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Current papers in developmental biology and gene function


Monday December 31st, 2018 - RNA and Transposons

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Haussmann, I. U., Ustaoglu, P., Brauer, U., Hemani, Y., Dix, T. C. and Soller, M. (2018). Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4. Nucleic Acids Res. PubMed ID: 30541104
Alternative splicing is a key feature of human genes, yet studying its regulation is often complicated by large introns. The Down Syndrome Cell Adhesion Molecule (Dscam) gene from Drosophila is one of the most complex genes generating vast molecular diversity by mutually exclusive alternative splicing. To resolve how alternative splicing in Dscam is regulated, a plasmid-based UAS reporter genes were developed for the Dscam variable exon 4 cluster; its alternative splicing was shown to recapitulated by GAL4-mediated expression in neurons. Gap-repair recombineering was developed to very efficiently manipulate these large reporter plasmids in Escherichia coli using restriction enzymes or sgRNA/Cas9 DNA scission to capitalize on the many benefits of plasmids in phiC31 integrase-mediated transgenesis. Using these novel tools, inclusion of Dscam exon 4 variables were shown to differ little in development and individual flies, and is robustly determined by sequences harbored in variable exons. It was further shown that introns drive selection of both proximal and distal variable exons. Since exon 4 cluster introns lack conserved sequences that could mediate robust long-range base-pairing to bring exons into proximity for splicing, the data argue for a central role of introns in mutually exclusive alternative splicing of Dscam exon 4 cluster.
Schor, I. E., Bussotti, G., Males, M., Forneris, M., Viales, R. R., Enright, A. J. and Furlong, E. E. M. (2018). Non-coding RNA expression, function, and variation during Drosophila embryogenesis. Curr Biol. PubMed ID: 30393032
Long non-coding RNAs (lncRNAs) can often function in the regulation of gene expression during development; however, their generality as essential regulators in developmental processes and organismal phenotypes remains unclear. A tailored investigation of lncRNA expression and function during Drosophila embryogenesis was performed, interrogating multiple stages, tissue specificity, nuclear localization, and genetic backgrounds. The results almost double the number of annotated lncRNAs expressed at these embryonic stages. lncRNA levels are generally positively correlated with those of their neighboring genes, with little evidence of transcriptional interference. Using fluorescent in situ hybridization, the spatiotemporal expression of 15 new lncRNAs is reported, revealing very dynamic tissue-specific patterns. Despite this, deletion of selected lncRNA genes had no obvious developmental defects or effects on viability under standard and stressed conditions. However, two lncRNA deletions resulted in modest expression changes of a small number of genes, suggesting that they fine-tune expression of non-essential genes. Several lncRNAs have strain-specific expression, indicating that they are not fixed within the population. This intra-species variation across genetic backgrounds may thereby be a useful tool to distinguish rapidly evolving lncRNAs with as yet non-essential roles.
Becker, K., Bluhm, A., Casas-Vila, N., Dinges, N., Dejung, M., Sayols, S., Kreutz, C., Roignant, J. Y., Butter, F. and Legewie, S. (2018). Quantifying post-transcriptional regulation in the development of Drosophila melanogaster. Nat Commun 9(1): 4970. PubMed ID: 30478415
Even though proteins are produced from mRNA, the correlation between mRNA levels and protein abundances is moderate in most studies, occasionally attributed to complex post-transcriptional regulation. To address this, a paired transcriptome/proteome time course dataset was prepared with 14 time points during Drosophila embryogenesis. Despite a limited mRNA-protein correlation (rho = 0.54), mathematical models describing protein translation and degradation explain 84% of protein time-courses based on the measured mRNA dynamics without assuming complex post transcriptional regulation, and allow for classification of most proteins into four distinct regulatory scenarios. By performing an in-depth characterization of the putatively post-transcriptionally regulated genes, it is postulated that the RNA-binding protein Hrb98DE is involved in post-transcriptional control of sugar metabolism in early embryogenesis, and this hypothesis was partially validated using Hrb98DE knockdown. In summary, a systems biology framework is presented for the identification of post-transcriptional gene regulation from large-scale, time-resolved transcriptome and proteome data.
Yoshitake, Y., Inomata, N., Sano, M., Kato, Y. and Itoh, M. (2018). The P element invaded rapidly and caused hybrid dysgenesis in natural populations of Drosophila simulans in Japan. Ecol Evol 8(19): 9590-9599. PubMed ID: 30386559
Transposable elements not only can change genomic positions and disperse across the gene pool, but also can jump to another species through horizontal transmission. Of late, the P element, a DNA transposon in insects, was shown to cross the genetic boundary from Drosophila melanogaster to D. simulans in Europe around 2006. To understand the dynamics of transposable elements, especially in the early stages of invasion, 63 lines were examined of D. simulans from 11 natural populations in Japan established in 1976-2015. Based on PCR analyses, P elements were demonstrated to exist in Japan in 2008 and later. One copy of the full-length P element was identified and mapped to a site on chromosome 3 L in a genome. All of 18 copies of P elements examined shared 'A' at the nucleotide position 2040, which is representative of the direct descendants of the original P element that invaded in D. simulans. It was also found that some lines having P elements can induce intensive gonadal dysgenesis in D. simulans at 29 degrees C. These results imply that P elements in D. simulans arrived at the east end of Asia just a few years later than or almost simultaneously to the initial invasion in Europe, Africa, and North America, suggesting a more astonishingly rapid spread than previously assumed.
Moon, S., Cassani, M., Lin, Y. A., Wang, L., Dou, K. and Zhang, Z. Z. (2018). A robust transposon-endogenizing response from germline stem cells. Dev Cell. PubMed ID: 30393075
The heavy occupancy of transposons in the genome implies that existing organisms have survived from multiple, independent rounds of transposon invasions. However, how and which host cell types survive the initial wave of transposon invasion remain unclear. This study shows that the germline stem cells can initiate a robust adaptive response that rapidly endogenizes invading P element transposons by activating the DNA damage checkpoint and piRNA production. That temperature modulates the P element activity in germline stem cells, establishing a powerful tool to trigger transposon hyper-activation. Facing vigorous invasion, Drosophila first shut down oogenesis and induce selective apoptosis. Interestingly, a robust adaptive response occurs in ovarian stem cells through activation of the DNA damage checkpoint. Within 4 days, the hosts amplify P element-silencing piRNAs, repair DNA damage, subdue the transposon, and reinitiate oogenesis. It is proposed that this robust adaptive response can bestow upon organisms the ability to survive recurrent transposon invasions throughout evolution.
Chang, T. H., Mattei, E., Gainetdinov, I., Colpan, C., Weng, Z. and Zamore, P. D. (2018). Maelstrom represses canonical Polymerase II transcription within bi-directional piRNA clusters in Drosophila melanogaster. Mol Cell. PubMed ID: 30527661
In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. This study reports that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. It is propose dthat by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins.

Friday, December 28th - Immune Response

Patrnogic, J., Castillo, J. C., Shokal, U., Yadav, S., Kenney, E., Heryanto, C., Ozakman, Y. and Eleftherianos, I. (2018). Pre-exposure to non-pathogenic bacteria does not protect Drosophila against the entomopathogenic bacterium Photorhabdus. PLoS One 13(10): e0205256. PubMed ID: 30379824
Immune priming in insects involves an initial challenge with a non-pathogenic microbe or exposure to a low dose of pathogenic microorganisms, which provides a certain degree of protection against a subsequent pathogenic infection. The protective effect of insect immune priming has been linked to the activation of humoral or cellular features of the innate immune response during the preliminary challenge, and these effects might last long enough to promote the survival of the infected animal. This study has examined the result of immune priming against two potent entomopathogenic bacteria, Photorhabdus luminescens and P. asymbiotica. Rearing D. melanogaster on diet containing a non-pathogenic strain of Escherichia coli alone or in combination with Micrococcus luteus was found to upregulate the antibacterial peptide immune response in young adult flies, but it does not prolong fly life span. Also, subsequent intrathoracic injection with P. luminescens or P. asymbiotica triggers the Immune deficiency and Toll signaling pathways in flies previously exposed to a live or heat-killed mix of the non-pathogenic bacteria, but the immune activation fails to promote fly survival against the pathogens. These findings suggest that immune priming in D. melanogaster is determined by the type of microbes involved as well as the mode of microbial exposure.
Green, N., Walker, J., Bontrager, A., Zych, M. and Geisbrecht, E. R. (2018). A tissue communication network coordinating innate immune response during muscle stress. J Cell Sci. PubMed ID: 30478194
Complex tissue communication networks function throughout an organism's lifespan to maintain tissue homeostasis. Using the genetic model Drosophila melanogaster, this study has defined a network of immune responses activated following the induction of muscle stresses, including hypercontraction, detachment, and oxidative stress. Of these stressors, loss of genes causing muscle detachment produce the strongest levels of JAK-STAT activation. In one of these mutants, fondue (fon), hemocyte recruitment and the accumulation of melanin was observed at muscle attachment sites (MASs), indicating a broad involvement of innate immune responses upon muscle detachment. Loss of fon results in pathogen-independent Toll signaling in the fat body and increased expression of the Toll-dependent antimicrobial peptide (AMP) Drosomycin. Interestingly, genetic interactions between fon and various Toll pathway components enhance muscle detachment. Finally, this study shows that JAK-STAT and Toll signaling are capable of reciprocal activation in larval tissues. A model of tissue communication is proposed for the integration of immune responses at the local and systemic level in response to altered muscle physiology.
Germani, F., Hain, D., Sternlicht, D., Moreno, E. and Basler, K. (2018). The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner. Elife 7. PubMed ID: 30451683
The Toll pathway regulates the cellular response to infection via the transcriptional upregulation of antimicrobial peptides. In Drosophila, apart from its role in innate immunity, this pathway has also been reported to be important for the elimination of loser cells in a process referred to as cell competition, which can be locally triggered by secreted factors released from winner cells. This work provides evidence that the inhibition of Toll signaling not only increases the fitness of loser cells, but also bestows a clonal growth advantage on wild-type cells. This growth advantage depends on basal infection levels since it is no longer present under axenic conditions but exacerbated upon intense pathogen exposure. Thus, the Toll pathway functions as a fine-tuned pro-apoptotic and anti-proliferative regulator, underlining the existence of a trade-off between innate immunity and growth during development.
Harsh, S., Ozakman, Y., Kitchen, S. M., Paquin-Proulx, D., Nixon, D. F. and Eleftherianos, I. (2018). Dicer-2 regulates resistance and maintains homeostasis against Zika virus infection in Drosophila. J Immunol. PubMed ID: 30305326
Zika virus (ZIKV) outbreaks pose a massive public health threat in several countries. This study has developed an in vivo model to investigate the host-ZIKV interaction in Drosophila. A strain of ZIKV replicates in wild-type flies without reducing their survival ability. ZIKV infection triggers RNA interference and that mutating Dicer-2 results in enhanced ZIKV load and increased susceptibility to ZIKV infection. Using a flavivirus-specific Ab, this study has found that ZIKV is localized in the gut and fat body cells of the infected wild-type flies and results in their perturbed homeostasis. In addition, Dicer-2 mutants display severely reduced insulin activity, which could contribute toward the increased mortality of these flies. This work establishes the suitability of Drosophila as the model system to study host-ZIKV dynamics, which is expected to greatly advance understanding of the molecular and physiological processes that determine the outcome of this disease.
Mondotte, J. A., Gausson, V., Frangeul, L., Blanc, H., Lambrechts, L. and Saleh, M. C. (2018). Immune priming and clearance of orally acquired RNA viruses in Drosophila. Nat Microbiol. PubMed ID: 30374170
Immune responses in insects are differentially triggered depending on the infection route used by the pathogen. In most studies involving Drosophila melanogaster and viruses, infection is done by injection, while oral infection, which is probably the most common route of viral entry in nature, remains unexplored. This study orally infected adults and larvae from wild-type and RNA interference (RNAi) mutant flies with different RNA viruses. In contrast with what is observed following virus injection, oral infections initiated at larval or adult stages are cleared in adult flies. Virus elimination occurred despite a larger infectious dose than for injected flies and evidence of viral replication. RNAi mutant flies suffered greater mortality relative to wild-type flies following oral infection, but they also eliminated the virus, implying that RNAi is not essential for viral clearance and that other immune mechanisms act during oral infections. It was further shown that information of infection by RNA viruses acquired orally leaves a trace under a DNA form, which confers protection against future reinfection by the same virus. Together, this work presents evidence of clearance and immune priming for RNA viruses in insects and challenges the current view of antiviral immunity in insects.
Abhyankar, V., Kaduskar, B., Kamat, S. S., Deobagkar, D. and Ratnaparkhi, G. S. (2018). Drosophila DNA/RNA methyltransferase contributes to robust host defense in ageing animals by regulating sphingolipid metabolism. J Exp Biol. PubMed ID: 30254027
Drosophila methyltransferase (Mt2) has been implicated in methylation of both DNA and tRNA. This study demonstrates that loss of Mt2 activity leads to an age dependent decline of immune function in the adult fly. A newly eclosed adult has mild immune defects that exacerbate in a fifteen-day old Mt2(-/-) fly. The age dependent effects appear to be systemic, including disturbances in lipid metabolism, changes in cell shape of hemocytes and significant fold changes in levels of transcripts related to host defense. Lipid imbalance, as measured by quantitative lipidomics, correlates with immune dysfunction with high levels of immunomodulatory lipids, sphingosine-1phosphate (S1P) and ceramides, along with low levels of storage lipids. Activity assays on fly lysates confirm the age dependent increase in S1P and concomitant reduction of S1P lyase activity. It is hypothesized that Mt2 functions to regulate genetic loci such as S1P lyase and this regulation is essential for robust host defense as the animal ages. This study uncovers novel links between age dependent Mt2 function, innate immune response and lipid homeostasis.

Thursday, December 27th - Chromatin

Glenn, S. E. and Geyer, P. K. (2018). Investigation of the developmental requirements of Drosophila HP1 and insulator protein partner. HIPP1. G3 (Bethesda). PubMed ID: 30514714
Drosophila Suppressor of Hairy-wing [Su(Hw)] is a multifunctional zinc finger DNA binding protein. Transcriptional regulation by Su(Hw) is essential in the ovary and testis, where Su(Hw) functions primarily as a repressor. Recently, the HP1a and Insulator Partner Protein 1 (HIPP1) was found to extensively co-localize with Su(Hw) and other insulator binding proteins in euchromatic regions of the genome, and with Heterochromatin Protein 1a (HP1a) in heterochromatic regions. As HIPP1 is the homologue of the human co-repressor Chromodomain Y-Like (CDYL), its requirement in was tested establishing transcriptional repression in flies. To this end, multiple Hipp1 null alleles and a tagged derivative of the endogenous gene (Hipp1(GFP)), were generated using CRISPR mutagenesis. HIPP1 is a widely expressed nuclear protein that is dispensable for viability, as well as female and male fertility. HIPP1 and HP1a display minimum co-localization in interphase cells, and HP1a-dependent transcriptional repression of several reporter genes is HIPP1-independent, indicating that HIPP1 is not essential for HP1a-dependent heterochromatin formation. Despite Su(Hw) having a major role in promoting HIPP1 occupancy in euchromatin, this study shows that HIPP1 is dispensable for the transcriptional and insulator functions of Su(Hw), indicating that HIPP1 is not a critical Su(Hw) cofactor. Further studies are needed to clarify the role of HIPP1 in Drosophila development.
Rosales-Vega, M., Hernandez-Becerril, A., Murillo-Maldonado, J. M., Zurita, M. and Vazquez, M. (2018). The role of the trithorax group TnaA isoforms in Hox gene expression, and in Drosophila late development. PLoS One 13(10): e0206587. PubMed ID: 30372466
Regulation of developmental gene expression in eukaryotes involves several levels. One of them is the maintenance of gene expression along the life of the animal once it is started by different triggers early in development. One of the questions in the field is when in developmental time, the animal start to use the different maintenance mechanisms. The trithorax group (TrxG) of genes was first characterized as essential for maintaining homeotic gene expression. The TrxG gene tonalli interacts genetically and physically with genes and subunits of the BRAHMA BAP chromatin remodeling complex and encodes TnaA proteins with putative E3 SUMO-ligase activity. In contrast to the phenocritic lethal phase of animals with mutations in other TrxG genes, tna mutant individuals die late in development. This study determined the requirements of TnaA for survival at pupal and adult stages, in different tna mutant genotypes where the lack of TnaA proteins, and the presence of adult homeotic loss-of-function phenotypes were corroborate. Also, whether the absence of TnaA in haltere and leg larval imaginal discs affects the presence of the homeotic proteins Ultrabithorax and Sex combs reduced respectively was investigated by using some of the characterized genotypes and more finely by generating TnaA defective clones induced at different stages of development. It was found that tna is not required for growth or survival of imaginal disc cells and that it is a fine modulator of homeotic gene expression.
Sokolova, M., Moore, H. M., Prajapati, B., Dopie, J., Merilainen, L., Honkanen, M., Matos, R. C., Poukkula, M., Hietakangas, V. and Vartiainen, M. K. (2018). Nuclear actin is required for transcription during Drosophila oogenesis. iScience 9: 63-70. PubMed ID: 30384134
Actin has been linked to processes spanning the whole gene expression cascade, from regulating specific transcription factors, such as myocardin-related transcription factor, to chromatin remodeling and RNA polymerase function. However, whether actin controls the transcription of only specific genes or has a global role in gene expression has remained elusive. A genome-wide analysis reveals, for the first time, that actin interacts with essentially all transcribed genes in Drosophila ovaries. Actin co-occupies the majority of gene promoters together with Pol II, and on highly expressed genes, these two proteins also associate with gene bodies. Mechanistically, actin is required for Pol II recruitment to gene bodies, and manipulation of nuclear transport factors for actin leads to the decreased expression of eggshell genes. Collectively, these results uncover a global role for actin in transcription and demonstrate the in vivo importance of balanced nucleocytoplasmic shuttling of actin in the transcriptional control of a developmental process.
Zhang, X., Miao, C., Nan, Z., Lyu, J., Xi, Y., Yang, X. and Ge, W. (2018). A positive role of Sin3A in regulating Notch signaling during Drosophila wing development. Cell Signal 53: 184-189. PubMed ID: 30316814
Notch is a transmembrane receptor that mediates intercellular signaling through a conserved signaling cascade in all animal species. Transcriptional and posttranscriptional regulation of Notch receptor are important for maintaining Notch signaling activity. This study shows that depletion of Drosophila Sin3A leads to loss of the adult wing margin and downregulation of Notch target gene expression in the developing wing disc. Sin3A regulates the Notch pathway downstream of Delta and upstream of Notch activation. The role of Sin3A in the Notch pathway is partly mediated by its ability to modulate Notch receptor transcription. Furthermore, the transcriptional activation of Notch receptor is autoregulated by Notch itself. Evidence is also provided that Sin3A is required for Notch activation mediated Notch transcription. Together, these data demonstrate that Sin3A activates Notch signaling by promoting Notch transcription and reveal a previously unknown autoregulatory mechanism for Notch signaling activation during Drosophila wing development.
Copur, O., Gorchakov, A., Finkl, K., Kuroda, M. I. and Muller, J. (2018). Sex-specific phenotypes of histone H4 point mutants establish dosage compensation as the critical function of H4K16 acetylation in Drosophila. Proc Natl Acad Sci U S A. PubMed ID: 30530664
Acetylation of histone H4 at lysine 16 (H4K16) modulates nucleosome-nucleosome interactions and directly affects nucleosome binding by certain proteins. In Drosophila, H4K16 acetylation by the dosage compensation complex subunit Mof is linked to increased transcription of genes on the single X chromosome in males. This study analyzed Drosophila containing different H4K16 mutations or lacking Mof protein. An H4K16A mutation causes embryonic lethality in both sexes, whereas an H4K16R mutation permits females to develop into adults but causes lethality in males. The acetyl-mimic mutation H4K16Q permits both females and males to develop into adults. Complementary analyses reveal that males lacking maternally deposited and zygotically expressed Mof protein arrest development during gastrulation, whereas females of the same genotype develop into adults. Together, this demonstrates the causative role of H4K16 acetylation by Mof for dosage compensation in Drosophila and uncovers a previously unrecognized requirement for this process already during the onset of zygotic gene transcription.
Durdevic, Z., Pillai, R. S. and Ephrussi, A. (2018). Transposon silencing in the Drosophila female germline is essential for genome stability in progeny embryos. Life Sci Alliance 1(5): e201800179. PubMed ID: 30456388
The Piwi-interacting RNA pathway functions in transposon control in the germline of metazoans. The conserved RNA helicase Vasa is an essential Piwi-interacting RNA pathway component, but has additional important developmental functions. This study addresses the importance of Vasa-dependent transposon control in the Drosophila female germline and early embryos. Transient loss of vasa expression during early oogenesis leads to transposon up-regulation in supporting nurse cells of the fly egg-chamber. Elevated transposon levels have dramatic consequences, as de-repressed transposons accumulate in the oocyte where they cause DNA damage. Suppression of Chk2-mediated DNA damage signaling in vasa mutant females restores oogenesis and egg production. Damaged DNA and up-regulated transposons are transmitted from the mother to the embryos, which sustain severe nuclear defects and arrest development. These findings reveal that the Vasa-dependent protection against selfish genetic elements in the nuage of nurse cell is essential to prevent DNA damage-induced arrest of embryonic development.

Wednesday, December 26th - Ovary and Testis

Anllo, L., Plasschaert, L. W., Sui, J. and DiNardo, S. (2018). Live imaging reveals hub cell assembly and compaction dynamics during morphogenesis of the Drosophila testis niche. Dev Biol. PubMed ID: 30553808
Adult stem cells are often found in specialized niches, where the constituent cells direct self-renewal of their stem cell pool. The niche is therefore crucial for both normal homeostasis and tissue regeneration. In many mammalian tissues, niche cells have classically been difficult to identify, which has hampered any understanding of how tissues first construct niches during development. Fortunately, the Drosophila germline stem cell (GSC) niche is well defined, allowing for unambiguous identification of both niche cells and resident stem cells. The testis niche this study followed pro-niche cells as they assemble and assume their final form. After ex vivo culture the niche appears fully functional, as judged by enrichment of adhesion proteins, the ability to activate STAT in adjacent GSCs, and to direct GSCs to divide orthogonally to the niche, just as they would in situ. Collectively, imaging has generated several novel insights on niche morphogenesis that could not be inferred from fixed images alone. Dynamic processes were identified that constitute an assembly phase and a compaction phase during morphogenesis. The compaction phase correlates with cell neighbor exchange among the assembled pro-niche cells, as well as a burst of divisions among newly recruited stem cells. Before compaction, an assembly phase involves the movement of pro-niche cells along the outer periphery of the gonad, using the extracellular matrix (ECM) to assemble at the anterior of the gonad. Finally, live-imaging in integrin mutants allows definition of the role of pro-niche cell-ECM interaction with regard to the new assembly and compaction dynamics revealed in this study.
Fedorova, E. V., Dorogova, N. V., Bolobolova, E. U., Fedorova, S. A., Karagodin, D. A., Ogienko, A. A., Khruscheva, A. S. and Baricheva, E. M. (2018). GAGA protein is required for multiple aspects of Drosophila oogenesis and female fertility. Genesis: e23269. PubMed ID: 30537428
Investigation of Drosophila oogenesis provides the opportunity to understand conservative genetic mechanisms underlying fertile female gamete development. This work showed that the Drosophila DNA-binding protein GAGA factor (GAF) had a multifunctional role in oogenesis and it is involved in the regulation of this process genetic program. This study examined the influence on Drosophila oogenesis of a number of mutations in the 5' region of the Trl gene that encodes GAF. Originally generated Trl mutations lead to a decrease in transcriptional gene activity and levels of GAF expression in both germline and follicular cells. Cytological (fluorescence and electron microscopy analysis) showed that GAF loss resulted in multiple oogenesis defects. Mutations affected the actin cytoskeleton, leading to decrease of cytoplasmic filaments in nurse cells and basal actin in follicular cells. GAF depletion also leads to abnormal follicular cells migration, both border and centripetal. In addition, mutant ovaries demonstrated abnormalities in germ cells, including mitochondria, endoplasmic reticulum, karyosome organization, yolk granule formation and selective transport. Loss of GAF also promoted excessive cell death and egg chamber degradation. In sum, these defects caused very high or full female sterility. Since one of the main GAF activities is regulation of transcription, the complex phenotypes of the Trl mutants might be the consequence of its multiple target genes misexpression.
Finegan, T. M., Na, D., Cammarota, C., Skeeters, A. V., Nadasi, T. J., Dawney, N. S., Fletcher, A. G., Oakes, P. W. and Bergstralh, D. T. (2018). Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium. EMBO J. PubMed ID: 30478193
This study investigated the cell behaviors that drive morphogenesis of the Drosophila follicular epithelium during expansion and elongation of early-stage egg chambers. Cell division was found not to be required for elongation of the early follicular epithelium, but drives the tissue toward optimal geometric packing. The orientation of cell divisions with respect to the planar tissue axis was examined, and a bias toward the primary direction of tissue expansion was found. However, interphase cell shapes demonstrate the opposite bias. Hertwig's rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. This observation cannot be explained by the anisotropic activity of the conserved Pins/Mud spindle-orienting machinery, which controls division orientation in the apical-basal axis and planar division orientation in other epithelial tissues. Rather, cortical tension at the apical surface translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin-to-adherens junctions. These findings demonstrate that division orientation in different axes-apical-basal and planar-is controlled by distinct, independent mechanisms in a proliferating epithelium.
Alpern, J. H. M., Asselin, M. M. and Moehring, A. J. (2018). Identification of a novel sperm class and its role in fertilization in Drosophila. J Evol Biol. PubMed ID: 30484924
In many species, males have evolved to produce a sterile sperm (parasperm) in conjunction with fertilizing sperm (eusperm). This study documents evidence of males depositing two morphologically distinct types of parasperm (1 and 2) into the female reproductive tract in Drosophila pseudoobscura. These parasperm differ in length, shape, amount produced, amount in long-term storage, and may have separate roles in ensuring male fertilization success. While both parasperm types protect eusperm from female spermicides, only parasperm 2, which has a corkscrew shape, is associated with sperm competition. Increased production of parasperm 2 is also negatively correlated with the eusperm and parasperm 1 production. Thus, selection may be acting on parasperm production in the presence of sperm competition. These findings show how both sperm competition and cryptic female choice may be acting in conjunction to influence the evolution of ejaculate composition. This identification and characterization of two distinct parasperm morphs will enhance the ability for further evaluation of parasperm's role in fertilization.
Velentzas, A. D., Velentzas, P. D., Katarachia, S. A., Anagnostopoulos, A. K., Sagioglou, N. E., Thanou, E. V., Tsioka, M. M., Mpakou, V. E., Kollia, Z., Gavriil, V. E., Papassideri, I. S., Tsangaris, G. T., Cefalas, A. C., Sarantopoulou, E. and Stravopodis, D. J. (2018). The indispensable contribution of s38 protein to ovarian-eggshell morphogenesis in Drosophila melanogaster. Sci Rep 8(1): 16103. PubMed ID: 30382186
Drosophila chorion represents a remarkable model system for the in vivo study of complex extracellular-matrix architectures. For its organization and structure, s38 protein is considered as a component of major importance, since it is synthesized and secreted during early choriogenesis. However, there is no evidence that proves its essential, or redundant, role in chorion biogenesis. This study shows that targeted downregulation of s38 protein, specifically in the ovarian follicle-cell compartment, via employment of an RNAi-mediated strategy, causes generation of diverse dysmorphic phenotypes, regarding eggshell's regionally and radially specialized structures. Downregulation of s38 protein severely impairs fly's fertility and is unable to be compensated by the s36 homologous family member, thus unveiling s38 protein's essential contribution to chorion's assembly and function. Altogether, s38 acts as a key skeletal protein being critically implicated in the patterning establishment of a highly structured tripartite endochorion. Furthermore, it seems that s38 loss may sensitize choriogenesis to stochastic variation in its coordination and timing.
Wittes, J. and Schupbach, T. (2018). A gene expression screen in Drosophila melanogaster identifies novel JAK/STAT and EGFR targets during oogenesis. G3 (Bethesda). PubMed ID: 30385460
JAK/STAT and epidermal growth factor receptor (EGFR) signaling pathways are conserved regulators of tissue patterning, morphogenesis, and other cell biological processes. During Drosophila oogenesis, these pathways determine the fates of epithelial follicle cells (FCs). JAK/STAT and EGFR together specify a population of cells called the posterior follicle cells (PFCs), which signal to the oocyte to establish the embryonic axes. In this study, whole genome expression analysis was performed to identify genes activated by JAK/STAT and/or EGFR. 317 genes were transcriptionally upregulated in egg chambers with ectopic JAK/STAT and EGFR activity in the FCs. The list was enriched for genes encoding extracellular matrix (ECM) components and ECM-associated proteins. 69 candidates were tested for a role in axis establishment using RNAi knockdown in the FCs. The signaling protein Semaphorin 1b is reported to become enriched in the PFCs in response to JAK/STAT and EGFR. ADAM metallopeptidase with thrombospondin type 1 motif A (AdamTS-A) was identified a novel target of JAK/STAT in the FCs that regulates egg chamber shape. AdamTS-A mRNA becomes enriched at the anterior and posterior poles of the egg chamber at stages 6 to 7 and is regulated by JAK/STAT. Altering AdamTS-A expression in the poles or middle of the egg chamber produces rounder egg chambers. It is proposed that AdamTS-A regulates egg shape by remodeling the basement membrane.

Monday, December 24th - Larval and adult CNS

Pauls, D., Blechschmidt, C., Frantzmann, F., El Jundi, B. and Selcho, M. (2018). A comprehensive anatomical map of the peripheral octopaminergic/tyraminergic system of Drosophila melanogaster. Sci Rep 8(1): 15314. PubMed ID: 30333565
The modulation of an animal's behavior through external sensory stimuli, previous experience and its internal state is crucial to survive in a constantly changing environment. In most insects, octopamine (OA) and its precursor tyramine (TA) modulate a variety of physiological processes and behaviors by shifting the organism from a relaxed or dormant condition to a responsive, excited and alerted state. Even though OA/TA neurons of the central brain are described on single cell level in Drosophila melanogaster, the periphery was largely omitted from anatomical studies. Given that OA/TA is involved in behaviors like feeding, flying and locomotion, which highly depend on a variety of peripheral organs, it is necessary to study the peripheral connections of these neurons to get a complete picture of the OA/TA circuitry. This study describes the anatomy of this aminergic system in relation to peripheral tissues of the entire fly. OA/TA neurons arborize onto skeletal muscles all over the body and innervate reproductive organs, the heart, the corpora allata, and sensory organs in the antennae, legs, wings and halteres underlining their relevance in modulating complex behaviors.
Pende, M., Becker, K., Wanis, M., Saghafi, S., Kaur, R., Hahn, C., Pende, N., Foroughipour, M., Hummel, T. and Dodt, H. U. (2018). High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster. Nat Commun 9(1): 4731. PubMed ID: 30413688
The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. This psper presents a novel approach combining tissue clearing, ultramicroscopy, and data analysis that enables the visualisation of neuronal networks with single-cell resolution from the larval stage up to the adult Drosophila. FlyClear, the signal preserving clearing technique, stabilises tissue integrity and fluorescence signal intensity for over a month and efficiently removes the overall pigmentation. An aspheric ultramicroscope set-up utilising an improved light-sheet generator allows visualization of long-range connections of peripheral sensory and central neurons in the visual and olfactory system. High-resolution 3D reconstructions with isotropic resolution from entire GFP-expressing flies are obtained by applying image fusion from orthogonal directions. This methodological integration of novel chemical, optical, and computational techniques allows a major advance in the analysis of global neural circuit organisation.
Vanderheyden, W. M., Goodman, A. G., Taylor, R. H., Frank, M. G., Van Dongen, H. P. A. and Gerstner, J. R. (2018). Astrocyte expression of the Drosophila TNF-alpha homologue, Eiger, regulates sleep in flies. PLoS Genet 14(10): e1007724. PubMed ID: 30379810
Sleep contributes to cognitive functioning and is sufficient to alter brain morphology and function. However, mechanisms underlying sleep regulation remain poorly understood. In mammals, tumor necrosis factor-alpha (TNFalpha) is known to regulate sleep, and cytokine expression may represent an evolutionarily ancient mechanism in sleep regulation. This study shows that the Drosophila TNFalpha homologue, Eiger, mediates sleep in flies. Knockdown of Eiger in astrocytes, but not in neurons, significantly reduces sleep duration, and total loss-of-function reduces the homeostatic response to sleep loss. In addition, neuronal, but not astrocyte, expression of the TNFalpha receptor superfamily member, Wengen, is necessary for sleep deprivation-induced homeostatic response and for mediating increases in sleep in response to human TNFalpha. These data identify a novel astrocyte-to-neuron signaling mechanism in the regulation of sleep homeostasis and show that the Drosophila cytokine, Eiger, represents an evolutionarily conserved mechanism of sleep regulation across phylogeny.
Vissers, J. H. A., Froldi, F., Schroder, J., Papenfuss, A. T., Cheng, L. Y. and Harvey, K. F. (2018). The Scalloped and Nerfin-1 transcription factors cooperate to maintain neuronal cell fate. Cell Rep 25(6): 1561-1576.e1567. PubMed ID: 30404010
The ability of cells to stably maintain their fate is governed by specific transcription regulators. This study shows that the Scalloped (Sd) and Nervous fingers-1 (Nerfin-1) transcription factors physically and functionally interact to maintain medulla neuron fate in the Drosophila melanogaster CNS. Using Targeted DamID, Sd and Nerfin-1 were found to occupy a highly overlapping set of target genes, including regulators of neural stem cell and neuron fate, and signaling pathways that regulate CNS development such as Notch and Hippo. Modulation of either Sd or Nerfin-1 activity causes medulla neurons to dedifferentiate to a stem cell-like state, and this is mediated at least in part by Notch pathway deregulation. Intriguingly, orthologs of Sd and Nerfin-1 have also been implicated in control of neuronal cell fate decisions in both worms and mammals. These data indicate that this transcription factor pair exhibits remarkable biochemical and functional conservation across metazoans.
Cosmanescu, F., Katsamba, P. S., Sergeeva, A. P., Ahlsen, G., Patel, S. D., Brewer, J. J., Tan, L., Xu, S., Xiao, Q., Nagarkar-Jaiswal, S., Nern, A., Bellen, H. J., Zipursky, S. L., Honig, B. and Shapiro, L. (2018). Neuron-subtype-specific expression, interaction affinities, and specificity determinants of DIP/Dpr cell recognition proteins. Neuron. PubMed ID: 30467080
Binding between DIP and Dpr neuronal recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system. Each lamina neuron was previously shown to express many Dprs. This study demonstrates, by contrast, that their synaptic partners typically express one or two DIPs, with binding specificities matched to the lamina neuron-expressed Dprs. A deeper understanding of the molecular logic of DIP/Dpr interaction requires quantitative studies on the properties of these proteins. A quantitative affinity-based DIP/Dpr interactome was generated for all DIP/Dpr protein family members. This revealed a broad range of affinities and identified homophilic binding for some DIPs and some Dprs. These data, along with full-length ectodomain DIP/Dpr and DIP/DIP crystal structures, led to the identification of molecular determinants of DIP/Dpr specificity. This structural knowledge, along with a comprehensive set of quantitative binding affinities, provides new tools for functional studies in vivo.
Tabuchi, M., Monaco, J. D., Duan, G., Bell, B., Liu, S., Liu, Q., Zhang, K. and Wu, M. N. (2018). Clock-generated temporal codes determine synaptic plasticity to control sleep. Cell 175(5): 1213-1227. PubMed ID: 30318147
Neurons use two main schemes to encode information: rate coding (frequency of firing) and temporal coding (timing or pattern of firing). While the importance of rate coding is well established, it remains controversial whether temporal codes alone are sufficient for controlling behavior. Moreover, the molecular mechanisms underlying the generation of specific temporal codes are enigmatic. This study shows in Drosophila clock neurons that distinct temporal spike patterns, dissociated from changes in firing rate, encode time-dependent arousal and regulate sleep. From a large-scale genetic screen, this study identified the molecular pathways mediating the circadian-dependent changes in ionic flux and spike morphology that rhythmically modulate spike timing. Remarkably, the daytime spiking pattern alone is sufficient to drive plasticity in downstream arousal neurons, leading to increased firing of these cells. These findings demonstrate a causal role for temporal coding in behavior and define a form of synaptic plasticity triggered solely by temporal spike patterns.

Friday, December 21st - Signaling

Bittova, L., Jedlicka, P., Dracinsky, M., Kirubakaran, P., Vondrasek, J., Hanus, R. and Jindra, M. (2018). Exquisite ligand stereoselectivity of a Drosophila juvenile hormone receptor contrasts with its broad agonist repertoire. J Biol Chem. PubMed ID: 30455350
The sesquiterpenoid juvenile hormone (JH) is vital to insect development and reproduction. Intracellular JH receptors have recently been established as basic helix-loop-helix transcription factor (bHLH)-PAS proteins in Drosophila melanogaster known as germ cell-expressed (Gce) and its duplicate paralog, methoprene-tolerant (Met). Upon binding JH, Gce/Met activates its target genes. Insects possess multiple native JH homologs whose molecular activities remain unexplored, and diverse synthetic compounds including insecticides exert JH-like effects. How the JH receptor recognizes its ligands is unknown. To determine what structural features define an active JH receptor agonist, several native JHs and their non-native geometric and optical isomers were tested for the ability to bind the Drosophila JH receptor Gce, to induce Gce-dependent transcription, and to affect the development of the fly. These results revealed high ligand stereoselectivity of the receptor. The geometry of the JH skeleton, dictated by two stereogenic double bonds, was the most critical feature, followed by the presence of an epoxide moiety at a terminal position. The optical isomerism at carbon C11 proved less important, even though Gce preferentially bound a natural JH enantiomer. The results of receptor-ligand binding and cell-based gene activation assays tightly correlated with the ability of different geometric JH isomers to induce gene expression and morphogenetic effects in the developing insects. Molecular modeling supported the requirement for the proper double-bond geometry of JH, which appears to be its major selective mechanism. The strict stereoselectivity of Gce towards the natural hormone contrasts with high potency of synthetic Gce agonists of disparate chemistries.
Chen, A. S., Wardwell-Ozgo, J., Shah, N. N., Wright, D., Appin, C. L., Vigneswaran, K., Brat, D. J., Kornblum, H. I. and Read, R. D. (2018). Drak/STK17A drives neoplastic glial proliferation through modulation of MRLC signaling. Cancer Res. PubMed ID: 30530503
Glioblastoma (GBM) and lower grade gliomas (LGG) are the most common primary malignant brain tumors and are resistant to current therapies. Genomic analyses reveal that signature genetic lesions in GBM and LGG include copy gain and amplification of chromosome 7, amplification, mutation, and overexpression of receptor tyrosine kinases (RTK) such as EGFR, and activating mutations in components of the PI-3 kinase (PI3K) pathway. In Drosophila melanogaster, constitutive co-activation of RTK and PI3K signaling in glial progenitor cells recapitulates key features of human gliomas. This study used this Drosophila glioma model to identify death-associated protein kinase (Drak), a cytoplasmic serine/threonine kinase orthologous to the human kinase STK17A, as a downstream effector of EGFR and PI3K signaling pathways. Drak was necessary for glial neoplasia, but not for normal glial proliferation and development, and Drak cooperated with EGFR to promote glial cell transformation. Drak phosphorylated Sqh, the Drosophila ortholog of MRLC (non-muscle myosin regulatory light chain), which was necessary for transformation. Moreover, Anillin, which is a binding partner of phosphorylated Sqh, was upregulated in a Drak-dependent manner in mitotic cells and co-localized with phosphorylated Sqh in neoplastic cells undergoing mitosis and cytokinesis, consistent with their known roles in non-muscle myosin-dependent cytokinesis. These functional relationships were conserved in human GBM. The results indicate that Drak/STK17A, its substrate Sqh/MRLC and the effector Anillin/ANLN regulate mitosis and cytokinesis in gliomas. This pathway may provide a new therapeutic target for gliomas.
Ahaley, S. S. (2018). Synaptojanin regulates Hedgehog signalling by modulating phosphatidylinositol 4-phosphate levels. J Biosci 43(5): 867-876. PubMed ID: 30541947
In Hedgehog (Hh) signalling, Hh ligand concentration gradient is effectively translated into a spatially distinct transcriptional program to give precisely controlled context dependent developmental outcomes. In the absence of Hh, the receptor Patched (Ptc) inhibits the signal transducer Smoothened (Smo) by maintaining low phosphatidylinositol 4-phosphate (PI(4)P) levels. Binding of Hh to its receptor Ptc promotes PI(4)P production, which in turn activates Smo. Using wingdiscs of Drosophila melanogaster, this study shows that Synaptojanin (Synj), a dual phosphatase, modulates PI(4)P levels and affects Smo activation, and thereby functions as an additional regulatory step in the Hh pathway. Reducing the levels of Synj in the wing-discs caused enhancement of a Hh dominant gain-of-function Moonrat phenotype in the adult wings. Synj downregulation augmented Hh signalling, which was associated with elevated PI(4)P levels and Smo activation. Synj did not control the absolute pathway activity but rather fine-tuned the response since its downregulation increased expression of decapentaplegic (dpp), a low-threshold target of the pathway while the high-threshold targets remained unaffected. This is the first report that identifies Synj as a negative regulator of Hh signalling, implying its importance and an additional regulatory step in Hh signal transduction.
Camp, D., Haage, A., Solianova, V., Castle, W. M., Xu, Q. A., Lostchuck, E., Goult, B. T. and Tanentzapf, G. (2018). Direct binding of Talin to Rap1 is required for cell-ECM adhesion in Drosophila. J Cell Sci. PubMed ID: 30446511
Attachment of cells to the Extracellular Matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin is necessary for linking integrins to the cytoskeleton and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. This study shows that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies null alleles of Talin. Moreover, this study shows that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion.
Chung, H. W., Nicholas Petersen, E., Cabanos, C., Murphy, K. R., Pavel, M. A., Hansen, A. S., Ja, W. W. and Hansen, S. B. (2018). A molecular target for an alcohol chain length cutoff. J Mol Biol. PubMed ID: 30529033
Despite the widespread consumption of ethanol, mechanisms underlying its anesthetic effects remain uncertain. n-Alcohols induce anesthesia up to a specific chain length and then lose potency-an observation known as the "chain length cutoff effect". This cutoff effect is thought to be mediated by alcohol binding sites on proteins such as ion channels, but where these sites are for long chain alcohols and how they mediate a cutoff remain poorly defined. In animals, the enzyme phospholipase D (PLD) has been shown to generate alcohol metabolites (e.g., phosphatidylethanol (PEtOH)) with a cutoff, but no phenotype has been shown connecting PLD to an anesthetic effect. This study shows loss of PLD blocks ethanol-mediated hyperactivity in D. melanogaster (fruit fly), demonstrating that PLD mediates behavioral responses to alcohol in vivo. Furthermore, the metabolite PEtOH directly competes for the endogenous PLD product phosphatidic acid (PA) at lipid binding sites within potassium channels (e.g. TWIK related K(+) channel type 1 (K2P2.1, TREK-1)). This gives rise to a PLD-dependent cutoff in a TREK-1. An alcohol pathway is proposed where PLD produces lipid-alcohol metabolites that bind to and regulate downstream effector molecules including lipid-regulated potassium channels.
Bigenzahn, J. W., Collu, G. M., Kartnig, F., Pieraks, M., Vladimer, G. I., Heinz, L. X., Sedlyarov, V., Schischlik, F., Fauster, A., Rebsamen, M., Parapatics, K., Blomen, V. A., Muller, A. C., Winter, G. E., Kralovics, R., Brummelkamp, T. R., Mlodzik, M. and Superti-Furga, G. (2018). LZTR1 is a regulator of RAS ubiquitination and signaling. Science 362(6419): 1171-1177. PubMed ID: 30442766
In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper-like transcription regulator 1 (LZTR1) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the Drosophila LZTR1 ortholog CG3711 resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of LZTR1 led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). It is proposed that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because LZTR1 disease mutations failed to revert loss-of-function phenotypes, these findings provide a molecular rationale for LZTR1 involvement in a variety of inherited and acquired human disorders.

Thursday, December 20th - Evolution

Mateo, L., Rech, G. E. and Gonzalez, J. (2018). Genome-wide patterns of local adaptation in Western European Drosophila melanogaster natural populations. Sci Rep 8(1): 16143. PubMed ID: 30385770
Signatures of spatially varying selection have been investigated both at the genomic and transcriptomic level in several organisms. In Drosophila melanogaster, the majority of these studies have analyzed North American and Australian populations, leading to the identification of several loci and traits under selection. However, several studies based mainly in North American populations showed evidence of admixture that likely contributed to the observed population differentiation patterns. Thus, disentangling demography from selection might be challenging when analyzing these populations. European populations could help identify loci under spatially varying selection provided that no recent admixture from African populations would have occurred. This work has individually sequence the genome of 42 European strains collected in populations from contrasting environments: Stockholm (Sweden) and Castellana Grotte (Southern Italy). Low levels of population structure were found and no evidence was found of recent African admixture in these two populations. Patterns of spatially varying selection affecting individual genes and gene sets were examined. Besides single nucleotide polymorphisms, the role of transposable elements in local adaptation. It is concluded that European populations are a good dataset to identify candidate loci under spatially varying selection was also investigated. The analysis of the two populations sequenced in this work in the context of all the available D. melanogaster data allowed pinpointing genes and biological processes likely to be relevant for local adaptation. Identifying and analyzing populations with low levels of population structure and admixture should help to disentangle selective from non-selective forces underlying patterns of population differentiation in other species as well.
Zhang, J. Y. and Zhou, Q. (2018). On the regulatory evolution of new genes throughout their life history. Mol Biol Evol. PubMed ID: 30395322
Every gene has a birthplace and an age, i.e., a cis-regulatory environment and an evolution lifespan since its origination, yet how the two shape the evolution trajectories of genes remains unclear. This basic question was addressed by comparing phylogenetically dated new genes in the context of both their ages and origination mechanisms. In both Drosophila and vertebrates, this study confirmed a clear 'out of the testis' transition from the specifically expressed young genes to the broadly expressed old housekeeping genes, observed only in testis but not in other tissues. Many new genes have gained important functions during embryogenesis, manifested as either specific activation at maternal-zygotic transition, or different spatiotemporal expressions from their parental genes. These expression patterns are largely driven by an age-dependent evolution of cis-regulatory environment. Retrogenes are more frequently born in a pre-existing repressive regulatory domain, and are more diverged in their enhancer repertoire than the DNA-based gene duplications. During evolution, new gene duplications gradually gain active histone modifications and undergo more enhancer turnovers when becoming older, but exhibit complex trends of gaining or losing repressive histone modifications in Drosophila or vertebrates respectively. Interestingly, vertebrate new genes exhibit an 'into the testis' epigenetic transition that older genes become more likely to be co-occupied by both active and repressive ('bivalent') histone modifications specifically in testis. These results uncover the regulatory mechanisms underpinning the stepwise acquisition of novel and complex functions by new genes, and illuminate the general evolution trajectory of genes throughout their life history.
Brengdahl, M., Kimber, C. M., Maguire-Baxter, J., Malacrino, A. and Friberg, U. (2018). Genetic quality affects the rate of male and female reproductive aging differently in Drosophila melanogaster. Am Nat 192(6): 761-772. PubMed ID: 30444654
Males and females often maximize fitness by pursuing different reproductive strategies, with males commonly assumed to benefit more from increased resource allocation into current reproduction. Such investment should trade off with somatic maintenance and may explain why males frequently live shorter than females. It also predicts that males should experience faster reproductive aging. This study investigated whether reproductive aging and life span respond to condition differently in male and female Drosophila melanogaster, as predicted if sexual selection has shaped male and female resource-allocation patterns. Condition through genetic quality was manipulated by comparing individuals inbred or outbred for a major autosome. While genetic quality had a similar effect on condition in both sexes, condition had a much larger general effect on male reproductive output than on female reproductive output, as expected when sexual selection on vigor acts more strongly on males. No differences were found in reproductive aging between the sexes in low condition, but in high condition reproductive aging is relatively faster in males. No corresponding sex-specific change was found for life span. The sex difference in reproductive aging appearing in high condition was specifically due to a decreased aging rate in females rather than any change in males. These results suggest that females age slower than males in high condition primarily because sexual selection has favored sex differences in resource allocation under high condition, with females allocating relatively more toward somatic maintenance than males.
Adrion, J. R., Begun, D. J. and Hahn, M. W. (2018). Patterns of transposable element variation and clinality in Drosophila. Mol Ecol. PubMed ID: 30484926
Natural populations often exist in spatially diverse environments and may experience variation in the strength and targets of natural selection across their ranges. Drosophila provides an excellent opportunity to study the effects of spatially varying selection in natural populations, as both D. melanogaster and D. simulans live across a wide range of environments in North America. This study characterized patterns of variation in transposable elements (TEs) from six populations of D. melanogaster and nine populations of D. simulans sampled from multiple latitudes across North America. A nearly two-fold excess of TEs was found in D. melanogaster compared to to D. simulans, with this difference largely driven by TEs segregating at the lowest and highest allele frequencies. No effect of latitude was found on either total TE abundance or average TE allele frequencies in either species. Moreover, it was shown that, as a class of mutations, the most common patterns of TE variation do not coincide with the sampled latitudinal gradient, nor are they consistent with local adaptation acting on environmental differences found in the most extreme latitudes. No cline was found in ancestry for North American D. melanogaster-for either TEs or SNPs-suggesting a limited role for demography in shaping patterns of TE variation. Though little evidence is found for widespread clinality among TEs in Drosophila, this does not necessarily imply a limited role for TEs in adaptation. The need is discussed for improved models of adaptation to large-scale environmental heterogeneity, and how these might be applied to TEs.
Archer, C. R., Recker, M., Duffy, E. and Hosken, D. J. (2018). Intralocus sexual conflict can resolve the male-female health-survival paradox. Nat Commun 9(1): 5048. PubMed ID: 30487539
At any given age, men are more likely to die than women, but women have poorer health at older ages. This is referred to as the "male-female, health-survival paradox", which is not fully understood. This study provides a general solution to the paradox that relies on intralocus sexual conflict, where alleles segregating in the population have late-acting positive effects on male fitness, but negative effects on female health. Using an evolutionary modelling framework, this study showed that male-benefit, female-detriment alleles can spread if they are expressed after female reproduction stops. Support is provided for the conflict based solution using experimental Drosophila data. The results show that selecting for increased late-life male reproductive effort can increase male fitness but have a detrimental effect on female fitness. Furthermore, It was shown that late-life male fertility is negatively genetically correlated with female health. This study suggests that intralocus sexual conflict could resolve the health-survival paradox.
Liang, Q., Peng, T., Sun, B., Tu, J., Cheng, X., Tian, Y., Fan, X., Yang, D., Gaur, U. and Yang, M. (2018). Gene expression patterns determine the differential numbers of dorsocentral macrochaetes between M. domestica and D. melanogaster. Genesis. PubMed ID: 30358076
The evolutionary differences in sensory bristle patterns on the thorax of dipterans are an excellent model for studying the patterns of evolutionary development. Drosophila melanogaster has two pairs of the large bristles, called macrochaetes, in the dorsocentral (DC) region of the notum, while Musca domestica retains six DC macrochaetes. To explore possible mechanism by which these two dipteran species have different numbers of DC bristles, the corresponding protein sequences, the gene expression levels and the spatial expression patterns of five genes (scute, pnr, ush, hairy and emc) were compared for bristle development between two species. The mRNA expression of the five genes differed significantly between D. melanogaster and M. domestica. The gene expression patterns exhibited a species-specific pattern during the larval development stage. It suggests that the function of these genes has been conserved in regulating the development of macrocheates between housefly and fruit fly, whereas the gene expression levels, especially spatial expression patterns lead to species-specificity in DC bristles.

Wednesday, December 19th - Stem Cells

Andriatsilavo, M., Stefanutti, M., Siudeja, K., Perdigoto, C. N., Boumard, B., Gervais, L., Gillet-Markowska, A., Al Zouabi, L., Schweisguth, F. and Bardin, A. J. (2018). Spen limits intestinal stem cell self-renewal. PLoS Genet 14(11): e1007773. PubMed ID: 30452449
Precise regulation of stem cell self-renewal and differentiation properties is essential for tissue homeostasis. Using the adult Drosophila intestine to study molecular mechanisms controlling stem cell properties, this study identified the gene split-ends (spen) in a genetic screen as a novel regulator of intestinal stem cell fate (ISC). Spen family genes encode conserved RNA recognition motif-containing proteins that are reported to have roles in RNA splicing and transcriptional regulation. This study demonstrates that spen acts at multiple points in the ISC lineage with an ISC-intrinsic function in controlling early commitment events of the stem cells and functions in terminally differentiated cells to further limit the proliferation of ISCs. Using two-color cell sorting of stem cells and their daughters, spen-dependent changes in RNA abundance and exon usage were identified and potential key regulators were found downstream of spen. This work identifies spen as an important regulator of adult stem cells in the Drosophila intestine, provides new insight to Spen-family protein functions, and may also shed light on Spen's mode of action in other developmental contexts.
Read, R. D. (2018). Pvr receptor tyrosine kinase signaling promotes post-embryonic morphogenesis and survival of glia and neural progenitor cells in Drosophila. Development. PubMed ID: 30327326
Stem cells reside in specialized microenvironments, called niches, that regulate their development and development of their progeny. However, the development and maintenance of niches are poorly understood. In the Drosophila brain, cortex glial cells provide a niche that promotes self-renewal and proliferation of neural stem cell-like cells (neuroblasts). In the central brain, neuroblasts and their progeny control post-embryonic morphogenesis of cortex glia through PDGF-like ligands, and PDGFR receptor tyrosine kinase (RTK) signaling in cortex glia is required for expression of DE-cadherin, which sustains neuroblasts. Thus, through an RTK-dependent feed-forward loop, neuroblasts and their glial niche actively maintain each other. When the EGFR RTK is constitutively activated in cortex glia, they overexpress PDGF orthologs to stimulate autocrine PDGFR signaling, which uncouples their growth and survival from neuroblasts, and drives neoplastic glial transformation and elimination of neuroblasts. These results provide fundamental insights into glial development and niche regulation, and show that niche-neural stem cell feed-forward signaling becomes hijacked to drive neural tumorigenesis (Read, 2018).
Kim-Yip, R. P. and Nystul, T. G. (2018). Wingless promotes EGFR signaling in follicle stem cells to maintain self-renewal. Development. PubMed ID: 30389852
Adult stem cell niche boundaries must be precisely maintained to facilitate segregation of stem cell and daughter cell fates. However, the mechanisms that govern this process in epithelial tissues are not fully understood. This study investigated the relationship between two signals, Wnt and EGFR, that are necessary for self-renewal of the epithelial follicle stem cells (FSCs) in the Drosophila ovary, but must be downregulated in cells that have exited the niche to allow for differentiation. Wingless produced by inner germarial sheath (IGS) cells was shown to act over a short distance to activate Wnt signaling in FSCs, and movement across the FSC niche boundary is limited. In addition, Wnt signaling was shown to function genetically upstream of EGFR signaling by activating expression of the EGF ligand, spitz, and constitutive activation of EGFR was shown to partially rescue the self-renewal defect caused by loss of Wnt signaling. Collectively, these findings support a model in which the Wnt and EGFR pathways operate in a signaling hierarchy to promote FSC self-renewal.
Martin, J. L., Sanders, E. N., Moreno-Roman, P., Jaramillo Koyama, L. A., Balachandra, S., Du, X. and O'Brien, L. E. (2018). Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation, and loss. Elife 7. PubMed ID: 30427308
Organ renewal is governed by the dynamics of cell division, differentiation, and loss. To study these dynamics in real time, this study presents a platform for extended live imaging of the adult Drosophila midgut, a premier genetic model for stem cell-based organs. A window cut into a living animal allows the midgut to be imaged while intact and physiologically functioning. This approach prolongs imaging sessions to 12-16 hours and yields movies that document cell and tissue dynamics at vivid spatiotemporal resolution. Applying a pipeline for movie processing and analysis, this study uncover new, intriguing cell behaviors: that mitotic stem cells dynamically re-orient, that daughter cells use slow kinetics of Notch activation to reach a fate-specifying threshold, and that enterocytes extrude via ratcheted constriction of a junctional ring. By enabling real-time study of midgut phenomena that were previously inaccessible, this platform opens a new realm for dynamic understanding of adult organ renewal.
Parasram, K., Bernardon, N., Hammoud, M., Chang, H., He, L., Perrimon, N. and Karpowicz, P. (2018). Intestinal stem cells exhibit conditional circadian clock function. Stem Cell Reports 11(5): 1287-1301. PubMed ID: 30428387
The circadian clock is a molecular pacemaker that produces 24-hr physiological cycles known as circadian rhythms. How the clock regulates stem cells is an emerging area of research with many outstanding questions. This study tested clock function in vivo at the single cell resolution in the Drosophila intestine, a tissue that is exquisitely sensitive to environmental cues and has circadian rhythms in regeneration. The results indicate that circadian clocks function in intestinal stem cells and enterocytes but are downregulated during enteroendocrine cell differentiation. Drosophila intestinal cells are principally synchronized by the photoperiod, but intestinal stem cell clocks are highly responsive to signaling pathways that comprise their niche, and the Wnt and Hippo signaling pathways positively regulate stem cell circadian clock function. These data reveal that intestinal stem cell circadian rhythms are regulated by cellular signaling and provide insight as to how clocks may be altered during physiological changes such as regeneration and aging.
Bohere, J., Mancheno-Ferris, A., Al Hayek, S., Zanet, J., Valenti, P., Akino, K., Yamabe, Y., Inagaki, S., Chanut-Delalande, H., Plaza, S., Kageyama, Y., Osman, D., Polesello, C. and Payre, F. (2018). Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis. Nat Commun 9(1): 5123. PubMed ID: 30504772
To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. This study shows that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells.

Monday, December 18th - Signaling

Pieper, K. E., Unckless, R. L. and Dyer, K. A. (2018). A fast-evolving X-linked duplicate of importin-alpha2 is overexpressed in sex-ratio drive in Drosophila neotestacea. Mol Ecol. PubMed ID: 30411843
Selfish genetic elements that manipulate gametogenesis to achieve a transmission advantage are known as meiotic drivers. Sex-ratio X-chromosomes (SR) are meiotic drivers that prevent the maturation of Y-bearing sperm in male carriers to result in the production of mainly female progeny. The spread of an SR chromosome can affect host genetic diversity and genome evolution, and can even cause host extinction if it reaches sufficiently high prevalence. Meiotic drivers have evolved independently many times, though only in a few cases is the underlying genetic mechanism known. This study use a combination of transcriptomics and population genetics to identify widespread expression differences between the standard (ST) and sex-ratio (SR) X-chromosomes of the fly Drosophila neotestacea. The X-chromosome was found to be enriched for differentially expressed transcripts, and many of these X-linked differentially expressed transcripts had elevated Ka /Ks values between ST and SR, indicative of potential functional differences. A set of candidate transcripts was identified, including a testis-specific, X-linked duplicate of the nuclear transport gene importin-alpha2 that is overexpressed in SR. Suggestions were found of positive selection in the lineage leading to the duplicate, and its molecular evolutionary patterns are consistent with relaxed purifying selection in ST. As these patterns are consistent with involvement in the mechanism of drive in this species, this duplicate is a strong candidate worthy of further functional investigation. Nuclear transport may be a common target for genetic conflict, as the mechanism of the autosomal Segregation Distorter drive system in D. melanogaster involves the same pathway.
Zoranovic, T., Manent, J., Willoughby, L., Matos de Simoes, R., La Marca, J. E., Golenkina, S., Cuiping, X., Gruber, S., Angjeli, B., Kanitz, E. E., Cronin, S. J. F., Neely, G. G., Wernitznig, A., Humbert, P. O., Simpson, K. J., Mitsiades, C. S., Richardson, H. E. and Penninger, J. M. (2018). A genome-wide Drosophila epithelial tumorigenesis screen identifies Tetraspanin 29Fb as an evolutionarily conserved suppressor of Ras-driven cancer. PLoS Genet 14(10): e1007688. PubMed ID: 30325918
Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. This study devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, the function of the top hit, the Tetraspanin family member Tsp29Fb was mechanistically characterized, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. This functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion.
Ressurreicao, M., Warrington, S. and Strutt, D. (2018). Rapid disruption of Dishevelled activity uncovers an intercellular role in maintenance of Prickle in core planar polarity protein complexes. Cell Rep 25(6): 1415-1424.e1416. PubMed ID: 30403998
Planar polarity, the coordinated polarization of cells in the plane of a tissue, is important for normal tissue development and function. Proteins of the core planar polarity pathway become asymmetrically localized at the junctions between cells to form intercellular complexes that coordinate planar polarity between cell neighbors. This study combined tools to rapidly disrupt the activity of the core planar polarity protein Dishevelled, with quantitative measurements of protein dynamics and levels, and mosaic analysis, to investigate Dishevelled function in maintenance of planar polarity. Mechanistic insight is provided into the hierarchical relationship of Dishevelled with other members of the core planar polarity complex. Notably, it was shown that removal of Dishevelled in one cell causes rapid release of Prickle into the cytoplasm in the neighboring cell. This release of Prickle generates a self-propagating wave of planar polarity complex destabilization across the tissue. Thus, Dishevelled actively maintains complex integrity across intercellular junctions.
Ripp, C., Loth, J., Petrova, I., Linnemannstons, K., Ulepic, M., Fradkin, L., Noordermeer, J. and Wodarz, A. (2018). Drosophila Ror is a nervous system-specific co-receptor for Wnt ligands. Biol Open 7(11). PubMed ID: 30341100
Wnt ligands are secreted glycoproteins that control many developmental processes and are crucial for homeostasis of numerous tissues in the adult organism. Signal transduction of Wnts involves the binding of Wnts to receptor complexes at the surface of target cells. These receptor complexes are commonly formed between a member of the Frizzled family of seven-pass transmembrane proteins and a co-receptor, which is usually a single-pass transmembrane protein. Among these co-receptors are several with structural homology to receptor tyrosine kinases, including Ror, PTK7, Ryk and MUSK. In vertebrates, Ror-2 and PTK7 are important regulators of planar cell polarity (PCP). By contrast, PCP phenotypes were not reported for mutations in off-track (otk) and off-track2 (otk2), encoding the Drosophila orthologs of PTK7. This study shows that Drosophila Ror is expressed in the nervous system and localizes to the plasma membrane of perikarya and neurites. A null allele of Ror is homozygous viable and fertile, does not display PCP phenotypes and interacts genetically with mutations in otk and otk2. Ror binds specifically to Wingless (Wg), Wnt4 and Wnt5 and also to Frizzled2 (Fz2) and Otk. These findings establish Drosophila Ror as a Wnt co-receptor expressed in the nervous system.
Pesch, Y. Y., Hesse, R., Ali, T. and Behr, M. (2018). A cell surface protein controls endocrine ring gland morphogenesis and steroid production. Dev Biol. PubMed ID: 30367846
Identification of signals for systemic adaption of hormonal regulation would help to understand the crosstalk between cells and environmental cues contributing to growth, metabolic homeostasis and development. Physiological states are controlled by precise pulsatile hormonal release, including endocrine steroids in human and ecdysteroids in insects. This study shows in Drosophila that regulation of genes that control biosynthesis and signaling of the steroid hormone ecdysone, a central regulator of developmental progress, depends on the extracellular matrix protein Obstructor-A (Obst-A). Ecdysone is produced by the prothoracic gland (PG), where sensory neurons projecting axons from the brain integrate stimuli for endocrine control. By defining the extracellular surface, Obst-A promotes morphogenesis and axonal growth in the PG. This process requires Obst-A-matrix reorganization by Clathrin/Wurst-mediated endocytosis. These data identifies the extracellular matrix as essential for endocrine ring gland function, which coordinates physiology, axon morphogenesis, and developmental programs. As Obst-A and Wurst homologs are found among all arthropods, it is proposed that this mechanism is evolutionary conserved.
Nonaka, S., Kawamura, K., Hori, A., Salim, E., Fukushima, K., Nakanishi, Y. and Kuraishi, T. (2018). Characterization of Spz5 as a novel ligand for Drosophila Toll-1 receptor. Biochem Biophys Res Commun. PubMed ID: 30361090
The Drosophila Toll-1 receptor is involved in embryonic development, innate immunity, and tissue homeostasis. Currently, as a ligand for the Toll-1 receptor, only Spatzle (Spz) has been identified and characterized. It has been previously reported that Drosophila larva-derived tissue extract contains ligand activity for the Toll-1 receptor, which differs from Spz based on the observation that larval extract prepared from spz mutants possessed full ligand activity. This study demonstrates that Spz5, a member of the Spz family of proteins, functions as a ligand for the Toll-1 receptor. Processing of Spz5 by Furin protease, which is known to be important for ligand activity of Spz5 to Toll-6, is not required for its function to the Toll-1 receptor. By generating a spz5 null mutant, it was further shown that the Toll-1 ligand activity of larva-derived extract is mainly derived from Spz5. Finally, a genetic interaction was found between spz and spz5 in terms of developmental processes. This study identified a novel ligand for the Drosophila Toll-1 receptor, providing evidence that Toll-1 is a multi-ligand receptor, similar to the mammalian Toll-like receptor.

Monday, December 17th - Behavior

Monier, M., Nobel, S., Isabel, G. and Danchin, E. (2018). Effects of a sex ratio gradient on female mate-copying and choosiness in Drosophila melanogaster. Curr Zool 64(2): 251-258. PubMed ID: 30402066
In many sexually reproducing species, individuals can gather information about potential mates by observing their mating success. This behavioral pattern, that is called mate-copying, has been reported in the fruit fly Drosophila melanogaster where females choosing between 2 males of contrasting phenotypes can build a preference for males of the phenotype they previously saw being chosen by a demonstrator female. As sex ratio is known to affect mate choice, the goal of this study was to test whether mate-copying is also affected by encountered sex ratios. Thus, a gradient of sex ratio was created during demonstrations of mate-copying experiments by changing the number of females observing from a central arena six simultaneous demonstrations unfolding in six peripheral compartments of a hexagonal device. Whether the sex ratio experienced by females during demonstrations affected their choosiness (male courtship duration and double courtship rate) was also tested in subsequent mate-choice tests. Experimental male:female sex ratio during demonstrations did not affect mate-copying indices, but positively affected the proportion of both males courting the female during mate-choice tests, as well as male courtship duration, the latter potentially explaining the former relationship. As expected, the sex ratio affected female choosiness positively, and Drosophila females seem to have evolved a mate-copying ability independently of sex ratio, and a capacity to adapt their choosiness to male availability. This suggests that, as in many animal species, individuals, especially females, can adapt their mate choice depending on the current sex ratio.
Schretter, C. E., Vielmetter, J., Bartos, I., Marka, Z., Marka, S., Argade, S. and Mazmanian, S. K. (2018). A gut microbial factor modulates locomotor behaviour in Drosophila. Nature 563(7731): 402-406. PubMed ID: 30356215
While research into the biology of animal behaviour has primarily focused on the central nervous system, cues from peripheral tissues and the environment have been implicated in brain development and function. There is emerging evidence that bidirectional communication between the gut and the brain affects behaviours including anxiety, cognition, nociception and social interaction. Coordinated locomotor behaviour is critical for the survival and propagation of animals, and is regulated by internal and external sensory inputs. However, little is known about how the gut microbiome influences host locomotion, or the molecular and cellular mechanisms involved. This study reports that germ-free status or antibiotic treatment results in hyperactive locomotor behaviour in the fruit fly Drosophila melanogaster. Increased walking speed and daily activity in the absence of a gut microbiome are rescued by mono-colonization with specific bacteria, including the fly commensal Lactobacillus brevis. The bacterial enzyme xylose isomerase from L. brevis recapitulates the locomotor effects of microbial colonization by modulating sugar metabolism in flies. Notably, thermogenetic activation of octopaminergic neurons or exogenous administration of octopamine, the invertebrate counterpart of noradrenaline, abrogates the effects of xylose isomerase on Drosophila locomotion. These findings reveal a previously unappreciated role for the gut microbiome in modulating locomotion, and identify octopaminergic neurons as mediators of peripheral microbial cues that regulate motor behaviour in animals.
Schneider, J., Murali, N., Taylor, G. W. and Levine, J. D. (2018). Can Drosophila melanogaster tell who's who?. PLoS One 13(10): e0205043. PubMed ID: 30356241
Drosophila melanogaster are known to live in a social but cryptic world of touch and odours, but the extent to which they can perceive and integrate static visual information is a hotly debated topic. Some researchers fixate on the limited resolution of D. melanogaster's optics, others on their seemingly identical appearance; yet there is evidence of individual recognition and surprising visual learning in flies. This study applied machine learning and showed that individual D. melanogaster are visually distinct. The striking similarity of Drosophila's visual system to current convolutional neural networks was used to theoretically investigate D. melanogaster's capacity for visual understanding. Despite their limited optical resolution, D. melanogaster's neuronal architecture has the capability to extract and encode a rich feature set that allows flies to re-identify individual conspecifics with surprising accuracy. These experiments provide a proof of principle that Drosophila inhabit a much more complex visual world than previously appreciated.
Stahl, B. A., Peco, E., Davla, S., Murakami, K., Caicedo Moreno, N. A., van Meyel, D. J. and Keene, A. C. (2018). The taurine transporter Eaat2 functions in ensheathing glia to modulate sleep and metabolic Rate. Curr Biol. PubMed ID: 30416062
Sleep is critical for many aspects of brain function and is accompanied by brain-wide changes in the physiology of neurons and synapses. Growing evidence suggests that glial cells contribute to diverse aspects of sleep regulation, including neuronal and metabolic homeostasis. Drosophila], displays all the behavioral and physiological characteristics of sleep. This study identified Excitatory amino acid transporter 2 (Eaat2) and found that its loss from glia, but not neurons, increases sleep. Eaat2 is expressed in ensheathing glia, where Eaat2 functions during adulthood to regulate sleep. Increased sleep in Eaat2-deficient flies is accompanied by reduction of metabolic rate during sleep bouts, an indicator of deeper sleep intensity. Eaat2 is a member of the conserved EAAT family of membrane transport proteins, raising the possibility that it affects sleep by controlling the movement of ions and neuroactive chemical messengers to and from ensheathing glia. In vitro, Eaat2 is a transporter of tauriner, which promotes sleep when fed to flies]. The acute effect of taurine on sleep is abolished in Eaat2 mutant flies. Together, these findings reveal a wake-promoting role for Eaat2 in ensheathing glia through a taurine-dependent mechanism.
Yang, D. (2018). Carnivory in the larvae of Drosophila melanogaster and other Drosophila species. Sci Rep 8(1): 15484. PubMed ID: 30341324
Drosophila melanogaster is widely used as a model organism for biological investigations, and food is a major aspect of its ecology and evolutionary biology. Previous studies have shown that this insect can use fruits, yeasts and insect carcasses as its food sources. This study demonstrates that this species is an omnivore, that its larvae can exploit not only fruits and yeast but also foods of animal origin (FAOs), and that larvae consume adult carcasses regularly. FAO-fed larvae develop into adulthood within a normal developmental time frame without the help of microbes. Yeast foods are better for Drosophila development than are foods of plant origin (FPOs) or FAO because in yeast foods, more eggs complete their life cycle, and the body size of emerged flies is much greater. Flies can use a mixture of yeast-FAO, which significantly boosts female fertility. Larvae digest FAOs externally. Larval D. virilis, D. hydei, and D. simulans are also omnivorous and demonstrate the same feeding habits as larval D. melanogaster. These findings prompt a reconsideratuib if previous conclusions about the original adaptations of D. melanogaster and other Drosophila species and have direct implications for diet-related studies using Drosophila as a model organism.
Liu, T., Wang, Y., Tian, Y., Zhang, J., Zhao, J. and Guo, A. (2018). The receptor channel formed by ppk25, ppk29 and ppk23 can sense the Drosophila female pheromone 7,11-HD. Genes Brain Behav: e12529. PubMed ID: 30345606
In Drosophila, pheromones play a crucial role in regulating courtship behaviors. In males, female aphrodisiac pheromones promote male-female courtship, and male antiaphrodisiac pheromones inhibit male-male courtship. Previous studies have reported that receptor proteins belonging to the pickpocket (ppk) family, ionotropic receptor family and gustatory receptor family are required for pheromone detection and normal courtship. However, none of them has been demonstrated to be sufficient for sensing pheromones after ectopic expression in originally unresponsive cells. "M" cells are activated by male antiaphrodisiac pheromones but not female aphrodisiac pheromones, and the activated cells inhibit male-male courtship. In this study, male flies with ectopic expression of ppk25, ppk29 and ppk23 in "M" cells showed decreased male-female courtship. Using an in vivo calcium imaging approach, this study found that the "M" cells expressing these three ppks were significantly activated by the female aphrodisiac pheromone 7,11-HD. The results indicate that a sodium channel consisting, at minimum, of ppk25, ppk29 and ppk23, can sense 7,11-HD, most likely as a receptor. These findings may provided insights into the molecular mechanisms of pheromonal functions.

Friday, December 14th - Cytoskeleton and Junctions

Wineland, D. M., Kelpsch, D. J. and Tootle, T. L. (2018). Multiple pools of nuclear actin. Anat Rec (Hoboken). PubMed ID: 30312534
While nuclear actin was reported ~50 years ago, it's in vivo prevalence and structure remain largely unknown. This study used Drosophila oogenesis, i.e. follicle development, to characterize nuclear actin. Three different reagents - DNase I, anti-actin C4, and anti-actin AC15 - were found to recognize distinct pools of nuclear actin. DNase I labels monomeric or G-actin, and, during follicle development, G-actin is present in the nucleus of every cell. Some G-actin is recognized by the C4 antibody. In particular, C4 nuclear actin colocalizes with DNase I to the nucleolus in anterior escort cells, follicle stem cells, some mitotic follicle cells, and a subset of nurse cells during early oogenesis. C4 also labels polymeric nuclear actin in the nucleoplasm of the germline stem cells, early cystoblasts, and oocytes. The AC15 antibody labels a completely distinct pool of nuclear actin from that of DNase I and C4. Specifically, AC15 nuclear actin localizes to the chromatin in the nurse and follicle cells during mid-to-late oogenesis. Within the oocyte, AC15 nuclear actin progresses from localizing to puncta surrounding the DNA, to forming a filamentous cage around the chromosomes. Together these findings reveal that nuclear actin is highly prevalent in vivo, and multiple pools of nuclear actin exist and can be recognized using different reagents. Additionally, these localization studies suggest that nuclear actin may regulate stemness, nucleolar structure and function, transcription, and nuclear structure. Such findings call for further studies to explore the prevalence, diversity, and functions of nuclear actin across tissues and organisms.
Chen, J., Sayadian, A. C., Lowe, N., Lovegrove, H. E. and St Johnston, D. (2018). An alternative mode of epithelial polarity in the Drosophila midgut. PLoS Biol 16(10): e3000041. PubMed ID: 30339698
Apical-basal polarity is essential for the formation and function of epithelial tissues, whereas loss of polarity is a hallmark of tumours. Studies in Drosophila have identified conserved polarity factors that define the apical (Crumbs, Stardust, Par-6, atypical protein kinase C [aPKC]), junctional (Bazooka [Baz]/Par-3), and basolateral (Scribbled [Scrib], Discs large [Dlg], Lethal [2] giant larvae [Lgl]) domains of epithelial cells. Because these conserved factors mark equivalent domains in diverse types of vertebrate and invertebrate epithelia, it is generally assumed that this system underlies polarity in all epithelia. This study shows that this is not the case, as none of these canonical factors are required for the polarisation of the endodermal epithelium of the Drosophila adult midgut. Furthermore, like vertebrate epithelia but not other Drosophila epithelia, the midgut epithelium forms occluding junctions above adherens junctions (AJs) and requires the integrin adhesion complex for polarity. Thus, Drosophila contains two types of epithelia that polarise by fundamentally different mechanisms. This diversity of epithelial types may reflect their different developmental origins, junctional arrangement, or whether they polarise in an apical-basal direction or vice versa. Since knock-outs of canonical polarity factors in vertebrates often have little or no effect on epithelial polarity and the Drosophila midgut shares several common features with vertebrate epithelia, this diversity of polarity mechanisms is likely to be conserved in other animals.
Singh, A., Saha, T., Begemann, I., Ricker, A., Nusse, H., Thorn-Seshold, O., Klingauf, J., Galic, M. and Matis, M. (2018). Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis. Nat Cell Biol 20(10): 1126-1133. PubMed ID: 30202051
Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis. However, unlike for actin-based mechanical forces, knowledge about the contribution of forces originating from other cytoskeletal components remains scarce. This study has establish microtubules as central components of cell mechanics during tissue morphogenesis. Individual cells were found to be mechanically autonomous during early Drosophila wing epithelium development. Each cell contains a polarized apical non-centrosomal microtubule cytoskeleton that bears compressive forces, whereby acute elimination of microtubule-based forces leads to cell shortening. It was further established that the Fat planar cell polarity (Ft-PCP) signalling pathway couples microtubules at adherens junctions (AJs) and patterns microtubule-based forces across a tissue via polarized transcellular stability, thus revealing a molecular mechanism bridging single cell and tissue mechanics. Together, these results provide a physical basis to explain how global patterning of microtubules controls cell mechanics to coordinate collective cell behaviour during tissue remodelling. These results also offer alternative paradigms towards the interplay of contractile and protrusive cytoskeletal forces at the single cell and tissue levels.
Garrido-Jimenez, S., Roman, A. C., Alvarez-Barrientos, A. and Carvajal-Gonzalez, J. M. (2018). Centriole planar polarity assessment in Drosophila wings. Development. PubMed ID: 30389850
In vertebrates, planar polarization of ciliary basal bodies has been associated with actin polymerization that acts downstream of the Frizzled-planar cell polarity (Fz-PCP) pathway. In Drosophila wing epithelial cells, which do not have cilia, centrioles also polarize in a Fz-PCP dependent manner, although the relationship with actin polymerization remains unknown. By combining existing and new quantitative methods, this study unexpectedly found that known PCP effectors linked to actin polymerization phenotypes affect neither final centriole polarization nor apical centriole distribution. But actin polymerization is required upstream of Fz-PCP to maintain the centrioles in restricted areas in the apical-most planes of those epithelial cells before and after the actin-based hair is formed. Furthermore, in the absence of proper core Fz-PCP signaling, actin polymerization is insufficient to drive this off-centred centriole migration. Altogether, the results reveal that there are at least two pathways controlling centriole positioning in Drosophila pupal wings - an upstream actin-dependent mechanism involved in centriole distribution which is PCP independent, and an unknown mechanism that links core Fz-PCP and centriole polarization.
Riparbelli, M. G., Persico, V. and Callaini, G. (2018). A transient microtubule-based structure uncovers a new intrinsic asymmetry between the mother centrioles in the early Drosophila spermatocytes. Cytoskeleton (Hoboken). PubMed ID: 30381895
Parent centrioles are characterized in most organisms by individual morphological traits and have distinct asymmetries that provide different functional properties. By contrast, mother and daughter centrioles are morphologically undistinguishable during Drosophila male gametogenesis. This study reports the presence of previously unrecognized microtubule-based structures that extend into the peripheral cytoplasm of the Drosophila polar spermatocytes at the onset of the first meiosis and are positive for the typical centriolar protein Sas-4 and for the kinesin-like protein Klp10A. These structures have a short lifespan and are no longer found in early apolar spermatocytes. Remarkably, each polar spermatocyte holds only one microtubule-based structure that is associated with one of the sister centriole pairs and specifically with the mother centriole. These findings reveal an inherent asymmetry between the parent centrioles at the onset of male meiosis and also uncover unexpected functional properties between the mother centrioles of the same cells.
Yan, C., Wang, F., Peng, Y., Williams, C. R., Jenkins, B., Wildonger, J., Kim, H. J., Perr, J. B., Vaughan, J. C., Kern, M. E., Falvo, M. R., O'Brien, E. T., Superfine, R., Tuthill, J. C., Xiang, Y., Rogers, S. L. and Parrish, J. Z. (2018). Microtubule acetylation is required for mechanosensation in Drosophila. Cell Rep 25(4): 1051-1065.e1056. PubMed ID: 30355484
At the cellular level, alpha-tubulin acetylation alters the structure of microtubules to render them mechanically resistant to compressive forces. How this biochemical property of microtubule acetylation relates to mechanosensation remains unknown, although prior studies have shown that microtubule acetylation influences touch perception. This study identified the major Drosophila alpha-tubulin acetylase (dTAT/CG3967) and show that it plays key roles in several forms of mechanosensation. dTAT is highly expressed in the larval peripheral nervous system (PNS), but it is largely dispensable for neuronal morphogenesis. Mutation of the acetylase gene or the K40 acetylation site in alpha-tubulin impairs mechanical sensitivity in sensory neurons and behavioral responses to gentle touch, harsh touch, gravity, and vibration stimuli, but not noxious thermal stimulus. Finally, this study showed that dTAT is required for mechanically induced activation of NOMPC, a microtubule-associated transient receptor potential channel, and functions to maintain integrity of the microtubule cytoskeleton in response to mechanical stimulation.

Thursday, December 13th - Adult Physiology

Aw, W. C., Towarnicki, S. G., Melvin, R. G., Youngson, N. A., et al. (2018). Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness. PLoS Genet 14(11): e1007735. PubMed ID: 30399141
Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. Four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) were fed to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised), and their frequency was assayed in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. It is hypothesised that the increased physical activity diverted energy from growth and cell division and thereby slowed development. If humans respond similarly, it is posited that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson's Disease.
Mateus, P. R., Nazario-Yepiz, N. O., Ibarra-Laclette, E., Ramirez Loustalot Laclette, M. and Markow, T. A. (2018). Developmental and transcriptomal responses to seasonal dietary shifts in the cactophilic Drosophila mojavensis of North America. J Hered. PubMed ID: 30371801
Drosophila mojavensis normally breeds in necrotic columnar cactus, but they also feed and breed in Opuntia fruit (prickly pear) which serves as a seasonal resource. The prickly pear fruits are much different chemically from cacti, mainly in their free sugars and lipid content, raising the question of the effects of this seasonal shift on fitness and on gene expression. Three isofemale strains of D. mojavensis collected from different parts of the species' range were raised on semi-natural medium of either cactus or prickly pear fruit and the development time, survival, body weights and desiccation resistance were measured. All these parameters were affected by diet and by interaction with strain and or sex. Interestingly, however, there appear to be tradeoffs: flies developed faster in prickly pear and the emerging adults were heavier, but those having grown in cactus were more resistant to desiccation. Gene expression of emerging male and female adult flies was evaluated using RNA-Seq. While more genes were down-regulated in prickly pear fruit than up-regulated in both sexes, the sexes did differ in expression patterns. The majority of the genes that were preferentially expressed comparing prickly pear fruit vs cactus underlie metabolism. Genes involved with carbohydrate and lipid metabolism, as well as with the amino acid serine, and their relationship to growth and development reflect the ways in which these dietary differences affect the flies.
Erkosar, B., Yashiro, E., Zajitschek, F., Friberg, U., Maklakov, A. A., van der Meer, J. R. and Kawecki, T. J. (2018). Host diet mediates a negative relationship between abundance and diversity of Drosophila gut microbiota. Ecol Evol 8(18): 9491-9502. PubMed ID: 30377517
Nutrient supply to ecosystems has major effects on ecological diversity, but it is unclear to what degree the shape of this relationship is general versus dependent on the specific environment or community. Although the diet composition in terms of the source or proportions of different nutrient types is known to affect gut microbiota composition, the relationship between the quantity of nutrients supplied and the abundance and diversity of the intestinal microbial community remains to be elucidated. This relationship was addressed using replicate populations of Drosophila melanogaster maintained over multiple generations on three diets differing in the concentration of yeast (the only source of most nutrients). While a 6.5-fold increase in yeast concentration led to a 100-fold increase in the total abundance of gut microbes, it caused a major decrease in their alpha diversity (by 45-60% depending on the diversity measure). This was accompanied by only minor shifts in the taxonomic affiliation of the most common operational taxonomic units (OTUs). Thus, nutrient concentration in host diet mediates a strong negative relationship between the nutrient abundance and microbial diversity in the Drosophila gut ecosystem.
Zheng, H., Zhang, Y., Chen, Y., Guo, P., Wang, X., Yuan, X., Ge, W., Yang, R., Yan, Q., Yang, X. and Xi, Y. (2018). Prominin-like, a homolog of mammalian CD133, suppresses dilp6 and TOR signaling to maintain body size and weight in Drosophila. FASEB J: fj201800123R. PubMed ID: 30307770
CD133 (AC133/prominin-1) has been identified as a stem cell marker and a putative cancer stem cell marker in many solid tumors. Its biologic function and molecular mechanisms remain largely elusive. This study shows that a fly mutant for prominin-like, a homolog of mammalian CD133, shows a larger body size and excess weight accompanied with higher fat deposits as compared with the wild type. The expression levels of prominin-like are mediated by ecdysone signaling where its protein levels increase dramatically in the fat body during metamorphosis. Prominin-like mutants exhibit higher Drosophila insulin-like peptide 6 (dilp6) levels during nonfeeding stages and increased Akt/ Drosophila target of rapamycin (dTOR) signaling. On an amino acid-restricted diet, prominin-like mutants exhibit a significantly larger body size than the wild type does, similar to that which occurs upon the activation of the dTOR pathway in the fat body. These data suggest that prominin-like functions by suppressing TOR and dilp6 signaling to control body size and weight. The identification of the physiologic function of prominin-like in Drosophila may provide valuable insight into the understanding of the metabolic function of CD133 in mammals.
Mills, B. B., Thomas, A. D. and Riddle, N. C. (2018). HP1B is a euchromatic Drosophila HP1 homolog with links to metabolism. PLoS One 13(10): e0205867. PubMed ID: 30346969
Heterochromatin Protein 1 (HP1) proteins are an important family of chromosomal proteins conserved among all major eukaryotic lineages. While HP1 proteins are best known for their role in heterochromatin, many HP1 proteins function in euchromatin as well. As a group, HP1 proteins carry out diverse functions, playing roles in the regulation of gene expression, genome stability, chromatin structure, and DNA repair. While the heterochromatic HP1 proteins are well studied, knowledge of HP1 proteins with euchromatic distribution is lagging behind. This study created the first mutations in HP1B, a Drosophila HP1 protein with euchromatic function, and the Drosophila homolog most closely related to mammalian HP1alpha, HP1beta, and HP1gamma. HP1B was found to be a non-essential protein in Drosophila, with mutations affecting fertility and animal activity levels. In addition, animals lacking HP1B show altered food intake and higher body fat levels. Gene expression analysis of animals lacking HP1B demonstrates that genes with functions in various metabolic processes are affected primarily by HP1B loss. These findings suggest that there is a link between the chromatin protein HP1B and the regulation of metabolism.
Scopelliti, A., Bauer, C., Yu, Y., Zhang, T., Kruspig, B., Murphy, D. J., Vidal, M., Maddocks, O. D. K. and Cordero, J. B. (2018). A neuronal relay mediates a nutrient responsive gut/fat body axis regulating energy homeostasis in adult Drosophila. Cell Metab. PubMed ID: 30344016
The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. This study shows that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon alpha, which signals via its neuronal receptor DLgr2/Rickets. Bursicon alpha/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon alpha/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, this work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis.

Wednesday, December 12th - Disease Models

Rao, D. S., Kronert, W. A., Guo, Y., Hsu, K. H., Sarsoza, F. and Bernstein, S. I. (2018). Reductions in ATPase activity, actin sliding velocity and myofibril stability yield muscle dysfunction in Drosophila models of myosin-based Freeman Sheldon syndrome. Mol Biol Cell: mbcE18080526. PubMed ID: 30379605
Using Drosophila melanogaster the first animal models were created for myosin-based Freeman Sheldon Syndrome, a dominant form of distal arthrogryposis defined by congenital facial and distal skeletal muscle contractures. Electron microscopy of homozygous mutant indirect flight muscles showed normal (Y583S) or altered (T178I, R672C) myofibril assembly, followed by progressive disruption of the myofilament lattice. In contrast, all alleles permitted normal myofibril assembly in the heterozygous state, but caused myofibrillar disruption during aging. The severity of myofibril defects in heterozygotes correlated with the level of flight impairment. Thus the Drosophila models mimic the human condition, in that Freeman Sheldon Syndrome mutations are dominant and display varied degrees of phenotypic severity. Molecular modeling indicates that the mutations disrupt communication between the nucleotide binding site of myosin and its lever arm that drives force production. Each mutant myosin showed reduced in vitro actin sliding velocity, with the two more severe alleles significantly decreasing the catalytic efficiency of actin-activated ATP hydrolysis. The observed reductions in actin motility and catalytic efficiency may serve as the mechanistic basis of the progressive myofibrillar disarray observed in the Drosophila models as well as the prolonged contractile activity responsible for skeletal muscle contractures in Freeman Sheldon Syndrome patients.
Steyaert, J., Scheveneels, W., Vanneste, J., Van Damme, P., Robberecht, W., Callaerts, P., Bogaert, E. and Van Den Bosch, L. (2018). FUS-induced neurotoxicity in Drosophila is prevented by downregulating nucleocytoplasmic transport proteins. Hum Mol Genet. PubMed ID: 30379317
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases characterized by the progressive loss of specific groups of neurons. Due to clinical, genetic and pathological overlap, both diseases are considered as the extremes of one disease spectrum and in a number of ALS and FTD patients, fused in sarcoma (FUS) aggregates are present. Even in families with a monogenetic disease cause, a striking variability is observed in disease presentation. This suggests the presence of important modifying genes. The identification of disease-modifying genes will contribute to defining clear therapeutic targets and to understanding the pathways involved in motor neuron death. This study established a novel in vivo screening platform in which new modifying genes of FUS toxicity can be identified. Expression of human FUS induced the selective apoptosis of crustacean cardioactive peptide (CCAP) neurons from the ventral nerve cord of fruit flies. No defects in the development of these neurons were observed nor were the regulatory CCAP neurons from the brain affected. The number of CCAP neurons from the ventral nerve cord was used as an in vivo read-out for FUS toxicity in neurons. Via a targeted screen, a potent modifying role of proteins involved in nucleocytoplasmic transport was discovered. Downregulation of Nucleoporin 154 and Exportin1 (XPO1) prevented FUS-induced neurotoxicity. Moreover, XPO1 interacted with FUS. Silencing XPO1 significantly reduced the propensity of FUS to form inclusions upon stress. Taken together, these findings point to an important role of nucleocytoplasmic transport proteins in FUS-induced ALS/FTD.
Ma, P., Yun, J., Deng, H. and Guo, M. (2018). Atg1 mediated autophagy suppresses tissue degeneration in pink1/parkin mutants by promoting mitochondrial fission in Drosophila. Mol Biol Cell: mbcE18040243. PubMed ID: 30354903
Mitochondria dysfunction is considered as a hallmark of multiple neuro-degenerative diseases, including Parkinson's disease (PD). PD familial genes, Pink1 and parkin function in a conserved pathway that regulates mitochondrial function including dynamics (fusion and fission). Mammalian cell culture studies suggested that pink1/ parkin pathway promotes mitophagy (mitochondrial autophagy). Mitophagy through mitochondrial fission and auto-lysosomal recycling was considered as a quality control system in organelle level. Whether this quality control machinery involves in PD pathogenesis in vivo, remains elusive. This study found elevating autophagy by Atg1 over-expression can significantly rescue mitochondrial defects and apoptotic cell death in pink1 and parkin mutants in Drosophila. Surprisingly, the rescue effect relied both on the autophagy-lysosome machinery and on drp1, a mitochondrial fission molecule. It was further shown that Atg1 promotes mitochondrial fission by post-transcriptional increasing of Drp1 protein level. In contrast, increasing fission (by drp1 overexpression) or inhibiting fusion (by mfn knocking down) rescue pink1 mutants when lysosomal or proteasomal machinery were impaired. Taken together, these results identified atg1 as a dual function node to control mitochondrial quality by promoting mitochondria fission and autophagy, which makes it a potential therapeutic target for treatment of mitochondrial dysfunction related diseases, including PD.
Guo, J. F., et al. (2018). Coding mutations in NUS1 contribute to Parkinson's disease. Proc Natl Acad Sci U S A 115(45): 11567-11572. PubMed ID: 30348779
Whole-exome sequencing has been successful in identifying genetic factors contributing to familial or sporadic Parkinson's disease (PD). However, this approach has not been applied to explore the impact of de novo mutations on PD pathogenesis. This study sequenced the exomes of 39 early onset patients, their parents, and 20 unaffected siblings to investigate the effects of de novo mutations on PD. 12 genes were identified with de novo mutations (MAD1L1, NUP98, PPP2CB, PKMYT1, TRIM24, CEP131, CTTNBP2, NUS1, SMPD3, MGRN1, IFI35, and RUSC2), which could be functionally relevant to PD pathogenesis. Further analyses of two independent case-control cohorts (1,852 patients and 1,565 controls in one cohort and 3,237 patients and 2,858 controls in the other) revealed that NUS1 harbors significantly more rare nonsynonymous variants in PD patients than in controls. Functional studies in Drosophila demonstrated that the loss of NUS1 could reduce the climbing ability, dopamine level, and number of dopaminergic neurons in 30-day-old flies and could induce apoptosis in fly brain. Together, these data suggest that de novo mutations could contribute to early onset PD pathogenesis and identify NUS1 as a candidate gene for PD.
Xiao, C. and Qiu, S. (2018). Downregulation of EDTP in glial cells suppresses polyglutamine protein aggregates and extends lifespan in Drosophila melanogaster. Neurosci Lett. PubMed ID: 30528881
Drosophila Egg-derived tyrosine phosphatase (EDTP) is a lipid phosphatase essential for oogenesis and muscle function. Loss-of-EDTP is lethal at early developmental stages. Hypomorphic mutation of EDTP causes impaired muscle performance and shortened lifespan. Mutation of MTMR14, a mammalian homolog to EDTP, is associated with muscle fatigue in rodents and a rare genetic disease called centronuclear myopathy in humans. Despite the deleterious consequences, downregulation of MTMR14 promotes autophagy. It is proposed that selective downregulation of EDTP/MTMR14 in non-muscle tissues improves the survivorship to cellular wastes and extend lifespan. This study shows that downregulation of EDTP in glial cells suppressed the expression of polyglutamine (polyQ) protein aggregates and improved survival. Downregulation of EDTP in glial cells also extended lifespan. These effects were not observed by targeting pan-neurons in the nervous system, suggesting the significance of tissue-specificity. Additionally, flies carrying an EDTP mutant had increased survival to prolonged anoxia and altered dynamics of polyQ expression. These data supported the proposal that selective downregulation of EDTP in non-muscle tissues improved survivorship to cellular protein aggregates and extended lifespan. These findings suggest that EDTP/MTMR14 could be a novel molecular target for the treatment of neurodegeneration.
Salazar, A. M., Resnik-Docampo, M., Ulgherait, M., Clark, R. I., Shirasu-Hiza, M., Jones, D. L. and Walker, D. W. (2018). Intestinal Snakeskin limits microbial dysbiosis during aging and promotes longevity. iScience 9: 229-243. PubMed ID: 30419503
Intestinal barrier dysfunction is an evolutionarily conserved hallmark of aging, which has been linked to microbial dysbiosis, altered expression of occluding junction proteins, and impending mortality. However, the interplay between intestinal junction proteins, age-onset dysbiosis, and lifespan determination remains unclear. This study shows that altered expression of Snakeskin (Ssk), a septate junction-specific protein, can modulate intestinal homeostasis, microbial dynamics, immune activity, and lifespan in Drosophila. Loss of Ssk leads to rapid and reversible intestinal barrier dysfunction, altered gut morphology, dysbiosis, and dramatically reduced lifespan. Remarkably, restoration of Ssk expression in flies showing intestinal barrier dysfunction rescues each of these phenotypes previously linked to aging. Intestinal up-regulation of Ssk protects against microbial translocation following oral infection with pathogenic bacteria. Furthermore, intestinal up-regulation of Ssk improves intestinal barrier function during aging, limits dysbiosis, and extends lifespan. These findings indicate that intestinal occluding junctions may represent prolongevity targets in mammals.

Tuesday, December 11th - Chromatin

Pindyurin, A. V., Ilyin, A. A., Ivankin, A. V., Tselebrovsky, M. V., Nenasheva, V. V., Mikhaleva, E. A., Pagie, L., van Steensel, B. and Shevelyov, Y. Y. (2018). The large fraction of heterochromatin in Drosophila neurons is bound by both B-type lamin and HP1a. Epigenetics Chromatin 11(1): 65. PubMed ID: 30384843
In most mammalian cell lines, chromatin located at the nuclear periphery is represented by condensed heterochromatin, as evidenced by microscopy observations and DamID mapping of lamina-associated domains (LADs) enriched in dimethylated Lys9 of histone H3 (H3K9me2). However, in Kc167 cell culture, the only Drosophilla cell type where LADs have previously been mapped, they are neither H3K9me2-enriched nor overlapped with the domains of heterochromatin protein 1a (HP1a). Using cell type-specific DamID this study mapped genome-wide LADs, HP1a and Polycomb (Pc) domains from the central brain, Repo-positive glia, Elav-positive neurons and the fat body of Drosophila third instar larvae. Strikingly, contrary to Kc167 cells of embryonic origin, in neurons and, to a lesser extent, in glia and the fat body, HP1a domains appear to overlap strongly with LADs in both the chromosome arms and pericentromeric regions. Accordingly, centromeres reside closer to the nuclear lamina in neurons than in Kc167 cells. As expected, active gene promoters are mostly not present in LADs, HP1a and Pc domains. These domains are occupied by silent or weakly expressed genes with genes residing in the HP1a-bound LADs expressed at the lowest level. In various differentiated Drosophila cell types, this study has discovered the existence of peripheral heterochromatin, similar to that observed in mammals. These findings support the model that peripheral heterochromatin matures enhancing the repression of unwanted genes as cells terminally differentiate.
Lee, H. and Oliver, B. (2018). Non-canonical Drosophila X chromosome dosage compensation and repressive topologically associated domains. Epigenetics Chromatin 11(1): 62. PubMed ID: 30355339
In animals with XY sex chromosomes, X-linked genes from a single X chromosome in males are imbalanced relative to autosomal genes. To minimize the impact of genic imbalance in male Drosophila, there is a dosage compensation complex (MSL) that equilibrates X-linked gene expression with the autosomes. There are other potential contributions to dosage compensation. Hemizygous autosomal genes located in repressive chromatin domains are often derepressed. If this homolog-dependent repression occurs on the X, which has no pairing partner, then derepression could contribute to male dosage compensation. This study asked whether different chromatin states or topological associations correlate with X chromosome dosage compensation, especially in regions with little MSL occupancy. These analyses demonstrated that male X chromosome genes that are located in repressive chromatin states are depleted of MSL occupancy; however, they show dosage compensation. The genes in these repressive regions were also less sensitive to knockdown of MSL components. These results suggest that this non-canonical dosage compensation is due to the same transacting derepression that occurs on autosomes. This mechanism would facilitate immediate compensation during the evolution of sex chromosomes from autosomes. This mechanism is similar to that of C. elegans, where enhanced recruitment of X chromosomes to the nuclear lamina dampens X chromosome expression as part of the dosage compensation response in XX individuals.
Scacchetti, A., Brueckner, L., Jain, D., Schauer, T., Zhang, X., Schnorrer, F., van Steensel, B., Straub, T. and Becker, P. B. (2018). CHRAC/ACF contribute to the repressive ground state of chromatin. Life Sci Alliance 1(1): e201800024. PubMed ID: 30456345
The chromatin remodeling complexes chromatin accessibility complex and ATP-utilizing chromatin assembly and remodeling factor (ACF) combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression remains unclear. This study explored the influence of Drosophila melanogaster chromatin accessibility complex/ACF on transcription by using complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of an Acf knock-out allele showed that only lowly expressed genes are derepressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together, these results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.
Mashtalir, N., D'Avino, A. R., Michel, B. C., Luo, J., Pan, J., Otto, J. E., Zullow, H. J., McKenzie, Z. M., Kubiak, R. L., St Pierre, R., Valencia, A. M., Poynter, S. J., Cassel, S. H., Ranish, J. A. and Kadoch, C. (2018). Modular organization and assembly of SWI/SNF family chromatin remodeling complexes. Cell 175(5): 1272-1288. PubMed ID: 30343899
Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. This study elucidates the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class were uncovered. Finally, human disease-associated mutations were mapped within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.
Nazer, E., Dale, R. K., Palmer, C. and Lei, E. P. (2018). Argonaute2 attenuates active transcription by limiting RNA Polymerase II elongation in Drosophila melanogaster. Sci Rep 8(1): 15685. PubMed ID: 30356106
Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. This study shows that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, differential ChIP-seq analysis was performed for RNA Polymerase II (Pol II) upon depletion of AGO2. Remarkably, specific accumulation of the elongating but not initiating form of Pol II was found after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (NELF) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.
Raich, N., Mahmoudi, S., Emre, D. and Karess, R. E. (2018). Mad1 influences interphase nucleoplasm organization and chromatin regulation in Drosophila. Open Biol 8(10). PubMed ID: 30333236
The Drosophila Mad1 spindle checkpoint protein helps organize several nucleoplasmic components, and flies lacking Mad1 present changes in gene expression reflecting altered chromatin conformation. In interphase, checkpoint protein Mad1 is usually described as localizing to the inner nuclear envelope by binding the nucleoporin Tpr, an interaction believed to contribute to proper mitotic regulation. Whether Mad1 has other nuclear interphase functions is unknown. This study found in Drosophila that Mad1 is present in nuclei of both mitotic and postmitotic tissues. Three proteins implicated in various aspects of chromatin organization co-immunoprecipitated with Mad1 from fly embryos: Mtor/Tpr, the SUMO peptidase Ulp1 and Raf2, a subunit of a Polycomb-like complex. In primary spermatocytes, all four proteins colocalized in a previously undescribed chromatin-associated structure called here a MINT (Mad1-containing IntraNuclear Territory). MINT integrity required all four proteins. In mad1 mutant spermatocytes, the other proteins were no longer confined to chromatin domains but instead dispersed throughout the nucleoplasm. mad1 flies also presented phenotypes indicative of excessive chromatin of heterochromatic character during development of somatic tissues. Together these results suggest that Drosophila Mad1, by helping organize its interphase protein partners in the nucleoplasm, contributes to proper chromatin regulation.

Monday, December 10th - Signaling

Pareek, G., Thomas, R. E., Vincow, E. S., Morris, D. R. and Pallanck, L. J. (2018). Lon protease inactivation in Drosophila causes unfolded protein stress and inhibition of mitochondrial translation. Cell Death Discov 5: 51. PubMed ID: 30374414
Mitochondrial dysfunction is a frequent participant in common diseases and a principal suspect in aging. To combat mitochondrial dysfunction, eukaryotes have evolved a large repertoire of quality control mechanisms. One such mechanism involves the selective degradation of damaged or misfolded mitochondrial proteins by mitochondrial resident proteases, including proteases of the ATPase Associated with diverse cellular Activities (AAA(+)) family. The importance of the AAA(+) family of mitochondrial proteases is exemplified by the fact that mutations that impair their functions cause a variety of human diseases, yet knowledge of the cellular responses to their inactivation is limited. To address this matter, flies were created and characterized with complete or partial inactivation of the Drosophila matrix-localized AAA(+) protease Lon. A Lon null allele was found to confer early larval lethality and that severely reducing Lon expression using RNAi results in shortened lifespan, locomotor impairment, and respiratory defects specific to respiratory chain complexes that contain mitochondrially encoded subunits. The respiratory chain defects of Lon knockdown (Lon (KD) ) flies appeared to result from severely reduced translation of mitochondrially encoded genes. This translational defect was not a consequence of reduced mitochondrial transcription, as evidenced by the fact that mitochondrial transcripts were elevated in abundance in Lon (KD) flies. Rather, the translational defect of Lon (KD) flies appeared to be derived from sequestration of mitochondrially encoded transcripts in highly dense ribonucleoparticles. The translational defect of Lon (KD) flies was also accompanied by a substantial increase in unfolded mitochondrial proteins. Together, these findings suggest that the accumulation of unfolded mitochondrial proteins triggers a stress response that culminates in the inhibition of mitochondrial translation. This work provides a foundation to explore the underlying molecular mechanisms.
Palmer, W. H., Joosten, J., Overheul, G. J., Jansen, P. W., Vermeulen, M., Obbard, D. J. and Van Rij, R. P. (2018). Induction and suppression of NF-kappaB signalling by a DNA virus of Drosophila. J Virol. PubMed ID: 30404807
Interactions between the insect immune system and RNA viruses have been extensively studied in Drosophila, where RNA interference, NF-kappaB and JAK-STAT pathways underlie antiviral immunity. In response to RNA interference, insect viruses have convergently evolved suppressors of this pathway that act by diverse mechanisms to permit viral replication. However, interactions between the insect immune system and DNA viruses have received less attention, primarily because few Drosophila-infecting DNA virus isolates are available. This study used a recently-isolated DNA virus of Drosophila melanogaster, Kallithea virus (family Nudiviridae), to probe known antiviral immune responses and virus evasion tactics in the context of DNA virus infection. Fly mutants for RNA interference and Immune deficiency (Imd), but not Toll, pathways are more susceptible to Kallithea virus infection. The Kallithea virus-encoded protein gp83 was identified as a potent inhibitor of Toll signalling, suggesting that Toll mediates antiviral defense against Kallithea virus infection, but that it is suppressed by the virus. This study found that Kallithea virus gp83 inhibits Toll signalling through the regulation of NF-kappaB transcription factors. Furthermore, gp83 of the closely related Drosophila innubila nudivirus (DiNV) suppresses D. melanogaster Toll signalling, suggesting an evolutionary conserved function of Toll in defense against DNA viruses. Together, these results provide a broad description of known antiviral pathways in the context of DNA virus infection and identify the first Toll pathway inhibitor in a Drosophila virus, extending the known diversity of insect virus-encoded immune inhibitors.
Khan, M., Shaukat, Z., Saint, R. and Gregory, S. L. (2018). Chromosomal instability causes sensitivity to protein folding stress and ATP depletion. Biol Open 7(10). PubMed ID: 30327366
Aneuploidy -- having an unbalanced genome - is poorly tolerated at the cellular and organismal level. It gives rise to proteotoxic stress as well as a stereotypical oxidative shift which makes these cells sensitive to internal and environmental stresses. Using Drosophila as a model, this study found that protein folding stress is exacerbated by redox stress that occurs in response to ongoing changes to ploidy (chromosomal instability, CIN). If de novo nucleotide synthesis is blocked, CIN cells are dependent on a high level of lysosome function to survive. Depletion of adenosine monophosphate (AMP) synthesis enzymes led to DNA damage in CIN cells, which showed elevated activity of the DNA repair enzyme activated poly(ADP ribose) polymerase (PARP). PARP activation causes depletion of its substrate, nicotinamide adenine dinucleotide (NAD+) and subsequent loss of Adenosine Tri-Phosphate (ATP), and this study found that adding ATP or nicotinamide (a precursor in the synthesis of NAD+) could rescue the observed phenotypes. These findings provide ways to interpret, target and exploit aneuploidy, which has the potential to offer tumour-specific therapies.
Kurshakova, M. M., Nabirochkina, E. N., Georgieva, S. G. and Kopytova, D. V. (2018). TRF4, the novel TBP-related protein of Drosophila melanogaster, is concentrated at the endoplasmic reticulum and copurifies with proteins participating in the processes associated with endoplasmic reticulum. J Cell Biochem. PubMed ID: 30426565
Understanding the functions of TBP-related factors is essential for studying chromatin assembly and transcription regulation in higher eukaryotes. The novel TBP-related protein-coding gene, trf4, was described in Drosophila melanogaster. trf4 is found only in Drosophila and has likely originated in Drosophila common ancestor. TRF4 protein has a distant homology with TBP and TRF2 in the region of TBP-like domain and is evolutionarily conserved among distinct Drosophila species, which indicates its functional significance. TRF4 is widely expressed in D. melanogaster with high levels of its expression being observed in testes. Interestingly enough, TRF4 has become a cytoplasmic protein having lost nuclear localization signal sequence. TRF4 is concentrated at the endoplasmic reticulum (ER) and copurifies with the proteins participating in the ER-associated processes. It is suggested that trf4 gene is an example of homolog neofunctionalization by protein subcellular relocalization pathway, where the subcellular relocalization of gene product of duplicated gene leads to the new functions in ER-associated processes.
Ma, Z., Li, P., Hu, X. and Song, H. (2018). Polarity protein Canoe mediates overproliferation via modulation of JNK, Ras-MAPK and Hippo signalling. Cell Prolif: e12529. PubMed ID: 30328653
Over the past decade an intriguing connection between cell polarity and tumorigenesis has emerged. Multiple core components of the junction complexes that help to form and maintain cell polarity display both pro- and anti-tumorigenic functions in a context-dependent manner, with the underlying mechanisms poorly understood. With transgenic fly lines that overexpress or knock down specific signalling components, this study performed genetic analysis to investigate the precise role of the polarity protein Canoe (Cno) in tumorigenesis and the downstream pathways. Overexpression of cno simultaneously activates JNK and Ras-MEK-ERK signalling, resulting in mixed phenotypes of both overproliferation and cell death in the Drosophila wing disc. Moderate alleviation of JNK activation eliminates the effect of Cno on cell death, leading to organ overgrowth and cell migration that mimic the formation and invasion of tumours. In addition, this study found that the Hippo pathway acts downstream of JNK and Ras signalling to mediate the effect of Cno on cell proliferation. This work reveals an oncogenic role of Cno and creates a new type of Drosophila tumour model for cancer research.
Negreiros, E., Herszterg, S., Hwa, K., Camara, A., Dias, W. B., Carneiro, K., Bier, E., Todeschini, A. and Araujo, H. (2018). N-linked glycosylation restricts the function of short gastrulation to bind and shuttle BMPs. Development. PubMed ID: 30355725
Disorders of N-linked glycosylation are increasingly reported in the literature. However, targets responsible for the associated developmental and physiological defects are largely unknown. Bone Morphogenetic Proteins (BMPs) act as highly dynamic complexes to regulate several functions during development. The range and strength of BMP activity depend on interactions with glycosylated protein complexes in the extracellular milieu. This study investigated the role of glycosylation for the function of the conserved extracellular BMP antagonist Short gastrulation (Sog). Conserved N-glycosylated sites are identified, and the effect of mutating these residues on BMP pathway activity in Drosophila is described. Functional analysis reveals that loss of individual Sog glycosylation sites enhances BMP antagonism and/or increases the spatial range of Sog effects in the tissue. Mechanistically, evidence is provided that N-terminal and stem glycosylation controls extracellular Sog levels and distribution. The identification of similar residues in vertebrate Chordin proteins suggests that N-glycosylation may be an evolutionarily conserved process that adds complexity to the regulation of BMP activity.

Friday, December 7th - Transcriptional Regulation

Li, M., Ma, Z., Roy, S., Patel, S. K., Lane, D. C., Duffy, C. R. and Cai, H. N. (2018). Selective interactions between diverse STEs organize the ANT-C Hox cluster. Sci Rep 8(1): 15158. PubMed ID: 30310129
This study used the SF1 insulator in the Drosophila Antennapedia homeotic gene complex (ANT-C) as a model to study the mechanism and regulation of chromatin looping events. Previously work has shown that SF1 tethers a transient chromatin loop in the early embryo that insulates the Hox gene Sex comb reduce from the neighbor non-Hox gene fushi tarazu for their independent regulation. To further probe the functional range and connectivity of SF1, high-resolution chromosomal conformation capture (3C) was used to search for SF1 looping partners across ANT-C. This study reports here the identification of three distal SF1 Tether Elements (STEs) located in the labial, Deformed and Antennapedia Hox gene regions, extending the range of SF1 looping network to the entire complex. These novel STEs are bound by four different combinations of insulator proteins and exhibit distinct behaviors in enhancer block, enhancer-bypass and boundary functions. Significantly, the six STEs identified so far map to all but one of the major boundaries between repressive and active histone domains, underlining the functional relevance of these long-range chromatin loops in organizing the Hox complex. Importantly, SF1 selectively captured with only 5 STEs out of ~20 sites that display similar insulator binding profiles, indicating that presence of insulator proteins alone is not sufficient to determine looping events. These findings suggest that selective interaction among diverse STE insulators organize the Drosophila Hox genes in the 3D nuclear space.
Hope, C. M., Webber, J. L., Tokamov, S. A. and Rebay, I. (2018). Tuned polymerization of the transcription factor Yan limits off-DNA sequestration to confer context-specific repression. Elife 7. PubMed ID: 30412049
During development, transcriptional complexes at enhancers regulate gene expression in complex spatiotemporal patterns. To achieve robust expression without spurious activation, the affinity and specificity of transcription factor-DNA interactions must be precisely balanced. Protein-protein interactions among transcription factors are also critical, yet how their affinities impact enhancer output is not understood. The Drosophila transcription factor Yan provides a well-suited model to address this, as its function depends on the coordinated activities of two independent and essential domains: the DNA-binding ETS domain and the self-associating SAM domain. To explore how protein-protein affinity influences Yan function, mutants were engineered that increase SAM affinity over four orders of magnitude. This produced a dramatic subcellular redistribution of Yan into punctate structures, reduced repressive output and compromised survival. Cell-type specification and genetic interaction defects suggest distinct requirements for polymerization in different regulatory decisions. It is concluded that tuned protein-protein interactions enable the dynamic spectrum of complexes that are required for proper regulation.
Combs, P. A. and Fraser, H. B. (2018). Spatially varying cis-regulatory divergence in Drosophila embryos elucidates cis-regulatory logic. PLoS Genet 14(11): e1007631. PubMed ID: 30383747
Spatial patterning of gene expression is a key process in development, yet how it evolves is still poorly understood. Both cis- and trans-acting changes could participate in complex interactions, so to isolate the cis-regulatory component of patterning evolution, allele-specific spatial gene expression patterns were measured in D. melanogaster x simulans hybrid embryos. RNA-seq of cryo-sectioned slices revealed 66 genes with strong spatially varying allele-specific expression. hunchback, a major regulator of developmental patterning, had reduced expression of the D. simulans allele specifically in the anterior tip of hybrid embryos. Mathematical modeling of hunchback cis-regulation suggested a candidate transcription factor binding site variant, which was verified as causal using CRISPR-Cas9 genome editing. In sum, even comparing morphologically near-identical species surprisingly extensive spatial variation was found in gene expression, suggesting not only that development is robust to many such changes, but also that natural selection may have ample raw material for evolving new body plans via changes in spatial patterning.
Wang, Y., Cho, D. Y., Lee, H., Fear, J., Oliver, B. and Przytycka, T. M. (2018). Reprogramming of regulatory network using expression uncovers sex-specific gene regulation in Drosophila. Nat Commun 9(1): 4061. PubMed ID: 30283019
Gene regulatory networks (GRNs) describe regulatory relationships between transcription factors (TFs) and their target genes. Computational methods to infer GRNs typically combine evidence across different conditions to infer context-agnostic networks. A method, Network Reprogramming using EXpression (NetREX), has been developed that constructs a context-specific GRN given context-specific expression data and a context-agnostic prior network. NetREX remodels the prior network to obtain the topology that provides the best explanation for expression data. Because NetREX utilizes prior network topology, PriorBoost, a method that evaluates a prior network in terms of its consistency with the expression data was developed. NetREX and PriorBoost were validated using the "gold standard" E. coli GRN from the DREAM5 network inference challenge; they were applied to construct sex-specific Drosophila GRNs. NetREX constructed sex-specific Drosophila GRNs that, on all applied measures, outperform networks obtained from other methods indicating that NetREX is an important milestone toward building more accurate GRNs.
Jishage, M., Yu, X., Shi, Y., Ganesan, S. J., Chen, W. Y., Sali, A., Chait, B. T., Asturias, F. J. and Roeder, R. G. (2018). Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1. Nat Struct Mol Biol 25(9): 859-867. PubMed ID: 30190596
Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. This study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function.
Nagy, O., Nuez, I., Savisaar, R., Peluffo, A. E., Yassin, A., Lang, M., Stern, D. L., Matute, D. R., David, J. R. and Courtier-Orgogozo, V. (2018). Correlated evolution of two copulatory organs via a single cis-regulatory nucleotide change. Curr Biol 28(21): 3450-3457.e3413. PubMed ID: 30344115
Diverse traits often covary between species. The possibility that a single mutation could contribute to the evolution of several characters between species is rarely investigated as relatively few cases are dissected at the nucleotide level. Drosophila santomea has evolved additional sex comb sensory teeth on its legs and has lost two sensory bristles on its genitalia. Evidence is presented that a single nucleotide substitution in an enhancer of the scute gene contributes to both changes. The mutation alters a binding site for the Hox protein Abdominal-B in the developing genitalia, leading to bristle loss, and for another factor in the developing leg, leading to bristle gain. This study suggests that morphological evolution between species can occur through a single nucleotide change affecting several sexually dimorphic traits.

Thursday, December 6th - RNA

van den Beek, M., da Silva, B., Pouch, J., Ali Chaouche, M. E. A., Carre, C. and Antoniewski, C. (2018). Dual-layer transposon repression in heads of Drosophila melanogaster. RNA. PubMed ID: 30217866
piRNA-mediated repression of transposable elements (TE) in the germline limits the accumulation of heritable mutations caused by their transposition in the genome. It is not clear whether the piRNA pathway plays a functional role in adult, non-gonadal tissues in Drosophila melanogaster. To address this question, the small RNA content of adult Drosophila melanogaster heads was analyzed. Varying amount of piRNA-sized, ping-pong positive molecules in heads correlates with contamination by gonadal tissue during RNA extraction, suggesting that most of piRNAs detected in head sequencing libraries originate from gonads. The heads of wild type and piwi mutants were sequenced to address whether piwi loss of function would affect the low amount of piRNA-sized, ping-pong negative molecules that are still detected in heads hand-checked to avoid gonadal contamination. Loss of piwi was found to not affect significantly these 24-28 RNA molecules. Instead, increased siRNA levels were found against the majority of Drosophila transposable element families. To determine the effect of this siRNA level change on transposon expression, the transcriptome of wild type, piwi, dicer-2 and piwi, dicer-2 double-mutant fly heads was sequenced. RNA expression levels of the majority of TE families in piwi or dicer-2 mutants remain unchanged and that TE transcript abundance increases significantly only in piwi, dicer-2 double-mutants. These results lead to the suggestion of a dual-layer model for TE repression in adult somatic tissues. Piwi-mediated transcriptional gene silencing (TGS) established during embryogenesis constitutes the first layer of TE repression whereas Dicer-2-dependent siRNA-mediated post-transcriptional gene silencing (PTGS) provide a backup mechanism to repress TEs that escape silencing by piwi-mediated TGS.
Kelleher, E. S., Jaweria, J., Akoma, U., Ortega, L. and Tang, W. (2018). QTL mapping of natural variation reveals that the developmental regulator bruno reduces tolerance to P-element transposition in the Drosophila female germline. PLoS Biol 16(10): e2006040. PubMed ID: 30376574
Transposable elements (TEs) are obligate genetic parasites that propagate in host genomes by replicating in germline nuclei, thereby ensuring transmission to offspring. Resistance to TE propagation is enacted by germline-specific small-RNA-mediated silencing pathways, such as the Piwi-interacting RNA (piRNA) pathway, and is studied extensively. However, it remains entirely unknown whether host genomes may also evolve tolerance by desensitizing gametogenesis to the harmful effects of TEs. In part, the absence of research on tolerance reflects a lack of opportunity, as small-RNA-mediated silencing evolves rapidly after a new TE invades, thereby masking existing variation in tolerance. This study has exploited the recent historical invasion of the Drosophila melanogaster genome by P-element DNA transposons in order to study tolerance of TE activity. In the absence of piRNA-mediated silencing, the genotoxic stress imposed by P-elements disrupts oogenesis and, in extreme cases, leads to atrophied ovaries that completely lack germline cells. By performing quantitative trait locus (QTL) mapping on a panel of recombinant inbred lines (RILs) that lack piRNA-mediated silencing of P-elements, multiple QTL were uncovered that are associated with differences in tolerance of oogenesis to P-element transposition. The most significant QTL was localized to a small 230-kb euchromatic region, with the logarithm of the odds (LOD) peak occurring in the bruno locus, which codes for a critical and well-studied developmental regulator of oogenesis. Genetic, cytological, and expression analyses suggest that bruno dosage modulates germline stem cell (GSC) loss in the presence of P-element activity. These observations reveal segregating variation in TE tolerance for the first time, and implicate gametogenic regulators as a source of tolerant variants in natural populations.
Lee, M., Nguyen, T. M. T. and Kim, K. (2018). In-depth study of lin-28 suggests selectively conserved let-7 independent mechanism in Drosophila. Gene. PubMed ID: 30415010
Lin-28 is a conserved RNA-binding protein that is involved in a wide range of developmental processes and pathogenesis. At the molecular level, Lin-28 blocks the maturation of let-7 and regulates translation of certain mRNA targets. In Drosophila, Lin-28 is reported to play a role in oogenesis, muscle formation, and the symmetric division of adult intestinal stem cells. This study characterized Drosophila Lin-28 through a detailed examination of its temporal and spatial expression. Lin-28 is specifically expressed in embryonic nervous and cardiac systems. However, loss or gain of lin-28 function does not cause any abnormality during embryonic development. Instead, the ubiquitous overexpression of Lin-28 leads to lethality from late larval stage to pupal stage, and eye-specific overexpression causes severe cell loss. The ectopic expression of human Lin28A has the same effect as Drosophila Lin-28, indicating functional conservation in Lin-28 orthologs. The effect of Lin-28 on let-7 biogenesis was examined through the mutant and overexpression analysis. Lin-28 does not block the production of let-7 in Drosophila, which suggests the let-7 independent pathway as a molecular mechanism of Lin-28.
Grobler, Y., Yun, C. Y., Kahler, D. J., Bergman, C. M., Lee, H., Oliver, B. and Lehmann, R. (2018). Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation. PLoS Pathog 14(11): e1007445. PubMed ID: 30422992
Infection by the bacterium Wolbachia provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. Genome-wide RNAi screening was coupled with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. A diverse set of host gene networks was identified to regulate Wolbachia levels and unexpectedly revealed that perturbations of host translation components such as the ribosome and translation initiation factors results in increased Wolbachia levels both in vitro using RNAi and in vivo using mutants and a chemical-based translation inhibition assay. This work provides evidence for Wolbachia-host translation interaction and strengthens general understanding of the Wolbachia-host intracellular relationship.
Zhu, L., Kandasamy, S. K., Liao, S. E. and Fukunaga, R. (2018). LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes. Nat Commun 9(1): 4031. PubMed ID: 30279526
Post-transcriptional regulation of gene expression plays an essential role during oocyte maturation. This study reports that Drosophila MARF1 (Meiosis Regulator And mRNA Stability Factor 1), which consists of one RNA-recognition motif and six tandem LOTUS domains with unknown molecular function, is essential for oocyte maturation. When tethered to a reporter mRNA, MARF1 post-transcriptionally silences reporter expression by shortening reporter mRNA poly-A tail length and thereby reducing reporter protein level. This activity is mediated by the MARF1 LOTUS domain, which binds the CCR4-NOT deadenylase complex. MARF1 binds cyclin A mRNA and shortens its poly-A tail to reduce Cyclin A protein level during oocyte maturation. This study identifies MARF1 as a regulator in oocyte maturation and defines the conserved LOTUS domain as a post-transcriptional effector domain that recruits CCR4-NOT deadenylase complex to shorten target mRNA poly-A tails and suppress their translation.
Zhang, G., Tu, S., Yu, T., Zhang, X. O., Parhad, S. S., Weng, Z. and Theurkauf, W. E. (2018). Co-dependent assembly of Drosophila piRNA precursor complexes and piRNA cluster heterochromatin. Cell Rep 24(13): 3413-3422. PubMed ID: 30257203
In Drosophila, the piRNAs that guide germline transposon silencing are produced from heterochromatic clusters marked by the HP1 homolog Rhino. Rhino is shown to promotes cluster transcript association with UAP56 and the THO complex, forming RNA-protein assemblies that are unique to piRNA precursors. UAP56 and THO are ubiquitous RNA-processing factors, and null alleles of uap56 and the THO subunit gene tho2 are lethal. However, uap56(sz15) and mutations in the THO subunit genes thoc5 and thoc7 are viable but sterile and disrupt piRNA biogenesis. The uap56(sz15) allele reduces UAP56 binding to THO, and the thoc5 and thoc7 mutations disrupt interactions among the remaining THO subunits and UAP56 binding to the core THO subunit Hpr1. These mutations also reduce Rhino binding to clusters and trigger Rhino binding to ectopic sites across the genome. Rhino thus promotes assembly of piRNA precursor complexes, and these complexes restrict Rhino at cluster heterochromatin.

Wednesday, December 5th - Behavior

Lam, V. H., Li, Y. H., Liu, X., Murphy, K. A., Diehl, J. S., Kwok, R. S. and Chiu, J. C. (2018). CK1alpha collaborates with DOUBLETIME to regulate PERIOD function in the Drosophila circadian clock. J Neurosci. PubMed ID: 30373768
The animal circadian timing system interprets environmental time cues and internal metabolic status to orchestrate circadian rhythms of physiology, allowing animals to perform necessary tasks in a time-of-day dependent manner. In Drosophila, PERIOD (PER), TIMELESS (TIM), CLOCK (CLK), and CYCLE (CYC) are core clock proteins that function in a transcriptional-translational feedback mechanism to regulate the circadian transcriptome. Post-translational modifications of core clock proteins provide precise temporal control over when they are active as regulators of clock-controlled genes. In particular, phosphorylation is a key regulatory mechanism that dictates the subcellular localization, stability, and transcriptional activity of clock proteins. Previously, Casein kinase 1alpha (CK1alpha) was identified as a kinase that phosphorylates mammalian PER1 and modulates its stability, but the mechanisms by which it modulates PER protein stability is still unclear. Using Drosophila as a model, this study shows that CK1alpha has an overall function of speeding up PER metabolism, and is required to maintain the 24-hour period of circadian rhythms. The results indicate that CK1alpha collaborates with the key clock kinase DOUBLETIME (DBT) in both the cytoplasm and the nucleus to regulate the timing of PER-dependent repression of the circadian transcriptome. Specifically, it was observed that CK1alpha promotes PER nuclear localization by antagonizing the activity of DBT to inhibit PER nuclear translocation. Furthermore, CK1alpha enhances DBT-dependent PER phosphorylation and degradation once PER moves into the nucleus.
Grabowska, M. J., Steeves, J., Alpay, J., van de Poll, M., Ertekin, D. and van Swinderen, B. (2018). Innate visual preferences and behavioral flexibility in Drosophila. J Exp Biol. PubMed ID: 30322983
Visual decision-making in animals is influenced by innate preferences as well as experience. Interaction between hard-wired responses and changing motivational states determines whether a visual stimulus is attractive, aversive, or neutral. It is however difficult to separate the relative contribution of nature versus nurture in experimental paradigms, especially for more complex visual parameters such as the shape of objects. Yhis study used a closed-loop virtual reality paradigm for walking Drosophila flies to uncover innate visual preferences for the shape and size of objects, in a recursive choice scenario allowing the flies to reveal their visual preferences over time. Drosophila flies were found to display a robust attraction / repulsion profile for a range of objects sizes in this paradigm, and that this visual preference profile remains evident under a variety of conditions and persists into old age. A level of flexibility in this behavior was also demonstrated: innate repulsion to certain objects could be transiently overridden if these were novel, although this effect was only evident in younger flies. Finally, a neuromodulatory circuit in the fly brain, Drosophila neuropeptide F (dNPF), can be recruited to guide visual decision-making. Optogenetic activation of dNPF-expressing neurons converted a visually repulsive object into a more attractive object. This suggests that dNPF activity in the Drosophila brain guides ongoing visual choices, to override innate preferences and thereby provide a necessary level of behavioral flexibility in visual decision-making.
Kutta, R. J., Archipowa, N. and Scrutton, N. S. (2018). The sacrificial inactivation of the blue-light photosensor cryptochrome from Drosophila melanogaster. Phys Chem Chem Phys. PubMed ID: 30417904
Drosophila melanogaster Cryptochrome functions as the primary blue-light receptor that mediates circadian photo entrainment. Absorption of a photon leads to reduction of the protein-bound FAD via consecutive electron transfer along a conserved tryptophan tetrad resembling the signalling state required for conformational changes and induction of subsequent signalling cascades. However, how the initial photochemistry and subsequent dark processes leading to downstream signalling are linked to each other at the molecular level is still poorly understood. This study investigated in detail the initial photochemical events in DmCRY by time-resolved and stationary absorption spectroscopy combined with quantum chemical and molecular dynamics calculations. The early events along the conserved tryptophan tetrad and the final deprotonation of the terminal tryptophanyl radical cation were resolved. These initial events lead to conformational changes, such as the known C-terminal tail release, Trp decomposition, and finally FAD release providing evidence that DmCRY does not undergo a photocycle. It is proposed that light is a negative regulator of DmCRY stability even under in vitro conditions where the proteasomal machinery is missing, that is in line with its biological function, i.e. entrainment of the circadian clock.
Kirszenblat, L., Ertekin, D., Goodsell, J., Zhou, Y., Shaw, P. J. and van Swinderen, B. (2018). Sleep regulates visual selective attention in Drosophila. J Exp Biol. PubMed ID: 30355611
Although sleep deprivation is known to impair attention in humans and other mammals, the underlying reasons are not well understood, and whether similar effects are present in non-mammalian species is not known. This study sought to investigate whether sleep is important for optimizing attention in an invertebrate species, the genetic model Drosophila melanogaster. A high-throughput paradigm was developed to measure visual attention in freely-walking Drosophila, using competing foreground/background visual stimuli. Whereas sleep-deprived flies could respond normally to either stimulus alone, they were more distracted by background cues in a visual competition task. Other stressful manipulations such as starvation, heat exposure, and mechanical stress had no effects on visual attention in this paradigm. In contrast to sleep deprivation, providing additional sleep using the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP) did not affect attention in wild-type flies, but specifically improved attention in the learning mutant dunce. These results reveal a key function of sleep in optimizing attention processes in Drosophila, and establish a behavioral paradigm that can be used to explore the molecular mechanisms involved.
Currier, T. A. and Nagel, K. I. (2018). Multisensory control of orientation in tethered flying Drosophila. Curr Biol. PubMed ID: 30393038
A longstanding goal of systems neuroscience is to quantitatively describe how the brain integrates sensory cues over time. This study developed a closed-loop orienting paradigm in Drosophila to study the algorithms by which cues from two modalities are integrated during ongoing behavior. Flies exhibit two behaviors when presented simultaneously with an attractive visual stripe and aversive wind cue. First, flies perform a turn sequence where they initially turn away from the wind but later turn back toward the stripe, suggesting dynamic sensory processing. Second, turns toward the stripe are slowed by the presence of competing wind, suggesting summation of turning drives. A model is developed in which signals from each modality are filtered in space and time to generate turn commands and then summed to produce ongoing orienting behavior. This computational framework correctly predicts behavioral dynamics for a range of stimulus intensities and spatial arrangements.
Harbison, S. T., Kumar, S., Huang, W., McCoy, L. J., Smith, K. R. and Mackay, T. F. C. (2018). Genome-wide association study of circadian behavior in Drosophila melanogaster. Behav Genet. PubMed ID: 30341464
Circadian rhythms influence physiological processes from sleep-wake cycles to body temperature and are controlled by highly conserved cycling molecules. Although the mechanistic basis of the circadian clock has been known for decades, the extent to which circadian rhythms vary in nature and the underlying genetic basis for that variation is not well understood. This study measured circadian period (T) and rhythmicity index in the Drosophila Genetic Reference Panel (DGRP) and observed extensive genetic variation in both. Seven DGRP lines had sexually dimorphic arrhythmicity and one line had an exceptionally long T. Genome-wide analyses identified 584 polymorphisms in 268 genes. Differences were observed among transcripts for nine genes predicted to interact among themselves and canonical clock genes in the long period line and a control. Mutations/RNAi knockdown targeting these genes also affected circadian behavior. These observations reveal that complex genetic interactions influence high levels of variation in circadian phenotypes.

Tuesday, December 2nd - CNS Development

Yin, J., Gibbs, M., Long, C., Rosenthal, J., Kim, H. S., Kim, A., Sheng, C., Ding, P., Javed, U. and Yuan, Q. (2018). Transcriptional regulation of lipophorin receptors supports neuronal adaptation to chronic elevations of activity. Cell Rep 25(5): 1181-1192. PubMed ID: 30380410
Activity-dependent modifications strongly influence neural development. However, molecular programs underlying their context and circuit-specific effects are not well understood. To study global transcriptional changes associated with chronic elevation of synaptic activity, cell-type-specific transcriptome profiling was performed of Drosophila ventral lateral neurons (LNvs) in the developing visual circuit, and activity-modified transcripts were identified that are enriched in neuron morphogenesis, circadian regulation, and lipid metabolism and trafficking. Using bioinformatics and genetic analyses, activity-induced isoform-specific upregulation was validated of Drosophila lipophorin receptors LpR1 and LpR2, the homologs of mammalian low-density lipoprotein receptor (LDLR) family proteins. Furthermore, these morphological and physiological studies uncovered critical functions of neuronal lipophorin receptors (LpRs) in maintaining the structural and functional integrities in neurons challenged by chronic elevations of activity. Together, these findings identify LpRs as molecular targets for activity-dependent transcriptional regulation and reveal the functional significance of cell-type-specific regulation of neuronal lipid uptake in experience-dependent plasticity and adaptive responses.
Kwon, M. J., Han, M. H., Bagley, J. A., Hyeon, D. Y., Ko, B. S., Lee, Y. M., Cha, I. J., Kim, S. Y., Kim, D. Y., Kim, H. M., Hwang, D., Lee, S. B. and Jan, Y. N. (2018). Coiled-coil structure-dependent interactions between polyQ proteins and Foxo lead to dendrite pathology and behavioral defects. Proc Natl Acad Sci U S A 115(45): E10748-e10757. PubMed ID: 30348793
Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. The molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. This study has demonstrated that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in Drosophila neurons. To functionally characterize coiled-coil structures in the Q repeat regions, three structural variants of SCA3 polyQ proteins were generated. Through comparative analysis of these variants, it was found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in Drosophila dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in Drosophila These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.
Chen, C. L., Hermans, L., Viswanathan, M. C., Fortun, D., Aymanns, F., Unser, M., Cammarato, A., Dickinson, M. H. and Ramdya, P. (2018). Imaging neural activity in the ventral nerve cord of behaving adult Drosophila. Nat Commun 9(1): 4390. PubMed ID: 30348941
To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because limb premotor and motor circuits have been largely inaccessible for large-scale recordings in intact, moving animals-a constraint that is true for both vertebrate and invertebrate models. This study introduces a method for 2-photon functional imaging from the ventral nerve cord (VNC) of behaving adult Drosophila melanogaster. This method was used to reveal patterns of activity across nerve cord populations during grooming and walking and to uncover the functional encoding of moonwalker ascending neurons (MANs), moonwalker descending neurons (MDNs), and a previously uncharacterized class of locomotion-associated A1 descending neurons. Finally, a genetic reagent was developed to destroy the indirect flight muscles and to facilitate experimental access to the VNC. Taken together, these approaches enable the direct investigation of circuits associated with complex limb movements.
Berry, J. A., Phan, A. and Davis, R. L. (2018). Dopamine neurons mediate learning and forgetting through bidirectional modulation of a memory trace. Cell Rep 25(3): 651-662. PubMed ID: 30332645
It remains unclear how memory engrams are altered by experience, such as new learning, to cause forgetting. This study reports that short-term aversive memory in Drosophila is encoded by and retrieved from the mushroom body output neuron MBOn-γ2α'1. Pairing an odor with aversive electric shock creates a robust depression in the calcium response of MBOn-gamma2alpha'1 and increases avoidance to the paired odor. Electric shock after learning, which activates the cognate dopamine neuron DAn-γ2α'1, restores the response properties of MBOn-γ2α'1 and causes behavioral forgetting. Conditioning with a second odor restores the responses of MBOn-γ2α'1 to a previously learned odor while depressing responses to the newly learned odor, showing that learning and forgetting can occur simultaneously. Moreover, optogenetic activation of DAn-γ2α'1 is sufficient for the bidirectional modulation of MBOn-γ2α'1 response properties. Thus, a single DAn can drive both learning and forgetting by bidirectionally modulating a cellular memory trace.
Heras, F. J. H., Vahasoyrinki, M. and Niven, J. E. (2018). Modulation of voltage-dependent K+ conductances in photoreceptors trades off investment in contrast gain for bandwidth. PLoS Comput Biol 14(11): e1006566. PubMed ID: 30399147
Modulation is essential for adjusting neurons to prevailing conditions and differing demands. Yet understanding how modulators adjust neuronal properties to alter information processing remains unclear, as is the impact of neuromodulation on energy consumption. This study combine two computational models, one Hodgkin-Huxley type and the other analytic, to investigate the effects of neuromodulation upon Drosophila melanogaster photoreceptors. Voltage-dependent K+ conductances in these photoreceptors: (i) activate upon depolarisation to reduce membrane resistance and adjust bandwidth to functional requirements; (ii) produce negative feedback to increase bandwidth in an energy efficient way; (iii) produce shunt-peaking thereby increasing the membrane gain bandwidth product; and (iv) inactivate to amplify low frequencies. Through their effects on the voltage-dependent K+ conductances, three modulators, serotonin, calmodulin and PIP2, trade-off contrast gain against membrane bandwidth. Serotonin shifts the photoreceptor performance towards higher contrast gains and lower membrane bandwidths, whereas PIP2 and calmodulin shift performance towards lower contrast gains and higher membrane bandwidths. These neuromodulators have little effect upon the overall energy consumed by photoreceptors, instead they redistribute the energy invested in gain versus bandwidth. This demonstrates how modulators can shift neuronal information processing within the limitations of biophysics and energy consumption.
Horne, J. A., Langille, C., McLin, S., Wiederman, M., Lu, Z., Xu, C. S., Plaza, S. M., Scheffer, L. K., Hess, H. F. and Meinertzhagen, I. A. (2018). A resource for the Drosophila antennal lobe provided by the connectome of glomerulus VA1v. Elife 7. PubMed ID: 30382940
Using focused ion beam-scanning electron microscope (FIB-SEM) this study reports the entire synaptic connectome of glomerulus VA1v of the right antennal lobe in Drosophila melanogaster. Within the glomerulus all neurons were densely reconstructed, including hitherto elusive local interneurons. The fruitless-positive, sexually dimorphic VA1v included >11,140 presynaptic sites with ~38,050 postsynaptic dendrites. These connected input olfactory receptor neurons (ORNs, 51 ipsilateral, 56 contralateral), output projection neurons (18 PNs), and local interneurons (56 of >150 previously reported LNs). ORNs are predominantly presynaptic and PNs predominantly postsynaptic; newly reported LN circuits are largely an equal mixture and confer extensive synaptic reciprocity, except the newly reported LN2V with input from ORNs and outputs mostly to monoglomerular PNs, however. PNs were more numerous than previously reported from genetic screens, suggesting that the latter failed to reach saturation. A matrix is reported of 192 bodies each having >50 connections; these form 88% of the glomerulus' pre/postsynaptic sites.

Monday, December 3rd. - Gonads

Sepil, I., Hopkins, B. R., Dean, R., Thezenas, M. L., Charles, P. D., Konietzny, R., Fischer, R., Kessler, B. and Wigby, S. (2018). Quantitative proteomics identification of seminal fluid proteins in male Drosophila melanogaster. Mol Cell Proteomics. PubMed ID: 30287546
Seminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and particularly in insects, modifying female physiology and behaviour. This study presents a new and complementary method, which provides added sensitivity to Sfp identification. Label-free quantitative proteomics were applied to Drosophila melanogaster male reproductive tissue - where Sfps are unprocessed, and highly abundant - and Sfps quantified before and immediately after mating, to infer those transferred during copulation. Female reproductive tracts were also analyzed immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. These data were consistent with 125 previously reported Sfps. This study found nine high-confidence novel candidate Sfps, which were both depleted in mated versus unmated males and identified within the reproductive tract of mated but not virgin females. 42 more candidates were identified that are likely Sfps based on their abundance, known expression and predicted characteristics, and this study revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for these candidate Sfps were lower than for previously identified Sfps, supporting the idea that this technique provides a deeper analysis of the Sfp proteome than previous studies. These results demonstrate a novel, high-sensitivity approach to the analysis of seminal fluid proteomes, whose application will further the understanding of reproductive biology.
Vedelek, V., Bodai, L., Grezal, G., Kovacs, B., Boros, I. M., Laurinyecz, B. and Sinka, R. (2018). Analysis of Drosophila melanogaster testis transcriptome. BMC Genomics 19(1): 697. PubMed ID: 30249207
The formation of matured and individual sperm involves a series of molecular and spectacular morphological changes of the developing cysts in Drosophila melanogaster testis. Recent advances in RNA Sequencing (RNA-Seq) technology help understanding the complexity of eukaryotic transcriptomes by dissecting different tissues and developmental stages of organisms. To gain a better understanding of cellular differentiation of spermatogenesis, RNA-Seq was applied to analyse the testis-specific transcriptome, including coding and non-coding genes. Three different parts of the wild-type testis were isolated by dissecting and cutting the different regions: 1.) the apical region, which contains stem cells and developing spermatocytes 2.) the middle region, with enrichment of meiotic cysts 3.) the basal region, which contains elongated post-meiotic cysts with spermatids. Total RNA was isolated from each region and analysed by next-generation sequencing. Data was collected from the annotated 17412 Drosophila genes and 5381 genes were identified with significant transcript accumulation differences between the regions, representing the main stages of spermatogenesis. The presence and region specific distribution is presented of 2061 lncRNAs in testis, with 203 significant differences. Using the available modENCODE RNA-Seq data, the tissue specificity indices was determined of Drosophila genes. Combining the indices with the current results, genes were identified with region-specific enrichment in testis. Searchable visualizations are presented that can facilitate the identification of new components that play role in the organisation and composition of different stages of spermatogenesis, including the less known, but complex regulation of post-meiotic stages.
Takahashi, K. H., Ishimori, M. and Iwata, H. (2018). HSP90 as a global genetic modifier for male genital morphology in Drosophila melanogaster. Evolution. PubMed ID: 30221481
The molecular chaperone protein HSP90 has been proposed to modulate genotype-phenotype relationship in a broad range of organisms. This study explores the proposed genetic modifier effect of HSP90 through a genomewide analysis. HSP90 is shown to function as a genetic modifier of genital morphology in Drosophila melanogaster. A large number of single-nucleotide polymorphisms (SNPs) with an HSP90-dependent effect were identfied by using genome wide association analysis. The SNPs were clasified into the ones under capacitance effect (smaller allelic effect under HSP90 inhibition) or the ones under potentiation effect (larger allelic effect under HSP90 inhibition). Although the majority of SNPs are under capacitance, there are a large number of SNPs under potentiation. This observation provides support for a model in which Hsp90 is not described exclusively as a "genetic capacitor," but is described more broadly as a "genetic modifier." Because the majority of the candidate genes estimated from SNPs with an HSP90-dependent effect in the current study has never been reported to interact with HSP90 directly, the global genetic modifier effect of HSP90 may be exhibited through epistatic interactions in gene regulatory networks.
Eagle, W. V. I., Yeboah-Kordieh, D. K., Niepielko, M. G. and Gavis, E. R. (2018). Distinct cis-acting elements mediate targeting and clustering of Drosophila polar granule mRNAs. Development. PubMed ID: 30333216
Specification and development of Drosophila germ cells depend on molecular determinants within the germ plasm, a specialized cytoplasmic domain at the posterior of the embryo. Localization of numerous mRNAs to the germ plasm occurs by their incorporation, as single-transcript ribonucleoprotein (RNP) particles, into complex RNP granules called polar granules. Incorporation of mRNAs into polar granules is followed by recruitment of additional like-transcripts to form discrete homotypic clusters. The cis-acting localization signals that target mRNAs to polar granules and promote homotypic clustering remain largely uncharacterized. This study shows that the polar granule component (pgc) and germ cell-less (gcl) 3' untranslated regions contain complex localization signals comprising multiple, independently weak and partially functionally redundant localization elements (LEs). Targeting of pgc to polar granules and self-assembly into homotypic clusters are functionally separable processes mediated by distinct classes of LEs. A sequence motif shared by other polar granule mRNAs was identifed that contributes to homotypic clustering. These results suggest that mRNA localization signal complexity may be a feature required by the targeting and self-recruitment mechanism that drives germ plasm mRNA localization.
Krishnakumar, P., Riemer, S., Perera, R., Lingner, T., Goloborodko, A., Khalifa, H., Bontems, F., Kaufholz, F., El-Brolosy, M. A. and Dosch, R. (2018). Functional equivalence of germ plasm organizers. PLoS Genet 14(11): e1007696. PubMed ID: 30399145
The proteins Oskar (Osk) in Drosophila and Bucky ball (Buc) in zebrafish act as germ plasm organizers. Both proteins recapitulate germ plasm activities but seem to be unique to their animal groups. This study discovered that Osk and Buc show similar activities during germ cell specification. Drosophila Osk induces additional PGCs in zebrafish. Surprisingly, Osk and Buc do not show homologous protein motifs that would explain their related function. Nonetheless, both proteins contain stretches of intrinsically disordered regions (IDRs), which seem to be involved in protein aggregation. IDRs are known to rapidly change their sequence during evolution, which might obscure biochemical interaction motifs. Indeed, this study showed that Buc binds to the known Oskar interactors Vasa protein and nanos mRNA indicating conserved biochemical activities. These data provide a molecular framework for two proteins with unrelated sequence but with equivalent function to assemble a conserved core-complex nucleating germ plasm.
Singh, A., Buehner, N. A., Lin, H., Baranowski, K. J., Findlay, G. D. and Wolfner, M. F. (2018). Long-term interaction between Drosophila sperm and sex peptide is mediated by other seminal proteins that bind only transiently to sperm. Insect Biochem Mol Biol 102: 43-51. PubMed ID: 30217614
Seminal fluid proteins elicit several post-mating physiological changes in mated Drosophila melanogaster females. Some of these changes persist for over a week after mating because the seminal protein that causes these changes, the Sex Peptide (SP), binds to sperm that are stored in the female reproductive tract. SP's sperm binding is mediated by a network of at least eight seminal proteins. Some of these network proteins (CG1656, CG1652, CG9997 and Antares) bind to sperm within 2h of mating, like SP. However, while SP remains bound to sperm at 4 days post-mating, none of the other network proteins are detectable at this time. It was also observed that the same network proteins are detectable at 2h post-mating in seminal receptacle tissue from which sperm have been removed, but are no longer detectable there by 4 days post-mating, suggesting short-term retention of these proteins in this female sperm storage organ. These results suggest that these network proteins act transiently to facilitate the conditions for SP's binding to sperm, perhaps by modifying SP or the sperm surface, but are not part of a long-acting complex that stably attaches SP to sperm.
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