The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues.

Exocytosis is a fundamental process in physiology, that ensures communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane.

Growth and Maturation in Development: A Fly's Perspective.

In mammals like humans, adult fitness is improved due to resource allocation, investing energy in the developmental growth process during the juvenile period, and in reproduction at the adult stage. Therefore, the attainment of their target body height/size co-occurs with the acquisition of maturation, implying a need for coordination between mechanisms that regulate organismal growth and maturation timing. Insects like Drosophila melanogaster also define their adult body size by the end of the juvenile larval period. Recent studies in the fly have shown evolutionary conservation of the regulatory pathways controlling growth and maturation, suggesting the existence of common coordinator mechanisms between them. In this review, we will present an overview of the significant advancements in the coordination mechanisms ensuring developmental robustness in Drosophila. We will include (i) the characterization of feedback mechanisms between maturation and growth hormones, (ii) the recognition of a relaxin-like peptide Dilp8 as a central processor coordinating juvenile regeneration and time of maturation, and (iii) the identification of a novel coordinator mechanism involving the AstA/KISS system.

Timing the Juvenile-Adult Neurohormonal Transition: Functions and Evolution.

Puberty and metamorphosis are two major developmental transitions linked to the reproductive maturation. In mammals and vertebrates, the central brain acts as a gatekeeper, timing the developmental transition through the activation of a neuroendocrine circuitry. In addition to reproduction, these neuroendocrine axes and the sustaining genetic network play additional roles in metabolism, sleep and behavior. Although neurohormonal axes regulating juvenile-adult transition have been classically considered the result of convergent evolution (i.e., analogous) between mammals and insects, recent findings challenge this idea, suggesting that at least some neuroendocrine circuits might be present in the common bilaterian ancestor Urbilateria. The initial signaling pathways that trigger the transition in different species appear to be of a single evolutionary origin and, consequently, many of the resulting functions are conserved with a few other molecular players being co-opted during evolution.

AstA Signaling Functions as an Evolutionary Conserved Mechanism Timing Juvenile to Adult Transition.

The onset of sexual maturation is the result of a hormonal cascade peaking with the production of steroid hormones. In animals undergoing a program of determinate growth, sexual maturation also coincides with the attainment of adult size. The exact signals that time the onset of maturation and the mechanisms coupling growth and maturation remain elusive. Here, we show that the Drosophila neuropeptide AstA and its receptor AstAR1 act as a brain trigger for maturation and juvenile growth. We first identified AstAR1 in an RNAi-based genetic screen as a key regulator of sexual maturation. Its specific knockdown in prothoracicotropic hormone (PTTH)-producing neurons delays the onset of maturation by impairing PTTH secretion. In addition to its role in PTTH neurons, AstAR1 is required in the brain insulin-producing cells (IPCs) to promote insulin secretion and systemic growth. AstAR1 function is mediated by the AstA neuropeptide that is expressed in two bilateral neurons contacting the PTTH neurons and the IPCs. Silencing brain AstA expression delays the onset of maturation, therefore extending the growth period. However, no pupal overgrowth is observed, indicating that, in these conditions, the growth-promoting function of AstAR1 is also impaired. These data suggest that AstA/AstAR1 acts to coordinate juvenile growth with maturation. Interesting, AstA/AstAR1 is homologous to KISS/GPR54, a ligand-receptor signal required for human puberty, suggesting that an evolutionary conserved neural circuitry controls the onset of maturation.

Prothoracicotropic hormone modulates environmental adaptive plasticity through the control of developmental timing.

Adult size and fitness are controlled by a combination of genetics and environmental cues. In Drosophila, growth is confined to the larval phase and final body size is impacted by the duration of this phase, which is under neuroendocrine control. The neuropeptide prothoracicotropic hormone (PTTH) has been proposed to play a central role in controlling the length of the larval phase through regulation of ecdysone production, a steroid hormone that initiates larval molting and metamorphosis. Here, we test this by examining the consequences of null mutations in the Ptth gene for Drosophila development. Loss of Ptth causes several developmental defects, including a delay in developmental timing, increase in critical weight, loss of coordination between body and imaginal disc growth, and reduced adult survival in suboptimal environmental conditions such as nutritional deprivation or high population density. These defects are caused by a decrease in ecdysone production associated with altered transcription of ecdysone biosynthetic genes. Therefore, the PTTH signal contributes to coordination between environmental cues and the developmental program to ensure individual fitness and survival.

Zonda is a novel early component of the autophagy pathway in Drosophila.

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation.

Neuroendocrine control of Drosophila larval light preference.

Animal development is coupled with innate behaviors that maximize chances of survival. Here, we show that the prothoracicotropic hormone (PTTH), a neuropeptide that controls the developmental transition from juvenile stage to sexual maturation, also regulates light avoidance in Drosophila melanogaster larvae. PTTH, through its receptor Torso, acts on two light sensors--the Bolwig's organ and the peripheral class IV dendritic arborization neurons--to regulate light avoidance. We found that PTTH concomitantly promotes steroidogenesis and light avoidance at the end of larval stage, driving animals toward a darker environment to initiate the immobile maturation phase. Thus, PTTH controls the decisions of when and where animals undergo metamorphosis, optimizing conditions for adult development.

Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.

Central role of the oxygen-dependent degradation domain of Drosophila HIFalpha/Sima in oxygen-dependent nuclear export.

The Drosophila HIFalpha homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFalpha/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension.

Regulation of the Drosophila hypoxia-inducible factor alpha Sima by CRM1-dependent nuclear export.

Hypoxia-inducible factor alpha (HIF-alpha) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-alpha protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia.

PVF1/PVR signaling and apoptosis promotes the rotation and dorsal closure of the Drosophila male terminalia.

The Drosophila adult male terminalia originate from the genital disc. During the pupal stages, the external parts of terminalia evert from two ventral stalks; the everted left and right dorsal halves fuse at the dorsal midline. At the same time the male terminalia perform a 360 clockwise rotation. Several mutations are known to affect the rotation of the male terminalia, while none is known to affect dorsal closure. We show here that the Pvf1 gene, encoding one of the three Drosophila homologues of the mammalian VEGF/PDGF growth factors, is required for both processes. Males either mutant for Pvf1 or bearing a dominant negative form of Pvr or stasis (stai), the unique PVF receptor, do not complete either rotation or dorsal closure. Pvf1 expression in the genital disc is restricted to the A8 cells. However, PVF1/PVR signaling influences A8, A9 and A10 cells, suggesting that the PVF1 protein diffuses from its source. Flies hemizygous for the apoptotic genes hid, reaper and grim, or mutant for puckered which encodes a phosphatase that down-regulates the n-Jun-N terminal kinase pathway, lead to the same phenotypes as mutations in PVF1/PVR. Our results indicate that PVF1/PVR signaling functions not only in apoptotic phenomena but are also required during rotation and dorsal closure of the Drosophila male genital disc.