the eye. This finding is consistent with clinical disease manifestations, where mutations (dominant or recessive) in rod photoreceptor cell-expressed genes commonly result in photoreceptor cell death and consequent RP. There are documented examples of RPE-defects [33,34], as well as a predominantly expressed ganglion cell transcript [35] that result in an RP-like phenotype. To date, retinal degeneration resulting from mutation in proteins originating from neurons or glia of the inner nuclear layer has not been described. Our data suggest that CLRN1, the protein implicated in Usher syndrome type 3 which caused progressive blindness and deafness in humans, is an inner retinal protein (at least in mice) produced by the retinas resident radial glia, Mu¨ ller cells. Retinitis pigmentosa (RP) is a group of blinding diseases affecting approximately 1.5 million people worldwide, and around 1 in 4000 births in the United States [36–38]. RP is sometimes referred to as rod-cone dystrophy, and is characterized by a loss of rod photoreceptor cells, narrowing of the visual field, and night blindness, which can eventually progress to cone dysfunction and total vision loss. Thus far, over 40 loci have been identified for non-syndromic RP [38] and 30 genes have been causally related (RetNet; http://www.sph.uth.tmc.edu/Retnet/). In contrast, cone or cone-rod dystrophies (21 loci, 15 identified genes) are due to primary defects in cone cells, and in turn, rods may eventually become vulnerable to progressive degeneration [39]. Usher syndrome (USH) is fundamentally characterized by combined deaf-blindness. There are over 50 syndromic/systemic loci involving RP (RetNet), about 25% of which are also associated with USH. USH syndromes are the most common cause of this combined deaf-blindness, accounting for approximately 50% [1,16], while contributing to about 14% of all RP cases [34]. PLoS Genetics | www.plosgenetics.org 9 August 2009 | Volume 5 | Issue 8 | e1000607