Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3Ј,5Ј monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein ␣ subunit (G␣ s *) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when G␣ s * was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of G␣ s * did not affect gross MB morphology, and wild-type G␣ s expression did not affect learning. Thus, olfactory learning depends on regulated G s signaling in Drosophila MBs.

Nitric oxide gas acts as a short-range signaling molecule in a vast array of important physiological processes, many of which include major changes in gene expression. How these genomic responses are induced, however, is poorly understood. Here, using genetic and chemical manipulations, we show that nitric oxide is produced in the Drosophila prothoracic gland, where it acts via the nuclear receptor ecdysone-induced protein 75 (E75), reversing its ability to interfere with its heterodimer partner, Drosophila hormone receptor 3 (DHR3). Manipulation of these interactions leads to gross alterations in feeding behavior, fat deposition, and developmental timing. These neuroendocrine interactions and consequences appear to be conserved in vertebrates.[Keywords: E75; DHR3; nitric oxide; Drosophila; metamorphosis; ecdysone; metabolism] Supplemental material is available for this article. In previous work, we showed that ecdysone-induced protein 75 (E75, also known as Eip75B; NR1D3) (Tweedie et al. 2009) contains heme constitutively bound to its ligand-binding domain (LBD), and that amino acids coordinately bound to the heme iron can be displaced in vitro by changes in redox state or the presence of nitric oxide (NO) gas (Pardee et al. 2004;Reinking et al. 2005;Marvin et al. 2009). In turn, these structural changes negate the ability of E75 to repress transcription and to reverse the positive transcriptional activity of its heterodimer partner, Drosophila hormone receptor 3 (DHR3; also known as DHR46, NR1F4) (Tweedie et al. 2009). Here, we look to see whether these interactions are relevant in vivo and, if so, what their roles are.One of the best-characterized roles of E75 and DHR3 in vivo is within the nuclear receptor (NR) transcriptional hierarchy that controls, and responds to, the production of the metamorphosis-inducing hormone ecdysone (diagrammed in Supplemental Fig. 1A). Upon binding ecdysone, the ecdysone receptor (EcR) acts as a heterodimer with a second NR, ultraspiracle (USP), to activate transcription of DHR3 and the E75 isoform E75A (Koelle et al. 1991;Lam et al. 1997Lam et al. , 1999White et al. 1997;Bialecki et al. 2002). DHR3 then promotes its own continued expression as well as that of the E75 splice variant E75B and the downstream NR gene bFtz-F1. bFTZ-F1, in turn, activates the expression of ecdysone synthetic enzyme genes (Lavorgna et al. 1993;Woodard et al. 1994;Broadus et al. 1999), resulting in another round of ecdysone production. The major site of larval ecdysone production is the prothoracic gland (PG) (diagrammed in Supplemental Fig. 1B). The PG is also a major site of NO synthase (Nos) expression (Wildemann and Bicker 1999). Hence, we looked to see whether E75 and NO are present and interactive in this tissue, with the hypothesis that activation of bFtz-F1 transcription by DHR3 requires inactivation of E75 isoforms by NO. As predicted, all of the above-listed genes are expressed in the PG toward the end of third instar development, and disruption of their expression or activity leads to molec...

Cell division and subsequent programmed cell death in imaginal discs of Drosophila larvae determine the final size of organs and structures of the adult fly. We show here that nitric oxide (NO) is involved in controlling the size of body structures during Drosophila development. We have found that NO synthase (NOS) is expressed at high levels in developing imaginal discs. Inhibition of NOS in larvae causes hypertrophy of organs and their segments in adult flies, whereas ectopic expression of NOS in larvae has the opposite effect. Blocking apoptosis in eye imaginal discs unmasks surplus cell proliferation and results in an increase in the number of ommatidia and component cells of individual ommatidia. These results argue that NO acts as an antiproliferative agent during Drosophila development, controlling the balance between cell proliferation and cell differentiation.

SUMMARYCandida albicans systemic dissemination in immunocompromised patients is thought to develop from initial gastrointestinal (GI) colonisation. It is unclear what components of the innate immune system are necessary for preventing C. albicans dissemination from the GI tract, but studies in mice have indicated that both neutropenia and GI mucosal damage are crucial for allowing widespread invasive C. albicans disease. Mouse models, however, provide limited applicability to genome-wide screens for pathogen or host factors – factors that might influence systemic dissemination following GI colonisation. For this reason we developed a Drosophila model to study intestinal infection by Candida. We found that commensal flora aided host survival following GI infection. Candida provoked extensive JNK-mediated death of gut cells and induced antimicrobial peptide expression in the fat body. From the side of the host, nitric oxide and blood cells influenced systemic antimicrobial responses. The secretion of SAP4 and SAP6 (secreted aspartyl proteases) from Candida was also essential for activating systemic Toll-dependent immunity.

Ubiquitin-mediated proteolysis regulates the steady-state abundance of proteins and controls cellular homoeostasis by abrupt elimination of key effector proteins. A multienzyme system targets proteins for destruction through the covalent attachment of a multiubiquitin chain. The specificity and timing of protein ubiquitination is controlled by ubiquitin ligases, such as the Skp1-Cullin-F box protein complex. Cullins are major components of SCF complexes, and have been implicated in degradation of key regulatory molecules including Cyclin E, beta-catenin and Cubitus interruptus. Here, we describe the genetic identification and molecular characterisation of the Drosophila Cullin-3 homologue. Perturbation of Cullin-3 function has pleiotropic effects during development, including defects in external sensory organ development, pattern formation and cell growth and survival. Loss or overexpression of Cullin-3 causes an increase or decrease, respectively, in external sensory organ formation, implicating Cullin-3 function in regulating the commitment of cells to the neural fate. We also find that Cullin-3 function modulates Hedgehog signalling by regulating the stability of full-length Cubitus interruptus (Ci155). Loss of Cullin-3 function in eye discs but not other imaginal discs promotes cell-autonomous accumulation of Ci155. Conversely, overexpression of Cullin-3 results in a cell-autonomous stabilisation of Ci155 in wing, haltere and leg, but not eye, imaginal discs suggesting tissue-specific regulation of Cullin-3 function. The diverse nature of Cullin-3 phenotypes highlights the importance of targeted proteolysis during Drosophila development.