Circadian-clock driven cone-like photoreceptor phagocytosis in the neural retina leucine zipper gene knockout mouse

PURPOSE Whereas much information is available on rod outer segment phagocytosis by the retinal pigmented epithelium (RPE), corresponding data for cones are quite limited, especially in laboratory models of normal rats and mice with very low cone numbers. To characterize the light and circadian control of cone photoreceptor phagocytosis in mice, we capitalized on the blue cone-like phenotype of neural retina leucine zipper gene (Nrl) null mice (Nrl(-/-)). METHODS Nrl(-/-) mice were maintained under standard cyclic light (12h:12h light-dark [LD] cycle; light=300 lux) for one month, then divided into two groups: 1) continued maintenance in LD (36 mice); or 2) transferred to constant darkness (DD; 21 mice) for 36 h. Animals were sacrificed every 3 h over 24 h, and their eyes were rapidly enucleated and fixed. Cryosections were stained using specific cone short-wavelength opsin antibodies. Phagosome numbers in the RPE were quantified with a morphometric system. We monitored the expression of c-mer proto-oncogene tyrosine kinase (MerTK) in wild-type and knockout mice using a specific MerTK antibody. RESULTS In LD, cone phagocytosis showed a statistically significant peak of activity 1 h after light onset, 2-3 fold higher than at other times. In constant darkness, the temporal phagocytic profile resembled that of LD (significant peak at 1 h of subjective day), but the number of phagosomes was decreased at all time points. Immunostaining of MerTK in wild-type and Nrl(-/-) mice showed expression at the apical surface of the RPE. CONCLUSIONS Cone-like outer segment phagocytosis in Nrl(-/-) mice shows a similar profile to that of rods in normal mice and other species. These data are the first to quantify blue cone-like photoreceptor phagocytosis under different lighting conditions in mice, and suggest this model may constitute a valuable system for investigating circadian regulation of cone function.