Nnier-Gal4 drives strong expression (Fig. 1A ). In contrast to wildtype controls, Rheb overexpressing flies showed a dark patch of pigment in the central posterior LED 209 web region of the thorax, and a broadening of the dorsal pigment stripe in abdominal segments A3 and A4 (Fig. S1A ). We noted that although Rheb wasTORC1 Controls Drosophila PigmentationFigure 1. Rheb drives increased pigmentation of the pupal and adult cuticle. The evolutionarily conserved TSC pathway regulates protein synthesis and cell growth through activation of TOR complex 1 (TORC1) (A) [12]. Uniform pigmentation of the adult male thorax in pannier-Gal4/+ (we will use the abbreviation “-G4” for Gal4 in this and subsequent figures) (B). Pattern of expression of pannier-Gal4, UAS-Rheb-GFP on the pupal thorax (C). “trident pattern” pigmentation in the posterior thorax UAS-Rheb, pannier-Gal4 adult male fly (D). MARCM clones of tsc1w243x and tsc2109 (E,F), exhibit posterior pigmentation (white arrowheads) in clones (clones marked with GFP, see L-O). UAS-TSC1 and UAS-TSC2 suppress the increased growth and pigmentation in pannier-Gal4, UAS-Rheb flies (G). UAS-TSC2RNAi enhances the increased growth and pigmentation in pannier-Gal4, UASRheb flies (H). pannier-Gal4, UAS-Rheb shows premature bristle pigmentation in a dorsal stripe in stage P11 pupa (I). Pupa, stage P10 in wildtype (J) and tsc1w243x MARCM clones (K-M), GFP-marked (arrowheads) tsc1w243x bristles pigment prematurely, red in M and O is autofluorescence of the cuticle. Premature pupal bristle pigmentation is suppressed in rheb2D1, tsc1R453x clones, marked by arrowheads (N,O) and GFP (green, O). Genotypes of flies: Y/w, UAS-dicer2; pannier-Gal4/+(B), Y/w, UAS-dicer2; UAS-Rheb-GFP/+, pannier-Gal4/+(C), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/+(D,I), w/yw, Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-Tau-GFP/+; FRT82B, tsc1w243x/FRT82B tub-Gal80 (E, K ), w/yw, Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-tauGFP/+; tsc2109 FRT80B/tub-Gal80 FRT80B (F). Y/w; UAS-Rheb/+, pannier-Gal4/UAS-tsc1,UAS-tsc2 (G), Y/w, UAS-dicer2; UAS-Rheb/+, pannier-Gal4/UAStsc2RNAi (H). w/yw, Ubx-flp; scabrous-Gal4,UAS-actin-GFP/+; FRT82B rheb2D1, tsc1R453x/FRT82B tub-Gal80 (N,O). doi:10.1371/journal.pone.0048720.gexpressed throughout the pannier-Gal4 expression domain (Fig. 1B), the darkening of the thoracic cuticle was KDM5A-IN-1 site almost exclusively confined to posterior-most region of the notum, a region referred to as the “trident”. In order to exclude the possibility that the cuticle darkening phenotype in this cross was due to a genetic background effect, we crossed pannier-Gal4 to two independent Pelement insertions of UAS-Rheb-GFP and to the rhebAV4 allele, which contains a UAS-bearing P element insertion within theUTR of Rheb (Fig. S1C). In all cases, we observed darkening of the cuticle on both the thorax and abdomen in a similar pattern. TSC2 functions as a GTPase activating protein (GAP) for Rheb, and along with its TSC1 binding partner, maintain Rheb in a GDP-bound, inactive state (Fig. 1A) [10?2]. In order to test whether the thoracic increased pigmentation phenotype is due to inappropriate activity of endogenous Rheb, we used the MARCM (mosaic analysis with a repressible cell marker) system [13] toTORC1 Controls Drosophila Pigmentationgenerate clones of either mutant tsc1 or tsc2. In adult flies of the appropriate genotypes, we observed regions of the thorax that contained ectopic pigmentation, as well as increased cell size, and duplicated bristles o.Nnier-Gal4 drives strong expression (Fig. 1A ). In contrast to wildtype controls, Rheb overexpressing flies showed a dark patch of pigment in the central posterior region of the thorax, and a broadening of the dorsal pigment stripe in abdominal segments A3 and A4 (Fig. S1A ). We noted that although Rheb wasTORC1 Controls Drosophila PigmentationFigure 1. Rheb drives increased pigmentation of the pupal and adult cuticle. The evolutionarily conserved TSC pathway regulates protein synthesis and cell growth through activation of TOR complex 1 (TORC1) (A) [12]. Uniform pigmentation of the adult male thorax in pannier-Gal4/+ (we will use the abbreviation “-G4” for Gal4 in this and subsequent figures) (B). Pattern of expression of pannier-Gal4, UAS-Rheb-GFP on the pupal thorax (C). “trident pattern” pigmentation in the posterior thorax UAS-Rheb, pannier-Gal4 adult male fly (D). MARCM clones of tsc1w243x and tsc2109 (E,F), exhibit posterior pigmentation (white arrowheads) in clones (clones marked with GFP, see L-O). UAS-TSC1 and UAS-TSC2 suppress the increased growth and pigmentation in pannier-Gal4, UAS-Rheb flies (G). UAS-TSC2RNAi enhances the increased growth and pigmentation in pannier-Gal4, UASRheb flies (H). pannier-Gal4, UAS-Rheb shows premature bristle pigmentation in a dorsal stripe in stage P11 pupa (I). Pupa, stage P10 in wildtype (J) and tsc1w243x MARCM clones (K-M), GFP-marked (arrowheads) tsc1w243x bristles pigment prematurely, red in M and O is autofluorescence of the cuticle. Premature pupal bristle pigmentation is suppressed in rheb2D1, tsc1R453x clones, marked by arrowheads (N,O) and GFP (green, O). Genotypes of flies: Y/w, UAS-dicer2; pannier-Gal4/+(B), Y/w, UAS-dicer2; UAS-Rheb-GFP/+, pannier-Gal4/+(C), Y/w, UAS-dicer2; UAS-Rheb/+; pannier-Gal4/+(D,I), w/yw, Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-Tau-GFP/+; FRT82B, tsc1w243x/FRT82B tub-Gal80 (E, K ), w/yw, Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-tauGFP/+; tsc2109 FRT80B/tub-Gal80 FRT80B (F). Y/w; UAS-Rheb/+, pannier-Gal4/UAS-tsc1,UAS-tsc2 (G), Y/w, UAS-dicer2; UAS-Rheb/+, pannier-Gal4/UAStsc2RNAi (H). w/yw, Ubx-flp; scabrous-Gal4,UAS-actin-GFP/+; FRT82B rheb2D1, tsc1R453x/FRT82B tub-Gal80 (N,O). doi:10.1371/journal.pone.0048720.gexpressed throughout the pannier-Gal4 expression domain (Fig. 1B), the darkening of the thoracic cuticle was almost exclusively confined to posterior-most region of the notum, a region referred to as the “trident”. In order to exclude the possibility that the cuticle darkening phenotype in this cross was due to a genetic background effect, we crossed pannier-Gal4 to two independent Pelement insertions of UAS-Rheb-GFP and to the rhebAV4 allele, which contains a UAS-bearing P element insertion within theUTR of Rheb (Fig. S1C). In all cases, we observed darkening of the cuticle on both the thorax and abdomen in a similar pattern. TSC2 functions as a GTPase activating protein (GAP) for Rheb, and along with its TSC1 binding partner, maintain Rheb in a GDP-bound, inactive state (Fig. 1A) [10?2]. In order to test whether the thoracic increased pigmentation phenotype is due to inappropriate activity of endogenous Rheb, we used the MARCM (mosaic analysis with a repressible cell marker) system [13] toTORC1 Controls Drosophila Pigmentationgenerate clones of either mutant tsc1 or tsc2. In adult flies of the appropriate genotypes, we observed regions of the thorax that contained ectopic pigmentation, as well as increased cell size, and duplicated bristles o.
