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Fig. 5
A GFP reporter for LWS1 indicates a switch from LWS2 to LWS1 in response to RA treatment.
A-C. Whole mount confocal images of retinas from LWS:PAC(H) embryos treated with DMSO (A) or 0.3 µM 9-cis RA (B) from 48 to 96 hpf. LWS2:RFP+ cones (red) are found in control retinas (A), while cones expressing LWS1:GFP alone (green, arrows) or co-expressing LWS2:RFP and LWS1:GFP (yellow, arrowheads) are found in retinas treated with RA (B); LWS1:GFP expression in transgenic retinas treated with RA tend to appear ventrally and peripherally (B). C. Percentage of retinas examined that contain LWS1:GFP+ cones for DMSO or 9-cis RA treatment 48 hpf to 4.5 dpf and from 75 hpf to 4.5 dpf. Error bars represent 95% binomial confidence interval. D. The pie charts show the frequency of 9-cis RA treated transgenic retinas expressing the indicated number of LWS1:GFP+ cones for treatment 48 hpf to 4.5 dpf (top) or 75 hpf to 4.5 dpf (bottom). E. Graphs show the percentage of LWS:PAC(H) transgenic retinas that contain widespread (covering more than half of the retina) LWS2:RFP+ cones at the indicated time points following treatment beginning at 48 hpf (n = 15, RA 4 dpf; 7, RA 4.5 dpf; 9, DMSO 4 dpf; 4 DMSO 4.5 dpf). Error bars represent 95% binomial confidence interval. F. Indirect immunofluorescence image of a transverse section of an LWS:PAC(H) embryo treated with 9-cis RA. The arrowhead indicates a cone co-expressing LWS1:GFP and LWS2:RFP. The arrow indicates a cone expressing LWS1:GFP. G. Graph indicating the numbers of LWS2:RFP (RFP), LWS1:GFP (GFP), or dual label (RFP/GFP) cones per section in retinas treated with DMSO or 9-cis RA (DMSO vs. 9-cis RA ***, p<0.001; 2-tailed Student’s t-test). H. Individual LWS1:GFP+ cones from retinas treated with 9-cis RA for the indicated time frames were examined for co-expression of LWS2:RFP. I. Dual in situ hybridization for LWS1 and LWS2 after treatment with 9-cis RA from 48 hpf to 4 dpf. Purple color reaction indicates LWS1 expression; pink fluorescent color indicates LWS2 expression; arrows at top show cones expressing LWS1 only; asterisks (*) show cones expressing LWS2 only; arrow at bottom shows cone that is dually labeled. J. Confocal images of pairs of cones at the end of mitosis from whole mount transgenic retinas treated with 9-cis RA from 48 hpf to 4.5 dpf. Apparent daughter cells of cone progenitors were observed expressing the same LWS opsin as well as pairs where one daughter cell also co-expresses a different LWS opsin. K. Confocal image of adult LWS:PAC(H) whole mounted retina showing isolated GFP+ (reporting LWS1) cones.