The Majority of the Photoreceptors in the rd7 Retina Represent a Morphologically Hybrid Cell Type
In order to assess the morphologic effects of the above gene expression changes, the ultrastructure of the photoreceptor cell bodies in the rd7 mutant was examined. The cell bodies were chosen for evaluation rather than the outer segments, since in the mouse, the ultrastructural differences between rod and cone somata are much greater than are the differences between the outer segments [26]. In the wild-type mouse, cone cell bodies are aligned along the scleral border of the ONL, and they are larger than those of rods. They have a smaller, more irregular mass of nuclear heterochromatin that is often broken up into multiple discrete clumps connected by thin threads. They also have more abundant electron-lucent euchromatin than rods. Lastly, they frequently have a patch of organelle-rich cytoplasm next to their nuclei, usually containing large mitochondria [26].
Analysis of toluidine blue-stained semi-thin sections revealed that such cone-like cells were present in greater abundance in the rd7 retina than in wild-type, and that their somata were scattered throughout the ONL (Figure 7). A comparison between the distribution of these cells and those expressing S-cone opsin strongly suggests that they represent the same cell population (compare Figure 7D and 7F). Analysis of the nuclear morphology of dissociated retinal cells stained for S-cone opsin by dissociated cell in situ hybridization confirmed that this is the case (unpublished data). These findings, along with the absence of rhodopsin staining in these cells (see Figure 3D–3F), suggest that these “cone-like” cells in the rd7 mutant retina may represent supernumerary normal cones with an abnormal localization of their cell bodies.
Figure 7  The rd7 Mutant Retina Contains a Morphologically Hybrid Photoreceptor Cell Type in Addition to Supernumerary S-Opsin–Positive Cones
(A and B) Toluidine blue-stained semi-thin sections of the outer nuclear layer (scleral edge oriented up).
(C and D) Hand-drawn diagrams of the cells in (A) and (B), respectively. Cells with the nuclear features of cones are highlighted in blue. Note that the number of such cells is greater in the mutant, and their cell bodies are scattered throughout the outer nuclear layer. In addition, the overall columnar architecture of the outer nuclear layer seen in the wild type is disrupted in this portion of the mutant retina. Other portions of the mutant retina with fewer supernumerary cone cells, however, retain the normal columnar appearance (unpublished data).
(E and F) Images of the outer nuclear layer (scleral edge up) stained by in situ hybridization for S-opsin. Note the typical pattern of staining at the scleral edge of the outer nuclear layer in the wild type. The rd7 mutant retina shows supernumerary S-opsin–positive cells scattered throughout the outer nuclear layer in a distribution very similar to the supernumerary cone cells seen in (B). Since images (E) and (F) derive from different retinas than those depicted in (A) and (B), the location of the individual cells do not correspond.
(G and H) Electron micrographs of the outer nuclear layer (10,000× magnification). Note the uniform distribution of rod cell bodies in the wild type (G). The cell bodies are nearly round and consist almost exclusively of a nucleus with a single, dense mass of heterochromatin. In the rd7 mutant (H), two types of cell are shown. The ovoid one with a lesser quantity of heterochromatin, paler euchromatin, and two juxtanuclear mitochondria (yellow arrow) represents a typical cone cell body. The adjacent cell with a more “rod-like” mass of heterochromatin and a single juxtanuclear mitochondrion (red arrow) represents one of the hybrid photoreceptors discussed more fully in the main text. In contrast to the cone cell body, the wild-type rod soma is small and nearly round. It has a single, large clump of dense heterochromatin, a thin rim of moderately electron-dense euchromatin, and very scant juxtanuclear cytoplasm without organelles [26,27]. The second cell population in the ONL of the rd7 retina has some of the nuclear features of normal rods, such as a single, dense mass of heterochromatin and moderately electron-dense euchromatin (Figure 7H); yet these cells also show features of cones. First, the euchromatin is, on average, more abundant in these cells than in wild-type rods (compare Figure 7G and 7H). In addition, comparison of the diagrammatic representation of the wild-type and rd7 ONLs suggests that the average area of the S-opsin–negative cells in rd7 is greater than in the wild-type (Figure 7C and 7D). In order to confirm this impression, we quantitated the area of 50 wild-type and 50 mutant rod-like cell bodies (see Materials and Methods for details). This experiment confirmed that the average area of the rod-like somata in rd7 is approximately 30% larger than that of wild-type rod somata (mean area in rd7 was 9.75 ± 1.36 (standard deviation) μm2, compared to wild-type rods, with 7.53 ± 0.72 μm2 ; n = 50; p = 7.6 × 10–16, Student's t-test). It is also notable that the standard deviation of the somal area is nearly twice as great in rd7 than in wild-type, confirming the subjective impression of greater variability in somal size and shape in the mutant compared to the wild-type (compare Figure 7C and 7D).
Lastly, 38% (19/50) of the rd7 photoreceptors selected for somal area quantitation had prominent juxtanuclear mitochondria (red arrow in Figure 7H; unpublished data). Such juxtanuclear organelles are only very rarely seen in normal rods (1.5%; six out of 399 cells counted), but are common in cones (yellow arrow in Figure 7H). In conclusion, it is clear that this second cell population in the rd7 retina has morphological features of both normal rods and cones consistent with the coexpression of many rod- and cone-specific genes in these cells.