Expression of Olfr558 in vessels of the choroid and retina.

<p>A and B. <i>In situ</i> hybridization of the mouse choroid slices with <i>Olfr558</i> probe (red) without (A) or with (B) the pretreatment with RNase (+RNase). The slices in A and B were adjacent to each other in the eye. Blue – nuclei staining (DAPI). C. <i>In situ</i> hybridization of a different choroid slice with <i>Olfr558</i> probe (red). Note that not all vessels express <i>Olfr558</i>; some vessels do not have <i>Olfr558</i> signal at all (white arrows). D. <i>Olfr558</i> is also expressed in a few vessels of the mouse retina. The retinal layers are indicated on the right – ganglionic layer (GL), inner plexiform layer (IPL), inner nuclear layer (INL) and outer nuclear layer (ONL).</p

Expression of Olfactory Signaling Genes in the Eye

<div><p>Purpose</p><p>To advance our understanding how the outer eye interacts with its environment, we asked which cellular receptors are expressed in the cornea, focusing on G protein-coupled receptors.</p><p>Methods</p><p>Total RNA from the mouse cornea was subjected to next-generation sequencing using the Illumina platform. The data was analyzed with TopHat and CuffLinks software packages. Expression of a representative group of genes detected by RNA-seq was further analyzed by RT-PCR and <i>in situ</i> hybridization using RNAscope technology and fluorescent microscopy.</p><p>Results</p><p>We generated more than 46 million pair-end reads from mouse corneal RNA. Bioinformatics analysis revealed that the mouse corneal transcriptome reconstructed from these reads represents over 10,000 gene transcripts. We identified 194 GPCR transcripts, of which 96 were putative olfactory receptors. RT-PCR analysis confirmed the presence of several olfactory receptors and related genes, including olfactory marker protein and the G protein associated with olfaction, Gαolf. <i>In situ</i> hybridization showed that mRNA for olfactory marker protein, Gαolf and possibly some olfactory receptors were found in the corneal epithelial cells. In addition to the corneal epithelium, Gαolf was present in the ganglionic and inner nuclear layers of the retina. One of the olfactory receptors, Olfr558, was present primarily in vessels of the eye co-stained with antibodies against alpha-smooth muscle actin, indicating expression in arterioles.</p><p>Conclusions</p><p>Several species of mRNA encoding putative olfactory receptors and related genes are expressed in the mouse cornea and other parts of the eye indicating they may play a role in sensing chemicals in the ocular environment.</p></div

Expression of Olfr558 in the mouse cornea.

<p>A. Representative images of <i>in situ</i> hybridization of the mouse cornea slices with <i>Olfr558</i> probe (red) without or with the pretreatment with RNase (+RNase). DAPI (blue) was used for nuclei staining. B. Quantification of in situ hybridization signals obtained without or with RNase pretreatment. All signal dots were counted in the entire corneal slice for each condition, and the average number of dots per slice (n = 6) is shown. Asterisks indicate significant difference (p<0.01).</p

Expression of G protein subunits and other olfactory genes in the cornea.

<p>The RT-PCR analysis of corneal RNA was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096435#pone-0096435-g001" target="_blank">Fig. 1</a> legend. For primers located within a single exon PCR was performed with (+) and without (–) the RT step. For primer pairs located in separate exons only results of PCR with (+) the RT are shown. Arrows indicate the positions of the predicted PCR fragment sizes for <i>Taar4</i> (140 bp), two splice variants of Gαolf (314 and 607 bp), <i>Gna15</i> (118 bp), <i>Reep5</i> (347 bp), <i>Rtp3</i> (180 bp), <i>Omp</i> (150 bp) and <i>Emx2</i> (133 bp). Molecular markers (M) are from a 100 bp DNA ladder; the lowest band is 100 bp.</p

Image_4_A Potential Compensatory Role of Panx3 in the VNO of a Panx1 Knock Out Mouse Model.JPEG

<p>Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared. Using qRT-PCR and Immunohistochemistry (IHC), we confirmed the loss of Panx1, found similar Panx2 expression levels in both models, and a significant upregulation of Panx3 in mice with a global ablation of Panx1. Specifically, Panx3 showed upregulated expression in nerve fibers of the non-sensory epithelial layer in juvenile and adult KO mice and in the sensory layer of adults, which overlaps with Panx1 expression areas in WT populations. Since both social behavior and evoked ATP release in the VNO was not compromised in KO animals, we hypothesized that Panx3 could compensate for the loss of Panx1. This led us to compare Panx1 and Panx3 channels in vitro, demonstrating similar dye uptake and ATP release properties. Outcomes of this study strongly suggest that Panx3 may functionally compensate for the loss of Panx1 in the VNO of the olfactory system, ensuring sustained chemosensory processing. This finding extends previous reports on the upregulation of Panx3 in arterial walls and the skin of Panx1 KO mice, suggesting that roles of Panx1 warrant uncharacterized safeguarding mechanisms involving Panx3.</p

Quantitative PCR analysis of Olfrs and OMP.

<p>Corneal RNA was isolated and subjected to qPCR as described in Materials and Methods. PCR products for <i>Gapdh</i> and <i>Omp</i>, become detectable at earlier PCR cycles than products for genes expressed at lower levels, such as <i>Olfr543</i>, <i>Olfr1444</i> and <i>Olfr558</i>. Note that qPCR curves for <i>Olfr543</i> and <i>Chrm3</i> are virtually identical, suggesting that Olfr543 and M3-muscarinic receptor are expressed at similar levels in the cornea. When PCR primers were located within a single exon (<i>Gapdh</i> and <i>Olfr558</i>, Fig. 3B) PCR products were detected even in the absence of the RT reaction. However, they were detected at much later cycles compared to the reactions with the RT step, thus, contributing less than 2% to the total PCR product.</p

Localization of OMP and Gαolf expression in the mouse corneal epithelium.

<p>Imaging of corneal slices was performed as described in Materials and Methods. A. <i>In situ</i> hybridization with OMP (green) and Gαolf (red) probes. The merged image also shows bright field and DAPI staining (blue). The corneal layers are indicated on the right: epithelium, stroma with keratocytes and Descemet’s membrane with the endothelium (DM+endo). B. A higher magnification image of the corneal epithelium stained with OMP and Gαolf probes. Epithelial cell layers are indicated on the right – basal cells (BC), wing cells (WC) and superficial surface cells (SC); str – the stroma. Note that Gaolf is mostly expressed in wing cells, whereas OMP is mostly in superficial cells. C. Expression of Gαolf (red) in the mouse retina. Retinal cell layers are indicated on the right – ganglionic layer (GL), inner nuclear layer (INL) and outer nuclear layer (ONL); OS – photoreceptor outer segments.</p

GPCR/Olfr related genes found in the corneal transcriptome.

<p>RT-PCR – the expression of the gene was (yes) or was not (no) confirmed by RT-PCR, or not tested (−); see Materials and Methods for details.</p

Image_3_A Potential Compensatory Role of Panx3 in the VNO of a Panx1 Knock Out Mouse Model.JPEG

<p>Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared. Using qRT-PCR and Immunohistochemistry (IHC), we confirmed the loss of Panx1, found similar Panx2 expression levels in both models, and a significant upregulation of Panx3 in mice with a global ablation of Panx1. Specifically, Panx3 showed upregulated expression in nerve fibers of the non-sensory epithelial layer in juvenile and adult KO mice and in the sensory layer of adults, which overlaps with Panx1 expression areas in WT populations. Since both social behavior and evoked ATP release in the VNO was not compromised in KO animals, we hypothesized that Panx3 could compensate for the loss of Panx1. This led us to compare Panx1 and Panx3 channels in vitro, demonstrating similar dye uptake and ATP release properties. Outcomes of this study strongly suggest that Panx3 may functionally compensate for the loss of Panx1 in the VNO of the olfactory system, ensuring sustained chemosensory processing. This finding extends previous reports on the upregulation of Panx3 in arterial walls and the skin of Panx1 KO mice, suggesting that roles of Panx1 warrant uncharacterized safeguarding mechanisms involving Panx3.</p

Olfactory receptor gene transcripts detected in the cornea by RT-PCR.

<p>Predicted exons –number of gene exons predicted by bioinformatics methods.</p><p>Found exons –number of exons from which transcripts were found after NGS.</p><p>RT-PCR – the expression of the gene was (yes) or was not (no) confirmed by RT-PCR; see Materials and Methods for details.</p><p>% identity – the percentage of identical amino acids in the mouse protein and its human homolog.</p