PMC:1186732 / 15906-21536 JSONTXT

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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1186732","sourcedb":"PMC","sourceid":"1186732","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1186732","text":"Novel Cone-Specific Genes Are Derepressed in rd7\nGiven that the majority of known cone-specific genes showed marked derepression in the rd7 mutant, additional candidate genes up-regulated on microarray analysis were evaluated for cone-specific expression. In situ hybridization was performed on an additional 45 up-regulated genes, confirming that 21 of them were derepressed. Of these, at least ten showed a definite cone-specific or cone-enriched pattern of expression in the wild-type retina (Figure 1, genes G26–G35). Several examples are given in Figure 4. Note that in the wild-type retina, there is a relatively weak pattern of scattered positive cells at the scleral edge of the ONL, consistent with a cone-specific pattern of expression. All of these genes show marked derepression in the rd7 retina. A number of these novel cone-specific genes showed a striking localization of their transcripts to the photoreceptor inner segment (e.g., Bub1b and Tcta). This localization manifests in a section in situ hybridization as a dark band of staining just beyond the outer edge of the ONL immediately underlying the outer segment layer. Although such a pattern of transcript localization is commonly seen in many rod-specific genes (e.g., Rho in Figure 2; Pcdh21, Rbp3, and Cnga1 in Table S2), it is not easily appreciated in cone-specific genes, possibly due to the relative scarcity of cones in the mouse. In the rd7 mutant retina in which such genes are widely derepressed, such a pattern of transcript localization often becomes apparent.\nFigure 4 Expression Patterns of Several Novel Cone Genes Up-Regulated in rd7\nIn the wild-type images (wt), note the scattered, weakly positive cells at the scleral edge of the outer nuclear layer in a cone distribution. All of the genes show marked up-regulation in the rd7 mutant. Bub1b and Tcta show transcript localization predominantly to the inner segment of the photoreceptors. Retinas are oriented such that the scleral edge is up. In addition to the ten genes that showed cone-specific expression in the wild-type retina, another 11 novel genes were derepressed in the rd7 retina by in situ hybridization (Figure 1, genes G36–G46). Some of these genes showed faint expression in a cone-like distribution (see Table S1, genes G36, G40, and G44), and one appeared to be expressed throughout the ONL but at greater levels in cones than in rods (Table S1, gene G37). The remainder of the up-regulated genes did not have detectable cone staining in the wild-type retina. Despite this apparent absence of cone staining, the pattern of derepression in rd7 suggests that these genes may also be novel cone-specific genes, albeit expressed at levels below the sensitivity threshold of our in situ hybridization assay.\nIn most cases, the novel cone genes identified in this study appear to have a type I pattern of derepression. However, due to the weakness of the signal in some cases, or transcript localization to the inner segment in others, it was not always possible to determine with confidence which of the two patterns of derepression (if either) each of these genes displayed. In terms of functional categorization, the novel cone genes cover a broad range including glucose metabolism (Pygm and Glo1), fatty acid metabolism (Elovl2), DNA repair (Smug1), cell cycle/chromosome segregation (Bub1b), carcinogenesis (Tcta), endothelial biology (Ece1), cytoskeletal function (Ebp4.1l1), and even otolith formation (Otop3).\nA relatively frequent finding among both previously identified cone-specific genes, as well as in some of those identified in the present study, is the occurrence of gene expression in an early photoreceptor precursor pattern (Figure 5). This pattern of expression consists of positive staining by in situ hybridization specifically at the scleral border of the retina during prenatal timepoints (in the range of E13–E18). Gnb3 and Thrb2 are two examples of known cone genes with this early pattern of expression (Figure 5). Two of the 11 novel cone genes identified in this study also have this early photoreceptor pattern of expression (Ece1 and Otop3). Intriguingly, three genes shown to be up-regulated in rd7 on microarray, but that had either no detectable signal by in situ hybridization at adult stages or no apparent change in expression by in situ hybridization between wild type and rd7, also showed this early photoreceptor pattern (Figure 1, genes G48–G50). The embryonic expression pattern of two of these genes is shown in Figure 5 (the embryonic in situ hybridization for the third, G50, can be found in Table S1). Although the significance of such early photoreceptor expression is not known, it is possible that these genes may also be cone-specific but are expressed at undetectably low levels in the adult.\nFigure 5 Some of the Genes Up-Regulated in rd7 Show an Early Photoreceptor Pattern of Expression\nGnb3 and Thrb2 are both previously characterized cone genes that show staining at the scleral edge of the embryonic mouse retina in cells that will differentiate into photoreceptors. Ece1 and Otop3 are two novel cone genes identified in this study that were up-regulated in rd7 and also showed an early photoreceptor pattern of expression. Prdm1 and RIKEN cDNA 1300018I05 (Figure 1, genes G48 and G49, respectively) are two other genes that had either undetectable signal (Prdm1) or no apparent change in expression pattern in adult rd7 mutants (RIKEN cDNA 1300018I05), but which also showed staining in the embryonic retina in a presumptive photoreceptor pattern. All images are from E17.5 retina except Gnb3, which was from 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