Fig. 5

Zebrafish outer segment phagocytosis (OSP) is regulated by external light sources. (A,E) Time course highlighting sample collection time (red x), and light-dark cycle. Samples were collected at ZT 4, ZT 9, and ZT 17 from 16 days post fertilization (dpf) wild-type zebrafish retinae. At each time point, one cohort had undergone a regular 14:10 light:dark cycle. The other had been raised in dark from ZT 10 the day prior to collection and were euthanized under red light. (B–D, G,H) Representative TEM images of wild-type retinae at 16 dpf, with phagosomes indicated (pink arrows). Scale bar; 5 μm. (I) Scatter plots of phagosome/μm of retinal pigment epithelium (RPE) in dark-reared and control retinas at ZT 4 and ZT 17 (OSP peaks) and ZT 9 (OSP trough). Fish raised in the dark for have ~35%–55% reductions in the number of RPE phagosomes containing engulfed OS material in comparison with controls at ZT 4 and ZT 17 respectively. Statistical analysis carried out by student's t test between individual groups. RPE of >570 μm was measured per animal. N = 3 animals (6 retinae) per group per time point. (J) Trend line demonstrating the level of RPE phagosomes present in the dark-raised wild-type eyes (black) compared with controls exposed to the regular light-dark cycle (orange) at defined time points throughout the day. A two-way anova, followed by Turkey's multiple comparison testing revealed no significant difference between CT 4 CT 9 or CT 17 (black), but a significant difference between ZT 4 versus ZT 9 (p = .0229), ZT 9 versus ZT 17 (p ≤ .0001), and ZT 4 versus ZT 17 (p = .003) (orange)