Figure 4. Analysis of Clrn1 in WT and photoreceptorless C3H/ HeJ aged mouse retinas. (A,B) Sixteen micron cryosections of WT (A) and degenerated C3H (B) mouse retinas counterstained with hematox- ylin. Note the virtual complete loss of photoreceptors in the 14 month C3H retina. (C) Real-time quantitative (q) RT–PCR was performed in order to correct for the RNA contribution of the outer retina in the WT cDNA sample. The data were normalized to the ganglion cell marker Thy1, and subsequently to expression levels in the WT sample. For comparison purposes, several additional known retinal transcripts were included in the qRT–PCR analysis. (D) Relative transcript expression levels in retinas from WT and photoreceptorless animals. Normalizing to a Clrn1 primer pair, the range in the abundance of non-Clrn1 transcripts was 100-fold fewer (Grm1) to 10,000-fold higher (Rhod). Scale bar = 50 mm in all images. ONL = outer nuclear layer, INL = inner nuclear layer, and GCL = ganglion cell layer. doi:10.1371/journal.pgen.1000607.g004 Retinal Structure in the Clrn1 Knockout Mouse Clrn1 KO mice show no phenotypic signs of retinal degenerative disease. Since the USH3 human combined blindness and deafness phenotype is progressive, we studied Clrn1 KO mice up to 2 years. At 2 years of age, Clrn1 KO mice continued to exhibit a normal retinal phenotype, both structurally and functionally. Figure 6 is toluidine blue stained 1 mm plastic embedded sections of 9 month old WT (+/+) and Clrn1 mutant (+/2,2/2) animals. Figure 6A– 6C show high magnification of the photoreceptors and their associated RPE, while Figure 6D–6F show the inner retina at the same magnification. Lower magnification offers a more gross histological view of mid-peripheral (Figure 6G–6I) retina, as well as retina immediately adjacent to the optic nerve head (asterisks, Figure 6J–6L). No abnormal histological phenomena were detected. We performed fluorescent confocal microscopy using a panel of antibodies to look for subtle structural or cellular changes in the Clarin 1 Is Necessary for Hearing but Not Vision Figure 5. Laser capture microdissection of WT mouse retina. (A–F) Images of a section of WT C57BL/6J retina taken during the laser capture process. (A) Retina before laser capture. (B) The ONL is being captured on the cap. (C) The ONL after capture, on the surface of the microfuge tube cap just prior to total RNA extraction. (D) The ONL has been removed, and the INL is being laser-captured. (E) The ONL and INL have both been removed (though some cells remain), and the GCL (ganglion cell layer) is being laser captured. (F) Retina remaining after cell layers have been laser captured and collected on microfuge tube caps. (G) RT–PCR was used to amplify Rp1 (a photoreceptor specific transcript) and Clrn1 in each sample. Rp1 was detected in both ONL samples as well as the INL sample due to carry-over of a few photoreceptors, but was not detected in the GCL. Note that Clrn1 transcripts were only detected in the INL. Scale bar = 50 mm. ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer, and RPE = retinal pigmented epithelium. doi:10.1371/journal.pgen.1000607.g005 Clrn1 KO retina (Figure 7). Eighteen month old littermate animals (WT and KO (2/2)) from early heterozygous matings were used. Antibodies used were: anti-Calbindin (horizontal and amacrine cells; Figure 7A–7D), anti-PKCa (rod bipolar cells; Figure 7E–7H), Rhodamine-PNA (cone photoreceptor sheaths; Figure 7I and 7J), anti-synaptophysin (OPL and IPL synaptic structures; Figure 7K and 7L), anti-GFAP and anti-vimentin (glial cells; Figure 7M–7P), and a chicken anti-CLRN1 antibody (Clarin160; Figure 7Q and 7R. See below, and Figure S4 and Figure S5). No differences between WT and Clrn1 KO (2/2) animals were found using these antibodies. Due to low levels of retinal CLRN1 expression, the anti-CLRN1 antibody labeling (Figure 7Q and 7R) represents non-specific signal in both WT and Clrn1 KO retinas. Labeling with the secondary antibodies alone showed no reactivity (Figure 7S and 7T). It is important to note that when an EGFP- CLRN1 fusion protein is overexpressed in 293 cells (see below, and Figure S4), specific labeling is readily observed using two different anti-CLRN1 antibodies, including the Clarin160 anti- body immunoreactivity shown here (Figure 7Q and 7R). Clarin 1 Antibody Analysis and Membrane Localization in Transfected Cells Using the pEGFP-C1 vector (Clontech), we generated and expressed the EGFP-CLRN1 fusion protein in HEK293 cells PLoS Genetics | www.plosgenetics.org 6 August 2009 | Volume 5 | Issue 8 | e1000607