Materials and Methods

Mutant mice.
Nr2e3rd7 mutant mice were obtained from Jackson Laboratories (Bar Harbor, Maine, United States; stock #004643) and maintained on a C57BL/6 background. All control mice were C57BL/6.

Microarray analysis.
Total retinal RNA samples were isolated from P0, P6, P14, and P21 Nr2e3 mutant mice using the Trizol reagent (Gibco, San Diego, California. United States). Total retinal RNA samples from age-matched wild-type C57BL/6 mice were used as controls. Individual total RNA samples were derived from four retinas (pooled from two animals). All microarray experiments were performed in triplicate, in each case with separate RNA preparations. Microarray experiments with cDNAs were performed with the P0, P6, and P14 derived samples. Probes were labeled with either Cy3 or Cy5 using the Array 900 kit from Genisphere (Hatfield, Pennsylvania, United States) starting with 5 μg of total RNA according to the manufacturer's instructions. Wild-type control probes were compared to mutant on the same microarray. In two of the three replicates, the mutant probe was labeled with Cy3 and the wild type with Cy5, and in the third replicate the dyes were swapped. Labeled probe was hybridized to microarray slides spotted with approximately 11,500 cDNA clones from the brain molecular anatomy project library (kind gift of B. Soares, University of Iowa; see http://trans.nih.gov/bmap/index.htm for details) and 500 cDNA clones from our lab collection. Slides were printed and hybridized as described [59,60]. After hybridization and washing of slides according to the manufacturer's instructions (Genisphere), the slides were scanned on an Axon Instruments (Union City, California, United States) GenePix 4000 scanner and images were analyzed using the accompanying GenePix Pro software package. The complete raw cDNA microarray data set are available in Tables S6–S14.
Two types of normalization were performed on the raw intensity scores derived from the GenePix Pro analysis. First, for a given experiment, the average intensity of all the spots in the weaker of the two channels (Cy3 or Cy5) was normalized to those in the stronger channel. Second, in a given set of experiments done in triplicate at a particular time point, the two experiments with the weaker average signal intensity over all spots were normalized to those in the third microarray with the strongest average signal intensity. All spots with signal levels equal to or below background were removed from the analysis. The resulting files contained on average about 6,000 spots. These files were then sorted according to Cy3/Cy5 signal intensity, and those spots with the 10% highest and 10% lowest intensity ratios (approximately 600 spots/experiment) were compared across the three experiments at a given time point using custom Perl scripts (available upon request from JCC). All spots which were present in the top 10% most up- or down-regulated genes in two out of three or three out of three experiments were recorded (the latter are listed in Table S3).
Microarray analysis of the P21 retinas was performed on Affymetrix mouse genome 430 2.0 GeneChip array (Affymetrix, Santa Clara, California, United States). A total of six microarray hybridizations were performed: three with probes derived from mutant RNA and three from wild-type. Probes were synthesized starting with 10 μg of total RNA for each sample according to manufacturer's instructions (Affymetrix). Hybridization, washing, and scanning of the microarrays were all performed at the Bauer Center for Genomics Research at Harvard University according to manufacturer's instructions (Affymetrix). Initial data analysis was carried out using the GeneChip Operating System (GCOS) software from Affymetrix. Pairwise comparisons were made between individual mutant microarray results and controls. All genes were removed from the analysis for which “absent” calls were made by the software for both the wild-type and mutant samples being compared. The remaining gene lists contained approximately 26,000 transcripts. These lists were then sorted according to the mutant-to-wild-type “signal log ratio” in order to identify the most markedly up- and down-regulated genes. The top 500 most up- and down-regulated transcripts (approximately 2% of all genes in each case) from each of the three pairwise comparisons between mutant and wild-type were compared using custom Perl scripts (available upon request from JCC) to identify those genes that were present in two or three out of three lists. Those genes that were up- or down-regulated in three out of three experiments were recorded (Tables S4 and S5). The complete pairwise Affymetrix microarray datasets are available in Tables S15–S17.

In Situ hybridization.
Section in situ hybridization was performed as previously described [61] using 20-μm cryosections from OCT-embedded tissue or 4-μm paraffin sections. All in situ hybridizations were performed with the mutant and wild-type control sections on the same glass slide. Riboprobes labeled with digoxygenin-tagged UTP (Roche, Basel, Switzerland) were detected with NBT/BCIP (Sigma, St. Louis, Missouri, United States). The sources of the individual riboprobes used in this study are described in Tables S1 and S2. Dissociated cell in situ hybridization was performed as described previously [62] using the same S-opsin digoxygenin-labeled probe used for section in situ hybridization. All images were captured on a compound microscope (Eclipse e1000; Nikon, Tokyo, Japan) using a CCD camera (DXM1200F, Nikon). S-opsin positive cells were quantitated on dissociated cell in situ hybridization as previously described [62]. Twenty fields were quantitated in this manner at 200× magnification for both rd7 and wild-type retinas.

Immunohistochemistry.
For antibody staining, cryosections were prepared and stained as described previously [63]. Primary antibodies used were a polyclonal anti-blue opsin raised in rabbit (1:300; Chemicon International, Temecula, California, United States; AB5407) and a mouse monoclonal anti-rhodopsin (1:200; rho4D2). Secondary antibodies used were Cy2- or Cy3-conjugated goat anti-rabbit and anti-mouse (1:500; Jackson Immunologicals, West Grove, Pennsylvania, United States). Following antibody staining, 4′-DAPI was applied to stain nuclei (Sigma), and the sections were coverslipped and mounted in Gel/Mount (Biomeda, Foster City, California, United States).

Electron microscopy.
This protocol was adapted from one used by Raviola [64] with some modifications derived from Carter-Dawson and Lavail [26]. Four adult wild-type and four mutant animals were deeply anesthetized by intraperitoneal injection of Avertin and the eyes were then removed. The cornea was gently punctured with sharp forceps and excised with iridectomy scissors. The eye was then transferred to a solution of 2% paraformaldehyde/2% glutaraldehyde in cacodylate buffer (0.1 M cacodylic acid; 0.1% calcium chloride). The lens was gently removed and the eyecup allowed to fix for 2 h at room temperature. The fixed eye was then placed on dental wax and sectioned in the midline with a fresh razor blade (half of the retinas were sectioned along the D-V axis and half along the nasal-temporal axis). The retinas (and attached retinal pigment epithelium) were carefully dissected away from the sclera, which was discarded.
The retinas were then rinsed four times for 15 min each with Sorenson's buffer (pH 7.4). They were then stained for 2 h at 4 °C with 1% osmium tetroxide in 1.5% potassium ferrocyanide. Next, the retinas were rinsed four times for 15 min in maleate buffer (pH 5.1) and then stained for 2 h at room temperature with 1% uranyl acetate in maleate buffer (pH 6.2). They were then washed four times for 15 min with maleate buffer (pH 5.1); once for 10 min with 70% ethanol; once for 10 min with 95% ethanol; and four times for 30 min with 100% ethanol. Next, the retinas were washed three times over one hour with propylene oxide and then embedded in TAAB 812 Resin (Marivac, Quebec, Canada) for 1–2 d in a 60 °C oven. Semi-thin sections were cut at a thickness of 0.5 μm and stained with 1% toluidine blue in 1% sodium borate buffer. Images of semi-thin sections from the mutant retina were taken within the ventral two-thirds of the retina where the majority of the supernumerary S-opsin–expressing cells reside. Wild-type images were taken in comparable regions. Sections for electron microscopy were cut at a thickness of 95 nm, placed on grids, and poststained with 2% uranyl acetate followed by 0.2% lead citrate. All sections were cut in a plane perpendicular to the plane of the photoreceptor layer. They were then visualized on a Jeol 1200EX electron microscope (Jeol, Tokyo, Japan). The electron microscopic images in Figure 7 derive from the ventral two-thirds of the wild-type and mutant retinas.
The area of the cell bodies of the rods and “rod-like” cells in the wild-type and mutant ONLs, respectively, were quantitated in the following manner. First, ten fields within the ONL were chosen at random at 1,000× magnification and then photographed at 4,000× magnification for each of the two genotypes. Such images typically contained 35–45 cell bodies. In order to quantitate only the area of those cell bodies that were cut as near to the midline of the cell as possible (i.e., in order to obtain the maximal cross-sectional area) the five cells with the largest apparent area in each photograph were chosen by eye and the outline of the cell membranes were traced onto white paper. These tracings were scanned along with the size bar from the electron micrographs, and the areas of the resulting digital images were quantitated using Scion Image software (NIH Image, http://rsb.info.nih.gov/nih-image). A total of 50 cells of each genotype were quantitated in this manner. In addition, in order to evaluate the percentage of rods in the wild-type retina that had any juxtanuclear mitochondria, all rods within all ten images were counted as were the number of cells showing juxtanuclear organelles. A total of six out of 399 wild-type rods (1.5%) possessed a juxtanuclear organelle. This analysis permitted us to evaluate the wild-type rods for juxtanuclear mitochondria at multiple planes of section; however, serial sections of individual cell bodies were not performed.