Dcas Supports Cell Polarization and Cell-Cell Adhesion Complexes in Development

Mammalian Cas proteins regulate cell migration, division and survival, and are often deregulated in cancer. However, the presence of four paralogous Cas family members in mammals (BCAR1/p130Cas, EFS/Sin1, NEDD9/HEF1/Cas-L, and CASS4/HEPL) has limited their analysis in development. We deleted the single Drosophila Cas gene, Dcas, to probe the developmental function of Dcas. Loss of Dcas had limited effect on embryonal development. However, we found that Dcas is an important modulator of the severity of the developmental phenotypes of mutations affecting integrins (If and mew) and their downstream effectors Fak56D or Src42A. Strikingly, embryonic lethal Fak56D-Dcas double mutant embryos had extensive cell polarity defects, including mislocalization and reduced expression of E-cadherin. Further genetic analysis established that loss of Dcas modified the embryonal lethal phenotypes of embryos with mutations in E-cadherin (Shg) or its signaling partners p120- and β-catenin (Arm). These results support an important role for Cas proteins in cell-cell adhesion signaling in development

Conversion of neurons and glia to external-cell fates in the external sensory organs of Drosophila hamlet mutants by a cousin-cousin cell-type respecification

The Drosophila external sensory organ forms in a lineage elaborating from a single precursor cell via a stereotypical series of asymmetric divisions. HAMLET transcription factor expression demarcates the lineage branch that generates two internal cell types, the external sensory neuron and thecogen. In HAMLET mutant organs, these internal cells are converted to external cells via an unprecedented cousin-cousin cell-fate respecification event. Conversely, ectopic HAMLET expression in the external cell branch leads to internal cell production. The fate-determining signals NOTCH and PAX2 act at multiple stages of lineage elaboration and HAMLET acts to modulate their activity in a branch-specific manner

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

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