Ernal cells of the mechanosensory organs in order to commence pigmentation, and in the second, after eclosion, a burst of pigmentation activity occurs that is controlled by a neuropeptide cascade, which is required for cuticular tanning and hardening ofTORC1 Controls Drosophila PigmentationFigure 4. Rheb activity drives increased TH levels in pupal epidermal cells. Western blot analysis reveals a robust increase in levels of TH protein, and more modest increase of Yellow protein, in Rheb overexpressing thoraces compared to pannier-Gal4 (pnr-G4) line alone (A). TH protein is expressed in a subset of anterior epidermal cells prior to the onset of pigmentation in the P10 stage pupal thorax (B). UAS-Rheb, pannier-Gal4 pupa showing increased numbers of TH protein expressing cells along the central dorsal region of the thorax (C), which is suppressed by either raptorRNAi (D), or s6k1RNAi (E). Overexpression of Rheb by pannier-Gal4 expands the expression of the TH4.1-LacZ reporter, as shown by b-gal labeling (gray, F, G). Genotypes of flies: Y/w, BTZ043 UAS-dicer2; pannier-Gal4/+ (A, B, G), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/+ (A, C, G), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/UAS-raptorRNAi(D), Y/w, UAS-dicer2; UAS-Rheb/UAS-s6k1RNAi; pannier-Gal4/+(E), Y/w, UAS-dicer2;+/TH4.1-LacZ, pannier-Gal4/+ and Y/w, UASdicer2; UAS-Rheb/TH4.1-LacZ; pannier-Gal4/+(F). doi:10.1371/journal.pone.0048720.gthe adult cuticle [15,19]. We show that Rheb promotes premature pigmentation of the mechanosensory bristles during the pupal stage, and also drives darkening of the posterior cuticle of the thorax after eclosion. It is unclear why this increased pigmentation is biased to the posterior region, known as the trident, but key pigmentation enzymes such as Yellow and Ebony, are expressed at different levels in this part of thorax, suggesting that this region may be more sensitive to changes in catecholamine levels. While our data indicates that Rheb activity increases TH protein levels, it is unclear whether this is through a transcriptional or post-transcriptional regulation. Previous studies have indicted that TH is translationally repressed during pupal eclosion, but the mechanism of this repression is not well understood [15]. TORC1, through the combined K162 web activities of both S6K and eIF4E activities,promotes recruitment of the initiation factor complex to mature mRNAs thereby increasing protein synthesis [24?6]. Although we saw increases in both protein levels of Yellow and TH when Rheb was overexpressed, TH levels were markedly higher, while its mRNA levels did not show an increase. These finding point to the possibility that TH translation may be limited by TORC1 activity in wildtype cells. High TORC1 activity promotes the unwinding of mRNAs with long and structured 59 UTRs by the helicase subunit of the initiation complex eIF4A [27]. The TH 59UTR is longer and predicted to be more structured than the yellow 59 UTR and knockdown of eIF4A blocks Rheb-induced hyperpigmentation (Fig. S2G, H). High Rheb levels could therefore increase translation rates of 11967625 TH without increasing the level of TH mRNA. We cannot exclude however that activation ofTORC1 Controls Drosophila PigmentationRheb may, directly or indirectly, also increase levels of transcription or stability of the TH mRNA that was not detected in our rtPCR experiments. The fact that we observe premature pigmentation in tsc1 clones is reminiscent of the precocious differentiation of tsc mutant photoreceptors.Ernal cells of the mechanosensory organs in order to commence pigmentation, and in the second, after eclosion, a burst of pigmentation activity occurs that is controlled by a neuropeptide cascade, which is required for cuticular tanning and hardening ofTORC1 Controls Drosophila PigmentationFigure 4. Rheb activity drives increased TH levels in pupal epidermal cells. Western blot analysis reveals a robust increase in levels of TH protein, and more modest increase of Yellow protein, in Rheb overexpressing thoraces compared to pannier-Gal4 (pnr-G4) line alone (A). TH protein is expressed in a subset of anterior epidermal cells prior to the onset of pigmentation in the P10 stage pupal thorax (B). UAS-Rheb, pannier-Gal4 pupa showing increased numbers of TH protein expressing cells along the central dorsal region of the thorax (C), which is suppressed by either raptorRNAi (D), or s6k1RNAi (E). Overexpression of Rheb by pannier-Gal4 expands the expression of the TH4.1-LacZ reporter, as shown by b-gal labeling (gray, F, G). Genotypes of flies: Y/w, UAS-dicer2; pannier-Gal4/+ (A, B, G), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/+ (A, C, G), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/UAS-raptorRNAi(D), Y/w, UAS-dicer2; UAS-Rheb/UAS-s6k1RNAi; pannier-Gal4/+(E), Y/w, UAS-dicer2;+/TH4.1-LacZ, pannier-Gal4/+ and Y/w, UASdicer2; UAS-Rheb/TH4.1-LacZ; pannier-Gal4/+(F). doi:10.1371/journal.pone.0048720.gthe adult cuticle [15,19]. We show that Rheb promotes premature pigmentation of the mechanosensory bristles during the pupal stage, and also drives darkening of the posterior cuticle of the thorax after eclosion. It is unclear why this increased pigmentation is biased to the posterior region, known as the trident, but key pigmentation enzymes such as Yellow and Ebony, are expressed at different levels in this part of thorax, suggesting that this region may be more sensitive to changes in catecholamine levels. While our data indicates that Rheb activity increases TH protein levels, it is unclear whether this is through a transcriptional or post-transcriptional regulation. Previous studies have indicted that TH is translationally repressed during pupal eclosion, but the mechanism of this repression is not well understood [15]. TORC1, through the combined activities of both S6K and eIF4E activities,promotes recruitment of the initiation factor complex to mature mRNAs thereby increasing protein synthesis [24?6]. Although we saw increases in both protein levels of Yellow and TH when Rheb was overexpressed, TH levels were markedly higher, while its mRNA levels did not show an increase. These finding point to the possibility that TH translation may be limited by TORC1 activity in wildtype cells. High TORC1 activity promotes the unwinding of mRNAs with long and structured 59 UTRs by the helicase subunit of the initiation complex eIF4A [27]. The TH 59UTR is longer and predicted to be more structured than the yellow 59 UTR and knockdown of eIF4A blocks Rheb-induced hyperpigmentation (Fig. S2G, H). High Rheb levels could therefore increase translation rates of 11967625 TH without increasing the level of TH mRNA. We cannot exclude however that activation ofTORC1 Controls Drosophila PigmentationRheb may, directly or indirectly, also increase levels of transcription or stability of the TH mRNA that was not detected in our rtPCR experiments. The fact that we observe premature pigmentation in tsc1 clones is reminiscent of the precocious differentiation of tsc mutant photoreceptors.
