and/or intracellular pathways mitigating the loss of USH gene expression in the mouse retina. USH proteins in the retina are generally thought to be localized to photoreceptor and/or RPE cells. USH proteins are commonly associated with photoreceptor connecting cilia, inner/outer segments, and synaptic regions [2,3,16]. Indeed, recent immunohistochemical evidence purports CLRN1 to be localized to photoreceptors [24], and it remains unclear how to harmonize these data with our findings presented here, where we show that Clrn1 is not expressed in photoreceptors. Nevertheless, it has been proposed that USH proteins form a network of directly associated proteins within the cell, localizing at or near the photoreceptor’s connecting cilium, at the apical part of the inner segment [3,16,57,58]. It follows then that damage to any one of the proteins within the complex could theoretically result in clinically similar USH-related RP phenotypes. Though some USH gene products localize to the synaptic terminal, there is little evidence for synaptic zone dysfunction in human electrophysio- logical recordings [10,59]. Indeed, our original hypothesis maintained that CLRN1 would be a protein facilitating structural maintenance of the synaptic connection between photoreceptors and second order neurons, horizontal and bipolar cells [12]. However, this hypothesis is not supported by data presented here. Developmental Expression of the Murine USH3A Gene Clrn1 We demonstrated by RT-PCR that Clrn1 is readily amplified from a variety of adult mouse tissues, all examined brain regions, and the retina. Two primary transcripts were expressed at an approximate 5:2 ratio; the primary transcript includes exons 1, 3, and 4, while lower levels of expression are observed for the splice variant including exons 1 and 4. This is consistent with the expression of Clrn1 splice variants in the inner ear [12]. The purpose of inclusion/exclusion of exons 3 and/or 2 in mature Clrn1 transcripts remains unclear; as is the significance of Clrn1 splice variant expression levels. Nevertheless, the expression levels of the splice variants appear to be consistently regulated. Based on topological predictions [12] the shorter splice variant (without exon 3) would lack two of the four predicted transmembrane domains if translated into a functional protein. Thus, it is interesting to consider the possibility that the shorter splice variant may exhibit distinct biophysical properties and/or functions from its longer, four transmembrane ‘‘full-length’’ counterpart. Our results show that although Clrn1 mRNA can be readily RT- PCR amplified from embryonic, juvenile and adult retina, the mRNA is developmentally down regulated in adult mice. These data suggest that CLRN1 (protein) expression is likely to decrease over the course of retinal maturation, and that CLRN1 is probably translated and expressed at comparably low levels in the adult mouse. It may be that CLRN1 is non-essential to the adult mouse retina, and that CLRN1 serves only a transient (and presumably non-deleterious) role during murine retinal development. We do not currently have CLRN1 expression data from developing or adult human retina. However, it is intriguing to postulate that the CLRN1 gene may be differentially regulated in humans. If, in fact, human regulation differs from the mouse with respect to Clrn1 gene expression, and human retina exhibits (or requires) robust and/or continuous expression into adulthood, it seems reasonable to postulate that humans may develop the characteristic age- related USH3 retinal phenotype whereas mice do not. In other words, humans, but not mice, may have maintained and/or evolved a need for continuous CLRN1 expression in the retina. Another important possibility to consider is that mice, but not humans, express a compensatory protein or utilize a redundant functional pathway that obviates the need for CLRN1 in the Clarin 1 Is Necessary for Hearing but Not Vision mouse retina. Finally, it also seems reasonable to suggest that degeneration in the mouse retina could be dependent upon the expression of gain-of-function mutant CLRN1 proteins, and not simply the loss of CLRN1 altogether, as in the case of the total KO. In this case, the mouse cochlea would remain sensitive to either the absence (KO) or mutation (point mutation or truncation) of CLRN1, whereas the retina would be insensitive in the KO condition, but would degenerate when mutant CLRN1 proteins are expressed. Further studies of animal models exhibiting specific gene alterations, as well as analysis of gene expression in humans, should help to clarify the normal and pathological roles of CLRN1 in these sensory tissues. The USH3A Gene, Clrn1, Is Expressed in Mu¨ ller Cells in the Inner Retina In situ hybridization and laser capture microdissection indicate that Clrn1 is expressed in the INL, and not in the ONL (photoreceptors), as our original hypothesis proposed. Further supporting this unusual finding is the fact that aged animals (C3H mice, RCS rats, and P23H-3 rats) that have completely lost their photoreceptors continue to express Clrn1 at WT levels. If these data hold true in humans, they imply that CLRN1 is expressed in the INL and that proteins expressed in the inner retina, and specifically in M u¨ller cells, can cause RP. The expression of Clrn1 in M u¨ller cells was surprising. All three sub-classifications of USH (types I, II, and III) exhibit late-onset retinitis pigmentosa (RP). This phenotype, in general, suggests that a slow but perpetual rod photoreceptor degeneration underlies disease progression. Our original hypothesis stated that Clrn1 would be expressed in photoreceptors, and that the protein, CLRN1, would be targeted to rod photoreceptor synapses and contribute to synaptic adhesion and/or structure. Although significant variability exists among affected individuals, on average, USH3 patients exhibit a 50% functional vision loss at a mean age of 28 [11] and commonly have ‘‘tubular vision’’ by the age of 30 [9]. This is in contrast to numerous other recessive RP (or rod-cone) diseases, in which the onset is often in the first to second decade of life [38]. The discovery that Clrn1 is expressed in a developmentally regulated manner in M u¨ller cells, and that Clrn1 is not expressed in photoreceptors, was unexpected. Taken together, our data suggest that an INL protein (CLRN1), when mutated in humans (but not mice), results in degeneration of rod photoreceptors. Assuming that Clrn1 is similarly localized to Mu¨ ller cells in the human retina, we believe this to be the first instance of an INL transcript resulting in inherited photoreceptor degeneration [10]. CLRN1 Protein Expression Immunocytochemical detection of CLRN1 protein in retinal tissues has not been successful. Two antibodies (one developed in rabbit, one in chicken) accurately detected an EGFP-CLRN1 fusion protein transiently expressed by HEK293 cells (Figure S4). Moreover, the CLRN1-B2 antibody specifically detected protein bands at approximately the correct (predicted) molecular weight(s) in human, rat, and mouse retinal homogenates (Figure S4L). Immunoprecipitation (IP) with the CLRN1-B2 antibody indicates that CLRN1 may form higher molecular weight multimers and/or participate in a multi-protein complex, similar to complexes previously proposed [16,60], although none of the other USH- related proteins appear to be co-expressed in Mu¨ ller glia. In side-by-side comparisons with Clrn1 KO tissue samples, neither of our CLRN1 antibodies that demonstrated good specificity in vitro (Figure S4) offered a reliable immunohistochem- ical signal in WT retinal tissue sections (Figure S5). Similar non- PLoS Genetics | www.plosgenetics.org 11 August 2009 | Volume 5 | Issue 8 | e1000607