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. 2018 Mar 16;7:e33680. doi: 10.7554/eLife.33680

Figure 2. Both regenerative and non-regenerative eyes are homeostatically maintained.

(A) Control and notum(RNAi) animals were fed dsRNA food every three days for 35 days then starved and individually tracked for 200 days and imaged every 30–40 days to monitor stability of the duplicated eyes. (B) Left, cartoon of eye differentiation showing production of photoreceptor neurons (PRN) and pigment cup cells (PC) from ovo+ progenitors. Two-eyed control and four-eyed notum(RNAi) animals were generated by 35 days of dsRNA feeding were then treated with control or ovo dsRNA for 60 days by feeding. ovo inhibition caused loss of both the ectopic and pre-existing eyes of notum(RNAi) animals (12/12 sets of eyes). (C) Two-eyed control and four-eyed notum(RNAi) animals were injected with BrdU following 35 days of RNAi feeding, fixed 14 days later and stained by FISH for opsin (magenta), tyrosinase (cyan) and immunostained with anti-BrdU (gray). The head regions of BrdU-labeled notum(RNAi) animals had BrdU +cells in the anterior eyes (11/12 animals) and the posterior eyes (12/12 animals), a similar frequency as control animal eyes (14/14 animals). C (bottom), quantification of BrdU+ opsin+ cells after 7 or 14 days of BrdU pulsing measured per eye (left) or across all eyes (right) for each condition. p-values from 2-tailed t-tests, **p<0.01. Cartoons depict location of eyes imaged with insets showing single and multichannel enlarged images of BrdU +eye cells.

Figure 2.

Figure 2—figure supplement 1. Quantification of BrdU-labeling in notum(RNAi) animals.

Figure 2—figure supplement 1.

Maximum projections of eye cells labeled with opsin and fixed 14 days after BrdU pulsing and quantified in Figure 2C, with double and single channel images indicated along with BrdU +opsin+ cells (yellow arrows). Anterior, top. Bars, 25 microns.

Figure 2—figure supplement 2. Regenerative and non-regenerative eye sizes respond to growth.

Figure 2—figure supplement 2.

4-eyed notum(RNAi) animals were generated by dsRNA treatment followed by 28 days of head fragment regeneration, imaged (0d) fed dsRNA for 24 days, re-imaged, and the area size of the eye pigment cups measured in microns^2.

Figure 2—figure supplement 3. Injury-induced gene expression can occur near non-regenerative eyes.

Figure 2—figure supplement 3.

Control RNAi and 4-eyed notum(RNAi) animals were prepared by homeostatic inhibition for 40 days, resected to remove either normal, anterior or posterior eyes as shown, then fixed at 0 hr or 6 hr and stained for expression of jun-1, fos-1, and gpc-1. Injury-induced gene expression was similar between control and notum(RNAi) animals and between removal of either anterior or posterior eyes.