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Friday, October 18th - Adult Physiology

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Eichenlaub, T., Villadsen, R., Freitas, F. C. P., Andrejeva, D., Aldana, B. I., Nguyen, H. T., Petersen, O. W., Gorodkin, J., Herranz, H. and Cohen, S. M. (2018). Warburg effect metabolism drives neoplasia in a Drosophila genetic model of epithelial cancer. Curr Biol. PubMed ID: 30293715
Summary:
Cancers develop in a complex mutational landscape. Genetic models of tumor formation have been used to explore how combinations of mutations cooperate to promote tumor formation in vivo. This study identified lactate dehydrogenase (LDH), a key enzyme in Warburg effect metabolism, as a cooperating factor that is both necessary and sufficient for epidermal growth factor receptor (EGFR)-driven epithelial neoplasia and metastasis in a Drosophila model. LDH is upregulated during the transition from hyperplasia to neoplasia, and neoplasia is prevented by LDH depletion. Elevated LDH is sufficient to drive this transition. Notably, genetic alterations that increase glucose flux, or a high-sugar diet, are also sufficient to promote EGFR-driven neoplasia, and this depends on LDH activity. This study provides evidence that increased LDHA expression promotes a transformed phenotype in a human primary breast cell culture model. Furthermore, analysis of publically available cancer data showed evidence of synergy between elevated EGFR and LDHA activity linked to poor clinical outcome in a number of human cancers. Altered metabolism has generally been assumed to be an enabling feature that accelerates cancer cell proliferation. These findings provide evidence that sugar metabolism may have a more profound role in driving neoplasia than previously appreciated.
MacMillan, H. A., Nazal, B., Wali, S., Yerushalmi, G. Y., Misyura, L., Donini, A. and Paluzzi, J. P. (2018). Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance. J Exp Biol. PubMed ID: 30104306
Summary:
For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization, and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of Capability (CAPA) (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been previously demonstrated to cause anti-diuresis in insects, including dipterans. This study provides evidence that low (fM) and high (microM) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K(+) clearance rates and reduce chill tolerance, high doses facilitate K(+) clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, the maximum achievable hormonal titres of CAPA was estimated, and evidence was further found that CAPA may function as an anti-diuretic hormone in Drosophila melanogaster. Evidence is provided of a neuropeptide that can negatively affect cold tolerance in an insect, and further evidence of CAPA functioning as an anti-diuretic peptide in this ubiquitous insect model.
Grangeteau, C., Yahou, F., Everaerts, C., Dupont, S., Farine, J. P., Beney, L. and Ferveur, J. F. (2018). Yeast quality in juvenile diet affects Drosophila melanogaster adult life traits. Sci Rep 8(1): 13070. PubMed ID: 30166573
Summary:
.Diet quality is critical for animal development and survival. Fungi can provide nutrients that are essential to organisms that are unable to synthetize them, such as ergosterol in Drosophila melanogaster. Drosophila studies examining the influence of yeast quality in the diet have generally either provided the diet over the whole life span (larva to adult) or during the adult stage and have rarely focussed on the juvenile diet. This study tested the effect of yeast quality in the larval diet on pre-adult development and adult weight, survival, reproduction and food preference. The yeast Saccharomyces cerevisiae was added in three forms in three treatments-live, heated or dried-to food used as the juvenile diet or was not added (empty treatment). Adults resulting from the larvae raised on these four juvenile diets were all maintained on a similar standard laboratory food diet. The data indicate that yeast quality in the juvenile diet affects larva-to-pupa-but not pupa-to-adult-development. Importantly, adult survival, food preference, mating behaviour and cuticular pheromones strongly varied with the juvenile diet. Therefore, the variation of yeast quality in the pre-adult Drosophila diet affects key adult life traits involved in food search, reproduction and survival.
Fiorino, A., Thompson, D., Yadlapalli, S., Jiang, C., Shafer, O. T., Reddy, P. and Meyhofer, E. (2018). Parallelized, real-time, metabolic-rate measurements from individual Drosophila. Sci Rep 8(1): 14452. PubMed ID: 30262912
Summary:
Significant recent evidence suggests that metabolism is intricately linked to the regulation and dysfunction of complex cellular and physiological responses ranging from altered metabolic programs in cancers and aging to circadian rhythms and molecular clocks. While the metabolic pathways and their fundamental control mechanisms are well established, the precise cellular mechanisms underpinning, for example, enzymatic pathway control, substrate preferences or metabolic rates, remain far less certain. Comprehensive, continuous metabolic studies on model organisms, such as the fruit fly Drosophila melanogaster, may provide a critical tool for deciphering these complex physiological responses. This study describes the development of a high-resolution calorimeter, which combines sensitive thermometry with optical imaging to concurrently perform measurements of the metabolic rate of ten individual flies, in real-time, with ~100 nW resolution. Using this calorimeter we have measured the mass-specific metabolic rates of flies of different genotypes, ages, and flies fed with different diets. This powerful new approach enables systematic studies of the metabolic regulation related to cellular and physiological function and disease mechanisms.
Mochanova, M., Tomcala, A., Svobodova, Z. and Kodrik, D. (2018). Role of adipokinetic hormone during starvation in Drosophila. Comp Biochem Physiol B Biochem Mol Biol 226: 26-35. PubMed ID: 30110658
Summary:
The role of adipokinetic hormone (Drome-AKH) in maintaining the levels of basic nutrients, under starvation conditions, was studied using Drosophila melanogaster mutants with AKH deficiency (Akh(1)) and AKH abundance (EE-Akh). The results showed lipids as the main energy reserve in Drosophila, and their physiological level and metabolism were shown to be under the control of AKH. AKH abundance in the body resulted in lower levels of triacylglycerols and diacylglycerols than in the controls, probably due to a more intensive metabolism; interestingly, there was a disproportional representation of fatty acids in triacylglycerols and diacylglycerols in Drosophila. Lower level of glycogen and its partial control by AKH suggest its lesser role as the storage substance. However, maintenance of free carbohydrate level in Drosophila seemed to be critical; when glycogen stores are exhausted, carbohydrates are synthesized from other sources. Protein levels and their alterations, under starvation, did not seem controlled by AKH. AKH-deficient flies were more resistant while AKH-abundant flies were more sensitive to starvation; females were found to be more resistant than males, regardless of the AKH level, probably due to higher body mass and higher amount of nutrients. However, in accordance with the level of all nutrients, that of AKH also gradually decreased with prolonged starvation.
Keebaugh, E. S., Yamada, R., Obadia, B., Ludington, W. B. and Ja, W. W. (2018). Microbial quantity impacts Drosophila nutrition, Development, and Lifespan. iScience 4: 247-259. PubMed ID: 30240744
Summary:
In Drosophila, microbial association can promote development or extend life. This study tested the impact of microbial association during malnutrition and shows that microbial quantity is a predictor of fly longevity. Although all tested microbes, when abundantly provided, can rescue lifespan on low-protein diet, the effect of a single inoculation seems linked to the ability of that microbial strain to thrive under experimental conditions. Microbes, dead or alive, phenocopy dietary protein, and the calculated dependence on microbial protein content is similar to the protein requirements determined from fly feeding studies, suggesting that microbes enhance host protein nutrition by serving as protein-rich food. Microbes that enhance larval growth are also associated with the ability to better thrive on fly culture medium. These results suggest an unanticipated range of microbial species that promote fly development and longevity and highlight microbial quantity as an important determinant of effects on physiology and lifespan during undernutrition.

Thursday, October 18th - Behavior

Allen, A. M., Anreiter, I., Vesterberg, A., Douglas, S. J. and Sokolowski, M. B. (2018). Pleiotropy of the Drosophila melanogaster foraging gene on larval feeding-related traits. J Neurogenet: 1-11. PubMed ID: 30303018
Summary:
Little is known about the molecular underpinning of behavioral pleiotropy. The Drosophila melanogaster foraging gene is highly pleiotropic, affecting many independent larval and adult phenotypes. Included in foraging's multiple phenotypes are larval foraging path length, triglyceride levels, and food intake. foraging has a complex structure with four promoters and 21 transcripts that encode nine protein isoforms of a cGMP dependent protein kinase (PKG). This study examined if foraging's complex molecular structure underlies the behavioral pleiotropy associated with this gene. Using a promotor analysis strategy, DNA fragments upstream of each of foraging's transcription start sites was cloned and four separate forpr-Gal4s were generated. Supporting the hypothesis of modular function, they had discrete, restricted expression patterns throughout the larva. Promoter specific expression was found in the larval fat body, salivary glands, and body muscle. The modularity of foraging's molecular structure was also apparent in the phenotypic rescues. Path length, triglyceride levels (bordered on significance), and food intake were rescued of forpr0 null larvae using different forpr-Gal4s to drive UAS-for(cDNA). The results refine the spatial expression responsible for foraging's associated phenotypes, as well as the sub-regions of the locus responsible for their expression. foraging's pleiotropy arises at least in part from the individual contributions of its four promoters.
Beauchamp, M., Bertolini, E., Deppisch, P., Steubing, J., Menegazzi, P. and Helfrich-Forster, C. (2018). Closely related fruit fly species living at different latitudes diverge in their circadian clock anatomy and rhythmic behavior. J Biol Rhythms: 748730418798096. PubMed ID: 30203704
Summary:
Differences have been reported in the expression pattern of the blue light-sensitive flavoprotein Cryptochrome (CRY) and the neuropeptide Pigment-dispersing factor (PDF) in the neuronal clock network of high-latitude Drosophila species, belonging to the Drosophila subgenus (virilis-repleta radiation), compared with cosmopolitan D. melanogaster flies, belonging to the Sophophora subgenus. Alterations in rhythmic patterns of activity due to these differences might have adaptive significance for colonizing high-latitude habitats and, hence, adjusting to long photoperiods. This study shows that these differing CRY/PDF expression patterns are only present in those species of the virilis-repleta radiation that colonized high latitudes. The cosmopolitan species D. mercatorum and D. hydei have a D. melanogaster-like clock network and behavior despite belonging to the virilis-repleta radiation. Similarly, two species of the holotropical Zaprionus genus, more closely related to the Drosophila subgenus than to the Sophophora subgenus, retain a D. melanogaster-like clock network and rhythmic behavior. It is therefore suggested that the D. melanogaster-like clock network is the "ancestral fly clock phenotype" and that alterations in the CRY/PDF clock neurochemistry have allowed some species of the virilis-repleta radiation to colonize high-latitude environments.
Crowley-Gall, A., Shaw, M. and Rollmann, S. M. (2018). Host Preference and Olfaction in Drosophila mojavensis. J Hered. PubMed ID: 30299456
Summary:
Many organisms live in complex environments that vary geographically in resource availability. This environmental heterogeneity can lead to changes within species in their phenotypic traits. For example, in many herbivorous insects, variation in host plant availability has been shown to influence insect host preference behavior. This behavior can be mediated in part through the insect olfactory system and the odor-evoked responses of olfactory receptor neurons (ORNs), which are in turn mediated by their corresponding odorant receptor genes. The desert dwelling fly Drosophila mojavensis is a model species for understanding the mechanisms underlying host preference in a heterogeneous environment. Depending on geographic region, one to multiple host plant species are available. Electrophysiological studies were conducted and variation was found in responses of ORNs to host plant volatiles both within and between 2 populations-particularly to the odorant 4-methylphenol. Flies from select localities within each population were found to lack a response to 4-methylphenol. Experiments then assessed the extent to which these electrophysiological differences were associated with difference in several odor-mediated behavioral responses. No association between the presence/absence of these odor-evoked responses and short range olfactory behavior or oviposition behavior was observed. However, differences in odor-induced feeding behavior in response to 4-methylphenol were found. Localities that exhibit an odor-evoked response to the odorant had increased feeding behavior in the presence of the odorant. This study sets the stage for future work examining the functional genetics underlying variation in odor perception.
Feng, G., Zhang, J., Li, M., Shao, L., Yang, L., Song, Q. and Ping, Y. (2018). Control of sleep onset by Shal/Kv4 channels in Drosophila circadian neurons. J Neurosci. PubMed ID: 30185460
Summary:
Sleep is highly conserved across animal species. Both wake- and sleep-promoting neurons are implicated in the regulation of wake-sleep transition at dusk in Drosophila However, little is known about how they cooperate and whether they act via different mechanisms. This study demonstrated that in female Drosophila, sleep onset was specifically delayed by blocking the Shaker cognate L channels (Shal, also known as voltage-gated K(+) channel 4, Kv4) in wake-promoting cells, including large ventral lateral neurons (l-LNvs) and pars intercerebralis (PI), but not in sleep-promoting dorsal neurons (DN1s). Delayed sleep onset was also observed in males by blocking Kv4 activity in wake-promoting neurons. Electrophysiological recordings show that Kv4 channels contribute A-type currents (IA) in LNvs and PI cells, but are much less conspicuous in DN1s. Interestingly, blocking Kv4 in wake-promoting neurons preferentially increased firing rates at dusk around ZT13, when the resting membrane potentials (RMPs) and firing rates were at lower levels. Furthermore, pigment-dispersing factor (PDF) is essential for the regulation of sleep onset by Kv4 in l-LNvs, and downregulation of PDF receptor (PDFR) in PI neurons advanced sleep onset, indicating Kv4 controls sleep onset via regulating PDF/PDFR signaling in wake-promoting neurons. It is proposed that Kv4 acts as a sleep onset controller by suppressing membrane excitability in a clock-dependent manner to balance the wake-sleep transition at dusk. These results have important implications for the understanding and treatment of sleep disorders such as insomnia.
Everaerts, C., Cazale-Debat, L., Louis, A., Pereira, E., Farine, J. P., Cobb, M. and Ferveur, J. F. (2018). Pre-imaginal conditioning alters adult sex pheromone response in Drosophila. PeerJ 6: e5585. PubMed ID: 30280017
Summary:
Pheromones are chemical signals that induce innate responses in individuals of the same species that may vary with physiological and developmental state. In Drosophila melanogaster, the most intensively studied pheromone is 11-cis-vaccenyl acetate (cVA), which is synthezised in the male ejaculatory bulb and is transferred to the female during copulation. Among other effects, cVA inhibits male courtship of mated females. This study found that male courtship inhibition depends on the amount of cVA and this effect is reduced in male flies derived from eggs covered with low to zero levels of cVA. This effect is not observed if the eggs are washed, or if the eggs are laid several days after copulation. This suggests that courtship suppression involves a form of pre-imaginal conditioning, which is shown to occur during the early larval stage. The conditioning effect could not be rescued by synthetic cVA, indicating that it largely depends on conditioning by cVA and other maternally-transmitted factor(s). These experiments suggest that one of the primary behavioral effects of cVA is more plastic and less stereotypical than had hitherto been realised.
Brankatschk, M., Gutmann, T., Knittelfelder, O., Palladini, A., Prince, E., Grzybek, M., Brankatschk, B., Shevchenko, A., Coskun, U. and Eaton, S. (2018). A temperature-dependent switch in feeding preference improves Drosophila development and survival in the cold. Dev Cell 46(6): 781-793.e784. PubMed ID: 30253170
Summary:
How cold-blooded animals acclimate to temperature and what determines the limits of their viable temperature range are not understood. This study shows that Drosophila alter their dietary preference from yeast to plants when temperatures drop below 15 degrees C and that the different lipids present in plants improve survival at low temperatures. Drosophila require dietary unsaturated fatty acids present in plants to adjust membrane fluidity and maintain motor coordination. Feeding on plants extends lifespan and survival for many months at temperatures consistent with overwintering in temperate climates. Thus, physiological alterations caused by a temperature-dependent dietary shift could help Drosophila survive seasonal temperature changes.

Wednesday, October 17th - Adult CNS development and function

Zong, W., Wang, Y., Tang, Q., Zhang, H. and Yu, F. (2018). Prd1 associates with the clathrin adaptor alpha-Adaptin and the kinesin-3 Imac/Unc-104 to govern dendrite pruning in Drosophila. PLoS Biol 16(8): e2004506. PubMed ID: 30142146
Summary:
Refinement of the nervous system depends on selective removal of excessive axons/dendrites, a process known as pruning. Drosophila ddaC sensory neurons prune their larval dendrites via endo-lysosomal degradation of the L1-type cell adhesion molecule (L1-CAM), Neuroglian (Nrg). This study identified a novel gene, pruning defect 1 (prd1), which governs dendrite pruning of ddaC neurons. Prd1 colocalizes with the clathrin adaptor protein alpha-Adaptin (alpha-Ada) and the kinesin-3 immaculate connections (Imac)/Uncoordinated-104 (Unc-104) in dendrites. Moreover, Prd1 physically associates with alpha-Ada and Imac, which are both critical for dendrite pruning. Prd1, alpha-Ada, and Imac promote dendrite pruning via the regulation of endo-lysosomal degradation of Nrg. Importantly, genetic interactions among prd1, alpha-adaptin, and imac indicate that they act in the same pathway to promote dendrite pruning. These findings indicate that Prd1, alpha-Ada, and Imac act together to regulate discrete distribution of alpha-Ada/clathrin puncta, facilitate endo-lysosomal degradation, and thereby promote dendrite pruning in sensory neurons.
Alyagor, I., Berkun, V., Keren-Shaul, H., Marmor-Kollet, N., David, E., Mayseless, O., Issman-Zecharya, N., Amit, I. and Schuldiner, O. (2018). Combining developmental and perturbation-seq uncovers transcriptional kodules orchestrating neuronal remodeling. Dev Cell 47(1): 38-52.e36. PubMed ID: 30300589
Summary:
Developmental neuronal remodeling is an evolutionarily conserved mechanism required for precise wiring of nervous systems. Despite its fundamental role in neurodevelopment and proposed contribution to various neuropsychiatric disorders, the underlying mechanisms are largely unknown. This study uncovered the fine temporal transcriptional landscape of Drosophila mushroom body gamma neurons undergoing stereotypical remodeling (see Hierarchical TF Networks Regulate Axon Pruning). The data reveal rapid and dramatic changes in the transcriptional landscape during development. Focusing on DNA binding proteins, eleven were identified that are required for remodeling. Furthermore, developing gamma neurons perturbed for three key transcription factors required for pruning were sequenced. A hierarchical network is described featuring positive and negative feedback loops. Superimposing the perturbation-seq on the developmental expression atlas highlights a framework of transcriptional modules that together drive remodeling. Overall, this study provides a broad and detailed molecular insight into the complex regulatory dynamics of developmental remodeling and thus offers a pipeline to dissect developmental processes via RNA profiling.
Barnhart, E. L., Wang, I. E., Wei, H., Desplan, C. and Clandinin, T. R. (2018). Sequential nonlinear filtering of local motion cues by global motion circuits. Neuron 100(1): 229-243.e223. PubMed ID: 30220510
Summary:
Many animals guide their movements using optic flow, the displacement of stationary objects across the retina caused by self-motion. How do animals selectively synthesize a global motion pattern from its local motion components? To what extent does this feature selectivity rely on circuit mechanisms versus dendritic processing? This study used in vivo calcium imaging to identify pre- and postsynaptic mechanisms for processing local motion signals in global motion detection circuits in Drosophila. Lobula plate tangential cells (LPTCs) detect global motion by pooling input from local motion detectors, T4/T5 neurons. T4/T5 neurons were shown to suppress responses to adjacent local motion signals whereas LPTC dendrites selectively amplify spatiotemporal sequences of local motion signals consistent with preferred global patterns. It is proposed that sequential nonlinear suppression and amplification operations allow optic flow circuitry to simultaneously prevent saturating responses to local signals while creating selectivity for global motion patterns critical to behavior.
Egea-Weiss, A., Renner, A., Kleineidam, C. J. and Szyszka, P. (2018). High precision of spike timing across olfactory receptor neurons allows rapid odor coding in Drosophila. iScience 4: 76-83. PubMed ID: 30240755
Summary:
In recent years, it has become evident that olfaction is a fast sense, and millisecond short differences in stimulus onsets are used by animals to analyze their olfactory environment. In contrast, olfactory receptor neurons are thought to be relatively slow and temporally imprecise. These observations have led to a conundrum: how, then, can an animal resolve fast stimulus dynamics and smell with high temporal acuity? Using parallel recordings from olfactory receptor neurons in Drosophila, this study found hitherto unknown fast and temporally precise odorant-evoked spike responses, with first spike latencies (relative to odorant arrival) down to 3 ms and with a SD below 1 ms. These data provide new upper bounds for the speed of olfactory processing and suggest that the insect olfactory system could use the precise spike timing for olfactory coding and computation, which can explain insects' rapid processing of temporal stimuli when encountering turbulent odor plumes.
Du, J., Zhang, Y., Xue, Y., Zhao, X., Zhao, X., Wei, Y., Li, Z., Zhang, Y. and Zhao, Z. (2018). Diurnal protein oscillation profiles in Drosophila head. FEBS Lett. PubMed ID: 30311939
Summary:
Circadian clocks control daily rhythms in physiology, metabolism, and behavior in most organisms. Proteome-wide analysis of protein oscillations is still lacking in Drosophila. In this study, the total protein and phosphorylated protein in Drosophila heads in a 24-hour daily time-course were assayed by using the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) method, and 10 and 7 oscillating proteins as well as 19 and 22 oscillating phosphoproteins in the w(1118) wild type and Clk(Jrk) mutant strains were separately identified. Lastly, a mini screen was performed to investigate the functions of some oscillating proteins in circadian locomotion rhythms. This study provides the first proteomic profiling of diurnally oscillating proteins in fly heads, thereby providing a basis for further mechanistic studies of these proteins in circadian rhythm.
Barajas-Azpeleta, R., Wu, J., Gill, J., Welte, R., Seidel, C., McKinney, S., Dissel, S. and Si, K. (2018). Antimicrobial peptides modulate long-term memory. PLoS Genet 14(10): e1007440. PubMed ID: 30312294
Summary:
Antimicrobial peptides act as a host defense mechanism and regulate the commensal microbiome. To obtain a comprehensive view of genes contributing to long-term memory. mRNA sequencing from single Drosophila heads was performed following behavioral training that produces long-lasting memory. Surprisingly, it was found that Diptericin B, an immune peptide with antimicrobial activity, is upregulated following behavioral training. Deletion and knock down experiments revealed that Diptericin B and another immune peptide, Gram-Negative Bacteria Binding Protein like 3, regulate long-term but not short-term memory or instinctive behavior in Drosophila. Interestingly, removal of DptB in the head fat body and GNBP-like3 in neurons results in memory deficit. That putative antimicrobial peptides influence memory provides an example of how some immune peptides may have been repurposed to influence the function of nervous system.
Dolan, M. J., Belliart-Guerin, G., Bates, A. S., Frechter, S., Lampin-Saint-Amaux, A., Aso, Y., Roberts, R. J. V., Schlegel, P., Wong, A., Hammad, A., Bock, D., Rubin, G. M., Preat, T., Placais, P. Y. and Jefferis, G. (2018). Communication from learned to innate olfactory processing centers Is required for memory retrieval in Drosophila. Neuron. PubMed ID: 30244885
Summary:
The behavioral response to a sensory stimulus may depend on both learned and innate neuronal representations. How these circuits interact to produce appropriate behavior is unknown. In Drosophila, the lateral horn (LH) and mushroom body (MB) are thought to mediate innate and learned olfactory behavior, respectively, although LH function has not been tested directly. Here we identify two LH cell types (PD2a1 and PD2b1) that receive input from an MB output neuron required for recall of aversive olfactory memories. These neurons are required for aversive memory retrieval and modulated by training. Connectomics data demonstrate that PD2a1 and PD2b1 neurons also receive direct input from food odor-encoding neurons. Consistent with this, PD2a1 and PD2b1 are also necessary for unlearned attraction to some odors, indicating that these neurons have a dual behavioral role. This provides a circuit mechanism by which learned and innate olfactory information can interact in identified neurons to produce appropriate behavior (Dolan, 2018).
Felsenberg, J., Jacob, P. F., Walker, T., Barnstedt, O., Edmondson-Stait, A. J., Pleijzier, M. W., Otto, N., Schlegel, P., Sharifi, N., Perisse, E., Smith, C. S., Lauritzen, J. S., Costa, M., Jefferis, G., Bock, D. D. and Waddell, S. (2018). Integration of parallel opposing memories underlies memory extinction. Cell. PubMed ID: 30245010
Summary:
Accurately predicting an outcome requires that animals learn supporting and conflicting evidence from sequential experience. In mammals and invertebrates, learned fear responses can be suppressed by experiencing predictive cues without punishment, a process called memory extinction. This study shows that extinction of aversive memories in Drosophila requires specific dopaminergic neurons, which indicate that omission of punishment is remembered as a positive experience. Functional imaging revealed co-existence of intracellular calcium traces in different places in the mushroom body output neuron network for both the original aversive memory and a new appetitive extinction memory. Light and ultrastructural anatomy are consistent with parallel competing memories being combined within mushroom body output neurons that direct avoidance. Indeed, extinction-evoked plasticity in a pair of these neurons neutralizes the potentiated odor response imposed in the network by aversive learning. Therefore, flies track the accuracy of learned expectations by accumulating and integrating memories of conflicting events.

Tuesday, October 16th - Disease Models

Chandran, S., Suggs, J. A., Wang, B. J., Han, A., Bhide, S., Cryderman, D. E., Moore, S. A., Bernstein, S. I., Wallrath, L. L. and Melkani, G. C. (2018). Suppression of myopathic lamin mutations by muscle-specific activation of AMPK and modulation of downstream signaling. Hum Mol Genet. PubMed ID: 30239736
Summary:
Laminopathies are diseases caused by dominant mutations in the human LMNA gene encoding A-type lamins. Lamins are intermediate filaments that line the inner nuclear membrane, provide structural support for the nucleus, and regulate gene expression. Human disease-causing LMNA mutations were modeled in Drosophila Lamin C (LamC) and expressed in indirect flight muscle (IFM). IFM-specific expression of mutant, but not wild-type LamC, caused held-up wings indicative of myofibrillar defects. Analyses of the muscles revealed cytoplasmic aggregates of nuclear envelope (NE) proteins, nuclear and mitochondrial dysmorphology, myofibrillar disorganization, and up-regulation of the autophagy cargo receptor p62. It was hypothesized that the cytoplasmic aggregates of NE proteins trigger signaling pathways that alter cellular homeostasis, causing muscle dysfunction. In support of this hypothesis, transcriptomics data from human muscle biopsy tissue revealed misregulation of the AMPK/4E-BP1/autophagy/proteostatic pathways. S6K mRNA levels were increased and AMPKalpha and mRNAs encoding downstream targets were decreased in muscles expressing mutant LMNA relative controls. The Drosophila laminopathy models were used to determine if altering the levels of these factors modulated muscle pathology. Muscle-specific over-expression of AMPKalpha and down-stream targets 4E-BP, Foxo and PGC1alpha, as well as inhibition of S6K, suppressed the held-up wing phenotype, myofibrillar defects, and LamC aggregation. These findings provide novel insights on mutant LMNA-based disease mechanisms and identify potential targets for drug therapy.
Chakraborty, J., von Stockum, S., Marchesan, E., Caicci, F., Ferrari, V., Rakovic, A., Klein, C., Antonini, A., Bubacco, L. and Ziviani, E. (2018). USP14 inhibition corrects an in vivo model of impaired mitophagy. EMBO Mol Med. PubMed ID: 30249595
Summary:
Mitochondrial autophagy or mitophagy is a key process that allows selective sequestration and degradation of dysfunctional mitochondria to prevent excessive reactive oxygen species, and activation of cell death. Recent studies revealed that ubiquitin-proteasome complex activity and mitochondrial membrane rupture are key steps preceding mitophagy, in combination with the ubiquitination of specific outer mitochondrial membrane (OMM) proteins. The deubiquitinating enzyme ubiquitin-specific peptidase 14 (USP14) has been shown to modulate both proteasome activity and autophagy. This study reports that genetic and pharmacological inhibition of USP14 promotes mitophagy, which occurs in the absence of the well-characterised mediators of mitophagy, PINK1 and Parkin. Critical to USP14-induced mitophagy is the exposure of the LC3 receptor Prohibitin 2 by mitochondrial fragmentation and mitochondrial membrane rupture. Genetic or pharmacological inhibition of USP14 in vivo corrected mitochondrial dysfunction and locomotion behaviour of PINK1/Parkin mutant Drosophila model of Parkinson's disease, an age-related progressive neurodegenerative disorder that is correlated with diminished mitochondrial quality control. This study identifies a novel therapeutic target that ameliorates mitochondrial dysfunction and in vivo PD-related symptoms.
Wang, X., Guo, X., Ma, Y., Wu, C., Li, W. and Xue, L. (2018). APLP2 Modulates JNK-Dependent Cell Migration in Drosophila. Biomed Res Int 2018: 7469714. PubMed ID: 30155482
Summary:
Amyloid precursor-like protein 2 (APLP2) belongs to the APP family and is widely expressed in human cells. Though previous studies have suggested a role of APLP2 in cancer progression, the exact role of APLP2 in cell migration remains elusive. This report shows that ectopic expression of APLP2 in Drosophila induces cell migration which is mediated by JNK signaling, as loss of JNK suppresses while gain of JNK enhances such phenotype. APLP2 is able to activate JNK signaling by phosphorylation of JNK, which triggers the expression of matrix metalloproteinase MMP1 required for basement membranes degradation to promote cell migration. The data presented in this study unraveled an in vivo role of APLP2 in JNK-mediated cell migration.
Aron, R., Pellegrini, P., Green, E. W., Maddison, D. C., Opoku-Nsiah, K., Wong, J. S., Daub, A. C., Giorgini, F. and Finkbeiner, S. (2018). Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease. Nat Commun 9(1): 3191. PubMed ID: 30266909
Summary:
Huntington's disease is a progressive neurodegenerative disorder caused by polyglutamine-expanded mutant huntingtin (mHTT). This study shows that the deubiquitinase Usp12 rescues mHTT-mediated neurodegeneration in Huntington's disease rodent and patient-derived human neurons, and in Drosophila. The neuroprotective role of Usp12 may be specific amongst related deubiquitinases, as the closely related homolog Usp46 does not suppress mHTT-mediated toxicity. Mechanistically, this study identifies Usp12 as a potent inducer of neuronal autophagy. Usp12 overexpression accelerates autophagic flux and induces an approximately sixfold increase in autophagic structures as determined by ultrastructural analyses, while suppression of endogenous Usp12 slows autophagy. Surprisingly, the catalytic activity of Usp12 is not required to protect against neurodegeneration or induce autophagy. These findings identify the deubiquitinase Usp12 as a regulator of neuronal proteostasis and mHTT-mediated neurodegeneration.
Stapper, Z. A. and Jahn, T. R. (2018). Changes in glutathione redox potential are linked to Abeta42-induced neurotoxicity. Cell Rep 24(7): 1696-1703. PubMed ID: 30110626
Summary:
Glutathione is the major low-molecular weight thiol of eukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer's disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. This study combined genetically encoded redox sensors with a Drosophila models of amyloid-beta (Abeta) aggregation. Changes in glutathione redox potential (EGSH) closely correlate with disease onset and progression. This redox imbalance was found to be specifically in neurons, but not in glia cells. EGSH changes and Abeta42 deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Abeta42-mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology.
Ast, A., et al. (2018). mHTT seeding activity: A marker of disease progression and neurotoxicity in models of Huntington's disease. Mol Cell 71(5): 675-688.e676. PubMed ID: 30193095
Summary:
Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). This paper reports the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, the neurotoxicity of mHTT seeds was assessed in an inducible Drosophila model transgenic for HTTex1. A strong correlation was found between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.

Monday, October 15th - Transcriptional Regulation

Lim, B., Fukaya, T., Heist, T. and Levine, M. (2018). Temporal dynamics of pair-rule stripes in living Drosophila embryos. Proc Natl Acad Sci U S A 115(33): 8376-8381. PubMed ID: 30061421
Summary:
Traditional studies of gene regulation in the Drosophila embryo centered primarily on the analysis of fixed tissues. These methods provided considerable insight into the spatial control of gene activity, such as the borders of eve stripe 2, but yielded only limited information about temporal dynamics. The advent of quantitative live-imaging and genome-editing methods permits the detailed examination of the temporal control of endogenous gene activity. This study presents evidence that the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) undergo dynamic shifts in gene expression. Sequential anterior shifting of the stripes along the anterior to posterior axis was observed, with stripe 1 exhibiting movement before stripe 2 and the more posterior stripes. Conversely, posterior stripes shift over greater distances (two or three nuclei) than anterior stripes (one or two nuclei). Shifting of the ftz and eve stripes are slightly offset, with ftz moving faster than eve. This observation is consistent with previous genetic studies, suggesting that eve is epistatic to ftz. The precision of pair-rule temporal dynamics might depend on enhancer-enhancer interactions within the eve locus, since removal of the endogenous eve stripe 1 enhancer via CRISPR/Cas9 genome editing led to precocious and expanded expression of eve stripe 2. These observations raise the possibility of an added layer of complexity in the positional information encoded by the segmentation gene regulatory network.
Du, L., Zhou, A., Sohr, A. and Roy, S. (2018). An efficient strategy for generating tissue-specific binary transcription systems in Drosophila by genome editing. J Vis Exp(139). PubMed ID: 30295654
Summary:
Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. This study presents a method to generate highly tissue-specific targeted expression system by employing a CRISPR/Cas-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented in this study for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.
Newcomb, S., Voutev, R., Jory, A., Delker, R. K., Slattery, M. and Mann, R. S. (2018). cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster leg. PLoS Genet 14(8): e1007568. PubMed ID: 30142157
Summary:
This study characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE many additional leg enhancers were identified, suggesting that this logic is broadly used by many genes during Drosophila limb development.
Vincent, B. J., Staller, M. V., Lopez-Rivera, F., Bragdon, M. D. J., Pym, E. C. G., Biette, K. M., Wunderlich, Z., Harden, T. T., Estrada, J. and DePace, A. H. (2018). Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos. PLoS Genet 14(9): e1007644. PubMed ID: 30192762
Summary:
Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. This study investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. Evidence was also found that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. These results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. How counter-repression may influence eve stripe 2 regulation and evolution is discussed.
Wang, L. H. and Baker, N. E. (2018). Spatial regulation of expanded transcription in the Drosophila wing imaginal disc. PLoS One 13(7): e0201317. PubMed ID: 30063727
Summary:
Growth and patterning are coordinated during development to define organ size and shape. The growth, proliferation and differentiation of Drosophila wings are regulated by several conserved signaling pathways. This study shows that the Salvador-Warts-Hippo (SWH) and Notch pathways converge on an enhancer in the expanded (ex) gene, which also responds to levels of the bHLH transcription factor Daughterless (Da). Separate cis-regulatory elements respond to Salvador-Warts-Hippo (SWH) and Notch pathways, to bHLH proteins, and to unidentified factors that repress ex transcription in the wing pouch and in the proneural region at the anterior wing margin. Senseless, a zinc-finger transcription factor acting in proneural regions, had a negative impact on ex transcription in the proneural region, but the transcriptional repressor Hairy had no effect. This study suggests that a complex pattern of ex transcription results from integration of a uniform SWH signal with multiple other inputs, rather than from a pattern of SWH signaling.
Wester, J., Lima, C. A. C., Machado, M. C. R., Zampar, P. V., Tavares, S. S. and Monesi, N. (2018). Characterization of a novel Drosophila melanogaster cis-regulatory module that drives gene expression to the larval tracheal system and adult thoracic musculature. Genesis: e23222. PubMed ID: 30096221
Summary:
This study characterized a Drosophila melanogaster cis-regulatory module (CRM) termed TT-CRM. The TT-CRM is 646 bp long and is located in one of the introns of CG32239 (RhoGEF64C) and resides about 3,500 bp upstream of CG13711 and about 620 bp upstream of CG12493. Analysis of 646 bp-lacZ lines revealed that TT-CRM drives gene expression not only to the larval, prepupal, and pupal tracheal system but also to the adult dorsal longitudinal muscles. The patterns of mRNA expression of the transgene and of the CGs that lie in the vicinity of TT-CRM were investigated both in dissected trachea and in adult thoraces. Through RT-qPCR it was observed that in the tracheal system the pattern of expression of 646 bp-lacZ is similar to the pattern of expression of CG32239 and CG13711, whereas in the thoracic muscles 646 bp-lacZ expression accompanies the expression of CG12493. Together, these results suggest new functions for two previously characterized D. melanogaster genes and also contribute to the initial characterization of a novel CRM that drives a dynamic pattern of expression throughout development.

Friday, October 12 - Behavior

Waters, A. J., Capriotti, P., Gaboriau, D. C. A., Papathanos, P. A. and Windbichler, N. (2018). Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster. Sci Rep 8(1): 13125. PubMed ID: 30177778
Summary:
The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. This study examined the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. 13 single guide RNAs (sgRNAs) were directed to the promoters of 7 evolutionary conserved genes, and 11 drivers were used to conduct a misactivation screen. Dominant-lethal activators of the eve locus were identified, and it was found that they disrupt development by strongly activating eve outside its native spatio-temporal context. The same set of sgRNAs were used to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined it was found that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, these results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy presented in this study allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.
Thimgan, M. S., Kress, N., Lisse, J., Fiebelman, C. and Hilderbrand, T. (2018). The acyl-CoA synthetase, pudgy, promotes sleep and is required for the homeostatic response to sleep deprivation. Front Endocrinol (Lausanne) 9: 464. PubMed ID: 30186232
Summary:
The regulation of sleep and the response to sleep deprivation rely on multiple biochemical pathways. A critical connection is the link between sleep and metabolism. Metabolic changes can disrupt sleep, and conversely decreased sleep can alter the metabolic environment. There is building evidence that lipid metabolism, in particular, is a critical part of mounting the homeostatic response to sleep deprivation. This study evaluated an acyl-CoA synthetase, pudgy (pdgy), for its role in sleep and response to sleep deprivation. When pdgy transcript levels are decreased through transposable element disruption of the gene, mutant flies showed lower total sleep times and increased sleep fragmentation at night compared to genetic controls. Consistent with disrupted sleep, mutant flies had a decreased lifespan compared to controls. pdgy disrupted fatty acid handling as pdgy mutants showed increased sensitivity to starvation and exhibited lower fat stores. Moreover, the response to sleep deprivation is reduced when compared to a control flies. When the transcript levels for pdgy were decreased using RNAi, the response to sleep deprivation was decreased compared to background controls. In addition, when pdgy transcription is rescued throughout the fly, the response to sleep deprivation is restored. These data demonstrate that the regulation and function of acyl-CoA synthetase plays a critical role in regulating sleep and the response to sleep deprivation. Endocrine and metabolic signals that alter transcript levels of pdgy impact sleep regulation or interfere with the homeostatic response to sleep deprivation.
Troup, M., Yap, M. H., Rohrscheib, C., Grabowska, M. J., Ertekin, D., Randeniya, R., Kottler, B., Larkin, A., Munro, K., Shaw, P. J. and van Swinderen, B. (2018). Acute control of the sleep switch in Drosophila reveals a role for gap junctions in regulating behavioral responsiveness. Elife 7. PubMed ID: 30109983
Summary:
Sleep is a dynamic process in most animals, involving distinct stages that probably perform multiple functions for the brain. Before sleep functions can be initiated, it is likely that behavioral responsiveness to the outside world needs to be reduced, even while the animal is still awake. Recent work in Drosophila has uncovered a sleep switch in the dorsal fan-shaped body (dFB) of the fly's central brain, but it is not known whether these sleep-promoting neurons also govern the acute need to ignore salient stimuli in the environment during sleep transitions. This study found that optogenetic activation of the sleep switch suppressed behavioral responsiveness to mechanical stimuli, even in awake flies, indicating a broader role for these neurons in regulating arousal. The dFB-mediated suppression mechanism and its associated neural correlates requires innexin6 expression, suggesting that the acute need to reduce sensory perception when flies fall asleep is mediated in part by electrical synapses.
Zhang, W., Guo, C., Chen, D., Peng, Q. and Pan, Y. (2018). Hierarchical control of Drosophila sleep, courtship, and feeding behaviors by male-specific P1 neurons. Neurosci Bull. PubMed ID: 30182322
Summary:
Animals choose among sleep, courtship, and feeding behaviors based on the integration of both external sensory cues and internal states; such choices are essential for survival and reproduction. These competing behaviors are closely related and controlled by distinct neural circuits, but whether they are also regulated by shared neural nodes is unclear. This study investigated how a set of male-specific P1 neurons controls sleep, courtship, and feeding behaviors in Drosophila males. Mild activation of P1 neurons was sufficient to affect sleep, but not courtship or feeding, while stronger activation of P1 neurons labeled by four out of five independent drivers induced courtship, but only the driver that targeted the largest number of P1 neurons affected feeding. These results reveal a common neural node that affects sleep, courtship, and feeding in a threshold-dependent manner, and provide insights into how competing behaviors can be regulated by a shared neural node.
Wienecke, C. F. R., Leong, J. C. S. and Clandinin, T. R. (2018). Linear summation underlies direction selectivity in Drosophila. Neuron 99(4): 680-688.e684. PubMed ID: 30057202
Summary:
While linear mechanisms lay the foundations of feature selectivity in many brain areas, direction selectivity in the elementary motion detector (EMD) of the fly has become a paradigm of nonlinear neuronal computation. This study has bridged this divide by demonstrating that linear spatial summation can generate direction selectivity in the fruit fly Drosophila. Using linear systems analysis and two-photon imaging of a genetically encoded voltage indicator, the emergence was measured of direction-selective (DS) voltage signals in the Drosophila OFF pathway. This study is a direct, quantitative investigation of the algorithm underlying directional signals, with the striking finding that linear spatial summation is sufficient for the emergence of direction selectivity. A linear stage of the fly EMD strongly resembles similar computations in vertebrate visual cortex, demands a reappraisal of the role of upstream nonlinearities, and implicates the voltage-to-calcium transformation in the refinement of feature selectivity in this system.
Jaeger, A. H., Stanley, M., Weiss, Z. F., Musso, P. Y., Chan, R. C., Zhang, H., Feldman-Kiss, D. and Gordon, M. D. (2018). A complex peripheral code for salt taste in Drosophila. Elife 7. PubMed ID: 30307393
Summary:
Each taste modality is generally encoded by a single, molecularly defined, population of sensory cells. However, salt stimulates multiple taste pathways in mammals and insects, suggesting a more complex code for salt taste. This study examined salt coding in Drosophila. After creating a comprehensive molecular map comprised of five discrete sensory neuron classes across the fly labellum, four were found to be sactivated by salt: two exhibiting characteristics of 'low salt' cells, and two 'high salt' classes. Behaviorally, low salt attraction depends primarily on 'sweet' neurons, with additional input from neurons expressing the ionotropic receptor IR94e. High salt avoidance is mediated by 'bitter' neurons and a population of glutamatergic neurons expressing Ppk23. Interestingly, the impact of these glutamatergic neurons depends on prior salt consumption. These results support a complex model for salt coding in flies that combinatorially integrates inputs from across cell types to afford robust and flexible salt behaviors.

Thursday, October 11th - RNA

Zeng, J., Kamiyama, T., Niwa, R. and King-Jones, K. (2018). The Drosophila CCR4-NOT complex is required for cholesterol homeostasis and steroid hormone synthesis. Dev Biol. PubMed ID: 30149007
Summary:
CCR4-NOT is a highly conserved protein complex that regulates gene expression at multiple levels. In yeast, CCR4-NOT functions in transcriptional initiation, heterochromatin formation, mRNA deadenylation and other processes. The range of functions for Drosophila CCR4-NOT is less clear, except for a well-established role as a deadenylase for maternal mRNAs during early embryogenesis. This study report here that CCR4-NOT has an essential function in the Drosophila prothoracic gland (PG), a tissue that predominantly produces the steroid hormone ecdysone. Interfering with the expression of the CCR4-NOT components twin, Pop2, Not1, and Not3 in a PG-specific manner resulted in larval arrest and a failure to initiate metamorphosis. Transcriptome analysis of PG-specific Pop2-RNAi samples revealed that Pop2 is required for the normal expression of ecdysone biosynthetic gene spookier (spok) as well as cholesterol homeostasis genes of the NPC2 family. Interestingly, dietary supplementation with ecdysone and its various sterol precursors showed that 7-dehydrocholesterol and cholesterol completely rescued the larval arrest phenotype, allowing Pop2-RNAi animals to reach pupal stage, and, to a low degree, even survival to adulthood, while the biologically active hormone, 20-Hydroxyecdysone (20E), was significantly less effective. Also, genetic evidence is presented that CCR4-NOT has a nuclear function where CCR4-NOT-depleted cells exhibit aberrant chromatin and nucleoli structures. In summary, these findings indicate that the Drosophila CCR4-NOT complex has essential roles in the PG, where it is required for Drosophila steroid hormone production and cholesterol homeostasis, and likely has functions beyond a mere mRNA deadenylase in Drosophila.
Kordyukova, M., Morgunova, V., Olovnikov, I., Komarov, P. A., Mironova, A., Olenkina, O. M. and Kalmykova, A. (2018). Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis. PLoS One 13(8): e0201787. PubMed ID: 30157274
Summary:
The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. The biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development was studied with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, this study showed that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, it was demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, it is suggested that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants.
Kaschula, R., Pinho, S. and Alonso, C. R. (2018). microRNA-dependent regulation of Hox gene expression sculpts fine-grain morphological patterns in a Drosophila appendage. Development. PubMed ID: 30143542
Summary:
Disruptions of normal Hox gene expression can lead to severe morphological defects revealing a link between the regulation of Hox expression and pattern formation. This study explored these links focusing on the impact of microRNA regulation on the expression of the Drosophila Hox gene Ultrabithorax (Ubx) during haltere development. Through the combination of bioinformatic and transcriptomic analyses the miR-310/313 cluster (miR-310C) as a candidate regulator of Ubx. Several experiments confirm this. First, miR-310C and Ubx protein show complementary expression patterns in haltere imaginal discs; second, artificial activation of miR-310C expression in haltere discs leads to Ubx-like phenotypes. Third, expression of a fluorescent reporter bearing Ubx 3'UTR sequences is reduced when co-expressed with miR-310C. Fourth, deletion of miR-310C leads to Ubx upregulation and changes the array of mechanosensory sensilla at the base of the haltere. Fifth, artificial increase of Ubx levels within the miR-310C expression domain phenocopies the mechanosensory defects observed in miR-310C mutants. It is proposed that miR-310C-mediated repression delimits Ubx fine-grain expression contributing to the sculpting of complex morphologies in the Drosophila haltere. This work reveals a novel role of microRNA regulation in the control of Hox gene expression with impact on morphology.
Theron, E., Maupetit-Mehouas, S., Pouchin, P., Baudet, L., Brasset, E. and Vaury, C. (2018). The interplay between the Argonaute proteins Piwi and Aub within Drosophila germarium is critical for oogenesis, piRNA biogenesis and TE silencing. Nucleic Acids Res. PubMed ID: 30113668
Summary:
Transposable elements (TEs) have invaded most genomes and constitute up to 50% of the human genome. Machinery based on small non-coding piRNAs has evolved to inhibit their expression at the transcriptional and post-transcriptional levels. Surprisingly, this machinery is weakened during specific windows of time in mice, flies or plants, allowing the expression of TEs in germline cells. The function of this de-repression remains unknown. In Drosophila, it has been shown that this developmental window is characterized by a reduction of Piwi expression in dividing germ cells. This study shows that the unique knock-down of Aub in these cells leads to female sterility. It correlates with defects in piRNA amplification, an increased Piwi expression and an increased silencing of transcriptionally silenced TEs. These defects are similar to those observed when Aub is depleted in the whole germline which underlies the crucial role of this developmental window for both oogenesis and TE silencing. This study further showed that, with age, some fertility is recovered which is concomitant to a decrease of Piwi and TE silencing. These data pinpoint the Pilp (Piwi-less pocket - a region of the oocyte lacking Piei) as a tremendously important step for female fertility and genome stability. They further show that such a restricted developmental niche of germ cells may sense environmental changes, such as aging, to protect the germline all along the life.
Tikhonov, M., Utkina, M., Maksimenko, O. and Georgiev, P. (2018). Conserved sequences in the Drosophila mod(mdg4) intron promote poly(A)-independent transcription termination and trans-splicing. Nucleic Acids Res. PubMed ID: 30102331
Summary:
Alternative splicing (AS) is a regulatory mechanism of gene expression that greatly expands the coding capacities of genomes by allowing the generation of multiple mRNAs from a single gene. In Drosophila, the mod(mdg4) locus is an extreme example of AS that produces more than 30 different mRNAs via trans-splicing that joins together the common exons and the 3' variable exons generated from alternative promoters. To map the regions required for trans-splicing, this study has developed an assay for measuring trans-splicing events and identified a 73-bp region in the last common intron that is critical for trans-splicing of three pre-mRNAs synthesized from different DNA strands. It was have also found that conserved sequences in the distal part of the last common intron induce polyadenylation-independent transcription termination and are enriched by paused RNA polymerase II (RNAP II). These results suggest that all mod(mdg4) mRNAs are formed by joining in trans the 5' splice site in the last common exon with the 3' splice site in one of the alternative exons.
Lin, C. J., Hu, F., Dubruille, R., Vedanayagam, J., Wen, J., Smibert, P., Loppin, B. and Lai, E. C. (2018). The hpRNA/RNAi pathway is essential to resolve intragenomic conflict in the Drosophila male germline. Dev Cell 46(3): 316-326.e315. PubMed ID: 30086302
Summary:
Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. This study reveals that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, D. simulans null mutants of Dcr-2 and AGO2 were generated. Strikingly, these core RNAi knockouts massively derepress Dox and MDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, these data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species.

Wednesday, October 10th - Adult Neural Development and Function

Mishra, D., Thorne, N., Miyamoto, C., Jagge, C. and Amrein, H. (2018). The taste of ribonucleosides: Novel macronutrients essential for larval growth are sensed by Drosophila gustatory receptor proteins. PLoS Biol 16(8): e2005570. PubMed ID: 30086130
Summary:
Animals employ various types of taste receptors to identify and discriminate between different nutritious food chemicals. These macronutrients are thought to fall into 3 major groups: carbohydrates/sugars, proteins/amino acids, and fats. This study reports that Drosophila larvae exhibit a novel appetitive feeding behavior towards ribose, ribonucleosides, and RNA. Members of the gustatory receptor (Gr) subfamily 28 (Gr28), expressed in both external and internal chemosensory neurons were identified as molecular receptors necessary for cellular and appetitive behavioral responses to ribonucleosides and RNA. Specifically, behavioral preference assays show that larvae are strongly attracted to ribose- or RNA-containing agarose in a Gr28-dependent manner. Moreover, Ca2+ imaging experiments reveal that Gr28a-expressing taste neurons are activated by ribose, RNA and some ribonucleosides and that these responses can be conveyed to Gr43aGAL4 fructose-sensing neurons by expressing single members of the Gr28 gene family. Lastly, a critical role in behavioral fitness for the Gr28 genes was established by showing that Gr28 mutant larvae exhibit low survival rates when challenged to find ribonucleosides in food. Together, this work identifies a novel taste modality dedicated to the detection of RNA and ribonucleosides, nutrients that are essential for survival during the accelerated growth phase of Drosophila larvae.
Pu, Y., Zhang, Y., Zhang, Y. and Shen, P. (2018). Two Drosophila Neuropeptide Y-like neurons define a reward module for transforming appetitive odor representations to motivation. Sci Rep 8(1): 11658. PubMed ID: 30076343
Summary:
Neuropeptides, many of which are conserved among vertebrate and invertebrate animals, are implicated in the regulation of motivational states that selectively facilitate goal-directed behaviors. After a brief presentation of appetitive odors, Drosophila larvae display an impulsive-like feeding activity in readily accessible palatable food. This innate appetitive response may require coordinated signaling activities of dopamine (DA) and neuropeptide F (NPF; a fly homolog of neuropeptide Y). This study provides anatomical and functional evidence, at single-cell resolution, that two NPF neurons define a reward module in the highest-order brain region for cognitive processing of food-related olfactory representations. First, laser lesioning of these NPF neurons abolished odor induction of appetitive arousal, while their genetic activation mimicked the behavioral effect of appetitive odors. Further, a circuit analysis shows that each of the two NPF neurons relays its signals to a subset of target neurons in the larval hindbrain-like region. Finally, the NPF neurons discriminatively responded to appetitive odor stimuli, and their odor responses were blocked by targeted lesioning of a pair of dopaminergic third-order olfactory neurons that appear to be presynaptic to the NPF neurons. Therefore, the two NPF neurons contribute to appetitive odor induction of impulsive-like feeding by selectively decoding DA-encoded ascending olfactory inputs and relaying NPF-encoded descending motivational outputs for behavioral execution.
Otto, N., Marelja, Z., Schoofs, A., Kranenburg, H., Bittern, J., Yildirim, K., Berh, D., Bethke, M., Thomas, S., Rode, S., Risse, B., Jiang, X., Pankratz, M., Leimkuhler, S. and Klambt, C. (2018). The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia. Nat Commun 9(1): 3514. PubMed ID: 30158546
Summary:
Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, a screen was carried out for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper (Sulfite oxidase) encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, the current findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity.
Wolff, T. and Rubin, G. M. (2018). Neuroarchitecture of the Drosophila central complex: A catalog of nodulus and asymmetrical body neurons and a revision of the protocerebral bridge catalog. J Comp Neurol. PubMed ID: 30084503
Summary:
The central complex, a set of neuropils in the center of the insect brain, plays a crucial role in spatial aspects of sensory integration and motor control. Stereotyped neurons interconnect these neuropils with one another and with accessory structures. Over 5000 Drosophila melanogaster GAL4 lines were screened for expression in two neuropils, the noduli (NO) of the central complex and the asymmetrical body (AB), and multicolor stochastic labelling was used to analyze the morphology, polarity and organization of individual cells in a subset of the GAL4 lines that showed expression in these neuropils. Nine NO and three AB cell types were identified and are described in this study. The morphology of the NO neurons suggests that they receive input primarily in the lateral accessory lobe and send output to each of the six paired noduli. The AB is demonstrated to be a bilateral structure which exhibits asymmetry in size between the left and right bodies. The AB neurons are shown to directly connect the AB to the central complex and accessory neuropils, that they target both the left and right ABs, and that one cell type preferentially innervates the right AB. It is proposed that the AB be considered a central complex neuropil in Drosophila. Finally, highly restricted GAL4 lines are presented for most identified protocerebral bridge, NO and AB cell types. These lines, generated using the split-GAL4 method, will facilitate anatomical studies, behavioral assays, and physiological experiments.
Sheng, C., Javed, U., Gibbs, M., Long, C., Yin, J., Qin, B. and Yuan, Q. (2018). Experience-dependent structural plasticity targets dynamic filopodia in regulating dendrite maturation and synaptogenesis. Nat Commun 9(1): 3362. PubMed ID: 30135566
Summary:
Highly motile dendritic protrusions are hallmarks of developing neurons. These exploratory filopodia sample the environment and initiate contacts with potential synaptic partners. To understand the role for dynamic filopodia in dendrite morphogenesis and experience-dependent structural plasticity, this study analyzed dendrite dynamics, synapse formation, and dendrite volume expansion in developing ventral lateral neurons (LNvs) of the Drosophila larval visual circuit. The findings reveal the temporal coordination between heightened dendrite dynamics with synaptogenesis in LNvs and illustrate the strong influence imposed by sensory experience on the prevalence of dendritic filopodia, which regulate the formation of synapses and the expansion of dendritic arbors. Using genetic analyses, Amphiphysin (Amph), a BAR (Bin/Amphiphysin/Rvs) domain-containing protein, was identified as a required component for tuning the dynamic state of LNv dendrites and promoting dendrite maturation. Taken together, this study establishes dynamic filopodia as the key cellular target for experience-dependent regulation of dendrite development.
Ravi, P., Trivedi, D. and Hasan, G. (2018). FMRFa receptor stimulated Ca2+ signals alter the activity of flight modulating central dopaminergic neurons in Drosophila melanogaster. PLoS Genet 14(8): e1007459. PubMed ID: 30110323
Summary:
Neuropeptide signaling influences animal behavior by modulating neuronal activity and thus altering circuit dynamics. Insect flight is a key innate behavior that very likely requires robust neuromodulation. Cellular and molecular components that help modulate flight behavior are therefore of interest and require investigation. In a genetic RNAi screen for G-protein coupled receptors that regulate flight bout durations, several receptors have been identified, including the receptor for the neuropeptide FMRFa (FMRFaR). To further investigate modulation of insect flight by FMRFa CRISPR-Cas9 mutants were generated in the gene encoding the Drosophila FMRFaR. The mutants exhibit significant flight deficits with a focus in dopaminergic cells. Expression of a receptor specific RNAi in adult central dopaminergic neurons resulted in progressive loss of sustained flight. Further, genetic and cellular assays demonstrated that FMRFaR stimulates intracellular calcium signaling through the IP3R and helps maintain neuronal excitability in a subset of dopaminergic neurons for positive modulation of flight bout durations.

Tuesday, September 9th - Signaling

Rizzo, M. J., Evans, J. P., Burt, M., Saunders, C. J. and Johnson, E. C. (2018). Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking. Biochem Biophys Res Commun 503(3): 1919-1926. PubMed ID: 30064912
Summary:
G protein-coupled receptors are the largest superfamily of cell surface receptors in the Metazoa and play critical roles in transducing extracellular signals into intracellular responses. This action is mediated through conformational changes in the receptor following ligand binding. A number of conserved motifs have critical roles in GPCR function, and this study focused on a highly conserved motif (WxFG) in extracellular loop one (EL1). A phylogenetic analysis documents the presence of the WxFG motif in approximately 90% of Class A GPCRs and the motif is represented in 17 of the 19 Class A GPCR subfamilies. Using site-directed mutagenesis, the conserved tryptophan residue was mutagenized in eight receptors which are members of disparate class A GPCR subfamilies from different taxa. The modification of the Drosophila leucokinin receptor shows that substitution of any non-aromatic amino acid for the tryptophan leads to a loss of receptor function. Additionally, leucine substitutions at this position caused similar signaling defects in the follicle-stimulating hormone receptor (FSHR), Galanin receptor (GALR1), AKH receptor (AKHR), corazonin receptor (CRZR), and muscarinic acetylcholine receptor (mACHR1). Visualization of modified receptors through the incorporation of a fluorescent tag revealed a severe reduction in plasma membrane expression, indicating aberrant trafficking of these modified receptors. Taken together, these results suggest a novel role for the WxFG motif in GPCR trafficking and receptor function.
Patrnogic, J., Heryanto, C. and Eleftherianos, I. (2018). Transcriptional up-regulation of the TGF-beta intracellular signaling transducer Mad of Drosophila larvae in response to parasitic nematode infection. Innate Immun: 1753425918790663. PubMed ID: 30049242
Summary:
The common fruit fly Drosophila melanogaster is an exceptional model for dissecting innate immunity. However, knowledge on responses to parasitic nematode infections still lags behind. Recent studies have demonstrated that the well-conserved TGF-beta signaling pathway participates in immune processes of the fly, including the anti-nematode response. To elucidate the molecular basis of TGF-beta anti-nematode activity, a transcript level analysis of different TGF-beta signaling components was performed following infection of D. melanogaster larvae with the nematode parasite Heterorhabditis gerrardi. No significant changes were found in the transcript level of most extracellular ligands in both bone morphogenic protein (BMP) and activin branches of the TGF-beta signaling pathway between nematode-infected larvae and uninfected controls. However, extracellular ligand, Scw, and Type I receptor, Sax, in the BMP pathway as well as the Type I receptor, Babo, in the activin pathway were substantially up-regulated following H. gerrardi infection. The results suggest that receptor up-regulation leads to transcriptional up-regulation of the intracellular component Mad in response to H. gerrardi following changes in gene expression of intracellular receptors of both TGF-beta signaling branches. These findings identify the involvement of certain TGF-beta signaling pathway components in the immune signal transduction of D. melanogaster larvae against parasitic nematodes.
Casal, J., Ibanez-Jimenez, B. and Lawrence, P. A. (2018). Planar cell polarity: the prickle gene acts independently on both the Ds/Ft and the Stan/Fz systems. Development. PubMed ID: 30154173
Summary:
Epithelial cells are polarised within the plane of the epithelium, forming oriented structures whose coordinated and consistent polarity (planar cell polarity, PCP) relates to the principal axes of the body or organ. In Drosophila at least two separate molecular systems generate and interpret intercellular polarity signals: Dachsous/Fat, and the "core" or Stan/Fz system. This study examined the prickle gene and its protein products Prickle and Spiny leg. Much research on PCP has focused on the asymmetric localisation of core proteins in the cell and as a result prickle was placed in the heart of the Stan/Fz system. This study asked if this view is correct and how the prickle gene relates to the two systems. This study found that prickle can affect, separately, both systems - however, neither Pk nor Sple are essential components of the Ds/Ft or the Stan/Fz system, nor do they act as a functional link between the two systems.
Rode, S., Ohm, H., Anhauser, L., Wagner, M., Rosing, M., Deng, X., Sin, O., Leidel, S. A., Storkebaum, E., Rentmeister, A., Zhu, S. and Rumpf, S. (2018). Differential requirement for translation initiation factor pathways during ecdysone-dependent neuronal remodeling in Drosophila. Cell Rep 24(9): 2287-2299. PubMed ID: 30157424
Summary:
Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, the requirements for translation initiation factors during dendrite pruning were assessed. The canonical cap-binding complex eIF4F was found to be dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. It is proposed that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes.
Sapar, M. L., Ji, H., Wang, B., Poe, A. R., Dubey, K., Ren, X., Ni, J. Q. and Han, C. (2018). Phosphatidylserine externalization results from and causes neurite degeneration in Drosophila. Cell Rep 24(9): 2273-2286. PubMed ID: 30157423
Summary:
Phagocytic clearance of degenerating dendrites or axons is critical for maintaining tissue homeostasis and preventing neuroinflammation. Externalized phosphatidylserine (PS) has been postulated to be an "eat-me" signal allowing recognition of degenerating neurites by phagocytes. This study shows that in Drosophila, PS is dynamically exposed on degenerating dendrites during developmental pruning and after physical injury, but PS exposure is suppressed when dendrite degeneration is genetically blocked. Ectopic PS exposure via phospholipid flippase knockout and scramblase overexpression induced PS exposure preferentially at distal dendrites and caused distinct modes of neurite loss that differ in larval sensory dendrites and in adult olfactory axons. Surprisingly, extracellular lactadherin that lacks the integrin-interaction domain induced phagocyte-dependent degeneration of PS-exposing dendrites, revealing an unidentified bridging function that potentiates phagocytes. These findings establish a direct causal relationship between PS exposure and neurite degeneration in vivo.
Sawant, K., Chen, Y., Kotian, N., Preuss, K. M. and McDonald, J. A. (2018). Rap1 GTPase promotes coordinated collective cell migration in vivo. Mol Biol Cell: mbcE17120752. PubMed ID: 30156466
Summary:
During development and in cancer, cells often move together in small to large collectives. In order to move as a unit, cells within collectives need to stay coupled together and coordinate their motility. How cell collectives remain interconnected and migratory, especially when moving through in vivo environments, is not well understood. The genetically tractable border cell group undergoes a highly polarized and cohesive cluster-type migration in the Drosophila ovary. This study reports that the small GTPase Rap1, through activation by PDZ-GEF, regulates border cell collective migration. Rap1 maintains cell contacts within the cluster, at least in part by promoting the organized distribution of E-cadherin at specific cell-cell junctions. Rap1 also restricts migratory protrusions to the front of the border cell cluster and promotes the extension of protrusions with normal dynamics. Further, Rap1 is required in the outer migratory border cells but not in the central non-migratory polar cells. Such cell specificity correlates well with the spatial distribution of the inhibitory Rapgap1 protein, which is higher in polar cells than in border cells. It is proposed that precisely regulated Rap1 activity reinforces connections between cells and polarizes the cluster, thus facilitating the coordinated collective migration of border cells.

Monday, October 8th - Behavior

Yang, Y. and Edery, I. (2018). Parallel clinal variation in the mid-day siesta of Drosophila melanogaster implicates continent-specific targets of natural selection. PLoS Genet 14(9): e1007612. PubMed ID: 30180162
Summary:
Similar to many diurnal animals, Drosophila melanogaster exhibits a mid-day siesta that is more robust as ambient temperature rises, an adaptive response aimed at minimizing exposure to heat. Mid-day siesta levels are partly regulated by the thermosensitive splicing of a small intron (termed dmpi8) found in the 3' untranslated region (UTR) of the circadian clock gene period (per). Using the well-studied D. melanogaster latitudinal cline along the eastern coast of Australia, this study showed that flies from temperate populations sleep less during the day compared to those from tropical regions. Combinations of four single nucleotide polymorphisms (SNPs) were identified in the 3' UTR of per that yield several different haplotypes. The two most abundant of these haplotypes exhibit a reciprocal tropical-temperate distribution in relative frequency. Intriguingly, transgenic flies with the major tropical isoform manifest increased daytime sleep and reduced dmpi8 splicing compared to those carrying the temperate variant. These results strongly suggest that for a major portion of D. melanogaster in Australia, thermal adaptation of daily sleep behavior included spatially varying selection on ancestrally derived polymorphisms in the per 3' UTR that differentially control dmpi8 splicing efficiency. Prior work showed that African flies from high altitudes manifest reduced mid-day siesta levels, indicative of parallel latitudinal and altitudinal adaptation across continents. However, geographical variation in per 3' UTR haplotypes was not observed for African flies, providing a compelling case for inter-continental variation in factors targeted by natural selection in attaining a parallel adaptation. It is proposed that the ability to calibrate mid-day siesta levels to better match local temperature ranges is a key adaptation contributing to the successful colonization of D. melanogaster beyond its ancestral range in the lowlands of Sub-Saharan Africa.
Mercier, D., Tsuchimoto, Y., Ohta, K. and Kazama, H. (2018). Olfactory landmark-based communication in interacting Drosophila. Curr Biol 28(16): 2624-2631.e2625. PubMed ID: 30078566
Summary:
To communicate with conspecifics, animals deploy various strategies to release pheromones, chemical signals modulating social and sexual behaviors. Importantly, a single pheromone induces different behaviors depending on the context and exposure dynamics. Therefore, to comprehend the ethological role of pheromones, it is essential to characterize how neurons in the recipients respond to temporally and spatially fluctuating chemical signals emitted by donors during natural interactions. In Drosophila melanogaster, the male pheromone 11-cis-vaccenyl acetate (cVA) activates specific olfactory receptor neurons (ORNs) to regulate diverse social and sexual behaviors in recipients. Physicochemical analyses have identified this chemical on an animal's body and in its local environment. However, because these methods are imprecise in capturing spatiotemporal dynamics, it is poorly understood how individual pheromone cues are released, detected, and interpreted by recipients. This study developed a system based on bioluminescence to monitor neural activity in freely interacting Drosophila, and investigated the active detection and perception of the naturally emitted cVA. Unexpectedly, neurons specifically tuned to cVA did not exhibit significant activity during physical interactions between males, and instead responded strongly to olfactory landmarks deposited by males. These landmarks mediated attraction through Or67d receptors and allured both sexes to the marked region. Importantly, the landmarks remained attractive even when a pair of flies was engaged in courtship behavior. In contrast, female deposits did not affect the exploration pattern of either sex. Thus, Drosophila use pheromone marking to remotely signal their sexual identity and to enhance social interactions.
Morley, E. L., Jonsson, T. and Robert, D. (2018). Auditory sensitivity, spatial dynamics, and amplitude of courtship song in Drosophila melanogaster. J Acoust Soc Am 144(2): 734. PubMed ID: 30180716
Summary:
Acoustic communication is an important component of courtship in Drosophila melanogaster. It takes the form of courtship song produced by males through the unilateral extension and vibration of a wing. Following the paradigm of sender-receiver matching, song content is assumed to match tuning in the auditory system, however, D. melanogaster audition is nonlinear and tuning dependent upon signal amplitude. At low stimulus amplitudes or in the absence of sound the antenna is tuned into song frequency, but as amplitude increases the antenna's resonance is shifted up by hundreds of Hertz. Accurate measurements of song amplitude have been elusive because of the strong dependency of amplitude upon the spatial geometry between sender and receiver. In this study, D. melanogaster auditory directional sensitivity and the geometric position between the courting flies are quantified. It is shown that singing occurs primarily from positions resulting in direct stimulation of the female antenna. Using this information, it is established that the majority of song is louder than theoretically predicted and at these sound levels the female antenna should not amplify or be tuned into song. The study implies that Drosophila hearing, and, in particular, its active mechanisms, could function in a broader context than previously surmised.
Park, A., Tran, T. and Atkinson, N. S. (2018). Monitoring food preference in Drosophila by oligonucleotide tagging. Proc Natl Acad Sci U S A 115(36): 9020-9025. PubMed ID: 30127010
Summary:
Drosophila melanogaster is a powerful model organism for dissecting the neurogenetic basis of appetitive and aversive behaviors. However, some methods used to assay food preference require or cause starvation. This can be problematic for fly ethanol research because it can be difficult to dissociate caloric preference for ethanol from pharmacological preference for the drug. BARCODE, a starvation-independent assay that uses trace levels of oligonucleotide tags was designed to differentially mark food types. In BARCODE, flies feed ad libitum, and relative food preference is monitored by qPCR of the oligonucleotides. Persistence of the ingested oligomers within the fly records the feeding history of the fly over several days. Using BARCODE, this study identified a sexually dimorphic preference for ethanol. Females are attracted to ethanol-laden foods, whereas males avoid consuming it. Furthermore, genetically feminizing male mushroom body lobes induces preference for ethanol. In addition, it was demonstrated that BARCODE can be used for multiplex diet measurements when animals are presented with more than two food choices.
Liu, G., Nath, T., Linneweber, G. A., Claeys, A., Guo, Z., Li, J., Bengochea, M., De Backer, S., Weyn, B., Sneyders, M., Nicasy, H., Yu, P., Scheunders, P. and Hassan, B. A. (2018). A simple computer vision pipeline reveals the effects of isolation on social interaction dynamics in Drosophila. PLoS Comput Biol 14(8): e1006410. PubMed ID: 30161262
Summary:
Isolation profoundly influences social behavior in all animals. Longer-term analysis of large groups of flies is hampered by the lack of effective and reliable tools. In this study a new imaging arena was built and the existing tracking algorithm was improved to reliably follow a large number of flies simultaneously. Next, based on the automatic classification of touch and graph-based social network analysis, an algorithm was designed to quantify changes in the social network in response to prior social isolation. It was observed that isolation significantly and swiftly enhanced individual and local social network parameters depicting near-neighbor relationships. The genome-wide molecular correlates of these behavioral changes were explored, and it was found that whereas behavior changed throughout the six days of isolation, gene expression alterations occurred largely on day one. These changes occurred mostly in metabolic genes, and the metabolic changes were varified by showing an increase of lipid content in isolated flies. In summary, this study describes a highly reliable tracking and analysis pipeline for large groups of flies that were use to unravel the behavioral, molecular and physiological impact of isolation on social network dynamics in Drosophila.
Luo, W., Guo, F., McMahon, A., Couvertier, S., Jin, H., Diaz, M., Fieldsend, A., Weerapana, E. and Rosbash, M. (2018). NonA and CPX link the circadian clockwork to locomotor activity in Drosophila. Neuron 99(4): 768-780.e763. PubMed ID: 30057203
Summary:
Drosophila NonA and its mammalian ortholog NONO are members of the Drosophila behavior and human splicing (DBHS) family. NONO also has a strong circadian connection: it associates with the circadian repressor protein Period (Per) and contributes to circadian timekeeping. This study investigated NonA, which is required for proper levels of evening locomotor activity as well as a normal free-running period in Drosophila. NonA is associated with the positive transcription factor Clock/Cycle (Clk/Cyc), interacts directly with complexin (cpx) pre-mRNA, and upregulates gene expression, including the gene cpx. Downregulation of cpx expression in circadian neurons phenocopies NonA downregulation, whereas cpx overexpression rescues the nonA RNAi phenotypes, indicating that cpx is an important NonA target gene. As the cpx protein contributes to proper neurotransmitter and neuropeptide release in response to calcium, these results and others indicate that this control is important for the normal circadian regulation of locomotor activity.
Li, C., Dong, H. and Zhao, K. (2018). A balance between aerodynamic and olfactory performance during flight in Drosophila. Nat Commun 9(1): 3215. PubMed ID: 30097572
Summary:
The ability to track odor plumes to their source (food, mate, etc.) is key to the survival of many insects. During this odor-guided navigation, flapping wings could actively draw odorants to the antennae to enhance olfactory sensitivity, but it is unclear if improving olfactory function comes at a cost to aerodynamic performance. This study computationally quantified the odor plume features around a fruit fly in forward flight and confirmed that the antenna is well positioned to receive a significant increase of odor mass flux (peak 1.8 times), induced by wing flapping, vertically from below the body but not horizontally. This anisotropic odor spatial sampling may have important implications for behavior and the algorithm during plume tracking. Further analysis also suggests that, because both aerodynamic and olfactory functions are indispensable during odor-guided navigation, the wing shape and size may be a balance between the two functions.
Mayseless, O., Berns, D. S., Yu, X. M., Riemensperger, T., Fiala, A. and Schuldiner, O. (2018). Developmental coordination during olfactory circuit remodeling in Drosophila. Neuron. PubMed ID: 30146303
Summary:
Developmental neuronal remodeling is crucial for proper wiring of the adult nervous system. While remodeling of individual neuronal populations has been studied, how neuronal circuits remodel-and whether remodeling of synaptic partners is coordinated-is unknown. This study found that the Drosophila anterior paired lateral (APL) neuron undergoes stereotypic remodeling during metamorphosis in a similar time frame as the mushroom body (MB) -neurons, with whom it forms a functional circuit. By simultaneously manipulating both neuronal populations, this study found that cell-autonomous inhibition of -neuron pruning resulted in the inhibition of APL pruning in a process that is mediated, at least in part, by Ca(2+)-Calmodulin and neuronal activity dependent interaction. Finally, ectopic unpruned MB axons display ectopic connections with the APL, as well as with other neurons, at the adult, suggesting that inhibiting remodeling of one neuronal type can affect the functional wiring of the entire micro-circuit.

Friday, October 5th - Signaling

Mazzotta, G. M., Bellanda, M., Minervini, G., Damulewicz, M., Cusumano, P., Aufiero, S., Stefani, M., Zambelli, B., Mammi, S., Costa, R. and Tosatto, S. C. E. (2018). Calmodulin enhances Cryptochrome binding to INAD in Drosophila photoreceptors. Front Mol Neurosci 11: 280. PubMed ID: 30177872
Summary:
Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila Cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. Previous work has shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. This study predicts and demonstrates a novel Ca(2+)-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses this study to identified and characterized a CaM binding motif in the dCRY C-terminus. Similarly, the CaM binding site of the scaffold protein INAD was also detailed, and it was demonstrated that CaM bridges dCRY and INAD to form a ternary complex in vivo. These results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM.
Li, B., Wong, C., Gao, S. M., Zhang, R., Sun, R., Li, Y. and Song, Y. (2018). The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating notch receptor trafficking. Elife 7. PubMed ID: 30176986
Summary:
The correct establishment and maintenance of unidirectional Notch signaling are critical for the homeostasis of various stem cell lineages. However, the molecular mechanisms that prevent cell-autonomous ectopic Notch signaling activation and deleterious cell fate decisions remain unclear. This study shows that the retromer complex directly and specifically regulates Notch receptor retrograde trafficking in Drosophila neuroblast lineages to ensure the unidirectional Notch signaling from neural progenitors to neuroblasts. Notch polyubiquitination mediated by E3 ubiquitin ligase Itch/Su(dx) is inherently inefficient within neural progenitors, relying on retromer-mediated trafficking to avoid aberrant endosomal accumulation of Notch and cell-autonomous signaling activation. Upon retromer dysfunction, hypo-ubiquitinated Notch accumulates in Rab7(+) enlarged endosomes, where it is ectopically processed and activated in a ligand-dependent manner, causing progenitor-originated tumorigenesis. These results therefore unveil a safeguard mechanism whereby retromer retrieves potentially harmful Notch receptors in a timely manner to prevent aberrant Notch activation-induced neural progenitor dedifferentiation and brain tumor formation.
Ji, S., Samara, N. L., Revoredo, L., Zhang, L., Tran, D. T., Muirhead, K., Tabak, L. A. and Ten Hagen, K. G. (2018). A molecular switch orchestrates enzyme specificity and secretory granule morphology. Nat Commun 9(1): 3508. PubMed ID: 30158631
Summary:
Regulated secretion is an essential process where molecules destined for export are directed to membranous secretory granules, where they undergo packaging and maturation. This study identified a gene (pgant9) that influences the structure and shape of secretory granules within the Drosophila salivary gland. Loss of pgant9, which encodes an O-glycosyltransferase, results in secretory granules with an irregular, shard-like morphology, and altered glycosylation of cargo. Interestingly, pgant9 undergoes a splicing event that acts as a molecular switch to alter the charge of a loop controlling access to the active site of the enzyme. The splice variant with the negatively charged loop glycosylates the positively charged secretory cargo and rescues secretory granule morphology. This study highlights a mechanism for dictating substrate specificity within the O-glycosyltransferase enzyme family. Moreover, these in vitro and in vivo studies suggest that the glycosylation status of secretory cargo influences the morphology of maturing secretory granules.
Jiang, Y., Seimiya, M., Schlumpf, T. B. and Paro, R. (2018). An intrinsic tumour eviction mechanism in Drosophila mediated by steroid hormone signalling. Nat Commun 9(1): 3293. PubMed ID: 30120247
Summary:
Polycomb group proteins are epigenetic regulators maintaining transcriptional memory during cellular proliferation. In Drosophila larvae, malfunction of Polyhomeotic (Ph), a member of the PRC1 silencing complex, results in neoplastic growth. This study reports an intrinsic tumour suppression mechanism mediated by the steroid hormone ecdysone during metamorphosis. Ecdysone alters neoplastic growth into a nontumorigenic state of the mutant ph cells which then become eliminated during adult stage. This study demonstrates that ecdysone exerts this function by inducing a heterochronic network encompassing the activation of the microRNA lethal-7, which suppresses its target gene chronologically inappropriate morphogenesis. This pathway can also promote remission of brain tumours formed in brain tumour mutants, revealing a restraining of neoplastic growth in different tumour types. Given the conserved role of let-7, the identification and molecular characterization of this innate tumour eviction mechanism in flies might provide important clues towards the exploitation of related pathways for human tumour therapy.
Lee, C. H., Kiparaki, M., Blanco, J., Folgado, V., Ji, Z., Kumar, A., Rimesso, G. and Baker, N. E. (2018). A regulatory response to ribosomal protein mutations controls translation, growth, and cell competition. Dev Cell 46(4): 456-469. PubMed ID: 30078730
Summary:
Ribosomes perform protein synthesis but are also involved in signaling processes, the full extent of which are still being uncovered. This study reports that phenotypes of mutating ribosomal proteins (Rps) are largely due to signaling. Using Drosophila, this study discovered that a bZip-domain protein, Xrp1, becomes elevated in Rp mutant cells. Xrp1 reduces translation and growth, delays development, is responsible for gene expression changes, and causes the cell competition of Rp heterozygous cells from genetic mosaics. Without Xrp1, even cells homozygously deleted for Rp genes persist and grow. Xrp1 induction in Rp mutant cells depends on a particular Rp with regulatory effects, RpS12, and precedes overall changes in translation. Thus, effects of Rp mutations, even the reductions in translation and growth, depend on signaling through the Xrp1 pathway and are not simply consequences of reduced ribosome production limiting protein synthesis. One benefit of this system may be to eliminate Rp-mutant cells by cell competition.
Potdar, S. and Sheeba, V. (2018). Wakefulness is promoted during day time by PDFR signalling to dopaminergic neurons in Drosophila melanogaster. eNeuro 5(4). PubMed ID: 30131970
Summary:
Circadian clocks modulate timing of sleep/wake cycles in animals; however, the underlying mechanisms remain poorly understood. In Drosophila melanogaster, large ventral lateral neurons (l-LNv) are known to promote wakefulness through the action of the neuropeptide pigment dispersing factor (PDF), but the downstream targets of PDF signalling remain elusive. In a screen using downregulation or overexpression (OEX) of the gene encoding PDF receptor (pdfr), this study found that a subset of dopaminergic neurons responds to PDF to promote wakefulness during the day. Moreover, this study found that small LNv (s-LNv) and dopaminergic neurons form synaptic contacts, and PDFR signalling inhibited dopaminergic neurons specifically during day time. It is proposed that these dopaminergic neurons that respond to PDFR signalling are sleep-promoting and that during the day when PDF levels are high, they are inhibited, thereby promoting wakefulness. Thus, this study has identified a novel circadian clock pathway that mediates wake promotion specifically during day time.

Thursday, October 4th - Adult Neural Development and Function

Feng, G., Zhang, J., Li, M., Shao, L., Yang, L., Song, Q. and Ping, Y. (2018). Control of sleep onset by Shal/Kv4 channels in Drosophila circadian neurons. J Neurosci. PubMed ID: 30185460
Summary:
Sleep is highly conserved across animal species. Both wake- and sleep-promoting neurons are implicated in the regulation of wake-sleep transition at dusk in Drosophila However, little is known about how they cooperate and whether they act via different mechanisms. This study demonstrated that in female Drosophila, sleep onset was specifically delayed by blocking the Shaker cognate L channels (Shal, also known as voltage-gated K(+) channel 4, Kv4) in wake-promoting cells, including large ventral lateral neurons (l-LNvs) and pars intercerebralis (PI), but not in sleep-promoting dorsal neurons (DN1s). Delayed sleep onset was also observed in males by blocking Kv4 activity in wake-promoting neurons. Electrophysiological recordings show that Kv4 channels contribute A-type currents (IA) in LNvs and PI cells, but are much less conspicuous in DN1s. Interestingly, blocking Kv4 in wake-promoting neurons preferentially increased firing rates at dusk around ZT13, when the resting membrane potentials (RMPs) and firing rates were at lower levels. Furthermore, pigment-dispersing factor (PDF) is essential for the regulation of sleep onset by Kv4 in l-LNvs, and downregulation of PDF receptor (PDFR) in PI neurons advanced sleep onset, indicating Kv4 controls sleep onset via regulating PDF/PDFR signaling in wake-promoting neurons. It is proposed that Kv4 acts as a sleep onset controller by suppressing membrane excitability in a clock-dependent manner to balance the wake-sleep transition at dusk. These results have important implications for the understanding and treatment of sleep disorders such as insomnia.
Jones, S. G., Nixon, K. C. J., Chubak, M. C. and Kramer, J. M. (2018). Mushroom body specific transcriptome analysis reveals dynamic regulation of learning and memory genes after acquisition of long-term courtship memory in Drosophila. G3 (Bethesda). PubMed ID: 30158319
Summary:
The formation and recall of long-term memory (LTM) requires neuron activity-induced gene expression. The complex spatial and temporal dynamics of memory formation creates significant challenges in defining memory-relevant gene expression changes. The Drosophila mushroom body (MB) is a signaling hub in the insect brain that integrates sensory information to form memories across several different experimental memory paradigms. This study performed transcriptome analysis in the MB at two time points after the acquisition of LTM: 1 hour and 24 hours. The MB transcriptome was compared to biologically paired whole head (WH) transcriptomes. In both, more transcript level changes were identified at 1 hour after memory acquisition (WH = 322, MB = 302) than at 24 hours (WH = 23, MB = 20). WH samples showed downregulation of developmental genes and upregulation of sensory response genes. In contrast, MB samples showed vastly different changes in transcripts involved in biological processes that are specifically related to LTM. MB-downregulated genes were highly enriched for metabolic function. MB-upregulated genes were highly enriched for known learning and memory processes, including calcium-mediated neurotransmitter release and cAMP signalling. The neuron activity inducible genes Hr38 and sr were also specifically induced in the MB. These results highlight the importance of sampling time and cell type in capturing biologically relevant transcript level changes involved in learning and memory. The data suggests that MB cells transiently upregulate known memory-related pathways after memory acquisition.
Franconville, R., Beron, C. and Jayaraman, V. (2018). Building a functional connectome of the Drosophila central complex. Elife 7. PubMed ID: 30124430
Summary:
The central complex is a highly conserved insect brain region composed of morphologically stereotyped neurons that arborize in distinctively shaped substructures. The region is implicated in a wide range of behaviors and several modeling studies have explored its circuit computations. Most studies have relied on assumptions about connectivity between neurons based on their overlap in light microscopy images. This study presents an extensive functional connectome of Drosophila melanogaster's central complex at cell-type resolution. Using simultaneous optogenetic stimulation, calcium imaging and pharmacology, the connectivity was tested between 70 presynaptic-to-postsynaptic cell-type pairs. Numerous inputs to the central complex were identified, but only a small number of output channels. Additionally, the connectivity of this highly recurrent circuit appears to be sparser than anticipated from light microscopy images. Finally, the connectivity matrix highlights the potentially critical role of a class of bottleneck interneurons. All data is provided for interactive exploration on a website.
Kinold, J. C., Pfarr, C. and Aberle, H. (2018). Sidestep-induced neuromuscular miswiring causes severe locomotion defects in Drosophila larvae. Development 145(17). PubMed ID: 30166331
Summary:
Mutations in motor axon guidance molecules cause aberrant projection patterns of motor nerves. As most studies in Drosophila have analysed these molecules in fixed embryos, the consequences for larval locomotion are entirely unexplored. This study took advantage of sidestep (side)-mutant larvae that display severe locomotion defects because of irreparable innervation errors. Mutations in side affected all motor nerve branches and all body wall regions. Innervation defects were non-stereotypical, showing unique innervation patterns in each hemisegment. Premature activation of Side in muscle precursors abrogated dorsal migration of motor nerves, resulting in larvae with a complete loss of neuromuscular junctions on dorsal-most muscles. High-speed videography showed that these larvae failed to maintain substrate contact and inappropriately raised both head and tail segments above the substrate, resulting in unique 'arching' and 'lifting' phenotypes. These results show that guidance errors in side mutants are maintained throughout larval life and are asymmetrical with respect to the bilateral body axis. Together with similar findings in mice, this study also suggests that miswiring could be an underlying cause of inherited movement disorders.
Li, J., Guajardo, R., Xu, C., Wu, B., Li, H., Li, T., Luginbuhl, D. J., Xie, X. and Luo, L. (2018). Stepwise wiring of the Drosophila olfactory map requires specific Plexin B levels. Elife 7. PubMed ID: 30136927
Summary:
The precise assembly of a neural circuit involves many consecutive steps. The conflict between a limited number of wiring molecules and the complexity of the neural network impels each molecule to execute multiple functions at different steps. This study examined the cell-type specific distribution of endogenous levels of axon guidance receptor Plexin B (PlexB) in the developing antennal lobe, the first olfactory processing center in Drosophila. Different classes of olfactory receptor neurons (ORNs) express PlexB at different levels in two wiring steps - axonal trajectory choice and subsequent target selection. In line with its temporally distinct patterns, the proper levels of PlexB control both steps in succession. Genetic interactions further revealed that the effect of high-level PlexB is antagonized by its canonical partner Sema2b. Thus, PlexB plays a multifaceted role in instructing the assembly of the Drosophila olfactory circuit through temporally-regulated expression patterns and expression level-dependent effects.
Loewen, C., Boekhoff-Falk, G., Ganetzky, B. and Chtarbanova, S. (2018). A novel mutation in brain tumor causes both neural over-proliferation and neurodegeneration in adult Drosophila. G3 (Bethesda). PubMed ID: 30126833
Summary:
A screen for neuroprotective genes in Drosophila melanogaster led to the identification of a mutation that causes extreme, progressive loss of adult brain neuropil in conjunction with massive brain overgrowth. The mutation was mapped to the brain tumor (brat) locus, which encodes a tripartite motif-NCL-1, HT2A, and LIN-41 (TRIM-NHL) RNA-binding protein with established roles limiting stem cell proliferation in developing brain and ovary. However, a neuroprotective role for brat in the adult Drosophila brain has not been described previously. The new allele, bratcheesehead(bratchs), carries a mutation in the coiled-coil domain of the TRIM motif, and is temperature-sensitive. mRNA and protein levels of neural stem cell genes are increased in heads of adult bratchs mutants; the over-proliferation phenotype initiates prior to adult eclosion. It is also reported that disruption of an uncharacterized gene coding for a presumptive prolyl-4-hydroxylase strongly enhances the over-proliferation and neurodegeneration phenotypes. Together, these results reveal an unexpected role for brat that could be relevant to human cancer and neurodegenerative diseases.

Wednesday, October 3rd - Chromatin

Hundertmark, T., Gartner, S. M. K., Rathke, C. and Renkawitz-Pohl, R. (2018). Nejire/dCBP-mediated histone H3 acetylation during spermatogenesis is essential for male fertility in Drosophila melanogaster. PLoS One 13(9): e0203622. PubMed ID: 30192860
Summary:
Spermatogenesis in many species including Drosophila melanogaster is accompanied by major reorganisation of chromatin in post-meiotic stages, involving a nearly genome-wide displacement of histones by protamines, Mst77F and Protamine-like 99C. A proposed prerequisite for the histone-to-protamine transition is massive histone H4 hyper-acetylation prior to the switch. This study investigated the pattern of histone H3 lysine acetylation and general lysine crotonylation in D. melanogaster spermiogenesis to elucidate a possible role of these marks in chromatin reorganisation. Lysine crotonylation was strongest prior to remodelling and the deposition of this mark depended on the acetylation status of the spermatid chromatin. In contrast to H4 acetylation, individual H3 acetylation marks displayed surprisingly distinct patterns during the histone-to-protamine transition. Nejire, a histone acetyl transferase, is expressed during the time of histone-to-protamine transition. Nejire knock down led to strongly reduced fertility, which correlated with misshaped spermatid nuclei and a lack of mature sperm. protA and prtl99C transcript levels were reduced after knocking down Nejire. ProtB-eGFP, Mst77F-eGFP and Prtl99C-eGFP were synthesized at the late canoe stage, while histones were often not detectable. However, in some cysts histones persist in parallel to protamines. Therefore, it was hypothesized that complete histone removal requires multiple histone modifications besides H3K18ac and H3K27ac. In summary, H3K18 and H3K27 acetylation during Drosophila spermatogenesis is dependent on Nejire or a yet uncharacterized acetyl transferase. Nejire is required for male fertility since Nejire contributes to efficient transcription of protA and prtl99C, but not Mst77F, in spermatocytes, and to maturation of sperm.
Deutschman, E., Ward, J. R., Ho, A. L. K. T., Alban, T. J., Zhang, D., Willard, B., Lemieux, M. E., Lathia, J. D. and Longworth, M. S. (2018). Comparing and contrasting the effects of Drosophila Condensin II subunit dCAP-D3 overexpression and depletion in vivo. Genetics. PubMed ID: 30068527
Summary:
The Condensin II complex plays important, conserved roles in genome organization throughout the cell cycle and in the regulation of gene expression. Previous studies have linked decreased Condensin II subunit expression with a variety of diseases. This study shows that elevated levels of Condensin II subunits are detected in somatic cancers. To evaluate potential biological effects of elevated Condensin II levels, the Condensin II subunit, dCAP-D3, was overexpressed in Drosophila melanogaster larval tissues and the effects were examined on the mitotic and interphase specific functions of Condensin II. Interestingly, while ubiquitous overexpression resulted in pupal lethality, tissue specific overexpression of dCAP-D3 caused formation of nucleoplasmic protein aggregates which slowed mitotic prophase progression, mimicking results observed when dCAP-D3 levels are depleted. Surprisingly, dCAP-D3 aggregate formation resulted in faster transitions from metaphase to anaphase. Overexpressed dCAP-D3 protein failed to precipitate other Condensin II subunits in non-dividing tissues, but did cause changes to gene expression which occurred in a manner opposite of what was observed when dCAP-D3 levels were depleted in both dividing and non-dividing tissues. These findings show that altering dCAP-D3 levels in either direction has detrimental effects on mitotic timing, the regulation of gene expression, and organism development. Taken together, these data suggest that the different roles for Condensin II throughout the cell cycle may be independent of each other and/or that dCAP-D3 may possess functions that are separate from those involving its association with the Condensin II complex. If conserved, these findings could have implications for tumors harboring elevated CAP-D3 levels.
Hua, B. L., Bell, G. W., Kashevsky, H., Von Stetina, J. R. and Orr-Weaver, T. L. (2018). Dynamic changes in ORC localization and replication fork progression during tissue differentiation. BMC Genomics 19(1): 623. PubMed ID: 30134926
Summary:
Genomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role. This study exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, sites of ORC binding were found to show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited. These studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation.
Fedotova, A., Aoki, T., Rossier, M., Mishra, R., Clendine, C., Kyrchanova, O., Wolle, D., Bonchuk, A., Maeda, R., Mutero, A., Cleard, F., Mogila, V., Karch, F., Georgiev, P. and Schedl, P. (2018). The BEN domain protein insensitive binds to the Fab-7 chromatin boundary to establish proper segmental identity in Drosophila. Genetics. PubMed ID: 30082280
Summary:
Boundaries (insulators) in the Drosophila bithorax complex (BX-C) delimit autonomous regulatory domains that orchestrate the parasegment (PS)-specific expression of the BX-C homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains, which control Abd-B expression in PS11 and PS12, respectively. This boundary is composed of multiple functionally redundant elements and has two key functions: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass. Tnis study shows that two BEN domain protein complexes, Insensitive and Elba, bind to multiple sequences located in the Fab-7 nuclease hypersensitive regions. Two of these sequences are recognized by both Insv and Elba and correspond to a CCAATTGG palindrome. Elba also binds to a related CCAATAAG sequence, while Insv does not. However, the third Insv recognition sequences is ~100 bp in length and contains the CCAATAAG sequence at one end. Both Insv and Elba are assembled into large complexes (~420 kD and ~265-290 kD, respectively) in nuclear extracts. Using a sensitized genetic background this study showed that the Insv protein is required for Fab-7 boundary function, and that PS11 identity is not properly established in insv mutants. This is the first demonstration that a BEN domain protein is important for the functioning of an endogenous fly boundary.
Mahajan, S., Wei, K. H., Nalley, M. J., Gibilisco, L. and Bachtrog, D. (2018). De novo assembly of a young Drosophila Y chromosome using single-molecule sequencing and chromatin conformation capture. PLoS Biol 16(7): e2006348. PubMed ID: 30059545
Summary:
While short-read sequencing technology has resulted in a sharp increase in the number of species with genome assemblies, these assemblies are typically highly fragmented. Repeats pose the largest challenge for reference genome assembly, and pericentromeric regions and the repeat-rich Y chromosome are typically ignored from sequencing projects. This study assemble the genome of Drosophila miranda using long reads for contig formation, chromatin interaction maps for scaffolding and short reads, and optical mapping and bacterial artificial chromosome (BAC) clone sequencing for consensus validation. Tne assembly recovers entire chromosomes and contains large fractions of repetitive DNA, including about 41.5 Mb of pericentromeric and telomeric regions, and >100 Mb of the recently formed highly repetitive neo-Y chromosome. While Y chromosome evolution is typically characterized by global sequence loss and shrinkage, the neo-Y increased in size by almost 3-fold because of the accumulation of repetitive sequences. This high-quality assembly allows reconstruction og the chromosomal events that have led to the unusual sex chromosome karyotype in D. miranda, including the independent de novo formation of a pair of sex chromosomes at two distinct time points, or the reversion of a former Y chromosome to an autosome.
Guio, L., Vieira, C. and Gonzalez, J. (2018). Stress affects the epigenetic marks added by natural transposable element insertions in Drosophila melanogaster. Sci Rep 8(1): 12197. PubMed ID: 30111890
Summary:
Transposable elements are emerging as an important source of cis-acting regulatory sequences and epigenetic marks that could influence gene expression. However, few studies have dissected the role of specific transposable element insertions on epigenetic gene regulation. Bari-Jheh is a natural transposon that mediates resistance to oxidative stress by adding cis-regulatory sequences that affect expression of nearby genes. This work, integrated publicly available ChIP-seq and piRNA data with chromatin immunoprecipitation experiments to get a more comprehensive picture of Bari-Jheh molecular effects. Bari-Jheh was shown to be enriched for H3K9me3 in nonstress conditions, and for H3K9me3, H3K4me3 and H3K27me3 in oxidative stress conditions, which is consistent with expression changes in adjacent genes. It was further shown that under oxidative stress conditions, H3K4me3 and H3K9me3 spread to the promoter region of Jheh1 gene. Finally, another insertion of the Bari1 family was associated with increased H3K27me3 in oxidative stress conditions suggesting that Bari1 histone marks are copy-specific. It is concluded that besides adding cis-regulatory sequences, Bari-Jheh influences gene expression by affecting the local chromatin state.

Tuesday, October 2nd - Signaling

Alpar, L., Bergantinos, C. and Johnston, L. A. (2018). Spatially restricted regulation of Spatzle/Toll signaling during cell competition. Dev Cell. PubMed ID: 30146479
Summary:
Cell competition employs comparisons of fitness to selectively eliminate cells sensed as less healthy. In Drosophila, apoptotic elimination of the weaker "loser" cells from growing wing discs is induced by a signaling module consisting of the Toll ligand Spatzle (Spz), several Toll-related receptors, and NF-kappaB factors. How this module is activated and restricted to competing disc cells is unknown. This study used Myc-induced cell competition to demonstrate that loser cell elimination requires local wing disc synthesis of Spz. Spz processing enzyme (SPE) and modular serine protease (ModSP) are identified as activators of Spz-regulated competitive signaling, and "winner" cells are shown to trigger elimination of nearby WT cells by boosting SPE production. Moreover, Spz requires both Toll and Toll-8 to induce apoptosis of wing disc cells. Thus, during cell competition, Spz-mediated signaling is strictly confined to the imaginal disc, allowing errors in tissue fitness to be corrected without compromising organismal physiology.
Cohen, E., Allen, S. R., Sawyer, J. K. and Fox, D. T. (2018). Fizzy-related dictates a cell cycle switch during organ repair and tissue growth responses in the Drosophila hindgut. Elife 7. PubMed ID: 30117808
Summary:
Ploidy-increasing cell cycles drive tissue growth in many developing organs. Such cycles, including endocycles, are increasingly appreciated to drive tissue growth following injury or activated growth signaling in mature organs. In these organs, the regulation and distinct roles of different cell cycles remains unclear. This study uncovered a programmed switch between cell cycles in the Drosophila hindgut pylorus. Using an acute injury model, mitosis was identified as the response in larval pyloric cells, whereas endocycles occur in adult pyloric cells. By developing a novel genetic method, DEMISE (Dual-Expression-Method-for-Induced-Site-specific-Eradication), it was shown that the cell cycle regulator Fizzy-related dictates the decision between mitosis and endocycles. After injury, both cycles accurately restore tissue mass and genome content. However, in response to sustained growth signaling, only endocycles preserve epithelial architecture. These data reveal distinct cell cycle programming in response to similar stimuli in mature vs. developmental states and reveal a tissue-protective role of endocycles.
Hou, X., Burstein, S. R. and Long, S. B. (2018).. Structures reveal opening of the store-operated calcium channel Orai. Elife 7. PubMed ID: 30160233
Summary:
The store-operated calcium (Ca(2+)) channel Orai governs Ca(2+) influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca(2+) stored in the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Previous work revealed the structure of Orai with a closed pore. In this study, using a gain-of-function mutation that constitutively activates the channel, an X-ray structure is presented of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca(2+) selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, a sequence is proposed for store-operated activation.
Peters, K. A., Detmar, E., Sepulveda, L., Del Valle, C., Valsquier, R., Ritz, A., Rogers, S. L. and Applewhite, D. A. (2018). A cell-based assay to investigate non-muscle Myosin II contractility via the Folded-gastrulation signaling pathway in Drosophila S2R+ cells. J Vis Exp(138). PubMed ID: 30176023
Summary:
A cell-based assay has been developed using Drosophila cells that recapitulates apical constriction initiated by Folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a Galpha12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

Monday, October 1st - RNA

Hirashima, T., Tanaka, R., Yamaguchi, M. and Yoshida, H. (2018). The ABD on the nascent polypeptide and PH domain are required for the precise Anillin localization in Drosophila syncytial blastoderm. Sci Rep 8(1): 12910. PubMed ID: 30150713
Summary:
Targeting proteins to regions where they are required is essential for proper development of organisms. For achievement of this, subcellular mRNA localization is one of the critical mechanisms. Subcellular mRNA localization is an evolutionarily conserved phenomenon from E. coli to human and contributes to limiting the regions at which its products function and efficiently supplies substrates for protein translation. During early Drosophila embryogenesis, while 71% of the 3370 mRNAs analyzed have shown prominent subcellular localization, the underlying molecular mechanisms have not been elucidated. This study revealed that anillin mRNA, one of the localized mRNAs in early Drosophila embryo, localizes to the tip of the pseudo-cleavage furrow in the Drosophila syncytial blastoderm using in situ hybridization combined with immunohistochemistry. Localization analyses with transgenic fly lines carrying a series of deletion mRNAs indicate that this localization is dependent on its own nascent polypeptides including the actin binding domain (ABD). In addition to the mRNA localization, it is revealed that the pleckstrin homology (PH) domain of Anillin protein is also required for its proper localization. Thus, this study indicates that the precise localization of Anillin protein is tightly regulated by the ABD on the nascent polypeptide and PH domain in the Drosophila syncytial blastoderm.
Monedero Cobeta, I., Stadler, C. B., Li, J., Yu, P., Thor, S. and Benito-Sipos, J. (2018). Specification of Drosophila neuropeptidergic neurons by the splicing component brr2. PLoS Genet 14(8): e1007496. PubMed ID: 30133436
Summary:
During embryonic development, a number of genetic cues act to generate neuronal diversity. While intrinsic transcriptional cascades are well-known to control neuronal sub-type cell fate, the target cells can also provide critical input to specific neuronal cell fates. Such signals, denoted retrograde signals, are known to provide critical survival cues for neurons, but have also been found to trigger terminal differentiation of neurons. One salient example of such target-derived instructive signals pertains to the specification of the Drosophila FMRFamide neuropeptide neurons, the Tv4 neurons of the ventral nerve cord. Tv4 neurons receive a BMP signal from their target cells, which acts as the final trigger to activate the FMRFa gene. A recent FMRFa-eGFP genetic screen identified several genes involved in Tv4 specification, two of which encode components of the U5 subunit of the spliceosome: brr2 (l(3)72Ab) and Prp8. This study focused on the role of RNA processing during target-derived signaling. brr2 and Prp8 were found to play crucial roles in controlling the expression of the FMRFa neuropeptide specifically in six neurons of the VNC (Tv4 neurons). Detailed analysis of brr2 revealed that this control is executed by two independent mechanisms, both of which are required for the activation of the BMP retrograde signaling pathway in Tv4 neurons: (1) Proper axonal pathfinding to the target tissue in order to receive the BMP ligand. (2) Proper RNA splicing of two genes in the BMP pathway: the thickveins (tkv) gene, encoding a BMP receptor subunit, and the
Burow, D. A., Martin, S., Quail, J. F., Alhusaini, N., Coller, J. and Cleary, M. D. (2018). Attenuated codon optimality contributes to neural-specific mRNA decay in Drosophila. Cell Rep 24(7): 1704-1712. PubMed ID: 30110627
Summary:
Tissue-specific mRNA stability is important for cell fate and physiology, but the mechanisms involved are not fully understood. This study found that zygotic mRNA stability in Drosophila correlates with codon content: optimal codons are enriched in stable transcripts associated with metabolic functions like translation, while non-optimal codons are enriched in unstable transcripts, including those associated with neural development. Bioinformatic analyses and reporter assays revealed that similar codons stabilize or destabilize mRNAs in the nervous system and other tissues, but the link between codon content and stability is attenuated in the nervous system. This study confirmed that optimal codons are decoded by abundant tRNAs while non-optimal codons are decoded by less abundant tRNAs in embryos and in the nervous system. It is concluded that codon optimality is a general determinant of zygotic mRNA stability, and attenuation of codon optimality allows trans-acting factors to exert greater influence over mRNA decay in the nervous system.
Dong, H., Zhu, M., Meng, L., Ding, Y., Yang, D., Zhang, S., Qiang, W., Fisher, D. W. and Xu, E. Y. (2018). Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice. Oncotarget 9(63): 32134-32148. PubMed ID: 30181804
Summary:
PUMILIO 2 (PUM2) is a member of Pumilio and FBF (PUF) family, an RNA binding protein family with phylogenetically conserved roles in germ cell development. The Drosophila Pumilio homolog is also required for dendrite morphogenesis and synaptic function via translational control of synaptic proteins, such as glutamate receptors. Now PUM2 influences synaptic function in vivo and, subsequently, seizures is not known. This study found that PUM2 is highly expressed in the brain, especially in the temporal lobe, and knockout of Pum2 (Pum2(-/-) ) resulted in significantly increased pyramidal cell dendrite spine and synapse density. In addition, multiple proteins associated with excitatory synaptic function, including glutamate receptor 2 (GLUR2), are up-regulated in Pum2(-/-) mice. The expression of GLUR2 protein but not mRNA is increased in the Pum2(-/-) mutant hippocampus, Glur2 transcripts are increased in mutant polysome fractions, and overexpression of PUM2 led to repression of reporter expression containing the 3'Untranslated Region (3'UTR) of Glur2, suggesting translation of GLUR2 was increased in the absence of Pum2. Overall, these studies provide a molecular mechanism for the increased temporal lobe excitability observed with PUM2 loss and suggest PUM2 might contribute to intractable temporal lobe epilepsy.
Volin, M., Zohar-Fux, M., Gonen, O., Porat-Kuperstein, L. and Toledano, H. (2018). microRNAs selectively protect hub cells of the germline stem cell niche from apoptosis. J Cell Biol. PubMed ID: 30093492
Summary:
Genotoxic stress such as irradiation causes a temporary halt in tissue regeneration. The ability to regain regeneration depends on the type of cells that survived the assault. Previous studies showed that this propensity is usually held by the tissue-specific stem cells. However, stem cells cannot maintain their unique properties without the support of their surrounding niche cells. This study shows that exposure of Drosophila melanogaster to extremely high levels of irradiation temporarily arrests spermatogenesis and kills half of the stem cells. In marked contrast, the hub cells that constitute a major component of the niche remain completely intact. It was further shown that this atypical resistance to cell death relies on the expression of certain antiapoptotic microRNAs (miRNAs) that are selectively expressed in the hub and keep the cells inert to apoptotic stress signals. It is proposed that at the tissue level, protection of a specific group of niche cells from apoptosis underlies ongoing stem cell turnover and tissue regeneration.
Pai, A. A., Paggi, J. M., Yan, P., Adelman, K. and Burge, C. B. (2018). Numerous recursive sites contribute to accuracy of splicing in long introns in flies. PLoS Genet 14(8): e1007588. PubMed ID: 30148878
Summary:
Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates that are inherently unstable, making it difficult to study. This study developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. A subset of recursive sites were validated by RT-PCR and sequencing. Recursive sites occur in most very long (> 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, it was observed that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns.
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