Phagocytosed Photoreceptor Outer Segment Particles Within the Retinal Pigment Epithelium Show Diurnal Rhythmicity and Variation Between Cone Subtypes in Larval Zebrafish

Phagocytosis of retinal rod and cone outer segment (OS) tips by the retinal pigment epithelium (RPE) occurs daily to prevent the accumulation of harmful compounds in the photoreceptors. Rhythmic bursts seen as increased numbers of phagocytosed OS particles in the RPE are known to appear once or twice a day depending on the animal species. Yet, the variation of this rhythmicity between the distinct photoreceptor types is not well understood. We used zebrafish to compare the phagosome numbers and their daily rhythms between the different cone subtypes. We immunolabeled the different cone opsins from the histological sections of the eyes of zebrafish larvae that were collected at seven different time points throughout a 24 h circadian cycle. Internalized OS particles were then quantified using confocal microscopy and image analysis. Interestingly, the results revealed the presence of OS particles of all cone subtypes in the RPE throughout the day in larval zebrafish. However, we observed a significant increase in the phagosome numbers from UV and blue cones at two time points, whereas the number of green cone OS particles was more constant, probably reflecting their more immature developmental stage. We also investigated whether the rhythmicity is regulated by external light by keeping the larvae in constant darkness before sample preparation. We found that the complete darkness condition diminished the phagosome numbers of all cone subtypes and abolished the daytime peaks in the UV and blue cones, indicating that the rhythmicity is strongly affected by the external light in the larval zebrafish. Our findings provide new understanding on the rhythmicity of cone OS phagocytosis and its regulation.