The TSC1/2 Complex Controls <em>Drosophila</em> Pigmentation through TORC1-Dependent Regulation of Catecholamine Biosynthesis

<div><p>In <em>Drosophila</em>, the pattern of adult pigmentation is initiated during late pupal stages by the production of catecholamines DOPA and dopamine, which are converted to melanin. The pattern and degree of melanin deposition is controlled by the expression of genes such as <em>ebony</em> and <em>yellow</em> as well as by the enzymes involved in catecholamine biosynthesis. In this study, we show that the conserved TSC/TORC1 cell growth pathway controls catecholamine biosynthesis in <em>Drosophila</em> during pigmentation. We find that high levels of Rheb, an activator of the TORC1 complex, promote premature pigmentation in the mechanosensory bristles during pupal stages, and alter pigmentation in the cuticle of the adult fly. Disrupting either melanin synthesis by RNAi knockdown of melanogenic enzymes such as <em>tyrosine hydroxylase</em> (TH), or downregulating TORC1 activity by Raptor knockdown, suppresses the Rheb-dependent pigmentation phenotype in vivo. Increased Rheb activity drives pigmentation by increasing levels of TH in epidermal cells. Our findings indicate that control of pigmentation is linked to the cellular nutrient-sensing pathway by regulating levels of a critical enzyme in melanogenesis, providing further evidence that inappropriate activation of TORC1, a hallmark of the human tuberous sclerosis complex tumor syndrome disorder, can alter metabolic and differentiation pathways in unexpected ways.</p> </div

Rheb drives increased pigmentation of the pupal and adult cuticle.

<p>The evolutionarily conserved TSC pathway regulates protein synthesis and cell growth through activation of TOR complex 1 (TORC1) (A) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048720#pone.0048720-Garami1" target="_blank">[12]</a>. Uniform pigmentation of the adult male thorax in <i>pannier-Gal4/+</i> (we will use the abbreviation “<i>-G4</i>” for Gal4 in this and subsequent figures) (B). Pattern of expression of <i>pannier-Gal4, UAS-Rheb-GFP</i> on the pupal thorax (C). “trident pattern” pigmentation in the posterior thorax <i>UAS-Rheb</i>, <i>pannier-Gal4</i> adult male fly (D). MARCM clones of <i>tsc1^w243x</i> and <i>tsc2¹⁰⁹</i> (E,F), exhibit posterior pigmentation (white arrowheads) in clones (clones marked with GFP, see L­O). <i>UAS-TSC1</i> and <i>UAS-TSC2</i> suppress the increased growth and pigmentation in <i>pannier-Gal4, UAS-Rheb</i> flies (G). <i>UAS-TSC2^RNAi</i> enhances the increased growth and pigmentation in <i>pannier-Gal4, UAS-Rheb</i> flies (H). <i>pannier-Gal4, UAS-Rheb</i> shows premature bristle pigmentation in a dorsal stripe in stage P11 pupa (I). Pupa, stage P10 in wildtype (J) and <i>tsc1^w243x</i> MARCM clones (K-M), GFP-marked (arrowheads) <i>tsc1^w243x</i> bristles pigment prematurely, red in M and O is autofluorescence of the cuticle. Premature pupal bristle pigmentation is suppressed in <i>rheb^2D1, tsc1^R453x</i> clones, marked by arrowheads (N,O) and GFP (green, O). Genotypes of flies: <i>Y/w, UAS-dicer2; pannier-Gal4/+</i>(B), <i>Y/w, UAS-dicer2; UAS-Rheb-GFP/+</i>, <i>pannier-Gal4/+</i>(C), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/+</i>(D,I), <i>w/yw, Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-Tau-GFP/+; FRT82B, tsc1^w243x/FRT82B tub-Gal80</i> (E, K–M), <i>w/yw, Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-tau-GFP/+; tsc2¹⁰⁹ FRT80B/tub-Gal80 FRT80B</i> (F). <i>Y/w; UAS-Rheb/+</i>, <i>pannier-Gal4/UAS-tsc1,UAS-tsc2</i> (G), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>, <i>pannier-Gal4/UAS-tsc2^RNAi</i> (H). <i>w/yw, Ubx-flp</i>; <i>scabrous-Gal4,UAS-actin-GFP/+; FRT82B rheb^2D1, tsc1^R453x</i>/<i>FRT82B tub-Gal80</i> (N,O).</p

TORC1 and S6 kinase-dependent pigmentation of the adult cuticle.

<p>Pigmentation and bristle growth phenotype in <i>UAS-Rheb-GFP</i>, <i>pannier-Gal4</i> is suppressed in <i>tor^Æ</i>^Pclones (A–D). In order to identify clones by expression of fluorescent markers, the epidermis was imaged in P9 pupae prior to the onset of pigmentation (A, B). Clones were identified by lack of Ubi-nls-RFP (red), and expression of Rheb was visualized by GFP (green). After live imaging of fluorescently marked clones (dotted lines) in the pupa, the adult fly was recovered to assess the effect of <i>tor</i> deletion on pigmentation induced by Rheb-GFP (C, D), the location of the clone was identified by it position relative to the large nuclei of macrochaete bristle cells in the pupa (white arrowheads). Expression of either <i>raptor^RNAi</i> (E), or <i>s6k1^RNAi</i> (F). <i>UAS-s6k1^TE</i>, <i>pannier-Gal4</i> flies show mild posterior pigmentation on the thorax (G). The increased pigmentation in the posterior thorax by <i>pannier-Gal4-</i>driven overexpression of both <i>s6k1^TE</i> and eIF4E was fully penetrant, but the darkening of the scutellum in this background was not consistently observed in all flies (H). Genotypes of flies: <i>yw, Ubx-FLP/w; Tor^ΔP FRT40A/Ubi-mRFP.nls FRT40A; pannier-Gal4, UAS-Rheb-GFP/+</i> (A–D). <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/UAS-raptor^RNAi</i> (E), <i>Y/w, UAS-dicer2; UAS-Rheb/UAS-s6k1^RNAi</i>; <i>pannier-Gal4/+</i>(F), <i>Y/w, UAS-dicer2; +/UAS-s6k1^TE</i>; <i>pannier-Gal4/+</i> (G), <i>Y/w, UAS-dicer2; +/UAS-s6k1^TE</i>; <i>pannier-Gal4/UAS-eIF4E</i> raised at 29°C (H).</p

Rheb activity drives increased TH levels in pupal epidermal cells.

<p>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 <i>pannier-Gal4</i> (<i>pnr-G4</i>) 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). <i>UAS-Rheb, pannier-Gal4</i> pupa showing increased numbers of TH protein expressing cells along the central dorsal region of the thorax (C), which is suppressed by either <i>raptor^RNAi</i> (D), or <i>s6k1^RNAi</i> (E). Overexpression of Rheb by <i>pannier-Gal4</i> expands the expression of the <i>TH^4.1-LacZ</i> reporter, as shown by β-gal labeling (gray, F, G). Genotypes of flies: <i>Y/w, UAS-dicer2; pannier-Gal4/+</i> (A, B, G), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/+</i> (A, C, G), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/UAS-raptor^RNAi</i>(D), <i>Y/w, UAS-dicer2; UAS-Rheb/UAS-s6k1^RNAi</i>; <i>pannier-Gal4/+</i>(E), <i>Y/w, UAS-dicer2;+/TH^4.1-LacZ, pannier-Gal4/+</i> and <i>Y/w, UAS-dicer2; UAS-Rheb/TH^4.1-LacZ</i>; <i>pannier-Gal4/+</i>(F).</p

TSC1/2 pathway regulates amino acid levels and function upstream of the catecholamine pathway.

<p>The <i>Drosophila</i> melanin biosynthesis pathway (modified from (Wittkopp, True and Carroll, 2002) enzymes in blue, substrates in black; phenol oxidases, aaNAT and NADA sclerotin have been excluded) (A). Pigmentation in MARCM clones of <i>tsc1^R453x</i> (B) is partially suppressed in a <i>yellow</i> background (C, arrowheads indicate clone regions in both B and C). Amino acid and metabolite analysis of heads collected from <i>UAS-Rheb/TM3, Sb</i> and <i>elav-Gal4/UAS-Rheb</i> flies, show statistically significant increases in glutamine, ammonia, lysine, 1-methylhistidine, and asparagine under conditions of neuronal Rheb-overexpression (Student’s T-test-*, D). <i>UAS-TH^RNAi</i> markedly suppressed the <i>UAS-Rheb</i>, <i>pannier-Gal4</i> pigmentation phenotype (E). Genotypes of flies: <i>w/yw,Ubx-flp; scabrous-Gal4,UAS-Pon-GFP,UAS-Tau-GFP/+;FRT82B, tsc1^R453x/FRT82B tub-Gal80</i> (B), y<i>w/yw,Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-Tau-GFP/+; FRT82B, tsc1^R453/FRT82B tub-Gal80</i> (C), <i>Y/w</i>; UAS-Rheb/TM3, Sb and <i>Y/w</i>; UAS-Rheb/<i>elav-Gal4</i> (D), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/UAS-TH^RNAi</i> (E).</p

Theta-burst-induced LTP in Tg<i>hCBS</i>60.4 mice.

<p>Comparison of averaged LTP expressed as percent change in the slope of fEPSP <i>vs</i> time, induced by theta-burst stimulation (TBS, arrow) of glutamate afferents and recorded in slices from Tg<i>hCBS</i>60.4 (12 slices/9 animals) and control (10 slices/7 animals) mice. In the insert, representative traces of fEPSPs, recorded before and 60 min after TBS, are superimposed.</p

Basal synaptic transmission in Tg<i>hCBS</i>60.4 mice.

<p>(A) Superimposed sample traces of evoked AMPA-R-mediated fEPSPs induced in a control (left) and a Tg<i>hCBS</i>60.4 mouse (right) by increased intensities of electrical stimulation of glutamate afferents (arrow). Traces are averages of 3 consecutive responses. (B) Comparison of synaptic efficacy as determined by the fEPSP/PFV ratio calculated at a stimulus intensity from 500 to 900 µA in Tg<i>hCBS</i>60.4 (23 slices/5 animals) and control (22 slices/5 animals) mice.</p

Involvement of CBS in sulfur-containing amino acid metabolism.

<p>From Stipanuk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029056#pone.0029056-Stipanuk1" target="_blank">[54]</a>.</p

Concentrations (nmoles/mg protein) of 30 amino acids and metabolite compounds in cerebellum extracts of Tg<i>hCBS</i>60.4 and control mice.

<p>Data correspond to mean ± S.E.M. BD: below detection. ND: not determined.</p

CBS expression and activity in brain regions of Tg<i>hCBS</i>60.4 mice.

<p>(A, top) Immunoblots for CBS and β-actin in cerebellum (left panel), hippocampus (middle panel) and forebrain (right panel). Total CBS proteins (human CBS plus mouse CBS) were detected using the anti-hCBS polyclonal antibody that recognises both the human and mouse proteins. (A, bottom) Quantification of CBS proteins normalized to β-actin and relative to control mice in the three brain regions (<i>n</i> = 3 Tg<i>hCBS</i>60.4 and <i>n</i> = 3 control) (B) CBS activity in cerebellum (left panel), hippocampus (middle panel) and forebrain (right panel). Units are nanomoles of cystathionine formed per milligram of protein extract per hour. For cerebellum and forebrain, data represent mean ± S.E.M. of activity measurements in each group (<i>n</i> = 9 Tg<i>hCBS</i>60.4 and <i>n</i> = 7 control; <i>n</i> = 8 Tg<i>hCBS</i>60.4 and <i>n</i> = 8 control, respectively). For hippocampus, data represent mean ± S.E.M. of three experimental assays performed on same pooled hippocampi (<i>n</i> = 7 Tg<i>hCBS</i>60.4 and <i>n</i> = 8 control). *for <i>p</i><0.05. ***for <i>p</i><0.001.</p