Rhythmicity of photoreceptor outer segment phagocytosis differs between the cone subtypes in the larval zebrafish

Phagocytosis of retinal rod and cone outer segment (OS) tips by the retinal pigment epithelium (RPE) occurs daily to prevent accumulation of harmful compounds in the photoreceptors. Rhythmic bursts of phagocytosis, seen as increased levels of phagocytosed OS particles in the RPE, are known to appear once or twice a day depending on the animal species. However, differences in the rhythmicity of phagocytosis between distinct photoreceptor types are not well understood. Here, we show that phagocytosis of cone subtype OSs does not have identical rhythmic profiles in young zebrafish larvae. We investigated this by immunolabelling histological sections from the eyes of larvae that were collected at seven different time points throughout a 24 h circadian cycle. Internalized OS particles were then quantified from confocal images. The results revealed that OSs of all cone subtypes are phagocytosed continuously at some levels in young zebrafish. Interestingly, we observed a significant increase in the OS phagosome numbers from UV and blue cones at two time points, whereas green and red cones were phagocytosed more evenly throughout the day. We also investigated whether this rhythmicity is regulated by the external light by keeping the larvae in constant darkness before sample preparation. We found that complete darkness condition dampened the peaks in OS phagosome numbers from UV and blue cones indicating that the rhythmicity is primarily driven by the external light rather than the intrinsic circadian clocks in young larval zebrafish. Our findings provide new understanding on the rhythmicity of cone OS phagocytosis and its regulation.