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Current papers in developmental biology and gene function


Tuesday July 31st, 2018 - Vesicles and Synapses

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Richhariya, S., Jayakumar, S., Kumar Sukumar, S. and Hasan, G. (2018). dSTIM- and Ral/Exocyst-Mediated Synaptic Release from Pupal Dopaminergic Neurons Sustains Drosophila Flight. eNeuro 5(3). PubMed ID: 29938216
Manifestation of appropriate behavior in adult animals requires developmental mechanisms that help in the formation of correctly wired neural circuits. Flight circuit development in Drosophila requires store-operated calcium entry (SOCE) through the STIM/Orai pathway. SOCE-associated flight deficits in adult Drosophila derive extensively from regulation of gene expression in pupal neurons, and one such SOCE-regulated gene encodes the small GTPase Ral. The cellular mechanism by which Ral helps in maturation of the flight circuit was not understood. This study shows that knockdown of components of a Ral effector, the exocyst complex, in pupal neurons also leads to reduced flight bout durations, and this phenotype derives primarily from dopaminergic neurons. Importantly, synaptic release from pupal dopaminergic neurons is abrogated upon knockdown of dSTIM, Ral, or exocyst components. Ral overexpression restores the diminished synaptic release of dStim knockdown neurons as well as flight deficits associated with dSTIM knockdown in dopaminergic neurons. These results identify Ral-mediated vesicular release as an effector mechanism of neuronal SOCE in pupal dopaminergic neurons with functional consequences on flight behavior.
Ros, O., et al (2018). A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse. PLoS Genet 14(6): e1007432. PubMed ID: 29912942
Axonal growth and guidance rely on correct growth cone responses to guidance cues. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover. Recent studies indicate that whereas axonal attraction requires exocytosis, chemorepulsion relies on endocytosis. Indeed, studies have shown that Netrin-1/Deleted in Colorectal Cancer (DCC) signaling triggers exocytosis through the SNARE Syntaxin-1 (STX1). However, limited in vivo evidence is available about the role of SNARE proteins in axonal guidance. To address this issue, this study systematically deleted SNARE genes in three species. Loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly, chick, and mouse embryos. Inactivation of VAMP2, Ti-VAMP, and SNAP25 led to additional abnormalities in axonal guidance. It was also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1. Finally, genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways. These data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo, by regulating both pre- and post-commissural guidance mechanisms.
Akbergenova, Y., Cunningham, K. L., Zhang, Y. V., Weiss, S. and Littleton, J. T. (2018). Characterization of developmental and molecular factors underlying release heterogeneity at Drosophila synapses. Elife 7. PubMed ID: 29989549
Neurons communicate through neurotransmitter release at specialized synaptic regions known as active zones (AZs). Using biosensors to visualize single synaptic vesicle fusion events at Drosophila neuromuscular junctions, this study analyzed the developmental and molecular determinants of release probability (Pr) for a defined connection with ~300 AZs. Pr was heterogeneous but represented a stable feature of each AZ. Pr remained stable during high frequency stimulation and retained heterogeneity in mutants lacking the Ca(2+) sensor Synaptotagmin 1. Pr correlated with both presynaptic Ca(2+) channel abundance and Ca(2+) influx at individual release sites. Pr heterogeneity also correlated with glutamate receptor abundance, with high Pr connections developing receptor subtype segregation. Intravital imaging throughout development revealed that AZs acquire high Pr during a multi-day maturation period, with Pr heterogeneity largely reflecting AZ age. The rate of synapse maturation was activity-dependent, as both increases and decreases in neuronal activity modulated glutamate receptor field size and segregation.
Bellec, K., Gicquel, I. and Le Borgne, R. (2018). Stratum recruits Rab8 at Golgi exit sites to regulate the basolateral sorting of Notch and Sanpodo. Development 145(13). PubMed ID: 29967125
In Drosophila, the sensory organ precursor (SOP or pI cell) divides asymmetrically to give birth to daughter cells, the fates of which are governed by the differential activation of the Notch pathway. Proteolytic activation of Notch induced by ligand is based on the correct polarized sorting and localization of the Notch ligand Delta, the Notch receptor and its trafficking partner Sanpodo (Spdo). This study has identified Stratum (Strat), a presumptive guanine nucleotide exchange factor for Rab GTPases, as a regulator of Notch activation. Loss of Strat causes cell fate transformations associated with an accumulation of Notch, Delta and Spdo in the trans-Golgi network (TGN), and an apical accumulation of Spdo. The strat mutant phenotype is rescued by the catalytically active as well as the wild-type form of Rab8, suggesting a chaperone function for Strat rather than that of exchange factor. Strat is required to localize Rab8 at the TGN, and rab8 phenocopies strat. It is proposed that Strat and Rab8 act at the exit of the Golgi apparatus to regulate the sorting and the polarized distribution of Notch, Delta and Spdo.

Monday, July 30th - Gonadogenesis

Armstrong, A. R. and Drummond-Barbosa, D. (2018). Insulin signaling acts in adult adipocytes via GSK-3beta and independently of FOXO to control Drosophila female germline stem cell numbers. Dev Biol. PubMed ID: 29729259
Tissue-specific stem cells are tied to the nutritional and physiological environment of adult organisms. Adipocytes have key endocrine and nutrient-sensing roles and have emerged as major players in relaying dietary information to regulate other organs. For example, previous studies in Drosophila melanogaster revealed that amino acid sensing as well as diet-dependent metabolic pathways function in adipocytes to influence the maintenance of female germline stem cells (GSCs). How nutrient-sensing pathways acting within adipocytes influence adult stem cell lineages, however, is just beginning to be elucidated. This study reports that insulin/insulin-like growth factor signaling in adipocytes promotes GSC maintenance, early germline cyst survival, and vitellogenesis. Further, adipocytes use distinct mechanisms downstream of insulin receptor activation to control these aspects of oogenesis, all of which are independent of FOXO. GSC maintenance is modulated by Akt1 through GSK-3beta, early germline cyst survival is downstream of adipocyte Akt1 but independent of GSK-3beta, and vitellogenesis is regulated through an Akt1-independent pathway in adipocytes. These results indicate that, in addition to employing different types of nutrient sensing, adipocytes can use distinct axes of a single nutrient-sensing pathway to regulate multiple stages of the GSC lineage in the ovary.
Albert, E. A., Puretskaia, O. A., Terekhanova, N. V., Labudina, A. and Bokel, C. (2018). Direct control of somatic stem cell proliferation factors by the Drosophila testis stem cell niche. Development. PubMed ID: 30002131
Niches have traditionally been characterized as signalling microenvironments that allow stem cells to maintain their fate. This definition implicitly assumes that the various niche signals are integrated towards a binary fate decision between stemness and differentiation. However, observations in multiple systems have demonstrated that stem cell properties such as proliferation and self renewal can be uncoupled at the level of niche signalling input, which is incompatible with this simplified view. This study has examined the role of the transcriptional regulator Zfh1, a shared target of the Hedgehog and Jak/Stat niche signalling pathways, in the somatic stem cells of the Drosophila testis. It was found that Zfh1 binds and downregulates salvador and kibra, two tumour suppressor genes of the Hippo/Wts/Yki pathway, thereby restricting Yki activation and proliferation to the Zfh1 positive stem cells. These observations provide an unbroken link from niche signal input to an individual aspect of stem cell behaviour that does not, at any step, involve a fate decision. The relevance of these findings is discussed for an overall concept of stemness and niche function.

Tang, X., Zhao, Y., Buchon, N. and Engstrom, Y. (2018). The POU/Oct transcription factor Nubbin controls the balance of intestinal stem cell maintenance and differentiation by isoform-specific regulation. Stem Cell Reports 10(5):1565-1578. PubMed ID: 29681543
Drosophila POU/Oct transcription factors are required for many developmental processes, but their putative regulation of adult stem cell activity has not been investigated. This stduy shows that Nubbin (Nub)/Pdm1, homologous to mammalian OCT1/POU2F1 and related to OCT4/POU5F1, is expressed in gut epithelium progenitor cells. The nub-encoded protein isoforms, Nub-PB and Nub-PD, play opposite roles in the regulation of intestinal stem cell (ISC) maintenance and differentiation. Depletion of Nub-PB in progenitor cells increased ISC proliferation by derepression of escargot expression. Conversely, loss of Nub-PD reduced ISC proliferation, suggesting that this isoform is necessary for ISC maintenance, analogous to mammalian OCT4/POU5F1 functions. Furthermore, Nub-PB is required in enteroblasts to promote differentiation, and it acts as a tumor suppressor of Notch RNAi-driven hyperplasia. It is suggested that a dynamic and well-tuned expression of Nub isoforms in progenitor cells is required for maintaining gut epithelium homeostasis.
Li, Q., Nirala, N. K., Nie, Y., Chen, H. J., Ostroff, G., Mao, J., Wang, Q., Xu, L. and Ip, Y. T. (2018). Ingestion of food particles regulates the mechanosensing Misshapen-Yorkie pathway in Drosophila intestinal growth. Dev Cell 45(4): 433-449.e436. PubMed ID: 29754801
The intestinal epithelium has a high cell turnover rate and is an excellent system to study stem cell-mediated adaptive growth. In the Drosophila midgut, the Ste20 kinase Misshapen, which is distally related to Hippo, has a niche function to restrict intestinal stem cell activity. Under low growth conditions, Misshapen is localized near the cytoplasmic membrane, is phosphorylated at the threonine 194 by the upstream kinase Tao, and is more active toward Warts, which in turn inhibits Yorkie. Ingestion of yeast particles causes a midgut distention and a reduction of Misshapen membrane association and activity. Moreover, Misshapen phosphorylation is regulated by the stiffness of cell culture substrate, changing of actin cytoskeleton, and ingestion of inert particles. These results together suggest that dynamic membrane association and Tao phosphorylation of Misshapen are steps that link the mechanosensing of intestinal stretching after food particle ingestion to control adaptive growth.

Friday, July 27th - Signaling

Rhodehouse, K., Cascino, K., Aseltine, L., Padula, A., Weinstein, R., Spina, J. S., Olivero, C. E. and Van Wynsberghe, P. M. (2018). The Doubletime Homolog KIN-20 Mainly Regulates let-7 Independently of Its Effects on the Period Homolog LIN-42 in Caenorhabditis elegans. G3 (Bethesda). PubMed ID: 29880558
Evolutionary Homolog Study:
The Caenorhabditis elegans (C. elegans) heterochronic pathway, which regulates developmental timing, is thought to be an ancestral form of the circadian clock in other organisms. An essential member of this clock is the Period protein whose homolog, lin-42, in C. elegans is an important heterochronic gene. LIN-42 functions as a transcriptional repressor of multiple genes including the conserved lin-4 and let-7 microRNAs. Like other Period proteins, levels of LIN-42 oscillate throughout development. In other organisms this cycling is controlled in part by phosphorylation. KIN-20 is the C. elegans homolog of the Drosophila Period protein kinase Doubletime. Worms containing a large deletion in kin-20 have a significantly smaller brood size and develop slower than wild type C. elegans. This study analyzed the effect of kin-20 on lin-42 phenotypes and microRNA expression. kin-20 RNAi enhances loss-of-function lin-42 mutant phenotypes, and kin-20 mutant worms express lower levels of LIN-42. It was also shown that kin-20 is important for post-transcriptional regulation of mature let-7 and lin-4 microRNA expression. In addition, the increased level of let-7 found in lin-42n1089 mutant worms is not maintained after kin-20 RNAi treatment. Instead, let-7 is further repressed when levels of kin-20 and lin-42 are both decreased. Altogether these results suggest that though kin-20 regulates lin-42 and let-7 microRNA, it mainly affects let-7 microRNA expression independently of lin-42. These findings further understanding of the mechanisms by which these conserved circadian rhythmic genes interact to ultimately regulate rhythmic processes, developmental timing and microRNA biogenesis in C. elegans.
Post, S., Karashchuk, G., Wade, J. D., Sajid, W., De Meyts, P. and Tatar, M. (2018). Drosophila Insulin-Like Peptides DILP2 and DILP5 Differentially Stimulate Cell Signaling and Glycogen Phosphorylase to Regulate Longevity. Front Endocrinol (Lausanne) 9: 245. PubMed ID: 29892262
Insulin and IGF signaling (IIS) is a complex system that controls diverse processes including growth, development, metabolism, stress responses, and aging. DILPs 1-7 are thought to act through a single Drosophila insulin/IGF receptor, InR, but it is unclear how the DILPs thereby mediate a range of physiological phenotypes. This study determined the distinct cell signaling effects of DILP2 and DILP5 stimulation upon Drosophila S2 cells. DILP2 and DILP5 induced similar transcriptional patterns but differed in signal transduction kinetics. DILP5 induced sustained phosphorylation of Akt, while DILP2 produced acute, transient Akt phosphorylation. Accordingly, this study used phosphoproteomic analysis to identify distinct patterns of non-genomic signaling induced by DILP2 and DILP5. Across all treatments and replicates, 5,250 unique phosphopeptides were identified, representing 1,575 proteins. Among these peptides, DILP2, but not DILP5, dephosphorylated Ser15 on glycogen phosphorylase (GlyP), and DILP2, but not DILP5, was subsequently shown to repress enzymatic GlyP activity in S2 cells. The functional consequences of this difference were evaluated in adult Drosophila dilp mutants: dilp2 null adults have elevated GlyP enzymatic activity relative to wild type, while dilp5 mutants have reduced GlyP activity. In flies with intact insulin genes, GlyP overexpression extended lifespan in a Ser15 phosphorylation-dependent manner. In dilp2 mutants, that are otherwise long-lived, longevity was repressed by expression of phosphonull GlyP that is enzymatically inactive. Overall, DILP2, unlike DILP5, signals to affect longevity in part through its control of phosphorylation to deactivate glycogen phosphorylase, a central modulator of glycogen storage and gluconeogenesis.
Portela, M., Yang, L., Paul, S., Li, X., Veraksa, A., Parsons, L. M. and Richardson, H. E. (2018). Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex. Sci Signal 11(533). PubMed ID: 29871910
Epithelial cell polarity is linked to the control of tissue growth and tumorigenesis. The tumor suppressor and cell polarity protein lethal-2-giant larvae (Lgl) promotes Hippo signaling and inhibits Notch signaling to restrict tissue growth in Drosophila melanogaster. Notch signaling is greater in lgl mutant tissue than in wild-type tissue because of increased acidification of endosomal vesicles, which promotes the proteolytic processing and activation of Notch by gamma-secretase. The increased Notch signaling and tissue growth defects of lgl mutant tissue depended on endosomal vesicle acidification mediated by the vacuolar adenosine triphosphatase (V-ATPase). Lgl promoted the activity of the V-ATPase by interacting with Vap33 (VAMP-associated protein of 33 kDa). Vap33 physically and genetically interacted with Lgl and V-ATPase subunits and repressed V-ATPase-mediated endosomal vesicle acidification and Notch signaling. Vap33 overexpression reduced the abundance of the V-ATPase component Vha44, whereas Lgl knockdown reduced the binding of Vap33 to the V-ATPase component Vha68-3. These data indicate that Lgl promotes the binding of Vap33 to the V-ATPase, thus inhibiting V-ATPase-mediated endosomal vesicle acidification and thereby reducing gamma-secretase activity, Notch signaling, and tissue growth. These findings implicate the deregulation of Vap33 and V-ATPase activity in polarity-impaired epithelial cancers.
Yashiro, R., Murota, Y., Nishida, K. M., Yamashiro, H., Fujii, K., Ogai, A., Yamanaka, S., Negishi, L., Siomi, H. and Siomi, M. C. (2018). Piwi nuclear localization and its regulatory mechanism in Drosophila ovarian somatic cells. Cell Rep 23(12): 3647-3657. PubMed ID: 29925005
In Drosophila ovarian somatic cells (OSCs), Piwi represses transposons transcriptionally to maintain genome integrity. Piwi nuclear localization requires the N terminus and PIWI-interacting RNA (piRNA) loading of Piwi. However, the underlying mechanism remains unknown. This study shows that Importinalpha (Impalpha) plays a pivotal role in Piwi nuclear localization and that Piwi has a bipartite nuclear localization signal (NLS). Impalpha2 and Impalpha3 are highly expressed in OSCs, whereas Impalpha1 is the least expressed. Loss of Impalpha2 or Impalpha3 forces Piwi to be cytoplasmic, which is rectified by overexpression of any Impalpha members. Extension of Piwi-NLS with an additional Piwi-NLS leads Piwi to be imported to the nucleus in a piRNA-independent manner, whereas replacement of Piwi-NLS with SV40-NLS fails. Limited proteolysis analysis suggests that piRNA loading onto Piwi triggers conformational change, exposing the N terminus to the environment. These results suggest that Piwi autoregulates its nuclear localization by exposing the NLS to Impalpha upon piRNA loading.

Thursday, July 26th - - Adult Neural Development and Function

Wissel, S., Harzer, H., Bonnay, F., Burkard, T. R., Neumuller, R. A. and Knoblich, J. A. (2018). Time-resolved transcriptomics in neural stem cells identifies a v-ATPase/Notch regulatory loop. J Cell Biol. PubMed ID: 29959232
Drosophila melanogaster neural stem cells (neuroblasts [NBs]) divide asymmetrically by differentially segregating protein determinants into their daughter cells. Although the machinery for asymmetric protein segregation is well understood, the events that reprogram one of the two daughter cells toward terminal differentiation are less clear. This study used time-resolved transcriptional profiling to identify the earliest transcriptional differences between the daughter cells on their way toward distinct fates. By screening for coregulated protein complexes, vacuolar-type H(+)-ATPase (v-ATPase) was identified among the first and most significantly down-regulated complexes in differentiating daughter cells. v-ATPase is essential for NB growth and persistent activity of the Notch signaling pathway. The data suggest that v-ATPase and Notch form a regulatory loop that acts in multiple stem cell lineages both during nervous system development and in the adult gut. This study provides a unique resource for investigating neural stem cell biology and demonstrate that cell fate changes can be induced by transcriptional regulation of basic, cell-essential pathways.
Utashiro, N., Williams, C. R., Parrish, J. Z. and Emoto, K. (2018). Prior activity of olfactory receptor neurons is required for proper sensory processing and behavior in Drosophila larvae. Sci Rep 8(1): 8580. PubMed ID: 29872087
Animal responses to their environment rely on activation of sensory neurons by external stimuli. In many sensory systems, however, neurons display basal activity prior to the external stimuli. This prior activity is thought to modulate neural functions, yet its impact on animal behavior remains elusive. This study reveals a potential role for prior activity in olfactory receptor neurons (ORNs) in shaping larval olfactory behavior. Prior activity in larval ORNs is shown to be mediated by the olfactory receptor complex (OR complex). Mutations of Orco, an odorant co-receptor required for OR complex function, cause reduced attractive behavior in response to optogenetic activation of ORNs. Calcium imaging reveals that Orco mutant ORNs fully respond to optogenetic stimulation but exhibit altered temporal patterns of neural responses. These findings together suggest a critical role for prior activity in information processing upon ORN activation in Drosophila larvae, which in turn contributes to olfactory behavior control.
Namiki, S., Dickinson, M. H., Wong, A. M., Korff, W. and Card, G. M. (2018). The functional organization of descending sensory-motor pathways in Drosophila. Elife 7. PubMed ID: 29943730
In most animals, the brain controls the body via a set of descending neurons (DNs) that traverse the neck. DN activity activates, maintains or modulates locomotion and other behaviors. Individual DNs have been well-studied in species from insects to primates, but little is known about overall connectivity patterns across the DN population. This study systematically investigated DN anatomy in Drosophila melanogaster and created over 100 transgenic lines targeting individual cell types. Roughly half of all Drosophila DNs were identified and connectivity was comprehensively mapped between sensory and motor neuropils in the brain and nerve cord, respectively. The nerve cord was found to be a layered system of neuropils reflecting the fly's capability for two largely independent means of locomotion -- walking and flight -- using distinct sets of appendages. These results reveal the basic functional map of descending pathways in flies and provide tools for systematic interrogation of neural circuits.
Youn, H., Kirkhart, C., Chia, J. and Scott, K. (2018). A subset of octopaminergic neurons that promotes feeding initiation in Drosophila melanogaster. PLoS One 13(6): e0198362. PubMed ID: 29949586
Octopamine regulates feeding behavioral responses in Drosophila melanogaster, however the molecular and circuit mechanisms have not been fully elucidated. This study investigated the role of a subset of octopaminergic neurons, the OA-VPM4 cluster, in sucrose acceptance behavior. Thermogenetic activation of Gal4 lines containing OA-VPM4 promoted proboscis extension to sucrose, while optogenetic inactivation reduced extension. Anatomically, the presynaptic terminals of OA-VPM4 are in close proximity to the axons of sugar-responsive gustatory sensory neurons. Moreover, RNAi knockdown of a specific class of octopamine receptor, OAMB, selectively in sugar-sensing gustatory neurons decreased the behavioral response to sucrose. By calcium imaging experiments, this study found that application of octopamine potentiates sensory responses to sucrose in satiated flies. Taken together, these findings suggest a model by which OA-VPM4 promotes feeding behavior by modulating the activity of sensory neurons.

Wednesday, July 25 - Evolution

Lajbner, Z., Pnini, R., Camus, M. F., Miller, J. and Dowling, D. K. (2018). Experimental evidence that thermal selection shapes mitochondrial genome evolution. Sci Rep 8(1): 9500. PubMed ID: 29934612
Mitochondria are essential organelles, found within eukaryotic cells, which contain their own DNA. Mitochondrial DNA (mtDNA) has traditionally been used in population genetic and biogeographic studies as a maternally-inherited and evolutionary-neutral genetic marker. However, it is now clear that polymorphisms within the mtDNA sequence are routinely non-neutral, and furthermore several studies have suggested that such mtDNA polymorphisms are also sensitive to thermal selection. These observations led to the formulation of the "mitochondrial climatic adaptation" hypothesis, for which all published evidence to date is correlational. This study used laboratory-based experimental evolution in the fruit fly, Drosophila melanogaster, to test whether thermal selection can shift population frequencies of two mtDNA haplogroups whose natural frequencies exhibit clinal associations with latitude along the Australian east-coast. Experimental evidence is presented that the thermal regime in which the laboratory populations were maintained drove changes in haplogroup frequencies across generations. These results strengthen the emerging view that intra-specific mtDNA variants are sensitive to selection, and suggest spatial distributions of mtDNA variants in natural populations of metazoans might reflect adaptation to climatic environments rather than within-population coalescence and diffusion of selectively-neutral haplotypes across populations.
Teixeira, J. R., Dias, G. B., Svartman, M., Ruiz, A. and Kuhn, G. C. S. (2018). Concurrent Duplication of Drosophila Cid and Cenp-C Genes Resulted in Accelerated Evolution and Male Germline-Biased Expression of the New Copies. J Mol Evol. PubMed ID: 29934734
Despite their essential role in the process of chromosome segregation in eukaryotes, kinetochore proteins are highly diverse across species, being lost, duplicated, created, or diversified during evolution. Surprisingly, the Drosophila CenH3 homolog Cid underwent four independent duplication events during evolution. Particularly interesting are the highly diverged Cid1 and Cid5 paralogs of the Drosophila subgenus, which are probably present in over one thousand species. Given that CenH3 and Cenp-C likely co-evolve as a functional unit, this study investigated the molecular evolution of Cenp-C in species of Drosophila. Yet another Cid duplication (leading to Cid6) was found within the Drosophila subgenus; and not only Cid, but also Cenp-C is duplicated in the entire subgenus. The Cenp-C paralogs, which were named Cenp-C1 and Cenp-C2, are highly divergent. Both Cenp-C1 and Cenp-C2 retain key motifs involved in centromere localization and function, while some functional motifs are conserved in an alternate manner between the paralogs. Interestingly, both Cid5 and Cenp-C2 are male germline-biased and evolved adaptively. However, it is currently unclear if the paralogs subfunctionalized or if the new copies acquired a new function. These findings point towards a specific inner kinetochore composition in a specific context (i.e., spermatogenesis), which could prove valuable for the understanding of how the extensive kinetochore diversity is related to essential cellular functions.
Marmion, R. A. and Yakoby, N. (2018). In locus analysis of patterning evolution in the BMPR2 Wishful thinking. Development. PubMed ID: 29884674
Proper tissue patterning is an essential step during organ formation. During this process, genes are expressed in distinct patterns, defining boundaries for future functional domains. The bone morphogenetic protein (BMP) signaling pathway sets the anterior domain during eggshell patterning. Previously, the Drosophila melanogaster BMPR2, Wishful thinking (WIT), was shown to be required for BMP signaling and patterning during eggshell formation. Expressed in a conserved anterior pattern, the width of wit patterning in the follicular epithelium is evolutionally divergent between Drosophila species. This study used genome editing to demonstrate how the gene pattern divergence is controlled in cis within the wit locus of D. virilis. Furthermore, unlike direct targets of BMP signaling, this study demonstrated how one transcription factor binding site shapes the pattern of WIT in D. melanogaster by negative regulation. However, changes in this site are not sufficient to explain the evolution of wit patterning, suggesting that a yet undiscovered positive regulatory element controls pattern divergence.
Parker, D. J., Wiberg, R. A. W., Trivedi, U., Tyukmaeva, V. I., Gharbi, K., Butlin, R. K., Hoikkala, A., Kankare, M. and Ritchie, M. G. (2018). Inter- and intra-specific genomic divergence in Drosophila montana shows evidence for cold adaptation. Genome Biol Evol. PubMed ID: 30010752
The genomes of species that are ecological specialists will likely contain signatures of genomic adaptation to their niche. This study describes the genome of Drosophila montana, which is the most extremely cold-adapted Drosophila species. Branch tests were used to identify genes showing accelerated divergence in contrasts between cold- and warm adapted species, and about 250 genes were identified that show differences, possibly driven by a lower synonymous substitution rate in cold-adapted species. Evidence was sought of accelerated divergence between D. montana and D. virilis, a previously sequenced relative, and no strong evidence was found for divergent selection on coding sequence variation. Divergent genes are involved in a variety of functions, including cuticular and olfactory processes. Three populations of D. montana were resequenced from its ecological and geographic range. Outlier loci were more likely to be found on the X chromosome and there was a greater than expected overlap between population outliers and those genes implicated in cold adaptation between Drosophila species, implying some continuity of selective process at these different evolutionary scales.
Hill, T. and Betancourt, A. J. (2018). Extensive exchange of transposable elements in the Drosophila pseudoobscura group. Mob DNA 9: 20. PubMed ID: 29946370
As species diverge, so does their transposable element (TE) content. Within a genome, TE families may eventually become dormant due to host-silencing mechanisms, natural selection and the accumulation of inactive copies. The transmission of active copies from a TE families, both vertically and horizontally between species, can allow TEs to escape inactivation if it occurs often enough, as it may allow TEs to temporarily escape silencing in a new host. Thus, the contribution of horizontal exchange to TE persistence has been of increasing interest. This study annotated TEs in five species with sequenced genomes from the D. pseudoobscura species group, and curated a set of TE families found in these species. Compared to host genes, many TE families showed lower neutral divergence between species, consistent with recent transmission of TEs between species. Despite these transfers, there are differences in the TE content between species in the group. It is concluded that the TE content is highly dynamic in the D. pseudoobscura species group, frequently transferring between species, keeping TEs active. This result highlights how frequently transposable elements are transmitted between sympatric species and, despite these transfers, how rapidly species TE content can diverge.
Hart, M. L. I., Vu, B. L., Bolden, Q., Chen, K. T., Oakes, C. L., Zoronjic, L. and Meisel, R. P. (2018). Genes relocated between Drosophila chromosome arms evolve under relaxed selective constraints relative to non-relocated genes. J Mol Evol. PubMed ID: 29926120
Gene duplication creates a second copy of a gene either in tandem to the ancestral locus or dispersed to another chromosomal location. Gene relocations may be as common as canonical dispersed duplications in which both the ancestral and derived copies are retained. Relocated genes appear to be under more selective constraints than the derived copies of canonical duplications, and they are possibly as conserved as single-copy non-relocated genes. To test this hypothesis, comparative genomics, population genetics, gene expression, and functional analyses were combined to assess the selection pressures acting on relocated, duplicated, and non-relocated single-copy genes in Drosophila genomes. Relocated genes were found to evolve faster than single-copy non-relocated genes, and there is no evidence that this faster evolution is driven by positive selection. In addition, relocated genes are less essential for viability and male fertility than single-copy non-relocated genes, suggesting that relocated genes evolve fast because of relaxed selective constraints. However, relocated genes evolve slower than the derived copies of canonical dispersed duplicated genes. We therefore conclude that relocated genes are under more selective constraints than canonical duplicates, but are not as conserved as single-copy non-relocated genes.

Tuesday, July 24th - RNA

Mo, X., Yang, X. and Yuan, Y. A. (2018). Structural insights into Drosophila-C3PO complex assembly and 'Dynamic Side Port' model in substrate entry and release. Nucleic Acids Res. PubMed ID: 29860349
In Drosophila and human, component 3 promoter of RISC (C3PO), a heteromeric complex, enhances RISC assembly and promotes RISC activity. This study reports crystal structure of full-length Drosophila C3PO (E126Q), an inactive C3PO mutant displaying much weaker RNA binding ability, at 2.1 A resolution. In addition, this study also reports the cryo-EM structures of full-length Drosophila C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT, sumo-TRAX + Translin) particles trapped at different conformations at 12, 19.7 and 12.8 A resolutions, respectively. Crystal structure of C3PO (E126Q) displays a half-barrel architecture consisting of two Trax/Translin heterodimers, whereas cryo-EM structures of C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT) adopt a closed football-like shape with a hollow interior cavity. Remarkably, both cryo-EM structures of Drosophila C3PO (E126Q) and Drosophila SUMO-C3PO (WT) particles contain a wide side port (approximately 25 A x approximately 30 A versus approximately 15 A x approximately 20 A) for RNA substrate entry and release, formed by a pair of anti-parallel packed long alpha1 helices of TRAX subunits. Notably, cryo-EM structure of SUMO-C3PO showed that four copies of extra densities belonging to N-terminal SUMO tag are located at the outside shell of SUMO-C3PO particle, which demonstrated that the stoichiometry of TRAX/Translin for the in vitro expressed and assembled full-length Drosophila-SUMO-C3PO particle is 4:4, suggesting Drosophila C3PO is composed by TRAX/translin at a ratio of 4:4. Remarkably, the comparison of the cryo-EM structures suggests that the C3PO side ports regulated by alpha1 helices of TRAX molecules are highly dynamic. Hence, it is propose that C3PO particles could adopt a 'Dynamic Side Port' model to capture/digest nucleic acid duplex substrate and release the digested fragments through the dynamic side ports.
Nelson, J. O., Forster, D., Frizzell, K. A., Luschnig, S. and Metzstein, M. M. (2018). Multiple nonsense-mediated mRNA processes require Smg5 in Drosophila. Genetics. PubMed ID: 29903866
The nonsense-mediated mRNA decay (NMD) pathway is a cellular quality control and post-transcriptional gene regulatory mechanism and is essential for viability in most multicellular organisms. A complex of proteins has been identified to be required for NMD function to occur, however there is an incomplete understanding of the individual contributions of each of these factors to the NMD process. Central to the NMD process are three proteins, Upf1 (SMG-2), Upf2 (SMG-3), and Upf3 (SMG-4), which are found in all eukaryotes, with Upf1 and Upf2 being absolutely required for NMD in all organisms in which their functions have been examined. The other known NMD factors, Smg1, Smg5, Smg6, and Smg7 are more variable in their presence in different orders of organisms and are thought to have a more regulatory role. This study presents the first genetic analysis of the NMD factor Smg5 in Drosophila. Surprisingly, it was found that unlike the other analyzed Smg genes in this organism, Smg5 is essential for NMD activity. This was found to be due in part to a requirement for Smg5 in both the activity of Smg6-dependent endonucleolytic cleavage, as well as an additional Smg6-independent mechanism. Redundancy between these degradation pathways explains why some Drosophila NMD genes are not required for all NMD-pathway activity. It was also found that while the NMD component Smg1 has only a minimal role in Drosophila NMD during normal conditions, it becomes essential when NMD activity is compromised by partial loss of Smg5 function. These findings suggest that not all NMD complex components are required for NMD function at all times, but instead are utilized in a context dependent manner in vivo.
Goodwin, P. R., Meng, A., Moore, J., Hobin, M., Fulga, T. A., Van Vactor, D. and Griffith, L. C. (2018). MicroRNAs regulate sleep and sleep homeostasis in Drosophila. Cell Rep 23(13): 3776-3786. PubMed ID: 29949763
To discover microRNAs that regulate sleep, a genetic screen was performed using a library of miRNA sponge-expressing flies, Sponges are transgenic miRNA inhibitors. 25 miRNAs were identified that regulate baseline sleep; 17 were sleep-promoting and 8 promoted wake. One miRNA was identified that is required for recovery sleep after deprivation and 8 miRNAs that limit the extent of recovery sleep. 65% of the hits belong to human-conserved families. Interestingly, the majority (75%), but not all, of the baseline sleep-regulating miRNAs are required in neurons. Sponges that target miRNAs in the same family, including the miR-92a/92b/310 family and the miR-263a/263b family, have similar effects. Finally, mutation of one of the screen's strongest hits, let-7, using CRISPR/Cas-9, phenocopies sponge-mediated let-7 inhibition. Cell-type-specific and temporally restricted let-7 sponge expression experiments suggest that let-7 is required in the mushroom body both during development and in adulthood. This screen sets the stage for understanding the role of miRNAs in sleep.
Aquilina, B. and Cauchi, R. J. (2018). Genetic screen identifies a requirement for SMN in mRNA localisation within the Drosophila oocyte. BMC Res Notes 11(1): 378. PubMed ID: 29895323
Spinal muscular atrophy (SMA) results from insufficient levels of the survival motor neuron (SMN) protein. Drosophila is conducive to large-scale genetic-modifier screens which can reveal novel pathways underpinning the disease mechanism. The ability of a large collection of genomic deletions to enhance SMN-dependent lethality was tested. To test this design, it was asked whether this study can identify loci containing genes identified in previous genetic screens. The objective was to find a common link between genes flagged in independent screens, which would allow exposing of novel functions for SMN in vivo. Out of 128 chromosome deficiency lines, 12 (9.4%) were found to consistently depress adult viability when crossed to SMN loss-of-function heterozygotes. In their majority, the enhancing deletions harboured genes that were previously identified as genetic modifiers, hence, validating the design of the screen. Importantly, gene overlap allowed flagging of genes with a role in post-transcriptional regulation of mRNAs that are crucial for determining the axes of the oocyte and future embryo. SMN was found to also be required for the correct localisation of gurken and oskar mRNAs in oocytes. These findings extend the role of SMN in oogenesis by identifying a key requirement for mRNA trafficking.
Sander, M., Eichenlaub, T. and Herranz, H. (2018). Oncogenic cooperation between Yorkie and the conserved microRNA miR-8 in the wing disc of Drosophila. Development. PubMed ID: 29945869
Tissue growth has to be carefully controlled to generate well-functioning organs. microRNAs are small noncoding RNAs that modulate the activity of target genes and play a pivotal role in animal development. Understanding the functions of microRNAs in development requires the identification of their target genes. This study finds that the conserved microRNA miR-8/miR-200 controls tissue growth and homeostasis in the Drosophila wing imaginal disc. Upregulation of miR-8 causes the repression of Yorkie, the effector of the Hippo pathway in Drosophila, and reduces tissue size. Remarkably, coexpression of Yorkie and miR-8 causes the formation of neoplastic tumors. Upregulation of miR-8 represses the growth inhibitor brinker, and depletion of brinker cooperates with Yorkie in the formation of neoplastic tumors. Hence, miR-8 modulates a positive growth regulator, Yorkie, and a negative growth regulator, brinker. Deregulation of this network can result in the loss of tissue homeostasis and the formation of tumors.
Sladewski, T. E., Billington, N., Ali, M. Y., Bookwalter, C. S., Lu, H., Krementsova, E. B., Schroer, T. A. and Trybus, K. M. (2018). Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex. Elife 7. PubMed ID: 29944116
This study investigated the role of full-length Drosophila Bicaudal D (BicD) binding partners in dynein-dynactin activation for mRNA transport on microtubules. Full-length BicD robustly activated dynein-dynactin motility only when both the mRNA binding protein Egalitarian (Egl) and K10 mRNA cargo were present, and electron microscopy showed that both Egl and mRNA were needed to disrupt a looped, auto-inhibited BicD conformation. BicD can recruit two dimeric dyneins, resulting in faster speeds and longer runs than with one dynein. Moving complexes predominantly contained two Egl molecules and one K10 mRNA. This mRNA-bound configuration makes Egl bivalent, likely enhancing its avidity for BicD and thus its ability to disrupt BicD auto-inhibition. Consistent with this idea, artificially dimerized Egl activates dynein-dynactin-BicD in the absence of mRNA. The ability of mRNA cargo to orchestrate the activation of the mRNP (messenger ribonucleotide protein) complex is an elegant way to ensure that only cargo-bound motors are motile.

Monday, July 23rd - Disease Models

Morris, M., Shaw, A., Lambert, M., Perry, H. H., Lowenstein, E., Valenzuela, D. and Velazquez-Ulloa, N. A. (2018). Developmental nicotine exposure affects larval brain size and the adult dopaminergic system of Drosophila melanogaster. BMC Dev Biol 18(1): 13. PubMed ID: 29898654
This study determined whether developmental nicotine exposure affects the nervous system of Drosophila melanogaster, focusing on changes to brain size and the dopaminergic system at two developmental stages. Flies were reared on control or nicotine food from egg to 3rd instar larvae or from egg to adult, and effectiveness of the nicotine treatment was determined. Immunohistochemistry was used to visualize the whole brain and dopaminergic neurons, using tyrosine hydroxylase as the marker. Brain area and tyrosine hydroxylase fluorescence were measured, and the number of dopaminergic neurons in brain clusters was measured. An increase in larval brain hemisphere area, a decrease in tyrosine hydroxylase fluorescence in adult central brains, and a decrease in the number of neurons in the PPM3 adult dopaminergic cluster were detected. Involvement of Dalpha7, one of the nicotinic acetylcholine receptor subunits, was examined, and it was found to be involved in eclosion, as previously described, but not involved in brain size. It is concluded that developmental nicotine exposure in Drosophila melanogaster affects brain size and the dopaminergic system. Prenatal nicotine exposure in mammals has also been shown to have effects on brain size and in the dopaminergic system. This study further establishes Drosophila melanogaster as model organism to study the effects of developmental nicotine exposure. The genetic and molecular tools available for Drosophila research will allow elucidation of the mechanisms underlying the effects of nicotine exposure during development.
Raj, K. and Sarkar, S. (2018). Tissue-specific upregulation of Drosophila Insulin Receptor (InR) mitigates poly(Q)-mediated neurotoxicity by restoration of cellular transcription machinery. Mol Neurobiol. PubMed ID: 29881950
Polyglutamine [poly(Q)] disorders are a class of trinucleotide repeat expansion neurodegenerative disorders which are dominantly inherited and progressively acquired with age. This group of disorders entail the characteristic formation of protein aggregates leading to widespread loss of neurons in different regions of the brain. Spinocerebellar ataxia type 3 and Huntington's Disease, the two most commonly occurring types of poly(Q) disorders, were examined in the present study. With the aim of elucidating novel genetic modifiers of poly(Q) disorders, the Drosophila Insulin receptor (InR) was identified as a potential suppressor of poly(Q)-induced neurotoxicity and degeneration. Targeted upregulation of InR could effectively mitigate poly(Q)-mediated neurodegeneration in fly models. A significant reduction in poly(Q)-mediated cellular stress and apoptosis was noted upon InR overexpression in poly(Q) background. It was further revealed that targeted upregulation of InR causes a substantial reduction in poly(Q) aggregate formation with the residual inclusion bodies localised to the cytoplasm. It was also demonstrated that InR achieves suppression of poly(Q) toxicity by replenishing the cellular pool of CREB binding protein and improving the histone acetylation status of the cell. This leads to restoration of the cellular transcriptional machinery which is otherwise severely compromised in poly(Q) disease conditions. Interestingly, there also appeared a possibility of autophagy-mediated rescue of poly(Q) phenotype due to upregulation of InR. Therefore, this study strongly suggests that modulation of the insulin signalling pathway could be an effective therapeutic intervention against poly(Q) disorders.
Manzo, E., O'Conner, A. G., Barrows, J. M., Shreiner, D. D., Birchak, G. J. and Zarnescu, D. C. (2018). Medium-chain fatty acids, beta-hydroxybutyric acid and genetic modulation of the carnitine shuttle are protective in a Drosophila model of ALS Based on TDP-43. Front Mol Neurosci 11: 182. PubMed ID: 29904341
ALS patients exhibit dyslipidemia, hypermetabolism and weight loss; in addition, cellular energetics deficits have been detected prior to denervation. Although evidence that metabolism is altered in ALS is compelling, the mechanisms underlying metabolic dysregulation and the contribution of altered metabolic pathways to disease remain poorly understood. This study used a Drosophila model of ALS based on TDP-43 that recapitulates hallmark features of the disease including locomotor dysfunction and reduced lifespan. A global, unbiased metabolomic profiling of larvae expressing TDP-43 (wild-type, TDPWT or disease-associated mutant, TDPG298S) and identified several lipid metabolism associated alterations. Among these, a significant increase was found in carnitine conjugated long-chain fatty acids and a significant decrease in carnitine, acetyl-carnitine and beta-hydroxybutyrate, a ketone precursor. Taken together these data suggest a deficit in the function of the carnitine shuttle and reduced lipid beta oxidation. To test this possibility a combined genetic and dietary approach was used in Drosophila. The findings indicate that components of the carnitine shuttle are misexpressed in the context of TDP-43 proteinopathy and that genetic modulation of CPT1 or CPT2 expression, two core components of the carnitine shuttle, mitigates TDP-43 dependent locomotor dysfunction, in a variant dependent manner. In addition, feeding medium-chain fatty acids or beta-hydroxybutyrate improves locomotor function, consistent with the notion that bypassing the carnitine shuttle deficit is neuroprotective. Taken together, these findings highlight the potential contribution of the carnitine shuttle and lipid beta oxidation in ALS and suggest strategies for therapeutic intervention based on restoring lipid metabolism in motor neurons.
Morozova, T. V., Hussain, Y., McCoy, L. J., Zhirnov, E. V., Davis, M. R., Pray, V. A., Lyman, R. A., Duncan, L. H., McMillen, A., Jones, A., Mackay, T. F. C. and Anholt, R. R. H. (2018). A Cyclin E centered genetic network contributes to alcohol-induced variation in Drosophila development. G3 (Bethesda). PubMed ID: 29871898
Prenatal exposure to ethanol causes a wide range of adverse physiological, behavioral and cognitive consequences. However, identifying allelic variants and genetic networks associated with variation in susceptibility to prenatal alcohol exposure is challenging in human populations, since time and frequency of exposure and effective dose cannot be determined quantitatively and phenotypic manifestations are diverse. This study harnessed the power of natural variation in the Drosophila melanogaster Genetic Reference Panel (DGRP) to identify genes and genetic networks associated with variation in sensitivity to developmental alcohol exposure. Development time from egg to adult and viability were measured of 201 DGRP lines reared on regular or ethanol- supplemented medium, and polymorphisms were identified associated with variation in susceptibility to developmental ethanol exposure. Genotype-dependent variation in sensorimotor behavior was measured after developmental exposure to ethanol using the startle response assay in a subset of 39 DGRP lines. Genes associated with development, including development of the nervous system, featured prominently among genes that harbored variants associated with differential sensitivity to developmental ethanol exposure. Many of them have human orthologs and mutational analyses and RNAi targeting functionally validated a high percentage of candidate genes. Analysis of genetic interaction networks identified Cyclin E (CycE) as a central, highly interconnected hub gene. Cyclin E encodes a protein kinase associated with cell cycle regulation and is prominently expressed in ovaries. Thus, exposure to ethanol during development of Drosophila melanogaster might serve as a genetic model for translational studies on fetal alcohol spectrum disorder.
Iyer, J., Singh, M. D., Jensen, M., Patel, P., Pizzo, L., Huber, E., Koerselman, H., Weiner, A. T., Lepanto, P., Vadodaria, K., Kubina, A., Wang, Q., Talbert, A., Yennawar, S., Badano, J., Manak, J. R., Rolls, M. M., Krishnan, A. and Girirajan, S. (2018). Pervasive genetic interactions modulate neurodevelopmental defects of the autism-associated 16p11.2 deletion in Drosophila melanogaster. Nat Commun 9(1): 2548. PubMed ID: 29959322
As opposed to syndromic Copy Number Variations (CNVs) caused by single genes, extensive phenotypic heterogeneity in variably-expressive CNVs complicates disease gene discovery and functional evaluation. This study proposes a complex interaction model for pathogenicity of the autism-associated 16p11.2 deletion, where CNV genes interact with each other in conserved pathways to modulate expression of the phenotype. Using multiple quantitative methods in Drosophila RNAi lines, a range of neurodevelopmental phenotypes were identified for knockdown of individual 16p11.2 homologs in different tissues. 565 pairwise knockdowns were tested in the developing eye, and 24 interactions were identified between pairs of 16p11.2 homologs and 46 interactions between 16p11.2 homologs and neurodevelopmental genes that suppress or enhance cell proliferation phenotypes compared to one-hit knockdowns. These interactions within cell proliferation pathways are also enriched in a human brain-specific network, providing translational relevance in humans. This study indicates a role for pervasive genetic interactions within CNVs towards cellular and developmental phenotypes.
Mohite, G. M., Dwivedi, S., Das, S., Kumar, R., Paluri, S., Mehra, S., Ruhela, N., S, A., Jha, N. N. and Maji, S. K. (2018). Parkinson's disease associated alpha-synuclein familial mutants promote dopaminergic neuronal death in Drosophila melanogaster. ACS Chem Neurosci. PubMed ID: 29906099
alpha-Synuclein (alpha-Syn) aggregation and amyloid formation are associated with loss of dopaminergic neurons in Parkinson's disease (PD). In addition, familial mutations in alpha-Syn are shown to be one of the definite causes of PD. Familial PD associated alpha-Syn G51D, H50Q and E46K mutations were extensively studied using the Drosophila model system. The data showed that flies expressing alpha-Syn familial mutants have a shorter lifespan and exhibit more climbing defects compared to wild-type (WT) flies in an age-dependent manner. The immuno-fluorescence studies of the brain from the old flies showed more dopaminergic neuronal cell death in all mutants compared to WT. This adverse effect of alpha-Syn familial mutations highly correlated with the sustained population of oligomer production/ retention in mutant flies. Furthermore, this was supported by in vitro studies, where significantly higher amount of oligomer was observed in mutants compared to WT. The data suggest that the sustained population of oligomer formation/ retention could be a major cause of cell death by alpha-Syn familial mutants.

Friday, July 20th

Sechi, S., Frappaolo, A., Fraschini, R., Capalbo, L., Gottardo, M., Belloni, G., Glover, D. M., Wainman, A. and Giansanti, M. G. (2017). Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster. Open Biol 7(1). Pubmed ID: 28100664
Cytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. This study provides evidence for the requirement of Rab1 during cleavage furrow ingression in cytokinesis. The gene omelette (omt) encodes the Drosophila orthologue of human Rab1 and is required for successful cytokinesis in both mitotic and meiotic dividing cells of Drosophila melanogaster. Rab1 protein colocalizes with the conserved oligomeric Golgi (COG) complex Cog7 subunit and the phosphatidylinositol 4-phosphate effector GOLPH3 at the Golgi stacks. Analysis by transmission electron microscopy and 3D-SIM super-resolution microscopy reveals loss of normal Golgi architecture in omt mutant spermatocytes indicating a role for Rab1 in Golgi formation. In dividing cells, Rab1 enables stabilization and contraction of actomyosin rings. It was further demonstrated that GTP-bound Rab1 directly interacts with GOLPH3 and controls its localization at the Golgi and at the cleavage site. It is proposed that Rab1, by associating with GOLPH3, controls membrane trafficking and contractile ring constriction during cytokinesis.
Palacios-Munoz, A. and Ewer, J. (2018). Calcium and cAMP directly modulate the speed of the Drosophila circadian clock. PLoS Genet 14(6): e1007433. PubMed ID: 29879123
Circadian clocks impose daily periodicities to animal behavior and physiology. At their core, circadian rhythms are produced by intracellular transcriptional/translational feedback loops (TTFL). TTFLs may be altered by extracellular signals whose actions are mediated intracellularly by calcium and cAMP. In mammals these messengers act directly on TTFLs via the calcium/cAMP-dependent transcription factor, CREB. In the fruit fly, Drosophila melanogaster, calcium and cAMP also regulate the periodicity of circadian locomotor activity rhythmicity, but whether this is due to direct actions on the TTFLs themselves or are a consequence of changes induced to the complex interrelationship between different classes of central pacemaker neurons is unclear. This question was investigated by focusing on the peripheral clock housed in the non-neuronal prothoracic gland (PG), which, together with the central pacemaker in the brain, controls the timing of adult emergence. Genetic manipulations that increased and decreased the levels of calcium and cAMP in the PG caused, respectively, a shortening and a lengthening of the periodicity of emergence. Importantly, knockdown of CREB in the PG caused an arrhythmic pattern of eclosion. Interestingly, the same manipulations directed at central pacemaker neurons caused arrhythmicity of eclosion and of adult locomotor activity, suggesting a common mechanism. These results reveal that the calcium and cAMP pathways can alter the functioning of the clock itself. In the PG, these messengers, acting as outputs of the clock or as second messengers for stimuli external to the PG, could also contribute to the circadian gating of adult emergence.
de Vreede, G., Morrison, H. A., Houser, A. M., Boileau, R. M., Andersen, D., Colombani, J. and Bilder, D. (2018). A Drosophila tumor suppressor gene prevents tonic TNF signaling through receptor N-glycosylation. Dev Cell 45(5): 595-605.e594. PubMed ID: 29870719
Drosophila tumor suppressor genes have revealed molecular pathways that control tissue growth, but mechanisms that regulate mitogenic signaling are far from understood. This study reports that the Drosophila TSG tumorous imaginal discs (tid), whose phenotypes were previously attributed to mutations in a DnaJ-like chaperone, are in fact driven by the loss of the N-linked glycosylation pathway component ALG3. tid/alg3 imaginal discs display tissue growth and architecture defects that share characteristics of both neoplastic and hyperplastic mutants. Tumorous growth is driven by inhibited Hippo signaling, induced by excess Jun N-terminal kinase (JNK) activity. Ectopic JNK activation is caused by aberrant glycosylation of a single protein, the fly tumor necrosis factor (TNF) receptor homolog, which results in increased binding to the continually circulating TNF. These results suggest that N-linked glycosylation sets the threshold of TNF receptor signaling by modifying ligand-receptor interactions and that cells may alter this modification to respond appropriately to physiological cues.
Patrnogic, J., Heryanto, C. and Eleftherianos, I. (2018). Wounding-induced upregulation of the Bone Morphogenic Protein signaling pathway in Drosophila promotes survival against parasitic nematode infection. Gene [Epub ahead of print]. PubMed ID: 29920363
The common fruit fly, Drosophila melanogaster is an outstanding model to analyze the regulation of conserved signaling pathways. This study examined whether signaling components in the Bone Morphogenic Protein (BMP) branch of the TGF-beta signaling pathway are involved in the response to wounding caused by either sterile injury or infection by parasitic nematodes in D. melanogaster adult flies. Following sterile injury, the BMP pathway Type I receptor sax and intracellular transcription factor Mad were found to be substantially upregulated. Also, inactivation of Mad or dpp promoted fly survival and increased antimicrobial peptide gene transcript levels upon sterile injury or H. bacteriophora nematode infection, respectively, but not against the bacterial pathogen Photorhabdus luminescens. These findings indicate the roles of certain BMP signaling components in the regulation of the fly immune response against sterile injury or nematode infection. In conclusion, this study highlights the ability of D. melanogaster to activate the BMP branch of TGF-beta signaling in order to modulate the response to injury in the absence or presence of pathogenic infection.
Li, S., Cho, Y. S., Wang, B., Li, S. and Jiang, J. (2018). Regulation of Smoothened ubiquitination and cell surface expression by a Cul4-DDB1-Gbeta E3 ubiquitin ligase complex. J Cell Sci. PubMed ID: 29930086
Hedgehog (Hh) transduces signal by promoting cell surface accumulation and activation of the G protein coupled receptor (GPCR)-family protein Smoothened (Smo) in Drosophila, but the molecular mechanism underlying the regulation of Smo trafficking has remained poorly understood. This study identified a Cul4-DDB1 E3 ubiquitin ligase complex as essential for Smo ubiquitination and cell surface clearance. The C-terminal intracellular domain of Smo was found to recruit Cul4-DDB1 through the beta subunit of trimeric G protein (Gbeta), and that Cul4-DDB1-Gbeta promotes the ubiquitination of both Smo and its binding partner G-protein-coupled-receptor-kinase 2 (Gprk2) and induces the internalization and degradation of Smo. Hh dissociates Cul4-DDB1 from Smo by recruiting the catalytic subunit of protein kinase A (PKA) to phosphorylate DDB1, which disrupts its interaction with Gbeta. Inactivation of the Cul4-DDB1 complex resulted in elevated Smo cell surface expression whereas excessive Cul4-DDB1 blocked Smo accumulation and attenuated Hh pathway activation. Taken together, this study identifies an E3 ubiquitin ligase complex targeting Smo for ubiquitination and provides new insight into how Hh signaling regulates Smo trafficking and cell surface expression.
Poon, C. L. C., Brumby, A. M. and Richardson, H. E. (2018). Src cooperates with oncogenic Ras in tumourigenesis via the JNK and PI3K pathways in Drosophila epithelial tissue. Int J Mol Sci 19(6). PubMed ID: 29861494
The Ras oncogene (Rat Sarcoma oncogene, a small GTPase) is a key driver of human cancer, however alone it is insufficient to produce malignancy, due to the induction of cell cycle arrest or senescence. In a Drosophila melanogaster genetic screen for genes that cooperate with oncogenic Ras (bearing the RasV12 mutation, or RasACT), this study identified the Drosophila Src (Sarcoma virus oncogene) family non-receptor tyrosine protein kinase genes, Src42A and Src64B, as promoting increased hyperplasia in a whole epithelial tissue context in the Drosophila eye. Moreover, overexpression of Src cooperated with RasACT in epithelial cell clones to drive neoplastic tumourigenesis. Src overexpression alone activated the Jun N-terminal Kinase (JNK) signalling pathway to promote actin cytoskeletal and cell polarity defects and drive apoptosis, whereas, in cooperation with RasACT, JNK led to a loss of differentiation and an invasive phenotype. Src + RasRasACT cooperative tumourigenesis was dependent on JNK as well as Phosphoinositide 3-Kinase (PI3K) signalling, suggesting that targeting these pathways might provide novel therapeutic opportunities in cancers dependent on Src and Ras signalling.

Thursday, July 19th - Immune Response

Bachtel, N. D., Hovsepian, G. A., Nixon, D. F. and Eleftherianos, I. (2018). Allatostatin C modulates nociception and immunity in Drosophila. Sci Rep 8(1): 7501. PubMed ID: 29760446
Bacterial induced inflammatory responses cause pain through direct activation of nociceptive neurons, and the ablation of these neurons leads to increased immune infiltration. This study investigated nociceptive-immune interactions in Drosophila and the role these interactions play during pathogenic bacterial infection. After bacterial infection, robust upregulation is found of ligand-gated ion channels and allatostatin receptors involved in nociception, which potentially leads to hyperalgesia. It was further found that Allatostatin-C Receptor 2 (AstC-R2) plays a crucial role in host survival during infection with the pathogenic bacterium Photorhabdus luminescens. Upon examination of immune signaling in AstC-R2 deficient mutants, it was demonstrated that Allatostatin-C Receptor 2 specifically inhibits the Immune deficiency pathway, and knockdown of AstC-R2 leads to overproduction of antimicrobial peptides related to this pathway and decreased host survival. This study provides mechanistic insights into the importance of microbe-nociceptor interactions during bacterial challenge. It is posited that Allatostatin C is an immunosuppressive substance released by nociceptors or Drosophila hemocytes that dampens IMD signaling in order to either prevent immunopathology or to reduce unnecessary metabolic cost after microbial stimulation. AstC-R2 also acts to dampen thermal nociception in the absence of infection, suggesting an intrinsic neuronal role in mediating these processes during homeostatic conditions. Further examination into the signaling mechanisms by which Allatostatin-C alters immunity and nociception in Drosophila may reveal conserved pathways which can be utilized towards therapeutically targeting inflammatory pain and chronic inflammation.
Li, D., Luan, Y., Wang, L., Qi, M., Wang, J., Xu, J., Arefin, B. and Li, M. (2018). Expression of the Shrimp wap gene in Drosophila elicits defense responses and protease inhibitory activity. Sci Rep 8(1): 8779. PubMed ID: 29884877
The wap gene encodes a single whey acidic protein (WAP) domain-containing peptide from Chinese white shrimp (Fenneropenaeus chinensis), which shows broad-spectrum antimicrobial activities and proteinase inhibitory activities in vitro. To explore the medical applications of the WAP peptide, a wap gene transgenic Drosophila melanogaster was constructed. In wap-expressing flies, high expression levels of wap gene (>100 times) were achieved, in contrast to those of control flies, by qRT-PCR analysis. The wap gene expression was associated with increased resistance to microbial infection and decreased bacterial numbers in the flies. In addition, the WAP protein extract from wap-expressing flies, compared with control protein extract from control flies, showed improved antimicrobial activities against broad Gram-positive and Gram-negative bacteria, including the clinical drug resistant bacterium of methicillin-resistant S. aureus (MRSA), improved protease inhibitor activities against crude proteinases and commercial proteinases, including elastase, subtilis proteinase A, and proteinase K in vitro, and improved growth rate and microbial resistance, as well as wound-healing in loach and mouse models. These results suggest that wap-expressing flies could be used as a food additive in aquaculture to prevent infections and a potential antibacterial for fighting drug-resistant bacteria.
Lindsay, S. A., Lin, S. J. H. and Wasserman, S. A. (2018). Short-Form Bomanins Mediate Humoral Immunity in Drosophila. J Innate Immun: 1-9. PubMed ID: 29920489
The Bomanins are a family of a dozen secreted peptides that mediate the innate response governed by the Drosophila Toll receptor. It was recently shown that deleting a cluster of 10 Bom genes blocks Toll-mediated defenses against a range of fungi and gram-positive bacteria. This study characterize the activity of individual Bom family members. Evidence is provided that the Boms overlap in function and that a single Bom gene encoding a mature peptide of just 16 amino acids can act largely or entirely independent of other family members to provide phenotypic rescue in vivo. It was further demonstrate that the Boms function in Drosophila humoral immunity, mediating the killing of the fungal pathogen Candida glabrata in an in vitro assay of cell-free hemolymph. In addition, the level of antifungal activity both in vivo and in vitro were found to be linked to the level of Bom gene expression. Although Toll dictates expression of the antimicrobial peptides (AMPs) Drosomycin and Metchnikowin, no evidence was found that Boms act by modifying the expression of the mature forms of these antifungal AMPs.
Kamareddine, L., Robins, W. P., Berkey, C. D., Mekalanos, J. J. and Watnick, P. I. (2018). The Drosophila immune deficiency pathway modulates enteroendocrine function and host metabolism. Cell Metab. PubMed ID: 29937377
Enteroendocrine cells (EEs) are interspersed between enterocytes and stem cells in the Drosophila intestinal epithelium. Like enterocytes, EEs express components of the immune deficiency (IMD) innate immune pathway, which activates transcription of genes encoding antimicrobial peptides. The discovery of large lipid droplets in intestines of IMD pathway mutants prompted an investigation of the role of the IMD pathway in the host metabolic response to its intestinal microbiota. This study provides evidence that the short-chain fatty acid acetate is a microbial metabolic signal that activates signaling through the enteroendocrine IMD pathway in a PGRP-LC-dependent manner. This, in turn, increases transcription of the gene encoding the endocrine peptide Tachykinin (Tk), which is essential for timely larval development and optimal lipid metabolism and insulin signaling. These findings suggest innate immune pathways not only provide the first line of defense against infection but also afford the intestinal microbiota control over host development and metabolism.
Martin, M., Hiroyasu, A., Guzman, R. M., Roberts, S. A. and Goodman, A. G. (2018). Analysis of Drosophila STING reveals an evolutionarily conserved antimicrobial function. Cell Rep 23(12): 3537-3550. PubMed ID: 29924997
The vertebrate protein STING, an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response and the induction of type I interferon during pathogenic infection. This study shows that a STING ortholog (dmSTING; CG1667 ) exists in Drosophila, which, similar to vertebrate STING, associates with cyclic dinucleotides to initiate an innate immune response. Following infection with Listeria monocytogenes, dmSTING activates an innate immune response via activation of the NF-kappaB transcription factor Relish, part of the immune deficiency (IMD) pathway. DmSTING-mediated activation of the immune response reduces the levels of Listeria-induced lethality and bacterial load in the host. Of significance, dmSTING triggers an innate immune response in the absence of a known functional cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) ortholog in the fly. Together, these results demonstrate that STING is an evolutionarily conserved antimicrobial effector between flies and mammals, and it comprises a key component of host defense against pathogenic infection in Drosophila.
Thuma, L., Carter, D., Weavers, H. and Martin, P. (2018). Drosophila immune cells extravasate from vessels to wounds using Tre1 GPCR and Rho signaling. J Cell Biol. PubMed ID: 29941473
Inflammation is pivotal to fight infection, clear debris, and orchestrate repair of injured tissues. Although Drosophila melanogaster have proven invaluable for studying extravascular recruitment of innate immune cells (hemocytes) to wounds, they have been somewhat neglected as viable models to investigate a key rate-limiting component of inflammation-that of immune cell extravasation across vessel walls-due to their open circulation. This study has now identified a period during pupal development when wing hearts pulse hemolymph, including circulating hemocytes, through developing wing veins. Wounding near these vessels triggers local immune cell extravasation, enabling live imaging and correlative light-electron microscopy of these events in vivo. RNAi knockdown of immune cell integrin blocks diapedesis, just as in vertebrates, and this study uncovered a novel role for Rho-like signaling through the GPCR Tre1, a gene previously implicated in the trans-epithelial migration of germ cells. This new Drosophila model complements current murine models and provides new mechanistic insight into immune cell extravasation.

Wednesday, July 18th - Behavior

Rouse, J., Watkinson, K. and Bretman, A. (2018). Flexible memory controls sperm competition responses in male Drosophila melanogaster. Proc Biol Sci 285(1879). Pubmed ID: 29848652
Males of many species use social cues to predict sperm competition (SC) and tailor their reproductive strategies, such as ejaculate or behavioural investment, accordingly. While these plastic strategies are widespread, the underlying mechanisms remain largely unknown. Plastic behaviour requires individuals to learn and memorize cues associated with environmental change before using this experience to modify behaviour. Drosophila melanogaster respond to an increase in SC threat by extending mating duration after exposure to a rival male. This behaviour shows lag times between environmental change and behavioural response suggestive of acquisition and loss of memory. Considering olfaction is important for a male's ability to assess the SC environment, it is hypothesized that an olfactory learning and memory pathway may play a key role in controlling this plastic behaviour. The role were assessed of genes and brain structures known to be involved in learning and memory. SC responses depend on anaesthesia-sensitive memory, specifically the genes rut and amn. The gamma lobes of the mushroom bodies are integral to the control of plastic mating behaviour. These results reveal the genetic and neural properties required for reacting to changes in the SC environment.
Song, W., Zhao, L., Tao, Y., Guo, X., Jia, J., He, L., Huang, Y., Zhu, Y., Chen, P. and Qin, H. (2018). The interruptive effect of electric shock on odor response requires mushroom bodies in Drosophila melanogaster. Genes Brain Behav: e12488. Pubmed ID: 29808570
Nociceptive stimulus involuntarily interrupts concurrent activities. This interruptive effect is related to the protective function of nociception that is believed to be under stringent evolutionary pressure. Background noxious electric shock (ES) dramatically interrupts Drosophila odor response behaviors in a T-maze, termed blocking odor-response by electric-shock (BOBE). ES could interrupt both odor avoidance and odor approach. To identify involved brain areas, focus placed on the odor avoidance to 3-OCT. By spatially abolishing neurotransmission with temperature sensitive Shibire(TS1), This study found that mushroom bodies (MBs) are necessary for BOBE. Among the three major MB Kenyon cell (KCs) subtypes, α/β neurons and γ neurons but not α'/β' neurons are required for normal BOBE. Specifically, abolishing the neurotransmission of either α/β surface (α/&betas;), α/β core (α/βc) or γ dorsal (γd) neurons alone is sufficient to abrogate BOBE. This pattern of MB subset requirement is distinct from that of aversive olfactory learning, indicating a specialized BOBE pathway. Consistent with this idea, BOBE wasn't diminished in several associative memory mutants and noxious ES interrupted both innate and learned odor avoidance. Overall, These results suggest that MB α/β and γ neurons are parts of a previously unappreciated central neural circuit that processes the interruptive effect of nociception.
Fei, Y., Zhu, D., Sun, Y., Gong, C., Huang, S. and Gong, Z. (2018). Repeated failure in reward pursuit alters innate Drosophila larval behaviors. Neurosci Bull. PubMed ID: 29951979
Animals always seek rewards and the related neural basis has been well studied. However, what happens when animals fail to get a reward is largely unknown, although this is commonly seen in behaviors such as predation. This study set up a behavioral model of repeated failure in reward pursuit (RFRP) in Drosophila larvae. In this model, the larvae were repeatedly prevented from reaching attractants such as yeast and butyl acetate, before finally abandoning further attempts. After giving up, they usually showed a decreased locomotor speed and impaired performance in light avoidance and sugar preference, which were named as phenotypes of RFRP states. In larvae that had developed RFRP phenotypes, the octopamine concentration was greatly elevated, while tbetah mutants devoid of octopamine were less likely to develop RFRP phenotypes, and octopamine feeding efficiently restored such defects. By down-regulating tbetah in different groups of neurons and imaging neuronal activity, neurons that regulated the development of RFRP states and the behavioral exhibition of RFRP phenotypes were mapped to a small subgroup of non-glutamatergic and glutamatergic octopaminergic neurons in the central larval brain. These results establish a model for investigating the effect of depriving an expected reward in Drosophila and provide a simplified framework for the associated neural basis.
Ojima, N., Hara, Y., Ito, H. and Yamamoto, D. (2018). Genetic dissection of stress-induced reproductive arrest in Drosophila melanogaster females. PLoS Genet 14(6): e1007434. PubMed ID: 29889831
By genetic manipulations, the roles played by insulin-producing cells (IPCs) in the brain and their target, the corpora allata (CA), was studied for reproductive dormancy in female Drosophila melanogaster that was induced by exposing them to a combination of low temperature (11 ° C), short-day photoperiod (10L:14D) and starvation (water only) for 7 days immediately after eclosion (dormancy-inducing conditions). Artificial inactivation of IPCs promotes, whereas artificial activation impedes, the induction of reproductive dormancy. A transcriptional reporter assay reveals that the IPC activity is reduced when the female flies are exposed to dormancy-inducing conditions. The photoperiod sensitivity of reproductive dormancy is lost in pigment-dispersing factor (pdf), but not cry, mutants, suggesting that light input to IPCs is mediated by pdf-expressing neurons. Genetic manipulations to upregulate and downregulate insulin signaling in the CA, a pair of endocrine organs that synthesize the juvenile hormone (JH), decrease and increase the incidence of reproductive dormancy, respectively. These results demonstrate that the IPC-CA axis constitutes a key regulatory pathway for reproductive dormancy.
Cande, J., Namiki, S., Qiu, J., Korff, W., Card, G. M., Shaevitz, J. W., Stern, D. L. and Berman, G. J. (2018). Optogenetic dissection of descending behavioral control in Drosophila. Elife 7. PubMed ID: 29943729
In most animals, the brain makes behavioral decisions that are transmitted by descending neurons to the nerve cord circuitry that produces behaviors. In insects, only a few descending neurons have been associated with specific behaviors. To explore how descending neurons control an insect's movements, this study developed a novel method to systematically assay the behavioral effects of activating individual neurons on freely behaving terrestrial D. melanogaster. A two-dimensional representation was calculated of the entire behavior space explored by these flies, and descending neurons with specific behaviors were associated by identifying regions of this space that were visited with increased frequency during optogenetic activation. Applying this approach across a large collection of descending neurons, it was found that (1) activation of most of the descending neurons drove stereotyped behaviors, (2) in many cases multiple descending neurons activated similar behaviors, and (3) optogenetically-activated behaviors were often dependent on the behavioral state prior to activation.
Marescotti, M., Lagogiannis, K., Webb, B., Davies, R. W. and Armstrong, J. D. (2018).. Monitoring brain activity and behaviour in freely moving Drosophila larvae using bioluminescence. Sci Rep 8(1): 9246. PubMed ID: 29915372
This study presents a bioluminescence method, based on the calcium-reporter Aequorin (AEQ), that exploits targeted transgenic expression patterns to identify activity of specific neural groups in the larval Drosophila nervous system. First, for intact but constrained larva, the choice of Aequorin transgene and method of delivery of the co-factor coelenterazine was refined, and the luminescence signal produced for different neural expression patterns and concentrations of co-factor was assayed using standard photo-counting techniques. An apparatus was developed that allows simultaneous measurement of this neural signal while video recording the crawling path of an unconstrained animal. The setup also enables delivery and measurement of an olfactory cue (CO2) and the ability was demonstrated to record synchronized changes in Kenyon cell activity and crawling speed caused by the stimulus. This approach is thus shown to be an effective and affordable method for studying the neural basis of behavior in Drosophila larvae.

Tuesday, July 17th - Oogenesis and Spermatogenesis

Niepielko, M. G., Eagle, W. V. I. and Gavis, E. R. (2018). Stochastic seeding coupled with mRNA self-recruitment generates heterogeneous Drosophila germ granules. Curr Biol 28(12): 1872-1881.e1873. PubMed ID: 29861136
The formation of ribonucleoprotein assemblies called germ granules is a conserved feature of germline development. In Drosophila, germ granules form at the posterior of the oocyte in a specialized cytoplasm called the germ plasm, which specifies germline fate during embryogenesis. mRNAs, including nanos (nos) and polar granule component (pgc), that function in germline development are localized to the germ plasm through their incorporation into germ granules, which deliver them to the primordial germ cells. Germ granules are nucleated by Oskar (Osk) protein and contain varying combinations and quantities of their constituent mRNAs, which are organized as spatially distinct, multi-copy homotypic clusters. The process that gives rise to such heterogeneous yet organized granules remains unknown. This study show that individual nos and pgc transcripts can populate the same nascent granule, and these first transcripts then act as seeds, recruiting additional like transcripts to form homotypic clusters. Within a granule, homotypic clusters grow independently of each other but depend on the simultaneous acquisition of additional Osk. Although granules can contain multiple clusters of a particular mRNA, granule mRNA content is dominated by cluster size. These results suggest that the accumulation of mRNAs in the germ plasm is controlled by the mRNAs themselves through their ability to form homotypic clusters; thus, RNA self-association drives germ granule mRNA localization. It is proposed that a stochastic seeding and self-recruitment mechanism enables granules to simultaneously incorporate many different mRNAs while ensuring that each becomes enriched to a functional threshold.
Morita, S., Ota, R. and Kobayashi, S. (2018). Downregulation of NHP2 promotes proper cyst formation in Drosophila ovary. Dev Growth Differ. PubMed ID: 29845608
In Drosophila ovary, germline stem cells (GSCs) divide to produce two daughter cells. One daughter is maintained as a GSC, whereas the other initiates cyst formation, a process involving four synchronous mitotic divisions that form 2-, 4-, 8-, and 16-cell cysts. This study found that reduction in the level of NHP2, a component of the H/ACA small nucleolar ribonucleoprotein complex that catalyzes rRNA pseudouridylation, promotes progression to 8-cell cysts. NHP2 protein was concentrated in the nucleoli of germline cells during cyst formation. NHP2 expression, as well as the nucleolar size, abruptly decreased during progression from 2-cell to 4-cell cysts. Reduction in NHP2 activity in the germline caused accumulation of 4- and 8-cell cysts and decreased the number of single cells. In addition, NHP2 knockdown impaired the transition to 16-cell cysts. Furthermore, a tumorous phenotype caused by Sex-lethal (Sxl) knockdown, which is characterized by accumulation of single and two-cell cysts, was partially rescued by NHP2 knockdown. When Sxl and NHP2 activities were concomitantly repressed, the numbers of four- and eight-cell cysts were increased. In addition, Sxl protein physically interacted with NHP2 mRNA in ovaries. Thus, it is reasonable to conclude that Sxl represses NHP2 activity at the post-transcriptional level to promote proper cyst formation. Because NHP2 knockdown did not affect global protein synthesis in the germarium, it is speculated that changes in NHP2-dependent pseudouridylation, which is involved in translation of specific mRNAs, must be intact in order to promote proper cyst formation.
Kubo, A., Matsuka, M., Minami, R., Kimura, F., Sakata-Niitsu, R., Kokuryo, A., Taniguchi, K., Adachi-Yamada, T. and Nakagoshi, H. (2018). Nutrient conditions sensed by the reproductive organ during development optimize male fecundity in Drosophila. Genes Cells. PubMed ID: 29846027
Nutrient conditions affect the reproductive potential and lifespan of many organisms through the insulin signaling pathway. Although this is well characterized in female oogenesis, nutrient-dependent regulation of fertility/fecundity in males is not known. Seminal fluid components synthesized in the accessory gland are required for high fecundity in Drosophila males. The accessory gland is composed of two types of binucleated cells: a main cell and a secondary cell (SC). The transcription factors Defective proventriculus (Dve) and Abdominal-B (Abd-B) are strongly expressed in adult SCs, whose functions are essential for male fecundity. Gene expression of both Dve and Abd-B was down-regulated under nutrient-poor conditions. In addition, nutrient conditions during the pupal stage affected the size and number of SCs. These morphological changes clearly correlated with fecundity, suggesting that SCs act as nutrient sensors. This study provides evidence that Dve associates nutrient conditions with optimal reproductive potential in a target of rapamycin signaling-dependent manner.
Neitzel, L. R., Broadus, M. R., Zhang, N., Sawyer, L., Wallace, H. A., Merkle, J. A., Jodoin, J. N., Sitaram, P., Crispi, E. E., Rork, W., Lee, L. A., Pan, D., Gould, K. L., Page-McCaw, A. and Lee, E. (2018). Characterization of a cdc14 null allele in Drosophila melanogaster. Biol Open. PubMed ID: 29945873
Cdc14 is an evolutionarily conserved serine/threonine phosphatase. Originally identified in S. cerevisiae as a cell cycle regulator, its role in other eukaryotic organisms remains unclear. In Drosophila melanogaster, Cdc14 is encoded by a single gene, thus facilitating its study. Cdc14 expression is highest in the testis of adult flies and cdc14 null flies are viable. cdc14 null female and male flies do not display altered fertility. cdc14 null males, however, exhibit decreased sperm competitiveness. Previous studies have shown that Cdc14 plays a role in ciliogenesis during zebrafish development. In Drosophila, sensory neurons are ciliated. The Drosophila cdc14 null mutants have defects in chemosensation and mechanosensation as indicated by decreased avoidance of repellant substances and decreased response to touch. In addition, it was shown that cdc14 null mutants have defects in lipid metabolism and resistance to starvation. These studies highlight the diversity of Cdc14 function in eukaryotes despite its structural conservation.
Ote, M. and Yamamoto, D. (2018). The Wolbachia protein TomO interacts with a host RNA to induce polarization defects in Drosophila oocytes. Arch Insect Biochem Physiol: e21475. Pubmed ID: 29851149
Wolbachia is an endosymbiont prevalent in arthropods. To maximize its transmission thorough the female germline, Wolbachia induces in infected hosts male-to-female transformation, male killing, parthenogenesis, and cytoplasmic incompatibility, depending on the host species and Wolbachia strain involved. However, the molecular mechanisms underlying these host manipulations by Wolbachia remain largely unknown. The Wolbachia strain wMel, an inhabitant of Drosophila melanogaster, impairs host oogenesis only when transplanted into a heterologous host, for example, Drosophila simulans. Egg polarity defects induced by wMel infection in D. simulans can be recapitulated in the natural host D. melanogaster by transgenic overexpression of a variant of the Wolbachia protein Toxic manipulator of oogenesis (TomO), TomOwMel(HS) , in the female germline. RNA immunoprecipitation assays demonstrated that TomO physically associates with orb mRNA, which, as a result, fails to interact with the translation repressor Cup. This leads to precocious translation of Orb, a posterior determinant, and thereby to the misspecification of oocytes and accompanying polarity defects. It is proposed that the ability of TomO to bind to orb mRNA might provide a means for Wolbachia to enter the oocyte located at the posterior end of the egg chamber, thereby accomplishing secure maternal transmission thorough the female germline.
Chung, C. Y., Hsiao, Y. M., Huang, T. Y., Chang, T. H. and Chang, C. C. (2018). Germline expression of the hunchback orthologues in the asexual viviparous aphids: A conserved feature within the Aphididae. Insect Mol Biol. PubMed ID: 29892979
In animals, differentiation of germline from soma usually takes place during embryogenesis. Genes and their products that are preferentially expressed in the embryonic germ cells are regarded as candidates for maintaining germline fate or promoting germline identity. In Drosophila, for example, the protein encoded by the germline gene vasa is specifically restricted to the germ cells while products of the gap gene hunchback (hb), a somatic gene, are preferentially expressed in the neuroblasts. This study reports the expression of both mRNA and protein encoded by Aphb, an hb orthologue in the asexual viviparous pea aphid Acyrthosiphon pisum, in germ cells as well as in neuroblasts. It is inferred that expression of Aphb mRNA in the germ cells during the formation of germaria is required for the anterior localization of Aphb in the protruding oocytes. Germarial expression and anterior localization of ApKruppel was also identified but, unlike Aphb, its expression was not detected in the migrating germ cells. Very similar patterns of hb expression were also identified in the green peach aphid Myzus persicae, suggesting that germline expression of hb is conserved within the Aphididae. To date, this pattern of hb germline expression has not been reported in other insects.

Monday, July 16th - Vesicles and Synapses

Crespo-Yanez, X., Aguilar-Gurrieri, C., Jacomin, A. C., Journet, A., Mortier, M., Taillebourg, E., Soleilhac, E., Weissenhorn, W. and Fauvarque, M. O. (2018). CHMP1B is a target of USP8/UBPY regulated by ubiquitin during endocytosis. PLoS Genet 14(6): e1007456. PubMed ID: 29933386
Integration and down-regulation of cell growth and differentiation signals rely on plasma membrane receptors endocytosis and sorting towards either recycling vesicles or degradative lysosomes via multivesicular bodies (MVB). In this process, the endosomal sorting complex-III required for transport (ESCRT-III) controls membrane deformation and scission triggering intraluminal vesicle (ILV) formation at early endosomes. This study shows that the ESCRT-III member CHMP1B can be ubiquitinated within a flexible loop known to undergo conformational changes during polymerization. This study demonstrates further that CHMP1B is deubiquitinated by the ubiquitin specific protease USP8 (syn. UBPY) and found fully devoid of ubiquitin in a ~500 kDa large complex that also contains its known ESCRT-III partner IST1. Moreover, EGF stimulation induces the rapid and transient accumulation of ubiquitinated forms of CHMP1B on cell membranes. Accordingly, CHMP1B ubiquitination is necessary for CHMP1B function in both EGF receptor trafficking in human cells and wing development in Drosophila. From these observations, it is proposed that CHMP1B is dynamically regulated by ubiquitination in response to EGF and that USP8 triggers CHMP1B deubiquitination possibly favoring its subsequent assembly into a membrane-associated ESCRT-III polymer.
Ke, H., Feng, Z., Liu, M., Sun, T., Dai, J., Ma, M., Liu, L. P., Ni, J. Q. and Pastor-Pareja, J. C. (2018). Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements. J Genet Genomics. PubMed ID: 29935791
This study reports an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, distribution of GFP-tagged Collagen IV was examined in live animals, and 88 gene hits were found for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4alphaEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in the hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. Additionally a characterization of a screening hit, trabuco (tbc), is presented, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, the success is discussed of this screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.
Neuman, S. D. and Bashirullah, A. (2018). Hobbit regulates intracellular trafficking to drive insulin-dependent growth during Drosophila development. Development 145(11). PubMed ID: 29891564
All animals must coordinate growth rate and timing of maturation to reach the appropriate final size. This study describes hobbit, a novel and conserved gene identified in a forward genetic screen for Drosophila animals with small body size. hobbit is highly conserved throughout eukaryotes, but its function remains unknown. hobbit mutant animals have systemic growth defects because they fail to secrete insulin. Other regulated secretion events also fail in hobbit mutant animals, including mucin-like 'glue' protein secretion from the larval salivary glands. hobbit mutant salivary glands produce glue-containing secretory granules that are reduced in size. Importantly, secretory granules in hobbit mutant cells lack essential membrane fusion machinery required for exocytosis, including Synaptotagmin 1 and the SNARE SNAP-24. These membrane fusion proteins instead accumulate inside enlarged late endosomes. Surprisingly, however, the Hobbit protein localizes to the endoplasmic reticulum. The results suggest that Hobbit regulates a novel step in intracellular trafficking of membrane fusion proteins. These studies also suggest that genetic control of body size, as a measure of insulin secretion, is a sensitive functional readout of the secretory machinery.
Zhao, H., Wang, J. and Wang, T. (2018). The V-ATPase V1 subunit A1 Is required for rhodopsin anterograde trafficking in Drosophila. Mol Biol Cell: mbcE17090546. Pubmed ID: 29742016
Synthesis and maturation of the light sensor, rhodopsin, is critical for the maintenance of light sensitivity and for photoreceptor homeostasis. In Drosophila, the main rhodopsin, Rh1, is synthesized in the endoplasmic reticulum and transported to the rhabdomere through the secretory pathway. In an unbiased genetic screen for factors involved in rhodopsin homeostasis, mutations in vha68-1, which encodes V-ATPase catalytic subunit A isoform 1 of the V1 component, were identified. Loss of vha68-1 in photoreceptor cells disrupted post-Golgi anterograde trafficking of Rh1, reduced light sensitivity, increased secretory vesicle pH, and resulted in incomplete Rh1 deglycosylation. In addition, vha68-1 was required for activity-independent photoreceptor cell survival. Importantly, vha68-1 mutants exhibited phenotypes similar to those exhibited by mutations in the V0 component of V-ATPase, vha100-1. These data demonstrate that V1 and V0 components of V-ATPase play key roles in post-Golgi trafficking of Rh1, and that Drosophila may represent an important animal model system for studying diseases associated with V-ATPase dysfunction.
Lund, V. K., Madsen, K. L. and Kjaerulff, O. (2018). Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes. Autophagy. PubMed ID: 29940804
Rab2 is a conserved Rab GTPase with a well-established role in secretory pathway function and phagocytosis. This study demonstrates that Drosophila Rab2 is recruited to late endosomal membranes, where it controls the fusion of LAMP-containing biosynthetic carriers and lysosomes to late endosomes. In contrast, the lysosomal GTPase Gie/Arl8 is only required for late endosome-lysosome fusion, but not for the delivery of LAMP to the endocytic pathway. Rab2 was also found to be required for the fusion of autophagosomes to the endolysosomal pathway, but not for the biogenesis of lysosome-related organelles. Surprisingly, Rab2 does not rely on HOPS-mediated vesicular fusion for recruitment to late endosomal membranes. This work suggests that Drosophila Rab2 is a central regulator of the endolysosomal and macroautophagic/autophagic pathways by controlling the major heterotypic fusion processes at the late endosome.
Li, X., Goel, P., Wondolowski, J., Paluch, J. and Dickman, D. (2018). A glutamate homeostat controls the presynaptic inhibition of neurotransmitter release. Cell Rep 23(6): 1716-1727. PubMed ID: 29742428
This study has interrogated the synaptic dialog that enables the bi-directional, homeostatic control of presynaptic efficacy at the glutamatergic Drosophila neuromuscular junction (NMJ). Homeostatic depression and potentiation use disparate genetic, induction, and expression mechanisms. Specifically, homeostatic potentiation is achieved through reduced CaMKII activity postsynaptically and increased abundance of active zone material presynaptically at one of the two neuronal subtypes innervating the NMJ, while homeostatic depression occurs without alterations in CaMKII activity and is expressed at both neuronal subtypes. Furthermore, homeostatic depression is only induced through excess presynaptic glutamate release and operates with disregard to the postsynaptic response. It is proposed that two independent homeostats modulate presynaptic efficacy at the Drosophila NMJ: one is an intercellular signaling system that potentiates synaptic strength following diminished postsynaptic excitability, while the other adaptively modulates presynaptic glutamate release through an autocrine mechanism without feedback from the postsynaptic compartment.

Friday, July 13th - Adult Neural Function

Chatterjee, A., Lamaze, A., De, J., Mena, W., Chelot, E., Martin, B., Hardin, P., Kadener, S., Emery, P. and Rouyer, F. (2018). Reconfiguration of a multi-oscillator network by light in the Drosophila circadian clock. Curr Biol. PubMed ID: 29910074
The brain clock that drives circadian rhythms of locomotor activity relies on a multi-oscillator neuronal network. In addition to synchronizing the clock with day-night cycles, light also reformats the clock-driven daily activity pattern. How changes in lighting conditions modify the contribution of the different oscillators to remodel the daily activity pattern remains largely unknown. Data in Drosophila indicate that light readjusts the interactions between oscillators through two different modes. This study shows that a morning s-LNv > DN1p circuit works in series, whereas two parallel evening circuits are contributed by LNds and other DN1ps. Based on the photic context, the master pacemaker in the s-LNv neurons swaps its enslaved partner-oscillator-LNd in the presence of light or DN1p in the absence of light-to always link up with the most influential phase-determining oscillator. When exposure to light further increases, the light-activated LNd pacemaker becomes independent by decoupling from the s-LNvs. The calibration of coupling by light is layered on a clock-independent network interaction wherein light upregulates the expression of the PDF neuropeptide in the s-LNvs, which inhibits the behavioral output of the DN1p evening oscillator. Thus, light modifies inter-oscillator coupling and clock-independent output-gating to achieve flexibility in the network. It is likely that the light-induced changes in the Drosophila brain circadian network could reveal general principles of adapting to varying environmental cues in any neuronal multi-oscillator system.
Lamba, P., Foley, L. E. and Emery, P. (2018). Neural network interactions modulate CRY-dependent photoresponses in Drosophila. J Neurosci. PubMed ID: 29875268
Light is one of the chief environmental cues that reset circadian clocks. In Drosophila, Cryptochrome (CRY) mediates acute photic resetting of circadian clocks by promoting the degradation of Timeless (TIM) in a cell-autonomous manner. Thus, even circadian oscillators in peripheral organs can independently perceive light in Drosophila. However, there is substantial evidence for non-autonomous mechanisms of circadian photoreception in the brain. Previous work has shown that the Morning (M) and Evening (E) oscillators are critical light-sensing neurons that cooperate to shift the phase of circadian behavior in response to light input. This study shows that light can efficiently phase-delay or phase-advance circadian locomotor behavior in male Drosophila even when either the M- or the E-oscillators are ablated, suggesting that behavioral phase shifts and their directionality are largely a consequence of the cell-autonomous nature of CRY-dependent photoreception. The observation that the Phase Response Curve (PRC) of brain and peripheral oscillators are remarkably similar further supports this idea. Nevertheless, the neural network modulates circadian photoresponses. This study shows that the M-oscillator neurotransmitter Pigment Dispersing Factor plays a critical role in the coordination between M- and E-oscillators after light exposure, and a potential role was uncovered for a subset of Dorsal Neurons in the control of phase advances. Thus, neural modulation of autonomous light detection might play an important role in the plasticity of circadian behavior.
Davie, K., Janssens, J., Koldere, D., De Waegeneer, M., Pech, U., Kreft, L., Aibar, S., Makhzami, S., Christiaens, V., Bravo Gonzalez-Blas, C., Poovathingal, S., Hulselmans, G., Spanier, K. I., Moerman, T., Vanspauwen, B., Geurs, S., Voet, T., Lammertyn, J., Thienpont, B., Liu, S., Konstantinides, N., Fiers, M., Verstreken, P. and Aerts, S. (2018). A single-cell transcriptome atlas of the aging Drosophila brain. Cell. PubMed ID: 29909982
The diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. This paper presents a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. The data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in this unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.
Konstantinides, N., Kapuralin, K., Fadil, C., Barboza, L., Satija, R. and Desplan, C. (2018). Phenotypic convergence: Distinct transcription factors regulate common terminal features. Cell. PubMed ID: 29909983
Transcription factors regulate the molecular, morphological, and physiological characteristics of neurons and generate their impressive cell-type diversity. To gain insight into the general principles that govern how transcription factors regulate cell-type diversity, large-scale single-cell RNA sequencing was used to characterize the extensive cellular diversity in the Drosophila optic lobes. 55,000 single cells were sequenced and assign to 52 clusters. Many clusters were validated and annotated using RNA sequencing of FACS-sorted single-cell types and cluster-specific genes. To identify transcription factors responsible for inducing specific terminal differentiation features, a "random forest" model was generated, and the transcription factors Apterous and Traffic-jam were shown to be required in many but not all cholinergic and glutamatergic neurons, respectively. In fact, the same terminal characters often can be regulated by different transcription factors in different cell types, arguing for extensive phenotypic convergence. These data provide a deep understanding of the developmental and functional specification of a complex brain structure.
Liou, N. F., Lin, S. H., Chen, Y. J., Tsai, K. T., Yang, C. J., Lin, T. Y., Wu, T. H., Lin, H. J., Chen, Y. T., Gohl, D. M., Silies, M. and Chou, Y. H. (2018). Diverse populations of local interneurons integrate into the Drosophila adult olfactory circuit. Nat Commun 9(1): 2232. PubMed ID: 29884811
Drosophila olfactory local interneurons (LNs) in the antennal lobe are highly diverse and variable. How and when distinct types of LNs emerge, differentiate, and integrate into the olfactory circuit is unknown. Through systematic developmental analyses, this study found that LNs are recruited to the adult olfactory circuit in three groups. Group 1 LNs are residual larval LNs. Group 2 are adult-specific LNs that emerge before cognate sensory and projection neurons establish synaptic specificity, and Group 3 LNs emerge after synaptic specificity is established. Group 1 larval LNs are selectively reintegrated into the adult circuit through pruning and re-extension of processes to distinct regions of the antennal lobe, while others die during metamorphosis. Precise temporal control of this pruning and cell death shapes the global organization of the adult antennal lobe. These findings provide a road map to understand how LNs develop and contribute to constructing the olfactory circuit.
Ludke, A., Raiser, G., Nehrkorn, J., Herz, A. V. M., Galizia, C. G. and Szyszka, P. (2018). Calcium in Kenyon cell somata as a substrate for an olfactory sensory memory in Drosophila. Front Cell Neurosci 12: 128. PubMed ID: 29867361
Animals can form associations between temporally separated stimuli. To do so, the nervous system has to retain a neural representation of the first stimulus until the second stimulus appears. The neural substrate of such sensory stimulus memories is unknown. This study searched for a sensory odor memory in the insect olfactory system and characterized odorant-evoked Ca(2+) activity at three consecutive layers of the olfactory system in Drosophila: in olfactory receptor neurons (ORNs) and projection neurons (PNs) in the antennal lobe, and in Kenyon cells (KCs) in the mushroom body. The post-stimulus responses in ORN axons, PN dendrites, PN somata, and KC dendrites are odor-specific, but they are not predictive of the chemical identity of past olfactory stimuli. However, the post-stimulus responses in KC somata carry information about the identity of previous olfactory stimuli. These findings show that the Ca(2+) dynamics in KC somata could encode a sensory memory of odorant identity and thus might serve as a basis for associations between temporally separated stimuli.

Thursday, July 12th - Larval and Adult Development

Drelon, C., Belalcazar, H. M. and Secombe, J. (2018). The histone demethylase KDM5 is essential for larval growth in Drosophila. Genetics. PubMed ID: 29764901
Regulated gene expression is necessary for developmental and homeostatic processes. The KDM5 family of transcriptional regulators are histone H3 lysine 4 demethylases that can function through both demethylase-dependent and independent mechanisms. While loss and overexpression of KDM5 proteins are linked to intellectual disability and cancer, respectively, their normal developmental functions remain less characterized. Drosophila melanogaster provides an ideal system to investigate KDM5 function, as it encodes a single ortholog in contrast to the four paralogs found in mammalian cells. To examine the consequences of complete loss of KDM5, a null allele of Drosophila kdm5, also known as little imaginal discs (lid), was generated, and it was shown to be essential for viability. Animals lacking KDM5 show a dramatically delayed larval development that coincides with decreased proliferation and increased cell death in wing imaginal discs. Interestingly, this developmental delay is independent of the well-characterized Jumonji C (JmjC) domain-encoded histone demethylase activity of KDM5, suggesting key functions for less characterized domains. Consistent with the phenotypes observed, transcriptome analyses of kdm5 null mutant wing imaginal discs revealed the dysregulation of genes involved in several cellular processes, including cell cycle progression and DNA repair. Together, these analyses reveal KDM5 as a key regulator of larval growth and offer an invaluable tool for defining the biological activities of KDM5 family proteins.
Kittelmann, S., Buffry, A. D., Franke, F. A., Almudi, I., Yoth, M., Sabaris, G., Couso, J. P., Nunes, M. D. S., Frankel, N., Gomez-Skarmeta, J. L., Pueyo-Marques, J., Arif, S. and McGregor, A. P. (2018). Gene regulatory network architecture in different developmental contexts influences the genetic basis of morphological evolution. PLoS Genet 14(5): e1007375. PubMed ID: 29723190
Convergent phenotypic evolution is often caused by recurrent changes at particular nodes in the underlying gene regulatory networks (GRNs). The genes at such evolutionary 'hotspots' are thought to maximally affect the phenotype with minimal pleiotropic consequences. This has led to the suggestion that if a GRN is understood in sufficient detail, the path of evolution may be predictable. The repeated evolutionary loss of larval trichomes among Drosophila species is caused by the loss of shavenbaby (svb) expression. svb is also required for development of leg trichomes, but the evolutionary gain of trichomes in the 'naked valley' on T2 femurs in Drosophila melanogaster is caused by reduced microRNA-92a (miR-92a) expression rather than changes in svb. The expression and function of components between the larval and leg trichome GRNs were compared to investigate why the genetic basis of trichome pattern evolution differs in these developmental contexts. Key differences were found between the two networks in both the genes employed, and in the regulation and function of common genes. These differences in the GRNs reveal why mutations in svb are unlikely to contribute to leg trichome evolution and how instead miR-92a represents the key evolutionary switch in this context. This work shows that variability in GRNs across different developmental contexts. Therefore, these findings have important implications for understanding the pathways and predictability of evolution.
Currea, J. P., Smith, J. L. and Theobald, J. C. (2018). Small fruit flies sacrifice temporal acuity to maintain contrast sensitivity. Vision Res 149: 1-8. PubMed ID: 29859226
Holometabolous insects, like fruit flies, grow primarily during larval development. Scarce larval feeding is common in nature and generates smaller adults. Despite the importance of vision to flies, eye size scales proportionately with body size, and smaller eyes confer poorer vision due to smaller optics. Variable larval feeding, therefore, causes within-species differences in visual processing, which have gone largely unnoticed due to ad libitum feeding in the lab that results in generally large adults. Do smaller eyes have smaller ommatidial lenses, reducing sensitivity, or broader inter-ommatidial angles, reducing acuity? And to what extent might neural processes adapt to these optical challenges with temporal and spatial summation? To understand this in the fruit fly, a distribution of body lengths (1.67-2.34mm; n=24) and eye lengths (0.33-0.44mm; n=24) was generated, resembling the distribution of wild-caught flies, by removing larvae from food during their third instar. Smaller eyes have substantially fewer and smaller ommatidia separated by slightly wider inter-ommatidial angles. This corresponds to a greater loss in contrast sensitivity than spatial acuity. Using a flight arena and psychophysics paradigm, it was found that smaller flies lose little spatial acuity, and recover contrast sensitivity by sacrificing temporal acuity at the neural level. Therefore, smaller flies sacrifice contrast sensitivity to maintain spatial acuity optically, but recover contrast sensitivity, almost completely, by sacrificing temporal acuity neurally.
Gonzalez-Itier, S., Contreras, E. G., Larrain, J., Glavic, A. and Faunes, F. (2018). A role for Lin-28 in growth and metamorphosis in Drosophila melanogaster. Mech Dev [Epub ahead of print]. PubMed ID: 29908237
Insect metamorphosis has been a classic model to understand the role of hormones in growth and timing of developmental transitions. In addition to hormones, transitions in some species are regulated by genetic programs, such as the heterochronic gene network discovered in C. elegans. However, the functional link between hormones and heterochronic genes is not clear. The heterochronic gene lin-28 is involved in the maintenance of stem cells, growth and developmental timing in vertebrates. This work used gain-of-function and loss-of-function experiments to study the role of Lin-28 in larval growth and the timing of metamorphosis of Drosophila melanogaster. During the late third instar stage, Lin-28 is mainly expressed in neurons of the central nervous system and in the intestine. Loss-of-function lin-28 mutant larvae are smaller and the larval-to-pupal transition is accelerated. This faster transition correlates with increased levels of ecdysone direct target genes such as Broad-Complex (BR-C) and Ecdysone Receptor (EcR). Overexpression of Lin-28 does not affect the timing of pupariation but most animals are not able to eclose, suggesting defects in metamorphosis. Overexpression of human Lin-28 results in delayed pupariation and the death of animals during metamorphosis. Altogether, these results suggest that Lin-28 is involved in the control of growth during larval development and in the timing and progression of metamorphosis.
Lee, G. J., Han, G., Yun, H. M., Lim, J. J., Noh, S., Lee, J. and Hyun, S. (2018). Steroid signaling mediates nutritional regulation of juvenile body growth via IGF-binding protein in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 29784791
Nutritional condition during the juvenile growth period considerably affects final adult size. The insulin/insulin-like growth factor signaling (IIS)/target of rapamycin (TOR) nutrient-sensing pathway is known to regulate growth and metabolism in response to nutritional conditions. However, there is limited information on how endocrine pathways communicate nutritional information to different metabolic organs to regulate organismal growth. This study shows that Imaginal morphogenesis protein-Late 2 (Imp-L2), a Drosophila homolog of insulin-like growth factor-binding protein 7 (IGFBP7), plays a key role in the nutritional control of organismal growth. Nutritional restriction during the larval growth period causes undersized adults, which is largely diminished by Imp-L2 mutation. A pathway was delineated in which nutritional restriction increases levels of the steroid hormone ecdysone, which, in turn, triggers ecdysone signaling-dependent Imp-L2 production from the fat body, a fly adipose organ, thereby attenuating peripheral IIS and body growth. Surprisingly, this endocrine pathway operates independent of the fat-body-TOR internal nutrient sensor. This study reveals a previously unrecognized endocrine circuit mediating nutrition-dependent juvenile growth.
Lloyd, D. L., Toegel, M., Fulga, T. A. and Wilkie, A. O. M. (2018). The Drosophila homologue of MEGF8 is essential for early development. Sci Rep 8(1): 8790. PubMed ID: 29884872
Mutations of the gene MEGF8 cause Carpenter syndrome in humans, and the mouse orthologue has been functionally associated with Nodal and Bmp4 signalling. This study has investigated the phenotype associated with loss-of-function of CG7466, a gene that encodes the Drosophila homologue of MEGF8. Three different frame-shift null mutations were generated in CG7466 using CRISPR/Cas9 gene editing. Heterozygous flies appeared normal, but homozygous animals had disorganised denticle belts and died as 2nd or 3rd instar larvae. Larvae were delayed in transition to 3rd instars and showed arrested growth, which was associated with abnormal feeding behaviour and prolonged survival when yeast food was supplemented with sucrose. RNAi-mediated knockdown using the Gal4-UAS system resulted in lethality with ubiquitous and tissue-specific Gal4 drivers, and growth defects including abnormal bristle number and orientation in a subset of escapers. It is concluded that CG7466 is essential for larval development and that diminished function perturbs denticle and bristle formation.

Wednesday, July 11th - Disease Models

Jantrapirom, S., Piccolo, L. L., Yoshida, H. and Yamaguchi, M. (2018). Depletion of Ubiquilin induces an augmentation in soluble ubiquitinated Drosophila TDP-43 to drive neurotoxicity in the fly. Biochim Biophys Acta. PubMed ID: 29936333
The proteostasis machinery has critical functions in metabolically active cells such as neurons. Ubiquilins (UBQLNs) may decide the fate of proteins, with its ability to bind and deliver ubiquitinated misfolded or no longer functionally required proteins to the ubiquitin-proteasome system (UPS) and/or autophagy. Missense mutations in UBQLN2 have been linked to X-linked dominant amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). Although aggregation-prone TAR DNA-binding protein 43 (TDP-43) has been recognized as a major component of the ubiquitin pathology, the mechanisms by which UBQLN involves in TDP-43 proteinopathy have not yet been elucidated in detail. Previous work has characterized new Drosophila Ubiquilin (dUbqn) knockdown model that produces learning/memory and locomotive deficits during the proteostasis impairment. The present study demonstrated that the depletion of dUbqn markedly affected the expression and sub-cellular localization of Drosophila TDP-43 (TBPH), resulting in a cytoplasmic ubiquitin-positive (Ub(+)) TBPH pathology. Although it was found that the knockdown of dUbqn widely altered and affected the turnover of a large number of proteins, this study showed that an augmented soluble cytoplasmic Ub(+)-TBPH is as a crucial source of neurotoxicity following the depletion of dUbqn. It was demonstrated that dUbqn knockdown-related neurotoxicity may be rescued by either restoring the proteostasis machinery or reducing the expression of TBPH. These novel results extend knowledge on the UBQLN loss-of-function pathomechanism and may contribute to the identification of new therapeutics for ALS-FTD and aging-related diseases.
Lavoy, S., Chittoor-Vinod, V. G., Chow, C. Y. and Martin, I. (2018). Genetic Modifiers of Neurodegeneration in a Drosophila Model of Parkinson's Disease. Genetics. PubMed ID: 29907646
Disease phenotypes can be highly variable among individuals with the same pathogenic mutation. There is increasing evidence that background genetic variation is a strong driver of disease variability in addition to the influence of environment. To understand the genotype-phenotype relationship that determines the expressivity of a pathogenic mutation, a large number of backgrounds must be studied. This can be efficiently achieved using model organism collections such as the Drosophila Genetic Reference Panel (DGRP). This study used the DGRP to assess the variability of locomotor dysfunction in a LRRK2 G2019S Drosophila melanogaster model of Parkinson's disease. Substantial variability was found in the LRRK2 G2019S locomotor phenotype in different DGRP backgrounds. A genome-wide association study for candidate genetic modifiers reveals 177 genes that drive wide phenotypic variation, including 19 top association genes. Genes involved in the outgrowth and regulation of neuronal projections are enriched in these candidate modifiers. RNAi functional testing of the top association and neuronal projection-related genes reveals that pros, pbl, ct and CG33506 significantly modify age-related dopamine neuron loss and associated locomotor dysfunction in the Drosophila LRRK2 G2019S model. These results demonstrate how natural genetic variation can be used as a powerful tool to identify genes that modify disease-related phenotypes. This study reports novel candidate modifier genes for LRRK2 G2019S that may be used to interrogate the link between LRRK2, neurite regulation and neuronal degeneration in Parkinson's disease.
El Fissi, N., Rojo, M., Aouane, A., Karatas, E., Poliacikova, G., David, C., Royet, J. and Rival, T. (2018). Mitofusin gain and loss of function drive pathogenesis in Drosophila models of CMT2A neuropathy. EMBO Rep. PubMed ID: 29898954
Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by dominant alleles of the mitochondrial pro-fusion factor Mitofusin 2 (MFN2; see Drosophila Marf). To address the consequences of these mutations on mitofusin activity and neuronal function, this study generate Drosophila models expressing in neurons the two most frequent substitutions (R94Q and R364W, the latter never studied before) and two others localizing to similar domains (T105M and L76P). All alleles trigger locomotor deficits associated with mitochondrial depletion at neuromuscular junctions, decreased oxidative metabolism and increased mtDNA mutations, but they differently alter mitochondrial morphology and organization. Substitutions near or within the GTPase domain (R94Q, T105M) result in loss of function and provoke aggregation of unfused mitochondria. In contrast, mutations within helix bundle 1 (R364W, L76P) enhance mitochondrial fusion, as demonstrated by the rescue of mitochondrial alterations and locomotor deficits by over-expression of the fission factor DRP1. In conclusion, this study shows that both dominant negative and dominant active forms of mitofusin can cause CMT2A-associated defects and propose for the first time that excessive mitochondrial fusion drives CMT2A pathogenesis in a large number of patients.
Dahl-Halvarsson, M., Olive, M., Pokrzywa, M., Ejeskar, K., Palmer, R. H., Uv, A. E. and Tajsharghi, H. (2018). Drosophila model of myosin myopathy rescued by overexpression of a TRIM-protein family member. Proc Natl Acad Sci U S A. PubMed ID: 29946036
Myosin is a molecular motor indispensable for body movement and heart contractility. Apart from pure cardiomyopathy, mutations in MYH7 encoding slow/beta-cardiac myosin heavy chain also cause skeletal muscle disease with or without cardiac involvement. Mutations within the alpha-helical rod domain of MYH7 are mainly associated with Laing distal myopathy. A Drosophila model of Laing distal myopathy was developed by genomic engineering of the Drosophila Mhc locus. Flies expressing only Mhc(K1728del) in indirect flight and jump muscles, and heterozygous Mhc(K1728del) animals, were flightless, with reduced movement and decreased lifespan. Sarcomeres of Mhc(K1728del) mutant indirect flight muscles and larval body wall muscles were disrupted with clearly disorganized muscle filaments. Homozygous Mhc(K1728del) larvae also demonstrated structural and functional impairments in heart muscle. The impaired jump and flight ability and the myopathy of indirect flight and leg muscles associated with Mhc(K1728del) were fully suppressed by expression of Abba/Thin, an E3-ligase that is essential for maintaining sarcomere integrity. This model of Laing distal myopathy in Drosophila recapitulates certain morphological phenotypic features seen in Laing distal myopathy patients with the recurrent K1729del mutation. These observations that Abba/Thin modulates these phenotypes suggest that manipulation of Abba/Thin activity levels may be beneficial in Laing distal myopathy.
Guo, C., Jeong, H. H., Hsieh, Y. C., Klein, H. U., Bennett, D. A., De Jager, P. L., Liu, Z. and Shulman, J. M. (2018). Tau activates transposable elements in Alzheimer's disease. Cell Rep 23(10): 2874-2880. PubMed ID: 29874575
Aging and neurodegenerative disease are characterized by genomic instability in neurons, including aberrant activation and mobilization of transposable elements (TEs). Integrating studies of human postmortem brain tissue and Drosophila melanogaster models, TE activation was investigated in association with Tau pathology in Alzheimer's disease (AD). Leveraging RNA sequencing from 636 human brains, differential expression was discovered for several retrotransposons in association with neurofibrillary tangle burden and highlight evidence for global TE transcriptional activation among the long interspersed nuclear element 1 and endogenous retrovirus clades. In addition, Tau-associated, active chromatin signatures were detected at multiple HERV-Fc1 genomic loci. To determine whether Tau is sufficient to induce TE activation, retrotransposons were profiled in Drosophila expressing human wild-type or mutant Tau throughout the brain. Heterogeneous response profiles were discovered, including both age- and genotype-dependent activation of TE expression by Tau. The results implicate TE activation and associated genomic instability in Tau-mediated AD mechanisms.
Al-Ramahi, I., Lu, B., Di Paola, S., Pang, K., de Haro, M., Peluso, I., Gallego-Flores, T., Malik, N. T., Erikson, K., Bleiberg, B. A., Avalos, M., Fan, G., Rivers, L. E., Laitman, A. M., Diaz-Garcia, J. R., Hild, M., Palacino, J., Liu, Z., Medina, D. L. and Botas, J. (2018). High-Throughput Functional Analysis Distinguishes Pathogenic, Nonpathogenic, and Compensatory Transcriptional Changes in Neurodegeneration. Cell Syst. PubMed ID: 29936182
Discriminating transcriptional changes that drive disease pathogenesis from nonpathogenic and compensatory responses is a daunting challenge. This is particularly true for neurodegenerative diseases, which affect the expression of thousands of genes in different brain regions at different disease stages. This study integrates functional testing and network approaches to analyze previously reported transcriptional alterations in the brains of Huntington disease (HD) patients. 312 genes were selected whose expression is dysregulated both in HD patients and in HD mice and then replicated and/or antagonized each alteration in a Drosophila HD model. High-throughput behavioral testing in this model and controls revealed that transcriptional changes in synaptic biology and calcium signaling are compensatory, whereas alterations involving the actin cytoskeleton and inflammation drive disease. Knockdown of disease-driving genes in HD patient-derived cells lowered mutant Huntingtin levels and activated macroautophagy, suggesting a mechanism for mitigating pathogenesis. This multilayered approach can thus untangle the wealth of information generated by transcriptomics and identify early therapeutic intervention points.

Tuesday, July 10th - Apoptosis and Autophagy

Billes, V., et al. (2018). Developmentally regulated autophagy is required for eye formation in Drosophila. Autophagy. PubMed ID: 29940806
This study demonstrates that autophagy, an evolutionarily conserved self-degradation process of eukaryotic cells, is essential for eye development in this organism. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. The results also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog lab (labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by labial. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought.
Feng, Y., Li, Z., Lv, L., Du, A., Lin, Z., Ye, X., Lin, Y. and Lin, X. (2018). Tankyrase regulates apoptosis by activating JNK signaling in Drosophila. Biochem Biophys Res Commun. PubMed ID: 29953853
Programmed cell death (PCD), or apoptosis, plays essential roles in various cellular and developmental processes, and deregulation of apoptosis causes many diseases. Thus, regulation of apoptotic process is very important. Drosophila tankyrase (DTNKS) is an evolutionarily conserved protein with poly (ADP-ribose) polymerase activity. In mammalian cells, tankyrases (TNKSs) have been reported to regulate cell death. To determine whether DTNKS plays function in inducing apoptosis in in vivo development, this study used Drosophila as a model system and generate transgenic flies expressing DTNKS. Ectopic expression of DTNKS promotes caspase-dependent apoptosis and knockdown of DTNKS by RNAi dramatically alleviates apoptotic defect caused by ectopic expression of pro-apoptotic proteins hid or rpr during eye development. Moreover, the result shows that ectopic expression of DTNKS triggers the activation of c-Jun N-terminal kinase (JNK) signaling, which is required for DTNKS-mediated apoptosis. Taken together, these findings have identified the role of DTNKS in regulating apoptosis by activating JNK signaling in Drosophila.
Denton, D., Xu, T., Dayan, S., Nicolson, S. and Kumar, S. (2018). Dpp regulates autophagy-dependent midgut removal and signals to block ecdysone production. Cell Death Differ. PubMed ID: 29959404
Animal development and homeostasis require the programmed removal of cells. Autophagy-dependent cell deletion is a unique form of cell death often involved in bulk degradation of tissues. In Drosophila the steroid hormone ecdysone controls developmental transitions and triggers the autophagy-dependent removal of the obsolete larval midgut. The production of ecdysone is exquisitely coordinated with signals from numerous organ systems to mediate the correct timing of such developmental programs. This study shows that blocking Dpp signaling induces premature autophagy, rapid cell death, and midgut degradation, whereas sustained Dpp signaling inhibits autophagy induction. Furthermore, Dpp signaling in the midgut prevents the expression of ecdysone responsive genes and impairs ecdysone production in the prothoracic gland. It is proposed that Dpp has dual roles: one within the midgut to prevent improper tissue degradation, and one in inter-organ communication to coordinate ecdysone biosynthesis and developmental timing.
Liu, Y., Gordesky-Gold, B., Leney-Greene, M., Weinbren, N. L., Tudor, M. and Cherry, S. (2018). Inflammation-induced, STING-dependent autophagy restricts Zika virus infection in the Drosophila brain. Cell Host Microbe. PubMed ID: 29934091
The emerging arthropod-borne flavivirus Zika virus (ZIKV) is associated with neurological complications. Innate immunity is essential for the control of virus infection, but the innate immune mechanisms that impact viral infection of neurons remain poorly defined. Using the genetically tractable Drosophila system, this study shows that ZIKV infection of the adult fly brain leads to NF-kappaB-dependent inflammatory signaling, which serves to limit infection. ZIKV-dependent NF-kappaB activation induces the expression of Drosophila stimulator of interferon genes (dSTING) in the brain. dSTING protects against ZIKV by inducing autophagy in the brain. Loss of autophagy leads to increased ZIKV infection of the brain and death of the infected fly, while pharmacological activation of autophagy is protective. These data suggest an essential role for an inflammation-dependent STING pathway in the control of neuronal infection and a conserved role for STING in antimicrobial autophagy, which may represent an ancestral function for this essential innate immune sensor.

Monday, July 9th - Embryonic Development

Hodar, C. and Cambiazo, V. (2018). The dorsoventral patterning of Musca domestica embryos: insights into BMP/Dpp evolution from the base of the lower cyclorraphan flies. Evodevo 9: 13. PubMed ID: 29796243
In the last few years, accumulated information has indicated that the evolution of an extra-embryonic membrane in dipterans was accompanied by changes in the gene regulatory network controlled by the BMP/Dpp pathway, which is responsible for dorsal patterning in these insects. However, only comparative analysis of gene expression levels between distant species with two extra-embryonic membranes, like A. gambiae or C. albipunctata, and D. melanogaster, has been conducted. Analysis of gene expression in ancestral species, which evolved closer to the amnioserosa origin, could provide new insights into the evolution of dorsoventral patterning in dipterans. This study describes the spatial expression of several key and downstream elements of the Dpp pathway and show the compared patterns of expression between Musca and Drosophila embryos, both dipterans with amnioserosa. Most of the analyzed genes showed a high degree of expression conservation, however, several differences were found in the gene expression pattern of M. domestica orthologs for sog and tolloid. Bioinformatics analysis of the promoter of both genes indicated that the variations could be related to the gain of several binding sites for the transcriptional factor Dorsal in the Md.tld promoter and Snail in the Md.sog enhancer. These altered expressions could explain the unclear formation of the pMad gradient in the M. domestica embryo, compared to the formation of the gradient in D. melanogaster. It is concluded that gene expression changes during the dorsal-ventral patterning in insects contribute to the differentiation of extra-embryonic tissues as a consequence of changes in the gene regulatory network controlled by BMP/Dpp. This work, in early M. domestica embryos, identified the expression pattern of several genes members involved in the dorsoventral specification of the embryo. These data can contribute to understanding the evolution of the BMP/Dpp pathway, the regulation of BMP ligands, and the formation of a Dpp gradient in higher cyclorraphan flies.
Jha, A., van Zanten, T. S., Philippe, J. M., Mayor, S. and Lecuit, T. (2018). Quantitative control of GPCR organization and signaling by endocytosis in epithelial morphogenesis. Curr Biol 28(10): 1570-1584.e1576. PubMed ID: 29731302
Tissue morphogenesis arises from controlled cell deformations in response to cellular contractility. During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Myosin II (MyoII) is activated by cell-surface G protein-coupled receptors (GPCRs), such as Smog and Mist, that activate G proteins, the small GTPase Rho1, and the kinase Rok. Quantitative control over GPCR and Rho1 activation underlies differences in deformation of mesoderm and ectoderm cells. The GPCR Smog activity is concentrated on two different apical plasma membrane compartments, i.e., the surface and plasma membrane invaginations. Using fluorescence correlation spectroscopy, the surface of the plasma membrane was probed, and it was shown that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is regulated by the kinase Gprk2 and beta-arrestin-2 that clears active Smog from the plasma membrane. When Fog concentration is high or endocytosis is low, Smog rearranges in homo-clusters and accumulates in plasma membrane invaginations that are hubs for Rho1 activation. Lastly, this study found higher Smog homo-cluster concentration and numerous apical plasma membrane invaginations in the mesoderm compared to the ectoderm, indicative of reduced endocytosis. Dynamic partitioning of active Smog at the surface of the plasma membrane or plasma membrane invaginations has a direct impact on Rho1 signaling. Plasma membrane invaginations accumulate high Rho1-guanosine triphosphate (GTP) suggesting they form signaling centers. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that regulate differential Rho1 and MyoII activation in the Drosophila embryo.
Duan, R., Kim, J. H., Shilagardi, K., Schiffhauer, E. S., Lee, D. M., Son, S., Li, S., Thomas, C., Luo, T., Fletcher, D. A., Robinson, D. N. and Chen, E. H. (2018). Spectrin is a mechanoresponsive protein shaping fusogenic synapse architecture during myoblast fusion. Nat Cell Biol 20(6): 688-698. PubMed ID: 29802406
Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage. This study reports that alpha/betaH-spectrin (betaH is also called karst) dynamically accumulates and dissolves at the fusogenic synapse between fusing Drosophila muscle cells, where an attacking fusion partner invades its receiving partner with actin-propelled protrusions to promote cell fusion. Using genetics, cell biology, biophysics and mathematical modelling, it was demonstrated that spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse. The transiently accumulated spectrin network functions as a cellular fence to restrict the diffusion of cell-adhesion molecules and a cellular sieve to constrict the invasive protrusions, thereby increasing the mechanical tension of the fusogenic synapse to promote cell membrane fusion. This study study reveals a function of spectrin as a mechanoresponsive protein and has general implications for understanding spectrin function in dynamic cellular processes.
Mortensen, R. D., Moore, R. P., Fogerson, S. M., Chiou, H. Y., Obinero, C. V., Prabhu, N. K., Wei, A. H., Crawford, J. M. and Kiehart, D. P. (2018). Identifying genetic players in cell sheet morphogenesis using a Drosophila deficiency screen for genes on Chromosome 2R involved in dorsal closure. G3 (Bethesda). PubMed ID: 29776969
Dorsal closure has emerged as a model for cell sheet morphogenesis. Dorsal closure is remarkably robust and many questions regarding the molecular mechanisms involved in this complex biological process remain. Thus, it is important to identify all genes that contribute to the kinematics and dynamics of closure. This study used a set of large deletions (deficiencies), which collectively remove 98.5% of the genes on the right arm of Drosophila melanogaster's 2nd chromosome to identify "dorsal closure deficiencies". Through two crosses, embryos homozygous for each deficiency were unambiguously identified, and they were time-lapse imaged for the duration of closure. Images were analyzed for defects in cell shapes and tissue movements. Embryos homozygous for 47 deficiencies have notable, diverse defects in closure, demonstrating that a number of discrete processes comprise closure and are susceptible to mutational disruption. Further analysis of these deficiencies will lead to the identification of at least 30 novel "dorsal closure genes". It is expected that many of these novel genes will identify links to pathways and structures already known to coordinate various aspects of closure. New processes and pathways should be discovered that contribute to closure.
Izquierdo, E., Quinkler, T. and De Renzis, S. (2018). Guided morphogenesis through optogenetic activation of Rho signalling during early Drosophila embryogenesis. Nat Commun 9(1): 2366. PubMed ID: 29915285
During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. This study used optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. Precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal-ventral or anterior-posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes.
Inaki, M., Hatori, R., Nakazawa, N., Okumura, T., Ishibashi, T., Kikuta, J., Ishii, M., Matsuno, K. and Honda, H. (2018). Chiral cell sliding drives left-right asymmetric organ twisting. Elife 7. PubMed ID: 29891026
Polarized epithelial morphogenesis is an essential process in animal development. While this process is mostly attributed to directional cell intercalation, it can also be induced by other mechanisms. Using live-imaging analysis and a three-dimensional vertex model, 'cell sliding,' a novel mechanism driving epithelial morphogenesis, was identified in which cells directionally change their position relative to their subjacent (posterior) neighbors by sliding in one direction. In Drosophila embryonic hindgut, an initial left-right (LR) asymmetry of the cell shape (cell chirality in three dimensions), which occurs intrinsically before tissue deformation, is converted through LR asymmetric cell sliding into a directional axial twisting of the epithelial tube. In a Drosophila inversion mutant showing inverted cell chirality and hindgut rotation, cell sliding occurs in the opposite direction to that in wild-type. Unlike directional cell intercalation, cell sliding does not require junctional remodeling. Cell sliding may also be involved in other cases of LR-polarized epithelial morphogenesis.

Friday, July 6th - Evolution

Catalan, A., Macias-Munoz, A. and Briscoe, A. D. (2018).. Evolution of sex-biased gene expression and dosage compensation in the eye and brain of Heliconius butterflies. Mol Biol Evol. PubMed ID: 29931127
Differences in behavior and life history traits between females and males are the basis of divergent selective pressures between sexes. It has been suggested that a way for the two sexes to deal with different life history requirements is through sex-biased gene expression. A comparative sex-biased gene expression analysis was performed of the combined eye and brain transcriptome from five Heliconius species, H. charithonia, H. sara, H. erato, H. melpomene and H. doris, representing five of the main clades from the Heliconius phylogeny. The degree of sexual dimorphism in gene expression is not conserved across Heliconius. Most of the sex-biased genes identified in each species are not sex-biased in any other, suggesting that sexual selection might have driven sexually dimorphic gene expression. Only three genes shared sex-biased expression across multiple species: ultraviolet opsin UVRh1 and orthologs of Drosophila Kruppel-homolog 1 and CG9492. It was also observed that in some species female-biased genes have higher evolutionary rates, but in others, male-biased genes show the fastest rates when compared with unbiased genes, suggesting that selective forces driving sex-biased gene evolution in Heliconius act in a sex- and species-specific manner. Furthermore, dosage compensation was found in all the Heliconius tested, providing additional evidence for the conservation of dosage compensation across Lepidoptera. Finally, sex-biased genes are significantly enriched on the Z, a pattern that could be a result of sexually antagonistic selection.
Said, I., Byrne, A., Serrano, V., Cardeno, C., Vollmers, C. and Corbett-Detig, R. (2018). Linked genetic variation and not genome structure causes widespread differential expression associated with chromosomal inversions. Proc Natl Acad Sci U S A 115(21): 5492-5497. Pubmed ID: 29735663
Chromosomal inversions are widely thought to be favored by natural selection because they suppress recombination between alleles that have higher fitness on the same genetic background or in similar environments. Nonetheless, few selected alleles have been characterized at the molecular level. Gene expression profiling provides a powerful way to identify functionally important variation associated with inversions and suggests candidate phenotypes. This study characterized differential expression patterns associated with two chromosomal inversions found in natural Drosophila melanogaster populations. To isolate the impacts of genome structure, synthetic chromosomal inversions were engineered on controlled genetic backgrounds with breakpoints that closely match each natural inversion. Synthetic inversions have negligible effects on gene expression. Nonetheless, natural inversions have broad-reaching regulatory impacts in cis and trans Furthermore, differentially expressed genes associated with both natural inversions are enriched for loci associated with immune response to bacterial pathogens. The results support the idea that inversions in D. melanogaster experience natural selection to maintain associations between functionally related alleles to produce complex phenotypic outcomes.
Dugand, R. J., Kennington, W. J. and Tomkins, J. L. (2018). Evolutionary divergence in competitive mating success through female mating bias for good genes. Sci Adv 4(5): eaaq0369. PubMed ID: 29806021
Despite heritable variation for univariate sexually selected traits, recent analyses exploring multivariate traits find evidence consistent with the lek paradox in showing no genetic variation available to choosy females, and therefore no genetic benefits of choice. This study used the preferences of Drosophila melanogaster females to exert bidirectional selection on competitive male mating success to test for the presence and nature of genetic variation underlying this multivariate trait. Male mating success diverged between selection regimens, and flies from success-selected lines had a smaller burden of deleterious, recessive mutations that affect egg-to-adult viability, were better sperm competitors (sperm offence), and did not demonstrate reduced desiccation resistance or components of female fitness (traits thought to trade off with attractiveness) relative to flies from failure-selected populations. Mating success remained subject to inbreeding depression in success-selected lines, suggesting that variation in mating success remains, thanks to numerous genes of small effect. Together, these results provide unique evidence for the evolutionary divergence in male mating success, demonstrating that genetic variation is not exhausted along the axis of precopulatory sexual selection and that female mating biases align with the avoidance of bad genes.
Shropshire, J. D., On, J., Layton, E. M., Zhou, H. and Bordenstein, S. R. (2018). One prophage WO gene rescues cytoplasmic incompatibility in Drosophila melanogaster. Proc Natl Acad Sci U S A. PubMed ID: 29686091
Wolbachia are maternally inherited, intracellular bacteria at the forefront of vector control efforts to curb arbovirus transmission. In international field trials, the cytoplasmic incompatibility (CI) drive system of wMel Wolbachia is deployed to replace target vector populations, whereby a Wolbachia-induced modification of the sperm genome kills embryos. However, Wolbachia in the embryo rescue the sperm genome impairment, and therefore CI results in a strong fitness advantage for infected females that transmit the bacteria to offspring. The two genes responsible for the wMel-induced sperm modification of CI, cifA and cifB, were recently identified in the eukaryotic association module of prophage WO, but the genetic basis of rescue is unresolved. This study used transgenic and cytological approaches to demonstrate that maternal cifA expression independently rescues CI and nullifies embryonic death caused by wMel Wolbachia in Drosophila melanogaster. Discovery of cifA as the rescue gene and previously one of two CI induction genes establishes a "Two-by-One" model that underpins the genetic basis of CI. Results highlight the central role of prophage WO in shaping Wolbachia phenotypes that are significant to arthropod evolution and vector control.

Thursday, July 5th - Chromatin

Jordan-Pla, A., Yu, S., Waldholm, J., Kallman, T., Ostlund Farrants, A. K. and Visa, N. (2018). SWI/SNF regulates half of its targets without the need of ATP-driven nucleosome remodeling by Brahma. BMC Genomics 19(1): 367. PubMed ID: 29776334
Brahma (BRM) is the only catalytic subunit of the SWI/SNF chromatin-remodeling complex of Drosophila melanogaster. The function of SWI/SNF in transcription has long been attributed to its ability to remodel nucleosomes, which requires the ATPase activity of BRM. However, recent studies have provided evidence for a non-catalytic function of BRM in the transcriptional regulation of a few specific genes. This study used RNA-seq and ChIP-seq to identify the BRM target genes in S2 cells, and a catalytically inactive BRM mutant (K804R) was used that is unable to hydrolyze ATP to investigate the magnitude of the non-catalytic function of BRM in transcription regulation. 49% of the BRM target genes in S2 cells are regulated through mechanisms that do not require BRM to have an ATPase activity. The catalytic and non-catalytic mechanisms of SWI/SNF regulation operate on two subsets of genes that differ in promoter architecture and are linked to different biological processes. This study shows that the non-catalytic role of SWI/SNF in transcription regulation is far more prevalent than previously anticipated and that the genes that are regulated by SWI/SNF through ATPase-dependent and ATPase-independent mechanisms have specialized roles in different cellular and developmental processes.
Weissmann, S., Cloos, P. A., Sidoli, S., Jensen, O. N., Pollard, S. and Helin, K. (2018). The tumor suppressor CIC directly regulates MAPK pathway genes via histone deacetylation. Cancer Res. Pubmed ID: 29844126
Oligodendrogliomas (ODG) are brain tumors accounting for approximately 10% of all central nervous system cancers. CIC is a transcription factor that is mutated in most patients with ODG; these mutations are believed to be a key oncogenic event in such cancers. Analysis of the Drosophila melanogaster orthologue of CIC, Capicua, indicates that CIC loss phenocopies activation of the EGFR/RAS/MAPK pathway, and studies in mammalian cells have demonstrated a role for CIC in repressing the transcription of the PEA3 subfamily of ETS transcription factors. This study addresses the mechanism by which CIC represses transcription and assesses the functional consequences of CIC inactivation. Genome-wide binding patterns of CIC in several cell types revealed that CIC target genes were enriched for MAPK effector genes involved in cell cycle regulation and proliferation. CIC binding to target genes was abolished by high MAPK activity, which led to their transcriptional activation. CIC interacted with the SIN3 deacetylation complex and, based on the results, it is suggested that CIC functions as a transcriptional repressor through the recruitment of histone deacetylases. Independent single amino acid substitutions found in ODG tumors prevented CIC from binding its target genes. Taken together, the current results show that CIC is a transcriptional repressor of genes regulated by MAPK signaling, and that ablation of CIC function leads to increased histone acetylation levels and transcription at these genes, ultimately fueling mitogen-independent tumor growth.
Perino, M., van Mierlo, G., Karemaker, I. D., van Genesen, S., Vermeulen, M., Marks, H., van Heeringen, S. J. and Veenstra, G. J. C. (2018). MTF2 recruits Polycomb Repressive Complex 2 by helical-shape-selective DNA binding. Nat Genet. Pubmed ID: 29808031
Polycomb-mediated repression of gene expression is essential for development, with a pivotal role played by trimethylation of histone H3 lysine 27 (H3K27me3), which is deposited by Polycomb Repressive Complex 2 (PRC2). The mechanism by which PRC2 is recruited to target genes has remained largely elusive, particularly in vertebrates. This study demonstrates that MTF2, one of the three vertebrate homologs of Drosophila melanogaster Polycomblike, is a DNA-binding, methylation-sensitive PRC2 recruiter in mouse embryonic stem cells. MTF2 directly binds to DNA and is essential for recruitment of PRC2 both in vitro and in vivo. Genome-wide recruitment of the PRC2 catalytic subunit EZH2 is abrogated in Mtf2 knockout cells, resulting in greatly reduced H3K27me3 deposition. MTF2 selectively binds regions with a high density of unmethylated CpGs in a context of reduced helix twist, which distinguishes target from non-target CpG islands. These results demonstrate instructive recruitment of PRC2 to genomic targets by MTF2.
Teo, R. Y. W., Anand, A., Sridhar, V., Okamura, K. and Kai, T. (2018). Heterochromatin protein 1a functions for piRNA biogenesis predominantly from pericentric and telomeric regions in Drosophila. Nat Commun 9(1): 1735. Pubmed ID: 29728561
In metazoan germline, Piwi-interacting RNAs (piRNAs) provide defence against transposons. Piwi-piRNA complex mediates transcriptional silencing of transposons in nucleus. Heterochromatin protein 1a (HP1a) has been proposed to function downstream of Piwi-piRNA complex in Drosophila. HP1a germline knockdown (HP1a-GLKD) leads to a reduction in the total and Piwi-bound piRNAs mapping to clusters and transposons insertions, predominantly in the regions close to telomeres and centromeres, resulting in derepression of a limited number of transposons from these regions. In addition, HP1a-GLKD increases the splicing of transcripts arising from clusters in above regions, suggesting HP1a also functions upstream to piRNA processing. Evolutionarily old transposons enriched in the pericentric regions exhibit significant loss in piRNAs targeting these transposons upon HP1a-GLKD. This study suggests that HP1a functions to repress transposons in a chromosomal compartmentalised manner.
Ma, Z., Wang, H., Cai, Y., Wang, H., Niu, K., Wu, X., Ma, H., Yang, Y., Tong, W., Liu, F., Liu, Z., Zhang, Y., Liu, R., Zhu, Z. J. and Liu, N. (2018). Epigenetic drift of H3K27me3 in aging links glycolysis to healthy longevity in Drosophila. Elife 7. PubMed ID: 29809154
Epigenetic alteration has been implicated in aging. However, the mechanism by which epigenetic change impacts aging remains to be understood. H3K27me3, a highly conserved histone modification signifying transcriptional repression, is marked and maintained by Polycomb Repressive Complexes (PRCs). This study explores the mechanism by which age-modulated increase of H3K27me3 impacts adult lifespan. Using Drosophila, it was revealed that aging leads to loss of fidelity in epigenetic marking and drift of H3K27me3 and consequential reduction in the expression of glycolytic genes with negative effects on energy production and redox state. This study shows that a reduction of H3K27me3 by PRCs-deficiency promotes glycolysis and healthy lifespan. While perturbing glycolysis diminishes the pro-lifespan benefits mediated by PRCs-deficiency, transgenic increase of glycolytic genes in wild-type animals extends longevity. Together, it is proposed that epigenetic drift of H3K27me3 is one of the molecular mechanisms that contribute to aging and that stimulation of glycolysis promotes metabolic health and longevity.
Caridi, C. P., D'Agostino, C., Ryu, T., Zapotoczny, G., Delabaere, L., Li, X., Khodaverdian, V. Y., Amaral, N., Lin, E., Rau, A. R. and Chiolo, I. (2018). Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature. PubMed ID: 29925946
Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, 'safe' repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. This study shows that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes.

Wednesday, July 4th - Signaling

Yamakawa, T., Atsumi, Y., Kubo, S., Yamagishi, A., Morita, I. and Matsuno, K. (2018). Insight into Notch signaling steps that involve pecanex from dominant-modifier screens in Drosophila. Genetics. Pubmed ID: 29853475
Notch signaling plays crucial roles in intercellular communications. In Drosophila, the pecanex (pcx) gene, which encodes an evolutionarily conserved multi-pass transmembrane protein, appears to be required to activate Notch signaling in some contexts, especially during neuroblast segregation in the neuroectoderm. Although Pcx has been suggested to contribute to endoplasmic reticulum homeostasis, its functions remain unknown. This study performed a genetic modifier screens of pcx. This study found that pcx heterozygotes lacking its maternal contribution exhibit cold-sensitive lethality, which is attributed to a reduction in Notch signaling at decreased temperatures. Using sets of deletions that uncover most of the second and third chromosomes, four enhancers and two suppressors of the pcx cold-sensitive lethality were uncovered. Among these, five genes encode known Notch-signaling components: big brain, Delta (Dl), neuralized (neur), Brother of Bearded A (BobA), a member of the Bearded (Brd) family, and N-ethylmaleimide-sensitive factor 2 (Nsf2). BobA suppresses Dl endocytosis during neuroblast segregation in the neuroectoderm, as Brd family genes reportedly do in the mesoderm for mesoectoderm specification. Analyses of Nsf2, a key regulator of vesicular fusion, suggested a novel role in neuroblast segregation, which is distinct from Nsf2's previously reported role in imaginal tissues. Finally, jim lovell, which encodes a potential transcription factor, may play a role in Notch signaling during neuroblast segregation. These resalts reveal new research avenues for Pcx functions and Notch signaling.
Bleichert, F., Leitner, A., Aebersold, R., Botchan, M. R. and Berger, J. M. (2018). Conformational control and DNA-binding mechanism of the metazoan origin recognition complex. Proc Natl Acad Sci U S A 115(26): E5906-e5915. PubMed ID: 29899147
In eukaryotes, the heterohexameric origin recognition complex (ORC) coordinates replication onset by facilitating the recruitment and loading of the minichromosome maintenance 2-7 (Mcm2-7) replicative helicase onto DNA to license origins. Drosophila ORC can adopt an autoinhibited configuration that is predicted to prevent Mcm2-7 loading; how the complex is activated and whether other ORC homologs can assume this state are not known. Using chemical cross-linking and mass spectrometry, biochemical assays, and electron microscopy (EM), this study shows that the autoinhibited state of Drosophila ORC is populated in solution, and that human ORC can also adopt this form. ATP binding to ORC supports a transition from the autoinhibited state to an active configuration, enabling the nucleotide-dependent association of ORC with both DNA and Cdc6. An unstructured N-terminal region adjacent to the conserved ATPase domain of Orc1 is shown to be required for high-affinity ORC-DNA interactions, but not for activation. ORC optimally binds DNA duplexes longer than the predicted footprint of the ORC ATPases associated with a variety of cellular activities (AAA(+)) and winged-helix (WH) folds; cryo-EM analysis of Drosophila ORC bound to DNA and Cdc6 indicates that ORC contacts DNA outside of its central core region, bending the DNA away from its central DNA-binding channel. The findings indicate that ORC autoinhibition may be common to metazoans and that ORC-Cdc6 remodels origin DNA before Mcm2-7 recruitment and loading.
Trevisan, T., Pendin, D., Montagna, A., Bova, S., Ghelli, A. M. and Daga, A. (2018). Manipulation of mitochondria dynamics reveals separate roles for form and function in mitochondria distribution. Cell Rep 23(6): 1742-1753. Pubmed ID: 29742430
Mitochondria shape is controlled by membrane fusion and fission mediated by mitofusins, Opa1, and Drp1, whereas mitochondrial motility relies on microtubule motors. These processes govern mitochondria subcellular distribution, whose defects are emphasized in neurons because of their polarized structure. This study examined how perturbation of the fusion/fission balance affects mitochondria distribution in Drosophila axons. Knockdown of Marf or Opa1 resulted in progressive loss of distal mitochondria and in a distinct oxidative phosphorylation and membrane potential deficit. Downregulation of Drp1 rescued the lethality and bioenergetic defect caused by neuronal Marf RNAi, but induced only a modest restoration of axonal mitochondria distribution. Surprisingly, Drp1 knockdown rescued fragmentation and fully restored aberrant distribution of axonal mitochondria produced by Opa1 RNAi; however, Drp1 knockdown did not improve viability or mitochondria function. These data show that proper morphology is critical for proper axonal mitochondria distribution independent of bioenergetic efficiency. The health of neurons largely depends on mitochondria function, but does not depend on shape or distribution.
Tran, N. L., Takaesu, N. T., Cornell, E. F. and Newfeld, S. J. (2018). CORL expression in the Drosophila central nervous system is regulated by stage specific interactions of intertwined activators and repressors. G3 (Bethesda). Pubmed ID: 29848623
CORL proteins (SKOR in mice and Fussel in humans) are a subfamily of central nervous system (CNS) specific proteins related to Sno/Ski oncogenes. Their developmental and homeostatic roles are largely unknown. Previous work has shown that Drosophila CORL (dCORL; fussel in Flybase) functions between the Activin receptor Baboon and Ecdysone Receptor-B1 (EcR-B1) activation in mushroom body neurons of third instar larval brains. To better understand dCORL regulation and function a series of reporter genes was generated. this study examined the embryonic and larval CNS and found that dCORL is regulated by stage specific interactions between intertwined activators and repressors spanning numerous reporters. The reporter AH.lacZ, which contains sequences 7-11kb upstream of dCORL exon1, reflects dCORL brain expression at all stages. Surprisingly, AH.lacZ is not present in EcR-B1 expressing mushroom body neurons. In larvae AH.lacZ is coexpressed with Elav and the transcription factor Drifter as well as in dILP2 insulin producing cells of the pars intercerebralis. The presence of dCORL in insulin producing cells suggests that dCORL functions non-autonomously in the regulation of EcR-B1 mushroom body activation via the modulation of insulin signaling. Overall, the high level of sequence conservation seen in all CORL/SKOR/Fussel family members and their common CNS-specificity suggest that similarly complex regulation and a potential function in insulin signaling are associated with SKOR/Fussel proteins in mammals (Tran, 2018).
Ahmed-de-Prado, S., Diaz-Garcia, S. and Baonza, A. (2018). JNK and JAK/STAT signalling are required for inducing loss of cell fate specification during imaginal wing discs regeneration in Drosophila melanogaster. Dev Biol. PubMed ID: 29870691
The regenerative process after tissue damage relies on a variety of cellular responses that includes compensatory cell proliferation and cell fate re-specification. The identification of the signalling networks regulating these cellular events is a central question in regenerative biology. Tissue regeneration models in Drosophila have shown that two of the signals that play a fundamental role during the early stages of regeneration are the c-Jun N-terminal kinase (JNK) and JAK/STAT signalling pathways. These pathways have been shown to be required for controlling regenerative proliferation, however their contribution to the processes of cellular reprogramming and cell fate re-specification that take place during regeneration are largely unknown. This study presents evidence for a previously unrecognised function of the cooperative activities of JNK and JAK/STAT signalling pathways in inducing loss of cell fate specification in imaginal discs. Co-activation of these signalling pathways induces both the cell fate changes in injured areas, as well as in adjacent cells. This function relies on the activity of the Caspase initiator encoded by the gene dronc.
Wang, X. C., Liu, Z. and Jin, L. H. (2018). Anchor negatively regulates BMP signalling to control Drosophila wing development. Eur J Cell Biol 97(4): 308-317. Pubmed ID: 29735293
G protein-coupled receptors play particularly important roles in many organisms. The novel Drosophila gene anchor is an orthologue of vertebrate GPR155. However, the roles of anchor in molecular functions and biological processes, especially in wing development, remain unknown. Knockdown of anchor resulted in an increased wing size and additional and thickened veins. These abnormal wing phenotypes were similar to those observed in BMP signalling gain-of-function experiments. The BMP signalling indicator p-Mad: was significantly increased in wing discs in which anchor RNAi was induced in larvae and accumulated abnormally in intervein regions in pupae. Furthermore, the expression of target genes of the BMP signalling pathway was examined using a lacZ reporter, and the results indicated that omb and sal were substantially increased in anchor-knockdown wing discs. An investigation of genetic interactions between Anchor and the BMP signalling pathway revealed that the thickened and ectopic vein tissues were rescued by knocking down BMP levels. These results suggested that Anchor functions to negatively regulate BMP signalling during wing development and vein formation.

Tuesday, July 3rd - Adult Neural Development and Function

Tsai, K. T., Hu, C. K., Li, K. W., Hwang, W. L. and Chou, Y. H. (2018). Circuit variability interacts with excitatory-inhibitory diversity of interneurons to regulate network encoding capacity. Sci Rep 8(1): 8027. Pubmed ID: 29795277
Local interneurons (LNs) in the Drosophila olfactory system exhibit neuronal diversity and variability, yet it is still unknown how these features impact information encoding capacity and reliability in a complex LN network. This study used two strategies to construct a diverse excitatory-inhibitory neural network beginning with a ring network structure and then introduced distinct types of inhibitory interneurons and circuit variability to the simulated network. The continuity of activity within the node ensemble (oscillation pattern) was used as a readout to describe the temporal dynamics of network activity. Inhibitory interneurons were found to enhance the encoding capacity by protecting the network from extremely short activation periods when the network wiring complexity is very high. In addition, distinct types of interneurons have differential effects on encoding capacity and reliability. Circuit variability may enhance the encoding reliability, with or without compromising encoding capacity. Therefore, this study has described how circuit variability of interneurons may interact with excitatory-inhibitory diversity to enhance the encoding capacity and distinguishability of neural networks. This work has evaluate the effects of different types and degrees of connection diversity on a ring model, which may simulate interneuron networks in the Drosophila olfactory system or other biological systems.
Shaw, R. E., Kottler, B., Ludlow, Z. N., Buhl, E., Kim, D., Morais da Silva, S., Miedzik, A., Coum, A., Hodge, J. J., Hirth, F. and Sousa-Nunes, R. (2018). In vivo expansion of functionally integrated GABAergic interneurons by targeted increase in neural progenitors. EMBO J. Pubmed ID: 29728368
A central hypothesis for brain evolution is that it might occur via expansion of progenitor cells and subsequent lineage-dependent formation of neural circuits. This study reports in vivo amplification and functional integration of lineage-specific circuitry in Drosophila Levels of the cell fate determinant Prospero were attenuated in specific brain lineages within a range that expanded not only progenitors but also neuronal progeny, without tumor formation. Resulting supernumerary neural stem cells underwent normal functional transitions, progressed through the temporal patterning cascade, and generated progeny with molecular signatures matching source lineages. Fully differentiated supernumerary gamma-amino butyric acid (GABA)-ergic interneurons formed functional connections in the central complex of the adult brain, as revealed by in vivo calcium imaging and open-field behavioral analysis. These results show that quantitative control of a single transcription factor is sufficient to tune neuron numbers and clonal circuitry, and provide molecular insight into a likely mechanism of brain evolution.
Wang, Q., Wang, Y. and Yu, F. (2018). Yif1 associates with Yip1 on Golgi and regulates dendrite pruning in sensory neurons during Drosophila metamorphosis. Development. Pubmed ID: 29769219
Pruning that selectively removes unnecessary neurites without causing neuronal death is essential for sculpting the mature nervous system during development. In Drosophila, ddaC sensory neurons specifically prune their larval dendrites with intact axons during metamorphosis. However, it remains unknown about an important role of ER-to-Golgi transport in dendrite pruning. In a clonal screen this study identified Yif1, an uncharacterized Drosophila homologue of Yif1p that is known as a regulator of ER-to-Golgi transport in yeast. This study shows that Yif1 is required for dendrite pruning of ddaC neurons but not for apoptosis of ddaF neurons. The Yif1-binding partner Yip1 was identified that is also crucial for dendrite pruning. Yif1 forms a protein complex with Yip1 in S2 cells and ddaC neurons. Yip1 and Yif1 colocalize on ER/Golgi and are required for the integrity of Golgi apparatus and outposts. Moreover, two GTPases Rab1 and Sar1, known to regulate ER-to-Golgi transport, were shown to be essential for dendrite pruning of ddaC neurons. Finally, these data reveal that ER-to-Golgi transport promotes endocytosis and downregulation of cell adhesion molecule Neuroglian and thereby dendrite pruning.
Ren, Q., Awasaki, T., Wang, Y. C., Huang, Y. F. and Lee, T. (2018). Lineage-guided Notch-dependent gliogenesis by Drosophila multi-potent progenitors. Development. Pubmed ID: 29764857
Macroglial cells in the central nervous system exhibit regional specialization and carry out region-specific functions. Diverse glial cells arise from specific progenitors in specific spatiotemporal patterns. This raises an interesting possibility that there exist glial precursors with distinct developmental fates, which govern region-specific gliogenesis. This study mapped the glial progeny produced by the Drosophila type II neuroblasts, which, like vertebrate radial glia cells, yield both neurons and glia via intermediate neural progenitors (INPs). Distinct type II neuroblasts produce different characteristic sets of glia. A single INP can make both astrocyte-like and ensheathing glia, which co-occupy a relatively restrictive subdomain. Blocking apoptosis uncovers further lineage distinctions in the specification, proliferation, and survival of glial precursors. Both the switch from neurogenesis to gliogenesis and the subsequent glial expansion depend on Notch signaling. Taken together, lineage origins preconfigure the development of individual glial precursors with involvement of serial Notch actions in promoting gliogenesis.
Apitz, H. and Salecker, I. (2018). Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila. Nat Commun 9(1): 2295. PubMed ID: 29895891
Visual motion detection in sighted animals is essential to guide behavioral actions ensuring their survival. In Drosophila, motion direction is first detected by T4/T5 neurons. Their axons innervate one of the four lobula plate layers. How T4/T5 neurons with layer-specific representation of motion-direction preferences are specified during development is unknown. This study shows that diffusible Wingless (Wg) between adjacent neuroepithelia induces its own expression to form secondary signaling centers. These activate Decapentaplegic (Dpp) signaling in adjacent lateral tertiary neuroepithelial domains dedicated to producing layer 3/4-specific T4/T5 neurons. T4/T5 neurons derived from the core domain devoid of Dpp signaling adopt the default layer 1/2 fate. Dpp signaling induces the expression of the T-box transcription factor Optomotor-blind (Omb), serving as a relay to postmitotic neurons. Omb-mediated repression of Dachshund transforms layer 1/2- into layer 3/4-specific neurons. Hence, spatio-temporal relay mechanisms, bridging the distances between neuroepithelial domains and their postmitotic progeny, implement T4/T5 neuron-subtype identity.
Jeanne, J. M., Fisek, M. and Wilson, R. I. (2018). The organization of projections from olfactory glomeruli onto higher-order neurons. Neuron 98(6): 1198-1213. PubMed ID: 29909998
Each odorant receptor corresponds to a unique glomerulus in the brain. Projections from different glomeruli then converge in higher brain regions, but the logic governing which glomeruli converge and which do not is not understood. This study used two-photon optogenetics to map glomerular connections onto neurons in the lateral horn, the region of the Drosophila brain that receives the majority of olfactory projections. This study has identified 39 morphological types of lateral horn neurons (LHNs) and shows that different types receive input from different combinations of glomeruli. Different LHN types do not have independent inputs; rather, certain combinations of glomeruli converge onto many of the same LHNs and so are over-represented. Notably, many over-represented combinations are composed of glomeruli that prefer chemically dissimilar ligands whose co-occurrence indicates a behaviorally relevant "odor scene." The pattern of glomerulus-LHN connections thus represents a prediction of what ligand combinations will be most salient.

Monday, July 2nd - Junctions and Cytoskeleton

Wang, Z., Bosveld, F. and Bellaiche, Y. (2018). Tricellular junction proteins promote disentanglement of daughter and neighbour cells during epithelial cytokinesis. J Cell Sci. Pubmed ID: 29739875
In epithelial tissue, new cell-cell junctions are formed upon cytokinesis. To understand junction formation during cytokinesis, this study explored in Drosophila epithelium, de novo formation of tricellular septate junctions (TCJs). Upon midbody formation, the membranes of the two daughter cells and of the neighbouring cells located below the adherens junction (AJ) remain entangled in a 4-cell structure apposed to the midbody. The septate junction protein Discs-Large and components of the TCJ, Gliotactin and Anakonda accumulate in this 4-cell structure. Subsequently, a basal movement of the midbody parallels the detachment of the neighbouring cell membranes from the midbody, the disengagement of the daughter cells from their neighbours and the reorganisation of TCJs between the two daughter cells and their neighbouring cells. While the movement of midbody is independent of the Alix and Shrub abscission regulators, the loss of Gliotactin or Anakonda function impedes both the resolution of the connection between the daughter-neighbour cells and midbody movement. TCJ proteins therefore control an additional step of cytokinesis necessary for the disentanglement of the daughter cells and their neighbours during cytokinesis.
Zhang, Y., Yu, J. C., Jiang, T., Fernandez-Gonzalez, R. and Harris, T. J. C. (2018). Collision of expanding actin caps with actomyosin borders for cortical bending and mitotic rounding in a syncytium. Dev Cell. Pubmed ID: 29804877
The early Drosophila embryo is a large syncytial cell that compartmentalizes mitotic spindles with furrows. Before furrow ingression, an Arp2/3 actin cap forms above each nucleus and is encircled by actomyosin. This study investigated how these networks transform a flat cortex into a honeycomb-like compartmental array. The growing caps circularize and ingress upon meeting their actomyosin borders, which become the furrow base. Genetic perturbations indicate that the caps physically displace their borders and, reciprocally, that the borders resist and circularize their caps. These interactions create an actomyosin cortex arrayed with circular caps. The Rac-GEF Sponge, Rac-GTP, Arp3, and actin coat the caps as a growing material that can drive cortical bending for initial furrow ingression. Additionally, laser ablations indicate that actomyosin contraction squeezes the cytoplasm, producing counterforces that swell the caps. Thus, Arp2/3 caps form clearances of the actomyosin cortex and control buckling and swelling of these clearances for metaphase compartmentalization.
Kelliher, M. T., Yue, Y., Ng, A., Kamiyama, D., Huang, B., Verhey, K. J. and Wildonger, J. (2018). Autoinhibition of kinesin-1 is essential to the dendrite-specific localization of Golgi outposts. J Cell Biol. PubMed ID: 29728423
Neuronal polarity relies on the selective localization of cargo to axons or dendrites. The molecular motor kinesin-1 moves cargo into axons but is also active in dendrites. This raises the question of how kinesin-1 activity is regulated to maintain the compartment-specific localization of cargo. In vivo structure-function analysis of endogenous Drosophila melanogaster kinesin-1 reveals a novel role for autoinhibition in enabling the dendrite-specific localization of Golgi outposts. Mutations that disrupt kinesin-1 autoinhibition result in the axonal mislocalization of Golgi outposts. Autoinhibition also regulates kinesin-1 localization. Uninhibited kinesin-1 accumulates in axons and is depleted from dendrites, correlating with the change in outpost distribution and dendrite growth defects. Genetic interaction tests show that a balance of kinesin-1 inhibition and dynein activity is necessary to localize Golgi outposts to dendrites and keep them from entering axons. These data indicate that kinesin-1 activity is precisely regulated by autoinhibition to achieve the selective localization of dendritic cargo.
Juan, T., Geminard, C., Coutelis, J. B., Cerezo, D., Poles, S., Noselli, S. and Furthauer, M. (2018). Myosin1D is an evolutionarily conserved regulator of animal left-right asymmetry. Nat Commun 9(1): 1942. PubMed ID: 29769531
Evolutionary Homolog Study

The establishment of left-right (LR) asymmetry is fundamental to animal development, but the identification of a unifying mechanism establishing laterality across different phyla has remained elusive. A cilia-driven, directional fluid flow is important for symmetry breaking in numerous vertebrates, including zebrafish. Alternatively, LR asymmetry can be established independently of cilia, notably through the intrinsic chirality of the acto-myosin cytoskeleton. This study shows that Myosin1D (Myo1D), a previously identified regulator of Drosophila LR asymmetry, is essential for the formation and function of the zebrafish LR organizer (LRO), Kupffer's vesicle (KV). Myo1D controls the orientation of LRO cilia and interacts functionally with the planar cell polarity (PCP) pathway component VanGogh-like2 (Vangl2; see Drosophila Van Gogh), to shape a productive LRO flow. These findings identify Myo1D as an evolutionarily conserved regulator of animal LR asymmetry, and show that functional interactions between Myo1D and PCP are central to the establishment of animal LR asymmetry.

Morelli, G., Even, A., Gladwyn-Ng, I., Le Bail, R., Shilian, M., Godin, J. D., Peyre, E., Hassan, B. A., Besson, A., Rigo, J. M., Weil, M., Brone, B. and Nguyen, L. (2018). p27(Kip1) modulates axonal transport by regulating alpha-Tubulin acetyltransferase 1 stability. Cell Rep 23(8): 2429-2442. PubMed ID: 29791853
The protein p27(Kip1; see Drosophila Dacapo) plays roles that extend beyond cell-cycle regulation during cerebral cortex development, such as the regulation of neuronal migration and neurite branching via signaling pathways that converge on the actin and microtubule cytoskeletons. Microtubule-dependent transport is essential for the maturation of neurons and the establishment of neuronal connectivity though synapse formation and maintenance. This study shows that p27(Kip1) controls the transport of vesicles and organelles along the axon of mice cortical projection neurons in vitro. Moreover, suppression of the p27(Kip1) ortholog, dacapo, in Drosophila melanogaster disrupts axonal transport in vivo, leading to the reduction of locomotor activity in third instar larvae and adult flies. At the molecular level, p27(Kip1) stabilizes the alpha-tubulin acetyltransferase 1, thereby promoting the acetylation of microtubules, a post-translational modification required for proper axonal transport.
Zur Lage, P., Stefanopoulou, P., Styczynska-Soczka, K., Quinn, N., Mali, G., von Kriegsheim, A., Mill, P. and Jarman, A. P. (2018). Ciliary dynein motor preassembly is regulated by Wdr92 in association with HSP90 co-chaperone, R2TP. J Cell Biol. Pubmed ID: 29743191
The massive dynein motor complexes that drive ciliary and flagellar motility require cytoplasmic preassembly, a process requiring dedicated dynein assembly factors (DNAAFs). How DNAAFs interact with molecular chaperones to control dynein assembly is not clear. By analogy with the well-known multifunctional HSP90-associated cochaperone, R2TP, several DNAAFs have been suggested to perform novel R2TP-like functions. However, the involvement of R2TP itself (canonical R2TP) in dynein assembly remains unclear. This study shows that in Drosophila melanogaster, the R2TP-associated factor, Wdr92, is required exclusively for axonemal dynein assembly, likely in association with canonical R2TP. Proteomic analyses suggest that in addition to being a regulator of R2TP chaperoning activity, Wdr92 works with the DNAAF Spag1 at a distinct stage in dynein preassembly. Wdr92/R2TP function is likely distinct from that of the DNAAFs proposed to form dynein-specific R2TP-like complexes. These findings thus establish a connection between dynein assembly and a core multifunctional cochaperone.
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