Abstract
Purpose :
Regulated, cyclical peaks of outer segment phagocytosis (OSP), by the retinal pigmented epithelium (RPE), maintain healthy photoreceptors. Defective OSP is implicated in inherited and age-related blindness. However, the mechanisms instigating OSP in photoreceptors, particularly in cones, remains poorly characterised, leaving a knowledge gap in retinal biology and disease. This study investigates extrinsic environmental and intrinsic molecular regulators of OSP in zebrafish.
Methods :
Real-time PCR was carried out on cDNA generated from 15 dpf zebrafish eyes collected under normal light-dark (14h:10h) or following 1 day under dark-dark conditions. For TEM analysis of shed OS, phagosomes were manually counted, and the density calculated as phagosomes per μm of RPE (n=3). Proteomic profiling of sibling eyes collected at ZT 4, ZT 9 and ZT 17 was performed by mass spectrometry (MS) (n=3).
Results :
Real-time PCR identified candidate OSP regulators including cerkl, mertka and abcf1 to maximise expression prior to one or both OSP peaks under a regular light-dark cycle (n=3). Dark:dark environmental conditions revealed the number of RPE phagosomes at circadian time (CT) 4 (0.12 phagosomes/um RPE) was significantly (p=0.003) reduced compared to the peak number (0.18 phagosomes/um RPE) at ZT 4 under a normal light dark cycle, suggesting an absence of circadian regulation. Preliminary data (n=2) suggests that the absence of light alters the transcription profiles of several known OSP regulators. Potential novel regulators of OSP were uncovered using proteome profiling of the zebrafish retina at ZT 4, ZT 9 and ZT 17 and these are being validated by immunostaining.
Conclusions :
The data provides a model for insights into the regulators of OSP in a cone-rich retina. In summary, the results shown here identify transcript and proteomic profiles of known and putative OSP regulators, revealing highly upregulated expression levels at time-points correlating to both dawn and dusk peaks of OSP. Exposure to light:dark transition is necessary to fully initiate the burst of OSP at peaks in the zebrafish retina, and may be involved in controlling expression profiles of molecular regulators. This data also holds biomedical significance as defective OSP may be part of the disease pathway for many retinal and macular degenerations.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.