Analysis of a cellular structure observed in the compound eyes of Drosophila white; yata mutants and white mutants

Abstract

We previously identified the Drosophila yata mutant, which showed phenotypes including progressive vacuolization of the white-coloured compound eye, progressive shrinkage of the brain and a shortened lifespan. The yata gene was shown to be involved in controlling intracellular trafficking of the Amyloid precursor protein-like protein, which is an orthologue of Amyloid precursor protein, which is a causative molecule of Alzheimer's disease. In this study, we examined the phenotype of the compound eye of the yata mutant using electron microscopy and confocal microscopy. We found that abnormal cellular structures that seemed to originate from bleb-like structures and contained vesicles and organelles, such as multivesicular bodies and autophagosomes, were observed in aged white; yata mutants and aged white mutants. These structures were not observed in newly eclosed flies and the presence of the structures was suppressed in flies grown under constant dark conditions after eclosion. The structures were not observed in newly eclosed red-eyed yata mutants or wild-type flies, but were observed in very aged red-eyed wild-type flies. Thus, our data suggest that the observed structures are formed as a result of changes associated with exposure to light after eclosion in white mutants, white; yata mutants and aged flies.

Keywords: Ageing; Autophagy; Bleb; Drosophila; Eye; Photoreceptor.

Fig. 1.

Abnormal cellular structures were found in aged white and white; yata mutants. (A) Electron micrographs of tangential sections of ommatidia at the R8 and R7 levels in flies with the indicated ages and genotypes are shown. In the Drosophila compound eye, each ommatidium contains eight photoreceptor neurons designated R1 to R8. Because R7 is located at the distal side of the retina while R8 is located at the proximal side just beneath R7, seven rhabdomeres can always be observed in tangential sections of an ommatidium. In day 29 white and white; yata mutants, abnormal cellular structures were found in the vicinity of rhabdomeres (arrows) at both the R8 and R7 levels. Scale bar: 2 μm. (B,C) The proportions of ommatidia at the R8 (B) and R7 (C) levels that contained at least one identified structure are plotted. Each dot represents data from one fly. The raw data are shown in Table S1. **P<0.01; Games-Howell test (B) and Tukey's test (C).

Fig. 2.

The identified structures observed in day 29 white; yata mutants. (A) Highly magnified image of the identified structures. An electron micrograph of a tangential section of an ommatidium at the R8 level is shown. Rhabdomeres are indicated by open arrowheads. The identified structures contained many vesicles, multivesicular bodies (arrows), electron-dense structures (filled arrowheads), vacuoles of various sizes (asterisks) and structures with double membranes (open arrows). Mitochondria that were found outside the identified structures are labelled with a hash. (B) An electron micrograph on a horizontal section of ommatidia is shown. Rhabdomeres are indicated by open arrowheads. The identified structures were found between rhabdomeres (arrows), lateral to rhabdomeres (filled arrowheads) and embedded in the cytoplasm of photoreceptor neurons (open arrows). (C) An electron micrograph of a horizontal section of ommatidia is shown. Rhabdomeres are indicated by open arrowheads. The identified structures occasionally caused rhabdomeres to twist (arrows). (D,E) Electron micrographs of horizontal sections of ommatidia are shown. The identified structures were observed to be bleb-like protrusions (arrows) near adherens junctions (filled arrowheads). A rhabdomere is indicated by an open arrowhead. Scale bars: 500 nm (A), 2 μm (B–E).

Fig. 3.

Accumulation of autophagosomes and late endosomes in the day 15 white; yata mutants. (A) R8-level retina of day 15 white; yata mutants stained with anti-Atg8a antibody (green) that labelled autophagosomes and anti-Rab7 antibody (magenta) that labelled late endosomes were observed in an image of a single plane by confocal microscopy. Accumulated autophagosomes and late endosomes (arrows) were observed near rhabdomeres labelled with phalloidin (blue). Relatively little accumulation was observed in day 15 white mutants. Scale bar: 5 μm. (B) Numbers of signals showing the accumulation of autophagosomes and late endosomes in the R8-level retinas of day 15 white and white; yata mutants are plotted. Each dot represents data from one fly. The raw data are shown in Table S1. **P<0.01; Mann–Whitney U-test.

Fig. 4.

Formation of the identified structures in flies reared under constant dark conditions after eclosion. (A) Electron micrographs showing tangential sections of ommatidia at the R8 and R7 levels from flies of the indicated age and genotypes reared under constant dark conditions. In day 29 white mutants, formation of the identified structures in ommatidia at both the R8 and R7 levels was completely suppressed. In day 29 white; yata mutants, some ommatidia at both the R8 and R7 levels contained the identified structures (arrows). Scale bar: 2 μm. (B,C) The proportions of ommatidia at the R8 (B) and R7 (C) levels that contained at least one identified structure are plotted. Data collected from flies reared under 12-h light/12-h dark conditions (L/D) and those reared under constant dark conditions after eclosion (D/D) are compared. Each dot represents data from one fly. The raw data are shown in Table S1. **P<0.01; Games-Howell test (B) and Tukey's test (C).

Fig. 5.

The identified structures were not observed in red-eyed wild-type and yata mutant flies on days 1 and 29. (A) Electron micrographs showing tangential sections of ommatidia at the R8 and R7 levels from flies of the indicated ages and genotypes. Scale bar: 2 μm. (B,C) The proportions of ommatidia at the R8 (B) and R7 (C) levels that contained at least one identified structure are plotted. Each dot represents data from one fly. The raw data are shown in Table S1. ND, not determined.

Fig. 6.

Lifespan shortening in yata mutants was not affected by the white mutation. The lifespans of (A) female and (B) male red-eyed and white-eyed yata mutants are shown. The white mutation did not enhance or rescue lifespan shortening observed in yata mutants. The lifespans of female and male red-eyed white mutant heterozygotes and white-eyed white mutants are also shown. The white mutation caused a slight shortening of the lifespans of both females and males. The numbers of flies examined were as follows: 99 (female, red eye, yata mutant), 104 (female, white eye, yata mutant), 137 (female, red eye, control), 125 (female, white eye, control), 145 (male, red eye, yata mutant), 127 (male, white eye, yata mutant), 128 (male, red eye, control) and 110 (male, white eye, control). *P<0.05, **P<0.01; log-rank test.

Fig. 7.

The identified structures were observed in very aged day 71 wild-type flies. (A) Electron micrographs showing tangential sections of ommatidia at the R8 and R7 levels from day 71 wild-type flies are shown. The identified structures were observed at low frequency in ommatidia at the R8 level (arrow). The identified structures were not observed in ommatidia at the R7 level. (B) Highly magnified image of the identified structure. An electron micrograph of a tangential section of an ommatidium at the R8 level is shown. The identified cellular structure contained many vesicles, multivesicular bodies (arrow) and electron-dense structures (filled arrowheads). A rhabdomere is indicated by an open arrowhead. (C) An electron micrograph of a tangential section of an ommatidium in a day 71 white mutant fly is shown. The tissue structure was severely damaged, although traces of rhabdomeres could be identified. Scale bars: 2 μm (A,C), 500 nm (B).