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Fig. 2 Constant darkness reduces rod outer segment shedding. (A) Schematic timeline for examining rod outer segment (ROS) renewal in 8 dpf, 3 days post heat shock (dpHS) Tg(Xla.rho:EGFP); Tg(hsp70:HA-mCherryTM) fish after rearing in normal 14-hour light/10-hour dark cycle (LD) or constant dark (DD). (B) Representative images of the photoreceptor layer from LD- and DD-reared larvae immunolabeled with anti-GFP (green), anti-HA (red), and anti-Rhodopsin (blue) antibodies. Arrows in DD image indicate accumulation of phagosome-like bodies. Images are projections of a subset of z-sections totaling 11.09 μm. Scale bar: 10 μm. Quantification of (C) shedding distance (DS) and (D) growth distance (DG) in LD- and DD-reared larvae. Lower and upper hinges of box correspond to first and third quartiles; middle corresponds to median; whiskers extend 1.5 * interquartile range above and below the hinges; dots represent outliers. Graphs represent (C) n = 4 fish/treatment (96 LD, 157 DD outer segments) and (D) n = 4 fish/treatment (128 LD, 196 DD outer segments). (E) Schematic timeline for examining ROS renewal in adult Tg(Xla.rho:EGFP); Tg(hsp70:HA-mCherryTM) fish heat-shocked and reared 6 days in LD or DD. (F) Representative images of the photoreceptor layer from LD- and DD-reared adults immunolabeled with anti-GFP (green), anti-HA (red), and anti-Rhodopsin (blue) antibodies. Images are projections of a subset of z-sections totaling 5.97 μm. Scale bar: 10 μm. Quantification of (G) DS and (H) DG in LD- and DD-reared adults. Graphs represent (G) n = 3 fish/treatment (129 LD, 142 DD outer segments) and (H) n = 3 fish/treatment (187 LD, 190 DD outer segments). *95% CI of difference compared to control does not span zero.