What's hot today:
Current papers in developmental biology and gene function


Wednesday July 31st, 2019 - Behavior

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Cheng, K. Y., Colbath, R. A. and Frye, M. A. (2019). Olfactory and neuromodulatory signals reverse visual object avoidance to approach in Drosophila. Curr Biol 29(12): 2058-2065. PubMed ID: 31155354
Behavioral reactions of animals to environmental sensory stimuli are sometimes reflexive and stereotyped but can also vary depending on contextual conditions. Engaging in active foraging or flight provokes a reversal in the valence of carbon dioxide responses from aversion to approach in Drosophila, whereas mosquitoes encountering this same chemical cue show enhanced approach toward a small visual object. Sensory plasticity in insects has been broadly attributed to the action of biogenic amines, which modulate behaviors such as olfactory learning, aggression, feeding, and egg laying. Octopamine acts rapidly upon the onset of flight to modulate the response gain of directionally selective motion-detecting neurons in Drosophila. How the action of biogenic amines might couple sensory modalities to each other or to locomotive states remains poorly understood. This study used a visual flight simulator equipped for odor delivery to confirm that flies avoid a small contrasting visual object in odorless air but that the same animals reverse their preference to approach in the presence of attractive food odor. An aversive odor does not reverse object aversion. Optogenetic activation of either octopaminergic neurons or directionally selective motion-detecting neurons that express octopamine receptors elicits visual valence reversal in the absence of odor. The results suggest a parsimonious model in which odor-activated octopamine release excites the motion detection pathway to increase the saliency of either a small object or a bar, eliciting tracking responses by both visual features.
Enriquez, T. and Colinet, H. (2019). Cold acclimation triggers major transcriptional changes in Drosophila suzukii. BMC Genomics 20(1): 413. PubMed ID: 31117947
Insects have the capacity to adjust their physiological mechanisms during their lifetime to promote cold tolerance and cope with sublethal thermal condition. The spotted wing drosophila, Drosophila suzukii is an invasive fruit pest that enhances its thermotolerance in response to thermal acclimation. This study promoted flies' cold tolerance by gradually increasing acclimation duration (i.e. pre-exposure from 2 h to 9 days at 10 degrees C), and then compared transcriptomic responses of cold hardy versus cold susceptible phenotypes using RNA sequencing. Cold tolerance of D. suzukii increased with acclimation duration; the longer the acclimation, the higher the cold tolerance. Cold-tolerant flies that were acclimated for 9 days were selected for transcriptomic analyses. RNA sequencing revealed a total of 2908 differentially expressed genes: 1583 were up- and 1325 were downregulated in cold acclimated flies. Ionic transport across membranes and signaling were highly represented GO-terms in acclimated flies. Neuronal activity and carbohydrate metabolism were also enriched GO-terms in acclimated flies. GO-terms related to oogenesis were underrepresented in acclimated flies. Involvement of a large cluster of genes related to ion transport in cold acclimated flies suggests adjustments in the capacity to maintain ion and water homeostasis. Down regulation of genes related to oogenesis in cold acclimated females likely reflects that females were conditioned at 10 degrees C, a temperature that prevents oogenesis. Overall, these results help to understand the molecular underpinnings of cold tolerance acquisition in D. suzukii. These data are of importance considering that the invasive success of D. suzukii in diverse climatic regions relates to its high thermal plasticity.
Bezerra Da Silva, C. S., Park, K. R., Blood, R. A. and Walton, V. M. (2019). Intraspecific competition affects the pupation behavior of spotted-wing Drosophila (Drosophila suzukii). Sci Rep 9(1): 7775. PubMed ID: 31123337
In Drosophila, intraspecific competition (IC) may cause stress, cannibalism, and affect survival and reproduction. By migrating to less crowded environments, individuals can escape IC. Larvae of spotted-wing drosophila (SWD, Drosophila suzukii) are often exposed to IC. They are known to pupate either attached to or detached from their hosts. This study hypothesized that SWD pupates detached from the larval host as a means to escape IC and increase their survival and fitness. Under laboratory conditions, IC resulted in increased pupation detached from the larval host in both cornmeal medium and blueberry fruit. Males were more prone to detached pupation than females. In blueberry, IC-exposed larvae pupated farther away from the fruit relative to singly-developed individuals. Detached pupation was associated to survival and fitness gains. For example, larvae that displayed detached pupation showed shorter egg-pupa development times, higher pupa-adult survival, and larger adult size relative to fruit-attached individuals. These findings demonstrate that SWD larvae select pupation sites based on IC, and that such a strategy is associated with improved survival and fitness. This information contributes to a better understanding of SWD basic biology and behavior, offering insights to the development of improved practices to manage this pest in the field.
Chakraborty, T. S., Gendron, C. M., Lyu, Y., Munneke, A. S., DeMarco, M. N., Hoisington, Z. W. and Pletcher, S. D. (2019). Sensory perception of dead conspecifics induces aversive cues and modulates lifespan through serotonin in Drosophila. Nat Commun 10(1): 2365. PubMed ID: 31147540
Sensory perception modulates health and aging across taxa. Understanding the nature of relevant cues and the mechanisms underlying their action may lead to novel interventions that improve the length and quality of life. This study found that in the vinegar fly, Drosophila melanogaster, exposure to dead conspecifics in the environment induced cues that were aversive to other flies, modulated physiology, and impaired longevity. The effects of exposure to dead conspecifics on aversiveness and lifespan required visual and olfactory function in the exposed flies. Furthermore, the sight of dead flies was sufficient to produce aversive cues and to induce changes in the head metabolome. Genetic and pharmacologic attenuation of serotonergic signaling eliminated the effects of exposure on aversiveness and lifespan. These results indicate that Drosophila have an ability to perceive dead conspecifics in their environment and suggest conserved mechanistic links between neural state, health, and aging; the roots of which might be unearthed using invertebrate model systems.
Parigi, A., Porter, C., Cermak, M., Pitchers, W. R. and Dworkin, I. (2019). The behavioral repertoire of Drosophila melanogaster in the presence of two predator species that differ in hunting mode. PLoS One 14(5): e0216860. PubMed ID: 31150415
The fruit fly, Drosophila melanogaster, has proven to be an excellent model organism for genetic, genomic and neurobiological studies. However, relatively little is known about the natural history of D. melanogaster. In particular, neither the natural predators faced by wild populations of D. melanogaster, nor the anti-predatory behaviors they may employ to escape and avoid their enemies have been documented. This study observed and described the influence of two predators that differ in their mode of hunting: zebra jumping spiders, Salticus scenicus (active hunters) and Chinese praying mantids, Tenodera sinensis (ambush predators) on the behavioral repertoire of Drosophila melanogaster. Three particularly interesting behaviors were documented: abdominal lifting, stopping, and retreat-which were performed at higher frequency by D. melanogaster in the presence of predators. While mantids had only a modest influence on the locomotory activity of D. melanogaster, a significant increase was observed in the overall activity of D. melanogaster in the presence of jumping spiders. Finally, considerable among-individual behavioral variation was observed in response to both predators.
Baik, L. S., Recinos, Y., Chevez, J. A., Au, D. D. and Holmes, T. C. (2019). Multiple phototransduction inputs integrate to mediate UV light-evoked avoidance/attraction behavior in Drosophila. J Biol Rhythms: 748730419847339. PubMed ID: 31140349
Short-wavelength light guides many behaviors that are crucial for an insect's survival. In Drosophila melanogaster, short-wavelength light induces both attraction and avoidance behaviors. How light cues evoke two opposite valences of behavioral responses remains unclear. This study comprehensively examined the effects of (1) light intensity, (2) timing of light (duration of exposure, circadian time of day), and (3) phototransduction mechanisms processing light information that determine avoidance versus attraction behavior assayed at high spatiotemporal resolution in Drosophila. External opsin-based photoreceptors signal for attraction behavior in response to low-intensity ultraviolet (UV) light. In contrast, the cell-autonomous neuronal photoreceptors, CRYPTOCHROME (CRY) and RHODOPSIN 7 (RH7), signal avoidance responses to high-intensity UV light. In addition to binary attraction versus avoidance behavioral responses to UV light, flies show distinct clock-dependent spatial preference within a light environment coded by different light input channels.

Tuesday, July 30th - Signaling

Wu, X., Zhang, Y., Chuang, K. H., Cai, X., Ajaz, H. and Zheng, X. (2019). The Drosophila Hedgehog receptor component Interference hedgehog (Ihog) mediates cell-cell interactions through trans-homophilic binding. J Biol Chem. PubMed ID: 31209108
Hedgehog (Hh) signaling is crucial for establishing complex cellular patterns in embryonic tissues and maintaining homeostasis in adult organs. In Drosophila, Interference hedgehog (Ihog) or its close paralogue Bother of Ihog (Boi) forms a receptor complex with Patched to mediate intracellular Hh signaling. Ihog proteins (Ihog and Boi) also contribute to cell segregation in wing imaginal discs through an unknown mechanism independent of their role in transducing the Hh signal. This study reports a molecular mechanism by which the Ihog proteins mediate cell-cell interactions. Ihog proteins were found to be enriched at the site of cell-cell contacts and engage in trans-homophilic interactions in a calcium-independent manner. The region that was identified as mediating the trans-Ihog-Ihog interaction overlaps with the Ihog-Hh interface on the first fibronectin repeat of the extracellular domain of Ihog. It was further demonstrated that Hh interferes with Ihog-mediated homophilic interactions by competing for Ihog binding. These results, thus, not only reveal a mechanism for Ihog-mediated cell-cell interactions, but also suggest a direct Hh-mediated regulation of both intracellular signaling and cell adhesion through Ihog.
Maier, D., Nagel, A. C., Kelp, A. and Preiss, A. (2019). Protein kinase D is dispensable for development and survival of Drosophila melanogaster. G3 (Bethesda). PubMed ID: 31142547
Members of the Protein Kinase D (PKD) family are involved in numerous cellular processes in mammals, including cell survival after oxidative stress, polarized transport of Golgi vesicles, as well as cell migration and invasion. PKD proteins belong to the PKC/CAMK class of serine/threonine kinases, and transmit diacylglycerol-regulated signals. Whereas three PKD isoforms are known in mammals, Drosophila melanogaster contains a single PKD homologue. Previous analyses using overexpression and RNAi studies indicated likewise multi-facetted roles for Drosophila PKD, including the regulation of secretory transport and actin-cytoskeletal dynamics. This study generated PKD null alleles that are homozygous viable without apparent phenotype. They largely match control flies regarding fertility, developmental timing and weight. Males, but not females, are slightly shorter lived and starvation sensitive. Furthermore, migration of pole cells in embryos and border cells in oocytes appears normal. PKD mutants tolerate heat, cold and osmotic stress like the control but are sensitive to oxidative stress, conforming to the described role for mammalian PKDs. A candidate screen to identify functionally redundant kinases uncovered genetic interactions of PKD with Pkcdelta, sqa and Drak mutants, further supporting the role of PKD in oxidative stress response, and suggesting its involvement in starvation induced autophagy and regulation of cytoskeletal dynamics. Overall, PKD appears dispensable for fly development and survival presumably due to redundancy, but influences environmental responses.
Harpprecht, L., Baldi, S., Schauer, T., Schmidt, A., Bange, T., Robles, M. S., Kremmer, E., Imhof, A. and Becker, P. B. (2019). A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling. Nucleic Acids Res. PubMed ID: 31147711
Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. This chromatin assembly system was found to contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to gammaH2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The gammaH2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range gammaH2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei.
Mahneva, O., Caplan, S. L., Ivko, P., Dawson-Scully, K. and Milton, S. L. (2019). NO/cGMP/PKG activation protects Drosophila cells subjected to hypoxic stress. Comp Biochem Physiol C Toxicol Pharmacol 223: 106-114. PubMed ID: 31150868
The anoxia-tolerant fruit fly, Drosophila melanogaster, has routinely been used to examine cellular mechanisms responsible for anoxic and oxidative stress resistance. Nitric oxide (NO), an important cellular signaling molecule, and its downstream activation of cGMP-dependent protein kinase G (PKG) has been implicated as a protective mechanism against ischemic injury in diverse animal models from insects to mammals. In Drosophila, increased PKG signaling results in increased survival of animals exposed to anoxic stress. To determine if activation of the NO/cGMP/PKG pathway is protective at the cellular level, the present study employed a pharmacological protocol to mimic hypoxic injury in Drosophila S2 cells. The commonly used S2 cell line was derived from a primary culture of late stage (20-24h old) Drosophila melanogaster embryos. Hypoxic stress was induced by exposure to either sodium azide (NaN3) or cobalt chloride (CoCl2). During chemical hypoxic stress, NO/cGMP/PKG activation protected against cell death and this mechanism involved modulation of downstream mitochondrial ATP-sensitive potassium ion channels (mitoKATP). The cellular protection afforded by NO/cGMP/PKG activation during ischemia-like stress may be an adaptive cytoprotective mechanism and modulation of this signaling cascade could serve as a potential therapeutic target for protection against hypoxia or ischemia-induced cellular injury.
Choi, J., Troyanovsky, R. B., Indra, I., Mitchell, B. J. and Troyanovsky, S. M. (2019). Scribble, Erbin, and Lano redundantly regulate epithelial polarity and apical adhesion complex. J Cell Biol. PubMed ID: 31147384
The basolateral protein Scribble (Scrib), a member of the LAP protein family, is essential for epithelial apicobasal polarity (ABP) in Drosophila. However, a conserved function for this protein in mammals is unclear. This study showed that the crucial role for Scrib in ABP has remained obscure due to the compensatory function of two other LAP proteins, Erbin and Lano. A combined Scrib/Erbin/Lano knockout disorganizes the cell-cell junctions and the cytoskeleton. It also results in mislocalization of several apical (Par6, aPKC, and Pals1) and basolateral (Llgl1 and Llgl2) identity proteins. These defects can be rescued by the conserved "LU" region of these LAP proteins. Structure-function analysis of this region determined that the so-called LAPSDb domain is essential for basolateral targeting of these proteins, while the LAPSDa domain is essential for supporting the membrane basolateral identity and binding to Llgl. In contrast to the key role in Drosophila, mislocalization of Llgl proteins does not appear to be critical in the scrib ABP phenotype.
He, L., Binari, R., Huang, J., Falo-Sanjuan, J. and Perrimon, N. (2019). In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer. Elife 8. PubMed ID: 31140975
Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, this study enhanced translation of a destabilized fluorescent protein and demonstrated the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, a dual-color transcriptional timer was generated that provides spatio-temporal information about signaling pathway activities. Finally, the use of this transcriptional timer to identify new genes with dynamic expression patterns was demonstrated.

Monday, July 29th - Adult Neural Development and Function

Dombrovski, M., Kim, A., Poussard, L., Vaccari, A., Acton, S., Spillman, E., Condron, B. and Yuan, Q. (2019). A plastic visual pathway regulates cooperative behavior in Drosophila larvae. Curr Biol 29(11): 1866-1876. PubMed ID: 31130457
Cooperative behavior emerges in biological systems through coordinated actions among individuals. Although widely observed across animal species, the cellular and molecular mechanisms underlying the establishment and maintenance of cooperative behaviors remain largely unknown. To characterize the circuit mechanisms serving the needs of independent individuals and social groups, this study investigated cooperative digging behavior in Drosophila larvae. Although chemical and mechanical sensations are important for larval aggregation at specific sites, an individual larva's ability to participate in a cooperative burrowing cluster relies on direct visual input as well as visual and social experience during development. In addition, vision modulates cluster dynamics by promoting coordinated movements between pairs of larvae. To determine the specific pathways within the larval visual circuit underlying cooperative social clustering, larval photoreceptors (PRs) and the downstream local interneurons (lOLPs) were examined using anatomical and functional studies. The results indicate that rhodopsin-6-expressing-PRs (Rh6-PRs) and lOLPs are required for both cooperative clustering and movement detection. Remarkably, visual deprivation and social isolation strongly impact the structural and functional connectivity between Rh6-PRs and lOLPs, while at the same time having no effect on the adjacent rhodopsin-5-expressing PRs (Rh5-PRs). Together, these findings demonstrate that a specific larval visual pathway involved in social interactions undergoes experience-dependent modifications during development, suggesting that plasticity in sensory circuits could act as the cellular substrate for social learning, a possible mechanism allowing an animal to integrate into a malleable social environment and engage in complex social behaviors.
Du, E. J., Ahn, T. J., Sung, H., Jo, H., Kim, H. W., Kim, S. T. and Kang, K. (2019). Analysis of phototoxin taste closely correlates nucleophilicity to type 1 phototoxicity. Proc Natl Acad Sci U S A 116(24): 12013-12018. PubMed ID: 31138707
Pigments often inflict tissue-damaging and proaging toxicity on light illumination by generating free radicals and reactive oxygen species (ROS). However, the molecular mechanism by which organisms sense phototoxic pigments is unknown. This study discovered that Transient Receptor Potential Ankyrin 1-A isoform [TRPA1A], previously shown to serve as a receptor for free radicals and ROS induced by photochemical reactions, enables Drosophila melanogaster to aphotically sense phototoxic pigments for feeding deterrence. Thus, TRPA1A detects both cause (phototoxins) and effect (free radicals and ROS) of photochemical reactions. A group of pigment molecules not only activates TRPA1(A) in darkness but also generates free radicals on light illumination. Such aphotic detection of phototoxins harboring the type 1 (radical-generating) photochemical potential requires the nucleophile-sensing ability of TRPA1. In addition, agTRPA1A from malaria-transmitting mosquitoes Anopheles gambiae heterologously produces larger current responses to phototoxins than Drosophila TRPA1A, similar to their disparate nucleophile responsiveness. Along with TRPA1A-stimulating capabilities, type 1 phototoxins exhibit relatively strong photo-absorbance and low energy gaps between the highest occupied molecular orbital and the lowest unoccupied molecular orbital. However, TRPA1A activation is more highly concordant to type 1 phototoxicity than are those photochemical parameters. Collectively, nucleophile sensitivity of TRPA1A allows flies to taste potential phototoxins for feeding deterrence, preventing postingestive photo-injury. Conversely, pigments need to bear high nucleophilicity (electron-donating propensity) to act as type 1 phototoxins, which is consistent with the fact that transferring photoexcited electrons from phototoxins to other molecules causes free radicals. Thus, identification of a sensory mechanism in Drosophila reveals a property fundamental to type 1 phototoxins.
Hartenstein, V., Yuan, M., Younossi-Hartenstein, A., Karandikar, A., Bernardo-Garcia, F. J., Sprecher, S. and Knust, E. (2019). Serial electron microscopic reconstruction of the Drosophila larval eye: Photoreceptors with a rudimentary rhabdomere of microvillar-like processes. Dev Biol. PubMed ID: 31158364
Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. This paper analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3mum diameter) alternating with thin segments (>0.1mum). Loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.
Kang, H., Zhao, J., Jiang, X., Li, G., Huang, W., Cheng, H. and Duan, R. (2019). Drosophila Netrin-B controls mushroom body axon extension and regulates courtship-associated learning and memory of a Drosophila fragile X syndrome model. Mol Brain 12(1): 52. PubMed ID: 31138234
Mushroom body (MB) is a prominent structure essential for olfactory learning and memory in the Drosophila brain. The development of the MB involves the appropriate guidance of axon lobes and sister axon branches. Appropriate guidance that accurately shapes MB development requires the integration of various guidance cues provided by a series of cell types, which guide axons to reach their final positions within the MB neuropils. Netrins are axonal guidance molecules that are conserved regulators of embryonic nerve cord patterning. However, whether they contribute to MB morphogenesis has not yet been evaluated. This study found that Netrin-B (NetB) is highly expressed in the MB lobes, regulating lobe length through genetic interactions with the receptors Frazzled and Uncoordinated-5 from 24 h after pupal formation onwards. Overexpression of NetB causes severe beta lobe fusion in the MB, which is similar to the MB defects seen in the Drosophila model of fragile X syndrome (FXS). These results further show that fragile-X mental retardation protein FMRP inhibits the translational activity of human ortholog Netrin-1 (NTN1). Knock-down of NetB significantly rescues the MB defects and ameliorates deficits in the learning and memory in FXS model Drosophila. These results indicate a critical role for NetB in MB lobe extension and identify NetB as a novel target of FMRP which contributes to learning and memory.
Trush, O., Liu, C., Han, X., Nakai, Y., Takayama, R., Murakawa, H., Carrillo, J. A., Takechi, H., Hakeda-Suzuki, S., Suzuki, T. and Sato, M. (2019). N-cadherin orchestrates self-organization of neurons within a columnar unit in the Drosophila medulla. J Neurosci. PubMed ID: 31175213
Columnar structure is a basic unit of the brain, but the mechanism underlying its development remains largely unknown. The medulla, the largest ganglion of the Drosophila melanogaster visual center, provides a unique opportunity to reveal the mechanisms of three-dimensional organization of the columns. In this study, using N-cadherin (Ncad) as a marker, the donut-like columnar structures along the two-dimensional layer in the larval medulla were revealed that evolves to form three distinct layers in pupal development. Column formation is initiated by three core neurons, R8, R7, and Mi1, which establish distinct concentric domains within a column. Ncad-dependent relative adhesiveness of the core columnar neurons regulates their relative location within a column along a two-dimensional layer in the larval medulla according to the differential adhesion hypothesis. The presence of mutual interactions is proposed among the three layers during formation of the three-dimensional structures of the medulla columns.
Yoon, Y., Park, J., Taniguchi, A., Kohsaka, H., Nakae, K., Nonaka, S., Ishii, S. and Nose, A. (2019). System level analysis of motor-related neural activities in larval Drosophila. J Neurogenet: 1-11. PubMed ID: 31172848
The way in which the central nervous system (CNS) governs animal movement is complex and difficult to solve solely by the analyses of muscle movement patterns. This problem is tackled by observing the activity of a large population of neurons in the CNS of larval Drosophila. Focus was placed on two major behaviors of the larvae - forward and backward locomotion - and the neuronal activity related to these behaviors during the fictive locomotion that occurs spontaneously in the isolated CNS. A genetically-encoded calcium indicator, GCaMP, and a nuclear marker were analyzed in all neurons, and then digitally scanned light-sheet microscopy was used to record (at a fast frame rate) neural activities in the entire ventral nerve cord (VNC). The experimental procedures and computational pipeline enabled systematic identification of neurons that showed characteristic motor activities in larval Drosophila. Cells were found whose activity was biased toward forward locomotion and others biased toward backward locomotion. In particular, neurons near the boundary of the subesophageal zone (SEZ) and thoracic neuromeres were identified that were strongly active during an early phase of backward but not forward fictive locomotion.

Friday, July 26th - Evolution

Orsted, M., Hoffmann, A. A., Sverrisdottir, E., Nielsen, K. L. and Kristensen, T. N. (2019). Genomic variation predicts adaptive evolutionary responses better than population bottleneck history. PLoS Genet 15(6): e1008205. PubMed ID: 31188830

The relationship between population size, inbreeding, loss of genetic variation and evolutionary potential of fitness traits is still unresolved, and large-scale empirical studies testing theoretical expectations are surprisingly scarce. This study presents a highly replicated experimental evolution setup with 120 lines of Drosophila melanogaster having experienced inbreeding caused by low population size for a variable number of generations. Genetic variation in inbred lines and in outbred control lines was assessed by genotyping-by-sequencing (GBS) of pooled samples consisting of 15 males per line. All lines were reared on a novel stressful medium for 10 generations during which body mass, productivity, and extinctions were scored in each generation. In addition, egg-to-adult viability was investigated in the benign and the stressful environments before and after rearing at the stressful conditions for 10 generations. Strong positive correlations were found between levels of genetic variation and evolutionary response in all investigated traits, and showed that genomic variation was more informative in predicting evolutionary responses than population history reflected by expected inbreeding levels. It was also found that lines with lower genetic diversity were at greater risk of extinction. For viability, the results suggested a trade-off in the costs of adapting to the stressful environments when tested in a benign environment. This work presents convincing support for long-standing evolutionary theory, and it provides novel insights into the association between genetic variation and evolutionary capacity in a gradient of diversity rather than dichotomous inbred/outbred groups.

Yun, L., Bayoumi, M., Yang, S., Chen, P. J., Rundle, H. D. and Agrawal, A. F. (2019). Testing for local adaptation in adult male and female fitness among populations evolved under different mate competition regimes. Evolution. PubMed ID: 31206649
Mating/fertilization success and fecundity are influenced by sexual interactions among individuals, the nature and frequency of which can vary among different environments. The extent of local adaptation for such adult fitness components is poorly understood. This study allowed 63 populations of Drosophila melanogaster to independently evolve in one of three mating environments that alter sexual interactions: one involved enforced monogamy, while the other two permitted polygamy in either structurally simple standard fly vials or in larger "cages" with added complexity. Adult male and female reproductive fitness were measured after 16 and 28 generations, respectively, via full reciprocal transplants. In males, reciprocal local adaptation was observed between the monogamy and simple polygamy treatments, consistent with the evolution of reproductively competitive males under polygamy that perform poorly under monogamy because they harm their only mate. However, males evolved in the complex polygamy treatment performed similarly or better than all other males in all mating environments, consistent with previous results showing higher genetic quality in this treatment. Differences in female fitness were more muted, suggesting selection on females was less divergent across the mating treatments and echoing a common pattern of greater phenotypic and expression divergence in males than females.
Silva, J. J. and Scott, J. G. (2019). Conservation of the voltage-sensitive sodium channel protein within the Insecta. Insect Mol Biol. PubMed ID: 31206812
The voltage-sensitive sodium channel (VSSC; see Paralytic) is essential for the generation and propagation of action potentials. VSSC kinetics can be modified by producing different splice variants. The functionality of VSSC depends on features such as the voltage-sensors, the selectivity filter and the inactivation loop. Mutations in Vssc conferring resistance to pyrethroid insecticides are known as knockdown resistance (kdr). This study analyzed the conservation of VSSC in both a broad scope and a narrow scope by three approaches: 1) Compare conservation of sequences and of differential exon use across orders of the Insecta, 2) determine which kdr mutations were possible with a single nucleotide mutation in nine populations or Aedes aegypti, and 3) examine the individual VSSC variation that exists within a population of Drosophila melanogaster. There is an increasing amount of transcript diversity possible from Diplura towards Diptera. The residues of the voltage-sensors, selectivity filter and inactivation loop are highly conserved. The majority of exon sequences were >88.6% similar. Strain specific differences in codon constraints exist for kdr mutations in nine strains of A. aegypti. Three Vssc mutations were found in one population of D. melanogaster. This study shows that overall Vssc is highly conserved across Insecta and within a population of an insect, but that important differences do exist.
Sztepanacz, J. L. and Houle, D. (2019). Cross-sex genetic covariances limit the evolvability of wing-shape within and among species of Drosophila. Evolution. PubMed ID: 31206655
The independent evolution of males and females is potentially constrained by both sexes inheriting the same alleles from their parents. This genetic constraint can limit the evolvability of complex traits; however, there are few studies of multivariate evolution that incorporate cross-sex genetic covariances in their predictions. Drosophila wing-shape has emerged as a model high-dimensional phenotype; wing-shape is highly evolvable in contemporary populations, and yet perplexingly stable across phylogenetic timescales. This study shows that cross-sex covariances in D. melanogaster, given by the B-matrix, may considerably bias wing-shape evolution. Using random skewers, this study shows that B would constrain the response to antagonistic selection by 90%, on average, but would double the response to concordant selection. Both cross-sex within-trait and cross-sex cross-trait covariances determined the predicted response to antagonistic selection, but only cross-sex within-trait covariances facilitated the predicted response to concordant selection. Similar patterns were observed in the direction of extant sexual dimorphism in D. melanogaster, and in directions of most and least dimorphic variation across the Drosophila phylogeny. These results highlight the importance of considering between-sex genetic covariances when making predictions about evolution on both macro- and micro- evolutionary timescales, and may provide one more explanatory piece in the puzzle of stasis.
Ahmed, O. M., Avila-Herrera, A., Tun, K. M., Serpa, P. H., Peng, J., Parthasarathy, S., Knapp, J. M., Stern, D. L., Davis, G. W., Pollard, K. S. and Shah, N. M. (2019). Evolution of mechanisms that control mating in Drosophila males. Cell Rep 27(9): 2527-2536. PubMed ID: 31141679
Genetically wired neural mechanisms inhibit mating between species because even naive animals rarely mate with other species. These mechanisms can evolve through changes in expression or function of key genes in sensory pathways or central circuits. Gr32a is a gustatory chemoreceptor that, in D. melanogaster, is essential to inhibit interspecies courtship and sense quinine. Similar to D. melanogaster, this study found that D. simulans Gr32a is expressed in foreleg tarsi, sensorimotor appendages that inhibit interspecies courtship, and it is required to sense quinine. Nevertheless, Gr32a is not required to inhibit interspecies mating by D. simulans males. However, and similar to its function in D. melanogaster, Ppk25, a member of the Pickpocket family, promotes conspecific courtship in D. simulans. Together, this study have identified distinct evolutionary mechanisms underlying chemosensory control of taste and courtship in closely related Drosophila species.
Hawkes, M. F., Duffy, E., Joag, R., Skeats, A., Radwan, J., Wedell, N., Sharma, M. D., Hosken, D. J. and Troscianko, J. (2019). Sexual selection drives the evolution of male wing interference patterns. Proc Biol Sci 286(1903): 20182850. PubMed ID: 31138076
The seemingly transparent wings of many insects have recently been found to display unexpected structural coloration. These structural colours (wing interference patterns: WIPs) may be involved in species recognition and mate choice, yet little is known about the evolutionary processes that shape them. Furthermore, to date investigations of WIPs have not fully considered how they are actually perceived by the viewers' colour vision. This study used multispectral digital imaging and a model of Drosophila vision to compare WIPs of male and female Drosophila simulans from replicate populations forced to evolve with or without sexual selection for 68 generations. WIPs modelled in Drosophila vision evolve in response to sexual selection and provide evidence that WIPs correlate with male sexual attractiveness. These findings add a new element to the otherwise well-described Drosophila courtship display and confirm that wing colours evolve through sexual selection.

Thursday, July 25th - Embryonic Development

McCleery, W. T., Veldhuis, J., Bennett, M. E., Lynch, H. E., Ma, X., Brodland, G. W. and Hutson, M. S. (2019). Elongated cells drive morphogenesis in a surface-wrapped finite-element model of germband retraction. Biophys J. PubMed ID: 31229244
During Drosophila embryogenesis, the germband first extends to curl around the posterior end of the embryo and then retracts back; however, retraction is not simply the reversal of extension. At a tissue level, extension is coincident with ventral furrow formation, and at a cellular level, extension occurs via convergent cell neighbor exchanges in the germband, whereas retraction involves only changes in cell shape. This study investigated this process using a whole-embryo, surface-wrapped cellular finite-element model. This model represents two key epithelial tissues-amnioserosa and germband-as adjacent sheets of two-dimensional cellular finite elements that are wrapped around an ellipsoidal three-dimensional approximation of an embryo. The model reproduces the detailed kinematics of in vivo retraction by fitting just one free model parameter, the tension along germband cell interfaces; all other cellular forces are constrained to follow ratios inferred from experimental observations. With no additional parameter adjustments, the model also reproduces quantitative assessments of mechanical stress using laser dissection and failures of retraction when amnioserosa cells are removed via mutations or microsurgery. Surprisingly, retraction in the model is robust to changes in cellular force values but is critically dependent on starting from a configuration with highly elongated amnioserosa cells. Their extreme cellular elongation is established during the prior process of germband extension and is then used to drive retraction. The amnioserosa is the one tissue whose cellular morphogenesis is reversed from germband extension to retraction, and this reversal coordinates the forces needed to retract the germband back to its pre-extension position and shape. In this case, cellular force strengths are less important than the carefully established cell shapes that direct them.
Yue, X., Li, D., Lv, J., Liu, K., Chen, J. and Zhang, W. (2019). Involvement of mind the gap in the organization of the tracheal apical extracellular matrix in Drosophila and Nilaparvata lugens. Insect Sci. PubMed ID: 31240817
The tracheal apical extracellular matrix (aECM) is vital for expansion of the tracheal lumen and supports the normal structure of the lumen to guarantee air entry and circulation in insects. Although it has been found that some cuticular proteins are involved in the organization of the aECM, unidentified factors still exist. This study found that mind the gap (Mtg), a predicted chitin binding protein, is required for the normal formation of the apical chitin matrix of airway tubes in the model holometabolous insect Drosophila melanogaster. Similar to chitin, the Mtg protein was linearly arranged in the tracheal dorsal trunk of the tracheae in Drosophila. Decreased mtg expression in the tracheae seriously affected the viability of larvae and caused tracheal chitin spiral defects in some larvae. Analysis of mtg mutant showed that mtg was required for normal development of tracheae in embryos. Irregular taenidial folds of some mtg mutant embryos were found on either lateral view of tracheal dorsal trunk or internal view of transmission electron microscopy analysis. These abnormal tracheae were not full-filled with gas and accompanied by a reduction in tracheal width, which are characteristic phenotypes of tracheal aECM defects. Furthermore, in the hemimetabolous brown planthopper (BPH) Nilaparvata lugens, downregulation of NlCPAP1-N (a homologue of mtg) also led to the formation of abnormal tracheal chitin spirals and death. These results suggest that mtg and its homologue are involved in the proper organization of the tracheal aECMs in flies and BPH, and that this function may be conserved in insects.
Jeon, H., O, J., Jin, S., Lim, J. and Choe, C. P. (2019). A role for buttonhead in the early head and trunk development in the beetle Tribolium castaneum. Dev Reprod 23(1): 63-72. PubMed ID: 31049473
The head gap gene buttonhead (btd) is required for the patterning of head segments in the early Drosophila embryo. Mutant phenotypes of btd display a gap-like phenotype in which antennal, intercalary, mandibular and the anterior portion of the maxillary segments are eliminated. In agreement with the phenotypes, btd is expressed in a stripe covering the head segments at the blastoderm stage. During the early phase of the germband extension, btd is expressed in stripes with single segmental periodicity, which is required for the formation of the peripheral nervous system. In contrast to the key role of btd in Drosophila embryonic development, it has been suggested that Tribolium ortholog of btd (Tc-btd) is dispensable for embryonic head development. In order for better understanding of the requirement of Tc-btd in the early Tribolium embryo, this study re-analyzed the expression patterns and functions of Tc-btd during embryonic segmentation. Tc-btd is expressed in segmental stripes at the stages of blastoderm and germband elongation. Up to 28.3% of embryos in which Tc-btd is knocked down display the loss of antennal, mandibular and the pregnathal regions in the head, with abdominal segments being disrupted in the trunk. These findings suggest that Tc-btd is required for the head and trunk development in the early Tribolium embryo.
Kwasnieski, J. C., Orr-Weaver, T. L. and Bartel, D. P. (2019). Early genome activation in Drosophila is extensive with an initial tendency for aborted transcripts and retained introns. Genome Res. PubMed ID: 31235656
Control of metazoan embryogenesis shifts from maternal to zygotic gene products as the zygotic genome becomes transcriptionally activated. In Drosophila, zygotic genome activation (ZGA) has been thought to occur in two phases, starting with a minor wave, in which a small number of genes become expressed, and progressing to the major wave, in which many more genes are activated. However, technical challenges have hampered the identification of early transcripts or obscured the onset of their transcription. This study developed an approach to isolate transcribed mRNAs and applied it over the course of Drosophila early genome activation. The results increase by 10-fold the genes reported to be activated during what has been thought of as the minor wave and shows that early genome activation is continuous and gradual. Transposable-element mRNAs are also produced, but discontinuously. Genes transcribed in the early and middle part of ZGA are short with few if any introns, and their transcripts are frequently aborted and tend to have retained introns, suggesting that inefficient splicing as well as rapid cell divisions constrain the lengths of early transcripts.
Macabenta, F. and Stathopoulos, A. (2019). Migrating cells control morphogenesis of substratum serving as track to promote directional movement of the collective. Development. PubMed ID: 31239242
In Drosophila embryos, caudal visceral mesoderm (CVM) cells undergo bilateral migration along the trunk visceral mesoderm (TVM) in order to form midgut muscles. Mutation of FGF receptor Heartless (Htl) has been shown to cause CVM migration defects, particularly midline crossing of the bilateral groups. This study shows that, additionally, htl mutants exhibit TVM defects including contralateral merging. Both CVM mismigration and TVM contralateral merging are attenuated by restoring FGF signaling specifically in the CVM, suggesting that migrating CVM cells influence TVM morphogenesis; whereas the inverse, supplying FGF to the TVM, does not rescue CVM mismigration. Additionally, this study shows that FGF regulates integrin expression in both tissues, but only providing a source of integrin specifically to the TVM attenuates the contralateral merging phenotype. Finally, it was demonstrated that the CVM influences cell shape in the TVM, and a loss of CVM results in TVM morphological defects. In summary, this study provides insight into how a migrating collective of cells can influence their tissue substrate and supports the view that morphogenesis of tissues during development is interdependent.
Rudolf, H., Zellner, C. and El-Sherif, E. (2019). Speeding up anterior-posterior patterning of insects by differential initialization of the gap gene cascade. Dev Biol. PubMed ID: 31075221
Recently, it was shown that anterior-posterior patterning genes in the red flour beetle Tribolium castaneum are expressed sequentially in waves. However, in the fruit fly Drosophila melanogaster, an insect with a derived mode of embryogenesis compared to Tribolium, anterior-posterior patterning genes quickly and simultaneously arise as mature gene expression domains that, afterwards, undergo slight posterior-to-anterior shifts. This raises the question of how a fast and simultaneous mode of patterning, like that of Drosophila, could have evolved from a rather slow sequential mode of patterning, like that of Tribolium. This paper proposes a mechanism for this evolutionary transition based on a switch from a uniform to a gradient-mediated initialization of the gap gene cascade by maternal Hb. The model is supported by computational analyses and experiments.

Wednesday, July 24th - Behavior

Reisenman, C. E. and Scott, K. (2019). Food-derived volatiles enhance consumption in Drosophila melanogaster. J Exp Biol. PubMed ID: 31085598
Insects use multiple sensory modalities when searching for and accepting a food source, in particular odor and taste cues. Food-derived odorants are generally involved in mediating long-and short-range attraction. Taste cues, on the other hand, act directly by contact with the food source, promoting the ingestion of nutritious food and the avoidance of toxic substances. It is possible, however, that insects integrate information from these sensory modalities during the process of feeding itself. Using a simple feeding assay, this study investigated whether odors modulate food consumption in the fruit fly Drosophila melanogaster. The presence of both single food-derived odorants and complex odor mixtures enhanced consumption of an appetitive food. Feeding enhancement depended on the concentration and the chemical identity of the odorant. Volatile cues alone were sufficient to mediate this effect, as feeding was also increased when animals were prevented from contacting the odor source. Both males and females, including virgin females, increased ingestion in the presence of food-derived volatiles. Moreover, the presence of food-derived odorants significantly increased the consumption of food mixtures containing aversive bitter compounds, suggesting that flies integrate diverse olfactory and gustatory cues to guide feeding decisions, including in situations in which animals are confronted with stimuli of opposite valence. Overall, these results show that food-derived olfactory cues directly modulate feeding in D. melanogaster, enhancing ingestion.
Stone, R. A., McGlinn, A. M., Chakraborty, R., Lee, D. C., Yang, V., Elmasri, A., Landis, E., Shaffer, J., Iuvone, P. M., Zheng, X., Sehgal, A. and Pardue, M. T. (2019). Altered ocular parameters from circadian clock gene disruptions. PLoS One 14(6): e0217111. PubMed ID: 31211778
The pathophysiology of refractive errors is poorly understood. Myopia (nearsightedness) in particular both blurs vision and predisposes the eye to many blinding diseases during adulthood. Based on past findings of diurnal variations in the dimensions of the eyes of humans and other vertebrates, altered diurnal rhythms of these ocular dimensions with experimentally induced myopia, and evolving evidence that ambient light exposures influence refractive development, this study assessed whether disturbances in circadian signals might alter the refractive development of the eye. In mice, retinal-specific knockout of the clock gene Bmal1 induces myopia and elongates the vitreous chamber, the optical compartment separating the lens and the retina. These alterations simulate common ocular findings in clinical myopia. In Drosophila melanogaster, knockouts of the clock genes cycle or period lengthen the pseudocone, the optical component of the ommatidium that separates the facet lens from the photoreceptors. Disrupting circadian signaling thus alters optical development of the eye in widely separated species. It is proposed that mechanisms of myopia include circadian dysregulation, a frequent occurrence in modern societies where myopia also is both highly prevalent and increasing at alarming rates. Addressing circadian dysregulation may improve understanding of the pathogenesis of refractive errors and introduce novel therapeutic approaches to ameliorate myopia development in children.
Kohsaka, H., Zwart, M. F., Fushiki, A., Fetter, R. D., Truman, J. W., Cardona, A. and Nose, A. (2019). Regulation of forward and backward locomotion through intersegmental feedback circuits in Drosophila larvae. Nat Commun 10(1): 2654. PubMed ID: 31201326
Animal locomotion requires spatiotemporally coordinated contraction of muscles throughout the body. This study investigate how contractions of antagonistic groups of muscles are intersegmentally coordinated during bidirectional crawling of Drosophila larvae. Two pairs of higher-order premotor excitatory interneurons present in each abdominal neuromere were identified that intersegmentally provide feedback to the adjacent neuromere during motor propagation. The two feedback neuron pairs are differentially active during either forward or backward locomotion but commonly target a group of premotor interneurons that together provide excitatory inputs to transverse muscles and inhibitory inputs to the antagonistic longitudinal muscles. Inhibition of either feedback neuron pair compromises contraction of transverse muscles in a direction-specific manner. These results suggest that the intersegmental feedback neurons coordinate contraction of synergistic muscles by acting as delay circuits representing the phase lag between segments. The identified circuit architecture also shows how bidirectional motor networks could be economically embedded in the nervous system.
Wu, S. F., Ja, Y. L., Zhang, Y. J. and Yang, C. H. (2019). Sweet neurons inhibit texture discrimination by signaling TMC-expressing mechanosensitive neurons in Drosophila. Elife 8. PubMed ID: 31184585
Integration of stimuli of different modalities is an important but incompletely understood process during decision making. This study shows that Drosophila are capable of integrating mechanosensory and chemosensory information of choice options when deciding where to deposit their eggs. Specifically, females switch from preferring the softer option for egg-laying when both options are sugar free to being indifferent between them when both contain sucrose. Such sucrose-induced indifference between options of different hardness requires functional sweet neurons, and, curiously, the Transmembrane Channel-like (TMC)-expressing mechanosensitive neurons that have been previously shown to promote discrimination of substrate hardness during feeding. Further, axons of sweet neurons directly contact axons of TMC-expressing neurons in the brain and stimulation of sweet neurons increases Ca(2+) influx into axons of TMC-expressing neurons. These results uncover one mechanism by which Drosophila integrate taste and tactile information when deciding where to deposit their eggs and reveal that TMC-expressing neurons play opposing roles in hardness discrimination in two different decisions.
Shao, L., Chung, P., Wong, A., Siwanowicz, I., Kent, C. F., Long, X. and Heberlein, U. (2019). A neural circuit encoding the experience of copulation in female Drosophila. Neuron. PubMed ID: 31072787
Female behavior changes profoundly after mating. In Drosophila, the mechanisms underlying the long-term changes led by seminal products have been extensively studied. However, the effect of the sensory component of copulation on the female's internal state and behavior remains elusive. This question was pursued by dissociating the effect of coital sensory inputs from those of male ejaculate. The sensory inputs of copulation were shown to cause a reduction of post-coital receptivity in females, referred to as the "copulation effect." Three layers of a neural circuit underlying this phenomenon were identified. Abdominal neurons expressing the mechanosensory channel Piezo convey the signal of copulation to female-specific ascending neurons, LSANs, in the ventral nerve cord. LSANs relay this information to neurons expressing myoinhibitory peptides in the brain. This study hereby provides a neural mechanism by which the experience of copulation facilitates females encoding their mating status, thus adjusting behavior to optimize reproduction.
Bu, B., He, W., Song, L. and Zhang, L. (2019). Nuclear envelope protein MAN1 regulates the Drosophila circadian clock via Period. Neurosci Bull. PubMed ID: 31230212
Almost all organisms exhibit ~24-h rhythms, or circadian rhythms, in a plentitude of biological processes. These rhythms are driven by endogenous molecular clocks consisting of a series of transcriptional and translational feedback loops. Previously, it has been shown that the inner nuclear membrane protein MAN1 regulates this clock and thus the locomotor rhythm in flies, but the mechanism remains unclear. This study further confirmed the previous findings and found that knocking down MAN1 in the pacemaker neurons of adult flies is sufficient to lengthen the period of the locomotor rhythm. Molecular analysis revealed that knocking down MAN1 led to reduced mRNA and protein levels of the core clock gene period (per), likely by reducing its transcription. Over-expressing per rescued the long period phenotype caused by MAN1 deficiency whereas per mutation had an epistatic effect on MAN1, indicating that MAN1 sets the pace of the clock by targeting per.

Tuesday, July 23rd - Signaling

Nagy, D., Cusumano, P., Andreatta, G., Anduaga, A. M., Hermann-Luibl, C., Reinhard, N., Gesto, J., Wegener, C., Mazzotta, G., Rosato, E., Kyriacou, C. P., Helfrich-Forster, C. and Costa, R. (2019). Peptidergic signaling from clock neurons regulates reproductive dormancy in Drosophila melanogaster. PLoS Genet 15(6): e1008158. PubMed ID: 31194738
With the approach of winter, many insects switch to an alternative protective developmental program called diapause. Drosophila melanogaster females overwinter as adults by inducing a reproductive arrest that is characterized by inhibition of ovarian development at previtellogenic stages. The insulin producing cells (IPCs) are key regulators of this process, since they produce and release insulin-like peptides that act as diapause-antagonizing hormones. This study shows that in D. melanogaster two neuropeptides, Pigment Dispersing Factor (PDF) and short Neuropeptide F (sNPF) inhibit reproductive arrest, likely through modulation of the IPCs. In particular, genetic manipulations of the PDF-expressing neurons, which include the sNPF-producing small ventral Lateral Neurons (s-LNvs), modulated the levels of reproductive dormancy, suggesting the involvement of both neuropeptides. This study expressed a genetically encoded cAMP sensor in the IPCs and challenged brain explants with synthetic PDF and sNPF. Bath applications of both neuropeptides increased cAMP levels in the IPCs, even more so when they were applied together, suggesting a synergistic effect. Bath application of sNPF additionally increased Ca2+ levels in the IPCs. These results indicate that PDF and sNPF inhibit reproductive dormancy by maintaining the IPCs in an active state.
Su, Y., Wang, T., Wu, N., Li, D., Fan, X., Xu, Z., Mishra, S. K. and Yang, M. (2019). Alpha-ketoglutarate extends Drosophila lifespan by inhibiting mTOR and activating AMPK. Aging (Albany NY) 11. PubMed ID: 31242135
Alpha-ketoglutarate (AKG) is a key metabolite of the tricarboxylic acid (TCA) cycle, an essential process influencing the mitochondrial oxidative respiration rate. Recent studies have shown that dietary AKG reduces mTOR pathway activation by inhibiting ATP synthase, thereby extending the lifespan of nematodes. Although AKG also extends lifespan in fruit flies, the antiaging mechanisms of AKG in these organisms remain unclear. This study explored changes in gene expression associated with the extension of Drosophila lifespan mediated by dietary AKG. Supplementation of the flies' diets with 5 &mi;M AKG extended their lifespan but reduced their reproductive performance. Dietary AKG also enhanced vertical climbing ability, but did not protect against oxidative stress or increase tolerance to starvation. AKG-reared flies were resistant to heat stress and demonstrated higher expression of heat shock protein genes (Hsp22 and Hsp70) than control flies. In addition, AKG significantly upregulated mRNA expression of cry, FoxO, HNF4, p300, Sirt1 and AMPKalpha, and downregulated expression of HDAC4, PI3K, TORC, PGC, and SREBP. The metabolic effects of AKG supplementation included a reduction in the ATP/ADP ratio and increased autophagy. Collectively, these observations indicate that AKG extends Drosophila lifespan by activating AMPK signaling and inhibiting the mTOR pathway.
Tian, A., Duwadi, D., Benchabane, H. and Ahmed, Y. (2019). Essential long-range action of Wingless/Wnt in adult intestinal compartmentalization. PLoS Genet 15(6): e1008111. PubMed ID: 31194729
Signal transduction activated by Wingless/Wnt ligands directs cell proliferation and fate specification in metazoans, and its overactivation underlies the development of the vast majority of colorectal cancers. In the conventional model, the secretion and movement of Wingless to cells distant from its source of synthesis are essential for long-range signaling in tissue patterning. However, this model was upended recently by an unanticipated finding: replacement of wild-type Drosophila Wingless with a membrane-tethered form produced viable adults with largely normal external morphology, which suggested that Wingless secretion and movement are dispensable for tissue patterning. This study tested this foundational principle in the adult intestine, where Wingless signaling gradients coincide with all major boundaries between compartments. The critical roles of Wingless during adult intestinal development, which include regulation of target gene activation, boundary formation, stem cell proliferation, epithelial cell fate specification, muscle differentiation, gut folding, and signaling crosstalk with the Decapentaplegic pathway, are all were disrupted by Wingless tethering. These findings provide new evidence that supports the requirement for the direct, long-range action of Wingless in tissue patterning, with relevance for animal development, tissue homeostasis and Wnt-driven disease.
Wisidagama, D. R., Thomas, S. M., Lam, G. and Thummel, C. S. (2019). Functional analysis of Aarf domain-containing kinase 1 in Drosophila melanogaster. Dev Dyn. PubMed ID: 31175694
The ADCK proteins are predicted mitochondrial kinases. Most studies of these proteins have focused on the Abc1/Coq8 subfamily, which contributes to Coenzyme Q biosynthesis. In contrast, little is known about ADCK1 despite its evolutionary conservation in yeast, Drosophila, Caenorhabditis elegans and mammals. This study shows that Drosophila ADCK1 mutants die as second instar larvae with double mouth hooks and tracheal breaks. Tissue-specific genetic rescue and RNAi studies show that ADCK1 is necessary and sufficient in the trachea for larval viability. In addition, tracheal-rescued ADCK1 mutant adults have reduced lifespan, are developmentally delayed, have reduced body size, and normal levels of basic metabolites. It is concluded that The larval lethality and double mouth hooks seen in ADCK1 mutants are often associated with reduced levels of the steroid hormone ecdysone, suggesting that this gene could contribute to controlling ecdysone levels or bioavailability. Similarly, the tracheal defects in these animals could arise from defects in intracellular lipid trafficking. These studies of ADCK1 provide a new context to define the physiological functions of this poorly understood member of the ADCK family of predicted mitochondrial proteins.
Song, S., Andrejeva, D., Freitas, F. C. P., Cohen, S. M. and Herranz, H. (2019). dTcf/Pangolin suppresses growth and tumor formation in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 31235567
Wnt/Wingless (Wg) signaling controls many aspects of animal development and is deregulated in different human cancers. The transcription factor dTcf/Pangolin (Pan) is the final effector of the Wg pathway in Drosophila and has a dual role in regulating the expression of Wg target genes. In the presence of Wg, dTcf/Pan interacts with beta-catenin/Armadillo (Arm) and induces the transcription of Wg targets. In absence of Wg, dTcf/Pan partners with the transcriptional corepressor TLE/Groucho (Gro) and inhibits gene expression. This study used the wing imaginal disk of Drosophila as a model to examine the functions that dTcf/Pan plays in a proliferating epithelium. A function is reported of dTcf/Pan in growth control and tumorigenesis. The results show that dTcf/Pan can limit tissue growth in normal development and suppresses tumorigenesis in the context of oncogene up-regulation. The conserved transcription factors Sox box protein 15 (Sox15) and Ftz transcription factor 1 (Ftz-f1) were identified as genes controlled by dTcf/Pan involved in tumor development. In conclusion, this study reports a role for dTcf/Pan as a repressor of normal and oncogenic growth and identifies the genes inducing tumorigenesis downstream of dTcf/Pan.
Sitaram, P., Lu, S., Harsh, S., Herrera, S. C. and Bach, E. A. (2019). Next-generation sequencing reveals increased anti-oxidant response and ecdysone signaling in STAT supercompetitors in Drosophila. G3 (Bethesda). PubMed ID: 31227525
Cell competition is the elimination of one viable population of cells (the losers) by a neighboring fitter population (the winners) and was discovered by studies in the Drosophila melanogaster wing imaginal disc. Supercompetition is a process in which cells with elevated STAT signaling or increased Myc become winners and outcompete wild-type neighbors. To identify the genes that are differentially regulated in STAT supercompetitors, these cells were purified from Drosophila wing imaginal discs and next-generation sequencing was performed. Their transcriptome was compared to those of control wing disc cells and Myc supercompetitors. Bioinformatics revealed that STAT and Myc supercompetitors have distinct transcriptomes with only 41 common differentially regulated genes. Furthermore, STAT supercompetitors have elevated reactive oxygen species, an anti-oxidant response and ecdysone signaling. Using a combination of methods, this study validated 13 differentially expressed genes. These data sets will be useful resources to the community.

Monday, July 22nd - RNA and transposons

Catrina, I. E., Bayer, L. V., Omar, O. S. and Bratu, D. P. (2019). Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers. J Vis Exp(148). PubMed ID: 31233020
Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA's life cycle. This study present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.
Mateos, M., Silva, N. O., Ramirez, P., Higareda-Alvear, V. M., Aramayo, R. and Erickson, J. W. (2019). Effect of heritable symbionts on maternally-derived embryo transcripts. Sci Rep 9(1): 8847. PubMed ID: 31222094
Maternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. This study investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. mRNA-seq was used to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. No consistent evidence was found of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, this study capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. Small but statistically significant signals of depurination of Drosophila rRNA were found in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.
Villanueva-Canas, J. L., Horvath, V., Aguilera, L. and Gonzalez, J. (2019). Diverse families of transposable elements affect the transcriptional regulation of stress-response genes in Drosophila melanogaster. Nucleic Acids Res. PubMed ID: 31175824
Although transposable elements are an important source of regulatory variation, their genome-wide contribution to the transcriptional regulation of stress-response genes has not been studied yet. Stress is a major aspect of natural selection in the wild, leading to changes in the transcriptional regulation of a variety of genes that are often triggered by one or a few transcription factors. In this work, advantage was taken of the wealth of information available for Drosophila melanogaster and humans to analyze the role of transposable elements in six stress regulatory networks: immune, hypoxia, oxidative, xenobiotic, heat shock, and heavy metal. Transposable elements were found to be enriched for caudal, dorsal, HSF, and tango binding sites in D. melanogaster and for NFE2L2 binding sites in humans. Taking into account the D. melanogaster population frequencies of transposable elements with predicted binding motifs and/or binding sites, this study has shown that those containing three or more binding motifs/sites are more likely to be functional. For a representative subset of these TEs, in vivo transgenic reporter assays were performed in different stress conditions. Overall, the results showed that TEs are relevant contributors to the transcriptional regulation of stress-response genes.
Duc, C., Yoth, M., Jensen, S., Mouniee, N., Bergman, C. M., Vaury, C. and Brasset, E. (2019). Trapping a somatic endogenous retrovirus into a germline piRNA cluster immunizes the germline against further invasion. Genome Biol 20(1): 127. PubMed ID: 31227013
For species survival, the germline must faithfully transmit genetic information to the progeny. Transposable elements (TEs) constitute a significant threat to genome stability due to their mobility. In the metazoan germline, their mobilization is limited by a class of small RNAs called PIWI-interacting RNAs (piRNAs) produced by dedicated genomic loci called piRNA clusters. Although the piRNA pathway is an adaptive genomic immunity system, it remains unclear how the germline gains protection from a new transposon invasion. To address this question, Drosophila lines harboring a deletion within flamenco, a major piRNA cluster specifically expressed in somatic follicular cells. This deletion leads to derepression of the retrotransposon ZAM in the somatic follicular cells and subsequent germline genome invasion. In this mutant line, de novo production of sense and antisense ZAM-derived piRNAs were identified that display a germinal molecular signature. These piRNAs originated from a new ZAM insertion into a germline dual-strand piRNA cluster and silence ZAM expression specifically in germ cells. Finally, it was found that ZAM trapping in a germinal piRNA cluster is a frequent event that occurs early during the isolation of the mutant line. Transposons can hijack the host developmental process to propagate whenever their silencing is lost. This study shows that the germline can protect itself by trapping invading somatic-specific TEs into germline piRNA clusters. This is the first demonstration of "auto-immunization" of a germline endangered by mobilization of a surrounding somatic TE.
Panhale, A., Richter, F. M., Ramirez, F., Shvedunova, M., Manke, T., Mittler, G. and Akhtar, A. (2019). CAPRI enables comparison of evolutionarily conserved RNA interacting regions. Nat Commun 10(1): 2682. PubMed ID: 31213602
RNA-protein complexes play essential regulatory roles at nearly all levels of gene expression. Using in vivo crosslinking and RNA capture, this study reports a comprehensive RNA-protein interactome in a metazoan at four levels of resolution: single amino acids, domains, proteins and multisubunit complexes. CAPRI, a method to map RNA-binding domains (RBDs) by simultaneous identification of RNA interacting crosslinked peptides and peptides adjacent to such crosslinked sites. CAPRI identifies more than 3000 RNA proximal peptides in Drosophila and human proteins with more than 45% of them forming new interaction interfaces. The comparison of orthologous proteins enables the identification of evolutionary conserved RBDs in globular domains and intrinsically disordered regions (IDRs). By comparing the sequences of IDRs through evolution, they were classified based on the type of motif, accumulation of tandem repeats, conservation of amino acid composition and high sequence divergence.
Vijayakumar, J., Perrois, C., Heim, M., Bousset, L., Alberti, S. and Besse, F. (2019). The prion-like domain of Drosophila Imp promotes axonal transport of RNP granules in vivo. Nat Commun 10(1): 2593. PubMed ID: 31197139
Prion-like domains (PLDs), defined by their low sequence complexity and intrinsic disorder, are present in hundreds of human proteins. Although gain-of-function mutations in the PLDs of neuronal RNA-binding proteins have been linked to neurodegenerative disease progression, the physiological role of PLDs and their range of molecular functions are still largely unknown. This study shows that the PLD of Drosophila Imp, a conserved component of neuronal ribonucleoprotein (RNP) granules, is essential for the developmentally-controlled localization of Imp RNP granules to axons and regulates in vivo axonal remodeling. Furthermore, it was demonstrate that Imp PLD restricts, rather than promotes, granule assembly, revealing a novel modulatory function for PLDs in RNP granule homeostasis. Swapping the position of Imp PLD compromises RNP granule dynamic assembly but not transport, suggesting that these two functions are uncoupled. Together, this study uncovers a physiological function for PLDs in the spatio-temporal control of neuronal RNP assemblies.

Friday, July 19th - Adult Physiology

Schmitt, R. E., Messick, M. R., Shell, B. C., Dunbar, E. K., Fang, H. F., Shelton, K. L., Venton, B. J., Pletcher, S. D. and Grotewiel, M. (2019). Dietary yeast influences ethanol sedation in Drosophila via serotonergic neuron function. Addict Biol: e12779. PubMed ID: 31169340
Abuse of alcohol is a major clinical problem with far-reaching health consequences. Understanding the environmental and genetic factors that contribute to alcohol-related behaviors is a potential gateway for developing novel therapeutic approaches for patients that abuse the drug. To this end, Drosophila melanogaster was used as a model to investigate the effect of diet, an environmental factor, on ethanol sedation. Providing flies with diets high in yeast, a routinely used component of fly media, increased their resistance to ethanol sedation. The yeast-induced resistance to ethanol sedation occurred in several different genetic backgrounds, was observed in males and females, was elicited by yeast from different sources, was readily reversible, and was associated with increased nutrient intake as well as decreased internal ethanol levels. Inhibition of serotonergic neuron function using multiple independent genetic manipulations blocked the effect of yeast supplementation on ethanol sedation, nutrient intake, and internal ethanol levels. These results demonstrate that yeast is a critical dietary component that influences ethanol sedation in flies and that serotonergic signaling is required for the effect of dietary yeast on nutrient intake, ethanol uptake/elimination, and ethanol sedation. These studies establish the fly as a model for diet-induced changes in ethanol sedation and raise the possibility that serotonin might mediate the effect of diet on alcohol-related behavior in other species.
Schmitt, R. E., Shell, B. C., Lee, K. M., Shelton, K. L., Mathies, L. D., Edwards, A. C. and Grotewiel, M. (2019). Convergent evidence from humans and Drosophila melanogaster implicates the transcription factor MEF2B/Mef2 in alcohol sensitivity. Alcohol Clin Exp Res. PubMed ID: 31241765
Self-rating of the effects of alcohol (SRE) measures level of response to ethanol in humans. Interestingly, there is a positive relationship between the SRE and risk for abusing alcohol, suggesting mechanistic connections between SRE and alcohol abuse. To identify candidate genes with a role in SRE and alcohol-related behavior more generally, this study coupled human genetic analyses with studies in Drosophila melanogaster. First, a gene-based analysis was performed of GWAS summary statistics for SRE in the Avon Longitudinal Study of Parents and Children (ALSPAC) sample. Based on prior findings in humans, orthology to fly genes and the availability of genetic reagents, a subset of genes was selected for studies on ethanol behavior in Drosophila. This study found 37 genes with nominal associations in the SRE GWAS. The role was explored of 6 orthologous genes in Drosophila ethanol sedation and rapid tolerance. The transcription factor Mef2 was found to be required for normal ethanol sedation in flies. Pan-neuronal expression of two independent Mef2 RNAi transgenes significantly reduced Mef2 expression and made flies resistant to ethanol sedation. Additionally, flies with multiple independent mutant alleles of Mef2 were also resistant to ethanol sedation, confirming a role for Mef2 in this behavior. Altered expression of Mef2 did not change ethanol rapid tolerance or cause a net change in internal ethanol concentrations. It is concluded that these studies indicate that MEF2B influences SRE in humans and that Mef2 impacts ethanol sedation in Drosophila.
Villanueva, J. E., Livelo, C., Trujillo, A. S., Chandran, S., Woodworth, B., Andrade, L., Le, H. D., Manor, U., Panda, S. and Melkani, G. C. (2019). Time-restricted feeding restores muscle function in Drosophila models of obesity and circadian-rhythm disruption. Nat Commun 10(1): 2700. PubMed ID: 31221967
Pathological obesity can result from genetic predisposition, obesogenic diet, and circadian rhythm disruption. Obesity compromises function of muscle, which accounts for a majority of body mass. Behavioral intervention that can counteract obesity arising from genetic, diet or circadian disruption and can improve muscle function holds untapped potential to combat the obesity epidemic. This study shows that Drosophila melanogaster subject to obesogenic challenges exhibits metabolic disease phenotypes in skeletal muscle; sarcomere disorganization, mitochondrial deformation, upregulation of Phospho-AKT level, aberrant intramuscular lipid infiltration, and insulin resistance. Imposing time-restricted feeding (TRF) paradigm in which flies were fed for 12 h during the day counteracts obesity-induced dysmetabolism and improves muscle performance by suppressing intramuscular fat deposits, Phospho-AKT level, mitochondrial aberrations, and markers of insulin resistance. Importantly, TRF was effective even in an irregular lighting schedule mimicking shiftwork. Hence, TRF is an effective dietary intervention for combating metabolic dysfunction arising from multiple causes.
Rose, E., Lee, D., Xiao, E., Zhao, W., Wee, M., Cohen, J. and Bergwitz, C. (2019). Endocrine regulation of MFS2 by branchless controls phosphate excretion and stone formation in Drosophila renal tubules. Sci Rep 9(1): 8798. PubMed ID: 31217461
How inorganic phosphate (Pi) homeostasis is regulated in Drosophila is currently unknown. This study identified Na+-dependent inorganic phosphate cotransporter (NaPi-T/MFS2) as a key Pi transporter in fly renal (Malpighian) tubules. Consistent with its role in Pi excretion, this study found that dietary Pi induces MFS2 expression. This results in the formation of Malpighian calcium-Pi stones, while RNAi-mediated knockdown of MFS2 increases blood (hemolymph) Pi and decreases formation of Malpighian tubule stones in flies cultured on high Pi medium. Conversely, microinjection of adults with the phosphaturic human hormone fibroblast growth factor 23 (FGF23) induces tubule expression of MFS2 and decreases blood Pi. This action of FGF23 is blocked by genetic ablation of MFS2. Furthermore, genetic overexpression of the fly FGF branchless (bnl) in the tubules induces expression of MFS2 and increases Malpighian tubule stones suggesting that bnl is the endogenous phosphaturic hormone in adult flies. Finally, genetic ablation of MFS2 increased fly life span, suggesting that Malpighian tubule stones are a key element whereby high Pi diet reduces fly longevity. In conclusion, MFS2 mediates excretion of Pi in Drosophila, which is as in higher species under the hormonal control of FGF-signaling.
Song, Y., Park, J. O., Tanner, L., Nagano, Y., Rabinowitz, J. D. and Shvartsman, S. Y. (2019). Energy budget of Drosophila embryogenesis. Curr Biol 29(12): R566-r567. PubMed ID: 31211973
Eggs of oviparous animals must be prepared to develop rapidly and robustly until hatching. The balance between sugars, fats, and other macromolecules must therefore be carefully considered when loading the egg with nutrients. Clearly, packing too much or too little fuel would lead to suboptimal conditions for development. While many studies have measured the overall energy utilization of embryos, little is known of the identity of the molecular-level processes that contribute to the energy budget in the first place. This study introduced Drosophila embryos as a platform to study the energy budget of embryogenesis. Through three orthogonal measurements - respiration, calorimetry, and biochemical assays - it was demonstrated that Drosophila melanogaster embryogenesis utilizes 10 mJ of energy generated by the oxidation of the maternal glycogen and triacylglycerol (TAG) stores. Normalized for mass, this is comparable to the resting metabolic rates of insects. Interestingly, alongside data from earlier studies, these results imply that protein, RNA, and DNA polymerization require less than 10% of the total ATPs produced in the early embryo.
Nie, Y., Yu, S., Li, Q., Nirala, N. K., Amcheslavsky, A., Edwards, Y. J. K., Shum, P. W., Jiang, Z., Wang, W., Zhang, B., Gao, N. and Ip, Y. T. (2019). Oncogenic pathways and loss of the Rab11 GTPase synergize to alter metabolism in Drosophila. Genetics. PubMed ID: 31213502
Colorectal cancer is a complex disease driven by well-established mutations such as APC and other yet to be identified pathways. The GTPase Rab11 regulates endosomal protein trafficking and previous work has shown that loss of Rab11 caused intestinal inflammation and hyperplasia in mice and flies. To test the idea that loss of Rab11 may promote cancer progression, archival human patient tissues were analyzed and 51 out of 70 colon cancer tissues had lower Rab11 protein staining. By using the Drosophila midgut model, this study has found that loss of Rab11 can lead to three changes that may relate to cancer progression. First is the disruption of enterocyte polarity based on staining of the FERM domain protein Coracle. Second is an increased proliferation due to an increased expression of the JAK-STAT pathway ligand Upd3. Third is an increased expression of ImpL2, which is an IGFBP7 homolog and can suppress metabolism. Furthermore, loss of Rab11 can act synergistically with the oncoprotein Ras(V12) to regulate these cancer related phenotypes.

Thursday July 18th - Disease Models

Saito, T., Oba, T., Shimizu, S., Asada, A., Iijima, K. M. and Ando, K. (2019). Cdk5 increases MARK4 activity and augments pathological tau accumulation and toxicity through tau phosphorylation at Ser262. Hum Mol Genet. PubMed ID: 31174206
Hyperphosphorylation of the microtubule-associated protein tau is associated with many neurodegenerative diseases, including Alzheimer's disease. Microtubule affinity-regulating kinases (MARK) 1-4 and cyclin-dependent kinase 5 (Cdk5) are tau kinases under physiological and pathological conditions. However, their functional relationship remains elusive. This study reports a novel mechanism by which Cdk5 activates MARK4 and augments tau phosphorylation, accumulation, and toxicity. MARK4 is highly phosphorylated at multiple sites in the brain and in cultured neurons, and inhibition of Cdk5 activity reduces phosphorylation levels of MARK4. MARK4 is known to be activated by phosphorylation at its activation loop by liver kinase B1 (LKB1). In contrast, Cdk5 increased phosphorylation of MARK4 in the spacer domain, but not in the activation loop, and enhanced its kinase activity, suggesting a novel mechanism by which Cdk5 regulates MARK4 activity. It was also demonstrated that co-expression of Cdk5 and MARK4 in mammalian cultured cells significantly increased the levels of tau phosphorylation at both Cdk5 target sites (SP/TP sites) and MARK target sites (Ser262), as well as the levels of total tau. Furthermore, using a Drosophila model of tau toxicity, it was demonstrated that Cdk5 promoted tau accumulation and tau-induced neurodegeneration via increasing tau phosphorylation levels at Ser262 by a fly ortholog of MARK, Par-1. This study suggests a novel mechanism by which Cdk5 and MARK4 synergistically increase tau phosphorylation and accumulation, consequently promoting neurodegeneration in disease pathogenesis.
Sakai, R., Suzuki, M., Ueyama, M., Takeuchi, T., Minakawa, E. N., Hayakawa, H., Baba, K., Mochizuki, H. and Nagai, Y. (2019). E46K mutant alpha-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type alpha-synuclein in Drosophila models of Parkinson's disease. PLoS One 14(6): e0218261. PubMed ID: 31242217
Alpha-synuclein (alphaSyn) plays key roles in the pathogenesis of Parkinson's disease (PD). The mechanisms underlying the variance in the clinical phenotypes of familial PD caused by missense mutations in the alphaSyn gene remain elusive. This study established novel Drosophila models expressing either wild-type (WT) alphaSyn or one of five alphaSyn mutants (A30P, E46K, H50Q, G51D, and A53T) using site-specific transgenesis, which express transgenes at equivalent levels. Expression of either WT or mutant alphaSyn in the compound eyes by the GMR-GAL4 driver caused mild rough eye phenotypes with no obvious difference among the mutants. Upon pan-neuronal expression by the nSyb-GAL4 driver, these alphaSyn-expressing flies showed a progressive decline in locomotor function. Notably, it was found that E46K, H50Q, G51D, and A53T alphaSyn-expressing flies showed earlier onset of locomotor dysfunction than WT alphaSyn-expressing flies, suggesting their enhanced toxic effects. Whereas mRNA levels of WT and mutant alphaSyn were almost equivalent, it was found that protein expression levels of E46K alphaSyn were higher than those of WT alphaSyn. In vivo chase experiments using the drug-inducible GMR-GeneSwitch driver demonstrated that degradation of E46K alphaSyn protein was significantly slower than WT alphaSyn protein, indicating that the E46K alphaSyn mutant gains resistance to degradation in vivo. It is therefore conclude that the novel site-specific transgenic fly models expressing either WT or mutant alphaSyn are useful to explore the mechanisms by which different alphaSyn mutants gain toxic functions in vivo.
Miguel, C., Cruz, J., Martin, D. and Franch-Marro, X. (2019). Dual role of FGF in proliferation and endoreplication of Drosophila tracheal adult progenitor cells. J Mol Cell Biol. PubMed ID: 31237953
Adult progenitor cells activation is a key event in the formation of adult organs. In Drosophila, formation of abdominal adult trachea depends on the specific activation of tracheal adult progenitors (tracheoblasts) at the Tr4 and Tr5 spiracular branches (SB). Proliferation of these tracheoblasts generate a pool of tracheal cells that migrate towards the posterior part of the trachea by the activation of the Branchless/Fibroblast growth factor (Bnl/FGF) signaling to form the abdominal adult trachea. This study shows that, in addition to migration, Bnl/FGF signaling, mediated by the transcription factor Pointed, is also required for tracheoblast proliferation. This tracheoblast activation relies on the expression of the FGF ligand bnl in their nearby branches. Finally, it was shown that, in absence of the transcription factor Cut (Ct), Bnl/FGF signaling induces endoreplication of tracheoblasts partially by promoting Fizzy-related expression. Altogether, these results suggest a dual role of Bnl/FGF signaling in tracheoblasts, inducing both proliferation and endoreplication, depending on the presence or absence of the transcription factor Ct, respectively.
Shim, K. H., Kim, S. H., Hur, J., Kim, D. H., Demirev, A. V. and Yoon, S. Y. (2019). Small-molecule drug screening identifies drug Ro 31-8220 that reduces toxic phosphorylated tau in Drosophila melanogaster. Neurobiol Dis: 104519. PubMed ID: 31233882
The intraneuronal aggregates of hyperphosphorylated and misfolded tau (neurofibrillary tangles, NFTs) cause a stereotypical spatiotemporal Alzheimer's disease (AD) progression. Hyperactivation of kinases including the conventional protein kinase C (PKC) is a defective molecular event accompanying associative memory loss, tau phosphorylation, and progression of AD. This study investigated the ability of small therapeutic compounds (a custom library) to improve tau-induced rough-eye phenotype in a Drosophila melanogaster model of frontotemporal dementia. Tau phosphorylation in vivo and selected hit compounds was also assessed. Among the potential hits, Ro 31-8220, described earlier as a potent PKCalpha inhibitor, was tested. Ro 31-8220 robustly improved the rough-eye phenotype, reduced phosphorylated tau species in vitro and in vivo, reversed tau-induced memory impairment, and improved the fly motor functions. In a human neuroblastoma cell line, Ro 31-8220 reduced the PKC activity and the tau phosphorylation pattern, bthe compound's wide range of biological activity is also acknowledged. Nevertheless, Ro 31-8220 is a novel therapeutic mitigator of tau-induced neurotoxocity.
Moskalev, A. A., Shaposhnikov, M. V., Zemskaya, N. V., Koval Lcapital A, C., Schegoleva, E. V., Guvatova, Z. G., Krasnov, G. S., Solovev, I. A., Sheptyakov, M. A., Zhavoronkov, A. and Kudryavtseva, A. V. (2019). Transcriptome analysis of long-lived Drosophila melanogaster E(z) mutants sheds light on the molecular mechanisms of longevity. Sci Rep 9(1): 9151. PubMed ID: 31235842
The E(z) histone methyltransferase heterozygous mutation in Drosophila is known to increase lifespan and stress resistance. However, the longevity mechanisms of E(z) mutants have not been revealed. Using genome-wide transcriptome analysis, this study demonstrated that lifespan extension, increase of resistance to hyperthermia, oxidative stress and endoplasmic reticulum stress, and fecundity enhancement in E(z) heterozygous mutants are accompanied by changes in the expression level of 239 genes. The results demonstrated sex-specific effects of E(z) mutation on gene expression, which, however, did not lead to differences in lifespan extension in both sexes. A mutation in an E(z) gene was shown to lead to perturbations in gene expression, most of which participates in metabolism, such as Carbohydrate metabolism, Lipid metabolism, Drug metabolism, Nucleotide metabolism. Age-dependent changes in the expression of genes involved in pathways related to immune response, cell cycle, and ribosome biogenesis were found.
Manzo, E., Lorenzini, I., Barrameda, D., O'Conner, A. G., Barrows, J. M., Starr, A., Kovalik, T., Rabichow, B. E., Lehmkuhl, E. M., Shreiner, D. D., Joardar, A., Lievens, J. C., Bowser, R., Sattler, R. and Zarnescu, D. C. (2019). Glycolysis upregulation is neuroprotective as a compensatory mechanism in ALS. Elife 8. PubMed ID: 31180318
Amyotrophic Lateral Sclerosis (ALS), is a fatal neurodegenerative disorder, with TDP-43 inclusions as a major pathological hallmark. Using a Drosophila model of TDP-43 proteinopathy this study found significant alterations in glucose metabolism including increased pyruvate, suggesting that modulating glycolysis may be neuroprotective. Indeed, a high sugar diet improves locomotor and lifespan defects caused by TDP-43 proteinopathy in motor neurons or glia, but not muscle, suggesting that metabolic dysregulation occurs in the nervous system. Overexpressing human glucose transporter GLUT-3 in motor neurons mitigates TDP-43 dependent defects in synaptic vesicle recycling and improves locomotion. Furthermore, PFK mRNA, a key indicator of glycolysis, is upregulated in flies and patient derived iPSC motor neurons with TDP-43 pathology. Surprisingly, PFK overexpression rescues TDP-43 induced locomotor deficits. These findings from multiple ALS models show that mechanistically, glycolysis is upregulated in degenerating motor neurons as a compensatory mechanism and suggest that increased glucose availability is protective.

Wednesday, July 17th - Adult Neural Development and Function

Soni, N., Chahda, J. S. and Carlson, J. R. (2019). Odor coding in the antenna of the tsetse fly Glossina morsitans. Proc Natl Acad Sci U S A. PubMed ID: 31221757
Tsetse flies transmit trypanosomiasis to humans and livestock across much of sub-Saharan Africa. Tsetse are attracted by olfactory cues emanating from their hosts. However, remarkably little is known about the cellular basis of olfaction in tsetse. This study has carried out a systematic physiological analysis of the Glossina morsitans antenna. Seven functional classes of olfactory sensilla were identified that respond to human or animal odorants, CO2, sex and alarm pheromones, or other odorants known to attract or repel tsetse. Sensilla differ in their response spectra, show both excitatory and inhibitory responses, and exhibit different response dynamics to different odor stimuli. Striking differences were found between the functional organization of the tsetse fly antenna and that of the fruit fly Drosophila melanogaster. One morphological type of sensilla has a different function in the 2 species: Trichoid sensilla respond to pheromones in Drosophila but respond to a wide diversity of compounds in G. morsitans. In contrast to Drosophila, all tested G. morsitans sensilla that show excitatory responses are excited by one odorant, 1-octen-3-ol, which is contained in host emanations. The response profiles of some classes of sensilla are distinct but strongly correlated, unlike the organization described in the Drosophila antenna. Taken together, this study defines elements that likely mediate the attraction of tsetse to its hosts and that might be manipulated as a means of controlling the fly and the diseases it transmits.
Tomlinson, A., Mavromatakis, Y. E. and Arias, R. (2019). The role of Sevenless in Drosophila R7 photoreceptor specification. Dev Biol. PubMed ID: 31207209
Sevenless (Sev) is a Receptor Tyrosine Kinase (RTK) that is required for the specification of the Drosophila R7 photoreceptor. Other Drosophila photoreceptors are specified by the action of another RTK; the Drosophila EGF Receptor (DER). Why Sev is required specifically in the R7 precursor has long remained unclear. To test the function of the two receptors, a Sev/DER chimera was generated in which the intracellular domain of Sev is replaced with that of DER. This chimerical receptor acts indistinguishably from Sev itself; a result that is entirely consistent with the two RTKs sharing identical transduction abilities. phyllopod (phyl) is the target gene of the RTK pathway, and R7 precursors were shown to be selectively lost when phyl gene function is mildly compromised and that other photoreceptors are removed when the gene function is further reduced. This result adds a key piece of evidence for the hyperactivation of the RTK pathway in the R7 precursor. To facilitate the hyperactivation of the RTK pathway, Sev is expressed at high levels. However, when DER was expressed at the levels at which Sev is expressed, strong gain-of-function effects result, consistent with ligand-independent activation of the receptor. This highlights another key feature of Sev; that it is expressed at high levels yet remains strictly ligand dependent. Finally, it was found that activated Sev can rescue R3/4 photoreceptors when their DER function is abrogated. These results are collectively consistent with Sev and DER activating the same transduction machinery, with Sev generating a pathway hyperactivation to overcome the N-imposed block to photoreceptor specification in R7 precursors.
Nielsen, A. K., Moller, I. R., Wang, Y., Rasmussen, S. G. F., Lindorff-Larsen, K., Rand, K. D. and Loland, C. J. (2019). Substrate-induced conformational dynamics of the dopamine transporter. Nat Commun 10(1): 2714. PubMed ID: 31221956
The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. This study probed the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, ligand-dependent slow cooperative fluctuations of helical stretches were detected in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. These results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.
Kono, K., Yoshiura, S., Fujita, I., Okada, Y., Shitamukai, A., Shibata, T. and Matsuzaki, F. (2019). Reconstruction of Par-dependent polarity in apolar cells reveals a dynamic process of cortical polarization. Elife 8. PubMed ID: 31172945
Cellular polarization is fundamental for various biological processes. The Par network system is conserved for cellular polarization. Its core complex consists of Par3, Par6, and aPKC. However, the general dynamic processes that occur during polarization are not well understood. This study reconstructed Par-dependent polarity using non-polarized Drosophila S2 cells expressing all three components endogenously in the cytoplasm. The results indicated that elevated Par3 expression induces cortical localization of the Par-complex at the interphase. Its asymmetric distribution goes through three steps: emergence of cortical dots, development of island-like structures with dynamic amorphous shapes, repeating fusion and fission, and polarized clustering of the islands. These findings also showed that these islands contain a meshwork of unit-like segments. Furthermore, Par-complex patches resembling Par-islands exist in Drosophila mitotic neuroblasts. Thus, this reconstruction system provides an experimental paradigm to study features of the assembly process and structure of Par-dependent cell-autonomous polarity.
Rimal, S. and Lee, Y. (2019). Molecular sensor of nicotine in taste of Drosophila melanogaster. Insect Biochem Mol Biol: 103178. PubMed ID: 31226410
Nicotine is an alkaloid and potent parasympathomimetic stimulant found in the leaves of many plants including Nicotiana tabacum, which functions as an anti-herbivore chemical and an insecticide. Chemoreceptors embedded in the gustatory receptor neurons (GRNs) enable animals to judge the quality of bitter compounds and respond to them. Various taste receptors such as gustatory receptors (GRs), ionotropic receptors (IRs), transient receptor potential channels (TRPs), and pickpocket channels (PPKs) have been shown to have important roles in taste sensation. However, the mechanism underlying nicotine taste sensation has not been resolved in the insect model. This study identified molecular receptors to detect the taste of nicotine and provide electrophysiological and behavioral evidence that gustatory receptors are required for avoiding nicotine-laced foods. GR10a, GR32a and GR33a are necessary for nicotine sensing while TRP family member receptors are not required. The results demonstrate that gustatory receptors are reasonable targets to develop new pesticides that maximize the insecticidal effects of nicotine.
Martelli, C. and Fiala, A. (2019). Slow presynaptic mechanisms that mediate adaptation in the olfactory pathway of Drosophila. Elife 8. PubMed ID: 31169499
The olfactory system encodes odor stimuli as combinatorial activity of populations of neurons whose response depends on stimulus history. How and on which timescales previous stimuli affect these combinatorial representations remains unclear. This study used in vivo optical imaging in Drosophila to analyze sensory adaptation at the first synaptic step along the olfactory pathway. Calcium signals in the axon terminals of olfactory receptor neurons (ORNs) do not follow the same adaptive properties as the firing activity measured at the antenna. While ORNs calcium responses are sustained on long timescales, calcium signals in the postsynaptic projection neurons (PNs) adapt within tens of seconds. It is proposed that this slow component of the postsynaptic response is mediated by a slow presynaptic depression of vesicle release and enables the combinatorial population activity of PNs to adjust to the mean and variance of fluctuating odor stimuli.

Tuesday, July 16th - Chromatin

Tian, K., Henderson, R. E., Parker, R., Brown, A., Johnson, J. E. and Bateman, J. R. (2019). Two modes of transvection at the eyes absent gene of Drosophila demonstrate plasticity in transcriptional regulatory interactions in cis and in trans. PLoS Genet 15(5): e1008152. PubMed ID: 31075100
For many genes, proper gene expression requires coordinated and dynamic interactions between multiple regulatory elements, each of which can either promote or silence transcription. In Drosophila, the complexity of the regulatory landscape is further complicated by the tight physical pairing of homologous chromosomes, which can permit regulatory elements to interact in trans, a phenomenon known as transvection. To better understand how gene expression can be programmed through cis- and trans-regulatory interactions, this study analyzed transvection effects for a collection of alleles of the eyes absent (eya) gene. Trans-activation of a promoter by the eya eye-specific enhancers is shown to be broadly supported in many allelic backgrounds, and the availability of an enhancer to act in trans can be predicted based on the molecular lesion of an eya allele. Furthermore, by manipulating promoter availability in cis and in trans, the eye-specific enhancers of eya were demonstrated to show plasticity in their promoter preference between two different transcriptional start sites, which depends on promoter competition between the two potential targets. Finally, it was shown that certain alleles of eya demonstrate pairing-sensitive silencing resulting from trans-interactions between Polycomb Response Elements (PREs), and genetic and genomic data support a general role for PcG proteins in mediating transcriptional silencing at eya. Overall, these data highlight how eya gene regulation relies upon a complex but plastic interplay between multiple enhancers, promoters, and PREs.
Thomas, C., Ji, Y., Wu, C., Datz, H., Boyle, C., MacLeod, B., Patel, S., Ampofo, M., Currie, M., Harbin, J., Pechenkina, K., Lodhi, N., Johnson, S. J. and Tulin, A. V. (2019). Hit and run versus long-term activation of PARP-1 by its different domains fine-tunes nuclear processes. Proc Natl Acad Sci U S A 116(20): 9941-9946. PubMed ID: 31028139
Poly(ADP-ribose) polymerase 1 (PARP-1; see Drosophila Hiiragi) is a multidomain multifunctional nuclear enzyme involved in the regulation of the chromatin structure and transcription. PARP-1 consists of three functional domains: the N-terminal DNA-binding domain (DBD) containing three zinc fingers, the automodification domain (A), and the C-terminal domain, which includes the protein interacting WGR domain (W) and the catalytic (Cat) subdomain responsible for the poly(ADP ribosyl)ating reaction. The mechanisms coordinating the functions of these domains and determining the positioning of PARP-1 in chromatin remain unknown. Using multiple deletional isoforms of PARP-1, lacking one or another of its three domains, as well as consisting of only one of those domains, this study demonstrates that different functions of PARP-1 are coordinated by interactions among these domains and their targets. Interaction between the DBD and damaged DNA leads to a short-term binding and activation of PARP-1. This "hit and run" activation of PARP-1 initiates the DNA repair pathway at a specific point. The long-term chromatin loosening required to sustain transcription takes place when the C-terminal domain of PARP-1 binds to chromatin by interacting with histone H4 in the nucleosome. This long-term activation of PARP-1 results in a continuous accumulation of pADPr, which maintains chromatin in the loosened state around a certain locus so that the transcription machinery has continuous access to DNA. Cooperation between the DBD and C-terminal domain occurs in response to heat shock (HS), allowing PARP-1 to scan chromatin for specific binding sites.
Syrzycka, M., Hallson, G., Fitzpatrick, K. A., Kim, I., Cotsworth, S., Hollebakken, R. E., Simonetto, K., Yang, L., Luongo, S., Beja, K., Coulthard, A. B., Hilliker, A. J., Sinclair, D. A. and Honda, B. M. (2019). Genetic and molecular analysis of essential genes in centromeric heterochromatin of the left arm of chromosome 3 in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 30948422
This study presents an updated genetic and molecular analysis of chromosome 3L centric heterochromatin (3L Het). A number of new, overlapping deficiencies (Dfs) were generated and characterized that remove regions of 3L Het. These Dfs were critically important reagents in subsequent genetic analysis for the isolation and characterization of lethal point mutations in the region. The assignment of these mutations to genetically-defined essential loci was followed by matching them to gene models derived from genome sequence data: this was done by using molecular mapping plus sequence analysis of mutant alleles, thereby aligning genetic and physical maps of the region. Putative essential gene sequences were identified in 3L Het by using RNA interference to target candidate gene sequences. At least 25, or just under 2/3 of loci in 3L Het, are essential for viability and/or fertility. This work contributes to the functional annotation of centric heterochromatin in Drosophila, and the genetic and molecular tools generated should help to provide important insights into the organization and functions of gene sequences in 3L Het.
Piacentini, L., Marchetti, M., Bucciarelli, E., Casale, A. M., Cappucci, U., Bonifazi, P., Renda, F. and Fanti, L. (2019). A role of the Trx-G complex in Cid/CENP-A deposition at Drosophila melanogaster centromeres. Chromosoma. PubMed ID: 31203392
Centromeres are epigenetically determined chromatin structures that specify the assembly site of the kinetochore, the multiprotein machinery that binds microtubules and mediates chromosome segregation during mitosis and meiosis. The centromeric protein A (CENP-A) and its Drosophila orthologue centromere identifier (Cid) are H3 histone variants that replace the canonical H3 histone in centromeric nucleosomes of eukaryotes. CENP-A/Cid is required for recruitment of other centromere and kinetochore proteins and its deficiency disrupts chromosome segregation. Despite the many components that are known to cooperate in centromere function, the complete network of factors involved in CENP-A recruitment remains to be defined. In Drosophila, the Trx-G proteins localize along the heterochromatin with specific patterns and some of them localize to the centromeres of all chromosomes. This study shows that the Trx, Ash1, and CBP proteins are required for the correct chromosome segregation and that Ash1 and CBP mediate for Cid/CENP-A recruitment at centromeres through post-translational histone modifications. This study found that centromeric H3 histone is consistently acetylated in K27 by CBP and that nej and ash1 silencing respectively causes a decrease in H3K27 acetylation and H3K4 methylation along with an impairment of Cid loading.
Zykova, T. Y., Levitsky, V. G. and Zhimulev, I. F. (2019). Architecture of promoters of house-keeping genes in polytene chromosome interbands of Drosophila melanogaster. Dokl Biochem Biophys 485(1): 95-100. PubMed ID: 31201623
This is the first study to investigate the molecular-genetic organization of polytene chromosome interbands located on both molecular and cytological maps of Drosophila genome. The majority of the studied interbands contained one gene with a single transcription initiation site; the remaining interbands contained one gene with several alternative promoters, two or more unidirectional genes, and "head-to-head" arranged genes. In addition, intricately arranged interbands containing three or more genes in both unidirectional and bidirectional orientation were found. Insulator proteins, ORC, P-insertions, DNase I hypersensitive sites, and other open chromatin structures were situated in the promoter region of the genes located in the interbands. This area is critical for the formation of the interband, an open chromatin region in which gene transcription and replication are combined.
Tettey, T. T., Gao, X., Shao, W., Li, H., Story, B. A., Chitsazan, A. D., Glaser, R. L., Goode, Z. H., Seidel, C. W., Conaway, R. C., Zeitlinger, J., Blanchette, M. and Conaway, J. W. (2019). A Role for FACT in RNA polymerase II promoter-proximal pausing. Cell Rep 27(13): 3770-3779. PubMed ID: 31242411
FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. This report describes a global analysis of FACT function in Pol II transcription in Drosophila. This study presents evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, this study identified a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.

Monday July 15th - Adult Nutrition and Physiology

Hehlert, P., Hofferek, V., Heier, C., Eichmann, T. O., Riedel, D., Rosenberg, J., Takacs, A., Nagy, H. M., Oberer, M., Zimmermann, R. and Kuhnlein, R. P. (2019). The alpha/beta-hydrolase domain-containing 4 and 5 (ABHD4/5)-related phospholipase Pummelig controls energy storage in Drosophila. J Lipid Res. PubMed ID: 31164391
Triglycerides (TGs) are the main energy storage form to accommodate for changing organismal energy demands. In Drosophila melanogaster, the TG lipase Brummer (Bmm; also known as DmATGL) is of central importance for body fat mobilization. The mammalian orthologue adipose triglyceride lipase (ATGL) becomes activated by the alpha/beta-hydrolase fold domain containing 5 (ABHD5; also called CGI-58), one member of the paralogous gene pair ABHD4 and ABHD5. In Drosophila, the pummelig (puml) gene encodes the single sequence-related protein to mammalian ABHD4/ABHD5 with unknown function. This study generate puml mutant flies, that are short-lived, store excess body fat on the expense of glycogen, and exhibit ectopic fat storage with altered TG fatty acid profile in the fly kidneys, called Malpighian tubules. TG accumulation in puml mutants is not associated with increased food intake but with elevated lipogenesis, while starvation-induced lipid mobilization is functional. Despite its structural similarity to mammalian ABHD5/CGI-58, Puml does not stimulate TG lipase activity of Bmm/DmATGL in vitro. However, a substrate screen identifies Puml as a phospholipase, that is localized on lipid droplets, mitochondria, and peroxisomes. In conclusion, this study identifies the ABHD4/5 family member Puml as a versatile phospholipase, which regulates Drosophila body fat storage and energy metabolism.
Murgier, J., Everaerts, C., Farine, J. P. and Ferveur, J. F. (2019). Live yeast in juvenile diet induces species-specific effects on Drosophila adult behaviour and fitness. Sci Rep 9(1): 8873. PubMed ID: 31222019
The presence and the amount of specific yeasts in the diet of saprophagous insects such as Drosophila can affect their development and fitness. However, the impact of different yeast species in the juvenile diet has rarely been investigated. This study measured the behavioural and fitness effects of three live yeasts (Saccharomyces cerevisiae = SC; Hanseniaspora uvarum = HU; Metschnikowia pulcherrima = MP) added to the diet of Drosophila melanogaster larvae. Beside these live yeast species naturally found in natural Drosophila populations or in their food sources, the inactivated "drySC" yeast widely used in Drosophila research laboratories was tested. All flies were transferred to drySC medium immediately after adult emergence, and several life traits and behaviours were measured. These four yeast diets had different effects on pre-imaginal development: HU-rich diet tended to shorten the "egg-to-pupa" period of development while MP-rich diet induced higher larval lethality compared to other diets. Pre- and postzygotic reproduction-related characters (copulatory ability, fecundity, cuticular pheromones) varied according to juvenile diet and sex. Juvenile diet also changed adult food choice preference and longevity. These results indicate that specific yeast species present in natural food sources and ingested by larvae can affect their adult characters crucial for fitness.
Tufi, R., Gleeson, T. P., von Stockum, S., Hewitt, V. L., Lee, J. J., Terriente-Felix, A., Sanchez-Martinez, A., Ziviani, E. and Whitworth, A. J. (2019). Comprehensive genetic characterization of mitochondrial Ca(2+) uniporter components reveals their different physiological requirements in vivo. Cell Rep 27(5): 1541-1550. PubMed ID: 31042479
Mitochondrial Ca(2+) uptake is an important mediator of metabolism and cell death. Identification of components of the highly conserved mitochondrial Ca(2+) uniporter has opened it up to genetic analysis in model organisms. This study reports a comprehensive genetic characterization of all known uniporter components conserved in Drosophila. While loss of pore-forming MCU or EMRE abolishes fast mitochondrial Ca(2+) uptake, this results in only mild phenotypes when young, despite shortened lifespans. In contrast, loss of the MICU1 gatekeeper is developmentally lethal, consistent with unregulated Ca(2+) uptake. Mutants for the neuronally restricted regulator MICU3 are viable with mild neurological impairment. Genetic interaction analyses reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that this results from uniporter-independent functions. These data reveal the interplay among components of the mitochondrial Ca(2+) uniporter and shed light on their physiological requirements in vivo.
Xu, Y., Borcherding, A. F., Heier, C., Tian, G., Roeder, T. and Kuhnlein, R. P. (2019). Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila. Sci Rep 9(1): 6989. PubMed ID: 31061470
Obesity is a progressive, chronic disease, which can be caused by long-term miscommunication between organs. It remains challenging to understand how chronic dysfunction in a particular tissue remotely impairs other organs to eventually imbalance organismal energy homeostasis. This study introduced RNAi Pulse Induction (RiPI) mediated by short hairpin RNA (shRiPI) or double-stranded RNA (dsRiPI) to generate chronic, organ-specific gene knockdown in the adult Drosophila fat tissue. Organ-restricted RiPI targeting Stromal interaction molecule (Stim), an essential factor of store-operated calcium entry (SOCE), results in progressive fat accumulation in fly adipose tissue. Chronic SOCE-dependent adipose tissue dysfunction manifests in considerable changes of the fat cell transcriptome profile, and in resistance to the glucagon-like Adipokinetic hormone (Akh) signaling. Remotely, the adipose tissue dysfunction promotes hyperphagia likely via increased secretion of Akh from the neuroendocrine system. Collectively, this study presents a novel in vivo paradigm in the fly, which is widely applicable to model and functionally analyze inter-organ communication processes in chronic diseases.
Musselman, L. P., Fink, J. L. and Baranski, T. J. (2019). Similar effects of high-fructose and high-glucose feeding in a Drosophila model of obesity and diabetes. PLoS One 14(5): e0217096. PubMed ID: 31091299
As in mammals, high-sucrose diets lead to obesity and insulin resistance in the model organism Drosophila melanogaster. To explore the relative contributions of glucose and fructose, sucrose's component monosaccharides, their effects on larval physiology were compared. Both sugars exhibited similar effects to sucrose, leading to obesity and hyperglycemia. There were no striking differences resulting from larvae fed high glucose versus high fructose. Some small but statistically significant differences in weight and gene expression were observed that suggest Drosophila is a promising model system for understanding monosaccharide-specific effects on metabolic homeostasis.
Xu, Y. and Wang, T. (2019). LOVIT is a putative vesicular histamine transporter required in Drosophila for vision. Cell Rep 27(5): 1327-1333. PubMed ID: 31042461
Classical fast neurotransmitters are loaded into synaptic vesicles and concentrated by the action of a specific vesicular transporter before being released from the presynaptic neuron. In Drosophila, histamine is distributed mainly in photoreceptors, where it serves as the main neurotransmitter for visual input. In a targeted RNAi screen for neurotransmitter transporters involved in concentrating photoreceptor synaptic histamine, thus study identified an SLC45 transporter protein, LOVIT (loss of visual transmission). LOVIT is prominently expressed in photoreceptor synaptic vesicles and is required for Drosophila visual neurotransmission. Null mutations of lovit severely reduced the concentration of histamine in photoreceptor terminals. These results demonstrate a LOVIT-dependent mechanism, maintaining the synaptic concentration of histamine, and provide evidence for a histamine vesicular transporter besides the vesicular monoamine transporter (VMAT) family.

Friday June 12th - Apoptosis and Autophagy

Tsakiri, E. N., Gumeni, S., Vougas, K., Pendin, D., Papassideri, I., Daga, A., Gorgoulis, V., Juhasz, G., Scorrano, L. and Trougakos, I. P. (2019). Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy. Autophagy: 1-17. PubMed ID: 31002009
The ubiquitin-proteasome pathway (UPP) is central to proteostasis network (PN) functionality and proteome quality control. Yet, the functional implication of the UPP in tissue homeodynamics at the whole organism level and its potential cross-talk with other proteostatic or mitostatic modules are not well understood. This study shows that knock down (KD) of proteasome subunits in Drosophila flies, induced, for most subunits, developmental lethality. Ubiquitous or tissue specific proteasome dysfunction triggered systemic proteome instability and activation of PN modules, including macroautophagy/autophagy, molecular chaperones and the antioxidant cncC (the fly ortholog of NFE2L2/Nrf2) pathway. Also, proteasome KD increased genomic instability, altered metabolic pathways and severely disrupted mitochondrial functionality, triggering a cncC-dependent upregulation of mitostatic genes and enhanced rates of mitophagy. Whereas, overexpression of key regulators of antioxidant responses (e.g., cncC or foxo) could not suppress the deleterious effects of proteasome dysfunction; these were alleviated in both larvae and adult flies by modulating mitochondrial dynamics towards increased fusion or by enhancing autophagy. These findings reveal the extensive functional wiring of genomic, proteostatic and mitostatic modules in higher metazoans. Also, they support the notion that age-related increase of proteotoxic stress due to decreased UPP activity deregulates all aspects of cellular functionality being thus a driving force for most age-related diseases.
Araki, M., Kurihara, M., Kinoshita, S., Awane, R., Sato, T., Ohkawa, Y. and Inoue, Y. H. (2019). Anti-tumour effects of antimicrobial peptides, components of the innate immune system, against haematopoietic tumours in Drosophila mxc mutants. Dis Model Mech 12(6). PubMed ID: 31160313
The innate immune response is the first line of defence against microbial infections. In Drosophila, two major pathways of the innate immune system (the Toll- and Imd-mediated pathways) induce the synthesis of antimicrobial peptides (AMPs) within the fat body. Recently, it has been reported that certain cationic AMPs exhibit selective cytotoxicity against human cancer cells; however, little is known about their anti-tumour effects. Drosophila mxc(mbn1) mutants exhibit malignant hyperplasia in a larval haematopoietic organ called the lymph gland (LG). Using RNA-seq analysis, this study found many immunoresponsive genes, including those encoding AMPs, to be upregulated in these mutants. Downregulation of these pathways by either a Toll or imd mutation enhanced the tumour phenotype of the mxc mutants. Conversely, ectopic expression of each of five different AMPs in the fat body significantly suppressed the LG hyperplasia phenotype in the mutants. Thus, it is proposed that the Drosophila innate immune system can suppress the progression of haematopoietic tumours by inducing AMP gene expression. Overexpression of any one of the five AMPs studied resulted in enhanced apoptosis in mutant LGs, whereas no apoptotic signals were detected in controls. Two AMPs, Drosomycin and Defensin, were taken up by circulating haemocyte-like cells, which were associated with the LG regions and showed reduced cell-to-cell adhesion in the mutants. By contrast, the AMP Diptericin was directly localised at the tumour site without intermediating haemocytes. These results suggest that AMPs have a specific cytotoxic effect that enhances apoptosis exclusively in the tumour cells.
Nonaka, S., Sono, M., Hoshi, C., Kanetani, T., Nakayama, H., Dohmae, N. and Nakanishi, Y. (2019). Transcription repressor-mediated control of engulfment receptor expression in Drosophila phagocytes. Exp Cell Res. PubMed ID: 31063732
Drosophila phagocytes have been shown to enhance their phagocytic activity after apoptotic cell engulfment accompanied by the activation of the transcription repressor Tailless and an increase in the levels of engulfment receptors. This study investigated the underlying mechanisms. Tailless phosphorylation levels decreased in Drosophila phagocytes following the stimulation with apoptotic cells. Anticipating the involvement of another transcription repressor, the possible involvement was examined of Kruppel, a bibliographically identified repressor whose expression is controlled by Tailless. The level of Kruppel in phagocytes decreased after the stimulation in a Tailless-dependent manner. The RNAi knockdown of Kruppel abrogated increases in the levels of engulfment receptors and phagocytic activity in stimulated phagocytes. The binding of Kruppel to the 5'-upstream regions of genes coding for engulfment receptors was demonstrated. These results suggest the following pathway: Tailless is activated by de-phosphorylation; Kruppel expression is inhibited by Tailless; the transcription of engulfment receptors-encoding genes is augmented due to a decrease of inhibition by Kruppel; and finally phagocytic activity is enhanced.
Binh, T. D., Pham, T. L. A., Men, T. T. and Kamei, K. (2019). Dysfunction of LSD-1 induces JNK signaling pathway-dependent abnormal development of thorax and apoptosis cell death in Drosophila melanogaster. Biochem Biophys Res Commun. PubMed ID: 31229267
Perilipins are evolutionarily conserved from insects to mammals. Lipid storage droplet-1 (LSD-1) is a member of the lipid droplet's surface-binding protein family and counterpart to mammalian perilipin 1. The role of LSD-1 has already been reported in lipid metabolism of Drosophila. However, the function of this gene during specific tissue development is still under investigation. This study found that LSD-1 is expressed in the notum of the wing imaginal disc, and notum-specific knockdown of Lsd-1 by pannir-GAL4 driver leads to split thorax phenotype in adults, suggesting an essential role of LSD-1 in development of Drosophila thorax. As overexpression of JNK homolog, bsk (basket) suppresses Lsd-1 knockdown phenotype, the role of LSD-1 in thorax development was proved to be dependent on the activity of the Drosophila c-Jun N-terminal kinase (JNK). The puckered (puc) expression led to significant decrease in the JNK activity in wing discs of Lsd-1 knockdown flies. In addition, depletion of Lsd-1 enhances apoptotic cell death in the wing notum area. Taken together, these data demonstrated that LSD-1 functions in Drosophila thorax development by regulating JNK pathway.
Mundorf, J., Donohoe, C. D., McClure, C. D., Southall, T. D. and Uhlirova, M. (2019). Ets21c Governs Tissue Renewal, Stress Tolerance, and Aging in the Drosophila Intestine. Cell Rep 27(10): 3019-3033.e3015. PubMed ID: 31167145
Homeostatic renewal and stress-related tissue regeneration rely on stem cell activity, which drives the replacement of damaged cells to maintain tissue integrity and function. The Jun N-terminal kinase (JNK) signaling pathway has been established as a critical regulator of tissue homeostasis both in intestinal stem cells (ISCs) and mature enterocytes (ECs), while its chronic activation has been linked to tissue degeneration and aging. This study shows that JNK signaling requires the stress-inducible transcription factor Ets21c to promote tissue renewal in Drosophila. Ets21c controls ISC proliferation as well as EC apoptosis through distinct sets of target genes that orchestrate cellular behaviors via intrinsic and non-autonomous signaling mechanisms. While its loss appears dispensable for development and prevents epithelial aging, ISCs and ECs demand Ets21c function to mount cellular responses to oxidative stress. Ets21c thus emerges as a vital regulator of proliferative homeostasis in the midgut and a determinant of the adult healthspan.
Roddie, H. G., Armitage, E. L., Coates, J. A., Johnston, S. A. and Evans, I. R. (2019). Simu-dependent clearance of dying cells regulates macrophage function and inflammation resolution. PLoS Biol 17(5): e2006741. PubMed ID: 31086359
Macrophages encounter and clear apoptotic cells during normal development and homeostasis, including at numerous sites of pathology. Clearance of apoptotic cells has been intensively studied, but the effects of macrophage-apoptotic cell interactions on macrophage behaviour are poorly understood. Using Drosophila embryos, this study exploited the ease of manipulating cell death and apoptotic cell clearance in this model to identify that the loss of the apoptotic cell clearance receptor Six-microns-under (Simu) leads to perturbation of macrophage migration and inflammatory responses via pathological levels of apoptotic cells. Removal of apoptosis ameliorates these phenotypes, while acute induction of apoptosis phenocopies these defects and reveals that phagocytosis of apoptotic cells is not necessary for their anti-inflammatory action. Furthermore, Simu is necessary for clearance of necrotic debris and retention of macrophages at wounds. Thus, Simu is a general detector of damaged self and represents a novel molecular player regulating macrophages during resolution of inflammation.

Thursday, July 11th - Gonads

Sakai, H., Oshima, H., Yuri, K., Gotoh, H., Daimon, T., Yaginuma, T., Sahara, K. and Niimi, T. (2019). Dimorphic sperm formation by Sex-lethal. Proc Natl Acad Sci U S A 116(21): 10412-10417. PubMed ID: 31036645
Sex is determined by diverse mechanisms and master sex-determination genes are highly divergent, even among closely related species. Therefore, it is possible that homologs of master sex-determination genes might have alternative functions in different species. This study focused on Sex-lethal (Sxl), which is the master sex-determination gene in Drosophila melanogaster and is necessary for female germline development. It has been widely shown that the sex-determination function of Sxl in Drosophilidae species is not conserved in other insects of different orders. The function of Sxl was studied in the lepidopteran insect Bombyx mori. In lepidopteran insects (moths and butterflies), spermatogenesis results in two different types of sperm: nucleated fertile eupyrene sperm and anucleate nonfertile parasperm, also known as apyrene sperm. Genetic analyses using Sxl mutants revealed that the gene is indispensable for proper morphogenesis of apyrene sperm. Similarly, analyses using Sxl mutants clearly demonstrate that apyrene sperm are necessary for eupyrene sperm migration from the bursa copulatrix to the spermatheca. Therefore, apyrene sperm is necessary for successful fertilization of eupyrene sperm in B. mori Although Sxl is essential for oogenesis in D. melanogaster, it also plays important roles in spermatogenesis in B. mori Therefore, the ancestral function of Sxl might be related to germline development.
Ke, Y. T. and Hsu, H. J. (2019). Generation of inducible gene-switched GAL4 expressed in the Drosophila female germline stem cell niche. G3 (Bethesda). PubMed ID: 31018943
The stem cell niche, a regulatory microenvironment, houses and regulates stem cells for maintenance of tissues throughout an organism's lifespan. While it is known that stem cell function declines with age, the role of niche cells in this decline is not completely understood. Drosophila exhibits a short lifespan with well-characterized ovarian germline stem cells (GSCs) and niche compartments, providing a good model with which to study stem cell biology. However, no inducible tools for temporal and spatial control of gene expression in the GSC-niche unit have been previously developed for aging studies. The current UAS-GAL4 systems are not ideal for aging studies because fly physiological aging may be affected by the temperature shifts used to manipulate GAL4 activity. Additionally, the actual needs of the aged niche may be masked by continuously driven gene expression. Since GeneSwitch GAL4 is conveniently activated by the steroid RU486 (mifepristone), an enhancer-trap screen was conducted to isolate GeneSwitch GAL4 lines with expression in the GSC-niche unit. Six lines were identified with expression in germarial somatic cells and two lines (#2305 and #2261) with expression in niche cap cells, the major constituent of the GSC niche. The use of lines #2305 or #2261 to overexpress Drosophila insulin-like peptide 2, which maintains GSC lifespan, in aged niche cap cells significantly delayed age-dependent GSC loss. These results support the notion that insulin signaling is beneficial for maintaining aged stem cells and also validate the utility of the GeneSwitch GAL4 lines for studying stem cell aging.
Mannix, K. M., Starble, R. M., Kaufman, R. S. and Cooley, L. (2019). Proximity labeling reveals novel interactomes in live Drosophila tissue. Development. PubMed ID: 31208963
Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10mum. While multiple proteins have been identified as components of ring canals (RCs), a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton is lacking. This study optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique high-confidence preys were identified. The RC-APEX baits produced almost entirely distinct interactomes that included both known RC proteins as well as uncharacterized proteins. The proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective preys. Further, an RNAi screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand understanding of RC biology.
La Marca, J. E., Diepstraten, S. T., Hodge, A., Wang, H., Hart, A. H., Richardson, H. E. and Somers, W. G. (2019). Strip and Cka negatively regulate JNK signalling during Drosophila spermatogenesis. Development. PubMed ID: 31164352
One fundamental property of a stem cell niche is the exchange of molecular signals between its component cells. Niche models, such as the Drosophila melanogaster testis, have been instrumental in identifying and studying the conserved genetic factors that contribute to niche molecular signalling. This study has identified jam packed (jam), an allele of Striatin interacting protein (Strip), which is a core member of the highly conserved Striatin-interacting phosphatase and kinase (STRIPAK) complex. In the developing Drosophila testis, Strip cell-autonomously regulates the differentiation and morphology of the somatic lineage, and non-cell-autonomously regulates the proliferation and differentiation of the germline lineage. Mechanistically, Strip acts in the somatic lineage with its STRIPAK partner, Connector of kinase to AP-1 (Cka), where they negatively regulate the c-Jun N-terminal kinase (JNK) signalling pathway. This study reveals a novel role for Strip/Cka in JNK pathway regulation during spermatogenesis within the developing Drosophila testis.
Luo, W., Veeran, S., Wang, J., Li, S., Li, K. and Liu, S. N. (2019). Dual roles of juvenile hormone signaling during early oogenesis in Drosophila. Insect Sci. PubMed ID: 31207060
Juvenile hormone (JH) signaling plays crucial roles in insect metamorphosis and reproduction. Function of JH signaling in germline stem cells (GSCs) remains largely unknown. This study found that number of GSCs significantly declined in the ovaries of Met, Gce and JHAMT mutants. Then JH signaling was inhibited in selected cell types of ovaries by expressing Met and Gce or Kr-h1 dsRNAs using different Gal4 drivers. Block of JH signaling in muscle cells have no effect on GSCs numbers. Block of JH signaling in cap cells reduced GSCs cells. Inductive expression Met and Gce dsRNA but not Kr-h1 by Nos-Gal4 increased GSCs cell. These results indicate that JH signaling plays an important role in GSCs maintenance.
Kober, L., Zimmermann, M., Kurz, M., Bayer, M. and Nagel, A. C. (2019). Loss of putzig in the germline impedes germ cell development by inducing cell death and new niche like microenvironments. Sci Rep 9(1): 9108. PubMed ID: 31235815
Germline stem cell development and differentiation is tightly controlled by the surrounding somatic cells of the stem cell niche. In Drosophila females, cells of the niche emit various signals including Dpp and Wg to balance stem cell renewal and differentiation. This study shows that the gene pzg is autonomously required in cells of the germline to sustain the interplay between niche and stem cells. Loss of pzg impairs stem cell differentiation and provokes the death of cells in the germarium. As a consequence of pzg loss, increased growth signalling activity predominantly of Dpp and Wg/Wnt, was observed, eventually disrupting the balance of germ cell self-renewal and differentiation. Whereas in the soma, apoptosis-induced compensatory growth is well established, the induction of self-renewal signals during oogenesis cannot compensate for dying germ cells, albeit inducing a new niche-like microenvironment. Instead, they impair the further development of germ cells and cause in addition a forward and feedback loop of cell death.

Wednesday, July 10th - Cytoskeleton and Junctions

Manning, L. A., Perez-Vale, K. Z., Schaefer, K. N., Sewell, M. T. and Peifer, M. (2019). The Drosophila Afadin and ZO-1 homologs Canoe and Polychaetoid act in parallel to maintain epithelial integrity when challenged by adherens junction remodeling. Mol Biol Cell: mbcE19040209. PubMed ID: 31188739
During morphogenesis cells must change shape and move without disrupting tissue integrity. This requires cell-cell junctions to allow dynamic remodeling while resisting force generated by the actomyosin cytoskeleton. Multiple proteins play roles in junctional-cytoskeletal linkage, but the mechanisms by which they act remain unclear. Drosophila Canoe maintains adherens junction-cytoskeletal linkage during gastrulation. Canoe's mammalian homolog Afadin plays similar roles in cultured cells, working in parallel with ZO-1 proteins, particularly at multicellular junctions. These insights were taken back into the fly embryo, exploring how cells maintain epithelial integrity when challenged by adherens junction remodeling during germband extension and dorsal closure. Canoe was found to help cells maintain junctional-cytoskeletal linkage when challenged by the junctional remodeling inherent in mitosis, cell intercalation and neuroblast invagination or by forces generated by the actomyosin cable at the leading edge. However, even in the absence of Canoe many cells retain epithelial integrity. This is explained by a parallel role played by the ZO-1 homolog Polychaetoid. In embryos lacking both Canoe and Polychaetoid, cell junctions fail early, with multicellular junctions especially sensitive, leading to widespread loss of epithelial integrity. These data suggest Canoe and Polychaetoid stabilize Bazooka/Par3 at cell-cell junctions, helping maintain balanced apical contractility and tissue integrity.
Lecompte, M., Cattaert, D., Vincent, A., Birman, S. and Cherif-Zahar, B. (2019). The Drosophila ammonium transporter Rh50 is required for integrity of larval muscles and neuromuscular system. J Comp Neurol. PubMed ID: 31273786
Rhesus glycoproteins (Rh50) have been shown to be ammonia transporters in many species from bacteria to human. They are involved in various physiological processes including acid excretion and pH regulation. Rh50 proteins can also provide a structural link between the cytoskeleton and plasma membranes that maintains cellular integrity. Although ammonia plays essential roles in the nervous system, in particular at glutamatergic synapses, a potential role for Rh50 proteins at synapses has not yet been investigated. To better understand the function of these proteins in vivo, the unique Rh50 gene of Drosophila melanogaster which encodes two isoforms, Rh50A and Rh50BC, was studied. Drosophila Rh50A is expressed in larval muscles and enriched in the postsynaptic regions of the glutamatergic neuromuscular junctions (NMJs). Rh50 inactivation by RNA interference (RNAi) selectively in muscle cells caused muscular atrophy in larval stages and pupal lethality. Interestingly, Rh50-deficiency in muscles specifically increased glutamate receptor subunit IIA (GluRIIA) level and the frequency of spontaneous excitatory postsynaptic potentials (EPSPs). This work therefore highlights a new role for Rh50 proteins in the maintenance of Drosophila muscle architecture and synaptic physiology which could be conserved in other species.
Sun, T., Song, Y., Dai, J., Mao, D., Ma, M., Ni, J. Q., Liang, X. and Pastor-Pareja, J. C. (2019). Spectraplakin Shot maintains perinuclear microtubule organization in Drosophila polyploid cells. Dev Cell. PubMed ID: 31006649
Polyploid cells endoreplicate their DNA through a modified cell cycle that skips mitosis as part of their differentiation programs. Upon cell-cycle exit and differentiation, non-centrosomal sites govern microtubule distribution in most cells. Little is known on how polyploid cells, differentiated but cycling, organize their microtubules. This study shows that microtubules in Drosophila adipocytes and other polyploid tissues form a dense perinuclear cortex responsible for nuclear size and position. Confirming a relation between this perinuclear cortex and the polyploid endocycle, polyploidization of normally diploid cells was sufficient for cortex formation. A critical component of the perinuclear microtubule organizer (pnMTOC) is Shortstop (Shot), absence of which caused collapse of the perinuclear network into a condensed organizer through kinesin-dependent microtubule sliding. Furthermore, this ectopic organizer was capable of directing partial assembly of a deeply disruptive cytokinesis furrow. In all, this study revealed the importance of perinuclear microtubule organization for stability of endocycling Drosophila cells.
Nakajima, Y. I., Lee, Z. T., McKinney, S. A., Swanson, S. K., Florens, L. and Gibson, M. C. (2019). Junctional tumor suppressors interact with 14-3-3 proteins to control planar spindle alignment. J Cell Biol. PubMed ID: 31088859
Proper orientation of the mitotic spindle is essential for cell fate determination, tissue morphogenesis, and homeostasis. During epithelial proliferation, planar spindle alignment ensures the maintenance of polarized tissue architecture, and aberrant spindle orientation can disrupt epithelial integrity. Nevertheless, in vivo mechanisms that restrict the mitotic spindle to the plane of the epithelium remain poorly understood. This study shows that the junction-localized tumor suppressors Scribbled (Scrib) and Discs large (Dlg) control planar spindle orientation via Mud and 14-3-3 proteins in the Drosophila wing disc epithelium. During mitosis, Scrib is required for the junctional localization of Dlg, and both affect mitotic spindle movements. Using coimmunoprecipitation and mass spectrometry, this study identified 14-3-3 proteins as Dlg-interacting partners and further reports that loss of 14-3-3s causes both abnormal spindle orientation and disruption of epithelial architecture as a consequence of basal cell delamination and apoptosis. Combined, these biochemical and genetic analyses indicate that 14-3-3s function together with Scrib, Dlg, and Mud during planar cell division.
Carrasco-Rando, M., Prieto-Sanchez, S., Culi, J., Tutor, A. S. and Ruiz-Gomez, M. (2019). A specific isoform of Pyd/ZO-1 mediates junctional remodeling and formation of slit diaphragms. J Cell Biol. PubMed ID: 31171632
The podocyte slit diaphragm (SD), responsible for blood filtration in vertebrates, is a major target of injury in chronic kidney disease. The damage includes severe morphological changes with destabilization of SDs and their replacement by junctional complexes between abnormally broadened foot processes. In Drosophila melanogaster, SDs are present in nephrocytes, which filter the fly's hemolymph. This study shows that a specific isoform of Polychaetoid/ZO-1, Pyd-P, is essential for Drosophila SDs, since, in pyd mutants devoid of Pyd-P, SDs do not form and the SD component Dumbfounded accumulates at ectopic septate-like junctions between abnormally aggregated nephrocytes. Reintroduction of Pyd-P leads to junctional remodeling and their progressive normalization toward SDs. This transition requires the coiled-coil domain of Pyd-P and implies formation of nonclathrin vesicles containing SD components and their trafficking to the nephrocyte external membrane, where SDs assemble. Analyses in zebrafish suggest a conserved role for Tjp1a/ZO-1 in promoting junctional remodeling in podocytes.
Ko, C. S., Tserunyan, V. and Martin, A. C. (2019). Microtubules promote intercellular contractile force transmission during tissue folding. J Cell Biol. PubMed ID: 31227595
During development, forces transmitted between cells are critical for sculpting epithelial tissues. Actomyosin contractility in the middle of the cell apex (medioapical) can change cell shape (e.g., apical constriction) but can also result in force transmission between cells via attachments to adherens junctions. How actomyosin networks maintain attachments to adherens junctions under tension is poorly understood. This study discovered that microtubules promote actomyosin intercellular attachments in epithelia during Drosophila melanogaster mesoderm invagination. First, live imaging was used to show a novel arrangement of the microtubule cytoskeleton during apical constriction: medioapical Patronin (CAMSAP) foci formed by actomyosin contraction organized an apical noncentrosomal microtubule network. Microtubules were required for mesoderm invagination but were not necessary for initiating apical contractility or adherens junction assembly. Instead, microtubules promoted connections between medioapical actomyosin and adherens junctions. These results delineate a role for coordination between actin and microtubule cytoskeletal systems in intercellular force transmission during tissue morphogenesis.

Tuesday July 9th - Signaling

Huang, Y., Lin, L., Liu, X., Ye, S., Yao, P. Y., Wang, W., Yang, F., Gao, X., Li, J., Zhang, Y., Zhang, J., Yang, Z., Liu, X., Yang, Z., Zang, J., Teng, M., Wang, Z., Ruan, K., Ding, X., Li, L., Cleveland, D. W., Zhang, R. and Yao, X. (2019). BubR1 phosphorylates CENP-E as a switch enabling the transition from lateral association to end-on capture of spindle microtubules. Cell Res. PubMed ID: 31201382
Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, powered congression of those chromosomes, their segregation in anaphase, and assembly of a spindle midzone at mitotic exit. The centromere-associated kinesin motor CENP-E, whose binding partner is BubR1, has been implicated in congression of misaligned chromosomes and the transition from lateral kinetochore-microtubule association to end-on capture. Although previously proposed to be a pseudokinase, this study reports the structure of the kinase domain of Drosophila melanogaster BubR1, revealing its folding into a conformation predicted to be catalytically active. BubR1 is shown to be a bona fide kinase whose phosphorylation of CENP-E switches it from a laterally attached microtubule motor to a plus-end microtubule tip tracker. Computational modeling is used to identify bubristatin as a selective BubR1 kinase antagonist that targets the alphaN1 helix of N-terminal extension and alphaC helix of the BubR1 kinase domain. Inhibition of CENP-E phosphorylation is shown to prevent proper microtubule capture at kinetochores and, surprisingly, proper assembly of the central spindle at mitotic exit. Thus, BubR1-mediated CENP-E phosphorylation produces a temporal switch that enables transition from lateral to end-on microtubule capture and organization of microtubules into stable midzone arrays.
Trostnikov, M. V., Roshina, N. V., Boldyrev, S. V., Veselkina, E. R., Zhuikov, A. A., Krementsova, A. V. and Pasyukova, E. G. (2019). Disordered expression of shaggy, the Drosophila gene encoding a serine-threonine protein kinase GSK3, affects the lifespan in a transcript-, stage-, and tissue-specific manner. Int J Mol Sci 20(9). PubMed ID: 31060255
GSK3 (glycogen synthase kinase 3) is a conserved protein kinase governing numerous regulatory pathways. In Drosophila melanogaster, GSK3 is encoded by shaggy (sgg), which forms 17 annotated transcripts corresponding to 10 protein isoforms. The goal was to demonstrate how differential sgg transcription affects lifespan, which GSK3 isoforms are important for the nervous system, and which changes in the nervous system accompany accelerated aging. Overexpression of three sgg transcripts affected the lifespan in a stage- and tissue-specific way: sgg-RA and sgg-RO affected the lifespan only when overexpressed in muscles and in embryos, respectively; the essential sgg-RB transcript affected lifespan when overexpressed in all tissues tested. In the nervous system, only sgg-RB overexpression affected lifespan, causing accelerated aging in a neuron-specific way, with the strongest effects in dopaminergic neurons and the weakest effects in GABAergic neurons. Pan-neuronal sgg-RB overexpression violated the properties of the nervous system, including the integrity of neuron bodies; the number, distribution, and structure of mitochondria; cytoskeletal characteristics; and synaptic activity. Such changes observed in young individuals indicated premature aging of their nervous system, which paralleled a decline in survival. These findings demonstrated the key role of GSK3 in ensuring the link between the pathology of neurons and lifespan.
Toshniwal, A. G., Gupta, S., Mandal, L. and Mandal, S. (2019). ROS inhibits cell growth by regulating 4EBP and S6K, independent of TOR, during development. Dev Cell 49(3): 473-489. PubMed ID: 31063760
Reactive oxygen species (ROS), despite having damaging roles, serve as signaling molecules regulating diverse biological and physiological processes. Employing in vivo genetic studies in Drosophila, this study shows that besides causing G1-S arrest by activation of Dacapo, ROS can simultaneously inhibit cell growth by regulating the expression of 4EBP and S6K. This is achieved by triggering a signaling cascade that includes Ask1, JNK, and FOXO independent of the Tsc-TOR growth regulatory pathway. Qualitative and quantitative differences in the types of ROS molecules generated dictate whether cells undergo G1-S arrest only or experience blocks in both cell proliferation and growth. Importantly, during normal development, this signaling cascade is triggered by ecdysone in late larval fat body cells to restrict their growth prior to pupation by antagonizing insulin signaling. The present work reveals an unexpected role of ROS in systemic control of growth in response to steroid hormone signaling to establish organismal size.
Lybrand, D. B., Naiman, M., Laumann, J. M., Boardman, M., Petshow, S., Hansen, K., Scott, G. and Wehrli, M. (2019). Destruction complex dynamics: Wnt/beta-catenin signaling alters Axin-GSK3beta interactions in in vivo. Development. PubMed ID: 31189665
The central regulator of the Wnt/beta-catenin pathway is the Axin/APC/GSK3beta destruction complex (DC), which in unstimulated conditions targets cytoplasmic beta-catenin for degradation. How Wnt activation inhibits the DC to permit beta-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo Using Bimolecular Fluorescence Complementation (BiFC) methods, this study has now analyzed the activity of the DC under near-physiological conditions in Drosophila. By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, the sequence of events was defined by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3beta interactions that prevent beta-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control beta-catenin's degradation and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signaling transduction in vivo.
Ung, P. M. U., Sonoshita, M., Scopton, A. P., Dar, A. C., Cagan, R. L. and Schlessinger, A. (2019). Integrated computational and Drosophila cancer model platform captures previously unappreciated chemicals perturbing a kinase network. PLoS Comput Biol 15(4): e1006878. PubMed ID: 31026276
Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). This study presents a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. This platform is demonstrated by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. Features from the hits were combined with those of known inhibitors to create a 'hybrid' molecule with improved suppression of dRetM955T transformation. This platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity.
El-Amine, N., Carim, S. C., Wernike, D. and Hickson, G. R. X. (2019). Rho-dependent control of the Citron kinase, Sticky, drives midbody ring maturation. Mol Biol Cell: mbcE19040194. PubMed ID: 31166845
Rho-dependent proteins control assembly of the cytokinetic contractile ring, yet it remains unclear how those proteins guide ring closure and how they promote subsequent formation of a stable midbody ring. Citron kinase is one important component required for midbody ring formation but its mechanisms of action and relationship with Rho are controversial. This study conducted a structure-function analysis of the Drosophila Citron kinase, Sticky, in Schneider's S2 cells. Two separable and redundant RhoGEF/Pebble-dependent inputs into Sticky recruitment to the nascent midbody ring are defined; each input was shown to be subsequently required for retention at, and for the integrity of, the mature midbody ring. The first input is via an actomyosin-independent interaction between Sticky and Anillin, a key scaffold also required for midbody ring formation. The second input requires the Rho-binding domain of Sticky, whose boundaries were defined in this study. Collectively, these results show how midbody ring biogenesis depends on the coordinated actions of Sticky, Anillin and Rho.

Monday, July 8th - Adult neural development and function

Ache, J. M., Namiki, S., Lee, A., Branson, K. and Card, G. M. (2019). State-dependent decoupling of sensory and motor circuits underlies behavioral flexibility in Drosophila. Nat Neurosci 22(7): 1132-1139. PubMed ID: 31182867
An approaching predator and self-motion toward an object can generate similar looming patterns on the retina, but these situations demand different rapid responses. How central circuits flexibly process visual cues to activate appropriate, fast motor pathways remains unclear. This study identified two descending neuron (DN) types that control landing and contribute to visuomotor flexibility in Drosophila. For each, silencing impairs visually evoked landing, activation drives landing, and spike rate determines leg extension amplitude. Critically, visual responses of both DNs are severely attenuated during non-flight periods, effectively decoupling visual stimuli from the landing motor pathway when landing is inappropriate. The flight-dependence mechanism differs between DN types. Octopamine exposure mimics flight effects in one, whereas the other probably receives neuronal feedback from flight motor circuits. Thus, this sensorimotor flexibility arises from distinct mechanisms for gating action-specific descending pathways, such that sensory and motor networks are coupled or decoupled according to the behavioral state.
Dason, J. S., Cheung, A., Anreiter, I., Montemurri, V. A., Allen, A. M. and Sokolowski, M. B. (2019). Drosophila melanogaster foraging regulates a nociceptive-like escape behavior through a developmentally plastic sensory circuit. Proc Natl Acad Sci U S A. PubMed ID: 31213548
Rover and sitter allelic variants of the Drosophila foraging (for) gene differ in parasitoid wasp susceptibility, suggesting a link between for and nociception. By optogenetically activating cells associated with each of for's promoters (pr1-pr4), this study shows that pr1 cells regulate larval escape behavior. In accordance with rover and sitter differences in parasitoid wasp susceptibility, rovers were found to have higher pr1 expression and increased sensitivity to nociception relative to sitters. The for null mutants display impaired responses to thermal nociception, which are rescued by restoring for expression in pr1 cells. Conversely, knockdown of for in pr1 cells phenocopies the for null mutant. To gain insight into the circuitry underlying this response, an intersectional approach and activity-dependent GFP reconstitution were used across synaptic partners (GRASP) to show that pr1 cells in the ventral nerve cord (VNC) are required for the nociceptive response, and multidendritic sensory nociceptive neurons synapse onto pr1 neurons in the VNC. Finally, activation of the pr1 circuit during development was shown to suppresses the escape response. These data demonstrate a role of for in larval nociceptive behavior. This function is specific to for pr1 neurons in the VNC, guiding a developmentally plastic escape response circuit.
Guajardo, R., Luginbuhl, D. J., Han, S., Luo, L. and Li, J. (2019). Functional divergence of Plexin B structural motifs in distinct steps of Drosophila olfactory circuit assembly. Elife 8. PubMed ID: 31225795
Plexins exhibit multitudinous, evolutionarily conserved functions in neural development. How Plexins employ their diverse structural motifs in vivo to perform distinct roles is unclear. Previously work has shown that Plexin B (PlexB) controls multiple steps during the assembly of the Drosophila olfactory circuit. This study systematically mutagenized structural motifs of PlexB and examined the function of these variants in these multiple steps: axon fasciculation, trajectory choice, and synaptic partner selection. The extracellular Sema domain was found to be essential for all three steps, the catalytic site of the intracellular RapGAP is engaged in none, and the intracellular GTPase-binding motifs are essential for trajectory choice and synaptic partner selection, but are dispensable for fasciculation. Moreover, extracellular PlexB cleavage serves as a regulatory mechanism of PlexB signaling. Thus, the divergent roles of PlexB motifs in distinct steps of neural development contribute to its functional versatility in neural circuit assembly.
Handler, A., Graham, T. G. W., Cohn, R., Morantte, I., Siliciano, A. F., Zeng, J., Li, Y. and Ruta, V. (2019). Distinct dopamine receptor pathways underlie the temporal sensitivity of associative learning. Cell 178(1): 60-75. PubMed ID: 31230716
Animals rely on the relative timing of events in their environment to form and update predictive associations, but the molecular and circuit mechanisms for this temporal sensitivity remain incompletely understood. This study shows that olfactory associations in Drosophila can be written and reversed on a trial-by-trial basis depending on the temporal relationship between an odor cue and dopaminergic reinforcement. Through the synchronous recording of neural activity and behavior, this study shows that reversals in learned odor attraction correlate with bidirectional neural plasticity in the mushroom body, the associative olfactory center of the fly. Two dopamine receptors, DopR1 and DopR2, contribute to this temporal sensitivity by coupling to distinct second messengers and directing either synaptic depression or potentiation. These results reveal how dopamine-receptor signaling pathways can detect the order of events to instruct opposing forms of synaptic and behavioral plasticity, allowing animals to flexibly update their associations in a dynamic environment.
Kanda, H., Shimamura, R., Koizumi-Kitajima, M. and Okano, H. (2019). Degradation of extracellular matrix by Matrix metalloproteinase 2 is essential for the establishment of the blood-brain barrier in Drosophila. iScience 16: 218-229. PubMed ID: 31195239
The blood-brain barrier (BBB) is an essential system that isolates the central nervous system from the internal environment. Increasing evidence has begun to reveal the molecules that are required for BBB integrity. However, how these components are regulated remains unclear. This study reports that a matrix metalloproteinase, Mmp2, is essential for the establishment of the BBB in Drosophila. In the absence of mmp2, the BBB becomes leaky, which allows the tracer to penetrate the brain. Moreover, the expression pattern of a junctional component, Neuroglian, is altered. It was also found that the regulation of the amounts of particular extracellular matrix components is critical for BBB establishment. Furthermore, the process of mesenchymal-epithelial transition of BBB-forming cells is perturbed in the absence of Mmp2. These data indicate that the presence of Mmp(s), which is typically considered to be a risk factor for BBB degradation, is essential for BBB integrity in Drosophila.
Ismail, J. N., Badini, S., Frey, F., Abou-Kheir, W. and Shirinian, M. (2019). Drosophila Tet is expressed in midline glia and is required for proper axonal development. Front Cell Neurosci 13: 252. PubMed ID: 31213988
Ten-Eleven Translocation (TET) proteins are important epigenetic regulators that play a key role in development and are frequently deregulated in cancer. Drosophila melanogaster has a single homologous Tet gene (dTet) that is highly expressed in the central nervous system during development. This study examined the expression pattern of dTet in the third instar larval CNS and discovered its presence in a specific set of glia cells: midline glia (MG). Moreover, dTet knockdown resulted in significant lethality, locomotor dysfunction, and alterations in axon patterning in the larval ventral nerve cord. Molecular analyses on dTet knockdown larvae showed a downregulation in genes involved in axon guidance and reduced expression of the axon guidance cue Slit. These findings point toward a potential role for dTet in midline glial function, specifically the regulation of axon patterning during neurodevelopment.

Friday, July 5th - Vesicles and Synapses

Johnson, D. M. and Andrew, D. J. (2019). Role of tbc1 in Drosophila embryonic salivary glands. BMC Mol Cell Biol 20(1): 19. PubMed ID: 31242864
CG4552/tbc1 was identified as a downstream target of Fork head (Fkh), the single Drosophila member of the FoxA family of transcription factors and a major player in salivary gland formation and homeostasis. Tbc1 and its orthologues have been implicated in phagocytosis, the innate immune response, border cell migration, cancer and an autosomal recessive form of non-degenerative Pontocerebellar hypoplasia. Recently, the mammalian Tbc1 orthologue, Tbc1d23, has been shown to bind both the conserved N-terminal domains of two Golgins (Golgin-97 and Golgin-245) and the WASH complex on endosome vesicles. Through this activity, Tbc1d23 has been proposed to link endosomally-derived vesicles to their appropriate target membrane in the trans Golgi (TGN). This paper provides an initial characterization of Drosophila orthologue. Like its mammalian orthologue, Tbc1 localizes to the trans Golgi. It also colocalizes with a subset of Rabs associated with both early and recycling endosomes. Animals completely missing tbc1 survive, but females have fertility defects. Consistent with the human disease, loss of tbc1 reduces optic lobe size and increases response time to mechanical perturbation. Loss and overexpression of tbc1 in the embryonic salivary glands leads to secretion defects and apical membrane irregularities. These findings support a role for tbc1 in endocytic/membrane trafficking, consistent with its activities in other systems.
Lorincz, P., Kenez, L. A., Toth, S., Kiss, V., Varga, A., Csizmadia, T., Simon-Vecsei, Z. and Juhasz, G. (2019). Vps8 overexpression inhibits HOPS-dependent trafficking routes by outcompeting Vps41/Lt. Elife 8. PubMed ID: 31194677
Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding class C core vacuole/endosome tethering (CORVET) that was suggested to transform into Rab7-binding homotypic fusion and vacuolar protein sorting (HOPS). Previous work has identified miniCORVET, containing Drosophila Vps8 and three shared core proteins that are required for endosome maturation upstream of HOPS in highly endocytic cells. This study shows that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Light and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation.
Johansson, J., Naszai, M., Hodder, M. C., Pickering, K. A., Miller, B. W., Ridgway, R. A., Yu, Y., Peschard, P., Brachmann, S., Campbell, A. D., Cordero, J. B. and Sansom, O. J. (2019). RAL GTPases drive intestinal stem cell function and regeneration through internalization of WNT signalosomes. Cell Stem Cell 24(4): 592-607.e597. PubMed ID: 30853556
Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, this study identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, this study has identified a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.
Metwally, E., Zhao, G., Li, W., Wang, Q. and Zhang, Y. Q. (2019). Calcium-activated Calpain specifically cleaves glutamate receptor IIA But Not IIB at the Drosophila neuromuscular junction. J Neurosci 39(15): 2776-2791. PubMed ID: 30705102
Calpains are calcium-dependent, cytosolic proteinases active at neutral pH. They do not degrade but cleave substrates at limited sites. Calpains are implicated in various pathologies, such as ischemia, injuries, muscular dystrophy, and neurodegeneration. Despite so, the physiological function of calpains remains to be clearly defined. Using the neuromuscular junction of Drosophila of both sexes as a model, RNAi screening was performed and it was uncovered that calpains negatively regulated protein levels of the glutamate receptor GluRIIA but not GluRIIB. Calpains enrich at the postsynaptic area, and the calcium-dependent activation of calpains induced cleavage of GluRIIA at Q788 of its C terminus. Further genetic and biochemical experiments revealed that different calpains genetically and physically interact to form a protein complex. The protein complex was required for the proteinase activation to downregulate GluRIIA. These data provide a novel insight into the mechanisms by which different calpains act together as a complex to specifically control GluRIIA levels and consequently synaptic function.
Mondragon, A. A., Yalonetskaya, A., Ortega, A. J., Zhang, Y., Naranjo, O., Elguero, J., Chung, W. S. and McCall, K. (2019). Lysosomal machinery drives extracellular acidification to direct non-apoptotic cell death. Cell Rep 27(1): 11-19.e13. PubMed ID: 30943394
Cell death is a fundamental aspect of development, homeostasis, and disease; yet, understanding of non-apoptotic forms of cell death is limited. One such form is phagoptosis, in which one cell utilizes phagocytosis machinery to kill another cell that would otherwise continue living. A non-autonomous requirement of phagocytosis machinery has been identified for the developmental programmed cell death of germline nurse cells in the Drosophila ovary; however, the precise mechanism of death remained elusive. This study shows that lysosomal machinery acting in epithelial follicle cells is used to non-autonomously induce the death of nearby germline cells. Stretch follicle cells recruit V-ATPases and chloride channels to their plasma membrane to extracellularly acidify the germline and release cathepsins that destroy the nurse cells. These results reveal a role for lysosomal machinery acting at the plasma membrane to cause the death of neighboring cells, providing insight into mechanisms driving non-autonomous cell death.
Urwyler, O., Izadifar, A., Vandenbogaerde, S., Sachse, S., Misbaer, A. and Schmucker, D. (2019). Branch-restricted localization of phosphatase Prl-1 specifies axonal synaptogenesis domains. Science 364(6439). PubMed ID: 31048465
Central nervous system (CNS) circuit development requires subcellular control of synapse formation and patterning of synapse abundance. This study identified the Drosophila membrane-anchored phosphatase of regenerating liver (Prl-1) as an axon-intrinsic factor that promotes synapse formation in a spatially restricted fashion. The loss of Prl-1 in mechanosensory neurons reduced the number of CNS presynapses localized on a single axon collateral and organized as a terminal arbor. Flies lacking all Prl-1 protein had locomotor defects. The overexpression of Prl-1 induced ectopic synapses. In mechanosensory neurons, Prl-1 modulates the insulin receptor (InR) signaling pathway within a single contralateral axon compartment, thereby affecting the number of synapses. The axon branch-specific localization and function of Prl-1 depend on untranslated regions of the prl-1 messenger RNA (mRNA). Therefore, compartmentalized restriction of Prl-1 serves as a specificity factor for the subcellular control of axonal synaptogenesis.
Bhuin, T. and Roy, J. K. (2019). Developmental expression, co-localization and genetic interaction of exocyst component Sec15 with Rab11 during Drosophila development. Exp Cell Res. PubMed ID: 31071318
Sec15, a component of an evolutionarily conserved octomeric exocyst complex, has been identified as an interactor of GTP-bound Rab11 in mammals and Drosophila which shows its role in secretion in yeast and intracellular vesicle transport. This paper reports the expression patterns of Drosophila Sec15 (DSec15) transcript and Sec15 protein during Drosophila development. At early embryonic stages, a profound level of maternally loaded DSec15 transcript and protein is found. At cellular blastoderm cells (stage 5 embryos); the expression is seen in pole cells, apical membrane and sub-apical region. The transcript is predominantly accumulated in mesoderm, tracheal pits, gut, LE cells, trachea, and ventral nerve cord as development proceeds. While, a robust expression of Sec15 is seen in amnioserosa (AS), lateral epidermis (LAE), developing trachea, gut, ventral nerve cord and epithelial cells. During larval development, the transcript is also found in all imaginal discs with a distinguished accumulation in the morphogenetic furrow of eye disc, gut, proventriculus and gastric ceacae, garland cells/nephrocytes, malpighian tubules, ovary and testis. Further, this study shows that Sec15 co-localizes with Rab11 during Drosophila embryonic and larval development. Finally, using a genetic approach, this study demonstrated that Sec15 interacts with Rab11 in producing blister during Drosophila wing development.
James, T. D., Zwiefelhofer, D. J. and Frank, C. A. (2019). Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling. Elife 8. PubMed ID: 31180325
Synapses and circuits rely on neuroplasticity to adjust output and meet physiological needs. Forms of homeostatic synaptic plasticity impart stability at synapses by countering destabilizing perturbations. The Drosophila melanogaster larval neuromuscular junction (NMJ) is a model synapse with robust expression of homeostatic plasticity. At the NMJ, a homeostatic system detects impaired postsynaptic sensitivity to neurotransmitter and activates a retrograde signal that restores synaptic function by adjusting neurotransmitter release. This process has been separated into temporally distinct phases, induction and maintenance. One prevailing hypothesis is that a shared mechanism governs both phases. This study shows the two phases are separable. Combining genetics, pharmacology, and electrophysiology, a signaling system consisting of PLCbeta, inositol triphosphate (IP3), IP3 receptors, and Ryanodine receptors was shown to be required only for the maintenance of homeostatic plasticity. It was also found that the NMJ is capable of inducing homeostatic signaling even when its sustained maintenance process is absent.

Wednesday, July 3rd - Disease Models

Donde, A., Sun, M., Jeong, Y. H., Wen, X., Ling, J., Lin, S., Braunstein, K., Nie, S., Wang, S., Chen, L. and Wong, P. C. (2019). Upregulation of ATG7 attenuates motor neuron dysfunction associated with depletion of TARDBP/TDP-43. Autophagy. PubMed ID: 31242080
A shared neuropathological hallmark in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is nuclear clearance and cytoplasmic aggregation of TARDBP/TDP-43 (TAR DNA binding protein). Previously work has shown that the ability of TARDBP to repress nonconserved cryptic exons was impaired in brains of patients with ALS and FTD, suggesting that its nuclear depletion contributes to neurodegeneration. However, the critical pathways impacted by the failure to repress cryptic exons that may contribute to neurodegeneration remain undefined. This report that transcriptome analysis of TARDBP-deficient neurons revealed downregulation of ATG7, a critical gene required for macroautophagy/autophagy. Mouse and Drosophila models lacking TARDBP/TBPH in motor neurons exhibiting age-dependent neurodegeneration and motor deficits showed reduction of ATG7 and accumulation of SQSTM1/p62 inclusions. Importantly, genetic upregulation of the autophagy pathway improved motor function and survival in TBPH-deficient flies. Together with the observation that ATG7 is reduced in ALS-FTD brain tissues, these findings identify the autophagy pathway as one key effector of nuclear depletion of TARDBP that contributes to neurodegeneration. It is thus suggested that the autophagy pathway is a therapeutic target for ALS-FTD and other disorders exhibiting TARDBP pathology.
Buhl, E., Higham, J. P. and Hodge, J. J. L. (2019). Alzheimer's disease-associated tau alters Drosophila circadian activity, sleep and clock neuron electrophysiology. Neurobiol Dis: 104507. PubMed ID: 31207389
Alzheimer's disease (AD) is the most common cause of dementia, which is associated with an enormous personal, social and economic burden worldwide. However, there are few current treatments with none of them targeting the underlying causes of the disease. Expression of the 0N4R isoform of tau has been associated with AD pathology and this study showa that expressing it in the Drosophila clock network gives rise to circadian and sleep phenotypes which closely match the behavioural changes seen in human AD patients. Tauopathic flies exhibited greater locomotor activity throughout the day and night and displayed a loss of sleep, particularly at night. Under constant darkness, the locomotor behaviour of tau-expressing flies was less rhythmic than controls indicating a defect in their intrinsic circadian rhythm. Current clamp recordings from wake-promoting, pigment dispersing factor (PDF)-positive large lateral ventral clock neurons (l-LNvs) revealed elevated spontaneous firing throughout the day and night which likely underlies the observed hyperactive circadian phenotype. Interestingly, expression of tau in only the PDF-positive pacemaker neurons, which are thought to be the most important for behaviour under constant conditions, was not sufficient or even necessary to affect circadian rhythmicity. This work establishes Drosophila as a model to investigate interactions between human pathological versions of tau and the machinery that controls neuronal excitability, allowing the identification of underlying mechanisms of disease that may reveal new therapeutic targets.
Farago, A., Zsindely, N. and Bodai, L. (2019). Mutant huntingtin disturbs circadian clock gene expression and sleep patterns in Drosophila. Sci Rep 9(1): 7174. PubMed ID: 31073199
Deficiency of the sleep-wake cycle can accelerate the progression of Huntington's disease (HD) and exacerbate symptoms making it a target of investigation to better understand the molecular pathology of the disorder. This study analyzed sleep defects in a Drosophila model of HD and investigated whether disturbed sleep coincides with alterations in the molecular mechanism controlling circadian rhythm. To analyze sleep defects, the daily activity was recorded of flies in 12:12 hours light:dark entrainment, and in regard to the underlying molecular mechanism circadian "clock" gene expression was measured. In HD flies reduced amount of sleep, sleep fragmentation and prolonged sleep latency were recorded. Changes were found in gene expression patterns of both transcriptional feedback loops of circadian regulation. Prolonged expression of the core feedback loop components period and timeless was detected, whilst the secondary feedback loop member vrille had lower expression rates in general. The results show that the Drosophila HD model recapitulates most of the sleep related symptoms reported in patients therefore it can be a potential tool to study the molecular background of sleep defects in HD. Altered expression of circadian "clock" genes suggests that disturbed sleep pattern in HD might be the consequence of disturbed circadian regulation.
Goodman, L. D., Prudencio, M., Srinivasan, A. R., Rifai, O. M., Lee, V. M., Petrucelli, L. and Bonini, N. M. (2019). eIF4B and eIF4H mediate GR production from expanded G4C2 in a Drosophila model for C9orf72-associated ALS. Acta Neuropathol Commun 7(1): 62. PubMed ID: 31023341
The discovery of an expanded (GGGGCC)n repeat (termed G4C2) within the first intron of C9orf72 in familial ALS/FTD has led to a number of studies showing that the aberrant expression of G4C2 RNA can produce toxic dipeptides through repeat-associated non-AUG (RAN-) translation. To reveal canonical translation factors that impact this process, an unbiased loss-of-function screen was performed in a G4C2 fly model that maintained the upstream intronic sequence of the human gene and contained a GFP tag in the GR reading frame. 11 of 48 translation factors were identified that impact production of the GR-GFP protein. Further investigations into two of these, eIF4B and eIF4H, revealed that downregulation of these factors reduced toxicity caused by the expression of expanded G4C2 and reduced production of toxic GR dipeptides from G4C2 transcripts. In patient-derived cells and in post-mortem tissue from ALS/FTD patients, eIF4H was found to be downregulated in cases harboring the G4C2 mutation compared to patients lacking the mutation and healthy individuals. Overall, these data define eIF4B and eIF4H as disease modifiers whose activity is important for RAN-translation of the GR peptide from G4C2-transcripts.
He, H., Huang, W., Wang, R., Lin, Y., Guo, Y., Deng, J., Deng, H., Zhu, Y., Allen, E. G., Jin, P. and Duan, R. (2019). Amyotrophic Lateral Sclerosis-associated GGGGCC repeat expansion promotes Tau phosphorylation and toxicity. Neurobiol Dis 130: 104493. PubMed ID: 31176718
Microtubule-associated protein Tau (MAPT) and GGGGCC (G4C2) repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) are the major known genetic causes of frontotemporal dementia (FTD) and other neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS). Although expanded G4C2 repeats and Tau traditionally are associated with different clinical presentations, pathological and genetic studies have suggested a strong association between them. This study demonstrates a strong genetic interaction between expanded G4C2 repeats and Tau. Co-expression of expanded G4C2 repeats and Tau could produce a synergistic deterioration of rough eyes, motor function, life span and neuromuscular junction morphological abnormalities in Drosophila. Mechanistically, compared with the normal allele containing (G4C2)3 repeats, the (G4C2)30 allele increased Tau phosphorylation levels and promoted Tau R406W aggregation. These results together suggest a potential crosstalk between expanded G4C2 repeats and Tau in modulating neurodegeneration.
Francois-Moutal, L., Scott, D. D., Felemban, R., Miranda, V. G., Sayegh, M. R., Perez-Miller, S., Khanna, R., Gokhale, V., Zarnescu, D. C. and Khanna, M. (2019). A small molecule targeting TDP-43's RNA recognition motifs reduces locomotor defects in a Drosophila model of ALS. ACS Chem Biol. PubMed ID: 31241884
RNA dysregulation likely contributes to disease pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. A pathological form of the transactive response (TAR) DNA Binding Protein (TDP-43) binds to RNA in stress granules and forms membraneless, amyloid-like, TDP-43 aggregates in the cytoplasm of ALS motor neurons. In was hypothesized in this study that by targeting the RNA recognition motifs (RRM) domains of TDP-43 that confer a pathogenic interaction between TDP-43 and RNA, motor neuron toxicity could be reduced. In silico docking of 50K compounds to the RRM domains of TDP-43 identified a small molecule (rTRD01) that (i) bound to TDP-43's RRM1 and RRM2 domains; (ii) partially disrupted TDP-43's interaction with the hexanucleotide RNA repeat of the disease-linked c9orf72 gene, but not with (UG)6 canonical binding sequence of TDP-43; and (iii) improved larval turning, an assay measuring neuromuscular coordination and strength, in an ALS fly model based on the overexpression of mutant TDP-43. These findings provide an instructive example of a chemical biology approach pivoted to discover small molecules targeting RNA-protein interactions in neurodegenerative diseases.

Tuesday, July 2nd - Evolution

Bracker, L. B., Schmid, C. A., Bolini, V. A., Holz, C. A., Prud'homme, B., Sirota, A. and Gompel, N. (2019). Quantitative and discrete evolutionary changes in the egg-laying behavior of single Drosophila females. Front Behav Neurosci 13: 118. PubMed ID: 31191270
This study focused on oviposition, the act of laying an egg, in flies of the genus Drosophila to describe the elementary behavioral steps or microbehaviors that a single female fly undertakes prior to and during egg laying. The hierarchy and relationships in time of these microbehaviors were analyzed in three closely related Drosophila species with divergent egg-laying preferences and uncovered cryptic differences in their behavioral patterns. Using high-speed imaging, the oviposition behavior of single females of Drosophila suzukii, Drosophila biarmipes and Drosophila melanogaster was quantified in depth in a novel behavioral assay. By computing transitions between microbehaviors, a common ethogram structure was identified underlying oviposition of all three species. Quantifying parameters such as relative time spent on a microbehavior and its average duration, however, revealed clear differences between species. In addition, the temporal dynamics and probability of transitions to different microbehaviors were analyzed relative to a central event of oviposition, ovipositor contact. Although the quantitative analysis highlights behavioral variability across flies, it reveals some interesting trends for each species in the mode of substrate sampling, as well as possible evolutionary differences. Larger datasets derived from automated video annotation will overcome this paucity of data in the future, and use the same framework to reappraise these observed differences. This study reveals a common architecture to the oviposition ethogram of three Drosophila species, indicating its ancestral state. It also indicates that Drosophila suzukii's behavior departs quantitatively and qualitatively from that of the outgroup species, in line with its known divergent ethology. Together, these results illustrate how a global shift in ethology breaks down in the quantitative reorganization of the elementary steps underlying a complex behavior.
Cortot, J., Farine, J. P., Houot, B., Everaerts, C. and Ferveur, J. F. (2019). Experimental introgression to evaluate the impact of sex specific traits on Drosophila melanogaster incipient speciation. G3 (Bethesda). PubMed ID: 31167833
Sex specific traits are involved in speciation but it is difficult to determine whether their variation initiates or reinforces sexual isolation. In some insects, speciation depends of the rapid change of expression in desaturase genes coding for sex pheromones. Two closely related desaturase genes are involved in Drosophila melanogaster pheromonal communication: desat1 affects both the production and the reception of sex pheromones while desat2 is involved in their production in flies of Zimbabwe populations. There is a strong asymmetric sexual isolation between Zimbabwe populations and all other "Cosmopolitan" populations: Zimbabwe females rarely copulate with Cosmopolitan males whereas Zimbabwe males readily copulate with all females. All populations express desat1 but only Zimbabwe strains show high desat2 expression. To evaluate the impact of sex pheromones, female receptivity and desat expression on the incipient speciation process between Zimbabwe and Cosmopolitan populations, the Zimbabwe genome was introgressed into a Cosmopolitan genome labeled with the white mutation, using a multi-generation procedure. The association between these sex-specific traits was determined during the procedure. The production of pheromones was largely dissociated between the sexes. The copulation frequency (but not latency) was highly correlated with the female-but not with the male-principal pheromones. Two stable white lines were finally obtained showing Zimbabwe-like sex pheromones, copulation discrimination and desat expression. This study indicates that the variation of sex pheromones and mating discrimination depend of distinct-yet overlapping-sets of genes in each sex suggesting that their cumulated effects participate to reinforce the speciation process.
Watanabe, T. (2019). Evolution of the neural sex-determination system in insects: does fruitless homolog regulate neural sexual dimorphism in basal insects?. Insect Mol Biol. PubMed ID: 31066110
In the brain of holometabolous insects such as the fruit fly Drosophila melanogaster, the fruitless gene produces sex-specific gene products under the control of the sex-specific splicing cascade and contributes to the formation of the sexually dimorphic circuits. Similar sex-specific gene products of fruitless homolog have been identified in other holometabolous insects such as the mosquitos and the parasitic wasp, suggesting the fruitless-dependent neural sex-determination system is widely conserved among holometabolous insects. However, it remains obscure whether the fruitless-dependent neural sex-determination system is present in basal hemimetabolous insects. To address this issue, the identification, characterization, and expression analyses of the fruitless homolog were conducted in the two-spotted cricket Gryllus bimaculatus as a model hemimetabolous insect. Gryllus fruitless gene encodes multiple isoforms with unique zinc finger domain, and does not encode a sex-specific gene product. Gryllus Fruitless protein is broadly expressed in the neurons and glial cells in the brain, and there was no prominent sex-related difference in the expression levels of Gryllus fruitless isoforms. The results suggest that the Gryllus fruitless gene is not involved in the neural sex-determination in the cricket brain.
Yukilevich, R. and Peterson, E. K. (2019). The evolution of male and female mating preferences in Drosophila speciation. Evolution. PubMed ID: 31077339
The relative importance of male and female mating preferences in causing sexual isolation between species remains a major unresolved question in speciation. Despite previous work showing that male courtship bias and/or female copulation bias for conspecifics occur in many taxa, the present study is one of the first large-scale works to study their relative divergence. To achieve this, data from the literature and present experiments were used across 66 Drosophila species pairs. The results revealed that male and female mate preferences are both ubiquitous in Drosophila but evolved largely independently, suggesting different underlying evolutionary and genetic mechanisms. Moreover, their relative divergence strongly depended on the geographical relationship of species. Between allopatric species, male courtship and female copulation preferences diverged at very similar rates, evolving approximately linearly with time of divergence. In sharp contrast, between sympatric species pairs, female preferences diverged much more rapidly than male preferences and were the only drivers of enhanced sexual isolation in sympatry and Reproductive Character Displacement (RCD). Not only does this result suggest that females are primarily responsible for such processes as reinforcement, but it also implies that evolved female preferences may reduce selection for further divergence of male courtship preferences in sympatry.
Freda, P. J., Ali, Z. M., Heter, N., Ragland, G. J. and Morgan, T. J. (2019). Stage-specific genotype-by-environment interactions for cold and heat hardiness in Drosophila melanogaster. Heredity (Edinb). PubMed ID: 31164731
Environments often vary across a life cycle, imposing fluctuating natural selection across development. Such fluctuating selection can favor different phenotypes in different life stages, but stage-specific evolutionary responses will depend on genetic variance, covariance, and their interaction across development and across environments. Thus, quantifying how genetic architecture varies with plastic responses to the environment and across development is vital to predict whether stage-specific adaptation will occur in nature. Additionally, the interaction of genetic variation and environmental plasticity (GxE) may be stage-specific, leading to a three-way interaction between genotype, environment, and development or GxDxE. To test for these patterns, larvae and adults of Drosophila melanogaster isogenic lines derived from a natural population were exposed to extreme heat and cold stress after developmental acclimation to cool (18 degrees C) and warm (25 degrees C) conditions, and genetic variance for thermal hardiness was measured. Significant GxE was observed that was specific to larvae and adults for cold and heat hardiness (GxDxE), but no significant genetic correlation across development for either trait at either acclimation temperature. However, cross-development phenotypic correlations for acclimation responses suggest that plasticity itself may be developmentally constrained, though rigorously testing this hypothesis requires more experimentation. These results illustrate the potential for stage-specific adaptation within a complex life cycle and demonstrate the importance of measuring traits at appropriate developmental stages and environmental conditions when predicting evolutionary responses to changing climates.
Green, J. E., Cavey, M., Medina Caturegli, E., Aigouy, B., Gompel, N. and Prud'homme, B. (2019). Evolution of ovipositor length in Drosophila suzukii is driven by enhanced cell size expansion and anisotropic tissue reorganization. Curr Biol 29(12): 2075-2082. PubMed ID: 31178315
Morphological diversity is dominated by variation in body proportion, which can be described with scaling relationships and mathematical equations, following the pioneering work of D'Arcy Thompson and Julian Huxley. Yet, the cellular processes underlying divergence in size and shape of morphological traits between species remain largely unknown. This study compared the ovipositors of two related species, Drosophila melanogaster and D. suzukii. D. suzukii has switched its egg-laying niche from rotting to ripe fruit. Along with this shift, the D. suzukii ovipositor has undergone a significant change in size and shape. Using an allometric approach, this study finds that, while adult ovipositor width has hardly changed between the species, D. suzukii ovipositor length is almost double that of D. melanogaster. This difference mostly arises in a 6-h time window during pupal development. It was observed that the developing ovipositors of the two species comprise an almost identical number of cells, with a similar profile of cell shapes and orientations. After cell division stops, the ovipositor area continues to grow in both species through the isotropic expansion of cell apical area and the anisotropic cellular reorganization of the tissue. Remarkably, it was found that the lengthening of the D. suzukii ovipositor compared to that of D. melanogaster results from the combination of the accelerated expansion of apical cell size and the enhanced anisotropic rearrangement of cells in the tissue. Therefore, the quantitative fine-tuning of morphogenetic processes can drive evolutionary changes in organ size and shape.

Monday, July 1st - Adult Neural Development and Function

Shyu, W. H., Lee, W. P., Chiang, M. H., Chang, C. C., Fu, T. F., Chiang, H. C., Wu, T. and Wu, C. L. (2019). Electrical synapses between mushroom body neurons are critical for consolidated memory retrieval in Drosophila. PLoS Genet 15(5): e1008153. PubMed ID: 31071084
Electrical synapses between neurons, also known as gap junctions, are direct cell membrane channels between adjacent neurons. Gap junctions play a role in the synchronization of neuronal network activity; however, their involvement in cognition has not been well characterized. Three-hour olfactory associative memory in Drosophila has two components: consolidated anesthesia-resistant memory (ARM) and labile anesthesia-sensitive memory (ASM). This study shows that knockdown of the gap junction gene innexin5 (inx5) in mushroom body (MB) neurons disrupted ARM, while leaving ASM intact. Whole-mount brain immunohistochemistry indicated that INX5 protein was preferentially expressed in the somas, calyxes, and lobes regions of the MB neurons. Adult-stage-specific knockdown of inx5 in αβ neurons disrupted ARM, suggesting a specific requirement of INX5 in αβ neurons for ARM formation. Hyperpolarization of αβ neurons during memory retrieval by expressing an engineered halorhodopsin (eNpHR) also disrupted ARM. Administration of the gap junction blocker carbenoxolone (CBX) reduced the proportion of odor responsive alphabeta neurons to the training odor 3 hours after training. Finally, the α-branch-specific 3-hour ARM-specific memory trace was also diminished with CBX treatment and in inx5 knockdown flies. Altogether, these results suggest INX5 gap junction channels in αβ neurons for ARM retrieval and also provide a more detailed neuronal mechanism for consolidated memory in Drosophila.
Alekseyenko, O. V., Chan, Y. B., Okaty, B. W., Chang, Y., Dymecki, S. M. and Kravitz, E. A. (2019). Serotonergic modulation of aggression in Drosophila involves GABAergic and cholinergic opposing pathways. Curr Biol. PubMed ID: 31231050
Pathological aggression is commonly associated with psychiatric and neurological disorders and can impose a substantial burden and cost on human society. Serotonin (5HT) has long been implicated in the regulation of aggression in a wide variety of animal species. In Drosophila, a small group of serotonergic neurons selectively modulates the escalation of aggression. This study has identified downstream targets of serotonergic input-two types of neurons with opposing roles in aggression control. The dendritic fields of both neurons converge on a single optic glomerulus LC12, suggesting a key pathway linking visual input to the aggression circuitry. The first type is an inhibitory GABAergic neuron: its activation leads to a decrease in aggression. The second neuron type is excitatory: its silencing reduces and its activation increases aggression. RNA sequencing (RNA-seq) profiling of this neuron type identified that it uses acetylcholine as a neurotransmitter and likely expresses 5HT1A, short neuropeptide F receptor (sNPFR), and the resistant to dieldrin (RDL) category of GABA receptors. Knockdown of RDL receptors in these neurons increases aggression, suggesting the possibility of a direct crosstalk between the inhibitory GABAergic and the excitatory cholinergic neurons. These data show further that neurons utilizing serotonin, GABA, ACh, and short neuropeptide F interact in the LC12 optic glomerulus. Parallel cholinergic and GABAergic pathways descending from this sensory integration area may be key elements in fine-tuning the regulation of aggression.
Devineni, A. V., Sun, B., Zhukovskaya, A. and Axel, R. (2019). Acetic acid activates distinct taste pathways in Drosophila to elicit opposing, state-dependent feeding responses. Elife 8. PubMed ID: 31205005
Taste circuits are genetically determined to elicit an innate appetitive or aversive response, ensuring that animals consume nutritious foods and avoid the ingestion of toxins. This study has examined the response of Drosophila melanogaster to acetic acid, a tastant that can be a metabolic resource but can also be toxic to the fly. The data reveal that flies accommodate these conflicting attributes of acetic acid by virtue of a hunger-dependent switch in their behavioral response to this stimulus. Fed flies show taste aversion to acetic acid, whereas starved flies show a robust appetitive response. These opposing responses are mediated by two different classes of taste neurons, the sugar- and bitter-sensing neurons. Hunger shifts the behavioral response from aversion to attraction by enhancing the appetitive sugar pathway as well as suppressing the aversive bitter pathway. Thus a single tastant can drive opposing behaviors by activating distinct taste pathways modulated by internal state.
Simon, F., Ramat, A., Louvet-Vallee, S., Lacoste, J., Burg, A., Audibert, A. and Gho, M. (2019). Shaping of Drosophila neural cell lineages through coordination of cell proliferation and cell fate by the BTB-ZF transcription factor Tramtrack-69. Genetics. PubMed ID: 31073020
Cell diversity in multicellular organisms relies on coordination between cell proliferation and the acquisition of cell identity. Using genetic approaches and correlative microscopy, this study shows that Tramtrack-69 (Ttk69, a BTB-ZF transcription factor ortholog of the human PLZF factor) plays an essential role in controlling this balance. In the Drosophila bristle cell lineage, producing the external sensory organs composed by a neuron and accessory cells, this study shows that ttk69 loss of function leads to supplementary neural-type cells at the expense of accessory cells. The data indicate that Ttk69 (1) promotes cell-cycle exit of newborn terminal cells by downregulating CycE, the principal cyclin involved in S-phase entry and (2) regulates cell fate acquisition and terminal differentiation by downregulating the expression of hamlet and upregulating that of Suppressor of Hairless, two transcription factors involved in neural-fate acquisition and accessory-cell differentiation, respectively. Thus, Ttk69 plays a central role in shaping neural cell lineages by integrating molecular mechanisms that regulate progenitor cell-cycle exit and cell-fate commitment.
Bielopolski, N., Amin, H., Apostolopoulou, A. A., Rozenfeld, E., Lerner, H., Huetteroth, W., Lin, A. C. and Parnas, M. (2019). Inhibitory muscarinic acetylcholine receptors enhance aversive olfactory learning in adult Drosophila. Elife 8. PubMed ID: 31215865
Olfactory associative learning in Drosophila is mediated by synaptic plasticity between the Kenyon cells of the mushroom body and their output neurons. Both Kenyon cells and their inputs from projection neurons are cholinergic, yet little is known about the physiological function of muscarinic acetylcholine receptors in learning in adult flies. This study shows that aversive olfactory learning in adult flies requires type A muscarinic acetylcholine receptors (mAChR-A), particularly in the gamma subtype of Kenyon cells. mAChR-A inhibits odor responses and is localized in Kenyon cell dendrites. Moreover, mAChR-A knockdown impairs the learning-associated depression of odor responses in a mushroom body output neuron. These results suggest that mAChR-A function in Kenyon cell dendrites is required for synaptic plasticity between Kenyon cells and their output neurons.
Ding, K., Han, Y., Seid, T. W., Buser, C., Karigo, T., Zhang, S., Dickman, D. K. and Anderson, D. J. (2019). Imaging neuropeptide release at synapses with a genetically engineered reporter. Elife 8. PubMed ID: 31241464
Research on neuropeptide function has advanced rapidly, yet there is still no spatio-temporally resolved method to measure the release of neuropeptides in vivo. This study introduce Neuropeptide Release Reporters (NPRRs): novel genetically-encoded sensors with high temporal resolution and genetic specificity. Using the Drosophila larval neuromuscular junction (NMJ) as a model, evidence is provided that NPRRs recapitulate the trafficking and packaging of native neuropeptides, and report stimulation-evoked neuropeptide release events as real-time changes in fluorescence intensity, with sub-second temporal resolution.
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