The insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-α /Sima

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-β subunit and an oxygen-regulated HIF-α subunit. We have previously defined a hypoxia-inducible transcriptional response in Drosophila melanogaster that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-α and HIF-β subunits, respectively. HIF-α/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. We show here for the first time that insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, we determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. We demonstrate that stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, we have analyzed in vivo the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein. Overexpression of dAKT and dPDK1 in normoxic embryos provoked a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. We conclude that regulation of HIF-α/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila.Fil:Dekanty, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Lavista-Llanos, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Irisarri, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Wappner, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

No functional contribution of the gustatory receptor, Gr64b, co-expressed in olfactory sensory neurons of Drosophila melanogaster

Chemosensation is essential for the survival of insects. Activities like searching for food, mating, and oviposition in the fruit fly, Drosophila melanogaster are to a great extent governed by chemical cues detected via olfaction and gustation. This chemical information is conveyed to higher brain centers via populations of diverse olfactory sensory neurons (OSNs) and gustatory sensory neurons (GSNs) expressing olfactory receptors (ORs) and gustatory receptors (GRs), respectively. ORs are exclusively expressed in the antenna and in the maxillary palps, while GRs are widely expressed in the labellum, tarsi, genitalia etc. Interestingly, 14 GRs were previously reported to be expressed in the antenna of D. melanogaster. However, the spatial expression pattern for all GRs and their functional role are still unclear. Recent data challenge the dogma that single OSNs express a single OR. In the present study, we studied the expression of 12 previously reported GRs among sensory structures on the fly antenna using the Gal4-UAS binary expression system. We observed antennal expression of nine out of the 12 reported. Out of these nine, consistent expression was only apparent for Gr64b, and we reconfirmed its presence in OSNs innervating three glomeruli in the antennal lobe. These glomeruli are known to be innervated by ab5A, ab5B and ab8A OSNs, respectively. Next, we generated double labeling crosses with Gr64b and observed co-expression of Gr64b with Or47a, which is expressed in the ab5B neuron. To elucidate the functional role of Gr64b co-expressed with Or47a, we challenged Or47a-expressing OSNs in wild type and Gr64b–/– mutant flies with odor stimulation using the single sensillum recording technique in two satiation states (fed and starved). Notably, we did not observe any significant odor sensitivity or specificity changes in Gr64b mutants as compared to wild type flies. Taken together, our results reveal co-expression of GRs with ORs in olfactory sensory neurons, while the functional contribution of the GR in this context remains obscure.</jats:p

Functional interaction between Drosophila olfactory sensory neurons and their support cells

Insects detect volatile chemicals using antennae, which house a vast variety of olfactory sensory neurons (OSNs) that innervate hair-like structures called sensilla where odor detection takes place. In addition to OSNs, the antenna also hosts various support cell types. These include the triad of trichogen, tormogen, and thecogen support cells that lie adjacent to their respective OSNs. The arrangement of OSN supporting cells occurs stereotypically for all sensilla and is widely conserved in evolution. While insect chemosensory neurons have received considerable attention, little is known about the functional significance of the cells that support them. For instance, it remains unknown whether support cells play an active role in odor detection, or only passively contribute to homeostasis, e.g., by maintaining sensillum lymph composition. To investigate the functional interaction between OSNs and support cells, we used optical and electrophysiological approaches in Drosophila. First, we characterized the distribution of various supporting cells using genetic markers. By means of an ex vivo antennal preparation and genetically-encoded Ca(2+) and K(+) indicators, we then studied the activation of these auxiliary cells during odor presentation in adult flies. We observed acute responses and distinct differences in Ca(2+) and K(+) fluxes between support cell types. Finally, we observed alterations in OSN responses upon thecogen cell ablation in mature adults. Upon inducible ablation of thecogen cells, we notice a gain in mechanical responsiveness to mechanical stimulations during single-sensillum recording, but a lack of change to the neuronal resting activity. Taken together, these results demonstrate that support cells play a more active and responsive role during odor processing than previously thought. Our observations thus reveal that support cells functionally interact with OSNs and may be important for the extraordinary ability of insect olfactory systems to dynamically and sensitively discriminate between odors in the turbulent sensory landscape of insect flight

Optimization of insect odorant receptor trafficking and functional expression via transient transfection in HEK293 cells

Insect odorant receptors show a limited functional expression in various heterologous expression systems including insect and mammalian cells. This may be in part due to the absence of key components driving the release of these proteins from the endoplasmic reticulum and directing them to the plasma membrane. In order to mitigate this problem we took advantage of small export signals within the human HCN1 and Rhodopsin that have been shown to promote protein release from the endoplasmic reticulum and the trafficking of post-Golgi vesicles, respectively. Moreover, we designed a new vector based on a bidirectional expression cassette to drive the functional expression of the insect odorant receptor co-receptor (Orco) and an odor-binding odorant receptor, simultaneously. We show that this new method can be used to reliably express insect odorant receptors in HEK293 cells via transient transfection and that is highly suitable for downstream applications using automated and high-throughput imaging platforms

Dopamine drives Drosophila sechellia adaptation to its toxic host

Many insect species are host-obligate specialists. The evolutionary mechanism driving the adaptation of a species to a toxic host is, however, intriguing. We analyzed the tight association of Drosophila sechellia to its sole host, the fruit of Morinda citrifolia, which is toxic to other members of the melanogaster species group. Molecular polymorphisms in the dopamine regulatory protein Catsup cause infertility in D. sechellia due to maternal arrest of oogenesis. In its natural host, the fruit compensates for the impaired maternal dopamine metabolism with the precursor l-DOPA, resuming oogenesis and stimulating egg production. l-DOPA present in morinda additionally increases the size of D. sechellia eggs, what in turn enhances early fitness. We argue that the need of l-DOPA for successful reproduction has driven D. sechellia to become an M. citrifolia obligate specialist. This study illustrates how an insect's dopaminergic system can sustain ecological adaptations by modulating ontogenesis and development. DOI: http://dx.doi.org/10.7554/eLife.03785.00

Odor-Induced Multi-Level Inhibitory Maps in Drosophila

Optical imaging of intracellular Ca2+ influx as a correlate of neuronal excitation represents a standard technique for visualizing spatiotemporal activity of neuronal networks. However, the information-processing properties of single neurons and neuronal circuits likewise involve inhibition of neuronal membrane potential. Here, we report spatially resolved optical imaging of odor-evoked inhibitory patterns in the olfactory circuitry of Drosophila using a genetically encoded fluorescent Cl- sensor. In combination with the excitatory component reflected by intracellular Ca2+ dynamics, we present a comprehensive functional map of both odor-evoked neuronal activation and inhibition at different levels of olfactory processing. We demonstrate that odor-evoked inhibition carried by Cl- influx is present both in sensory neurons and second-order projection neurons (PNs), and is characterized by stereotypic, odor-specific patterns. Cl--mediated inhibition features distinct dynamics in different neuronal populations. Our data support a dual role of inhibitory neurons in the olfactory system: global gain control across the neuronal circuitry and glomerulus-specific inhibition to enhance neuronal information processing

Odor-Induced Multi-Level Inhibitory Maps in Drosophila

Grabe V, Schubert M, Strube-Bloss M, et al. Odor-Induced Multi-Level Inhibitory Maps in Drosophila. eNeuro. 2019;7(1): ENEURO.0213-19.2019

Mate discrimination among subspecies through a conserved olfactory pathway.

Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner