Figure 3. In situ hybridization and immunohistochemistry localize Clrn1 expression to Mu¨ ller cells in P7 mouse retina. (A–C) A section of WT retina labeled for Clrn1 (A) and the neural marker TuJ1 (C). The merged images (B) show the lack of overlap with the neural specific marker. TuJ1 labels all inner retinal neurons, but not the Clrn1-positive cells. (D–F) Higher magnification views of the boxed region in (B). Arrowhead points out a TuJ1-positive cell that is not Clrn1- positive, while the arrow points to a cell that is Clrn1-positive but not TuJ1-positive. (G,H) Peripheral retina labeled with the Clrn1 riboprobe Clarin 1 Is Necessary for Hearing but Not Vision (G) and subsequently with an antibody to Sox9 (H), a marker of retinal progenitors and M u¨ller glia. We find double labeled cells throughout the peripheral retina. (I) In the central retina, where the last progenitors are turning into M u¨ller glia, a thinner row of Clrn1-positive cells remains. (J) The section shown in (I) was double immunolabeled (after ISH) with antibodies to Glast (green) and to Sox9 (red). Arrows (I and J) identify triple-labeled M u¨ller cells. Scale bars = 50 mm in all images. Scale bars: In A applies to A–C; in D applies to D–F; in G applies to G–J. ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. doi:10.1371/journal.pgen.1000607.g003 respectively. Rp1, another photoreceptor-specific transcript ex- presses about 200-fold higher levels, while Thy1 expresses about 15 times the number of Clrn1 transcripts. Finally, Vim mRNA is expressed at a roughly similar level, while significantly fewer cFos, Slc1a3, and Grm1 transcripts are expressed in the WT mouse retina. A similar genetic experiment (Figure S3) was performed using the Proline-23-Histidine line 3 (P23H-3) rhodopsin mutant and Royal College of Surgeons (RCS) mutant rats [30,31]. Analogous to the mouse, histological analysis shows a near complete loss of photoreceptors in the P23H-3 rat (Figure S3B) compared with the WT retina (Figure S3A). In both rat retinal degeneration models Rp1 expression is almost completely lost, indicating a near total loss of photoreceptors (Figure S3C and S3D). When equivalent total amounts of whole retinal cDNA were PCR amplified (without normalization), P23H-3 and RCS retinal samples show a relative increase in Clrn1 expression compared to control retinas. The apparent increase in Clrn1 expression is due to the loss of the photoreceptor RNA contribution to the total RNA samples from mutant animals when equal amounts (from WT and mutant retinas) were included in the amplification reactions. This apparent change in expression is quantified by qRT-PCR (Figure S3E) and represents the average of two retinas. These data suggest that approximately 75% of total retinal RNA in WT animals is produced by photoreceptors, resulting in a ,4-fold apparent increase in Clrn1 expression when photoreceptors are absent from the mutant retinal samples. In a complementary experiment, we used LCM (Figure 5) to isolate mRNAs from individual nuclear layers from WT mouse retinal sections. Micrographs (Figure 5A–5F) show the progression of laser capture sample collection. Red spots (Figure 5A–5E) show the location of the laser beam used to select each of the three individual nuclear layers: ONL, INL, and GCL. Figure 5A shows the retinal section before laser irradiation and Figure 5B shows the retina after the tissue was laser-selected (but not removed). Following removal, the selected/collected tissue (photoreceptors; ONL) was observed on the underside of the tube cap (Figure 5C). Figure 5D and 5E represent capture of the INL and GCL, respectively, while Figure 5F indicates the retinal tissue that remained after the collected cells were physically removed from the tissue section. Using LCM, we collected total RNA from each of the three major retinal nuclear layers (ONL, INL, GCL), and subsequently subjected the isolated total RNA to RT-PCR analysis (Figure 5G). Total RNA was extracted and amplified using two rounds of RT-PCR for Rp1 (a photoreceptor specific transcript) and Clrn1 (Figure 5G). As expected, robust Rp1 expression was detected in both ONL samples and absent in the GCL. We believe that the presence of some Rp1 transcripts in the INL sample is the result of a few photoreceptors inadvertently adhering to the cap of INL sample. Nevertheless, Clrn1 was only observed in the INL sample, and was completely absent from both ONL and GCL samples, further supporting the restricted INL localization of Clrn1 transcripts (Figure 2, Figure 3, and Figure 4). PLoS Genetics | www.plosgenetics.org 5 August 2009 | Volume 5 | Issue 8 | e1000607