Figure 2. Developmental time course of Clrn1 in situ hybrid- ization in the mouse retina. (A–C) Clrn1 was not detected in sections of E18.5 (A), newborn (B), or P3 (C) mouse retina. (D) Clrn1 in situ labeling in the INL (arrows) of a P7 retina. (E) Clrn1 in situ showing labeling across the entire central-to-peripheral extent of the retina at P7. (F) Section from a Clrn1 deficient mouse retina showing lack of labeling. (G,H) Clrn1 anti-sense (G) and sense control (H) hybridizations showing expression at P7 (arrows). (I,J) Clrn1 anti-sense (I) and sense control (J) sections from a P12 retina, showing a lower level of expression by this age (arrows). (K,L) Clrn1 expression at 4 weeks of age. No signal above the sense control (L) could be detected at this age. All sections were hybridized with the same probe and reacted for similar lengths of time. Scale bars = 100 mm for all images except (E), where the scale bar = 200 mm. Scale bars: In A applies to A–C; in D applies to D and F; in G applies to G–J; in K applies to K–L. NBL = neuroblastic layer, CB = ciliary body, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer, and RPE = retinal pigmented epithelium. doi:10.1371/journal.pgen.1000607.g002 (Figure 2A), P0 (Figure 2B), or P3 (Figure 2C). However, antisense riboprobe labeling of INL cells was unambiguous at P7 (Figure 2D and 2E). As expected, retinal tissue from the KO animal (Figure 2F) showed no labeling. Clrn1 ISH was clearly detectable at P7 in the INL (Figure 2G). However, Clrn1 expression is subsequently down-regulated (Figure 2I) and not detectable by 4 weeks of age in WT mouse retina (Figure 2K), although all ages show expression by end-point PCR (Figure 1). Hybridization with a sense (control) riboprobe showed no labeling at any age (Figure 2H, 2J, and 2L). ISH with a Clrn1 riboprobe on adult human retina did not generate a significant signal (data not shown). We were unable to obtain human retina at the equivalent stage of fetal development (third trimester) to the peak of labeling observed in the mouse retina (P7). Clarin 1 Is Necessary for Hearing but Not Vision To follow up on inner retinal localization of Clrn1 by ISH in WT retinal tissue (Figure 3), we performed immunohistochemistry to clarify which retinal cell type(s) express Clrn1. The blue-purple ISH reaction product represents Clrn1 mRNA, whereas red and green fluorescence indicates immunohistochemical staining. An antibody (TuJ1) to tubulin type III labeled all neuronal elements in the retina (shown in red; Figure 3B, 3C, 3E, and 3F). Dual labeling indicates that TuJ1 and Clrn1 lack co-localization, and thus are not co-expressed in neuronal cells. We also double-labeled P7 retinal sections for Clrn1 ISH and Sox9 immunohistochemistry (Figure 3G–3J). Sox9 is a transcription factor that is widely expressed in retinal progenitor cells during development. Sox9 expression is maintained in Mu¨ ller cells but lost in maturing neurons [25]. In peripheral retina, the antibody to Sox9 (red) labeled a band of retinal progenitor and M u¨ller glia (Figure 3G and 3H). In the more differentiated central retina, a narrower band of Clrn1 ISH-positive cells (Figure 3I) is apparent, and some of these cells also stain positively for Sox9 (Figure 3J; red) and Glast (Figure 3J; green). These data add additional support to the identification of Mu¨ ller cells as the predominant source of Clrn1 expression in the retina. Additionally, staining of P7 mouse retina shows co-labeling of Clrn1 ISH with a Glast antibody (Figure S2). Double labeling with Glast, a M u¨ller glial cell marker, indicates that some Glast-positive cells also express Clrn1 mRNA. Surprisingly, these data show that Clrn1 mRNA (and presumably the protein product(s) of the Clrn1 gene) is expressed in retinal glia and not in neurons, including photoreceptors. We performed two additional experiments (Figure 4 and Figure 5) in order to validate our ISH data shown in Figure 2 and Figure 3. We first used a genetic approach and examined a well-studied model of photoreceptor degeneration: the C3H/HeJ (C3H) mouse, which harbors the photoreceptor Pde6brd1 mutation [26]. The mutation results in a rapid degeneration and loss of nearly all photoreceptor cells [27,28]. A histological comparison of a 6-month-old WT C57BL/6J and a 14-month-old C3H mouse is shown in Figure 4A and 4B, respectively. Note the maintenance of the GCL and INL, but near complete loss of the ONL in the C3H animal (Figure 4B). We performed qRT-PCR and normalized the WT and C3H samples to the ganglion cell-specific transcript, Thy1, as well as to 28S RNA. We also measured the expression levels (Figure 4C) of six genes in addition to Clrn1 and Thy1: cFos, Slc1a3, Grm1, Rho, Rp1, and Vim. When compared to expression levels in the WT animal, the loss of photoreceptors resulted in reduced expression of the immediate early gene transcription factor cFos, as well as Slc1a3 and Grm1, a glutamate transporter and receptor, respectively. Not surprisingly, extremely low levels of the photoreceptor-specific transcripts Rho and Rp1 were measured in the C3H mutants. These data confirm our histological observa- tions that photoreceptors are for all intents and purposes absent or completely degenerated in the C3H animals. Importantly, as measured with two independent primer pairs, Clrn1 exhibited essentially no difference in expression in the C3H retinas (Figure 4C; Clrn1 (1), Clrn1 (2)) compared to the WT sample. The vimentin (Vim) transcript (Figure 4C) appeared to be slightly up-regulated in both degenerated C3H retinas, a finding consistent with other models of photoreceptor degeneration [29]. Figure 4D shows relative WT expression levels of the 28S/Thy1- normalized transcripts (Figure 4C). In Figure 4D, the averaged raw Ct (threshold cycle number) data for each transcript were normalized such that Clrn1 expression was assigned an expression level of 1. Though not quantitative, these data provide approximations of the relative abundance of genes in WT animals compared in Figure 4C. Hence, 28S and Rho express roughly 10,000 and 8,000 times the number of Clrn1 transcripts, PLoS Genetics | www.plosgenetics.org 4 August 2009 | Volume 5 | Issue 8 | e1000607