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{"project":"2_test","denotations":[{"id":"16110338-11487564-85798515","span":{"begin":2986,"end":2988},"obj":"11487564"},{"id":"16110338-9786999-85798516","span":{"begin":3733,"end":3735},"obj":"9786999"},{"id":"16110338-11055434-85798517","span":{"begin":3820,"end":3822},"obj":"11055434"},{"id":"T57828","span":{"begin":2986,"end":2988},"obj":"11487564"},{"id":"T56243","span":{"begin":3733,"end":3735},"obj":"9786999"},{"id":"T93871","span":{"begin":3820,"end":3822},"obj":"11055434"}],"text":"Two Distinct Patterns of Cone Gene Derepression in rd7\nIn order to confirm these microarray results, an in situ hybridization analysis of the putative up-regulated cone genes was carried out in which the rd7 mutant retina was compared with age-matched, wild-type controls. We found that the majority of the cone-specific genes that were up-regulated in microarray experiments were derepressed when assessed by in situ hybridization (Figure 2). There were two major patterns of cone gene derepression. The more common pattern (type I) manifested itself as ectopic gene expression throughout the ONL, consistent with gene expression in all photoreceptors (Figure 2; upper left photomicrographs). Typical examples of this pattern of derepression are shown in Figure 2, and many more are available in Table S1. This pattern of expression contrasts sharply with the usual pattern of cone gene expression, which consists of scattered cells localized to the scleral edge of the ONL (Figure 2).\nFigure 2 Cone and Rod Gene Expression in the rd7 Mutant at P14\nThe upper sets of photomicrographs demonstrate examples of type I and type II cone gene derepression in the rd7 mutant retina as explained in the main text. The bottom left images show several rod-specific genes that are essentially unchanged in the rd7 background at P14. The bottom right images show the expression pattern of three photoreceptor transcription factors in the rd7 mutant. Abbreviations in the lower left hand corner of each pair of panels represent the gene symbols summarized in Figure 1. The second category of cone gene derepression (type II) consisted of a patchy, salt-and-pepper pattern of ectopic expression in which individual positive cells were scattered throughout the ONL (Figure 2, upper right photomicrographs; Table S1). Although numerous positive cells were present in the rd7 retina (particularly in the ventral portion), there were clearly many interspersed cells that showed a complete absence of expression. In order to rule out the possibility that these scattered positive cells were simply the normal complement of cones that had failed to localize their cell bodies to the scleral edge of the ONL, the number of positive cells in the rd7 retina was quantitated by dissociated cell in situ hybridization.\nDissociated cell in situ hybridization was performed using a probe for the S-cone opsin gene (Opn1sw), which shows type II derepression (Figures 2 and 3A–3C). S-opsin was expressed in 3.2% of retinal cells in the rd7 mutant (66 S-opsin–positive cells out of 2,056 6-diamidino-2-phenylindole [DAPI]-positive cells). This value is approximately 2-fold greater than the percentage of S-opsin–positive cells identified in wild-type control retinas, 1.65% (54 S-opsin-positive cells out of 3,271 DAPI-positive cells), and accords well with the previously reported value of 2- to 3-fold more S-opsin–positive cells in rd7 compared to wild type arrived at by antibody staining of tissue sections [17].\nFigure 3 S-Opsin Dissociated Cell In Situ Hybridization and S-Opsin/Rhodopsin Antibody Staining on rd7 Mutant Retina\n(A–C) A dissociated cell in situ hybridization with an S-opsin probe (red) on dissociated rd7 mutant retinal cells stained with DAPI (blue). (C) shows the merged images.\n(D–F) The outer nuclear layer of an rd7 mutant retina stained by antibody for S-opsin (red) and rhodopsin (green). The scleral edge of the outer nuclear layer is up. DAPI staining is in blue. (F) shows the merged images. Insets are higher-power images of the outer segments showing non-overlap of S-opsin and rhodopsin staining in the mutant. Previous studies have estimated that the total number of cones in the mouse retina is 2% of all retinal cells [20], and that S-opsin is largely repressed in cones in the dorsal third of the retina [21]. The estimate of 1.65% S-opsin–positive cells in the wild-type retina is in agreement with these data. The fact that only 3.2% of all retinal cells are S-opsin–positive in the rd7 mutant also confirms that the majority of the photoreceptors (which make up just over 70% of the cells in the adult mouse retina) do not express this gene. In order to assess whether these supernumerary S-opsin–expressing cells coexpressed rod-specific markers, a double antibody staining for S-opsin and rhodopsin was performed. This study showed mutually exclusive domains of expression of S-opsin and rhodopsin in the photoreceptor outer segments (Figure 3D–3F). This finding suggests that the supernumerary S-opsin–expressing cells in the rd7 retina may represent normal “blue” cones."}

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PMC:1186732 / 11312-15904 JSON TXT