High-content screening of 1351 bioactive small molecules for ROS renewal. (a) Work-flow for high-content, image-based screen. Tg(Xla.rho:EGFP); Tg(hsp70:HA-mCherry^TM); alb^−/− fish were heat shocked at 6 dpf to induce expression of HA-mCherry^TM followed by rearing in 20 μM compound until 10 dpf when tissues were fixed and processed for immunolabeling and confocal microscopy. (b) Confocal z-stack images were acquired of the GFP (green) and HA-mCherry^TM stripe (red)-expressing rods labeled with anti-Rhodopsin antibody (blue) in two regions of interest: the photoreceptor layer and ciliary margin (white-hatched boxes). (c) ROS renewal was measured within the three-dimensional z-stacks with the growth distance (D^G) representing the distance from the base of the ROS to the mCherry^TM stripe and the shedding distance (D^S) representing the distance from the mCherry^TM stripe to the tip of the outer segment. Scale bar is 10 μm. (d,e) Qualitative changes in the photoreceptor layer were documented, including the (d) accumulation of phagosomes and (e) Rhodopsin mislocalization. (f) Qualitative changes in the ciliary margin were documented, such as the size of the proliferative zone and addition of new rods during compound treatment, which can be identified by lack of the mCherry^TM stripe.

Phagosomes accumulate following treatment with PI3K/Akt/mTor inhibitors. (a) Accumulation of phagosomes, which are packets of shed ROS that can be detected with Rhodopsin immunolabeling, following disrupted RPE phagocytosis or digestion. (b) Signaling pathways targeted by compounds that resulted in phagosome accumulation. (c) Compounds that target the PI3K/Akt/mTor pathway lead to phagosome accumulation as compared to the DMSO control. Merged images show GFP-expressing rods in green, HA-mCherry^TM stripe in red, and Rhodopsin immunolabeling in blue. Scale bar is 10 μm.

Rhodopsin mislocalizes following treatment with JAK1/2 and microtubule inhibitors. (a) Rhodopsin normally localizes to the ROS and is mislocalized when detected in the inner segment, cell body, or synaptic region with Rhodopsin immunolabeling. (b) Pathways targeted by compounds that lead to Rhodopsin mislocalization. (c) Rhodopsin localizes to the ROS in DMSO treated larva. (d) Rhodopsin is mislocalized to the inner segment, as well as some puncta in the cell body (arrows), following treatment with the JAK1/2 inhibitor CYT387. (e) Rhodopsin is mislocalized to the inner segment and cell body following treatment with Vinflunine, which disrupts microtubules. Merged images show GFP-expressing rods in green, HA-mCherry^TM stripe in red, and Rhodopsin immunolabeling in blue. Scale bar is 10 μm.

Notch signaling, cell cycle, and DNA damage pathways are among those targeted by compounds that disrupt the peripheral rim of the retina. (a) The peripheral rim of the retina consists of (1) a proliferative zone, the CMZ, where stem and precursor cells reside, (2) a region of newly-formed retinal neurons and glia including GFP-positive rods, and (3) rods that have the mCherry^TM stripe because they developed prior to heat shock. (b) Pathways targeted by compounds that disrupt the peripheral rim of the retina. (c) The peripheral rim from a control DMSO-treated larva segmented into region (1) with dotted white outline, (2) with GFP (green) and Rhodopsin (blue)-positive but mCherry^TM stripe-negative rods, and (3) with GFP, Rhodopsin, and mCherry^TM (red)-positive rods. (d) The proliferative zone (1) is nearly absent following treatment with IKK inhibitor TPCA-1. (e) The proliferative zone (1) is reduced in size and newly formed rods (2) are packed tightly with γ-Secretase inhibitor (DAPT and MK-0752) treatment. (f) The proliferative zone (1) is dramatically reduced with Aurora Kinase inhibitor (Alisertib and CCT129202) treatment. (g) The proliferative zone is dramatically reduced with topoisomerase inhibitor (Irinotecan and SN-38) treatment. Scale bar is 10 μm.