Crx is necessary for the formation of photoreceptor terminals
In a previous study, we demonstrated that forced expression of a dominant-negative allele of Crx in developing rods blocked formation of both rod spherules in the outer plexiform layer (OPL) and outer segments [7]. To expand on these studies, the ultrastructure of photoreceptor synapses was examined in Crx-/- retinas. In Crx+/+ retinas at P21, newly mature rod spherules were abundant (Figure 7A). The sperules were blunt or club-shaped structures with a single ribbon associated with a single invaginating synapse (Figure 7A, arrow indicates one example; Figure 8A and 8B). Two processes from horizontal cells were situated on either side of the synaptic ridge (Figure 8B, labelled H) and one or more dendrites of rod bipolar cells occupied a central position (Figure 8B, bipolar labelled B). Cone terminals are large, flat pedicles that exhibit many invaginating synapses containing separate sets of horizontal and bipolar cell processes. Each pedicle contains numerous ribbons. These terminals were much less common than spherules in Crx+/+ retinas at P21 (Figure 7, box). In the OPL of Crx-/- retinas, photoreceptor terminals were highly disorganized at P21 (Figure 7B, arrows). Processes containing synaptic vesicles and ribbon-like structures were apparent, suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. In addition, many terminals appeared to contain multiple ribbons (Figure 8C and 8D, r) not tethered to the plasma membrane and occasionally in perinuclear positions (Figure 8D).
Figure 7  Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas. (A) In Crx+/+ retina at P21, newly formed rod spherules were abundant (arrow demonstrates one example). The spherules were club-shaped and contained a single invaginating synapse with one ribbon complex. Cone terminals form large, flat pedicles that contain many invaginating synapses with separate ribbon structures. These terminals were more scarce, but apparent in Crx+/+ retinas at P21 (one example in box). (B) In the outer plexiform layer (OPL) of Crx-/- retinas, photoreceptor terminals appeared highly disorganized at P21 (arrows). Well-formed pedicles and spherules were not evident. Putative terminals containing ribbon-like structures were apparent, suggesting at least limited generation of synapse components. Many terminals appeared to contain multiple ribbon-like structures, instead of a singule ribbon. For example, terminal 1 and 2 contained two ribbons each, whereas terminal 3 appeared to contain only one. opl, outer plexiform layer. Scale bar = 2 μm.
Figure 8  Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas at P21. (A) Crx+/+ rod spherules contained a single invaginating synapse with one ribbon complex. The spherule was a blunt or club-shaped structure. (B) Crx+/+ rod terminals contained a single ribbon structure (r). Two processes, from horizontal cells (h), contacted the rod laterally. An additional process, the postsynaptic bipolar dendrite (b), was situated more centrally. Terminals were filled with synaptic vesicles. One coated vesicle originatinf from the cell membrane was observed (arrow). (C) In the OPL of Crx-/- retinas, photoreceptor terminals appeared highly disorganized. Putative terminals containing synaptic vesicles and ribbon-like structures were apparent (arrows), suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. Further, many terminals appeared to contain multiple ribbon-like structures (r). The majority of these ribbons were not associated with the synaptic membrane, but instead were found free floating and, in some instances, were perinuclear (D, arrow). H, horizontal cell; B, bipolar cell; N, nucleus; r, ribbon. (A) Scale bar = 500 nm, (B) Scale bar = 250 nm, (C and D) Scale bar = 500 nm.