Author Summary Usher syndrome (USH) is a progressive disease affecting two primary senses: vision and hearing. Often by the third decade of life, affected persons have lost the majority of their rod photoreceptors, which leads to night blindness and peripheral vision loss. Similarly, hearing loss often progresses into the third or fourth decade. By the fourth decade, patients typically approach legal blindness and hearing impairment continues to decline. The proteins that when mutated cause USH are frequently found in primary sensory cells, photoreceptor and hair cells, that directly respond to light and sound, respectively. Similar to other forms of USH, the mRNA coding for the protein responsible for USH type 3 (CLRN1) is expressed in cochlear hair cells of the inner ear. However, as demon- strated in the current study, and unlike other USH disease proteins in the retina, we show that the Clrn1 is expressed in glial cells in the retina (Mu¨ ller cells) and is not expressed in the photoreceptors themselves. For reasons that remain unclear, the Clrn1 knockout mouse does not have a retinal degeneration phenotype but does become deaf soon after birth. In the current paper, we characterize the expression pattern in the retina and analyze the effects of removing the Clrn1 gene on vision and hearing. with the two founder mutations being N48K and Y176X [17]; the former amino acid change eliminates the only predicted N-linked glycosylation site, while the latter results in a premature CLRN1 truncation. Several mouse models of Usher syndrome types I and II have been characterized [20], with only the Usherin (Ush2a) mouse demonstrating a significant retinal degeneration phenotype. The Ush2a mutant mouse exhibits a very slow, late-onset retinal degeneration [21]. It remains unclear as to why mutations in USH genes other than Usherin do not result in a retinal degeneration phenotype in mice. Clarin 1 is a unique protein with homology to other four-pass transmembrane proteins, including members of the Claudin, Connexin, and CACNG protein families [12]. Based on homology between CLRN1 and stargazin, as well as its structural similarity to synaptic cleft and gap junction proteins, CLRN1 was predicted to be localized to the region around specialized ribbon synaptic junctions in both photoreceptor and hair cells. Three recent studies have provided some insight into the primary cochlear defect [22] and CLRN1’s cytoskeletal cellular binding partners [23,24]. In the current study, we characterized the expression pattern of Clrn1 in the murine retina and analyzed the phenotype of the Clrn1 KO mouse. We show here that the loss of CLRN1 results in progressive degeneration and loss of cochlear hair cells and early-onset hearing impairment. Moreover, and in contrast to recent immunohisto- chemical evidence [24], we find no evidence that Clrn1 is expressed in murine photoreceptors and no ‘‘USH3-like’’ retinal degeneration phenotype in the Clrn1 KO mouse. We believe that CLRN1 may represent the first M u¨ller cell-expressed protein which, when mutated, results in an RP phenotype in humans. Results Molecular Expression of Clrn1 RT-PCR analysis of Clrn1 expression in mouse retina is shown in Figure 1. A diagram of the mouse Clrn1 gene structure is illustrated in Figure 1A. Clrn1 transcripts (Ensemble; http://www.ensembl.org/ Clarin 1 Is Necessary for Hearing but Not Vision Mus_musculus/Gene/Summary?g = ENSMUSG00000043850) are encoded by four exons spanning 40 kilobases on chromosome 3. Also shown in Figure 1A is the intron-spliced four-exon cDNA structure of the gene. All known transcripts include exons 1 and 4, and the two primary transcripts in the retina and cochlea both lack exon 2 (shown in light gray). The longer transcript includes exons 1, 3, and 4 (NM_153385; http://www.ncbi.nlm.nih.gov/nuccore/68533238), while the shorter splice variant contains exons 1 and 4 only (NM_153386; http://www.ncbi.nlm.nih.gov/nuccore/68533243) (see also [22,24]). Primers (Figure S1) used to amplify Clrn1 in all figures are noted above (forward; sense; F1-F4) and below (reverse; antisense; R1-R6) the cDNA diagram. Figure 1B shows adult mouse retinal cDNA amplified with a common Clrn1 forward (F2) and exon-specific reverse (R2-R6) primers, annealing to each of the 4 exons. Two major Clrn1 splice variants are readily amplified from retinal cDNA using primers that span all four exons (lanes 1 and 5). The two Clrn1 splice variants are distinguished by the inclusion of an alternatively spliced exon 3 (180 bp), and previously both transcripts have been shown to be expressed in the mouse inner ear [12,22,24]. The ‘‘full-length’’ transcript contains exons 1, 3, and 4 (upper bands, lanes 1 and 5), while the shorter splice variant includes exons 1 and 4 only (lower bands, lanes 1 and 5). Reverse primers annealing to exons 1 (lane 2), 3 (lane 4), and 4 (lane 5) readily amplify Clrn1 transcripts, whereas a primer annealing to the infrequently included exon 2 (previously referred to as exon 1 [12]) shows very low, but detectable, amplification (lane 3). Together these data indicate that exon 2 is routinely spliced out of the mature transcripts. Quantitative (q) RT-PCR (Figure 1C, graph) utilizing dual- labeled fluorescent probes that anneal to specific exon-exon junctions (exons 1/3 or 1/4) allowed assessment of the relative expression levels of the two major transcripts. Six retinas from WT C57BL/6J animals (3 males, 3 females) were individually processed for both real-time RT-PCR and end-point (30–40 cycles) RT-PCR. Quantitatively (Figure 1C), the 1/4 exon splice variant (lacking exons 2 and 3) is expressed at approximately 40% of full-length (exons 1, 3, and 4) transcript expression levels. The gel image (Figure 1C, inset) shows an ethidium bromide stained agarose gel reflecting Clrn1 end-point RT-PCR processed in parallel for the six animals. Clrn1 was readily amplified in all postnatal (P) to adult retinal samples throughout mouse development (Figure 1D). Using a pair of primers that span exon 1 (F2) to exon 4 (R2), the two major transcripts were readily detected from the day of birth (P0) through the first postnatal week (P7), as well as in a P12 and the adult mouse. Prenatal Clrn1 expression was detectable at embryonic (E) day 12 (Figure 1E) using semi-quantitative RT-PCR and primers that span introns. Earlier embryonic stages were not examined. Similar to findings in the inner ear [12], Clrn1 expression is noted at all developmental ages tested, with slightly higher expression levels observed in the E15 animal (both embryo and whole head samples; Figure 1E). More specifically, embryonic eyes and dissected retinas were examined by RT-PCR (Figure 1F). Clrn1 is clearly expressed in the eyes (and retinas) of animals before birth. Omission of the reverse transcriptase (RT) enzyme resulted in no amplification (Figure 1F). Clrn1 expression was also examined in the mouse brain (Figure 1G). All brain regions tested showed Clrn1 amplification. In contrast to neural and sensory tissue samples, amplification of Clrn1 transcripts was not apparent in all body tissues or organs (Figure 1H). Clrn1 was present in kidney, liver, skeletal and heart muscle, and below detection in pancreas, spleen, small intestine, and thymus samples. These data are complemen- tary to previously published data [12–14]. PLoS Genetics | www.plosgenetics.org 2 August 2009 | Volume 5 | Issue 8 | e1000607