CLRN1 Is Nonessential in the Mouse Retina but Is Required for Cochlear Hair Cell Development Scott F. Geller1*, Karen I. Guerin1,2, Meike Visel1, Aaron Pham1, Edwin S. Lee1, Amiel A. Dror3, Karen B. Avraham3, Toshinori Hayashi4, Catherine A. Ray4, Thomas A. Reh 4 , Olivia Bermingham-McDonogh 4 , William J. Triffo5, Shaowen Bao1, Juha Isosomppi6, Hanna Va¨ stinsalo6, Eeva-Marja Sankila6,7, John G. Flannery1,2* 1 Helen Wills Neuroscience Institute, University of California, Berkeley, California, United States of America, 2 Vision Science, University of California, Berkeley, California, United States of America, 3 Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, 4 Department of Biological Structure, School of Medicine, University of Washington, Seattle, Washington, United States of America, 5 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 6 Folkha¨lsan Institute of Genetics, Biomedicum Helsinki and Department of Medical Genetics, University of Helsinki, Helsinki, Finland, 7 Helsinki University Eye Hospital, Helsinki, Finland Abstract Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5–6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT–PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT– PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Mu¨ ller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration. Citation: Geller SF, Guerin KI, Visel M, Pham A, Lee ES, et al. (2009) CLRN1 Is Nonessential in the Mouse Retina but Is Required for Cochlear Hair Cell Development. PLoS Genet 5(8): e1000607. doi:10.1371/journal.pgen.1000607 Editor: Thomas B. Friedman, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, United States of America Received April 7, 2009; Accepted July 17, 2009; Published August 14, 2009 Copyright: 2009 Geller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors gratefully acknowledge financial support for this work: Foundation Fighting Blindness (JGF), European Commission FP6 Integrated Projects EuroHear LSHG-CT-20054-512063 and EUMODIC 037188 (KBA), and Hope for Vision (TAR and OBMcD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: drgeller@gmail.com (SFG); flannery@berkeley.edu (JGF) Introduction Usher syndrome (USH) is characterized by bilateral combined blindness and deafness, and is the most common cause of the dual sensory deficit [1]. To date, there are 11 chromosomal loci with 9 genes identified, and over 100 mutations directly associated with USH (for reviews see [2–4]). Although significant variability is present and misdiagnosis is prevalent [5–7], USH has for many years been sub-categorized into USH1, USH2, and USH3, based upon the patient’s phenotypic severity and age of onset, as well as the presence or absence of vestibular function [2,8]. For example, USH1-associated mutations in myosin VIIa, cadherin, protocad- herin, harmonin, and SANS result in congenital deafness and progressive retinitis pigmentosa (RP) beginning before puberty. In contrast, mutations in the USH2 genes, USH2A and GPR98, result in moderate to severe congenital hearing loss, normal vestibular function, and progressive RP. USH3 exhibits progressive post- lingual hearing loss with a late-onset RP [9], which has recently been shown to progress more rapidly than USH2A [10,11]. Although having the lowest clinical incidence of the three Usher syndromes, USH3 has the greatest therapeutic ‘window’ for treatment due to the relatively late onset and slow progression of vision loss. More importantly in terms of therapy, human USH3 hearing loss is progressive, in contrast to congenital hearing deficits in USH1 and USH2. A single USH3 gene, CLRN1 (USH3A), has been identified [12–15] to date, although there is a putative additional locus on chromosome 20 [2,16]. The incidence of USH3 is increased in Finland [9] and amongst Ashkenazi Jews in various regions [17] due to distinct founder mutations. Twelve CLRN1 mutations have been documented [6,10,12–14,17–19], PLoS Genetics | www.plosgenetics.org 1 August 2009 | Volume 5 | Issue 8 | e1000607