Clarin 1 Is Necessary for Hearing but Not Vision Figure 8. Cochlear hair cells of Clrn1 KO (2/2) mice degenerate with age and exhibit longer stereocilia. (A) Illustration of the inner ear, demonstrating the cochlea and auditory sensory epithelium. (B–E) Immunofluorescent confocal images of P15 and mouse inner-ear epithelia harvested from the middle turn of the cochlea, shown in side view. Filamentous actin was detected with rhodamine-Phalloidin (red), and anti-myosin VI labeling is shown in green. (B,C) At P15, the auditory epithelium in Clrn1 KO (2/2) (C) mice appears morphologically similar to WT animals (B). (D) WT animals maintain organized stereocilia on inner hair cells (IHCs). (E) Subtle IHC stereociliar abnormalities such as increased bundling and lengthening (arrowheads) are evident in KO animals at P15. (F,G) Quantitative analysis of IHC bundle lengths reveal considerably longer stereocilia (F) in P15 Clrn1 KO animals, distributed over a broader range of lengths (G, upper graph; n = 80), which is distinguishable from the highly uniform lengths of WT stereocilia (G, lower graph; n = 40). (H,I) Shown from the side view at P120, and compared to WT labeling (H), staining of Clrn1 KO auditory epithelia (I) indicates extensive degeneration and loss of IHCs. At this age, a disruption of supporting cell apical surfaces is apparent (I, arrowheads), and the surviving IHCs in the auditory epithelia of Clrn1 KO mice exhibit shortened hair cell bodies (I, arrows). (J,K) From a top view, a clear disruption and loss of both IHCs and OHCs is evident in the Clrn1 KO mutant (K, arrowheads). (L,M) IHCs from WT cochleas (L) possess highly ordered and symmetrical stereocilia, while Clrn1 KO IHCs (M) exhibit profoundly disorganized bundles of elongated and possibly fused stereocilia (arrowheads). * = P,0.001. Error bars = 1 SD. Scale bars: In B (25 mm) applies to B and C; in D (7.5 mm) applies to D and E, in H (25 mm) applies to H and I, in J (20 mm) applies to J and K, in L (5 mm) applies to L and M. doi:10.1371/journal.pgen.1000607.g008 Nine of the 11 Usher syndrome loci have been genetically characterized, with 2 genes (USH1E and USH1H) not yet identified [4]. Numerous transgenic and knockout mouse models of RP have been developed, in which a mutation or knockout of a photoreceptor or RPE specific gene results is an approximation of the human clinical phenotype, often exhibiting an abbreviated time course; among these are Rho [40], Pde6b [41], RDS [42], Rom1 [43], Crx [44], Tub [45], Gucy2e [46], RPE65 [47], and others. To date, knockout of Usher gene product has been largely unsuccessful in generating animal models with a retinal degener- ation phenotype in mice, with only the Usherin (Ush2a) mouse demonstrating a significant disease. [20,21]. Surprisingly, the majority of these USH animal models exhibit pronounced hearing defects [48–52]. Small alterations in the ERG amplitude and waveform have been noted in some of these mice [53–55] but the implications of these changes remain unclear. Overall, relatively few tetraspanins have been identified as having essential functions, and mouse knockouts of individual tetraspanins has resulted in mild phenotypes [56]. The lack of genotype-phenotype correlation in the retina of USH mouse models remains unclear. In the absence of direct evidence of a redundant pathway in mice, it is reasonable to suggest that there is compensation via undiscovered gene products PLoS Genetics | www.plosgenetics.org 10 August 2009 | Volume 5 | Issue 8 | e1000607