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Current papers in developmental biology and gene function


Tuesday June 25th - Enhancers & Transcriptional Regulation

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Haberle, V., Arnold, C. D., Pagani, M., Rath, M., Schernhuber, K. and Stark, A. (2019). Transcriptional cofactors display specificity for distinct types of core promoters. Nature. PubMed ID: 31092928
Transcriptional cofactors (COFs) communicate regulatory cues from enhancers to promoters and are central effectors of transcription activation and gene expression. Although some COFs have been shown to prefer certain promoter types over others, the extent to which different COFs display intrinsic specificities for distinct promoters is unclear. This study used a high-throughput promoter-activity assay in Drosophila melanogaster S2 cells to screen 23 COFs for their ability to activate 72,000 candidate core promoters (CPs). Differential activation of CPs was observed, indicating distinct regulatory preferences or 'compatibilities' between COFs and specific types of CPs. These functionally distinct CP types are differentially enriched for known sequence elements, such as the TATA box, downstream promoter element (DPE) or TCT motif, and display distinct chromatin properties at endogenous loci. Notably, the CP types differ in their relative abundance of H3K4me3 and H3K4me1 marks, suggesting that these histone modifications might distinguish trans-regulatory factors rather than promoter- versus enhancer-type cis-regulatory elements. The existence was confirmed of distinct COF-CP compatibilities in two additional Drosophila cell lines and in human cells, for which COFs were found that prefer TATA-box or CpG-island promoters, respectively. Distinct compatibilities between COFs and promoters can explain how different enhancers specifically activate distinct sets of genes, alternative promoters within the same genes, and distinct transcription start sites within the same promoter. Thus, COF-promoter compatibilities may underlie distinct transcriptional programs in species as divergent as flies and humans.
Chayengia, M., Veikkolainen, V., Jevtic, M. and Pyrowolakis, G. (2019). Sequence environment of BMP-dependent activating elements controls transcriptional responses to Dpp signaling in Drosophila. Development. PubMed ID: 31110028
Intercellular signaling pathways activate transcription factors, which along with tissue-specific co-factors regulate expression of target genes. Responses to TGF-β/BMP signals are mediated by Smad proteins, which form complexes and accumulate in the nucleus to directly bind and regulate enhancers of BMP-targets upon signaling. In Drosophila, gene activation by BMP signaling often requires, in addition to direct input by Smads, the signal-dependent removal of the transcriptional repressor Brinker. Previous studies on enhancers of BMP activated genes have defined a BMP responsive motif, the AE, which integrates activatory and repressive input by the Smad complex and Brk, respectively. This study addresses whether sequence variations within the core AE sequences might endow the motif with additional properties accounting for qualitative and quantitative differences in BMP responses including tissue-specificity of transcriptional activation and differential sensitivity to Smad and Brk inputs. By analyzing and cross-comparing three distinct BMP responsive enhancers from the genes wit and dad in two different epithelia, the wing imaginal disc and the follicular epithelium, this study demonstrates that differences in the AEs neither contribute to the observed tissue-restriction of BMP responses nor to differences in the utilization of the Smad and Brk branches for transcriptional activation. These results rather suggest that the cis-environment of the BMP response elements not only dictate tissue specificity but also differential sensitivity to the two BMP mediators.
Wu, W. H., Kuo, T. H., Kao, C. W., Girardot, C., Hung, S. J., Liu, T., Furlong, E. E. M. and Liu, Y. H. (2019). Expanding the mesodermal transcriptional network by genome-wide identification of Zinc finger homeodomain 1 (Zfh1) targets. FEBS Lett. PubMed ID: 31093969
The Drosophila transcription factor (TF) Zfh1 has distinct roles compared to the cell lineage-determining TFs in almost all mesoderm-derived tissues. This study links Zfh1 to the well-characterized mesodermal transcriptional network. Five enhancers were identified integrating upstream regulatory inputs from mesodermal TFs and directing zfh1 expression in mesoderm. Most downstream Zfh1-target genes are co-bound by mesodermal TFs, suggesting that Zfh1 and mesodermal TFs act on the same sets of co-regulated genes during the development of certain mesodermal tissues. Furthermore, this study demonstrates that Zfh1 is critical for the expression of a hemocyte marker gene peroxidasin and helps restrict the activity of a hemocyte-specific enhancer of serpent to hemocyte-deriving head mesoderm, suggesting a potential role of Zfh1 in hemocyte development.
Yamada, S., Whitney, P. H., Huang, S. K., Eck, E. C., Garcia, H. G. and Rushlow, C. A. (2019). The Drosophila pioneer factor Zelda modulates the nuclear microenvironment of a Dorsal target enhancer to potentiate transcriptional output. Curr Biol 29(8): 1387-1393.e1385. PubMed ID: 30982648
Connecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long-term goal of developmental biology. The dorsal-ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (Dl), a homolog of the nuclear factor kappaB (NF-kappaB) family of transcriptional activators found in humans. A second maternally deposited factor, Zelda (Zld), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors, such as Dl. This study utilized the MS2 live imaging system to evaluate the expression of the Dl target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. These experiments indicate that Zld modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. The results further show that this effective rate increase is due to an increased accumulation of Dl at the site of transcription, suggesting that transcription factor "hubs" induced by Zld functionally regulate transcription.

Monday, June 24th - Cell Cycle

Perkins, A. T., Greig, M. M., Sontakke, A. A., Peloquin, A. S., McPeek, M. A. and Bickel, S. E. (2019). Increased levels of superoxide dismutase suppress meiotic segregation errors in aging oocytes. Chromosoma. PubMed ID: 31037468
The risk of meiotic segregation errors increases dramatically during a woman's thirties, a phenomenon known as the maternal age effect. In addition, several lines of evidence indicate that meiotic cohesion deteriorates as oocytes age. One mechanism that may contribute to age-induced loss of cohesion is oxidative damage. In support of this model, it has been reported that the knockdown of the reactive oxygen species (ROS)-scavenging enzyme, superoxide dismutase (SOD), during meiotic prophase causes premature loss of arm cohesion and segregation errors in Drosophila oocytes. If age-dependent oxidative damage causes meiotic segregation errors, then the expression of extra SOD1 (cytosolic/nuclear) or SOD2 (mitochondrial) in oocytes may attenuate this effect. To test this hypothesis, flies were generatee that contain a UAS-controlled EMPTY, SOD1, or SOD2 cassette, and expression was induced using a Gal4 driver that turns on during meiotic prophase. The fidelity of chromosome segregation was compared in aged and non-aged Drosophila oocytes for all three genotypes. As expected, p{EMPTY} oocytes subjected to aging exhibited a significant increase in nondisjunction (NDJ) compared with non-aged oocytes. In contrast, the magnitude of age-dependent NDJ was significantly reduced when expression of extra SOD1 or SOD2 was induced during prophase. These findings support the hypothesis that a major factor underlying the maternal age effect in humans is age-induced oxidative damage that results in premature loss of meiotic cohesion. Moreover, this work raises the exciting possibility that antioxidant supplementation may provide a preventative strategy to reduce the risk of meiotic segregation errors in older women.
Oliveira, M. S., Freitas, J., Pinto, P. A. B., de Jesus, A., Tavares, J., Pinho, M., Domingues, R. G., Henriques, T., Lopes, C., Conde, C., Sunkel, C. E. and Moreira, A. (2019). The cell cycle kinase Polo is controlled by a conserved 3'UTR regulatory sequence in Drosophila melanogaster. Mol Cell Biol. PubMed ID: 31085682
Alternative polyadenylation generates transcriptomic diversity, although the physiological impact and regulatory mechanisms involved are still poorly understood. The cell cycle kinase Polo is controlled by alternative polyadenylation in the 3'UTR with critical physiological consequences. This study characterized the molecular mechanisms required for polo alternative polyadenylation. A conserved upstream sequence element (USE) close to polo proximal polyA signal. Transgenic flies without this sequence show incorrect selection of polo polyA signals with consequent downregulation of polo expression levels and insufficient/defective activation of Polo kinetochores targets, Mps1 and Aurora B. Deletion of the USE results in abnormal mitoses in neuroblasts, revealing a role for this sequence in vivo. Hephaestus was shown to bind to the USE RNA, and hephaestus mutants displayed defects in polo alternative polyadenylation concomitant with a striking reduction in Polo protein levels, leading to mitotic errors and aneuploidy. Bioinformatic analyses show that the USE is preferentially localized upstream of non-canonical polyadenylation signals in Drosophila melanogaster genes. Taken together, these results revealed the molecular mechanisms involved in polo alternative polyadenylation with remarkable physiological functions in Polo expression and activity at the kinetochores, and disclosed a new in vivo function for USEs in Drosophila melanogaster.
Hughes, S. E., Hemenway, E., Guo, F., Yi, K., Yu, Z. and Hawley, R. S. (2019). The E3 ubiquitin ligase Sina regulates the assembly and disassembly of the synaptonemal complex in Drosophila females. PLoS Genet 15(5): e1008161. PubMed ID: 31107865
During early meiotic prophase, homologous chromosomes are connected along their entire lengths by a proteinaceous tripartite structure known as the synaptonemal complex (SC). Although the components that comprise the SC are predominantly studied in this canonical ribbon-like structure, they can also polymerize into repeated structures known as polycomplexes. In Drosophila oocytes, the ability of SC components to assemble into canonical tripartite SC requires the E3 ubiquitin ligase Seven in absentia (Sina). In sina mutant oocytes, SC components assemble into large rod-like polycomplexes instead of proper SC. Thus, the wild-type Sina protein inhibits the polymerization of SC components, including those of the lateral element, into polycomplexes. These polycomplexes persist into meiotic stages when canonical SC has been disassembled, indicating that Sina also plays a role in controlling SC disassembly. Polycomplexes induced by loss-of-function sina mutations associate with centromeres, sites of double-strand breaks, and cohesins. Perhaps as a consequence of these associations, centromere clustering is defective and crossing over is reduced. These results suggest that while features of the polycomplexes can be recognized as SC by other components of the meiotic nucleus, polycomplexes nonetheless fail to execute core functions of canonical SC.
Ohhara, Y., Nakamura, A., Kato, Y. and Yamakawa-Kobayashi, K. (2019). Chaperonin TRiC/CCT supports mitotic exit and entry into endocycle in Drosophila. PLoS Genet 15(4): e1008121. PubMed ID: 31034473
Endocycle is a commonly observed cell cycle variant through which cells undergo repeated rounds of genome DNA replication without mitosis. Endocycling cells arise from mitotic cells through a switch of the cell cycle mode, called the mitotic-to-endocycle switch (MES), to initiate cell growth and terminal differentiation. However, the underlying regulatory mechanisms of MES remain unclear. This study used the Drosophila steroidogenic organ, called the prothoracic gland (PG), to study regulatory mechanisms of MES, which is critical for the PG to upregulate biosynthesis of the steroid hormone ecdysone. PG cells undergo MES through downregulation of mitotic cyclins, which is mediated by Fizzy-related (Fzr). Moreover, a RNAi screen was performed to further elucidate the regulatory mechanisms of MES, and the evolutionarily conserved chaperonin TCP-1 ring complex (TRiC) was identified as a novel regulator of MES. Knockdown of TRiC subunits in the PG caused a prolonged mitotic period, probably due to impaired nuclear translocation of Fzr, which also caused loss of ecdysteroidogenic activity. These results indicate that TRiC supports proper MES and endocycle progression by regulating Fzr folding. We propose that TRiC-mediated protein quality control is a conserved mechanism supporting MES and endocycling, as well as subsequent terminal differentiation.
Oon, C. H. and Prehoda, K. E. (2019). Asymmetric recruitment and actin-dependent cortical flows drive the neuroblast polarity cycle. Elife 8. PubMed ID: 31066675
During the asymmetric divisions of Drosophila neuroblasts, the Par polarity complex cycles between the cytoplasm and an apical cortical domain that restricts differentiation factors to the basal cortex. This study used rapid imaging of the full cell volume to uncover the dynamic steps that underlie transitions between neuroblast polarity states. Initially, the Par proteins aPKC and Bazooka form discrete foci at the apical cortex. Foci grow into patches that together comprise a discontinuous, unorganized structure. Coordinated cortical flows that begin near metaphase and are dependent on the actin cytoskeleton rapidly transform the patches into a highly organized apical cap. At anaphase onset, the cap disassembles as the cortical flow reverses direction toward the emerging cleavage furrow. Following division, cortical patches dissipate into the cytoplasm allowing the neuroblast polarity cycle to begin again. This work demonstrates how neuroblasts use asymmetric recruitment and cortical flows to dynamically polarize during asymmetric division cycles.
Djabrayan, N. J., Smits, C. M., Krajnc, M., Stern, T., Yamada, S., Lemon, W. C., Keller, P. J., Rushlow, C. A. and Shvartsman, S. Y. (2019). Metabolic regulation of developmental cell cycles and zygotic transcription. Curr Biol 29(7): 1193-1198.e1195. PubMed ID: 30880009
The thirteen nuclear cleavages that give rise to the Drosophila blastoderm are some of the fastest known cell cycles. Surprisingly, the fertilized egg is provided with at most one-third of the dNTPs needed to complete the thirteen rounds of DNA replication. The rest must be synthesized by the embryo, concurrent with cleavage divisions. What is the reason for the limited supply of DNA building blocks? It is proposed that frugal control of dNTP synthesis contributes to the well-characterized deceleration of the cleavage cycles and is needed for robust accumulation of zygotic gene products. In support of this model, it was demonstrated that when the levels of dNTPs are abnormally high, nuclear cleavages fail to sufficiently decelerate, the levels of zygotic transcription are dramatically reduced, and the embryo catastrophically fails early in gastrulation. This work reveals a direct connection between metabolism, the cell cycle, and zygotic transcription.

Friday, June 21st - Immune Response

Melcarne, C., Ramond, E., Dudzic, J., Bretscher, A. J., Kurucz, E., Ando, I. and Lemaitre, B. (2019). Two Nimrod receptors, NimC1 and Eater, synergistically contribute to bacterial phagocytosis in Drosophila melanogaster. FEBS J. PubMed ID: 30993828
Eater and NimC1 are transmembrane receptors of the Drosophila Nimrod family, specifically expressed in hemocytes, the insect blood cells. Previous ex vivo and in vivo RNAi studies have pointed to their role in the phagocytosis of bacteria. This study has created a novel NimC1 null mutant to re-evaluate the role of NimC1, alone or in combination with Eater, in the cellular immune response. NimC1 was shown to function as an adhesion molecule ex vivo, but in contrast to Eater it is not required for hemocyte sessility in vivo. Ex vivo phagocytosis assays and electron microscopy experiments confirmed that Eater is the main phagocytic receptor for Gram-positive, but not Gram-negative bacteria, and contributes to microbe tethering to hemocytes. Surprisingly, NimC1 deletion did not impair phagocytosis of bacteria, nor their adhesion to the hemocytes. However, phagocytosis of both types of bacteria was almost abolished in NimC1(1) ;eater(1) hemocytes. This indicates that both receptors contribute synergistically to the phagocytosis of bacteria, but that Eater can bypass the requirement for NimC1. Finally, it was uncovered that NimC1, but not Eater, is essential for uptake of latex beads and zymosan particles. It is concluded that Eater and NimC1 are the two main receptors for phagocytosis of bacteria in Drosophila, and that each receptor likely plays distinct roles in microbial uptake.
Wen, Y., He, Z., Xu, T., Jiao, Y., Liu, X., Wang, Y. F. and Yu, X. Q. (2019). Ingestion of killed bacteria activates antimicrobial peptide genes in Drosophila melanogaster and protects flies from septic infection. Dev Comp Immunol 95: 10-18. PubMed ID: 30731096
Drosophila melanogaster possesses a sophisticated and effective immune system composed of humoral and cellular immune responses, and production of antimicrobial peptides (AMPs) is an important defense mechanism. Expression of AMPs is regulated by the Toll and IMD (immune deficiency) pathways. Production of AMPs can be systemic in the fat body or a local event in the midgut and epithelium. So far, most studies focus on systemic septic infection in adult flies and little is known about AMP gene activation after ingestion of killed bacteria. This study investigated activation of AMP genes in the wild-type w(1118), MyD88 and Imd mutant flies after ingestion of heat-killed Escherichia coli and Staphylococcus aureus. Ingestion of E. coli activated most AMP genes, including drosomycin and diptericin, in the first to third instar larvae and pupae, while ingestion of S. aureus induced only some AMP genes in some larval stages or in pupae. In adult flies, ingestion of killed bacteria activated AMP genes differently in males and females. Interestingly, ingestion of killed E. coli and S. aureus in females conferred resistance to septic infection by both live pathogenic Enterococcus faecalis and Pseudomonas aeruginosa, and ingestion of E. coli in males conferred resistance to P. aeruginosa infection. These results indicated that E. coli and S. aureus can activate both the Toll and IMD pathways, and systemic and local immune responses work together to provide Drosophila more effective protection against infection.
Vaz, F., Kounatidis, I., Covas, G., Parton, R. M., Harkiolaki, M., Davis, I., Filipe, S. R. and Ligoxygakis, P. (2019). Accessibility to peptidoglycan is important for the recognition of gram-positive bacteria in Drosophila. Cell Rep 27(8): 2480-2492.e2486. PubMed ID: 31116990
In Drosophila, it is thought that peptidoglycan recognition proteins (PGRPs) SA and LC structurally discriminate between bacterial peptidoglycans with lysine (Lys) or diaminopimelic (DAP) acid, respectively, thus inducing differential antimicrobial transcription response. This study finds that accessibility to PG at the cell wall plays a central role in immunity to infection. When wall teichoic acids (WTAs) are genetically removed from S. aureus (Lys type) and Bacillus subtilis (DAP type), thus increasing accessibility, the binding of both PGRPs to either bacterium is increased. PGRP-SA and PFRP-LC double mutant flies are more susceptible to infection with both WTA-less bacteria. In addition, WTA-less bacteria grow better in PGRP-SA/-LC double mutant flies. Finally, infection with WTA-less bacteria abolishes any differential activation of downstream antimicrobial transcription. These results indicate that accessibility to cell wall PG is a major factor in PGRP-mediated immunity and may be the cause for discrimination between classes of pathogens.
Mihajlovic, Z., Tanasic, D., Bajgar, A., Perez-Gomez, R., Steffal, P. and Krejci, A. (2019). Lime is a new protein linking immunity and metabolism in Drosophila. Dev Biol. PubMed ID: 31085193
he proliferation, differentiation and function of immune cells in vertebrates, as well as in the invertebrates, is regulated by distinct signalling pathways and crosstalk with systemic and cellular metabolism. This study has identified the Lime gene (Linking Immunity and Metabolism, CG18446) as one such connecting factor, linking hemocyte development with systemic metabolism in Drosophila. Lime is expressed in larval plasmatocytes and the fat body and regulates immune cell type and number by influencing the size of hemocyte progenitor populations in the lymph gland and in circulation. Lime mutant larvae exhibit low levels of glycogen and trehalose energy reserves and they develop low number of hemocytes. The low number of hemocytes in Lime mutants can be rescued by Lime overexpression in the fat body. It is well known that immune cell metabolism is tightly regulated with the progress of infection and it must be supported by systemic metabolic changes. This study demonstrated that Lime mutants fails to induce such systemic metabolic changes essential for the larval immune response. Indeed, Lime mutants are not able to sustain high numbers of circulating hemocytes and are compromised in the number of lamellocytes produced during immune system challenge, using a parasitic wasp infection model. It is therefore proposed the Lime gene as a novel functional link between systemic metabolism and Drosophila immunity.
Duxbury, E. M., Day, J. P., Maria Vespasiani, D., Thuringer, Y., Tolosana, I., Smith, S. C., Tagliaferri, L., Kamacioglu, A., Lindsley, I., Love, L., Unckless, R. L., Jiggins, F. M. and (2019). Host-pathogen coevolution increases genetic variation in susceptibility to infection. Elife 8. PubMed ID: 31038124
It is common to find considerable genetic variation in susceptibility to infection in natural populations. This study has investigated whether natural selection increases this variation by testing whether host populations show more genetic variation in susceptibility to pathogens that they naturally encounter than novel pathogens. In a large cross-infection experiment involving four species of Drosophila and four host-specific viruses, greater genetic variation was always found in susceptibility to viruses that had coevolved with their host. The genetic architecture of resistance was examined in one host species, finding that there are more major-effect genetic variants in coevolved host-pathogen interactions. It is concluded that selection by pathogens has increased genetic variation in host susceptibility, and much of this effect is caused by the occurrence of major-effect resistance polymorphisms within populations.
Nazario-Toole, A. E. and Wu, L. P. (2019). Assessing the cellular immune response of the fruit fly, Drosophila melanogaster, using an in vivo phagocytosis assay. J Vis Exp(146). PubMed ID: 31033954
In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. This study demonstrates an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Thursday, June 20th - Adult Neural Development and Function

Mazaud, D., Kottler, B., Goncalves-Pimentel, C., Proelss, S., Tuchler, N., Deneubourg, C., Yuasa, Y., Diebold, C., Jungbluth, H., Lai, E. C., Hirth, F., Giangrande, A. and Fanto, M. (2019). Transcriptional regulation of the Glutamate/GABA/Glutamine cycle in adult glia controls motor activity and seizures in Drosophila. J Neurosci. PubMed ID: 31064860
The fruitfly Drosophila melanogaster has been extensively used as a genetic model for the maintenance of nervous system's functions. Glial cells are of utmost importance in regulating the neuronal functions in the adult organism and in the progression of neurological pathologies. Through a microRNA-based screen in adult Drosophila glia, this study uncovered the essential role of a major glia developmental determinant, repo, in the adult fly. This study reports that Repo expression is continuously required in adult glia to transcriptionally regulate the highly conserved function of neurotransmitter recycling in both males and females. Transient loss of Repo dramatically shortens fly lifespan, triggers motor deficits and increases the sensibility to seizures, partly due to the impairment of the Glutamate/GABA/Glutamine cycle. These findings highlight the pivotal role of transcriptional regulation of genes involved in the Glutamate/GABA/Glutamine cycle in glia to control neurotransmitter levels in neurons and their behavioural output. The mechanism here identified in Drosophila exemplifies how adult functions can be modulated at the transcriptional level and suggest an active synchronised regulation of genes involved in the same pathway. The process of neurotransmitter recycling is of essential importance in human epileptic and psychiatric disorders and these findings may thus have important consequences for the understanding of the role that transcriptional regulation of neurotransmitter recycling in astrocytes has in human disease.
Haberkern, H., Basnak, M. A., Ahanonu, B., Schauder, D., Cohen, J. D., Bolstad, M., Bruns, C. and Jayaraman, V. (2019). Visually guided behavior and optogenetically induced learning in head-fixed flies exploring a virtual landscape. Curr Biol 29(10): 1647-1659. PubMed ID: 31056392
Studying the intertwined roles of sensation, experience, and directed action in navigation has been facilitated by the development of virtual reality (VR) environments for head-fixed animals, allowing for quantitative measurements of behavior in well-controlled conditions. VR has long featured in studies of Drosophila melanogaster, but these experiments have typically allowed the fly to change only its heading in a visual scene and not its position. This study explores how flies move in two dimensions (2D) using a visual VR environment that more closely captures an animal's experience during free behavior. Flies' 2D interaction with landmarks cannot be automatically derived from their orienting behavior under simpler one-dimensional (1D) conditions. Using novel paradigms, this study demonstrated that flies in 2D VR adapt their behavior in response to optogenetically delivered appetitive and aversive stimuli. Much like free-walking flies after encounters with food, head-fixed flies exploring a 2D VR respond to optogenetic activation of sugar-sensing neurons by initiating a local search, which appears not to rely on visual landmarks. Visual landmarks can, however, help flies to avoid areas in VR where they experience an aversive, optogenetically generated heat stimulus. By coupling aversive virtual heat to the flies' presence near visual landmarks of specific shapes, selective learned avoidance of those landmarks was elicited. Thus, this study demonstrates that head-fixed flies adaptively navigate in 2D virtual environments, but their reliance on visual landmarks is context dependent. These behavioral paradigms set the stage for interrogation of the fly brain circuitry underlying flexible navigation in complex multisensory environments.
Feng, C., Thyagarajan, P., Shorey, M., Seebold, D. Y., Weiner, A. T., Albertson, R. M., Rao, K. S., Sagasti, A., Goetschius, D. J. and Rolls, M. M. (2019). Patronin-mediated minus end growth is required for dendritic microtubule polarity. J Cell Biol. PubMed ID: 31076454
Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, persistent minus end growth is observed, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, it was hypothesized that minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, dendrite development and regeneration were analyzed. Minus ends extended into growing dendrites in the presence of Patronin. In sum, these data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules.
Matsuno, M., Horiuchi, J., Ofusa, K., Masuda, T. and Saitoe, M. (2019). Inhibiting glutamate activity during consolidation suppresses age-related long-term memory impairment in Drosophila. iScience 15: 55-65. PubMed ID: 31030182
In Drosophila, long-term memory (LTM) formation requires increases in glial gene expression. Klingon (Klg), a cell adhesion molecule expressed in both neurons and glia, induces expression of the glial transcription factor, Repo. However, glial signaling downstream of Repo has been unclear. This study demonstrates that Repo increases expression of the glutamate transporter, EAAT1, and EAAT1 is required during consolidation of LTM. The expressions of Klg, Repo, and EAAT1 decrease upon aging, suggesting that age-related impairments in LTM are caused by dysfunction of the Klg-Repo-EAAT1 pathway. Supporting this idea, overexpression of Repo or EAAT1 rescues age-associated impairments in LTM. Pharmacological inhibition of glutamate activity during consolidation improves LTM in klg mutants and aged flies. Altogether, these results indicate that LTM formation requires glial-dependent inhibition of glutamate signaling during memory consolidation, and aging disrupts this process by inhibiting the Klg-Repo-EAAT1 pathway.
Meier, M. and Borst, A. (2019). Extreme compartmentalization in a Drosophila amacrine cell. Curr Biol 29(9): 1545-1550. PubMed ID: 31031119
A neuron is conventionally regarded as a single processing unit. It receives input from one or several presynaptic cells, transforms these signals, and transmits one output signal to its postsynaptic partners. Exceptions exist: amacrine cells in the mammalian retina or interneurons in the locust mesothoracic ganglion are thought to represent many electrically isolated microcircuits within one neuron. An extreme case of such an amacrine cell has recently been described in the Drosophila visual system. This cell, called CT1, reaches into two neuropils of the optic lobe, where it visits each of 700 repetitive columns, thereby covering the whole visual field. Due to its unusual morphology, CT1 has been suspected to perform local computations, but this has never been proven. Using 2-photon calcium imaging and visual stimulation, this study found highly compartmentalized retinotopic response properties in neighboring terminals of CT1, with each terminal acting as an independent functional unit. Model simulations demonstrate that this extreme case of compartmentalization is at the biophysical limit of neural computation.
Nakano, R., Iwamura, M., Obikawa, A., Togane, Y., Hara, Y., Fukuhara, T., Tomaru, M., Takano-Shimizu, T. and Tsujimura, H. (2019). Cortex glia clear dead young neurons via Drpr/dCed-6/Shark and Crk/Mbc/dCed-12 signaling pathways in the developing Drosophila optic lobe. Dev Biol. PubMed ID: 31063730
The molecular and cellular mechanism for clearance of dead neurons was explored in the developing Drosophila optic lobe. During development of the optic lobe, many neural cells die through apoptosis, and corpses are immediately removed in the early pupal stage. Most of the cells that die in the optic lobe are young neurons that have not extended neurites. This study shows that clearance was carried out by cortex glia via a phagocytosis receptor, Draper (Drpr). drpr expression in cortex glia from the second instar larval to early pupal stages was required and sufficient for clearance. Drpr that was expressed in other subtypes of glia did not mediate clearance. Shark and Ced-6 mediated clearance of Drpr. The Crk/Mbc/dCed-12 pathway was partially involved in clearance, but the role was minor. Suppression of the function of Pretaporter, CaBP1 and phosphatidylserine delayed clearance, suggesting a possibility for these molecules to function as Drpr ligands in the developing optic lobe.

Wednesday, June 19th - Adult Physiology

Dennis, E. J., Goldman, O. V. and Vosshall, L. B. (2019). Aedes aegypti mosquitoes use their legs to sense DEET on contact. Curr Biol 29(9): 1551-1556. PubMed ID: 31031114
DEET (N, N-diethyl-meta-toluamide) is the most effective and widely used insect repellent, but its mechanism of action is both complex and controversial. DEET acts on insect smell and taste, and its olfactory mode of action requires the odorant co-receptor orco. Previously it was observed that orco mutant female Aedes aegypti mosquitoes are strongly attracted to humans even in the presence of DEET, but they are rapidly repelled after contacting DEET-treated skin. DEET inhibits food ingestion by Drosophila melanogaster flies, and this repellency is mediated by bitter taste neurons in the proboscis. Similar neurons were identified in the mosquito proboscis, leading to the hypothesis that DEET repels on contact by activating an aversive bitter taste pathway. To understand the basis of DEET contact chemorepellency, behavioral experiments were carried out, and it was discovered that DEET acts by three distinct mechanisms: smell, ingestion, and contact. Like bitter tastants, DEET is a feeding deterrent when ingested, but its bitterness per se does not fully explain DEET contact chemorepellency. Mosquitoes blood fed on human arms treated with high concentrations of bitters, but rapidly avoided DEET-treated skin and did not blood feed. Insects detect tastants both through their proboscis and legs. DEET contact chemorepellency was found to be mediated exclusively by the tarsal segments of the legs and not the proboscis. This work establishes mosquito legs as the behaviorally relevant contact sensors of DEET. These results will inform the search for molecular mechanisms mediating DEET contact chemorepellency and novel contact-based insect repellents.
Klepsatel, P., Girish, T. N., Dircksen, H. and Galikova, M. (2019). Reproductive fitness of Drosophila is maximised by optimal developmental temperature. J Exp Biol 222(Pt 10). PubMed ID: 31064855
Whether the character of developmental plasticity is adaptive or non-adaptive has often been a matter of controversy. Although thermal developmental plasticity has been studied in Drosophila for several traits, it is not entirely clear how it affects reproductive fitness. This study, therefore, investigated how developmental temperature affects reproductive performance (early fecundity and egg-to-adult viability) of wild-caught Drosophila melanogaster. Competing hypotheses on the character of developmental thermal plasticity were characterized using a full-factorial design with three developmental and adulthood temperatures within the natural thermal range of this species. To account for potential intraspecific differences, flies were examined from tropical (India) and temperate (Slovakia) climate zones. The results show that flies from both populations raised at an intermediate developmental temperature (25 ° C) have comparable or higher early fecundity and fertility at all tested adulthood temperatures, while lower (17 ° C) or higher developmental temperatures (29 ° C) did not entail any advantage under the tested thermal regimes. Importantly, the superior thermal performance of flies raised at 25 ° C is apparent even after taking two traits positively associated with reproductive output into account: body size and ovariole number. Thus, in D. melanogaster, development at a given temperature does not necessarily provide any advantage in this thermal environment in terms of reproductive fitness. These findings strongly support the optimal developmental temperature hypothesis, which states that in different thermal environments, the highest fitness is achieved when an organism is raised at its optimal developmental temperature.
Matsuo, N., Nagao, K., Suito, T., Juni, N., Kato, U., Hara, Y. and Umeda, M. (2019). Different mechanisms for selective transport of fatty acids using a single class of lipoprotein in Drosophila. J Lipid Res. PubMed ID: 31085629
In mammals, lipids are selectively transported to specific sites using multiple classes of lipoprotein. However, in Drosophila, a single class of lipoprotein, lipophorin, carries more than 95% of the lipids in the hemolymph. Although a unique ability of the insect lipoprotein system for cargo transport has been demonstrated, it remains unclear how this single class of lipoprotein achieves the selective transport of lipids. In this study, a comparative analysis was carried out of the fatty acid composition among lipophorin, the central nervous system (CNS), and CNS-derived cell lines, and the transport mechanism of fatty acids was investigated, particularly focusing on the transport of poly-unsaturated fatty acids (PUFAs) in Drosophila. PUFAs were shown to be selectively incorporated into the acyl chains of lipophorin phospholipids and effectively transported to CNS through lipophorin receptor (LpR)-mediated endocytosis of lipophorin. In addition, this study demonstrated that C14-fatty acids are selectively incorporated into the diacylglycerols (DAGs) of lipophorin, and that C14-fatty acid-containing DAGs are spontaneously transferred from lipophorin to the phospholipid bilayer. These results suggest that PUFA-containing phospholipids and C14-fatty acids-containing DAGs in lipophorin could be transferred to different sites by different mechanisms to achieve selective transport of fatty acids using a single class of lipoprotein.
Molaei, M., Vandehoef, C. and Karpac, J. (2019). NF-kappaB shapes metabolic adaptation by attenuating Foxo-mediated lipolysis in Drosophila. Dev Cell. PubMed ID: 31080057
Metabolic and innate immune signaling pathways have co-evolved to elicit coordinated responses. However, dissecting the integration of these ancient signaling mechanisms remains a challenge. Using Drosophila, this study uncovered a role for the innate immune transcription factor nuclear factor kappaB (NF-kappaB)/Relish in governing lipid metabolism during metabolic adaptation to fasting. Relish was found to be required to restrain fasting-induced lipolysis, and thus conserve cellular triglyceride levels during metabolic adaptation, through specific repression of ATGL/Brummer lipase gene expression in adipose tissue (fat body). Fasting-induced changes in Brummer expression and, consequently, triglyceride metabolism are adjusted by Relish-dependent attenuation of Foxo transcriptional activation function, a critical metabolic transcription factor. Relish limits Foxo function by influencing fasting-dependent histone deacetylation and subsequent chromatin modifications within the Bmm locus. These results highlight that the antagonism of Relish and Foxo functions are crucial in the regulation of lipid metabolism during metabolic adaptation, which may further influence the coordination of innate immune-metabolic responses.
Nagarajan-Radha, V., Rapkin, J., Hunt, J. and Dowling, D. K. (2019). Interactions between mitochondrial haplotype and dietary macronutrient ratios confer sex-specific effects on longevity in Drosophila melanogaster. J Gerontol A Biol Sci Med Sci. PubMed ID: 31044222
Recent studies have demonstrated that modifications to the ratio of dietary macronutrients affect longevity in a diverse range of species. However, the degree to which levels of natural genotypic variation shape these dietary effects on longevity remains unclear. The mitochondria have long been linked to the ageing process. The mitochondria possess their own genome, and previous studies have shown that mitochondrial genetic variation affects longevity in insects. Furthermore, the mitochondria are the sites in which dietary nutrients are oxidized to produce adenosine triphosphate, suggesting a capacity for dietary quality to mediate the link between mitochondrial genotype and longevity. This study measured longevity of male and female fruit flies, across a panel of genetic strains of Drosophila melanogaster, which vary only in their mitochondrial haplotype, when fed one of two isocaloric diets that differed in their protein-to-carbohydrate ratio. The mitochondrial haplotype affected the longevity of flies, but the pattern of these effects differed across the two diets in males, but not in females. The implications of these results are discussed in relation to an evolutionary theory linking maternal inheritance of mitochondria to the accumulation of male-harming mitochondrial mutations, and to the theory exploring the evolution of phenotypic plasticity to novel environments.
Deepashree, S., Niveditha, S., Shivanandappa, T. and Ramesh, S. R. (2019). Oxidative stress resistance as a factor in aging: evidence from an extended longevity phenotype of Drosophila melanogaster. Biogerontology. PubMed ID: 31054025
Longevity of a species is a multifactorial quantitative trait influenced by genetic background, sex, age and environment of the organism. Extended longevity phenotypes (ELP) from experimental evolution in the laboratory can be used as model systems to investigate the mechanisms underlying aging and senescence. This study investigated the hypothesis that enhanced oxidative stress resistance and elevated antioxidant defense system play a positive role in longevity using an ELP of Drosophila melanogaster. An ELP of D. melanogaster isolated and characterized through artificial selection (inbred laboratory strain of Oregon K) was employed in this study. This ELP, designated long lifespan (LLS) flies, shows marked extension in lifespan when compared to the progenitor population (normal lifespan, NLS) and makes a suitable model to study the role of mitochondrial genome in longevity because of its least heterogeneity. In this study, sensitivity to ethanol with age was employed as a measure of resistance to oxidative stress in NLS and LLS flies. Effect of age and oxidative stress on longevity was examined by employing NLS and LLS flies of different age groups against ethanol-induced oxidative stress. Results show that the lower mortality against ethanol was associated with enhanced oxidative stress resistance, higher antioxidant defenses, lower reactive oxygen species (ROS) levels, enhanced alcohol dehydrogenase activity and better locomotor ability attributes of LLS flies. In addition, age-related changes like locomotor impairments, decreased antioxidant defenses, higher ROS levels and sensitivity to oxidative stress were delayed in LLS flies when compared to NLS. This study supports the hypothesis that higher oxidative stress resistance and enhanced antioxidant defenses are significant factors in extending longevity.

Tuesday, June 18th - Behavior

Corfas, R. A., Sharma, T. and Dickinson, M. H. (2019). Diverse food-sensing neurons trigger idiothetic local search in Drosophila. Curr Biol 29(10): 1660-1668. PubMed ID: 31056390
Foraging animals may benefit from remembering the location of a newly discovered food patch while continuing to explore nearby. For example, after encountering a drop of yeast or sugar, hungry flies often perform a local search. That is, rather than remaining on the food or simply walking away, flies execute a series of exploratory excursions during which they repeatedly depart and return to the resource. Fruit flies, Drosophila melanogaster, can perform this food-centered search behavior in the absence of external landmarks, instead relying on internal (idiothetic) cues. This path-integration behavior may represent a deeply conserved navigational capacity in insects, but its underlying neural basis remains unknown. This study used optogenetic activation to screen candidate cell classes and found that local searches can be initiated by diverse sensory neurons. Optogenetically induced searches resemble those triggered by actual food, are modulated by starvation state, and exhibit key features of path integration. Flies perform tightly centered searches around the fictive food site, even within a constrained maze, and they can return to the fictive food site after long excursions. Together, these results suggest that flies enact local searches in response to a wide variety of food-associated cues and that these sensory pathways may converge upon a common neural system for navigation. Using a virtual reality system, this study demonstrated that local searches can be optogenetically induced in tethered flies walking on a spherical treadmill, laying the groundwork for future studies to image the brain during path integration.
Frighetto, G., Zordan, M. A., Castiello, U. and Megighian, A. (2019). Action-based attention in Drosophila melanogaster. J Neurophysiol. PubMed ID: 31042449
The mechanism of action selection is a widely shared fundamental process required by animals to interact with the environment and adapt to it. A key step in this process is the filtering of many "distracting" sensory inputs which may disturb action selection. Because it has been suggested that, beyond sharing common mechanisms, action selection may also be processed by shared circuits in vertebrates and invertebrates, it was asked whether invertebrates showed the ability to filter out "distracting" stimuli to maintain a goal-directed action, as seen in vertebrates.In this experiment action selection was studied in wild-type Drosophila melanogaster, by investigating their reaction to the abrupt appearance of a visual distractor during an ongoing locomotor action directed to a specific visual target. Flies tended to shift the original trajectory towards the distractor, thus acknowledging it's presence, but did not appear to commit to it, suggesting that an inhibition process took place in order to continue to carry out the original goal-directed action. To some extent flies appeared to take into account the level of salience of the abrupt distractor appearance as a basis for the ensuing motor program. However, they did not engage in a complete change in their initial motor program in favour of the distractor. These results provide interesting insights into the selection-for-action mechanism, in a context requiring action-centered attention which might have appeared rather early in the course of evolution.
Kirszenblat, L., Yaun, R. and van Swinderen, B. (2019). Visual experience drives sleep need in Drosophila. Sleep. PubMed ID: 31100151
Sleep optimizes waking behavior, however, waking experience may also influence sleep. This study used the fruit fly Drosophila melanogaster to investigate the relationship between visual experience and sleep in wild-type and mutant flies. The classical visual mutant, optomotor-blind (omb), which has undeveloped horizontal system/vertical system (HS/VS) motion-processing cells and are defective in motion and visual salience perception, showed dramatically reduced and less consolidated sleep compared to wild-type flies. In contrast, optogenetic activation of the HS/VS motion-processing neurons in wild-type flies led to an increase in sleep following the activation, suggesting an increase in sleep pressure. Surprisingly, exposing wild-type flies to repetitive motion stimuli for extended periods did not increase sleep pressure. However, exposing flies to more complex image sequences from a movie led to more consolidated sleep, particularly when images were randomly shuffled through time. These results suggest that specific forms of visual experience that involve motion circuits and complex, nonrepetitive imagery, drive sleep need in Drosophila.
Krause, T., Spindler, L., Poeck, B. and Strauss, R. (2019). Drosophila acquires a long-lasting body-size memory from visual feedback. Curr Biol. PubMed ID: 31104933
Implicit knowledge of peripersonal space in humans is first acquired during infancy but will be continuously updated throughout life. In contrast, body size of holometabolous insects does not change after metamorphosis; nevertheless, they do have to learn their body reaches at least once. The body size of Drosophila imagines can vary by about 15% depending on environmental factors like food quality and temperature. To investigate how flies acquire knowledge about and memorize their body size, their decisions to either refrain from or initiate climbing over gaps exceeding their body size were studied. Naive (dark-reared) flies overestimate their size and have to learn it from the parallax motion of the retinal images of objects in their environment while walking. Naive flies can be trained in a striped arena and manipulated to underestimate their size, but once consolidated, this memory seems to last for a lifetime. Consolidation of this memory is stress sensitive only in the first 2 h after training but cannot be retrieved for the next 12 h. This study has identified a set of intrinsic, lateral neurons of the protocerebral bridge of the central complex that depend on dCREB2 transcriptional activity for long-term memory consolidation and maintenance.
May, C. E., Vaziri, A., Lin, Y. Q., Grushko, O., Khabiri, M., Wang, Q. P., Holme, K. J., Pletcher, S. D., Freddolino, P. L., Neely, G. G. and Dus, M. (2019). High dietary sugar reshapes sweet taste to promote feeding behavior in Drosophila melanogaster. Cell Rep 27(6): 1675-1685.e1677. PubMed ID: 31067455
Recent studies find that sugar tastes less intense to humans with obesity, but whether this sensory change is a cause or a consequence of obesity is unclear. To tackle this question, the effects of a high sugar diet on sweet taste sensation and feeding behavior were studied in Drosophila melanogaster. On this diet, fruit flies have lower taste responses to sweet stimuli, overconsume food, and develop obesity. Excess dietary sugar, but not obesity or dietary sweetness alone, caused taste deficits and overeating via the cell-autonomous action of the sugar sensor O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in the sweet-sensing neurons. Correcting taste deficits by manipulating the excitability of the sweet gustatory neurons or the levels of OGT protected animals from diet-induced obesity. This work demonstrates that the reshaping of sweet taste sensation by excess dietary sugar drives obesity and highlights the role of glucose metabolism in neural activity and behavior.
Dai, X., Zhou, E., Yang, W., Zhang, X., Zhang, W. and Rao, Y. (2019). D-Serine made by serine racemase in Drosophila intestine plays a physiological role in sleep. Nat Commun 10(1): 1986. PubMed ID: 31064979
Natural D-serine (D-Ser) has been detected in animals more than two decades ago, but little is known about the physiological functions of D-Ser. This study has revealed sleep regulation by endogenous D-Ser. Sleep was decreased in mutants defective in D-Ser synthesis or its receptor the N-methyl-D-aspartic receptor 1 (NMDAR1), but increased in mutants defective in D-Ser degradation. D-Ser but not L-Ser rescued the phenotype of mutants lacking serine racemase (SR), the key enzyme for D-Ser synthesis. Pharmacological and triple gene knockout experiments indicate that D-Ser functions upstream of NMDAR1. Expression of SR was detected in both the nervous system and the intestines. Strikingly, reintroduction of SR into specific intestinal epithelial cells rescued the sleep phenotype of sr mutants. These results have established a novel physiological function for endogenous D-Ser and a surprising role for intestinal cells.

Monday, June 17th - Signaling

Chowdhury, M., Li, C. F., He, Z., Lu, Y., Liu, X. S., Wang, Y. F., Ip, Y. T., Strand, M. R. and Yu, X. Q. (2019). Toll family members bind multiple Spatzle proteins and activate antimicrobial peptide gene expression in Drosophila. J Biol Chem. PubMed ID: 31088910
The Toll signaling pathway in Drosophila melanogaster regulates several immune-related functions, including the expression of antimicrobial peptide (AMP) genes. The canonical Toll receptor (Toll-1) is activated by the cytokine Spatzle (Spz-1), but Drosophila encodes eight other Toll genes and five other Spz genes whose interactions with one another and associated functions are less well understood. In vitro assays were conducted in the Drosophila S2 cell line with the Toll/interleukin-1 receptor (TIR) homology domains of each Toll family member to determine if they can activate a known target of Toll-1, the promoter of the antifungal peptide gene drosomycin. All TIR family members activated the drosomycin promoter, with Toll-1 and Toll-7 TIRs producing the highest activation. The Toll-1 and Toll-7 ectodomains bind Spz-1, -2, and -5 and also vesicular stomatitis virus (VSV) virions; Spz-1, -2, -5, and VSV all activated the promoters of drosomycin and several other AMP genes in S2 cells expressing full-length Toll-1 or Toll-7. In vivo experiments indicated that Toll-1 and Toll-7 mutants could be systemically infected with two bacterial species (Enterococcus faecalis and Pseudomonas aeruginosa), the opportunistic fungal pathogen Candida albicans and VSV with different survival in adult females and males compared with wild-type fly survival. These results suggest that all Toll family members can activate several AMP genes. These results further indicate that Toll-1 and Toll-7 bind multiple Spz proteins and also VSV, but differentially affect adult survival after systemic infection, potentially because of sex-specific differences in Toll-1 and Toll-7 expression.
Chen, Z. and Zou, Y. (2019). Anterior-Posterior patterning of Drosophila wing disc I: A baseline mathematical model. Math Biosci. PubMed ID: 31085191
Wing imaginal disc of Drosophila is one of the commonly used model systems for the studies of patterning, growth, and scaling. Development of the wing disc involves many interacting components as well as a variety of compound processes whose underlying mechanisms are still under investigation. For instance, it remains unclear about how to form compound expersmentally-measured patterns of Decapentaplegic (Dpp) type-I receptor Thickveins (Tkv), as well as phosphorylated Mothers Against Dpp (pMad) which is the indicator of Dpp signaling activities. This work proposes a baseline mathematical model that integrates established experimental facts to investigate the formation of pMad and Tkv gradients. This model is validated by the accurate reproduction of complex asymmetric profiles of Tkv and pMad in both anterior and posterior compartments of wing disc. Moreover, using this model as a numerical platform, specific roles played by engrailed (En), Hedgehog (Hh) and Dpp were examined in the establishment of Tkv and pMad profiles. It turns out that all En, Hh, Dpp play an essential role in the formation of pMad and Tkv patterns. In particular, the proposed model supports the crucial part of the downregulation of Tkv by Dpp. Besides, dual negative regulations of Tkv by both Hh and Dpp simultaneously prevent the Dpp signaling from interfering and expand the effective range of Dpp gradients. Finally, parameter sensitivity was carried out to ensure that these results and conclusions are robust against specific choices of parameter values.
Xu, Y., Anjaneyulu, M., Donelian, A., Yu, W., Greenberg, M. L., Ren, M., Owusu-Ansah, E. and Schlame, M. (2019). Assembly of the complexes of oxidative phosphorylation triggers the remodeling of cardiolipin. Proc Natl Acad Sci U S A. PubMed ID: 31110016
Cardiolipin (CL) is a mitochondrial phospholipid with a very specific and functionally important fatty acid composition, generated by tafazzin. However, in vitro Tafazzin catalyzes a promiscuous acyl exchange that acquires specificity only in response to perturbations of the physical state of lipids. To identify the process that imposes acyl specificity onto CL remodeling in vivo, this study analyzed a series of deletions and knockdowns in Saccharomyces cerevisiae and Drosophila melanogaster, including carriers, membrane homeostasis proteins, fission-fusion proteins, cristae-shape controlling and MICOS proteins, and the complexes I-V. Among those, only the complexes of oxidative phosphorylation (OXPHOS) affected the CL composition. Rather than any specific complex, it was the global impairment of the OXPHOS system that altered CL and at the same time shortened its half-life. The knockdown of OXPHOS expression had the same effect on CL as the knockdown of tafazzin in Drosophila flight muscles, including a change in CL composition and the accumulation of monolyso-CL. Thus, the assembly of OXPHOS complexes induces CL remodeling, which, in turn, leads to CL stabilization. It is hypothesized that protein crowding in the OXPHOS system imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid-protein complexes.
Garrido-Jimenez, S., Roman, A. C. and Carvajal-Gonzalez, J. M. (2019). Diminished expression of Fat and Dachsous PCP proteins impaired centriole planar polarization in Drosophila. Front Genet 10: 328. PubMed ID: 31031805
Proper ciliary basal body positioning within a cell is key for cilia functioning. Centriole and basal body positioning depends on signaling pathways such as the planar cell polarity pathway (PCP) governed by Frizzled (Fz-PCP). There have been described two PCP pathways controlled by different protein complexes, the Frizzled-PCP and the Fat-PCP pathway. Centriole planar polarization in non-dividing cells is a dynamic process that depends on the Fz-PCP pathway to properly occur during development from flies to humans. However, the function of the Ft-PCP pathway in centrioles polarization is elusive. This study presents a descriptive initial analysis of centrioles polarization in Fat-PCP loss of function (LOF) conditions. Fat (Ft) and Dachsous (Ds) LOF showed a marked centrioles polarization defect similar to what has previously been reported in Fz-PCP alterations. Altogether, the data suggest that centriole planar polarization in Drosophila wings depends on both Ft-PCP and Fz-PCP pathways. Further analyses in single and double mutant conditions will be required to address the functional connection between PCP and centriole polarization in flies.
Zhang, C. H., Zhang, M. J., Shi, X. X., Mao, C. and Zhu, Z. R. (2019). Alkaline ceramidase mediates the oxidative stress response in Drosophila melanogaster through Sphingosine. J Insect Sci 19(3). PubMed ID: 31115476
Alkaline ceramidase (Dacer) in Drosophila melanogaster was demonstrated to be resistant to paraquat-induced oxidative stress. However, the underlying mechanism for this resistance remained unclear. This study has shown that sphingosine feeding triggered the accumulation of hydrogen peroxide (H2O2). Dacer-deficient D. melanogaster (Dacer mutant) has higher catalase (CAT) activity and CAT transcription level, leading to higher resistance to oxidative stress induced by paraquat. By performing a quantitative proteomic analysis, this study identified 79 differentially expressed proteins in comparing Dacer mutant to wild type. Three oxidoreductases, including two cytochrome P450 (CG3050, CG9438) and an oxoglutarate/iron-dependent dioxygenase (CG17807), were most significantly upregulated in Dacer mutant. It is presumed that altered antioxidative activity in Dacer mutant might be responsible for increased oxidative stress resistance. This work provides a novel insight into the oxidative antistress response in D. melanogaster.
Katow, H., Takahashi, T., Saito, K., Tanimoto, H. and Kondo, S. (2019). Tango knock-ins visualize endogenous activity of G protein-coupled receptors in Drosophila. J Neurogenet: 1-8. PubMed ID: 31084242
G protein-coupled receptors (GPCRs) represent a family of seven-pass transmembrane protein receptors whose ligands include neuropeptides and small-molecule neuromodulators such as dopamine and serotonin. These neurotransmitters act at long distances and are proposed to define the ground state of the nervous system. The Drosophila genome encodes approximately 50 neuropeptides and their functions in physiology and behavior are now under intensive studies. Key information currently lacking in the field is the spatiotemporal activation patterns of endogenous GPCRs. This study reports application of the Tango system, a reporter assay to detect GPCR activity, to endogenous GPCRs in the fly genome. A method was developed to integrate the sensor component of the Tango system to the C-terminus of endogenous genes by using genome editing techniques. Tango sensors in the Sex-peptide receptor (SPR) locus were shown to allow sensitive detection of mating-dependent SPR activity in the female reproductive organ. The method is easily applicable to any GPCR and will provide a way to systematically characterize GPCRs in the fly brain.

Friday, June 14th - Evolution

Cooper, J. C., Lukacs, A., Reich, S., Schauer, T., Imhof, A. and Phadnis, N. (2019). Altered localization of hybrid incompatibility proteins in Drosophila. Mol Biol Evol. PubMed ID: 31038678
Understanding the molecular basis of hybrid incompatibilities is a fundamental pursuit in evolutionary genetics. In crosses between Drosophila melanogaster females and Drosophila simulans males, an interaction between at least three genes is necessary for hybrid male lethality: Hmr mel, Lhr sim, and gfzf sim. While HMR and LHR physically bind each other and function together in a single complex, the connection between gfzf and either of these proteins remains mysterious. This study shows that GFZF localizes to many regions of the genome in both D. melanogaster and D. simulans, including at telomeric retrotransposon repeats. GFZF localization at telomeres is significantly different between these two species, reflecting the rapid evolution of telomeric retrotransposon copy number composition between the two species. It was next shown that GFZF and HMR normally do not co-localize in D. melanogaster. In inter-species hybrids, however, HMR shows extensive mis-localization to GFZF sites, thus uncovering a new molecular interaction between these hybrid incompatibility factors. Spreading of HMR to GFZF sites requires gfzf sim but not Lhr sim, suggesting distinct roles for these factors in the hybrid incompatibility. Finally, this study found that over-expression of HMR and LHR within species is sufficient to mis-localize HMR to GFZF binding sites, indicating that HMR has a natural low affinity for GFZF sites. Together, these studies provide the first insights into the different properties of gfzf between D. melanogaster and D. simulans, and uncover a molecular interaction between gfzf and Hmr in the form of altered protein localization.
Finet, C., Slavik, K., Pu, J., Carroll, S. B. and Chung, H. (2019). Birth-and-death evolution of the fatty acyl-CoA reductase (FAR) gene family and diversification of cuticular hydrocarbon synthesis in Drosophila. Genome Biol Evol. PubMed ID: 31076758
The birth-and-death evolutionary model proposes that some members of a multigene family are phylogenetically stable and persist as a single copy over time whereas other members are phylogenetically unstable and undergo frequent duplication and loss. Functional studies suggest that stable genes are likely to encode essential functions, while rapidly evolving genes reflect phenotypic differences in traits that diverge rapidly among species. One such class of rapidly diverging traits are insect cuticular hydrocarbons (CHCs), which play dual roles in chemical communications as short-range recognition pheromones as well as protecting the insect from desiccation. Insect CHCs diverge rapidly between related species leading to ecological adaptation and/or reproductive isolation. Because the CHC and essential fatty acid biosynthetic pathways share common genes, it was hypothesized that genes involved in the synthesis of CHCs would be evolutionary unstable, while those involved in fatty acid-associated essential functions would be evolutionary stable. To test this hypothesis, the evolutionary history was investigated of the fatty acyl-CoA reductases (FARs) gene family that encodes enzymes in CHC synthesis. A unique dataset was compiled of 200 FAR proteins across 12 Drosophila species. A broad diversity in FAR content was uncovered that is generated by gene duplications, subsequent gene losses, and alternative splicing. FARs expressed in oenocytes and presumably involved in CHC synthesis are more unstable than FARs from other tissues. Taken together, this study provides empirical evidence that a comparative approach investigating the birth-and-death evolution of gene families can identify candidate genes involved in rapidly diverging traits between species.
Ruzicka, F., Hill, M. S., Pennell, T. M., Flis, I., Ingleby, F. C., Mott, R., Fowler, K., Morrow, E. H. and Reuter, M. (2019). Genome-wide sexually antagonistic variants reveal long-standing constraints on sexual dimorphism in fruit flies. PLoS Biol 17(4): e3000244. PubMed ID: 31022179
The evolution of sexual dimorphism is constrained by a shared genome, leading to 'sexual antagonism', in which different alleles at given loci are favoured by selection in males and females. Despite its wide taxonomic incidence, little is known about the identity, genomic location, and evolutionary dynamics of antagonistic genetic variants. To address these deficits, this study used sex-specific fitness data from 202 fully sequenced hemiclonal Drosophila melanogaster fly lines to perform a genome-wide association study (GWAS) of sexual antagonism. Approximately 230 chromosomal clusters of candidate antagonistic single nucleotide polymorphisms (SNPs) were identified. In contradiction to classic theory, no clear evidence was found that the X chromosome is a hot spot for sexually antagonistic variation. Characterising antagonistic SNPs functionally, a large excess of missense variants were found, but there was very little enrichment in terms of gene function. The evolutionary persistence of antagonistic variants was also assessed by examining extant polymorphism in wild D. melanogaster populations and closely related species. Remarkably, antagonistic variants are associated with multiple signatures of balancing selection across the D. melanogaster distribution range and in their sister species D. simulans, indicating widespread and evolutionarily persistent (about 1 million years) genomic constraints on the evolution of sexual dimorphism. Based on these results, it is proposed that antagonistic variation accumulates because of constraints on the resolution of sexual conflict over protein coding sequences, thus contributing to the long-term maintenance of heritable fitness variation.
Green, L., Battlay, P., Fournier-Level, A., Good, R. T. and Robin, C. (2019). Cis- and trans-acting variants contribute to survivorship in a naive Drosophila melanogaster population exposed to ryanoid insecticides. Proc Natl Acad Sci U S A 116(21): 10424-10429. PubMed ID: 31064874
Insecticide resistance is a paradigm of microevolution, and insecticides are responsible for the strongest cases of recent selection in the genome of Drosophila melanogaster. This study used a naive population and a novel insecticide class to examine the ab initio genetic architecture of a potential selective response. Genome-wide association studies (GWAS) of chlorantraniliprole susceptibility reveal variation in a gene of major effect, Stretchin Myosin light chain kinase (Strn-Mlck), which was validated with linkage mapping and transgenic manipulation of gene expression. It is proposed that allelic variation in Strn-Mlck alters sensitivity to the calcium depletion attributable to chlorantraniliprole's mode of action. GWAS also reveal a network of genes involved in neuromuscular biology. In contrast, phenotype to transcriptome associations identify differences in constitutive levels of multiple transcripts regulated by cnc, the homolog of mammalian Nrf2. This suggests that genetic variation acts in trans to regulate multiple metabolic enzymes in this pathway. The most outstanding association is with the transcription level of Cyp12d1 which is also affected in cis by copy number variation. Transgenic overexpression of Cyp12d1 reduces susceptibility to both chlorantraniliprole and the closely related insecticide cyantraniliprole. This systems genetics study reveals multiple allelic variants segregating at intermediate frequency in a population that is completely naive to this new insecticide chemistry and it foreshadows a selective response among natural populations to these chemicals.
Poikela, N., Kinnunen, J., Wurdack, M., Kauranen, H., Schmitt, T., Kankare, M., Snook, R. R. and Hoikkala, A. (2019). Strength of sexual and postmating prezygotic barriers varies between sympatric populations with different histories and species abundances. Evolution. PubMed ID: 30957216
The impact of different reproductive barriers on species or population isolation may vary in different stages of speciation depending on evolutionary forces acting within species and through species' interactions. Genetic incompatibilities between interacting species are expected to reinforce prezygotic barriers in sympatric populations and lead to cascade reinforcement between conspecific populations living within and outside the areas of sympatry. These predictions were tested and this work studied whether and how the strength and target of reinforcement between Drosophila montana and Drosophila flavomontana vary between sympatric populations with different histories and species abundances. All barriers between D. montana females and D. flavomontana males were nearly complete, while in the reciprocal cross strong postzygotic isolation was accompanied by prezygotic barriers whose strength varied according to population composition. Sexual isolation between D. flavomontana females and D. montana males was increased in long-established sympatric populations, where D. flavomontana is abundant, while postmating prezygotic (PMPZ) barriers were stronger in populations where this species is a new invader and still rare and where female discrimination against heterospecific males was lower. Strengthening of sexual and PMPZ barriers in this cross also induced cascade reinforcement of respective barriers between D. flavomontana populations, which is a classic signature of reinforcement process.
Durmaz, E., Rajpurohit, S., Betancourt, N., Fabian, D. K., Kapun, M., Schmidt, P. and Flatt, T. (2019). A clinal polymorphism in the insulin signaling transcription factor foxo contributes to life-history adaptation in Drosophila. Evolution. PubMed ID: 31111462
A fundamental aim of adaptation genomics is to identify polymorphisms that underpin variation in fitness traits. In D. melanogaster latitudinal life-history clines exist on multiple continents and make an excellent system for dissecting the genetics of adaptation. Previous work has identified numerous clinal SNPs in insulin/insulin-like growth factor signaling (IIS), a pathway known from mutant studies to affect life history. However, the effects of natural variants in this pathway remain poorly understood. This study investigated how two clinal alternative alleles at foxo, a transcriptional effector of IIS, affect fitness components (viability, size, starvation resistance, fat content). This polymorphism from the North American cline was assessed by reconstituting outbred populations, fixed for either the low- or high-latitude allele, from inbred DGRP lines. Since diet and temperature modulate IIS, alleles were phenotyped across two temperatures (18 ° C, 25 ° C) and two diets differing in sugar source and content. Consistent with clinal expectations, the high-latitude allele conferred larger body size and reduced wing loading. Alleles also differed in starvation resistance and expression of InR, a transcriptional target of FOXO. Allelic reaction norms were mostly parallel, with few GxE interactions. Together, these results suggest that variation in IIS makes a major contribution to clinal life-history adaptation.

Thursday, June 13th - Gonads

Lieber, T., Jeedigunta, S. P., Palozzi, J. M., Lehmann, R. and Hurd, T. R. (2019). Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline. Nature. PubMed ID: 31092924
Mitochondria contain their own genomes that, unlike nuclear genomes, are inherited only in the maternal line. Owing to a high mutation rate and low levels of recombination of mitrochondrial DNA (mtDNA), special selection mechanisms exist in the female germline to prevent the accumulation of deleterious mutations. However, the molecular mechanisms that underpin selection are poorly understood. This study visualized germline selection in Drosophila using an allele-specific fluorescent in situ-hybridization approach to distinguish wild-type from mutant mtDNA. Selection first manifests in the early stages of Drosophila oogenesis, triggered by reduction of the pro-fusion protein Mitofusin. This leads to the physical separation of mitochondrial genomes into different mitochondrial fragments, which prevents the mixing of genomes and their products and thereby reduces complementation. Once fragmented, mitochondria that contain mutant genomes are less able to produce ATP, which marks them for selection through a process that requires the mitophagy proteins Atg1 and BNIP3. A reduction in Atg1 or BNIP3 decreases the amount of wild-type mtDNA, which suggests a link between mitochondrial turnover and mtDNA replication. Fragmentation is not only necessary for selection in germline tissues, but is also sufficient to induce selection in somatic tissues in which selection is normally absent. It is postulated that there is a generalizable mechanism for selection against deleterious mtDNA mutations, which may enable the development of strategies for the treatment of mtDNA disorders.
Woo, W. K., Dzaki, N., Thangadurai, S. and Azzam, G. (2019). Ectopic miR-975 induces CTP synthase directed cell proliferation and differentiation in Drosophila melanogaster. Sci Rep 9(1): 6096. PubMed ID: 30988367
CTP synthase (CTPSyn) is an essential metabolic enzyme, synthesizing precursors required for nucleotides and phospholipids production. Previous studies have also shown that CTPSyn is elevated in various cancers. In many organisms, CTPSyn compartmentalizes into filaments called cytoophidia. In Drosophila melanogaster, only its isoform C (CTPSynIsoC) forms cytoophidia. In the fruit fly's testis, cytoophidia are normally seen in the transit amplification regions close to its apical tip, where the stem-cell niche is located, and development is at its most rapid. This study reports that CTPSynIsoC overexpression causes the lengthening of cytoophidia throughout the entirety of the testicular body. A bulging apical tip is found in approximately 34% of males overexpressing CTPSynIsoC. Immunostaining shows that this bulged phenotype is most likely due to increased numbers of both germline cells and spermatocytes. Through a microRNA (miRNA) overexpression screen, ectopic miR-975 was found to concurrently increase both the expression levels of CTPSyn and the length of its cytoophidia. The bulging testes phenotype was also recovered at a penetration of approximately 20%. However, qPCR assays reveal that CTPSynIsoC and miR-975 overexpression each provokes a differential response in expression of a number of cancer-related genes, indicating that the shared CTPSyn upregulation seen in either case is likely the cause of observed testicular overgrowth. This study presents the first instance of consequences of miRNA-asserted regulation upon CTPSyn in D. melanogaster, and further reaffirms the enzyme's close ties to germline cells overgrowth.
Camara, N., Whitworth, C., Dove, A. and Van Doren, M. (2019). Doublesex controls specification and maintenance of the gonad stem cell niches in Drosophila. Development. PubMed ID: 31043421
Sex-specific development of the gonads is a key aspect of sexual dimorphism that is regulated by Doublesex/Mab3 Related Transcription Factors (DMRTs) in diverse animal species. This study found that in mutants for Drosophila dsx, important components of the male and female gonad stem cell niches (hubs and terminal filaments/cap cells, respectively) still form. Initially, gonads in all dsx mutants (both XX and XY) initiate the male program of development, but later half of these gonads switch to form female stem cell niche structures. One individual can have both male-type and female-type gonad niches, however male and female niches are usually not observed in the same gonad, indicating that cells make a "group decision" about which program to follow. It is concluded that dsx does not act in an instructive manner to regulate male vs. female niche formation, as these structures form in the absence of dsx function. Instead, dsx acts to "tip the balance" between the male or female programs, which are then executed independent of dsx. bric a brac acts downstream of dsx to control the male vs. female niche decision. These results indicate that, in both flies and mammals, the sexual fate of the somatic gonad is remarkably plastic and is controlled by a combination of autonomous and non-autonomous cues.
Benitez, M., Tatapudy, S., Liu, Y., Barber, D. L. and Nystul, T. G. (2019). Drosophila anion exchanger 2 is required for proper ovary development and oogenesis. Dev Biol. PubMed ID: 31071312
Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type. Previous work has shown that the pHi of cells in the follicle stem cell (FSC) lineage in the Drosophila ovary increases progressively during differentiation from an average of 6.8 in the FSCs, to 7.0 in newly produced daughter cells, to 7.3 in more differentiated cells. Two major regulators of pHi in this lineage are Drosophila sodium-proton exchanger 2 (dNhe2) and a previously uncharacterized gene, CG8177, that is homologous to mammalian anion exchanger 2 (AE2). Based on this homology, the gene was named anion exchanger 2 (ae2). This study generated null alleles of ae2 and found that homozygous mutant flies are viable but have severe defects in ovary development and adult oogenesis. Specifically, it was found that ae2 null flies have smaller ovaries, reduced fertility, and impaired follicle formation. In addition, the follicle formation defect can be suppressed by a decrease in dNhe2 copy number and enhanced by the overexpression of dNhe2, suggesting that this phenotype is due to the dysregulation of pHi. These findings support the emerging idea that pHi dynamics regulate cell fate decisions and these studies provide new genetic tools to investigate the mechanisms by which this occurs.
Dreyer, A. P. and Shingleton, A. W. (2019). Insulin-insensitivity of male genitalia maintains reproductive success in Drosophila. Biol Lett 15(5): 20190057. PubMed ID: 31088279
For most arthropod species, male genital size is relatively implastic in response to variation in developmental nutrition, such that the genitals in large well-fed males are similar in size to those in small poorly-fed males. In Drosophila melanogaster, reduced nutritional plasticity of the male genitalia is a consequence of low insulin sensitivity through a tissue-specific reduction in the expression of FOXO, a negative growth regulator . Despite an understanding of the proximate developmental mechanisms regulating organ size, the ultimate evolutionary mechanisms that may have led to reduced FOXO expression in the genitalia have not been fully elucidated. This study shows that restoring FOXO activity in the developing genitalia reduces the male genital size and decreases various aspects of male reproductive success. These data support the hypothesis that sexual selection has acted on the male genitalia to limit their nutritional plasticity through a reduction in FOXO expression, linking proximate with ultimate mechanisms of genital evolution.
Fingerhut, J. M., Moran, J. V. and Yamashita, Y. M. (2019). Satellite DNA-containing gigantic introns in a unique gene expression program during Drosophila spermatogenesis. PLoS Genet 15(5): e1008028. PubMed ID: 31071079
Intron gigantism, where genes contain megabase-sized introns, is observed across species, yet little is known about its purpose or regulation. This study identify a unique gene expression program utilized for the proper expression of genes with intron gigantism. Two Drosophila genes with intron gigantism, kl-3 and kl-5, are transcribed in a spatiotemporal manner over the course of spermatocyte differentiation, which spans ~90 hours. The introns of these genes contain megabases of simple satellite DNA repeats that comprise over 99% of the gene loci, and these satellite-DNA containing introns are transcribed. Two RNA-binding proteins were identified that specifically localize to kl-3 and kl-5 transcripts and are needed for the successful transcription or processing of these genes. It is proposed that genes with intron gigantism require a unique gene expression program, which may serve as a platform to regulate gene expression during cellular differentiation.

Wednesday, June 12th - Embryonic Development

D'Angelo, A., Dierkes, K., Carolis, C., Salbreux, G. and Solon, J. (2019). In vivo force application reveals a fast tissue softening and external friction increase during early embryogenesis. Curr Biol 29(9): 1564-1571. PubMed ID: 31031116
During development, cell-generated forces induce tissue-scale deformations to shape the organism. The pattern and extent of these deformations depend not solely on the temporal and spatial profile of the generated force fields but also on the mechanical properties of the tissues that the forces act on. It is thus conceivable that, much like the cell-generated forces, the mechanical properties of tissues are modulated during development in order to drive morphogenesis toward specific developmental endpoints. Although many approaches have recently emerged to assess effective mechanical parameters of tissues, they could not quantitatively relate spatially localized force induction to tissue-scale deformations in vivo. This study presents a method that overcomes this limitation. The approach is based on the application of controlled forces on a single microparticle embedded in an individual cell of an embryo. Combining measurements of bead displacement with the analysis of induced deformation fields in a continuum mechanics framework, material properties were quantified of the tissue, and their changes over time were followed. In particular, a rapid change was uncovered in tissue response occurring during Drosophila cellularization, resulting from a softening of the blastoderm and an increase of external friction. The microtubule cytoskeleton is a major contributor to epithelial mechanics at this stage. Developmentally controlled modulations was identified in perivitelline spacing that can account for the changes in friction. Overall, this method allows for the measurement of key mechanical parameters governing tissue-scale deformations and flows occurring during morphogenesis.
Surkova, S., Sokolkova, A., Kozlov, K., Nuzhdin, S. V. and Samsonova, M. (2019). Quantitative analysis reveals genotype- and domain- specific differences between mRNA and protein expression of segmentation genes in Drosophila. Dev Biol 448(1): 48-58. PubMed ID: 30629954
In many biological systems gene expression at mRNA and protein levels is not identical. This study characterizes differences between mRNA and protein expression of Drosophila segmentation genes at the level of individual gene expression domains. Quantitative imaging data was obtained on expression of gap genes gt and hb and pair-rule gene eve for Drosophila wild type embryos, Kr null mutants and Kr+/Kr- heterozygotes. To compare mRNA and protein expression, several criteria were used, including difference in amplitude and positions of expression domains, pattern shape and positional variability. For a number of gene expression domains, examples are shown where protein expression does not repeat mRNA expression even after a temporal delay. Time delays were calculated between eve pattern formation at the level of mRNA and protein for wild type embryos, Kr mutants and Kr+Kr- heterozygotes. In wild type embryos, the amplitudes of eve stripes 3 and 7 do not differ significantly at the level of mRNA, however, stripe 3 is higher than stripe 7 at the protein level. It was further shown that hb mRNA and protein expression in both anterior and posterior domains significantly differs at specific time points. The formation of hb PS4 stripe at the mRNA level proceeds five times faster than at the level of protein. With regard to spatial expression, the offset between posterior gt mRNA and protein domains is much larger in Kr mutants than in wild type embryos and heterozygotes. Finally, differences were analyzed in positional variability of eve stripe 7 expression in Kr mutants and Kr+/Kr- heterozygotes at the level of mRNA and protein. These results enable further perspectives to uncover mechanisms underlying discrepancies between mRNA and protein expression in early embryo.
Yang, R., Li, E., Kwon, Y. J., Mani, M. and Beitel, G. J. (2019). QuBiT: a quantitative tool for analyzing epithelial tubes reveals unexpected patterns of organization in the Drosophila trachea. Development. PubMed ID: 30967427
Biological tubes are essential for animal survival, and their functions are critically dependent on tube shape. Analyzing the contributions of cell shape and organization to the morphogenesis of small tubes has been hampered by the limitations of existing programs in quantifying cell geometry on highly curved tubular surfaces and calculating tube-specific parameters. This study describes QuBiT (Quantitative Tool for Biological Tubes) and used it to analyze morphogenesis of embryonic Drosophila trachaea (airway). In the main tube, previously unknown anterior-to-posterior (A-P) gradients were found of cell apical orientation and aspect ratio and periodicity in the organization of apical cell surfaces. Inferred cell intercalation during development dampens an A-P gradient of the number of cells per cross-section of the tube, but does not change the patterns of cell connectivity. Computationally "unrolling" the apical surface of WT trachea and the hindgut reveals previously unrecognized spatial patterns of the apical marker Uninflatable and a non-redundant role for the Na(+)/K(+) ATPase in apical marker organization. These unexpected findings demonstrate the importance of a computational tool for analyzing small diameter biological tubes.
Ventos-Alfonso, A., Ylla, G. and Belles, X. (2019). Zelda and the maternal-to-zygotic transition in cockroaches. FEBS J. PubMed ID: 30993896
In the endopterygote Drosophila melanogaster, Zelda is an activator of the zygotic genome during the maternal-to-zygotic transition (MZT). Zelda binds cis-regulatory elements (TAGteam heptamers), making chromatin accessible for gene transcription. This study examined Zelda in the cockroach Blattella germanica, a hemimetabolan, short germ-band, and polyneopteran species. B. germanica Zelda has the complete set of functional domains, which is typical of species displaying ancestral features concerning embryogenesis. Interestingly, D. melanogaster TAGteam heptamers were found in the B. germanica genome. The canonical one, CAGGTAG, is present at a similar proportion in the genome of these two species and in the genome of other insects, suggesting that the genome admits as many CAGGTAG motifs as its length allows. Zelda-depleted embryos of B. germanica show defects involving blastoderm formation and abdomen development, and genes contributing to these processes are down-regulated. It is concluded that in B. germanica Zelda strictly activates the zygotic genome, within the MZT, a role conserved in more derived endopterygote insects. In B. germanica, zelda is expressed during MZT, whereas in D. melanogaster and T. castaneum it is expressed beyond this transition. In these species and A. mellifera, Zelda has functions even in postembryonic development. The expansion of zelda expression beyond the MZT in endopterygotes might be related with the evolutionary innovation of holometabolan metamorphosis.
Draper, I., Saha, M., Stonebreaker, H., Salomon, R. N., Matin, B. and Kang, P. B. (2019). The impact of Megf10/Drpr gain-of-function on muscle development in Drosophila. FEBS Lett 593(7): 680-696. PubMed ID: 30802937
Recessive mutations in multiple epidermal growth factor-like domains 10 (MEGF10) underlie a rare congenital muscle disease known as MEGF10 myopathy. MEGF10 and its Drosophila homolog Draper (Drpr) are transmembrane receptors expressed in muscle and glia. Drpr deficiency is known to result in muscle abnormalities in flies. In the current study, flies that ubiquitously overexpress Drpr, or mouse Megf10, display developmental arrest. The phenotype is reproduced with overexpression in muscle, but not in other tissues, and with overexpression during intermediate stages of myogenesis, but not in myoblasts. Tubular muscle subtypes are particularly sensitive to Megf10/Drpr overexpression. Complementary genetic analyses show that Megf10/Drpr and Notch may interact to regulate myogenesis. These findings provide a basis for investigating MEGF10 in muscle development using Drosophila.
McDaniel, S. L., Gibson, T. J., Schulz, K. N., Fernandez Garcia, M., Nevil, M., Jain, S. U., Lewis, P. W., Zaret, K. S. and Harrison, M. M. (2019). Continued activity of the pioneer factor Zelda is required to drive zygotic genome activation. Mol Cell 74(1): 185-195.e184. PubMed ID: 30797686
Reprogramming cell fate during the first stages of embryogenesis requires that transcriptional activators gain access to the genome and remodel the zygotic transcriptome. Nonetheless, it is not clear whether the continued activity of these pioneering factors is required throughout zygotic genome activation or whether they are only required early to establish cis-regulatory regions. To address this question, an optogenetic strategy was developed to rapidly and reversibly inactivate the master regulator of genome activation in Drosophila, Zelda. Using this strategy, continued Zelda activity was shown to be required throughout genome activation. Zelda was shown to bind DNA in the context of nucleosomes; this might allow Zelda to occupy the genome despite the rapid division cycles in the early embryo. These data identify a powerful strategy to inactivate transcription factor function during development and suggest that reprogramming in the embryo may require specific, continuous pioneering functions to activate the genome.

Tuesday, June 11th - RNA and Transposons

Ri, H., Lee, J., Sonn, J. Y., Yoo, E., Lim, C. and Choe, J. (2019). Drosophila CrebB is a substrate of the nonsense-mediated mRNA decay pathway that sustains circadian behaviors. Mol Cells 42(4): 301-312. PubMed ID: 31091556
Post-transcriptional regulation underlies the circadian control of gene expression and animal behaviors. However, the role of mRNA surveillance via the nonsense-mediated mRNA decay (NMD) pathway in circadian rhythms remains elusive. This study reports that Drosophila NMD pathway acts in a subset of circadian pacemaker neurons to maintain robust 24 h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide Pigment-Dispersing Factor (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. It was further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.
Chang, C. H., Chavan, A., Palladino, J., Wei, X., Martins, N. M. C., Santinello, B., Chen, C. C., Erceg, J., Beliveau, B. J., Wu, C. T., Larracuente, A. M. and Mellone, B. G. (2019). Islands of retroelements are major components of Drosophila centromeres. PLoS Biol 17(5): e3000241. PubMed ID: 31086362
Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in understanding of their functional organization and evolution. This study identified the sequence composition and organization of the centromeres of Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, this study demonstrates that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. A specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. This work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.
Chang, Y. H., Keegan, R. M., Prazak, L. and Dubnau, J. (2019). Cellular labeling of endogenous retrovirus replication (CLEVR) reveals de novo insertions of the gypsy retrotransposable element in cell culture and in both neurons and glial cells of aging fruit flies. PLoS Biol 17(5): e3000278. PubMed ID: 31095565
Evidence is rapidly mounting that transposable element (TE) expression and replication may impact biology more widely than previously thought. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by the use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, reporter constructs demonstrating engineered long interspersed nuclear element (LINE-1; L1) mobilization have been in use for quite some time, and strategies similar to L1 retrotransposition reporter assays have been developed to report replication of Ty1 elements in yeast and mouse intracisternal A particle (IAP) long terminal repeat (LTR) retrotransposons in cultivated cells. This study describes a novel approach termed cellular labeling of endogenous retrovirus replication (CLEVR), which reports replication of the gypsy element within specific cells in vivo in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly.The gypsy-CLEVR replication rate is increased when the short interfering RNA (siRNA) silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR retrotransposons, endogenous retroviruses (ERVs), and exogenous retroviruses.
Asaoka, M., Hanyu-Nakamura, K., Nakamura, A. and Kobayashi, S. (2019). Maternal Nanos inhibits Importin-alpha2/Pendulin-dependent nuclear import to prevent somatic gene expression in the Drosophila germline. PLoS Genet 15(5): e1008090. PubMed ID: 31091233
Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. This study shows that Nos represses somatic gene expression by inhibiting translation of maternal importin-alpha2 (impalpha2) mRNA. Mis-expression of Impalpha2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, it is proposed that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a 'double-lock' mechanism to inhibit somatic gene expression in germline progenitors.
Malik, S., Jang, W., Park, S. Y. Y., Kim, J. Y. Y., Kwon, K. S. and Kim, C. (2019). The target specificity of the RNA binding protein Pumilio is determined by distinct co-factors. Biosci Rep. PubMed ID: 31097674
Puf family proteins are translational regulators essential to a wide range of biological processes, including cell fate specification, stem cell self-renewal, and neural function. Yet, despite being associated with hundreds of RNAs, the underlying mechanisms of Puf target specification remain to be fully elucidated. In Drosophila, Pumilio - a sole Puf family protein - is known to collaborate with cofactors Nanos (Nos) and Brain Tumor (Brat); however, their roles in target specification are not clearly defined. This study identified Bag-of-marbles (Bam) as a new Pum cofactor in repression of Mothers against dpp (Mad) mRNAs, for which Nos is known to be dispensable. Notably, the data show that Nos (but not Bam) was required for Pum association with hunchback (hb) mRNAs, a well-known target of Pum and Nos. In contrast, Bam (but not Nos) is required for Pum association with Mad mRNAs. These findings show for the first time that Pum target specificity is determined not independently but in collaboration with cofactors.
Ruscica, V., Bawankar, P., Peter, D., Helms, S., Igreja, C. and Izaurralde, E. (2019). Direct role for the Drosophila GIGYF protein in 4EHP-mediated mRNA repression. Nucleic Acids Res. PubMed ID: 31114929
The eIF4E-homologous protein (4EHP) is a translational repressor that competes with eIF4E for binding to the 5'-cap structure of specific mRNAs, to which it is recruited by protein factors such as the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins (GIGYF). GIGYF proteins are not merely facilitating 4EHP recruitment to transcripts but are actually required for the repressor activity of the complex. This study investigated the role of GIGYF protein in post-transcriptional mRNA regulation. When in complex with 4EHP, Dm GIGYF not only elicits translational repression but also promotes target mRNA decay via the recruitment of additional effector proteins. Rhe RNA helicase Me31B/DDX6, the decapping activator HPat and the CCR4-NOT deadenylase complex as binding partners of GIGYF proteins. Recruitment of Me31B and HPat via discrete binding motifs conserved among metazoan GIGYF proteins is required for downregulation of mRNA expression by the 4EHP-GIGYF complex. These findings are consistent with a model in which GIGYF proteins additionally recruit decapping and deadenylation complexes to 4EHP-containing RNPs to induce translational repression and degradation of mRNA targets.

Monday, June 10th - Adult Physiology

Enriquez, T. and Colinet, H. (2019). Cold acclimation triggers major transcriptional changes in Drosophila suzukii. BMC Genomics 20(1): 413. PubMed ID: 31117947
Insects have the capacity to adjust their physiological mechanisms during their lifetime to promote cold tolerance and cope with sublethal thermal conditions, a phenomenon referred to as thermal acclimation. The spotted wing drosophila, Drosophila suzukii, is an invasive fruit pest that, like many other species, enhances its thermotolerance in response to thermal acclimation. This study promoted flies' cold tolerance by gradually increasing acclimation duration (i.e. pre-exposure from 2 h to 9 days at 10 ° C), and then compared transcriptomic responses of cold hardy versus cold susceptible phenotypes using RNA sequencing. Cold tolerance of D. suzukii increased with acclimation duration; the longer the acclimation, the higher the cold tolerance. Cold-tolerant flies that were acclimated for 9 days were selected for transcriptomic analyses. RNA sequencing revealed a total of 2908 differentially expressed genes: 1583 were up- and 1325 were downregulated in cold acclimated flies. Functional annotation revealed many enriched GO-terms among which ionic transport across membranes and signaling were highly represented in acclimated flies. Neuronal activity and carbohydrate metabolism were also enriched GO-terms in acclimated flies. Results also revealed many GO-terms related to oogenesis which were underrepresented in acclimated flies. It is concluded that involvement of a large cluster of genes related to ion transport in cold acclimated flies suggests adjustments in the capacity to maintain ion and water homeostasis. These processes are key mechanisms underlying cold tolerance in insects. Down regulation of genes related to oogenesis in cold acclimated females likely reflects that females were conditioned at 10 ° C, a temperature that prevents oogenesis.
Massey, J. H., Akiyama, N., Bien, T., Dreisewerd, K., Wittkopp, P. J., Yew, J. Y. and Takahashi, A. (2019). Pleiotropic effects of ebony and tan on pigmentation and cuticular hydrocarbon composition in Drosophila melanogaster. Front Physiol 10: 518. PubMed ID: 31118901
Pleiotropic genes are genes that affect more than one trait. For example, many genes required for pigmentation in the fruit fly Drosophila melanogaster also affect traits such as circadian rhythms, vision, and mating behavior. This paper presents evidence that two pigmentation genes, ebony and tan, which encode enzymes catalyzing reciprocal reactions in the melanin biosynthesis pathway, also affect cuticular hydrocarbon (CHC) composition in D. melanogaster females. More specifically, ebony loss-of-function mutants have a CHC profile that is biased toward long (>25C) chain CHCs, whereas tan loss-of-function mutants have a CHC profile that is biased toward short (<25C) chain CHCs. Moreover, pharmacological inhibition of dopamine synthesis, a key step in the melanin synthesis pathway, reversed the changes in CHC composition seen in ebony mutants, making the CHC profiles similar to those seen in tan mutants. These observations suggest that genetic variation affecting ebony and/or tan activity might cause correlated changes in pigmentation and CHC composition in natural populations. This possibility was tested using the Drosophila Genetic Reference Panel (DGRP) and CHC composition was found to covary with pigmentation as well as levels of ebony and tan expression in newly eclosed adults in a manner consistent with the ebony and tan mutant phenotypes. These data suggest that the pleiotropic effects of ebony and tan might contribute to covariation of pigmentation and CHC profiles in Drosophila.
Gleason, J. M., Roy, P. R., Everman, E. R., Gleason, T. C. and Morgan, T. J. (2019). Phenology of Drosophila species across a temperate growing season and implications for behavior. PLoS One 14(5): e0216601. PubMed ID: 31095588
Drosophila community composition is complex in temperate regions with different abundance of flies and species across the growing season. Monitoring Drosophila populations provides insights into the phenology of both native and invasive species. Over a single growing season, Drosophila were collected at regular intervals, and the number of individuals was determined of the nine species that were found in Kansas, USA. Species varied in their presence and abundance through the growing season with peak diversity occurring after the highest seasonal temperatures. Models were developed for the abundance of the most common species, Drosophila melanogaster, D. simulans, D. algonquin, and the recent invasive species, D. suzukii. These models revealed that temperature played the largest role in abundance of each species across the season. For the two most commonly studied species, D. melanogaster and D. simulans, the best models indicate shifted thermal optima compared to laboratory studies, implying that fluctuating temperature may play a greater role in the physiology and ecology of these insects than indicated by laboratory studies, and should be considered in global climate change studies.
Smith, G. A., Lin, T. H., Sheehan, A. E., Van der Goes van Naters, W., Neukomm, L. J., Graves, H. K., Bis-Brewer, D. M., Zuchner, S. and Freeman, M. R. (2019). Glutathione S-transferase regulates mitochondrial populations in axons through increased glutathione oxidation. Neuron. PubMed ID: 31101394
Mitochondria are essential in long axons to provide metabolic support and sustain neuron integrity. A healthy mitochondrial pool is maintained by biogenesis, transport, mitophagy, fission, and fusion, but how these events are regulated in axons is not well defined. This study shows that the Drosophila glutathione S-transferase (GST) Gfzf prevents mitochondrial hyperfusion in axons. Gfzf loss altered redox balance between glutathione (GSH) and oxidized glutathione (GSSG) and initiated mitochondrial fusion through the coordinated action of Mfn and Opa1. Gfzf functioned epistatically with the thioredoxin peroxidase Jafrac1 and the thioredoxin reductase 1 TrxR-1 to regulate mitochondrial dynamics. Altering GSH:GSSG ratios in mouse primary neurons in vitro also induced hyperfusion. Mitochondrial changes caused deficits in trafficking, the metabolome, and neuronal physiology. Changes in GSH and oxidative state are associated with neurodegenerative diseases like Alzheimer's. This demonstration that GSTs are key in vivo regulators of axonal mitochondrial length and number provides a potential mechanistic link.
Beyenbach, K. W. (2019). Voltages and resistances of the anterior Malpighian tubule of Drosophila melanogaster. J Exp Biol 222(Pt 10). PubMed ID: 31043456
The small size of Malpighian tubules in the fruit fly Drosophila melanogaster has discouraged measurements of the transepithelial electrical resistance. The present study introduces two methods for measuring the transepithelial resistance in isolated D . melanogaster Malpighian tubules using conventional microelectrodes and PClamp hardware and software. The first method uses three microelectrodes to measure the specific transepithelial resistance normalized to tubule length or luminal surface area for comparison with resistances of other epithelia. The second method uses only two microelectrodes to measure the relative resistance for comparing before and after effects in a single Malpighian tubule. Knowledge of the specific transepithelial resistance allows the first electrical model of electrolyte secretion by the main segment of the anterior Malpighian tubule of D . melanogaster. The electrical model is remarkably similar to that of the distal Malpighian tubule of Aedes aegypti when tubules of Drosophila and Aedes are studied in vitro under the same experimental conditions. Thus, despite 189 millions of years of evolution separating these two genera, the electrophysiological properties of their Malpighian tubules remains remarkably conserved.
Feingold, D., Knogler, L., Starc, T., Drapeau, P., O'Donnell, M. J., Nilson, L. A. and Dent, J. A. (2019). secCl is a cys-loop ion channel necessary for the chloride conductance that mediates hormone-induced fluid secretion in Drosophila. Sci Rep 9(1): 7464. PubMed ID: 31097722
Organisms use circulating diuretic hormones to control water balance (osmolarity), thereby avoiding dehydration and managing excretion of waste products. The hormones act through G-protein-coupled receptors to activate second messenger systems that in turn control the permeability of secretory epithelia to ions like chloride. In insects, the chloride channel mediating the effects of diuretic hormones was unknown. Surprisingly, this study found a pentameric, cys-loop chloride channel, secCl (CG7589), a type of channel normally associated with neurotransmission, mediating hormone-induced transepithelial chloride conductance. This discovery is important because: 1) it describes an unexpected role for pentameric receptors in the membrane permeability of secretory epithelial cells, and 2) it suggests that neurotransmitter-gated ion channels may have evolved from channels involved in secretion.

Friday, June 7th - Adult Development

Simoes da Silva, C. J., Sospedra, I., Aparicio, R. and Busturia, A. (2019). The microRNA-306/abrupt regulatory axis controls wing and haltere growth in Drosophila. Mech Dev: 103555. PubMed ID: 31112748
Growth control relies on extrinsic and intrinsic mechanisms that regulate and coordinate the size and pattern of organisms. This control is crucial for a homeostatic development and healthy physiology. The gene networks acting in this process are large and complex: factors involved in growth control are also important in diverse biological processes and these networks include multiple regulators that interact and respond to intra- and extra-cellular inputs that may ultimately converge in the control of the cell cycle. This work reports a study of the function of the Drosophila abrupt gene, coding for a BTB-ZF protein and previously reported to be required for wing vein pattern, in the control of haltere and wing growth. Inactivation of abrupt reduces the size of the wing and haltere. The microRNA miR-306 controls abrupt expression and that miR-306 and abrupt genetically interact to control wing size. Moreover, the reduced appendage size due to abrupt inactivation is rescued by overexpression of Cyclin-E and by inactivation of dacapo. These findings define a miR-306-abrupt regulatory axis that controls wing and haltere size, whereby miR-306 maintains appropriate levels of abrupt expression which, in turn, regulates the cell cycle. Thus, these results uncover a novel function of abrupt in the regulation of the size of Drosophila appendages during development and contribute to the understanding of the coordination between growth and pattern as well as to the understanding of abrupt oncogenic function in flies.
Zhang, M., Ji, Y., Zhang, X., Ma, P., Wang, Y., Moussian, B. and Zhang, J. (2019). The putative chitin deacetylases serpentine and vermiform have non-redundant functions during Drosophila wing development. Insect Biochem Mol Biol. PubMed ID: 31108167
The chitin modifying deacetylases (CDA) CDA1 and CDA2 have been reported to play partially redundant roles during insect cuticle formation and molting and tracheal morphogenesis in various insect species. In order to distinguish possible functional differences between these two enzymes, their function was analyzed during wing development in the fruit fly Drosophila melanogaster. In tissue-specific RNA interference experiments, this study demonstrate that DmCDA1 (Serpentine, Serp) and DmCDA2 (Vermiform, Verm) have distinct functions during Drosophila adult wing cuticle differentiation. Chitosan staining revealed that Serp is the major enzyme responsible for chitin deacetylation during wing cuticle formation, while Verm does not seem to be needed for this process. Indeed, it is questionable whether Verm is a chitin deacetylase at all. Atomic force microscopy suggested that Serp and Verm have distinct roles in establishing the shape of nanoscale bumps at the wing surface. Moreover, the data indicate that Verm but not Serp is required for the laminar arrangement of chitin. Both enzymes participate in the establishment of the cuticular inward barrier against penetration of xenobiotics. Taken together, correct differentiation of the wing cuticle involves both Serp and Verm in parallel in largely non-overlapping functions.
Xu, J., Yu, Y., Chen, K. and Huang, Y. (2019). Intersex regulates female external genital and imaginal disc development in the silkworm. Insect Biochem Mol Biol 108: 1-8. PubMed ID: 30831220
As a component of the mediator complex, the intersex (ix) gene product is involved in the sex determination pathway of the Drosophila melanogaster. IX functions together with the female-specific product of doublesex (dsx) at the bottom of the hierarchy to implement female sexual differentiation. This study analyzed the functions of the ix gene in the model lepidopteran insect Bombyx mori. Bmix was found to be expressed in many tissues and is highly expressed in early pupal stages. The transgene-based CRISPR/Cas9 system was used to generate mutants of the Bmix gene. The Bmix female mutants were sterile and had irregular external genitalia, whereas in the mutant males external genitalia were normal. Mutants of both sexes had normal gonad development and normal splicing of the Bmdsx pre-mRNA, suggesting that Bmix functions independently of Bmdsx. Interestingly, both male and female mutants had defective development of the imaginal disc including wing, antenna, and leg. RNA-seq and gene expression analyses indicated that genes involved in WNT, Hippo, and Hedgehog signaling pathways and wing development genes Bmawd and Bmfng were up-regulated or down-regulated in the Bmix mutants compared with wild-type animals. These data provide insights into the multiple functions of Bmix in female external genital and imaginal disc development in the silkworm.
Yamada, T., Habara, O., Yoshii, Y., Matsushita, R., Kubo, H., Nojima, Y. and Nishimura, T. (2019). The role of glycogen in development and adult fitness in Drosophila. Development 146(8). PubMed ID: 30918052
The polysaccharide glycogen is an evolutionarily conserved storage form of glucose. However, the physiological significance of glycogen metabolism on homeostatic control throughout the animal life cycle remains incomplete. This study describes Drosophila mutants that have defective glycogen metabolism. Null mutants of glycogen synthase (GlyS) and glycogen phosphorylase (GlyP) displayed growth defects and larval lethality, indicating that glycogen plays a crucial role in larval development. Unexpectedly, however, a certain population of larvae developed into adults with normal morphology. Semi-lethality in glycogen mutants during the larval period can be attributed to the presence of circulating sugar trehalose. Homozygous glycogen mutants produced offspring, indicating that glycogen stored in oocytes is dispensable for embryogenesis. GlyS and GlyP mutants showed distinct metabolic defects in the levels of circulating sugars and triglycerides in a life stage-specific manner. In adults, glycogen as an energy reserve is not crucial for physical fitness and lifespan under nourished conditions, but glycogen becomes important under energy stress conditions. This study provides a fundamental understanding of the stage-specific requirements for glycogen metabolism in the fruit fly.
He, S., Zhang, G., Wang, J., Gao, Y., Sun, R., Cao, Z., Chen, Z., Zheng, X., Yuan, J., Luo, Y., Wang, X., Zhang, W., Zhang, P., Zhao, Y., He, C., Tao, Y., Sun, Q. and Chen, D. (2019). 6mA-DNA-binding factor Jumu controls maternal-to-zygotic transition upstream of Zelda. Nat Commun 10(1): 2219. PubMed ID: 31101825
A long-standing question in the field of embryogenesis is how the zygotic genome is precisely activated by maternal factors, allowing normal early embryonic development. N6-methyladenine (6mA) DNA modification has been shown to be highly dynamic in early Drosophila embryos and forms an epigenetic mark. However, little is known about how 6mA-formed epigenetic information is decoded. This study reports that the Fox-family protein Jumu binds 6mA-marked DNA and acts as a maternal factor to regulate the maternal-to-zygotic transition. zelda encoding the pioneer factor Zelda was found to be marked by 6mA. Genetic assays suggest that Jumu controls the proper zygotic genome activation (ZGA) in early embryos, at least in part, by regulating zelda expression. Thus, these findings not only support that the 6mA-formed epigenetic marks can be read by specific transcription factors, but also uncover a mechanism by which the Jumu regulates ZGA partially through Zelda in early embryos.
Windner, S. E., Manhart, A., Brown, A., Mogilner, A. and Baylies, M. K. (2019). Nuclear scaling is coordinated among individual nuclei in multinucleated muscle fibers. Dev Cell 49(1): 48-62.e43. PubMed ID: 30905770
Optimal cell performance depends on cell size and the appropriate relative size, i.e., scaling, of the nucleus. How nuclear scaling is regulated and contributes to cell function is poorly understood, especially in skeletal muscle fibers, which are among the largest cells, containing hundreds of nuclei. Here, this study presents a Drosophila in vivo system to analyze nuclear scaling in whole multinucleated muscle fibers, genetically manipulate individual components, and assess muscle function. Despite precise global coordination, it was found that individual nuclei within a myofiber establish different local scaling relationships by adjusting their size and synthetic activity in correlation with positional or spatial cues. While myonuclei exhibit compensatory potential, even minor changes in global nuclear size scaling correlate with reduced muscle function. This study provides the first comprehensive approach to unraveling the intrinsic regulation of size in multinucleated muscle fibers. These insights to muscle cell biology will accelerate the development of interventions for muscle diseases.

Thursday, June 6th - Chromatin

Piwko, P., Vitsaki, I., Livadaras, I. and Delidakis, C. (2019). The role of insulators in transgene transvection in Drosophila. Genetics. PubMed ID: 30948430
Transvection is the phenomenon where a transcriptional enhancer activates a promoter located on the homologous chromosome. It has been amply documented in Drosophila where homologues are closely paired in most, if not all, somatic nuclei, but it has been known to rarely occur in mammals as well. This study has taken advantage of site-directed transgenesis to insert reporter constructs into the same genetic locus in Drosophila and have evaluated their ability to engage in transvection by testing many heterozygous combinations. Transvection was shown to require the presence of an insulator element on both homologues. Homotypic trans-interactions between four different insulators can support transvection: the gypsy insulator (GI), Wari, Fab-8 and 1A2; GI and Fab-8 are more effective than Wari or 1A2. In the presence of insulators, transvection displays the characteristics that have been previously described: it requires homologue pairing, but can happen at any of several loci in the genome; a solitary enhancer confronted with an enhancerless reporter is sufficient to drive transcription; it is weaker than the action of the same enhancer-promoter pair in cis and it is further suppressed by cis-promoter competition. Though necessary, the presence of homotypic insulators is not sufficient for transvection; their position, number and orientation matters. A single GI adjacent to both enhancer and promoter is the optimal configuration. The identity of enhancers and promoters in the vicinity of a trans-interacting insulator pair is also important, indicative of complex insulator-enhancer-promoter interactions.
Tsurumi, A., Xue, S., Zhang, L., Li, J. and Li, W. X. (2019). Genome-wide Kdm4 histone demethylase transcriptional regulation in Drosophila. Mol Genet Genomics. PubMed ID: 31020413
The histone lysine demethylase 4 (Kdm4/Jmjd2/Jhdm3) family is highly conserved across species and reverses di- and tri-methylation of histone H3 lysine 9 (H3K9) and lysine 36 (H3K36) at the N-terminal tail of the core histone H3 in various metazoan species including Drosophila, C.elegans, zebrafish, mice and humans. Previous studies have shown that the Kdm4 family plays a wide variety of important biological roles in different species, including development, oncogenesis and longevity by regulating transcription, DNA damage response and apoptosis. Only two functional Kdm4 family members have been identified in Drosophila, compared to five in mammals, thus providing a simple model system. Drosophila Kdm4 loss-of-function mutants do not survive past the early 2nd instar larvae stage and display a molting defect phenotype associated with deregulated ecdysone hormone receptor signaling. To further characterize and identify additional targets of Kdm4, a genome-wide approach was employed to investigate transcriptome alterations in Kdm4 mutants versus wild-type during early development. Evidence was found of increased deregulated transcripts, presumably associated with a progressive accumulation of H3K9 and H3K36 methylation through development. Gene ontology analyses found significant enrichment of terms related to the ecdysteroid hormone signaling pathway important in development, as expected, and additionally previously unidentified potential targets that warrant further investigation. Since Kdm4 is highly conserved across species, these results may be applicable more widely to other organisms and the genome-wide dataset may serve as a useful resource for further studies.
Yang, L., Ma, Z., Wang, H., Niu, K., Cao, Y., Sun, L., Geng, Y., Yang, B., Gao, F., Chen, Z., Wu, Z., Li, Q., Shen, Y., Zhang, X., Jiang, H., Chen, Y., Liu, R., Liu, N. and Zhang, Y. (2019). Ubiquitylome study identifies increased histone 2A ubiquitylation as an evolutionarily conserved aging biomarker. Nat Commun 10(1): 2191. PubMed ID: 31113955
he long-lived proteome constitutes a pool of exceptionally stable proteins with limited turnover. Previous studies on ubiquitin-mediated protein degradation primarily focused on relatively short-lived proteins; how ubiquitylation modifies the long-lived proteome and its regulatory effect on adult lifespan is unclear. This study profiles the age-dependent dynamics of long-lived proteomes in Drosophila by mass spectrometry using stable isotope switching coupled with antibody-enriched ubiquitylome analysis. The data describe landscapes of long-lived proteins in somatic and reproductive tissues of Drosophila during adult lifespan, and reveal a preferential ubiquitylation of older long-lived proteins. An age-modulated increase of ubiquitylation was found on long-lived histone 2A protein in Drosophila, which is evolutionarily conserved in mouse, monkey, and human. A reduction of ubiquitylated histone 2A in mutant flies is associated with longevity and healthy lifespan. Together, these data reveal an evolutionarily conserved biomarker of aging that links epigenetic modulation of the long-lived histone protein to lifespan.
Ji, J., Tang, X., Hu, W., Maggert, K. A. and Rong, Y. S. (2019). The processivity factor Pol32 mediates nuclear localization of DNA polymerase delta and prevents chromosomal fragile site formation in Drosophila development. PLoS Genet 15(5): e1008169. PubMed ID: 31100062
The Pol32 protein is one of the universal subunits of DNA polymerase delta (Pol delta), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. This study characterized Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol delta localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol delta would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32's role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. This study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism.
Akmammedov, A., Geigges, M. and Paro, R. (2019). Bivalency in Drosophila embryos is associated with strong inducibility of Polycomb target genes. Fly (Austin). PubMed ID: 31094269
Polycomb group (PcG) and Trithorax group (TrxG) proteins orchestrate development of a multicellular organism by faithfully maintaining cell fate decisions made early in embryogenesis. An important chromatin mark connected to PcG/TrxG regulation are bivalent domains, the simultaneous presence of H3K27me3 and H3K4me3 on a given locus, originally identified in mammalian embryonic stem cells but considered to be absent in invertebrates. This study provides evidence for existence of bivalency in fly embryos. Using a recently described PcG reporter fly line, a strong reporter inducibility was observed in embryo and its sharp decrease in larval and adult stages. Analysis of the chromatin landscape of the reporter revealed a strong signal for the repressive PcG mark, H3K27me3, in all three developmental stages and, surprisingly, a strong signal for a transcriptionally activating H3K4me3 mark in embryo. Using re-ChIP experiments, bivalent domains were also uncovered at endogenous PcG targets like the Hox genes.
Levendosky, R. F. and Bowman, G. D. (2019). Asymmetry between the two acidic patches dictates the direction of nucleosome sliding by the ISWI chromatin remodeler. Elife 8. PubMed ID: 31094676
The acidic patch is a functionally important epitope on each face of the nucleosome that affects chromatin remodeling. Although related by 2-fold symmetry of the nucleosome, each acidic patch is uniquely positioned relative to a bound remodeler. An open question is whether remodelers are distinctly responsive to each acidic patch. Previously a method was reporthed for homogeneously producing asymmetric nucleosomes with distinct H2A/H2B dimers. This methodology was used to show that the Chd1 remodeler from Saccharomyces cerevisiae and ISWI remodelers from human and Drosophila have distinct spatial requirements for the acidic patch. Unlike Chd1, which is equally affected by entry- and exit-side mutations, ISWI remodelers strongly depend on the entry-side acidic patch. Remarkably, asymmetry in the two acidic patches stimulates ISWI to slide mononucleosomes off DNA ends, overriding the remodeler's preference to shift the histone core toward longer flanking DNA.

Wednesday, June 5th - Signal Transduction

Dlugos, C. P., et al. (2019). Nephrin signaling results in integrin beta1 activation. J Am Soc Nephrol. PubMed ID: 31097607
Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, it was hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix. To investigate Nephrin's role in transmitting signals to the Integrin receptor complex, genetic studies were conducted in Drosophila nephrocytes, and findings from Drosophila were validated in a cultured human podocyte model. Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified c3g as necessary for nephrocyte function. In vivo, nephrocyte-specific gene silencing of sns or c3g compromised nephrocyte filtration and caused nephrocyte diaphragm defects. Nephrocytes with impaired Sns or C3G expression displayed an altered localization of Integrin and the Integrin-associated protein Talin. Furthermore, gene silencing of c3g partly rescued nephrocyte diaphragm defects of an sns overexpression phenotype, pointing to genetic interaction of sns and c3g in nephrocytes. Activated Nephrin recruited phosphorylated C3G and resulted in activation of Integrin beta1 in cultured podocytes. These findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin beta1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix.
Blount, J. R., Meyer, D. N., Akemann, C., Johnson, S. L., Gurdziel, K., Baker, T. R. and Todi, S. V. (2019). Unanchored ubiquitin chains do not lead to marked alterations in gene expression in Drosophila melanogaster. Biol Open. PubMed ID: 31097444
The small protein modifier, ubiquitin regulates various aspects of cellular biology through its chemical conjugation onto proteins. Ubiquitination of proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Ubiquitin chains can be attached onto other proteins or can exist as unanchored species - i.e. free from another protein. Unanchored ubiquitin chains are thought to be deleterious to the cell and rapidly disassembled into mono-ubiquitin. A recent study examined the toxicity and utilization of unanchored poly-ubiquitin in Drosophila melanogaster. Free poly-ubiquitin species were found to be largely innocuous to flies, and free poly-ubiquitin can be controlled by being degraded by the proteasome or by being conjugated onto another protein as a single unit. To explore whether an organismal defense is mounted against unanchored chains, RNA-Seq analyses was conducted to examine the transcriptomic impact of free poly-ubiquitin in the fly. Approximately 90 transcripts were found whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was essentially devoid of ubiquitin-, proteasome- or autophagy-related components. The seeming absence of a large and multipronged response to unanchored poly-ubiquitin supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell.
Kramer, R., Rode, S. and Rumpf, S. (2019). Rab11 is required for neurite pruning and developmental membrane protein degradation in Drosophila sensory neurons. Dev Biol 451(1): 68-78. PubMed ID: 30871987
Neurons, with their distinct neurites, require elaborate membrane trafficking pathways and regulation to uphold neurite identity and to be able to respond to neuronal or developmental stimuli. In a survey of trafficking regulators required for developmental dendrite pruning in Drosophila sensory neurons, the small GTPase Rab11 was identified as a regulator of recycling endosomes. Dendrite pruning requires the developmentally regulated degradation of the cell adhesion molecule Neuroglian, and loss of Rab11 causes defects in the developmental degradation of Neuroglian and another target, the ion channel Ppk26. Rab11 often links vesicles to molecular motors, and this study finds that loss of the microtubule motor dynein also leads to defective Neuroglian and Ppk26 degradation. Loss of Rab11 also leads to defects in larval dendrite elaboration, and Neuroglian and Ppk26 localization is already altered at this stage. These data highlight the importance of membrane protein recycling during development.
Camilleri-Robles, C., Serras, F. and Corominas, M. (2019). Role of D-GADD45 in JNK-dependent apoptosis and regeneration in Drosophila. Genes (Basel) 10(5). PubMed ID: 31109086
The GADD45 proteins are induced in response to stress and have been implicated in the regulation of several cellular functions, including DNA repair, cell cycle control, senescence, and apoptosis. This study investigated the role of D-GADD45 during Drosophila development and regeneration of the wing imaginal discs. Higher expression of D-GADD45 was found to result in JNK-dependent apoptosis, while its temporary expression does not have harmful effects. Moreover, D-GADD45 is required for proper regeneration of wing imaginal discs. These findings demonstrate that a tight regulation of D-GADD45 levels is required for its correct function both, in development and during the stress response after cell death.
Kim, L. H., Hong, S. T. and Choi, K. W. (2019). Protein phosphatase 2A interacts with Verthandi/Rad21 to regulate mitosis and organ development in Drosophila. Sci Rep 9(1): 7624. PubMed ID: 31110215
Rad21/Scc1 is a subunit of the cohesin complex implicated in gene regulation as well as sister chromatid cohesion. The level of Rad21/Scc1 must be controlled for proper mitosis and gene expression during development. This study has identified the PP2A catalytic subunit encoded by microtubule star (mts) as a regulator of Drosophila Rad21/Verthandi (Vtd). Mutations in mts and vtd cause synergistic mitotic defects, including abnormal spindles and loss of nuclei during nuclear division in early embryo. Depletion of Mts and Vtd in developing wing synergistically reduces the Cut protein level, causing severe defects in wing growth. Mts and PP2A subunit Twins (Tws) interact with Vtd protein. Loss of Mts or Tws reduces Vtd protein level. Reduced proteasome function suppresses mitotic defects caused by mutations in mts and vtd. Taken together, this work provides evidence that PP2A is required for mitosis and wing growth by regulating the Vtd level through the proteasomal pathway.
Moyer, T. C. and Holland, A. J. (2019). PLK4 promotes centriole duplication by phosphorylating STIL to link the procentriole cartwheel to the microtubule wall. Elife 8. PubMed ID: 31115335
Centrioles play critical roles in organizing the assembly of the mitotic spindle and templating the formation of primary cilia. Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 (PLK4). Although significant progress has been made in understanding centriole composition, there is only limited knowledge of how PLK4 activity controls specific steps in centriole formation. This study shows that PLK4 phosphorylates its centriole substrate STIL on a conserved site, S428, to promote STIL binding to CPAP. This phospho-dependent binding interaction is conserved in Drosophila and facilitates the stable incorporation of both STIL and CPAP into the centriole. It is proposed that procentriole assembly requires PLK4 to phosphorylate STIL in two different regions: phosphorylation of residues in the STAN motif allow STIL to bind SAS6 and initiate cartwheel assembly, while phosphorylation of S428 promotes the binding of STIL to CPAP, linking the cartwheel to microtubules of the centriole wall.

Tuesday, June 4th - Disease Models

Wu, W., Han, Y., Fan, X., Li, Q. and Sun, L. (2019). Protective mechanism of Wnt4 gene on Parkinson's disease (PD) transgenic Drosophila. Int J Neurosci 129(7): 703-714. PubMed ID: 30526176
The main pathological change of Parkinson's disease (PD) is progressive degeneration and necrosis of dopaminergic neurons in the midbrain, forming a Lewy body in many of the remaining neurons. Studies have found that in transgenic Drosophila, mutations in the PTEN-inducible kinase 1 (PINK1) gene may cause indirect flight muscle defects in Drosophila, and mitochondrial structural dysfunction as well. In this study, Wnt4 gene overexpression and knockdown were performed in PINK1 mutant PD transgenic Drosophila, and the protective effect of Wnt4 gene on PD transgenic Drosophila and its possible mechanism were explored. The Wnt4 gene was screened in the previous experiment; And by using the PD transgenic Drosophila model of the MHC-Gal4/UAS system, the PINK1 gene could be specifically activated in the Drosophila muscle tissue. In PINK1 mutation transgenic fruit flies, the Wnt4 gene to study its implication on PD transgenic fruit flies' wing normality and flight ability. Overexpression of Wnt4 gene significantly reduced abnormality rate of PD transgenic Drosophila and improved its flight ability, and then, increased ATP concentration, enhanced mitochondrial membrane potential and normalized mitochondrial morphology were found. All of these findings suggested Wnt4 gene may have a protective effect on PD transgenic fruit flies. Furthermore, in Wnt4 gene overexpression PD transgenic Drosophila, down-regulation autophagy and apoptosis-related proteins Ref(2)P, Pro-Caspase3, and up-regulation of Beclin1, Atg8a, Bcl2 protein were confirmed by Western Blotting. The results imply that the restoring of mitochondrial function though Wnt4 gene overexpression in the PINK1 mutant transgenic Drosophila may be related to autophagy and/or apoptosis.
Bialistoky, T., Manry, D., Smith, P., Ng, C., Kim, Y., Zamir, S., Moyal, V., Kalifa, R., Schedl, P., Gerlitz, O. and Deshpande, G. (2019). Functional analysis of Niemann-Pick disease type C family protein, NPC1a, in Drosophila melanogaster. Development 146(10). PubMed ID: 31092503
During embryonic gonad coalescence, primordial germ cells (PGCs) follow a carefully choreographed migratory route circumscribed by guidance signals towards somatic gonadal precursor cells (SGPs). In Drosophila melanogaster, SGP-derived Hedgehog (Hh), which serves as a guidance cue for the PGCs, is potentiated by mesodermally restricted HMGCoA-reductase (Hmgcr) and the ABC transporter Multi-drug-resistant-49 (Mdr49). Given the importance of cholesterol modification in the processing and long-distance transmission of the Hh ligand, this study analyzed the involvement of the Niemann-Pick disease type C-1a (NPC1a) protein, a cholesterol transporter, in germ cell migration and Hedgehog signaling. Mesoderm-specific inactivation of Npc1a results in germ cell migration defects. Similar to Mdr49, PGC migration defects in the Npc1a embryos are ameliorated by a cholesterol-rich diet. Consistently, reduction in Npc1a weakens the ability of ectopic HMG Coenzyme A reductase (Hmgcr) to induce germ cell migration defects. Moreover, compromising Npc1a levels influences Hh signaling adversely during wing development, a process that relies upon long-range Hh signaling. Last, doubly heterozygous embryos (Mdr49/Npc1a) display enhanced germ cell migration defects when compared with single mutants (Npc1a/+ or Mdr49/+), supporting cooperative interaction between the two.
Mondin, V. E., Ben El Kadhi, K., Cauvin, C., Jackson-Crawford, A., Belanger, E., Decelle, B., Salomon, R., Lowe, M., Echard, A. and Carreno, S. (2019). PTEN reduces endosomal PtdIns(4,5)P2 in a phosphatase-independent manner via a PLC pathway. J Cell Biol. PubMed ID: 31118240
The tumor suppressor PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2. This paper describes an unexpected discovery that in Drosophila melanogaster PTEN reduces PtdIns(4,5)P2 levels on endosomes, independently of its phosphatase activity. This new PTEN function requires the enzymatic action of dPLCXD, an atypical phospholipase C. Importantly, this novel PTEN/dPLCXD pathway can compensate for depletion of dOCRL, a PtdIns(4,5)P2 phosphatase. Mutation of OCRL1, the human orthologue of dOCRL, causes oculocerebrorenal Lowe syndrome, a rare multisystemic genetic disease. Both OCRL1 and dOCRL loss have been shown to promote accumulation of PtdIns(4,5)P2 on endosomes and cytokinesis defects. This study shows that PTEN or dPLCXD overexpression prevents these defects. In addition, chemical activation of this pathway was found to restore normal cytokinesis in human Lowe syndrome cells and rescues OCRL phenotypes in a zebrafish Lowe syndrome model. These findings identify a novel PTEN/dPLCXD pathway that controls PtdIns(4,5)P2 levels on endosomes. They also point to a potential new strategy for the treatment of Lowe syndrome.
Ly, S. and Naidoo, N. (2019). Loss of DmGluRA exacerbates age-related sleep disruption and reduces lifespan. Neurobiol Aging 80: 83-90. PubMed ID: 31103635
Declines in sleep amount and quality-characterized by excessive daytime sleepiness and an inability to sleep at night-are common features of aging. Sleep dysfunction is also associated with age-related ailments and diseases, suggesting that sleep is functionally relevant to the aging process. Metabotropic glutamate receptors (mGluRs)-which are critical regulators of neurotransmission and synaptic plasticity-have been implicated in both age-related disease and sleep regulation. This study examined sleep and aging effect of complete genetic loss of mGluR signaling in Drosophila melanogaster. Genetic knockdown of the sole Drosophila mGluR-known as DmGluRA-reduced daytime wakefulness and nighttime sleep, recapitulating age-related sleep changes that occur across species. Furthermore, loss of DmGluRA significantly reduced lifespan and exacerbated age-related sleep loss in older flies. Thus, this study has identified DmGluRA as a novel regulator of sleep whose loss results in an age-relevant sleep phenotype that is associated with shortened lifespan. This is the first evidence that mGluR signaling regulates sleep/wake in a manner that is relevant to the aging process.
Xu, F., Kula-Eversole, E., Iwanaszko, M., Hutchison, A. L., Dinner, A. and Allada, R. (2019). Circadian clocks function in concert with heat shock organizing protein to modulate mutant Huntingtin aggregation and toxicity. Cell Rep 27(1): 59-70. PubMed ID: 30943415
Neurodegenerative diseases commonly involve the disruption of circadian rhythms. Studies indicate that mutant Huntingtin (mHtt), the cause of Huntington's disease (HD), disrupts circadian rhythms often before motor symptoms are evident. Yet little is known about the molecular mechanisms by which mHtt impairs circadian rhythmicity and whether circadian clocks can modulate HD pathogenesis. To address this question, a Drosophila HD model was used. Both environmental and genetic perturbations of the circadian clock were found to alter mHtt-mediated neurodegeneration. To identify potential genetic pathways that mediate these effects, a behavioral platform was applied to screen for clock-regulated HD suppressors, identifying a role for Heat Shock Protein 70/90 Organizing Protein (Hop). Hop knockdown paradoxically reduces mHtt aggregation and toxicity. These studies demonstrate a role for the circadian clock in a neurodegenerative disease model and reveal a clock-regulated molecular and cellular pathway that links clock function to neurodegenerative disease.
Westfall, S., Lomis, N. and Prakash, S. (2019). A novel synbiotic delays Alzheimer's disease onset via combinatorial gut-brain-axis signaling in Drosophila melanogaster. PLoS One 14(4): e0214985. PubMed ID: 31009489
The gut-brain-axis (GBA) describing the bidirectional communication between the gut microbiota and brain was recently implicated in Alzheimer's disease (AD). The current study describes a novel synbiotic containing three metabolically active probiotics and a novel polyphenol-rich prebiotic which has beneficial impacts on the onset and progression of AD. In a transgenic humanized Drosophila melanogaster model of AD, the synbiotic increased survivability and motility and rescued amyloid beta deposition and acetylcholinesterase activity. Such drastic effects were due to the synbiotic's combinatorial action on GBA signaling pathways including metabolic stability, immune signaling, oxidative and mitochondrial stress possibly through pathways implicating PPARgamma. Overall, this study shows that the therapeutic potential of GBA signaling is best harnessed in a synbiotic that simultaneously targets multiple risk factors of AD.

Monday, June 3rd - Adult Neural Development and Function

He, Z., Luo, Y., Shang, X., Sun, J. S. and Carlson, J. R. (2019). Chemosensory sensilla of the Drosophila wing express a candidate ionotropic pheromone receptor. PLoS Biol 17(5): e2006619. PubMed ID: 31112532
The Drosophila wing has been proposed to be a taste organ. A differential RNA-seq analysis was carried out of a row of sensilla on the anterior wing margin; expression was found of many genes associated with pheromone and chemical perception. To ask whether these sensilla might receive pheromonal input, a dye-transfer paradigm was carried out; large, hydrophobic molecules comparable to pheromones were found to be transferred from one fly to the wing margin of another. One gene, Ionotropic receptor (IR)52a, is coexpressed in neurons of these sensilla with fruitless, a marker of sexual circuitry; IR52a is also expressed in legs. Mutation of IR52a and optogenetic silencing of IR52a+ neurons decrease levels of male sexual behavior. Optogenetic activation of IR52a+ neurons induces males to show courtship toward other males and, remarkably, toward females of another species. Surprisingly, IR52a is also required in females for normal sexual behavior. Optogenetic activation of IR52a+ neurons in mated females induces copulation, which normally occurs at very low levels. IR52a acts in both males and females, and can promote male-male as well as male-female interactions. Moreover, IR52a+ neurons can override the circuitry that normally suppresses sexual behavior toward unproductive targets. Circuit mapping and Ca2+ imaging using the trans-Tango system reveals second-order projections of IR52a+ neurons in the subesophageal zone (SEZ), some of which are sexually dimorphic. Optogenetic activation of IR52a+ neurons in the wing activates second-order projections in the SEZ. Taken together, this study provides a molecular description of the chemosensory sensilla of a greatly understudied taste organ and defines a gene that regulates the sexual circuitry of the fly.
Hou, K., Jiang, H., Karim, M. R., Zhong, C., Xu, Z., Liu, L., Guan, M., Shao, J. and Huang, X. (2019). A critical E-box in Barhl1 3' enhancer is essential for auditory hair cell differentiation. Cells 8(5). PubMed ID: 31096644
Barhl1, a mouse homologous gene of Drosophila BarH class homeobox genes, is highly expressed within the inner ear and crucial for the long-term maintenance of auditory hair cells that mediate hearing and balance, yet little is known about the molecular events underlying Barhl1 regulation and function in hair cells. Through data mining and in vitro report assay, this study firstly found that Barhl1 is a direct target gene of Atoh1 and one E-box (E3) in the Barhl1 3' enhancer that is crucial for Atoh1-mediated Barhl1 activation. Then a mouse embryonic stem cell (mESC) line was generated carrying disruptions on this E3 site E-box (CAGCTG) using CRISPR/Cas9 technology and this E3 mutated mESC line was further subjected to an efficient stepwise hair cell differentiation strategy in vitro. Disruptions on this E3 site caused dramatic loss of Barhl1 expression and significantly reduced the number of induced hair cell-like cells, while no changes were identified in differentiation toward early primitive ectoderm-like cells and otic progenitors. Finally, through RNA-seq profiling and gene ontology (GO) enrichment analysis, this E3 box was found to be indispensable for Barhl1 expression to maintain hair cell development and normal functions. The transcriptional profiles were compared of induced cells from CDS mutated and E3 mutated mESCs, respectively, and very consistent results were obtained except the Barhl1 transcript itself. These observations indicated that Atoh1-mediated Barhl1 expression could have important roles during auditory hair cell development. In brief, these findings delineate the detail molecular mechanism of Barhl1 expression regulation in auditory hair cell differentiation.
Boto, T., Stahl, A., Zhang, X., Louis, T. and Tomchik, S. M. (2019). Independent contributions of discrete dopaminergic circuits to cellular plasticity, memory strength, and valence in Drosophila. Cell Rep 27(7): 2014-2021.e2012. PubMed ID: 31091441
Dopaminergic neurons play a key role in encoding associative memories, but little is known about how these circuits modulate memory strength. This study reports that different sets of dopaminergic neurons projecting to the Drosophila mushroom body (MB) differentially regulate valence and memory strength. PPL2 neurons increase odor-evoked calcium responses to a paired odor in the MB and enhance behavioral memory strength when activated during olfactory classical conditioning. When paired with odor alone, they increase MB responses to the paired odor but do not drive behavioral approach or avoidance, suggesting that they increase the salience of the odor without encoding strong valence. This contrasts with the role of dopaminergic PPL1 neurons, which drive behavioral reinforcement but do not alter odor-evoked calcium responses in the MB when stimulated. These data suggest that different sets of dopaminergic neurons modulate olfactory valence and memory strength via independent actions on a memory-encoding brain region.
Malloy, C. A., Somasundaram, E., Omar, A., Bhutto, U., Medley, M., Dzubuk, N. and Cooper, R. L. (2019). Pharmacological identification of cholinergic receptor subtypes: modulation of locomotion and neural circuit excitability in Drosophila larvae. Neuroscience. PubMed ID: 31102763
(In Drosophila melanogaster acetylcholine (ACh) is the neurotransmitter used in peripheral sensory neurons and is a primary excitatory neurotransmitter and neuromodulator within the central nervous system (CNS). The receptors are divided into two broad subtypes: the ionotropic nicotinic acetylcholine receptors (nAChRs) and the metabotropic muscarinic acetylcholine receptors (mAChRs). A behavioral and electrophysiological approach was used to assess cholinergic modulation of locomotion and sensory-CNS-motor circuit excitability. Intact and semi-intact 3rd instar larvae were exposed to ACh receptor agonists and antagonists to observe their roles in behavior and regulation of neural circuit excitability and to investigate AChR pharmacological properties in vivo. This was combined with targeted AChR RNAi-mediated knockdown to identify specific receptor subtypes facilitating ACh modulation of circuit efficacy. A contribution by both mAChRs and nAChRs was identified in regulation of locomotor behavior and reveal they play a role in modulation of the excitability of a sensory-CNS-motor circuit. A conspicuous role was identified for mAChR-A and mAChR-C in motor neurons in modulation of their input-output efficacy.
Frechter, S., Bates, A. S., Tootoonian, S., Dolan, M. J., Manton, J. D., Jamasb, A. R., Kohl, J., Bock, D. and Jefferis, G. S. (2019). Functional and anatomical specificity in a higher olfactory centre. Elife 8. PubMed ID: 31112127
Most sensory systems are organized into parallel neuronal pathways that process distinct aspects of incoming stimuli. In the insect olfactory system, second order projection neurons target both the mushroom body, required for learning, and the lateral horn (LH), proposed to mediate innate olfactory behavior. Mushroom body neurons form a sparse olfactory population code, which is not stereotyped across animals. In contrast, odor coding in the LH remains poorly understood. This study combined genetic driver lines, anatomical and functional criteria to show that the Drosophila LH has ~1400 neurons and >165 cell types. Genetically labeled LHNs have stereotyped odor responses across animals and on average respond to three times more odors than single projection neurons. LHNs are better odor categorizers than projection neurons, likely due to stereotyped pooling of related inputs. These results reveal some of the principles by which a higher processing area can extract innate behavioral significance from sensory stimuli.
Dolan, M. J., Frechter, S., Bates, A. S., Dan, C., Huoviala, P., Roberts, R. J., Schlegel, P., Dhawan, S., Tabano, R., Dionne, H., Christoforou, C., Close, K., Sutcliffe, B., Giuliani, B., Li, F., Costa, M., Ihrke, G., Meissner, G. W., Bock, D. D., Aso, Y., Rubin, G. M. and Jefferis, G. S. (2019). Neurogenetic dissection of the Drosophila lateral horn reveals major outputs, diverse behavioural functions, and interactions with the mushroom body. Elife 8. PubMed ID: 31112130
Animals exhibit innate behaviours to a variety of sensory stimuli including olfactory cues. In Drosophila, one higher olfactory centre, the lateral horn (LH), is implicated in innate behaviour. However, structural and functional understanding of the LH is scant, in large part due to a lack of sparse neurogenetic tools for this region. This study generate a collection of split-GAL4 driver lines providing genetic access to 82 LH cell types. These were ysed to create an anatomical and neurotransmitter map of the LH and link this to EM connectomics data. ~30% of LH projections converge with outputs from the mushroom body, site of olfactory learning and memory. Using optogenetic activation, LH cell types were identified that drive changes in valence behavior or specific locomotor programs. In summary, this study has generated a resource for manipulating and mapping LH neurons, providing new insights into the circuit basis of innate and learned olfactory behavior.
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