Figure 1.
RpS6 mutant suppresses the small rough eye phenotype of cycEJP, but not through restoring CycE protein levels.
(A) Scanning electron micrographs (SEM) of female adult eyes with genotypes as indicated. Orientation of eyes: anterior (left) posterior (right). Scale bar 100 µm. (B) Confocal images of 3rd instar eye imaginal discs stained for CycE and DNA with genotypes as indicated. White boxes mark the band of cycE cells. Images were taken at 40× magnification. Orientation of eye discs: anterior (left), posterior (right). Scale bar equals 50 µm. (C) Confocal images of 3rd instar eye imaginal discs stained for BrdU incorporation and DNA with genotypes as indicated. White boxes mark the band of S phase cells. Images were taken at 40× magnification. Orientation of eye discs: anterior (left), posterior (right). Scale bar equals 50 µm. (D) Confocal images of 3rd instar eye imaginal discs stained for cells in the SMW (PH3) and DNA with genotypes as indicated. White boxes mark the band of cells in SMW. Images were taken at 40× magnification with 0.7× optical zoom. Orientation of eye discs: anterior (left), posterior (right). Scale bar equals 50 µm. (E) Graph quantifying the number of cells in the SMW. Results are represented as the mean +/− standard error. Statistical analysis applied: unpaired t-test, where ** = p<0.01, NS = not significant and n = 3.
Figure 2.
Reducing RpS6 by RNAi in the whole fly, but not specifically in the eye, suppresses cycEJP.
(A) Light micrographs of female adults bearing the genotypes as indicated. GMR-Gal4 drives expression in differentiated eye photoreceptor cells. Ey-Gal4 drives expression in all eye cells. (B) Graph showing the relative mRNA levels of RpS6 from the RpS6 mutant, eye specific reductions of RpS6 (GMR-Gal4 and Ey-Gal4) and ubiquitous reductions of RpS6 (Actin-Gal4) as measured by qRT-PCR. RNA samples were extracted from ten 3rd instar larvae or thirty 3rd instar eye imaginal discs. Samples were normalised to equal amounts of RNA (1 µg). Results are represented as the mean +/− standard error (n = 3). Statistical analysis applied: One-way ANOVA, where * = p<0.05, *** = p<0.001. (C–E) Light micrographs of female adult eyes bearing the genotypes indicated. Act-Gal4 drives expression in all cells. Orientation of eyes: anterior (left), posterior (right). (F) Graph of average eye area. (GMR>+) n = 13, (GMR>+, cycEJP/cycEJP) n = 13, (GMR>RpS6 RNAi) n = 15, (GMR>RpS6 RNAi; cycEJP/cycEJP) n = 19, (Ey>+) n = 21, (Ey>+; cycEJP/cycEJP) n = 11, (Ey>RpS6 RNAi) n = 17, (Ey>RpS6 RNAi; cycEJP/cycEJP) n = 12, (Act>+) n = 16, (Act>+; cycEJP/cycEJP) n = 42, (Act>RpS6 RNAi) n = 31, (Act>RpS6 RNAi; cycEJP/cycEJP) n = 49. Results are represented as the mean +/− standard error. Statistical analysis applied: One-way ANOVA, where *** = p<0.001 and NS = not significant.
Figure 3.
RpS6 mutant larvae have smaller prothoracic glands and an ecdysone dependent developmental delay.
(A) Confocal images of 3rd instar prothoracic glands marked with GFP with genotypes indicated. Magnification 40×. Scale bar 50 µM. (B) Graph of average PG size. Results are represented as the mean +/− standard error. Statistical analysis applied: unpaired t-test, where * = p<0.05 (n = 3). (C) Graph representing the time to eclosion after egg deposition (AED) of genotypes indicated raised in the presence or absence of ecdysone (20E). AmnC651-Gal4 drives expression in the prothoracic gland. (D) Light micrographs of female adult eyes bearing the genotypes indicated raised in the presence or absence of ecdysone (20E). Orientation of eyes: anterior (left), posterior (right).
Figure 4.
Reducing Rps by RNAi in the PG results in developmental delay and small prothoracic glands.
(A) Confocal images of 3rd instar prothoracic glands marked with GFP at day 5 with genotypes indicated. AmnC651-Gal4 drives expression in the prothoracic gland. Dp110DN is a dominant-negative form of PI3K. Magnification 40×. Scale bar 50 µM. (B) qRT-PCR of relative mRNA levels of an ecdysone responsive gene E74B. RNA samples were extracted from 3rd instar larvae. Samples were normalised to Actin5C mRNA levels. (AmnC651>RpS6 RNAi) n = 4, (AmnC651>RpS13 RNAi) n = 2, (AmnC651>RpL38 RNAi) n = 2. Results are represented as the mean +/− standard error. Statistical analysis applied: unpaired t-test, where *** = p<0.001. (C) Light micrographs of 5 days AED larvae (i–v) or 13 days AED adult (vi) or delayed larvae (vii–x) with genotypes marked.
Figure 5.
Addition of 20-hydroxyecdysone can partially rescue Amn>RpS6 RNAi larval lethality.
(A) Light micrographs of day 8 pupae/larvae or day 15 female adults bearing the genotypes indicated. The larvae were fed 0.75 mg/mL of 20E or equivalent concentration of 7.5% (v/v) ETOH. (B) qRT-PCR of relative mRNA levels of an ecdysone responsive gene E74B from larvae with or without 0.75 mg/mL of 20E. RNA samples were extracted from 3rd instar larvae. Samples were normalised to Actin5C mRNA levels. Results are represented as the mean +/− standard error (n = 3). Statistical analysis applied: unpaired t-test, where *** = p<0.001.
Figure 6.
Reducing RpS6 in the PG is associated with tissue overgrowth and suppresses cycEJP.
(A) Light micrographs of larvae, whole adult flies, adult eyes and wings bearing the genotypes: (i–iv) control (AmnC651; Tubulin-Gal80TS>+) and (v–viii) delaying reduction of RpS6 in the PG until 2nd instar (AmnC651; Tubulin-Gal80TS>RpS6 RNAi). (B) Graph of average wing area. (AmnC651; Tubulin-Gal80TS>+) n = 7, (AmnC651; Tubulin-Gal80TS>RpS6 RNAi) n = 10. Results are represented as the mean +/− standard error. Statistical analysis applied: unpaired t-test, where *** = p<0.001. (C) Light micrographs of female adult flies bearing the genotypes indicated raised in the presence or absence of ecdysone (20E). (D) Light micrographs of female adult eyes bearing the genotypes indicated. Orientation of eyes: anterior (left), posterior (right). (E) Graph of average eye area. (AmnC651; Tubulin-Gal80TS>+) n = 26, (AmnC651; Tubulin-Gal80TS>+; cycEJP/cycEJP) n = 26, (AmnC651; Tubulin-Gal80TS>RpS6 RNAi) n = 31, (AmnC651; Tubulin-Gal80TS>RpS6 RNAi; cycEJP/cycEJP) n = 19. Results are represented as the mean +/− standard error. Statistical analysis applied: unpaired t-test, where *** = p<0.001. (F) Light micrographs of female adult eyes bearing the genotypes indicated. Orientation of eyes: anterior (left), posterior (right).
Figure 7.
Restoring RpS6 expression in the PG inhibits the suppression of cycEJP by the RpS6WG1288 mutant.
(A,B) Light micrographs of female adult eyes bearing the genotypes indicated. Orientation of eyes: anterior (left), posterior (right). AmnC651-Gal4 and Phm-Gal4 drive expression in the prothoracic gland. (C) Graph of average eye area. (AmnC651>+; cycEJP/cycEJP) n = 7, (RpS6WG1288/+; cycEJP/cycEJP; Phm>+) n = 6, (RpS6WG1288/AmnC651>RpS6; cycEJP/cycEJP) n = 14. Results are represented as the mean +/− standard error. Statistical analysis applied: unpaired t-test, where *** = p<0.001. (D) Graph representing the time to eclosion after egg deposition (AED) of genotypes indicated. P0206-Gal4 is a ring gland specific driver.
Figure 8.
The ecdysone model of cycEJP suppression and Minute overgrowth phenotype.
(A) Diagram of the two effects of Rp reductions in Drosophila. First is the intrinsic effect of reducing Rps in the prothoracic gland (PG). The second is an extrinsic effect on the target tissue. The final size of the adult fly is the net consequence of both effects. (B) Model for suppression of cycEJP via altered PG size and ecdysone activity. In wild-type PGs, ecdysone titres accumulate and allow normal growth of the eye imaginal disc (depicted by the grey gradient). In cycEJP eye discs, while the PG size is normal, the eye discs have reduced proliferation/growth due to the cycEJP mutation. Reduction of RpS6 reduces PG size and ecdysone activity to cause an extended larval growth period, allowing extra time for the cycEJP eye discs to grow.