The elevated "zero-maze" is a modification of the elevated plus-maze model of anxiety in rats which incorporates both traditional and novel ethological measures in the analysis of drug effects. The novel design comprises an elevated annular platform with two opposite enclosed quadrants and two open, removing any ambiguity in interpretation of time spent on the central square of the traditional design and allowing uninterrupted exploration. Using this model, the reference benzodiazepine anxiolytics, diazepam (0.125-0.5 mg/kg) and chlordiazepoxide (0.5-2.0 mg/kg) significantly increased the percentage of time spent in the open quadrants (% TO) and the frequency of head dips over the edge of the platform (HDIPS), and reduced the frequency of stretched attend postures (SAP) from the closed to open quadrants. In contrast, the anxiogenic drug m-chlorophenyl-piperazine (mCPP; 0.25-1.0 mg/kg) induced the opposite effects, decreasing %TO and HDIPS, and increasing SAP. The 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.001-0.1 mg/kg) had no effects on either %TO or HDIPS, but did decrease SAP at 0.01 mg/kg although not at higher or lower doses. Similarly, the 5-HT3 receptor antagonist, ondansetron (0.0001-1.0 mg/kg) decreased SAP and increased %TO at 0.01 mg/kg, but not at other doses. The present data suggest that a combination of the novel "zero-maze" design and a detailed ethological analysis provides a sensitive model for the detection of anxiolytic/anxiogenic drug action.

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Regulating ribosome number is thought to control cellular growth. Synthesis of ribosomal RNA (rRNA) is a limiting step in ribosome biogenesis and rates of rRNA synthesis are generally altered depending on the growth status of a cell. Although studies in unicellular systems have addressed the mechanisms by which this occurs, few studies have applied a genetic approach to examine growth-dependent control of rRNA synthesis in metazoans. Here, we show that in Drosophila melanogaster Myc (dMyc) is a regulator of rRNA synthesis. Expression of dMyc is both necessary and sufficient to control rRNA synthesis and ribosome biogenesis during larval development. Stimulation of rRNA synthesis by dMyc is mediated through a rapid, coordinated increase in the levels of the Pol I transcriptional machinery. In addition, the growth effects of dMyc in larval wing imaginal discs require de novo rRNA synthesis. We suggest that during animal development, the control of rRNA synthesis and ribosome biogenesis is an essential Myc function.

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Endocycles are variant cell cycles comprised of DNA Synthesis (S)- and Gap (G)- phases but lacking mitosis1,2. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world’s biomass3,4. DNA replication in endocycling Drosophila cells is triggered by Cyclin E/Cyclin Dependent Kinase 2 (CycE/Cdk2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase5,6. How CycE/Cdk2 is periodically silenced to allow re-replication has not been established. Here, using genetic tests in parallel with computational modeling, we show that Drosophila’s endocycles are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4Cdt2 ubiquitin ligase, and this in turn mediates the destruction of E2F17. We propose that it is the transient loss of E2F1 during S-phases that creates the window of low Cdk activity required for preRC formation. In support of this model over-expressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by de-regulating target genes including CycE, as well as Cdk1 and mitotic Cyclins. Moreover, we find that altering cell growth by changing nutrition or TOR signaling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants1,2,8, suggesting that elements of this mechanism act in most growth-dependent cell cycles.

Almost all animals show sex differences in body size. For example, in Drosophila, females are larger than males. Although Drosophila is widely used as a model to study growth, the mechanisms underlying this male-female difference in size remain unclear. Here, we describe a novel role for the sex determination gene transformer (tra) in promoting female body growth. Normally, Tra is expressed only in females. We find that loss of Tra in female larvae decreases body size, while ectopic Tra expression in males increases body size. Although we find that Tra exerts autonomous effects on cell size, we also discovered that Tra expression in the fat body augments female body size in a non cell-autonomous manner. These effects of Tra do not require its only known targets doublesex and fruitless. Instead, Tra expression in the female fat body promotes growth by stimulating the secretion of insulin-like peptides from insulin producing cells in the brain. Our data suggest a model of sex-specific growth in which body size is regulated by a previously unrecognized branch of the sex determination pathway, and identify Tra as a novel link between sex and the conserved insulin signaling pathway.

SUMMARYCell competition is a conserved mechanism that regulates organ size and shares properties with the early stages of cancer. In Drosophila, wing cells with increased Myc or with optimum ribosome function become supercompetitors that kill their wild-type neighbors (called losers) up to several cell diameters away. Here, we report that modulating STAT activity levels regulates competitor status. Cells lacking STAT become losers that are killed by neighboring wild-type cells. By contrast, cells with hyper-activated STAT become supercompetitors that kill losers located at a distance in a manner that is dependent on hid but independent of Myc, Yorkie, Wingless signaling, and of ribosome biogenesis. These results indicate that STAT, Wingless and Myc are major parallel regulators of cell competition, which may converge on signals that non-autonomously kill losers. As hyper-activated STATs are causal to tumorigenesis and stem cell niche occupancy, our results have therapeutic implications for cancer and regenerative medicine.

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.Neurotrophins have long been recognized for their role in regulating neuronal survival, cell growth, differentiation, and neuronal plasticity. The archetypal neurotrophin, nerve growth factor (NGF), elicits most of these effects by binding and activating the receptor tyrosine kinase (RTK), TrkA, which leads to the activation of several well-defined signaling cascades. Of these, the phosphoinositide 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK) pathways are two of the most extensively studied. PI3-Ks have been implicated in multiple biological responses including membrane trafficking, proliferation, differentiation, and survival (67). These kinases consist of a family of proteins which phosphorylate phosphatidylinositol (PI) at the D3 position and have been categorized into three classes based on their lipid substrate specificity in vitro (96). The lipid products of PI3-Ks {PI 3-phosphate [PI(3)P], PI(3,4)P, PI(3,5)P, and PI(3,4,5)P} are known to act as second messengers and mediate most of the known functions of PI3-Ks in cells (45).The mitogen-activated protein kinase family members, ERK1 and ERK2, can also be activated by a wide variety of stimuli to promote a diverse array of cellular functions (77). In addition to their established rol...