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Figures
- Fig. 1.
Eyes of Prp31 mutant flies have no gross morphological abnormalities at eclosion. (A) Schematic of chromosome arm 3L. Prp31 and scarlet (st) are situated 2 cM apart (3–42 and 3–44, respectively; cytological positions 71B6 and 73A3, respectively; www.flybase.org). In both Prp31 mutant alleles the marker st1 from the original mutagenised chromosome (ru st e ca) is retained. The three deficiencies used cover the Prp31 locus, but not the st locus. (B) Schematic overview of the Drosophila Prp31 protein. The figure is drawn to scale using IBS (Liu et al., 2015). Domains described here are indicated. The two Prp31 alleles studied here carry non-conservative missense mutations, G90R in Prp31P17 and P277L in Prp31P18. (C–F) Representative bright-field images of Toluidine-blue stained semi-thin sections of eyes of w* (C), w*;; st1/+ (D) Prp31P18 /+ (E) and Prp31P17 /+ (F). Complete genotypes can be found in Table S1. Upon eclosion, flies were kept for 2 days under regular light conditions. Note that the number and stereotypic arrangement of photoreceptor cells within the mutant ommatidia are not affected. Scale bar, 10 µm.
- Fig. 2.
PRCs of heterozygous Prp31P17 and Prp31P18 flies undergo light-dependent degeneration. (A–D) Representative bright-field images of Toluidine-blue stained semi-thin sections of eyes of w* (A), w*;; st1/+ (B), Prp31P18/+ (C), and Prp31P17 /+ (D). Complete genotypes can be found in Table S1. Upon eclosion, flies were kept for 2 days under regular light conditions and then subjected to a degeneration paradigm of 7 days of continuous, high intensity light exposure. Whereas in w* (A) most ommatidia (red outlines) display seven rhabdomeres indicative of the seven PRCs, w*;; st1/+ and Prp31 mutant ommatidia (B–D, red outlines) display fewer rhabdomeres per ommatidium indicative of degeneration. Scale bar, 10 µm. (E) Quantification of retinal degeneration as indicated by the number of surviving rhabdomeres observed upon high intensity, continuous light exposure. Y-axis: percent frequency of ommatidia displaying one to seven rhabdomeres. Genotypes are indicated below. Number on top of each graph indicates the mean percentage of ommatidia displaying the full complement of seven rhabdomeres. Bars represent mean±s.e.m. (a minimum of n=60 ommatidia from eyes of three biological replicates). Statistical significance of differences in this parameter, between genotype pairs, is indicated in Table S2.
- Fig. 3.
RNAi-mediated knockdown of Prp31 results in light-dependent retinal degeneration. (A,B) Representative bright-field images of Toluidine-blue stained semi-thin sections of eyes of Rh1-Gal4> (A; control) and Rh1-Gal4>UAS Prp31RNAi (B). Complete genotypes can be found in Table S1. Upon eclosion, flies were kept for 2 days under regular light conditions and then subjected to a degeneration paradigm of 7 days of continuous, high-intensity light exposure. In case of Rh1-Gal4>UAS Prp31RNAi, fewer ommatidia with seven rhabdomeres are seen. Scale bar, 10 µm. (C) Quantification of retinal degeneration as indicated by the number of surviving rhabdomeres observed upon high intensity, continuous light exposure. Y-axis: percent frequency of ommatidia displaying one to seven rhabdomeres. Genotypes are indicated below. Number on top of each graph indicates the mean percentage of ommatidia displaying the full complement of seven rhabdomeres. Bars represent mean±s.e.m. (a minimum of n=60 ommatidia from eyes of three biological replicates). Whilst 71% of control ommatidia have seven rhabdomeres/ommatidium, this number is significantly reduced to 48% upon knocking-down Prp31 by RNAi (P<0.05, shown in Table S2).
- Fig. 4.
Flies heterozygous for deficiencies that remove the Prp31, but not the scarlet locus, undergo light-dependent degeneration. (A–D) Representative bright-field images of Toluidine-blue stained semi-thin sections of eyes of males of cn bw (A), Df (3L) Exel 6262/+ (B), Df (3L) ED217/+ (C) and Df (3L) ED218/+ (D). Complete genotypes can be found in Table S1. Upon eclosion, flies were kept for 2 days under regular light conditions and then subjected to a degeneration paradigm of 7 days of continuous, high intensity light exposure. Red circles outline individual ommatidia. Scale bar, 10 µm. (E) Quantification of retinal degeneration as indicated by the number of surviving rhabdomeres observed upon high intensity, continuous light exposure. Y-axis: percent frequency of ommatidia displaying one to seven rhabdomeres. Genotypes are indicated below. Number on top of each graph indicates the mean percentage of ommatidia displaying the full complement of seven rhabdomeres. Bars represent mean±s.e.m. (a minimum of n=60 ommatidia from eyes of three biological replicates). Statistical significance of differences in this parameter between genotype pairs is indicated in Table S2.
- Fig. 5.
PRCs of heterozygous Prp31P18 flies undergo light-dependent degeneration in the presence of st overexpression. (A–C) Representative bright-field images of Toluidine-blue stained semi-thin sections of eyes of GMR-Gal4> (A; control) and GMR-Gal4>; Prp31P18 /+ (B; mutant control) and GMR-Gal4 >;UAS-st ; Prp31P18 /+ (C; mutant and st overexpression). Complete genotypes can be found in Table S1. Upon eclosion, flies were kept for 2 days under regular light conditions and then subjected to a degeneration paradigm of 7 days of continuous, high-intensity light exposure. In the presence of st overexpression, PRCs of heterozygous Prp31P18 flies undergo degeneration to the same extent (C) as without st overexpression (B). Scale bar, 10 µm. (D) Quantification of retinal degeneration as indicated by the number of surviving rhabdomeres observed upon high intensity, continuous light exposure. Y-axis: percent frequency of ommatidia displaying one to seven rhabdomeres. Genotypes are indicated below. Number on top of each graph indicates the percentage of the mean number of ommatidia displaying the full complement of seven rhabdomeres. Bars represent mean±s.e.m. (a minimum of n=60 ommatidia from eyes of three biological replicates). Statistical significance of differences in this parameter between genotype pairs is indicated in Table S2.
- Fig. 6.
Reduction of Prp31 results in increased Rhodopsin 1 accumulation. Representative confocal images of 1 µm optical sections from 12 µm cross-sections (A–E), or whole mounts (A′–C′) of eyes of adult males with the genotypes indicated, stained with anti-Rh1. Images were taken using the same settings for mutant conditions and their respective controls. Rhabdomeres are shown in cross sections (A–E) and along their length (A′–C′) with the distal end directed towards the top and the proximal end directed towards the bottom. Red arrowheads indicate Rh1 staining in the rhabdomere and blue arrowheads indicate intracellular Rh1. Rh1 staining is more intense in the rhabdomeric membrane of Prp31P18/+ (C,C′) as compared to controls, w* (A,A′) and w*;; st1/+ (B,B′). Increased Rh1 immunostaining intensity is also observed in Prp31P18/ Prp31P18 (E) as compared to its genetic control (D). Scale bars, 10 µm. (F) Representative western blot for β-Tubulin and Rh1 from head lysates of w*;;Prp31P18 /+ and its genetic control w*;;st1/+, and for w*;;Prp31P18 / Prp31P18 and its genetic control w*;;st1/ st1. Complete genotypes can be found in Table S1. (G) Quantification of Rh1 levels normalised to Tubulin. Graph displays mean±s.e.m. of Rh1 levels calculated from intensity measurements of blots after normalisation compared to that of loading control (Tubulin) with each dot representing one biological replicate. On average, Rh1 levels are increased by 320% (P<0.05, Student's Unpaired t-test) in w*;;Prp31P18/+ as compared to control, w*;;st1/+ and by 140% (P<0.05, Student's Unpaired t-test) in w*;;Prp31P18 / Prp31P18 as compared to its genetic control w*;;st1/ st1. Complete genotypes can be found in Table S1.
- Fig. 7.
Carotenoid-depleted diet limits the extent of light-induced degeneration in heterozygous Prp31 mutants. (A–C′) Representative images of 1 µm confocal optical sections from 12 µm cryosections of male eyes. Genotypes indicated. Tissues were immunostained for Rh1 (white) and labelled with phalloidin (magenta) and DAPI (green), to stain the rhabdomeres and nuclei, respectively. (A–C) Overlay of two (A) and three (B,C) channels, (A′–C′) images showing the extracted channel (Rh1). Reduction in Rh1 levels and change in its localisation from the rhabdomeres to a peri-nuclear localisation is observed in control (B,B′) and mutant (C,C′) flies fed on a carotenoid-depleted diet (B–C′) as opposed to flies fed on standard food (A–A′). Arrowheads indicate Rh1 localisation in the rhabdomere as opposed to peri-nuclear localisation. Scale bar, 5 µm. (D–F) Representative bright-field images of Toluidine-blue stained semi-thin sections of eyes of w* (D), w*;Prp31P18/+ (E) and w*;;crb11A22 (F) adults. Complete genotypes can be found in Table S1. Animals were raised on a carotenoid-depleted diet. Upon eclosion, they were aged for 2 days under regular light conditions and then subjected to a degeneration paradigm of exposure for 7 days to continuous, high-intensity light. Scale bar, 10 µm. (G) Quantification of retinal degeneration as indicated by the number of surviving rhabdomeres observed upon high intensity, continuous light exposure. Y-axis: percent frequency of ommatidia displaying one to seven rhabdomeres. Genotypes are indicated below. Number on top of each graph indicates the mean percentage of ommatidia displaying the full complement of seven rhabdomeres. Bars represent mean±s.e.m. (a minimum of n=60 ommatidia from eyes of three biological replicates). Statistical significance of differences in this parameter between genotype pairs is indicated in Table S2.
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