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Monday, October 25th - Signaling

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Simon, E., Jimenez-Jimenez, C., Seijo-Barandiaran, I., Aguilar, G., Sanchez-Hernandez, D., Aguirre-Tamaral, A., Gonzalez-Mendez, L., Ripoll, P. and Guerrero, I. (2021). Glypicans define unique roles for the Hedgehog co-receptors boi and ihog in cytoneme-mediated gradient formation. Elife 10. PubMed ID: 34355694
Summary:
The conserved family of Hedgehog (Hh) signaling proteins plays a key role in cell-cell communication in development, tissue repair, and cancer progression, inducing distinct concentration-dependent responses in target cells located at short and long distances. One simple mechanism for long distance dispersal of the lipid modified Hh is the direct contact between cell membranes through filopodia-like structures known as cytonemes. This study has analyzed in Drosophila the interaction between the glypicans Dally and Dally-like protein, necessary for Hh signaling, and the adhesion molecules and Hh coreceptors Ihog and Boi. This study describes that glypicans are required to maintain the levels of Ihog, but not of Boi. It was also shown that the overexpression of Ihog, but not of Boi, regulates cytoneme dynamics through their interaction with glypicans, the Ihog fibronectin III domains being essential for this interaction. These data suggest that the regulation of glypicans over Hh signaling is specifically given by their interaction with Ihog in cytonemes. Contrary to previous data, this study also shows that there is no redundancy of Ihog and Boi functions in Hh gradient formation, being Ihog, but not of Boi, essential for the long-range gradient.
Rivas, G. B. S., Zhou, J., Merlin, C. and Hardin, P. E. (2021). CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the putative Drosophila ortholog of CLOCK INTERACTING PROTEIN CIRCADIAN. Curr Biol. PubMed ID: 34331859
Summary:
The Drosophila circadian clock is driven by a transcriptional feedback loop in which CLOCK-CYCLE (CLK-CYC) binds E-boxes to transcribe genes encoding the PERIOD-TIMELESS (PER-TIM) repressor, which releases CLK-CYC from E-boxes to inhibit transcription. CLOCKWORK ORANGE (CWO) reinforces PER-TIM repression by binding E-boxes to maintain PER-TIM bound CLK-CYC off DNA, but also promotes CLK-CYC transcription through an unknown mechanism. To determine how CWO activates CLK-CYC transcription, CWO target genes were identified that are upregulated in the absence of CWO repression, conserved in mammals, and preferentially expressed in brain pacemaker neurons. Among the genes identified was a putative ortholog of mouse Clock Interacting Protein Circadian (Cipc), which represses CLOCK-BMAL1 transcription. Reducing or eliminating Drosophila Cipc expression shortens period, while overexpressing Cipc lengthens period. Cipc represses CLK-CYC transcription in vivo, but not uniformly, as per is strongly repressed, tim less so, and vri hardly at all. Long period rhythms in cwo mutant flies are largely rescued when Cipc expression is reduced or eliminated, indicating that increased Cipc expression mediates the period lengthening of cwo mutants. These results suggest a mechanism for CWO-dependent CLK-CYC activation: CWO inhibition of CIPC repression promotes CLK-CYC transcription. This mechanism may be conserved since cwo and Cipc perform analogous roles in the mammalian circadian clock.
Zhang, C., van Leeuwen, W., Blotenburg, M., Aguilera-Gomez, A., Brussee, S., Grond, R., Kampinga, H. H. and Rabouille, C. (2021). Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells. J Cell Sci 134(17). PubMed ID: 34350957
Summary:
The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. This study addresses which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt.
Velarde, S. B., Quevedo, A., Estella, C. and Baonza, A. (2021). Dpp and Hedgehog promote the glial response to neuronal apoptosis in the developing Drosophila visual system. PLoS Biol 19(8): e3001367. PubMed ID: 34379617
Summary:
Damage in the nervous system induces a stereotypical response that is mediated by glial cells. This study used the eye disc of Drosophila melanogaster as a model to explore the mechanisms involved in promoting glial cell response after neuronal cell death induction. These cells rapidly respond to neuronal apoptosis by increasing in number and undergoing morphological changes, which will ultimately grant them phagocytic abilities. This glial response is controlled by the activity of Decapentaplegic (Dpp) and Hedgehog (Hh) signalling pathways. These pathways are activated after cell death induction, and their functions are necessary to induce glial cell proliferation and migration to the eye discs. The latter of these 2 processes depend on the function of the c-Jun N-terminal kinase (JNK) pathway, which is activated by Dpp signalling. Evidence is presented that a similar mechanism controls glial response upon apoptosis induction in the leg discs, suggesting that these results uncover a mechanism that might be involved in controlling glial cells response to neuronal cell death in different regions of the peripheral nervous system (PNS).
Prince, E., Kretzschmar, J., Trautenberg, L. C., Broschk, S. and Brankatschk, M. (2021). DIlp7-Producing Neurons Regulate Insulin-Producing Cells in Drosophila. Front Physiol 12: 630390. PubMed ID: 34385929
Summary:
Cellular Insulin signaling shows a remarkable high molecular and functional conservation. Insulin-producing cells respond directly to nutritional cues in circulation and receive modulatory input from connected neuronal networks. Neuronal control integrates a wide range of variables including dietary change or environmental temperature. Although it is shown that neuronal input is sufficient to regulate Insulin-producing cells, the physiological relevance of this network remains elusive. In Drosophila melanogaster, Insulin-like peptide7-producing neurons are wired with Insulin-producing cells. The former cells regulate the latter to facilitate larval development at high temperatures, and to regulate systemic Insulin signaling in adults feeding on calorie-rich food lacking dietary yeast. These results demonstrate a role for neuronal innervation of Insulin-producing cells important for fruit flies to survive unfavorable environmental conditions.
Zhao, P., Huang, P., Xu, T., Xiang, X., Sun, Y., Liu, J., Yan, C., Wang, L., Gao, J., Cui, S., Wang, X., Zhan, L., Song, H., Liu, J., Song, W. and Liu, Y. (2021). Fat body Ire1 regulates lipid homeostasis through the Xbp1s-FoxO axis in Drosophila.. iScience 24(8): 102819. PubMed ID: 34381963
Summary:
The endoplasmic reticulum (ER)-resident transmembrane protein kinase/RNase Ire1 is a conserved sensor of the cellular unfolded protein response and has been implicated in lipid homeostasis, including lipid synthesis and transport, across species. This study reports a novel catabolic role of Ire1 in regulating lipid mobilization in Drosophila. Ire1 is activated by nutrient deprivation, and, importantly, fat body-specific Ire1 deficiency leads to increased lipid mobilization and sensitizes flies to starvation, whereas fat body Ire1 overexpression results in the opposite phenotypes. Genetic interaction and biochemical analyses revealed that Ire1 regulates lipid mobilization by promoting Xbp1s-associated FoxO degradation and suppressing FoxO-dependent lipolytic programs. These results demonstrate that Ire1 is a catabolic sensor and acts through the Xbp1s-FoxO axis to hamper the lipolytic response during chronic food deprivation. These findings offer new insights into the conserved Ire1 regulation of lipid homeostasis.

Friday, October 22nd - Enhancers and Transcriptional Regulation

Mishra, A. K., Fritsch, C., Voutev, R., Mann, R. S. and Sprecher, S. G. (2021). Homothorax controls a binary Rhodopsin switch in Drosophila ocelli. PLoS Genet 17(7): e1009460. PubMed ID: 34314427
Summary:
Visual perception of the environment is mediated by specialized photoreceptor (PR) neurons of the eye. Each PR expresses photosensitive opsins, which are activated by a particular wavelength of light. In most insects, the visual system comprises a pair of compound eyes that are mainly associated with motion, color or polarized light detection, and a triplet of ocelli that are thought to be critical during flight to detect horizon and movements. It is widely believed that the evolutionary diversification of compound eye and ocelli in insects occurred from an ancestral visual organ around 500 million years ago. Concurrently, opsin genes were also duplicated to provide distinct spectral sensitivities to different PRs of compound eye and ocelli. In the fruit fly Drosophila melanogaster, Rhodopsin1 (Rh1) and Rh2 are closely related opsins that originated from the duplication of a single ancestral gene. However, in the visual organs, Rh2 is uniquely expressed in ocelli whereas Rh1 is uniquely expressed in outer PRs of the compound eye. It is currently unknown how this differential expression of Rh1 and Rh2 in the two visual organs is controlled to provide unique spectral sensitivities to ocelli and compound eyes. This study shows that Homothorax (Hth) is expressed in ocelli and confers proper rhodopsin expression. Hth was shown to control a binary Rhodopsin switch in ocelli to promote Rh2 expression and repress Rh1 expression. Genetic and molecular analysis of rh1 and rh2 supports that Hth acts through their promoters to regulate Rhodopsin expression in the ocelli. Finally, this study also showed that when ectopically expressed in the retina, hth is sufficient to induce Rh2 expression only at the outer PRs in a cell autonomous manner. It is therefore proposed that the diversification of rhodpsins in the ocelli and retinal outer PRs occurred by duplication of an ancestral gene, which is under the control of Homothorax.
Yue, X., Liang, Y., Wei, Z., Lv, J., Cai, Y., Fan, X., Zhang, W. and Chen, J. (2021). Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila. Insect Sci. PubMed ID: 34351065
Summary:
Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. This study reports the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell-based RNAi screen identified 537 candidate kkv regulators on a genome-wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and we identified six genes as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy-related 101 (Atg101) was knocked down in Drosophila at the 3rd-instar larval stage; whereas disoriented taenidial folds were observed on larval tracheae when Lines (lin) or Autophagy-related 3 (Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr and Abd-A showed the potential complex mechanism of kkv transcription are regulated by an interaction network with bithorax complex components. Our study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis.
Mika, K., Cruchet, S., Chai, P. C., Prieto-Godino, L. L., Auer, T. O., Pradervand, S. and Benton, R. (2021). Olfactory receptor-dependent receptor repression in Drosophila. Sci Adv 7(32). PubMed ID: 34362730
Summary:
In olfactory systems across phyla, most sensory neurons express a single olfactory receptor gene selected from a large genomic repertoire. This study describes previously unknown receptor gene-dependent mechanisms that ensure singular expression of receptors encoded by a tandem gene array [Ionotropic receptor 75c (Ir75c), Ir75b, and Ir75a, organized 5' to 3'] in Drosophila melanogaster. Transcription from upstream genes in the cluster runs through the coding region of downstream loci and inhibits their expression in cis, most likely via transcriptional interference. Moreover, Ir75c blocks accumulation of other receptor proteins in trans through a protein-dependent, posttranscriptional mechanism. These repression mechanisms operate in endogenous neurons, in conjunction with cell type-specific gene regulatory networks, to ensure unique receptor expression. These data provide evidence for inter-olfactory receptor regulation in invertebrates and highlight unprecedented, but potentially widespread, mechanisms for ensuring exclusive expression of chemosensory receptors, and other protein families, encoded by tandemly arranged genes.
Neville, M. C., Eastwood, A., Allen, A. M., de Haan, A., Nojima, T. and Goodwin, S. F. (2021). Generation and characterization of fruitless P1 promoter mutant in Drosophila melanogaster. J Neurogenet: 1-10. PubMed ID: 34338589
Summary:
The identification of mutations in the gene fruitless (fru) paved the way for understanding the genetic basis of male sexual behavior in the vinegar fly Drosophila melanogaster. D. melanogaster males perform an elaborate courtship display to the female, ultimately leading to copulation. Mutations in fru have been shown to disrupt most aspects of the male's behavioral display, rendering males behaviorally sterile. The fru genomic locus encodes for multiple transcription factor isoforms from several promoters; only those under the regulation of the most distal P1 promoter are under the control of the sex determination hierarchy and play a role in male-specific behaviors. This study used CRISPR/Cas9-based targeted genome editing of the fru gene, to remove the P1 promoter region. Removal of the P1 promoter was shown to lead to a dramatic decrease in male courtship displays towards females and male-specific sterility. The analysis of fru P1-dependent behaviors was expanded, examining male's response to courtship song and general activity levels during 12-hour light:dark cycles. This novel allele expands the mutant repertoire available for future studies of fru P1-derived function in D. melanogaster. The fruΔP1 mutant will be useful for future studies of fru P1-derived function, as it can be homozygosed without disrupting additional downstream promoter function and can be utilized in heterozygous combinations with other extant fru alleles.
Akhtar, J., Renaud, Y., Albrecht, S., Ghavi-Helm, Y., Roignant, J. Y., Silies, M. and Junion, G. (2021). m(6)A RNA methylation regulates promoter- proximal pausing of RNA polymerase II. Mol Cell 81(16): 3356-3367. PubMed ID: 34297910
Summary:
RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. This study shows that the m(6)A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m(6)A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m(6)A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m(6)A catalytic domain. Collectively, these data reveal an important link between RNAP II pausing and the m(6)A RNA modification, thus adding another layer to m(6)A-mediated gene regulation.
Pimmett, V. L., Dejean, M., Fernandez, C., Trullo, A., Bertrand, E., Radulescu, O. and Lagha, M. (2021). Quantitative imaging of transcription in living Drosophila embryos reveals the impact of core promoter motifs on promoter state dynamics. Nat Commun 12(1): 4504. PubMed ID: 34301936
Summary:
Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. This study investigated two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, it was demonstrated that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, these results provide a rationale for promoter switching during zygotic genome activation.

Wednesday, October 20th - Adult neural development and function

Krill, J. L. and Dawson-Scully, K. (2021). Characterization of a novel stimulus-induced glial calcium wave in Drosophila larval peripheral segmental nerves and its role in PKG-modulated thermoprotection. J Neurogenet: 1-15. PubMed ID: 34309496
Summary:
Insects, as poikilotherms, have adaptations to deal with wide ranges in temperature fluctuation. Allelic variations in the foraging gene that encodes a cGMP dependent protein kinase, were discovered to have effects on behavior in Drosophila by Dr. Marla Sokolowski in 1980. This single gene has many pleiotropic effects and influences feeding behavior, metabolic storage, learning and memory and has been shown to affect stress tolerance. PKG regulation affects motoneuronal thermotolerance in Drosophila larvae as well as adults. While the focus of thermotolerance studies has been on the modulation of neuronal function, other cell types have been overlooked. Because glia are vital to neuronal function and survival, this study determine if glia play a role in thermotolerance as well. In this investigation, a novel calcium wave was discovered at the larval NMJ and set out to characterize the wave's dynamics and the potential mechanism underlying the wave prior to determining what effect, if any, PKG modulation has on the thermotolerance of glia cells. Using pharmacology, it was determined that calcium buffering mechanisms of the mitochondria and endoplasmic reticulum play a role in the propagation of our novel glial calcium wave. By coupling pharmacology with genetic manipulation using RNA interference (RNAi), it was found that PKG modulation in glia alters thermoprotection of function as well as glial calcium wave dynamics.
Vinsland, E., Baskaran, P., Mihaylov, S. R., Hobbs, C., Wood, H., Bouybayoune, I., Shah, K., Houart, C., Tee, A. R., Murn, J., Fernandes, C. and Bateman, J. M. (2021). The zinc finger/RING domain protein Unkempt regulates cognitive flexibility. Sci Rep 11(1): 16299. PubMed ID: 34381067
Summary:
Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila. Unkempt also directly interacts with the mTOR complex I component Raptor. This study described the generation and characterisation of mice with a conditional knockout of Unkempt (Unk(cKO)) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. It was also shown that nervous system development occurs normally in Unk(cKO) mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that Unk(cKO) mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory.
Pauls, D., Selcho, M., Raderscheidt, J., Amatobi, K. M., Fekete, A., Krischke, M., Hermann-Luibl, C., Ozbek-Unal, A. G., Ehmann, N., Itskov, P. M., Kittel, R. J., Helfrich-Forster, C., Kuhnlein, R. P., Mueller, M. J. and Wegener, C. (2021). Endocrine signals fine-tune daily activity patterns in Drosophila. Curr Biol. PubMed ID: 34329588
Summary:
Animals need to balance competitive behaviors to maintain internal homeostasis. The underlying mechanisms are complex but typically involve neuroendocrine signaling. Using Drosophila, this study systematically manipulated signaling between energy-mobilizing endocrine cells producing adipokinetic hormone (AKH), octopaminergic neurons, and the energy-storing fat body to assess whether this neuroendocrine axis involved in starvation-induced hyperactivity also balances activity levels under ad libitum access to food. The results suggest that AKH signals via two divergent pathways that are mutually competitive in terms of activity and rest. AKH increases activity via the octopaminergic system during the day, while it prevents high activity levels during the night by signaling to the fat body. This regulation involves feedback signaling from octopaminergic neurons to AKH-producing cells (APCs). APCs are known to integrate a multitude of metabolic and endocrine signals. The results add a new facet to the versatile regulatory functions of APCs by showing that their output contributes to shape the daily activity pattern under ad libitum access to food.
Sakurai, A., Littleton, J. T., Kojima, H. and Yoshihara, M. (2021). Alteration in information flow through a pair of feeding command neurons underlies a form of Pavlovian conditioning in the Drosophila brain. Curr Biol. PubMed ID: 34352215
Summary:
Pavlovian conditioning is a broadly used learning paradigm where defined stimuli are associated to induce behavioral switching. To define a causal relationship between activity change in a single neuron and behavioral switching, this study took advantage of a "command neuron" that connects cellular function to behavior. To examine the cellular and molecular basis of Pavlovian conditioning, previous work identified a pair of feeding command neurons termed "feeding neurons" in the adult Drosophila brain using genetic screening and opto- and thermo-genetic techniques. The feeding neuron is activated by sweet signals like sucrose and induces the full complement of feeding behaviors, such as proboscis extension and food pumping. Ablation or inactivation of the pair of feeding neurons abolishes feeding behavior, suggesting that this single pair of neurons is indispensable for natural feeding behaviors. This study describes a novel conditioning protocol to associate a signal-mediating rod removal from legs (conditioned stimulus [CS]) to feeding behavior induced by sucrose stimulation (unconditioned stimulus [US]). Calcium imaging of the feeding neuron demonstrated it acquires responsiveness to CS during conditioning, with inactivation of the feeding neuron during conditioning suppressing plasticity. These results suggest conditioning alters signals flowing from the CS into the feeding circuit, with the feeding neuron functioning as a key integrative hub for Hebbian plasticity.
Mano, O., Creamer, M. S., Badwan, B. A. and Clark, D. A. (2021). Predicting individual neuron responses with anatomically constrained task optimization. Curr Biol. PubMed ID: 34324832
Summary:
Artificial neural networks trained to solve sensory tasks can develop statistical representations that match those in biological circuits. However, it remains unclear whether they can reproduce properties of individual neurons. This study investigated how artificial networks predict individual neuron properties in the visual motion circuits of the fruit fly Drosophila. Anatomically constrained networks were trained to predict movement in natural scenes, solving the same inference problem as fly motion detectors. Units in the artificial networks adopted many properties of analogous individual neurons, even though they were not explicitly trained to match these properties. Among these properties was the split into ON and OFF motion detectors, which is not predicted by classical motion detection models. The match between model and neurons was closest when models were trained to be robust to noise. These results demonstrate how anatomical, task, and noise constraints can explain properties of individual neurons in a small neural network.
Wu, B., He, L., Xiao, Y., Du, J., Wang, X. and Zhao, Z. (2021). Juvenile hormone receptor MET regulates sleep and neuronal morphology via glial-neuronal crosstalk. J Genet Genomics. PubMed ID: 34376377
Summary:
Juvenile hormone (JH) is one of the most important hormones in insects since it is essential for insect development. The mechanism by which JH affects the central nervous system still remains a mystery. This study demonstrates that one of the JH receptors, Methoprene-tolerant (Met), is important for the control of neurite development and sleep behavior in Drosophila. With the identification of Met-expressing glial cells, the mechanism that Met negatively controls the mushroom body (MB) β lobes fusion and positively maintains pigment-dispersing factor sLNvs projection pruning has been established. Furthermore, despite the developmental effects, Met can also maintain nighttime sleep in a development-independent manner through the α/β lobe of MB. Combining analyses of neuronal morphology and entomological behavior, this study advances understanding of how the JH receptor regulates the nervous system.

Tuesday, October 19th - Cytoskeleton

Shin, J. H. and Jeong, C. W. (2021). Zipper Is Necessary for Branching Morphogenesis of the Terminal Cells in the Drosophila melanogaster's Tracheal System. Biology (Basel) 10(8). PubMed ID: 34439961
Summary:
Branching morphogenesis and seamless tube formation in Drosophila melanogaster are essential for the development of vascular and tracheal systems, and instructive in studying complex branched structures such as human organs. Zipper is a myosin II's actin-binding heavy chain; hence, it is important for contracting actin, cell proliferation, and cell sheet adhesion for branching of the tracheal system in post-larval development of the D. melanogaster. Nevertheless, the specific role of Zipper in the larva is still in question. This paper intended to investigate the specific role of Zipper in branching morphogenesis and lumenogenesis in early developmental stages. It did so by checking the localization of the protein in the cytoplasm of the terminal cells and also by analyzing the morphology of zipper RNAi loss-of-function mutants in regard to branching and lumen formation in the terminal cells. A rescue experiment of RNAi mutants was also performed to check the sufficiency of Zipper in branching morphogenesis. Confocal imaging showed the localization of Zipper in the cytoplasm of the terminal cells, and respective quantitative analyses demonstrated that zipper RNAi terminal cells develop significantly fewer branches. Such a result hinted that Zipper is required for the regulation of branching in the terminal cells of D. melanogaster. Nevertheless, Zipper is not significantly involved in the formation of seamless tubes. One hypothesis is that Zipper's contractility at the lateral epidermis' leading edge allows cell sheet movement and respective elongation; as a result of such an elongation, further branching may occur in the elongated region of the cell, hence defining branching morphogenesis in the terminal cells of the tracheal system.
Vakaloglou, K. M., Mouratidou, M., Keramidioti, A. and Zervas, C. G. (2021). Differential Expression of Drosophila Transgelins Throughout Development. Front Cell Dev Biol 9: 648568. PubMed ID: 34322481
Summary:
Transgelins are a conserved family of actin-binding proteins involved in cytoskeletal remodeling, cell contractility, and cell shape. In both mammals and Drosophila, three genes encode transgelin proteins. Transgelins exhibit a broad and overlapping expression pattern, which has obscured the precise identification of their role in development. This study reports the first systematic developmental analysis of all Drosophila transgelin proteins, namely, Mp20, CG5023, and Chd64 in the living organism. Drosophila transgelins display overall higher sequence identity with mammalian TAGLN-3 and TAGLN-2 than with TAGLN. Detailed examination in different developmental stages revealed that Mp20 and CG5023 are predominantly expressed in mesodermal tissues with the onset of myogenesis and accumulate in the cytoplasm of all somatic muscles and heart in the late embryo. Notably, at postembryonic developmental stages, Mp20 and CG5023 are detected in the gut's circumferential muscles with distinct subcellular localization: Z-lines for Mp20 and sarcomere and nucleus for CG5023. Only CG5023 is strongly detected in the adult fly in the abdominal, leg, and synchronous thoracic muscles. Chd64 protein is primarily expressed in endodermal and ectodermal tissues and has a dual subcellular localization in the cytoplasm and the nucleus. During the larval-pupae transition, Chd64 is expressed in the brain, eye, legs, halteres, and wings. In contrast, in the adult fly, Chd64 is expressed in epithelia, including the alimentary tract and genitalia. Based on the non-overlapping tissue expression, it is predicted that Mp20 and CG5023 mostly cooperate to modulate muscle function, whereas Chd64 has distinct roles in epithelial, neuronal, and endodermal tissues.
Bu, S., Yong, W. L., Lim, B. J. W., Kondo, S. and Yu, F. (2021). A systematic analysis of microtubule-destabilizing factors during dendrite pruning in Drosophila. EMBO Rep: e52679. PubMed ID: 34338441
Summary:
It has long been thought that microtubule disassembly, one of the earliest cellular events, contributes to neuronal pruning and neurodegeneration in development and disease. However, how microtubule disassembly drives neuronal pruning remains poorly understood. A systematic investigation was undertaken of various microtubule-destabilizing factors and identify exchange factor for Arf6 (Efa6) and Stathmin (Stai) as new regulators of dendrite pruning in ddaC sensory neurons during Drosophila metamorphosis. Efa6 was shown to be both necessary and sufficient to regulate dendrite pruning. Interestingly, Efa6 and Stai facilitate microtubule turnover and disassembly prior to dendrite pruning without compromising the minus-end-out microtubule orientation in dendrites. Moreover, the pharmacological and genetic manipulations strongly support a key role of microtubule disassembly in promoting dendrite pruning. Thus, this systematic study highlights the importance of two selective microtubule destabilizers in dendrite pruning and substantiates a causal link between microtubule disassembly and neuronal pruning.
Menard, L. M., Wood, N. B. and Vigoreaux, J. O. (2021). Contiguity and Structural Impacts of a Non-Myosin Protein within the Thick Filament Myosin Layers. Biology (Basel) 10(7). PubMed ID: 34356468
Summary:
Myosin dimers arranged in layers and interspersed with non-myosin densities have been described by cryo-EM 3D reconstruction of the thick filament in Lethocerus (Hemiptera) at 5.5 Å resolution. One of the non-myosin densities, denoted the 'red density', is hypothesized to be flightin, an LMM-binding protein essential to the structure and function of Drosophila indirect flight muscle (IFM). Each independent red density winds its way through the myosin dimers, such that it links four dimers in a layer and one dimer in a neighboring layer. This area in which three distinct interfaces within the myosin rod are contacted at once and the red density extends to the thick filament core is designated the "multiface". Present within the multiface is a contact area inclusive of E1563 and R1568. Mutations in the corresponding Drosophila residues interfere with flightin accumulation and phosphorylation. This study examined the LMM area in direct apposition to the red density and identified potential binding residues spanning up to ten helical turns. The red density is associated within an expanse of the myosin coiled-coil that is unwound by the third skip residue and the coiled-coil is re-oriented while in contact with the red density. These findings suggest a mechanism by which Flightin induces ordered assembly of myosin dimers through its contacts with multiple myosin dimers and brings about reinforcement on the level of a single myosin dimer by stabilization of the myosin coiled-coil.
Panzade, S. and Matis, M. (2021). The Microtubule Minus-End Binding Protein Patronin Is Required for the Epithelial Remodeling in the Drosophila Abdomen. Front Cell Dev Biol 9: 682083. PubMed ID: 34368132
Summary:
In the developing Drosophila abdomen, the epithelial tissue displays extensive cytoskeletal remodeling. In stark contrast to the spatio-temporal control of the actin cytoskeleton, the regulation of microtubule architecture during epithelial morphogenesis has remained opaque. In particular, its role in cell motility remains unclear. This study shows that minus-end binding protein Patronin is required for organizing microtubule arrays in histoblast cells that form the Drosophila abdomen. Loss of Patronin results in a dorsal cleft, indicating the compromised function of histoblasts. It was further shown that Patronin is polarized in these cells and is required for the formation of highly dynamic non-centrosomal microtubules in the migrating histoblasts. Thus, this study demonstrates that regulation of microtubule cytoskeleton through Patronin mediates epithelium remodeling.
Kucera, O., Siahaan, V., Janda, D., Dijkstra, S. H., Pilatova, E., Zatecka, E., Diez, S., Braun, M. and Lansky, Z. (2021). Anillin propels myosin-independent constriction of actin rings. Nat Commun 12(1): 4595. PubMed ID: 34321459
Summary:
Constriction of the cytokinetic ring, a circular structure of actin filaments, is an essential step during cell division. Mechanical forces driving the constriction are attributed to myosin motor proteins, which slide actin filaments along each other. However, in multiple organisms, ring constriction has been reported to be myosin independent. How actin rings constrict in the absence of motor activity remains unclear. This study demonstrates that anillin, a non-motor actin crosslinker, indispensable during cytokinesis, autonomously propels the contractility of actin bundles. Anillin generates contractile forces of tens of pico-Newtons to maximise the lengths of overlaps between bundled actin filaments. The contractility is enhanced by actin disassembly. When multiple actin filaments are arranged into a ring, this contractility leads to ring constriction. These results indicate that passive actin crosslinkers can substitute for the activity of molecular motors to generate contractile forces in a variety of actin networks, including the cytokinetic ring.

Monday, October 18th - Disease Models

Liu, Q., Bautista-Gomez, J., Higgins, D. A., Yu, J. and Xiong, Y. (2021). Dysregulation of the AP2M1 phosphorylation cycle by LRRK2 impairs endocytosis and leads to dopaminergic neurodegeneration. Sci Signal 14(693). PubMed ID: 34315807
Summary:
Mutations in the kinase LRRK2 and impaired endocytic trafficking are both implicated in the pathogenesis of Parkinson's disease (PD). Expression of the PD-associated LRRK2 mutant in mouse dopaminergic neurons was shown to disrupt clathrin-mediated endocytic trafficking. This study explored the molecular mechanism linking LRRK2 to endocytosis and found that LRRK2 bound to and phosphorylated the μ2 subunit of the adaptor protein AP2 (AP2M1), a core component of the clathrin-mediated endocytic machinery. Analysis of human SH-SY5Y cells and mouse neurons and tissues revealed that loss of LRRK2 abundance or kinase function resulted in decreased phosphorylation of AP2M1, which is required for the initial formation of clathrin-coated vesicles (CCVs). In contrast, overexpression of LRRK2 or expression of a Parkinson's disease-associated gain-of-function mutant LRRK2 (G2019S) inhibited the uncoating of AP2M1 from CCVs at later stages and prevented new cycles of CCV formation. Thus, the abundance and activity of LRRK2 must be calibrated to ensure proper endocytosis. Dysregulated phosphorylation of AP2M1 from the brain but not thyroid tissues of LRRK2 knockout and G2019S-knockin mice suggests a tissue-specific regulatory mechanism of endocytosis. Furthermore, this study found that LRRK2-dependent phosphorylation of AP2M1 mediated dopaminergic neurodegeneration in a Drosophila model of PD. Together, these findings provide a mechanistic link between LRRK2, AP2, and endocytosis in the pathogenesis of PD.
Mokashi, S. S., Shankar, V., MacPherson, R. A., Hannah, R. C., Mackay, T. F. C. and Anholt, R. R. H. (2021). Developmental Alcohol Exposure in Drosophila: Effects on Adult Phenotypes and Gene Expression in the Brain. Front Psychiatry 12: 699033. PubMed ID: 34366927
Summary:
Fetal alcohol exposure can lead to developmental abnormalities, intellectual disability, and behavioral changes, collectively termed fetal alcohol spectrum disorder (FASD). In 2015, the Centers for Disease Control found that 1 in 10 pregnant women report alcohol use and more than 3 million women in the USA are at risk of exposing their developing fetus to alcohol. Drosophila exposed to alcohol undergo physiological and behavioral changes that resemble human alcohol-related phenotypes. This study shows that adult flies that developed on ethanol-supplemented medium have decreased viability, reduced sensitivity to ethanol, and disrupted sleep and activity patterns. To assess the effects of exposure to alcohol during development on brain gene expression, single cell RNA sequencing was performed, and cell clusters were resolved with differentially expressed genes which represent distinct neuronal and glial populations. Differential gene expression showed extensive sexual dimorphism with little overlap between males and females. Gene expression differences following developmental alcohol exposure were similar to previously reported differential gene expression following cocaine consumption, suggesting that common neural substrates respond to both drugs. Genes associated with glutathione metabolism, lipid transport, glutamate and GABA metabolism, and vision feature in sexually dimorphic global multi-cluster interaction networks. These results provide a blueprint for translational studies on alcohol-induced effects on gene expression in the brain that may contribute to or result from FASD in human populations.
Mituzaite, J., Petersen, R., Claridge-Chang, A. and Baines, R. A. (2021). Characterization of Seizure Induction Methods in Drosophila. eNeuro 8(4). PubMed ID: 34330816
Summary:
Epilepsy is one of the most common neurologic disorders. Around one third of patients do not respond to current medications. This lack of treatment indicates a need for better understanding of the underlying mechanisms and, importantly, the identification of novel targets for drug manipulation. The fruit fly Drosophila melanogaster has a fast reproduction time, powerful genetics, and facilitates large sample sizes, making it a strong model of seizure mechanisms. To better understand behavioral and physiological phenotypes across major fly seizure genotypes this study systematically measured seizure severity and secondary behavioral phenotypes at both the larval and adult stage. Comparison of several seizure-induction methods; specifically electrical, mechanical and heat induction, show that larval electroshock is the most effective at inducing seizures across a wide range of seizure-prone mutants tested. Locomotion in adults and larvae was found to be non-predictive of seizure susceptibility. Recording activity in identified larval motor neurons revealed variations in action potential (AP) patterns, across different genotypes, but these patterns did not correlate with seizure susceptibility. To conclude, while there is wide variation in mechanical induction, heat induction, and secondary phenotypes, electroshock is the most consistent method of seizure induction across known major seizure genotypes in Drosophila. ]
Melentev, P. A., Ryabova, E. V., Surina, N. V., Zhmujdina, D. R., Komissarov, A. E., Ivanova, E. A., Boltneva, N. P., Makhaeva, G. F., Sliusarenko, M. I., Yatsenko, A. S., Mohylyak, II, Matiytsiv, N. P., Shcherbata, H. R. and Sarantseva, S. V. (2021). Loss of swiss cheese in Neurons Contributes to Neurodegeneration with Mitochondria Abnormalities, Reactive Oxygen Species Acceleration and Accumulation of Lipid Droplets in Drosophila Brain. Int J Mol Sci 22(15). PubMed ID: 34361042
Summary:
Various neurodegenerative disorders are associated with human NTE/PNPLA6 dysfunction. Mechanisms of neuropathogenesis in these diseases are far from clearly elucidated. Hereditary spastic paraplegia belongs to a type of neurodegeneration associated with NTE/PNLPLA6 and is implicated in neuron death. This study used Drosophila melanogaster to investigate the consequences of neuronal knockdown of swiss cheese (sws)-the evolutionarily conserved ortholog of human NTE/PNPLA6-in vivo. Adult flies with the knockdown show longevity decline, locomotor and memory deficits, severe neurodegeneration progression in the brain, reactive oxygen species level acceleration, mitochondria abnormalities and lipid droplet accumulation. These results suggest that SWS/NTE/PNPLA6 dysfunction in neurons induces oxidative stress and lipid metabolism alterations, involving mitochondria dynamics and lipid droplet turnover in neurodegeneration pathogenesis. It is proposed that there is a complex mechanism in neurological diseases such as hereditary spastic paraplegia, which includes a stress reaction, engaging mitochondria, lipid droplets and endoplasmic reticulum interplay.
Hodgson, J. A., Parvy, J. P., Yu, Y., Vidal, M. and Cordero, J. B. (2021). Drosophila Larval Models of Invasive Tumorigenesis for In Vivo Studies on Tumour/Peripheral Host Tissue Interactions during Cancer Cachexia. Int J Mol Sci 22(15). PubMed ID: 34361081
Summary:
Cancer cachexia is a common deleterious paraneoplastic syndrome that represents an area of unmet clinical need, partly due to its poorly understood aetiology and complex multifactorial nature. This study interrogated multiple genetically defined larval Drosophila models of tumourigenesis against key features of human cancer cachexia. The results indicate that cachectic tissue wasting is dependent on the genetic characteristics of the tumour and demonstrate that host malnutrition or tumour burden are not sufficient to drive wasting. This study shows that JAK/STAT and TNF-α/Egr signalling are elevated in cachectic muscle and promote tissue wasting. Furthermore, a dual driver system is introduced that allows independent genetic manipulation of tumour and host skeletal muscle. Overall, this study presents a novel Drosophila larval paradigm to study tumour/host tissue crosstalk in vivo, which may contribute to future research in cancer cachexia and impact the design of therapeutic approaches for this pathology.
Migunova, E., Theophilopoulos, J., Mercadante, M., Men, J., Zhou, C. and Dubrovsky, E. B. (2021). ELAC2/RNaseZ-linked cardiac hypertrophy in Drosophila melanogaster. Dis Model Mech 14(8). PubMed ID: 34338278
Summary:
>A severe form of infantile cardiomyopathy (CM) has been linked to mutations in ELAC2, a highly conserved human gene. It encodes Zinc phosphodiesterase ELAC protein 2 (ELAC2), which plays an essential role in the production of mature tRNAs. To establish a causal connection between ELAC2 variants and CM, this study used the Drosophila melanogaster model organism, which carries the ELAC2 homolog RNaseZ. Even though RNaseZ and ELAC2 have diverged in some of their biological functions, this study demonstrates the use of the fly model to study the mechanism of ELAC2-related pathology. Transgenic lines harboring RNaseZ with CM-linked mutations in the background of endogenous RNaseZ knockout. Importantly, it was found that the phenotype of these flies is consistent with the pathological features in human patients. Specifically, expression of CM-linked variants in flies caused heart hypertrophy and led to reduction in cardiac contractility associated with a rare form of CM. This study provides first experimental evidence for the pathogenicity of CM-causing mutations in the ELAC2 protein, and the foundation to improve our understanding and diagnosis of this rare infantile disease.

Friday, October 15th - Chromatin

Vorobyeva, N. E., Erokhin, M., Chetverina, D., Krasnov, A. N. and Mazina, M. Y. (2021). Su(Hw) primes 66D and 7F Drosophila chorion genes loci for amplification through chromatin decondensation. Sci Rep 11(1): 16963. PubMed ID: 34417521
Summary:
Suppressor of Hairy wing [Su(Hw)] is an insulator protein that participates in regulating chromatin architecture and gene repression in Drosophila. Previous studies have shown that Su(Hw) is also required for pre-replication complex (pre-RC) recruitment on Su(Hw)-bound sites (SBSs) in Drosophila S2 cells and pupa. This study describes the effect of Su(Hw) on developmentally regulated amplification of 66D and 7F Drosophila amplicons in follicle cells (DAFCs), widely used as models in replication studies. Su(Hw) binding co-localizes with all known DAFCs in Drosophila ovaries, whereas disruption of Su(Hw) binding to 66D and 7F DAFCs causes a two-fold decrease in the amplification of these loci. The complete loss of Su(Hw) binding to chromatin impairs pre-RC recruitment to all amplification regulatory regions of 66D and 7F loci at early oogenesis (prior to DAFCs amplification). These changes coincide with a considerable Su(Hw)-dependent condensation of chromatin at 66D and 7F loci. Although this study observed the Brm, ISWI, Mi-2, and CHD1 chromatin remodelers at SBSs genome wide, their remodeler activity does not appear to be responsible for chromatin decondensation at the 66D and 7F amplification regulatory regions. This study has discovered that, in addition to the CBP/Nejire and Chameau histone acetyltransferases, the Gcn5 acetyltransferase binds to 66D and 7F DAFCs at SBSs and this binding is dependent on Su(Hw). It is proposed that the main function of Su(Hw) in developmental amplification of 66D and 7F DAFCs is to establish a chromatin structure that is permissive to pre-RC recruitment.
Yang, Y., Zhao, T., Li, Z., Qian, W., Peng, J., Wei, L., Yuan, D., Li, Y., Xia, Q. and Cheng, D. (2021). Histone H3K27 methylation-mediated repression of Hairy regulates insect developmental transition by modulating ecdysone biosynthesis. Proc Natl Acad Sci U S A 118(35). PubMed ID: 34429358
Summary:
Insect development is cooperatively orchestrated by the steroid hormone ecdysone and juvenile hormone (JH). The polycomb repressive complex 2 (PRC2)-mediated histone H3K27 trimethylation (H3K27me3) epigenetically silences gene transcription and is essential for a range of biological processes, but the functions of H3K27 methylation in insect hormone action are poorly understood. This study demonstrates that H3K27 methylation-mediated repression of Hairy transcription in the larval prothoracic gland (PG) is required for ecdysone biosynthesis in Bombyx and Drosophila H3K27me3 levels in the PG are dynamically increased during the last larval instar. H3K27me3 reduction induced by the down-regulation of PRC2 activity via inhibitor treatment in Bombyx or PG-specific knockdown of the PRC2 component Su(z)12 in Drosophila diminishes ecdysone biosynthesis and disturbs the larval-pupal transition. Mechanistically, H3K27 methylation targets the JH signal transducer Hairy to repress its transcription in the PG; PG-specific knockdown or overexpression of the Hairy gene disrupts ecdysone biosynthesis and developmental transition; and developmental defects caused by PG-specific Su(z)12 knockdown can be partially rescued by Hairy down-regulation. The application of JH mimic to the PG decreases both H3K27me3 levels and Su(z)12 expression. Altogether, this study reveals that PRC2-mediated H3K27 methylation at Hairy in the PG during the larval period is required for ecdysone biosynthesis and the larval-pupal transition and provides insights into epigenetic regulation of the crosstalk between JH and ecdysone during insect development.
Stephenson, R. A., Thomalla, J. M., Chen, L., Kolkhof, P., White, R. P., Beller, M. and Welte, M. A. (2021). Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones. Development 148(15). PubMed ID: 34355743
Summary:
Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B, H2A and H2Av accumulate on lipid droplets (LDs), which are cytoplasmic fat storage organelles. Without LD binding, maternally provided H2B, H2A and H2Av are absent; however, how LDs ensure histone storage is unclear. Using quantitative imaging, this study uncovered when during oogenesis these histones accumulate, and which step of accumulation is LD dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. These findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites.
Zhuravlev, A. V., Zakharov, G. A., Anufrieva, E. V., Medvedeva, A. V., Nikitina, E. A. and Savvateeva-Popova, E. V. (2021). Chromatin Structure and "DNA Sequence View": The Role of Satellite DNA in Ectopic Pairing of the Drosophila X Polytene Chromosome. Int J Mol Sci 22(16). PubMed ID: 34445413
Summary:
Chromatin 3D structure plays a crucial role in regulation of gene activity. Previous studies have envisioned spatial contact formations between chromatin domains with different epigenetic properties, protein compositions and transcription activity. This leaves specific DNA sequences that affect chromosome interactions. The Drosophila melanogaster polytene chromosomes are involved in non-allelic ectopic pairing. The mutant strain agnts3, a Drosophila model for Williams-Beuren syndrome, has an increased frequency of ectopic contacts (FEC) compared to the wild-type strain Canton-S (CS). Ectopic pairing can be mediated by some specific DNA sequences. Using Homology Segment Analysis software, this study estimated the correlation between FEC and frequency of short matching DNA fragments (FMF) for all sections of the X chromosome of Drosophila CS and agnts3 strains. With fragment lengths of 50 nucleotides (nt), CS showed a specific FEC-FMF correlation for 20% of the sections involved in ectopic contacts. The correlation was unspecific in agnts3, which may indicate the alternative epigenetic mechanisms affecting FEC in the mutant strain. Most of the fragments that specifically contributed to FMF were related to 1.688 or 372-bp middle repeats. Thus, middle repetitive DNA may serve as an organizer of ectopic pairing.
Tilly, B. C., Chalkley, G. E., van der Knaap, J. A., Moshkin, Y. M., Kan, T. W., Dekkers, D. H., Demmers, J. A. and Verrijzer, C. P. (2021). In vivo analysis reveals that ATP-hydrolysis couples remodeling to SWI/SNF release from chromatin. Elife 10. PubMed ID: 34313222
Summary:
ATP-dependent chromatin remodelers control the accessibility of genomic DNA through nucleosome mobilization. However, the dynamics of genome exploration by remodelers, and the role of ATP hydrolysis in this process remain unclear. This study used live-cell imaging of Drosophila polytene nuclei to monitor Brahma (BRM) remodeler interactions with its chromosomal targets. In parallel, local chromatin condensation and its effect on BRM association were measured. Surprisingly, only a small portion of BRM is bound to chromatin at any given time. BRM binds decondensed chromatin but is excluded from condensed chromatin, limiting its genomic search space. BRM-chromatin interactions are highly dynamic, whereas histone-exchange is limited and much slower. Intriguingly, loss of ATP hydrolysis enhanced chromatin retention and clustering of BRM, which was associated with reduced histone turnover. Thus, ATP hydrolysis couples nucleosome remodeling to remodeler release, driving a continuous transient probing of the genome.
Steffen, P. A., Altmutter, C., Dworschak, E., Junttila, S., Gyenesei, A., Zhu, X., Kockmann, T. and Ringrose, L. (2021). The Trithorax group protein ASH1 requires a combination of BAH domain and AT hooks, but not the SET domain, for mitotic chromatin binding and survival. Chromosoma. PubMed ID: 34331109
Summary:
The Drosophila Trithorax group (TrxG) protein ASH1 remains associated with mitotic chromatin through mechanisms that are poorly understood. ASH1 dimethylates histone H3 at lysine 36 via its SET domain. This study has identified domains of the TrxG protein ASH1 that are required for mitotic chromatin attachment in living Drosophila. Quantitative live imaging demonstrates that ASH1 requires AT hooks and the BAH domain but not the SET domain for full chromatin binding in metaphase, and that none of these domains are essential for interphase binding. Genetic experiments show that disruptions of the AT hooks and the BAH domain together, but not deletion of the SET domain alone, are lethal. Transcriptional profiling demonstrates that intact ASH1 AT hooks and the BAH domain are required to maintain expression levels of a specific set of genes, including several involved in cell identity and survival. This study identifies in vivo roles for specific ASH1 domains in mitotic binding, gene regulation, and survival that are distinct from its functions as a histone methyltransferase.

Thursday, October 14th - Adult physiology

Ding, M., Zheng, L., Li, Q. F., Wang, W. L., Peng, W. D. and Zhou, M. (2021). Exercise-Training Regulates Apolipoprotein B in Drosophila to Improve HFD-Mediated Cardiac Function Damage and Low Exercise Capacity. Front Physiol 12: 650959. PubMed ID: 34305631
Summary:
Apolipoprotein B plays an essential role in systemic lipid metabolism, and it is closely related to cardiovascular diseases. Exercise-training can regulate systemic lipid metabolism, improve heart function, and improve exercise capacity, but the molecular mechanisms involved are poorly understood. This study used a Drosophila model to demonstrate that exercise-training regulates the expression of apoLpp (a homolog of apolipoprotein B) in cardiomyocytes, thereby resisting heart insufficiency and low exercise capacity caused by obesity. The apoLpp is an essential lipid carrier produced in the heart and fat body of Drosophila. In a Drosophilla genetic screen, low expression of apoLpp reduced obesity and cardiac dysfunction induced by a high-fat diet (HFD). Cardiac-specific inhibition indicated that reducing apoLpp in the heart during HFD reduced the triglyceride content of the whole-body and reduced heart function damage caused by HFD. In exercise-trained flies, the result was similar to the knockdown effect of apoLpp. Therefore, the inhibition of apoLpp plays an important role in HFD-induced cardiac function impairment and low exercise capacity. Although the apoLpp knockdown of cardiomyocytes alleviated damage to heart function, it did not reduce the arrhythmia and low exercise capacity caused by HFD. Exercise-training can improve this condition more effectively, and the possible reason for this difference is that exercise-training regulates climbing ability in ways to promote metabolism. Exercise-training during HFD feeding can down-regulate the expression of apoLpp, reduce the whole-body TG levels, improve cardiac recovery, and improve exercise capacity. Exercise-training can downregulate the expression of apoLpp in cardiomyocytes to resist cardiac function damage and low exercise capacity caused by HFD. The results revealed the relationship between exercise-training and apoLpp and their essential roles in regulating heart function and climbing ability.
Kohashi, K., Mori, Y., Narumi, R., Kozawa, K., Kamasaki, T., Ishikawa, S., Kajita, M., Kobayashi, R., Tamori, Y. and Fujita, Y. (2021). Sequential oncogenic mutations influence cell competition. Curr Biol. PubMed ID: 34314674
Summary:
At the initial stage of carcinogenesis, newly emerging transformed cells are often eliminated from epithelial layers via cell competition with the surrounding normal cells. For instance, when surrounded by normal cells, oncoprotein RasV12-transformed cells are extruded into the apical lumen of epithelia. During cancer development, multiple oncogenic mutations accumulate within epithelial tissues. However, it remains elusive whether and how cell competition is also involved in this process. Using a mammalian cell culture model system, this study investigated what happens upon the consecutive mutations of Ras and tumor suppressor protein Scribble. When Ras mutation occurs under the Scribble-knockdown background, apical extrusion of Scribble/Ras double-mutant cells is strongly diminished. In addition, at the boundary with Scribble/Ras cells, Scribble-knockdown cells frequently undergo apoptosis and are actively engulfed by the neighboring Scribble/Ras cells. The comparable apoptosis and engulfment phenotypes are also observed in Drosophila epithelial tissues between Scribble/Ras double-mutant and Scribble single-mutant cells. Furthermore, mitochondrial membrane potential is enhanced in Scribble/Ras cells, causing the increased mitochondrial reactive oxygen species (ROS). Suppression of mitochondrial membrane potential or ROS production diminishes apoptosis and engulfment of the surrounding Scribble-knockdown cells, indicating that mitochondrial metabolism plays a key role in the competitive interaction between double- and single-mutant cells. Moreover, mTOR (mechanistic target of rapamycin kinase) acts downstream of these processes. These results imply that sequential oncogenic mutations can profoundly influence cell competition, a transition from loser to winner. Further studies would open new avenues for cell competition-based cancer treatment, thereby blocking clonal expansion of more malignant populations within tumors.
Suthakaran, N., Chandran, S., Iacobelli, M. and Binninger, D. (2021). Hypoxia Tolerance Declines with Age in the Absence of Methionine Sulfoxide Reductase (MSR) in Drosophila melanogaster. Antioxidants (Basel) 10(7). PubMed ID: 34356368
Summary:
Unlike the mammalian brain, Drosophila melanogaster can tolerate several hours of hypoxia without any tissue injury by entering a protective coma known as spreading depression. However, when oxygen is reintroduced, there is an increased production of reactive oxygen species (ROS) that causes oxidative damage. Methionine sulfoxide reductase (MSR) acts to restore functionality to oxidized methionine residues. The present study characterized in vivo effects of MSR deficiency on hypoxia tolerance throughout the lifespan of Drosophila. Flies subjected to sudden hypoxia that lacked MSR activity exhibited a longer recovery time and a reduced ability to survive hypoxic/re-oxygenation stress as they approached senescence. However, when hypoxia was induced slowly, MSR deficient flies recovered significantly quicker throughout their entire adult lifespan. In addition, the wildtype and MSR deficient flies had nearly 100% survival rates throughout their lifespan. Neuroprotective signaling mediated by decreased apoptotic pathway activation, as well as gene reprogramming and metabolic downregulation are possible reasons for why MSR deficient flies have faster recovery time and a higher survival rate upon slow induction of spreading depression. These data are the first to suggest important roles of MSR and longevity pathways in hypoxia tolerance exhibited by Drosophila.
Bitner, K., Rutledge, G. A., Kezos, J. N. and Mueller, L. D. (2021). The effects of adaptation to urea on feeding rates and growth in Drosophila larvae. Ecol Evol 11(14): 9516-9529. PubMed ID: 34306639
Summary:
A collection of forty populations were used to study the phenotypic adaptation of Drosophila melanogaster larvae to urea-laced food. Fifteen populations subjected to direct selection for urea tolerance and five controls were studied. In addition, another twenty populations were studied that had not been exposed to urea but were subjected to stress or demographic selection. This study describes the differentiation in these population for six phenotypes: (1) larval feeding rates, (2) larval viability in urea-laced food, (3) larval development time in urea-laced food, (4) adult starvation times, (5) adult desiccation times, and (6) larval growth rates. No significant differences were observed for desiccation resistance. The demographically/stress-selected populations had longer times to starvation than urea-selected populations. The urea-adapted populations showed elevated survival and reduced development time in urea-laced food relative to the control and nonadapted populations. The urea-adapted populations also showed reduced larval feeding rates relative to controls. We show that there is a strong linear relationship between feeding rates and growth rates at the same larval ages feeding rates were measured. This suggests that feeding rates are correlated with food intake and growth. This relationship between larval feeding rates, food consumption, and efficiency has been postulated to involve important trade-offs that govern larval evolution in stressful environments. These results support the idea that energy allocation is a central organizing theme in adaptive evolution.
Keith, S. A., Bishop, C., Fallacaro, S. and McCartney, B. M. (2021). Arc1 and the microbiota together modulate growth and metabolic traits in Drosophila. Development 148(15). PubMed ID: 34323271
Summary:
Perturbations to animal-associated microbial communities (the microbiota) have deleterious effects on various aspects of host fitness, but the molecular processes underlying these impacts are poorly understood. This study identified a connection between the microbiota and the neuronal factor Arc1 that affects growth and metabolism in Drosophila. Arc1 was found to exhibit tissue-specific microbiota-dependent expression changes, and germ-free flies bearing a null mutation of Arc1 exhibited delayed and stunted larval growth, along with a variety of molecular, cellular and organismal traits indicative of metabolic dysregulation. Remarkably, this study showed that the majority of these phenotypes can be fully suppressed by mono-association with a single Acetobacter sp. isolate, through mechanisms involving both bacterial diet modification and live bacteria. Additionally, evidence is provided that Arc1 function in key neuroendocrine cells of the larval brain modulates growth and metabolic homeostasis under germ-free conditions. These results reveal a role for Arc1 in modulating physiological responses to the microbial environment, and highlight how host-microbe interactions can profoundly impact the phenotypic consequences of genetic mutations in an animal host.
Amichot, M. and Tares, S. (2021). The Foraging Gene, a New Environmental Adaptation Player Involved in Xenobiotic Detoxification. Int J Environ Res Public Health 18(14). PubMed ID: 34299961
Summary:
Foraging is vital for animals, especially for food. In Drosophila melanogaster, this behavior is controlled by the foraging gene (for) which encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG). In wild populations of Drosophila, rover individuals that exhibit long foraging trails and sitter individuals that exhibit short ones coexist and are characterized by high and low levels of PKG activity, respectively. This study, therefore, postulated that rover flies are more exposed to environmental stresses, including xenobiotics contamination, than sitter flies. Test were performed to see whether these flies differed in their ability to cope with xenobiotics by exposing them to insecticides from different chemical families. Toxicological tests were performed, and the activity and expression levels of different classes of detoxification enzymes was measured. It was shown that a link exists between the for gene and certain cytochrome P450-dependent activities and that the expression of the insecticide-metabolizing cytochrome P450 Cyp6a2 is controlled by the for gene. An unsuspected regulatory pathway of P450s expression involving the for gene in Drosophila is revealed, and its involvement in adaptation to chemicals in the environment was investigated. This work can serve as a basis for reconsidering adaptation to xenobiotics in light of the behavior of species, including humans.

Wednesday October 13th - Signaling

Klemz, S., Wallach, T., Korge, S., Rosing, M., Klemz, R., Maier, B., Fiorenza, N. C., Kaymak, I., Fritzsche, A. K., Herzog, E. D., Stanewsky, R. and Kramer, A. (2021). Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity. Genes Dev 35(15-16): 1161-1174. PubMed ID: 34301769
Summary:
In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, a systematic RNAi screen was performed in human cells and protein phosphatase 4 (PPP4) was identified with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila. Genetic depletion of PPP4 (Pp4-19C in Drosophila) shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 (Cycle in Drosophila) and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.
Li, X., Fetter, R., Schwabe, T., Jung, C., Liu, L., Steller, H. and Gaul, U. (2021). The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation. Elife 10. PubMed ID: 34382936
Summary:
The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previous work identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. This study identified cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. PKA is enriched at the basal side of the SPG cell, and this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. It is proposed that Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity.
Insolera, R., Lorincz, P., Wishnie, A. J., Juhsz, G. and Collins, C. A. (2021). Mitochondrial fission, integrity and completion of mitophagy require separable functions of Vps13D in Drosophila neurons. PLoS Genet 17(8): e1009731. PubMed ID: 34383748
Summary:
A healthy population of mitochondria, maintained by proper fission, fusion, and degradation, is critical for the long-term survival and function of neurons. In this study, the discovery of mitophagy intermediates in fission-impaired Drosophila neurons brings new perspective into the relationship between mitochondrial fission and mitophagy. Neurons lacking either the ataxia disease gene Vps13D or the dynamin related protein Drp1 contain enlarged mitochondria that are engaged with autophagy machinery and also lack matrix components. Reporter assays combined with genetic studies imply that mitophagy both initiates and is completed in Drp1 impaired neurons, but fails to complete in Vps13D impaired neurons, which accumulate compromised mitochondria within stalled mito-phagophores. These findings imply that in fission-defective neurons, mitophagy becomes induced, and that the lipid channel containing protein Vps13D has separable functions in mitochondrial fission and phagophore elongation.
Martins, T., Meng, Y., Korona, B., Suckling, R., Johnson, S., Handford, P. A., Lea, S. M. and Bray, S. J. (2021). The conserved C2 phospholipid-binding domain in Delta contributes to robust Notch signalling. EMBO Rep: e52729. PubMed ID: 34347930
Summary:
Accurate Notch signalling is critical for development and homeostasis. Fine-tuning of Notch-ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N-terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. This study shows that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called β1-2 loop that is involved in phospholipid binding. Mutations in the β1-2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, five residues within the β1-2 loop of endogenous Delta were deleted. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss-of-function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine-tuning the balance of trans and cis ligand-receptor interactions.
Pojer, J. M., Manning, S. A., Kroeger, B., Kondo, S. and Harvey, K. F. (2021). The Hippo pathway uses different machinery to control cell fate and organ size. iScience 24(8): 102830. PubMed ID: 34355153
Summary:
The Hippo pathway is a conserved signaling network that regulates organ growth and cell fate. One such cell fate decision is that of R8 photoreceptor cells in the Drosophila eye, where Hippo specifies whether cells sense blue or green light. This study shows that only a subset of proteins that control organ growth via the Hippo pathway also regulate R8 cell fate choice, including the STRIPAK complex, Tao, Pez, and 14-3-3 proteins. Furthermore, key Hippo pathway proteins were primarily cytoplasmic in R8 cells rather than localized to specific membrane domains, as in cells of growing epithelial organs. Additionally, Warts was the only Hippo pathway protein to be differentially expressed between R8 subtypes, while central Hippo pathway proteins were expressed at dramatically lower levels in adult and pupal eyes than in growing larval eyes. Therefore, this study reveals several important differences in Hippo signaling in the contexts of organ growth and cell fate.
Enomoto, M., Takemoto, D. and Igaki, T. (2021). Interaction between Ras and Src clones causes interdependent tumor malignancy via Notch signaling in Drosophila. Dev Cell 56(15): 2223-2236.e2225. PubMed ID: 34324859
Summary:
Cancer tissue often comprises multiple tumor clones with distinct oncogenic alterations such as Ras or Src activation, yet the mechanism by which tumor heterogeneity drives cancer progression remains elusive. This study shows in Drosophila imaginal epithelium that clones of Ras- or Src-activated benign tumors interact with each other to mutually promote tumor malignancy. Mechanistically, Ras-activated cells upregulate the cell-surface ligand Delta while Src-activated cells upregulate its receptor Notch, leading to Notch activation in Src cells. Elevated Notch signaling induces the transcriptional repressor Zfh1/ZEB1, which downregulates E-cadherin and cell death gene hid, leading to Src-activated invasive tumors. Simultaneously, Notch activation in Src cells upregulates the cytokine Unpaired/IL-6, which activates JAK-STAT signaling in neighboring Ras cells. Elevated JAK-STAT signaling upregulates the BTB-zinc-finger protein Chinmo, which downregulates E-cadherin and thus generates Ras-activated invasive tumors. These findings provide a mechanistic explanation for how tumor heterogeneity triggers tumor progression via cell-cell interactions.

Tuesday, October 12th - RNA

Jung, J. E., Lee, J. Y., Park, H. R., Kang, J. W., Kim, Y. H. and Lee, J. H. (2021). MicroRNA-133 Targets Phosphodiesterase 1C in Drosophila and Human Oral Cancer Cells to Regulate Epithelial-Mesenchymal Transition. J Cancer 12(17): 5296-5309. PubMed ID: 34335946
Summary:
Non-coding microRNAs (miRNAs) have been proposed to play diverse roles in cancer biology, including epithelial-mesenchymal transition (EMT) crucial for cancer progression. Previous comparative studies revealed distinct expression profiles of miRNAs relevant to tumorigenesis and progression of oral cancer. With putative targets of these miRNAs mostly validated in vitro, it remains unclear whether similar miRNA-target relationships exist in vivo. This study employed a hybrid approach, utilizing both Drosophila melanogaster and human oral cancer cells, to validate projected miRNA-target relationships relevant to EMT. Notably, overexpression of dme-miR-133 resulted in significant tissue growth in Drosophila larval wing discs. The RT-PCR analysis successfully validated a subset of its putative targets, including Pde1c. Subsequent experiments performed in oral cancer cells confirmed conserved targeting of human PDE1C by hsa-miR-133. Furthermore, the elevated level of miR-133 and its targeting of PDE1C was positively correlated with enhanced migrative ability of oral cancer cells treated with LPS, along with the molecular signature of a facilitated EMT process induced by LPS and TGF-β. The analysis on the RNAseq data also revealed a negative correlation between the expression level of hsa-miR-133 and the survival of oral cancer patients. Taken together, this mammal-to-Drosophila-to-mammal approach successfully validates targeting of PDE1C by miR-133 both in vivo and in vitro, underlying the promoted EMT phenotypes and potentially influencing the prognosis of oral cancer patients. This hybrid approach will further aid to widen our scope in investigation of intractable human malignancies, including oral cancer.
Cao, D. (2021). Reverse complementary matches simultaneously promote both back-splicing and exon-skipping. BMC Genomics 22(1): 586. PubMed ID: 34344317
Summary:
Circular RNAs (circRNAs) play diverse roles in different biological and physiological environments and are always expressed in a tissue-specific manner. Especially, circRNAs are enriched in the brain tissues of almost all investigated species, including humans, mice, Drosophila, etc. Through large-scale neuron isolation from the first larval (L1) stage of C. elegans followed by RNA sequencing with ribosomal RNA depletion, the neuronal circRNA data in C. elegans were obtained. Hundreds of novel circRNAs were annotated with high accuracy. circRNAs were highly expressed in the neurons of C. elegans and were positively correlated to the levels of their cognate linear mRNAs. Disruption of reverse complementary match (RCM) sequences in circRNA flanking introns effectively abolished circRNA formation. In the zip-2 gene, deletion of either upstream or downstream RCMs almost eliminated the production of both the circular and the skipped transcript. Interestingly, the 13-nt RCM in zip-2 is highly conserved across five nematode ortholog genes, which show conserved exon-skipping patterns. Finally, through in vivo one-by-one mutagenesis of all the splicing sites and branch points required for exon-skipping and back-splicing in the zip-2 gene, this study showed that back-splicing still happened without exon-skipping, and vice versa. Through protocol optimization, total RNA obtained from sorted neurons is increased to hundreds of nanograms. circRNAs highly expressed in the neurons of C. elegans are more likely to be derived from genes also highly expressed in the neurons. RCMs are abundant in circRNA flanking introns, and RCM-deletion is an efficient way to knockout circRNAs. More importantly, these RCMs are not only required for back-splicing but also promote the skipping of exon(s) to be circularized. Finally, RCMs in circRNA flanking introns can directly promote both exon-skipping and back-splicing, providing a new explanation for the correlation between them.
Kim, K., Koo, J., Yoon, J. S. and Palli, S. R. (2021). Coleopteran-specific StaufenC functions like Drosophila melanogaster Loquacious-PD in dsRNA processing. RNA Biol: 1-11. PubMed ID: 34376105
Summary:
In Drosophila melanogaster, PD isoform of the double-stranded RNA binding protein (dsRBP) Loquacious (Loqs-PD) facilitates dsRNA cleavage to siRNA by Dicer-2. StaufenC (StauC) was discovered as a coleopteran-specific dsRBP required for dsRNA processing in coleopteran insects. This study shows that StauC is essential for the high RNAi efficiency observed in coleopterans. Knockdown of StauC but not the homologs of Loqs-PD and R2D2 evoked a long-lasting insensitivity to RNAi in the coleopteran cell line, Ledp-SL1. The dsRNA insensitivity induced by StauC knockdown could not be overcome merely by an increase in dose or time of exposure to dsRNA or expression of Loquacious or R2D2. Furthermore, StauC but not Loqs and R2D2 are required for processing of dsRNA into siRNA. StauC overexpression also partly restored the impaired RNAi caused by the knockdown of Loqs-PD in D. melanogaster Kc cells. However, StauC was unable to compensate for the loss-of-the function of Dcr-2 or R2D2. Overall, these data suggest that StauC functions like Lops-PD in processing dsRNA to siRNA.
Joosten, J., Overheul, G. J., Van Rij, R. P. and Miesen, P. (2021). Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA in Aedes aegypti mosquitoes. Nucleic Acids Res. PubMed ID: 34331446
Summary:
In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3' end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3' ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. This study reports that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3' ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. A reporter system was designed to study viral piRNA 3' end formation, and targeting viral RNA by abundant endogenous piRNAs was found to trigger the production of responder and trailer piRNAs. Using this reporter, the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3' end formation, were identified. These results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.
Gebert, D., Neubert, L. K., Lloyd, C., Gui, J., Lehmann, R. and Teixeira, F. K. (2021). Large Drosophila germline piRNA clusters are evolutionarily labile and dispensable for transposon regulation. Mol Cell. PubMed ID: 34352205
Summary:
PIWI proteins and their guiding Piwi-interacting small RNAs (piRNAs) are crucial for fertility and transposon defense in the animal germline. In most species, the majority of piRNAs are produced from distinct large genomic loci, called piRNA clusters. It is assumed that germline-expressed piRNA clusters, particularly in Drosophila, act as principal regulators to control transposons dispersed across the genome. Using synteny analysis, this study showed that large clusters are evolutionarily labile, arise at loci characterized by recurrent chromosomal rearrangements, and are mostly species-specific across the Drosophila genus. By engineering chromosomal deletions in D. melanogaster, it was demonstrated that the three largest germline clusters, which account for the accumulation of >40% of all transposon-targeting piRNAs in ovaries, are neither required for fertility nor for transposon regulation in trans. Further evidence is provided that dispersed elements, rather than the regulatory action of large Drosophila germline clusters in trans, may be central for transposon defense.
Iwasaki, Y. W., Sriswasdi, S., Kinugasa, Y., Adachi, J., Horikoshi, Y., Shibuya, A., Iwasaki, W., Tashiro, S., Tomonaga, T. and Siomi, H. (2021). Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci. EMBO J: e108345. PubMed ID: 34337769
Summary:
PIWI-interacting RNAs (piRNAs) are germline-specific small RNAs that form effector complexes with PIWI proteins (Piwi-piRNA complexes) and play critical roles for preserving genomic integrity by repressing transposable elements (TEs). Drosophila Piwi transcriptionally silences specific targets through heterochromatin formation and increases histone H3K9 methylation (H3K9me3) and histone H1 deposition at these loci, with nuclear RNA export factor variant Nxf2 serving as a co-factor. Using ChEP and DamID-seq, this study has uncovered a Piwi/Nxf2-dependent target association with nuclear lamins. Hi-C analysis of Piwi or Nxf2-depleted cells reveals decreased intra-TAD and increased inter-TAD interactions in regions harboring Piwi-piRNA target TEs. Using a forced tethering system, the functional effects were analyzed of Piwi-piRNA/Nxf2-mediated recruitment of piRNA target regions to the nuclear periphery. Removal of active histone marks is followed by transcriptional silencing, chromatin conformational changes, and H3K9me3 and H1 association. These data show that the Piwi-piRNA pathway can induce stepwise changes in nuclear architecture and chromatin state at target loci for transcriptional silencing.

Monday October 11th - Disease Models

Goodman, L. D., Cope, H., Nil, Z., Ravenscroft, T. A., Charng, W. L., Lu, S., Tien, A. C., Pfundt, R., Koolen, D. A., Haaxma, C. A., Veenstra-Knol, H. E., Wassink-Ruiter, J. S. K., Wevers, M. R., Jones, M., Walsh, L. E., Klee, V. H., Theunis, M., Legius, E., Steel, D., Barwick, K. E. S., Kurian, M. A., Mohammad, S. S., Dale, R. C., Terhal, P. A., van Binsbergen, E., Kirmse, B., Robinette, B., Cogné, B., Isidor, B., Grebe, T. A., Kulch, P., Hainline, B. E., Sapp, K., Morava, E., Klee, E. W., Macke, E. L., Trapane, P., Spencer, C., Si, Y., Begtrup, A., Moulton, M. J., Dutta, D., Kanca, O., Wangler, M. F., Yamamoto, S., Bellen, H. J. and Tan, Q. K. (2021). TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila. Am J Hum Genet 108(9): 1669-1691. PubMed ID: 34314705
Summary:
Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. This study identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. Fly dTnpo (orthologous to TNPO2) was found to be expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.
Castelli, L. M., Cutillo, L., Souza, C. D. S., Sanchez-Martinez, A., Granata, I., Lin, Y. H., Myszczynska, M. A., Heath, P. R., Livesey, M. R., Ning, K., Azzouz, M., Shaw, P. J., Guarracino, M. R., Whitworth, A. J., Ferraiuolo, L., Milo, M. and Hautbergue, G. M. (2021). SRSF1-dependent inhibition of C9ORF72-repeat RNA nuclear export: genome-wide mechanisms for neuroprotection in amyotrophic lateral sclerosis. Mol Neurodegener 16(1): 53. PubMed ID: 34376242
Summary:
Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. This study investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of a gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. This study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. This study also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1-3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.
Gondal, M. N., Butt, R. N., Shah, O. S., Sultan, M. U., Mustafa, G., Nasir, Z., Hussain, R., Khawar, H., Qazi, R., Tariq, M., Faisal, A. and Chaudhary, S. U. (2021). A Personalized Therapeutics Approach Using an In Silico Drosophila Patient Model Reveals Optimal Chemo- and Targeted Therapy Combinations for Colorectal Cancer. Front Oncol 11: 692592. PubMed ID: 34336681
Summary:
In silico models of biomolecular regulation in cancer, annotated with patient-specific gene expression data, can aid in the development of novel personalized cancer therapeutic strategies. Drosophila melanogaster is a well-established animal model that is increasingly being employed to evaluate such preclinical personalized cancer therapies. This study reports five Boolean network models of biomolecular regulation in cells lining the Drosophila midgut epithelium and annotate them with colorectal cancer patient-specific mutation data to develop an in silico Drosophila Patient Model (DPM). Cell-type-specific RNA-seq gene expression data from the FlyGut-seq database were employed to annotate and then validate these networks. Next, three literature-based colorectal cancer case studies were used to evaluate cell fate outcomes from the model. Results obtained from analyses of the proposed DPM help: (i) elucidate cell fate evolution in colorectal tumorigenesis, (ii) validate cytotoxicity of nine FDA-approved CRC drugs, and (iii) devise optimal personalized treatment combinations. The personalized network models helped identify synergistic combinations of paclitaxel-regorafenib, paclitaxel-bortezomib, docetaxel-bortezomib, and paclitaxel-imatinib for treating different colorectal cancer patients. Follow-on therapeutic screening of six colorectal cancer patients from cBioPortal using this drug combination demonstrated a 100% increase in apoptosis and a 100% decrease in proliferation. In conclusion, this work outlines a novel roadmap for decoding colorectal tumorigenesis along with the development of personalized combinatorial therapeutics for preclinical translational studies.
Ortega-Arellano, H. F., Jimenez-Del-Rio, M. and Velez-Pardo, C. (2021). Melatonin Increases Life Span, Restores the Locomotor Activity, and Reduces Lipid Peroxidation (LPO) in Transgenic Knockdown Parkin Drosophila melanogaster Exposed to Paraquat or Paraquat/Iron. Neurotox Res. PubMed ID: 34339012
Summary:
Parkinson's disease (PD) is a complex progressive neurodegenerative disorder involving impairment of bodily movement caused by the specific destruction of dopaminergic (DAergic) neurons. Mounting evidence suggests that PD might be triggered by an interplay between environmental neurotoxicants (e.g., paraquat, PQ), heavy metals (e.g., iron), and gene alterations (e.g., PARKIN gene). Unfortunately, there are no therapies currently available that protect, slow, delay, or prevent the progression of PD. Melatonin (Mel, N-acetyl-5-methoxy tryptamine) is a natural hormone with pleiotropic functions including receptor-independent pathways which might be useful in the treatment of PD. Therefore, as a chemical molecule, it has been shown that Mel prolonged the lifespan and locomotor activity, and reduced lipid peroxidation (LPO) in wild-type Canton-S flies exposed to PQ, suggesting antioxidant and neuroprotective properties. However, it is not yet known whether Mel can protect or prevent the genetic model parkin deficient in flies against oxidative stress (OS) stimuli. This study shows that Mel (0.5, 1, 3 mM) significantly extends the life span and locomotor activity of TH > parkin-RNAi/ + Drosophila melanogaster flies (> 15 days) compared to untreated flies. Knock-down (K-D) parkin flies treated with PQ (1 mM) or PQ (1 mM)/iron (1 mM) significantly diminished the survival index and climbing abilities (e.g., 50% of flies were dead and locomotor impairment by days 4 and 3, respectively). Remarkably, Mel reverted the noxious effect of PQ or PQ/iron combination in K-D parkin. Indeed, Mel protects TH > parkin-RNAi/  + Drosophila melanogaster flies against PQ- or PQ/iron-induced diminish survival, locomotor impairment, and LPO (e.g., 50% of flies were death and locomotor impairment by days 6 and 9, respectively). Similarly, Mel prevented K-D parkin flies against both PQ and PQ/iron. Taken together, these findings suggest that Mel can be safely used as an antioxidant and neuroprotectant agent against OS-stimuli in selective individuals at risk to suffer early-onset Parkinsonism and PD.
Tan, S., Kermasson, L., Hilcenko, C., Kargas, V., Traynor, D., Boukerrou, A. Z., Escudero-Urquijo, N., Faille, A., Bertrand, A., Rossmann, M., Goyenechea, B., Jin, L., Moreil, J., Alibeu, O., Beaupain, B., Bole-Feysot, C., Fumagalli, S., Kaltenbach, S., Martignoles, J. A., Masson, C., Nitschke, P., Parisot, M., Pouliet, A., Radford-Weiss, I., Tores, F., de Villartay, J. P., Zarhrate, M., Koh, A. L., Phua, K. B., Reversade, B., Bond, P. J., Bellanne-Chantelot, C., Callebaut, I., Delhommeau, F., Donadieu, J., Warren, A. J. and Revy, P. (2021). Somatic genetic rescue of a germline ribosome assembly defect. Nat Commun 12(1): 5044. PubMed ID: 34413298
Summary:
Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. This study established that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 (see Drosophila eIF6) from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. This study identified diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. These data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS. x
Hall, H., Cooper, B. R., Qi, G., Wijeratne, A. B., Mosley, A. L. and Weake, V. M. (2021). Quantitative Proteomic and Metabolomic Profiling Reveals Altered Mitochondrial Metabolism and Folate Biosynthesis Pathways in the Aging Drosophila Eye. Mol Cell Proteomics 20: 100127. PubMed ID: 34332122
Summary:
Aging is associated with increased risk of ocular disease, suggesting that age-associated molecular changes in the eye increase its vulnerability to damage. Although there are common pathways involved in aging at an organismal level, different tissues and cell types exhibit specific changes in gene expression with advanced age. Drosophila melanogaster is an established model system for studying aging and neurodegenerative disease that also provides a valuable model for studying age-associated ocular disease. Flies, like humans, exhibit decreased visual function and increased risk of retinal degeneration with age. This study profiled the aging proteome and metabolome of the Drosophila eye and compared these data with age-associated transcriptomic changes from both eyes and photoreceptors to identify alterations in pathways that could lead to age-related phenotypes in the eye. Of note, the proteomic and metabolomic changes observed in the aging eye are distinct from those observed in the head or whole fly, suggesting that tissue-specific changes in protein abundance and metabolism occur in the aging fly. This integration of the proteomic, metabolomic, and transcriptomic data reveals that changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of the immune response, may underpin many of the events observed in the aging Drosophila eye. It is proposed that targeting these pathways in the genetically tractable Drosophila system may help to identify potential neuroprotective approaches for neurodegenerative and age-related ocular diseases. Data are available via ProteomeXchange with identifier PXD027090.
Gavory, G., Baril, C., Laberge, G., Bidla, G., Koonpaew, S., Sonea, T., Sauvageau, G. and Therrien, M. (2021). A genetic screen in Drosophila uncovers the multifaceted properties of the NUP98-HOXA9 oncogene. PLoS Genet 17(8): e1009730. PubMed ID: 34383740
Summary:
Acute myeloid leukemia (AML) underlies the uncontrolled accumulation of immature myeloid blasts. Several cytogenetic abnormalities have been associated with AML. Among these is the NUP98-HOXA9 (NA9) translocation that fuses the Phe-Gly repeats of nucleoporin NUP98 (see Drosophila Nup98-96) to the homeodomain of the transcription factor HOXA9 (see Drosophila Abd-B). The mechanisms enabling NA9-induced leukemia are poorly understood. A genetic screen in Drosophila was conducted for modifiers of NA9. The screen uncovered 29 complementation groups, including genes with mammalian homologs known to impinge on NA9 activity. Markedly, the modifiers encompassed a diversity of functional categories, suggesting that NA9 perturbs multiple intracellular events. Unexpectedly, this study discovered that NA9 promotes cell fate transdetermination and that this phenomenon is greatly influenced by NA9 modifiers involved in epigenetic regulation. Together, this work reveals a network of genes functionally connected to NA9 that not only provides insights into its mechanism of action, but also represents potential therapeutic targets.
Yamashita, K., Oi, A., Kosakamoto, H., Yamauchi, T., Kadoguchi, H., Kuraishi, T., Miura, M. and Obata, F. (2021). Activation of innate immunity during development induces unresolved dysbiotic inflammatory gut and shortens lifespan. Dis Model Mech 14(9). PubMed ID: 34448472
Summary:
An early-life inflammatory response is associated with risks of age-related pathologies. How transient immune signalling activity during animal development influences life-long fitness is not well understood. Using Drosophila as a model, this study found that activation of innate immune pathway Immune deficiency (Imd) signalling in the developing larvae increases adult starvation resistance, decreases food intake and shortens organismal lifespan. Interestingly, lifespan is shortened by Imd activation in the larval gut and fat body, whereas starvation resistance and food intake are altered by that in neurons. The adult flies that developed with Imd activation show sustained Imd activity in the gut, despite complete tissue renewal during metamorphosis. The larval Imd activation increases an immunostimulative bacterial species, Gluconobacter sp., in the gut microbiome, and this dysbiosis is persistent to adulthood. Removal of gut microbiota by antibiotics in the adult fly mitigates intestinal immune activation and rescues the shortened lifespan. This study demonstrates that early-life immune activation triggers long-term physiological changes, highlighted as an irreversible alteration in gut microbiota, prolonged inflammatory intestine and concomitant shortening of the organismal lifespan.

Friday, October 8th - Evolution

Perez-Pereira, N., Pouso, R., Rus, A., Vilas, A., Lopez-Cortegano, E., Garcia-Dorado, A., Quesada, H. and Caballero, A. (2021). Long-term exhaustion of the inbreeding load in Drosophila melanogaster. Heredity (Edinb). PubMed ID: 34400819
Summary:
Inbreeding depression, the decline in fitness of inbred individuals, is a ubiquitous phenomenon of great relevance in evolutionary biology and in the fields of animal and plant breeding and conservation. Inbreeding depression is due to the expression of recessive deleterious alleles that are concealed in heterozygous state in noninbred individuals, the so-called inbreeding load. Genetic purging reduces inbreeding depression by removing these alleles when expressed in homozygosis due to inbreeding. It is generally thought that fast inbreeding (such as that generated by full-sib mating lines) removes only highly deleterious recessive alleles, while slow inbreeding can also remove mildly deleterious ones. However, a question remains regarding which proportion of the inbreeding load can be removed by purging under slow inbreeding in moderately large populations. This study reports results of two long-term slow inbreeding Drosophila experiments (125-234 generations), each using a large population and a number of derived lines with effective sizes about 1000 and 50, respectively. The inbreeding load was virtually exhausted after more than one hundred generations in large populations and between a few tens and over one hundred generations in the lines. This result is not expected from genetic drift alone, and is in agreement with the theoretical purging predictions. Computer simulations suggest that these results are consistent with a model of relatively few deleterious mutations of large homozygous effects and partially recessive gene action.
Ahlawat, N., Geeta Arun, M., Maggu, K. and Prasad, N. G. (2021). Enemies make you stronger: Coevolution between fruit fly host and bacterial pathogen increases postinfection survivorship in the host. Ecol Evol 11(14): 9563-9574. PubMed ID: 34306643
Summary:
Multiple laboratory studies have evolved hosts against a nonevolving pathogen to address questions about evolution of immune responses. However, an ecologically more relevant scenario is one where hosts and pathogens can coevolve. Such coevolution between the antagonists, depending on the mutual selection pressure and additive variance in the respective populations, can potentially lead to a different pattern of evolution in the hosts compared to a situation where the host evolves against a nonevolving pathogen. This study used Drosophila melanogaster as the host and Pseudomonas entomophila as the pathogen. The host populations were allowed either to evolve against a nonevolving pathogen or tocoevolve with the same pathogen. It was found that the coevolving hosts on average evolved higher survivorship against the coevolving pathogen and ancestral (nonevolving) pathogen relative to the hosts evolving against a nonevolving pathogen. The coevolving pathogens evolved greater ability to induce host mortality even in nonlocal (novel) hosts compared to infection by an ancestral (nonevolving) pathogen. Thus, these results clearly show that the evolved traits in the host and the pathogen under coevolution can be different from one-sided adaptation. In addition, the results also show that the coevolving host-pathogen interactions can involve certain general mechanisms in the pathogen, leading to increased mortality induction in nonlocal or novel hosts.
Muirhead, C. A. and Presgraves, D. C. (2021). Satellite DNA-mediated diversification of a sex-ratio meiotic drive gene family in Drosophila. Nat Ecol Evol. PubMed ID: 34489561
Summary:
Sex chromosomes are susceptible to the evolution of selfish meiotic drive elements that bias transmission and distort progeny sex ratios. Conflict between such sex-ratio drivers and the rest of the genome can trigger evolutionary arms races resulting in genetically suppressed 'cryptic' drive systems. The Winters cryptic sex-ratio drive system of Drosophila simulans comprises a driver, Distorter on the X (Dox) and an autosomal suppressor, Not much yang, a retroduplicate of Dox that suppresses via production of endogenous small interfering RNAs (esiRNAs). This study reports that over 22 Dox-like (Dxl) sequences originated, amplified and diversified over the ~250,000-year history of the three closely related species, D. simulans, D. mauritiana and D. sechellia. The Dxl sequences encode a rapidly evolving family of protamines. Dxl copy numbers amplified by ectopic exchange among euchromatic islands of satellite DNAs on the X chromosome and separately spawned four esiRNA-producing suppressors on the autosomes. These results reveal the genomic consequences of evolutionary arms races and highlight complex interactions among different classes of selfish DNAs.
Finet, C., Kassner, V. A., Carvalho, A. B., Chung, H., Day, J. P., Day, S., Delaney, E. K., De Ré, F. C., Dufour, H. D., Dupim, E., Izumitani, H. F., Gauterio, T. B., Justen, J., Katoh, T., Kopp, A., Koshikawa, S., Longdon, B., Loreto, E. L., Nunes, M. D. S., Raja, K. K. B., Rebeiz, M., Ritchie, M. G., Saakyan, G., Sneddon, T., Teramoto, M., Tyukmaeva, V., Vanderlinde, T., Wey, E. E., Werner, T., Williams, T. M., Robe, L. J., Toda, M. J. and Marletaz, F. (2021). DrosoPhyla: Resources for Drosophilid Phylogeny and Systematics. Genome Biol Evol 13(8). PubMed ID: 34343293
Summary:
The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4,400 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the genera, subgenera, and species groups in the Drosophilidae. To clarify these relationships, a set of new genomic markers was developed and a multilocus data set of 17 genes was assembled from 704 species of Drosophilidae. A species tree was inferred with highly supported groups for this family. Additionally, it was possible to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.
Villa, R., Jagtap, P. K. A., Thomae, A. W., Campos Sparr, A., Forne, I., Hennig, J., Straub, T. and Becker, P. B. (2021). Divergent evolution toward sex chromosome-specific gene regulation in Drosophila. Genes Dev 35(13-14): 1055-1070. PubMed ID: 34140353
Summary:
The dosage compensation complex (DCC) of Drosophila identifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from the D. melanogaster model: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. This study found that in D. virilis, a species separated from melanogaster by 40 million years of evolution, all principles are active but contribute differently to X specificity. In melanogaster, the DCC subunit MSL2 evolved intrinsic DNA-binding selectivity for rare PionX sites, which mark the X chromosome. In virilis, PionX motifs are abundant and not X-enriched. Accordingly, MSL2 lacks specific recognition. Here, roX2 RNA plays a more instructive role, counteracting a nonproductive interaction of CLAMP and modulating DCC binding selectivity. Remarkably, roX2 triggers a stable chromatin binding mode characteristic of DCC. Evidently, X-specific regulation is achieved by divergent evolution of protein, DNA, and RNA components.
Matsuo, Y., Nose, A. and Kohsaka, H. (2021). Interspecies variation of larval locomotion kinematics in the genus Drosophila and its relation to habitat temperature. BMC Biol 19(1): 176. PubMed ID: 34470643
Summary:
Speed and trajectory of locomotion are the characteristic traits of individual species. Locomotion kinematics may have been shaped during evolution towards increased survival in the habitats of each species. Although kinematics of locomotion is thought to be influenced by habitats, the quantitative relation between the kinematics and environmental factors has not been fully revealed. Comparative analyses of larval locomotion was performed in 11 Drosophila species. Larval locomotion kinematics was found to be divergent among the species. The diversity is not correlated to the body length but is correlated instead to the habitat temperature of the species. Phylogenetic analyses using Bayesian inference suggest that the evolutionary rate of the kinematics is diverse among phylogenetic tree branches. The results of this study imply that the kinematics of larval locomotion has diverged in the evolutionary history of the genus Drosophila and evolved under the effects of the ambient temperature of habitats.

Thursday, October 7th - Adult neural development and function

Deng, Q., Tan, Y. S., Chew, L. Y. and Wang, H. (2021). Msps governs acentrosomal microtubule assembly and reactivation of quiescent neural stem cells. Embo j: e104549. PubMed ID: 34368973
Summary:
The ability of stem cells to switch between quiescence and proliferation is crucial for tissue homeostasis and regeneration. Drosophila quiescent neural stem cells (NSCs) extend a primary cellular protrusion from the cell body prior to their reactivation. However, the structure and function of this protrusion are not well established. This study shows that in the protrusion of quiescent NSCs, microtubules are predominantly acentrosomal and oriented plus-end-out toward the tip of the primary protrusion. This study has identified Mini Spindles (Msps)/XMAP215 as a key microtubule regulator in quiescent NSCs that governs NSC reactivation via regulating acentrosomal microtubule growth and orientation. Quiescent NSCs form membrane contact with the neuropil and E-cadherin, a cell adhesion molecule, localizes to these NSC-neuropil junctions. Msps and a plus-end directed motor protein Kinesin-2 promote NSC cell cycle re-entry and target E-cadherin to NSC-neuropil contact during NSC reactivation. Together, this work establishes acentrosomal microtubule organization in the primary protrusion of quiescent NSCs and the Msps-Kinesin-2 pathway that governs NSC reactivation, in part, by targeting E-cad to NSC-neuropil contact sites.
Das, R., Bhattacharjee, S., Letcher, J. M., Harris, J. M., Nanda, S., Foldi, I., Lottes, E. N., Bobo, H. M., Grantier, B. D., Mihaly, J., Ascoli, G. A. and Cox, D. N. (2021). Formin 3 directs dendritic architecture via microtubule regulation and is required for somatosensory nociceptive behavior. Development 148(16). PubMed ID: 34322714
Summary:
Dendrite shape impacts functional connectivity and is mediated by organization and dynamics of cytoskeletal fibers. Identifying the molecular factors that regulate dendritic cytoskeletal architecture is therefore important in understanding the mechanistic links between cytoskeletal organization and neuronal function. This study identified Formin 3 (Form3) as an essential regulator of cytoskeletal architecture in nociceptive sensory neurons in Drosophila larvae. Time course analyses reveal that Form3 is cell-autonomously required to promote dendritic arbor complexity. form3 is required for the maintenance of a population of stable dendritic microtubules (MTs), and mutants exhibit defects in the localization of dendritic mitochondria, satellite Golgi, and the TRPA channel Painless. Form3 directly interacts with MTs via FH1-FH2 domains. Mutations in human inverted formin 2 (INF2; ortholog of form3) have been causally linked to Charcot-Marie-Tooth (CMT) disease. CMT sensory neuropathies lead to impaired peripheral sensitivity. Defects in form3 function in nociceptive neurons result in severe impairment of noxious heat-evoked behaviors. Expression of the INF2 FH1-FH2 domains partially recovers form3 defects in MTs and nocifensive behavior, suggesting conserved functions, thereby providing putative mechanistic insights into potential etiologies of CMT sensory neuropathies.
Chew, L. Y., Zhang, H., He, J. and Yu, F. (2021). The Nrf2-Keap1 pathway is activated by steroid hormone signaling to govern neuronal remodeling. Cell Rep 36(5): 109466. PubMed ID: 34348164
Summary:
The evolutionarily conserved Nrf2-Keap1 pathway is a key antioxidant response pathway that protects cells/organisms against detrimental effects of oxidative stress. Impaired Nrf2 function is associated with cancer and neurodegenerative diseases in humans. However, the function of the Nrf2-Keap1 pathway in the developing nervous systems has not been established. This study demonstrates a cell-autonomous role of the Nrf2-Keap1 pathway, composed of CncC/Nrf2, Keap1, and MafS, in governing neuronal remodeling during Drosophila metamorphosis. Nrf2-Keap1 signaling is activated downstream of the steroid hormone ecdysone. Mechanistically, the Nrf2-Keap1 pathway is activated via cytoplasmic-to-nuclear translocation of CncC in an importin- and ecdysone-signaling-dependent manner. Moreover, Nrf2-Keap1 signaling regulates dendrite pruning independent of its canonical antioxidant response pathway, acting instead through proteasomal degradation. This study reveals an epistatic link between the Nrf2-Keap1 pathway and steroid hormone signaling and demonstrates an antioxidant-independent but proteasome-dependent role of the Nrf2-Keap1 pathway in neuronal remodeling.
Jiang, L. and Litwin-Kumar, A. (2021). Models of heterogeneous dopamine signaling in an insect learning and memory center. PLoS Comput Biol 17(8): e1009205. PubMed ID: 34375329
Summary:
The Drosophila mushroom body exhibits dopamine dependent synaptic plasticity that underlies the acquisition of associative memories. Recordings of dopamine neurons in this system have identified signals related to external reinforcement such as reward and punishment. However, other factors including locomotion, novelty, reward expectation, and internal state have also recently been shown to modulate dopamine neurons. This heterogeneity is at odds with typical modeling approaches in which these neurons are assumed to encode a global, scalar error signal. How is dopamine dependent plasticity coordinated in the presence of such heterogeneity? This study developed a modeling approach that infers a pattern of dopamine activity sufficient to solve defined behavioral tasks, given architectural constraints informed by knowledge of mushroom body circuitry. Model dopamine neurons exhibit diverse tuning to task parameters while nonetheless producing coherent learned behaviors. Notably, reward prediction error emerges as a mode of population activity distributed across these neurons. These results provide a mechanistic framework that accounts for the heterogeneity of dopamine activity during learning and behavior.
Han, R., Huang, H. P., Chuang, C. L., Yen, H. H., Kao, W. T., Chang, H. Y. and Lo, C. C. (2021). Coordination through Inhibition: Control of Stabilizing and Updating Circuits in Spatial Orientation Working Memory. eNeuro 8(5). PubMed ID: 34385152
Summary:
Spatial orientation memory plays a crucial role in animal navigation. Recent studies of tethered Drosophila melanogaster (fruit fly) in a virtual reality setting showed that the head direction is encoded in the form of an activity bump, i.e., localized neural activity, in the torus-shaped ellipsoid body (EB). However, how this system is involved in orientation working memory is not well understood. This study investigated this question using free moving flies (D. melanogaster) in a spatial orientation memory task by manipulating two EB subsystems, C and P circuits, which are hypothesized for stabilizing and updating the activity bump, respectively. To this end, two types of inhibitory ring neurons (EIP and P) which innervate EB were suppressed or activated, and it was discovered that manipulating the two inhibitory neuron types produced distinct behavioral deficits, suggesting specific roles of the inhibitory neurons in coordinating the stabilization and updating functions of the EB circuits. The neural mechanisms underlying such control circuits were further elucidated using a connectome-constrained spiking neural network model.
Izadifar, A., Courchet, J., Virga, D. M., Verreet, T., Hamilton, S., Ayaz, D., Misbaer, A., Vandenbogaerde, S., Monteiro, L., Petrovic, M., Sachse, S., Yan, B., Erfurth, M. L., Dascenco, D., Kise, Y., Yan, J., Edwards-Faret, G., Lewis, T., Polleux, F. and Schmucker, D. (2021). Axon morphogenesis and maintenance require an evolutionary conserved safeguard function of Wnk kinases antagonizing Sarm and Axed. Neuron. PubMed ID: 34384519
Summary:
The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. This study reports a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. This study uncovered that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons.

Wednesday, October 6th - Gonads

Hayashi, Y., Shibata, A., Kamimura, K. and Kobayashi, S. (2021). Heparan sulfate proteoglycan molecules, syndecan and perlecan, have distinct roles in the maintenance of Drosophila germline stem cells. Dev Growth Differ 63(6): 295-305. PubMed ID: 34324711
Summary:
The Drosophila female germline stem cell (GSC) niche provides an excellent model for understanding the stem cell niche in vivo. The GSC niche is composed of stromal cells that provide growth factors for the maintenance of GSCs and the associated extracellular matrix (ECM). Although the function of stromal cells/growth factors has been well studied, the function of the ECM in the GSC niche is largely unknown. This study investigated the function of syndecan and perlecan, molecules of the heparan sulfate proteoglycan (HSPG) family, as the main constituents of the ECM. Both of these genes were expressed in niche stromal cells, and knockdown of them in stromal cells decreased GSC number, indicating that these genes are important niche components. Interestingly, genetic analysis revealed that the effects of syndecan and perlecan on the maintenance of GSC were distinct. While the knockdown of perlecan in the GSC niche increased the number of cystoblasts, a phenotype suggestive of delayed differentiation of GSCs, the same was not true in the context of syndecan. Notably, the overexpression of syndecan and perlecan did not cause an expansion of the GSC niche, opposing the results reported in the context of glypican, another HSPG gene. Altogether, these data suggest that HSPG genes contribute to the maintenance of GSCs through multiple mechanisms, such as the control of signal transduction, and ligand distribution/stabilization. Therefore, this study paves the way for a deeper understanding of the ECM functions in the stem cell niche.
Hodge, S. H., Watts, A., Marley, R., Baines, R. A., Hafen, E. and MacDougall, L. K. (2021). Twitchy, the Drosophila orthologue of the ciliary gating protein FBF1/dyf-19, is required for coordinated locomotion and male fertility. Biol Open 10(8). PubMed ID: 34357392
Summary:
Primary cilia are compartmentalised from the rest of the cell by a ciliary gate comprising transition fibres and a transition zone. The ciliary gate allows the selective import and export of molecules such as transmembrane receptors and transport proteins. These are required for the assembly of the cilium, its function as a sensory and signalling centre, and to maintain its distinctive composition. Certain motile cilia can also form within the cytosol as exemplified by human and Drosophila sperm. The role of transition fibre proteins has not been well described in the cytoplasmic cilia. Drosophila have both compartmentalised primary cilia, in sensory neurons, and sperm flagella that form within the cytosol. This study describes phenotypes for twitchy the Drosophila orthologue of a transition fibre protein, mammalian FBF1/C. elegans dyf-19. Loss-of-function mutants in twitchy are adult lethal and display a severely uncoordinated phenotype. Twitchy flies are too uncoordinated to mate but RNAi-mediated loss of twitchy specifically within the male germline results in coordinated but infertile adults. Examination of sperm from twitchy RNAi-knockdown flies shows that the flagellar axoneme forms, elongates and is post-translationally modified by polyglycylation but the production of motile sperm is impaired. These results indicate that twitchy is required for the function of both sensory cilia that are compartmentalised from the rest of the cell and sperm flagella that are formed within the cytosol of the cell. Twitchy is therefore likely to function as part of a molecular gate in sensory neurons but may have a distinct function in sperm cells.
Rivard, E. L., Ludwig, A. G., Patel, P. H., Grandchamp, A., Arnold, S. E., Berger, A., Scott, E. M., Kelly, B. J., Mascha, G. C., Bornberg-Bauer, E. and Findlay, G. D. (2021). A putative de novo evolved gene required for spermatid chromatin condensation in Drosophila melanogaster. PLoS Genet 17(9): e1009787. PubMed ID: 34478447
Summary:
Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, a functional genetic screen was conducted of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas (CG13541), required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis.
Witt, E., Shao, Z., Hu, C., Krause, H. M. and Zhao, L. (2021). Single-cell RNA-sequencing reveals pre-meiotic X-chromosome dosage compensation in Drosophila testis. PLoS Genet 17(8): e1009728. PubMed ID: 34403408
Summary:
Dosage compensation equalizes X-linked expression between XY males and XX females. In male fruit flies, expression levels of the X-chromosome are increased approximately two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis; however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, testis single-cell RNA-sequencing (scRNA-seq) data was analyzed from two Drosophila melanogaster strains. Evidence was found that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. Low or undetectable levels of germline expression of most msl genes, mle, roX1 and roX2 were found via scRNA-seq and RNA-FISH, and no evidence was found of germline nuclear roX1/2 localization. These results suggest that, although dosage compensation occurs in somatic and pre-meiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation mechanism. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in a database.
Gilmutdinov, R. A., Kozlov, E. N., Yakovlev, K. V., Olenina, L. V., Kotov, A. A., Barr, J., Zhukova, M. V., Schedl, P. and Shidlovskii, Y. V. (2021). The 3'UTR of the orb2 gene encoding the Drosophila CPEB translation factor plays a critical role in spermatogenesis. Development. PubMed ID: 34473243
Summary:
CPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins in Drosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later process orb2 mRNAs and proteins are localized within the developing spermatid. To evaluate the role of orb2 mRNA 3'UTR in spermatogenesis, the CRISPR/Cas9 system was used to generate a deletion of the orb2 3'UTR, orb2R. This deletion disrupts the process of spermatid differentiation but has no apparent effect on meiosis. Differentiation abnormalities include defects in the initial polarization of the 64-cell spermatid cysts, mislocalization of mRNAs and proteins in the elongating spermatid tails, altered morphology of the elongating spermatid tails, and defects in the assembly of the individualization complex. These disruptions in differentiation appear to arise because orb2 mRNAs and proteins are not properly localized within the 64-cell spermatid cyst.
Slaidina, M., Gupta, S., Banisch, T. U. and Lehmann, R. (2021). A single-cell atlas reveals unanticipated cell type complexity in Drosophila ovaries. Genome Res. PubMed ID: 34389661
Summary:
Organ function relies on the spatial organization and functional coordination of numerous cell types. The Drosophila ovary is a widely used model system to study the cellular activities underlying organ function, including stem cell regulation, cell signaling and epithelial morphogenesis. However, the relative paucity of cell type-specific reagents hinders investigation of molecular functions at the appropriate cellular resolution. This study used single-cell RNA sequencing to characterize all cell types of the stem cell compartment and early follicles of the Drosophila ovary. Transcriptional signatures were computed, and specific markers were identified for nine states of germ cell differentiation, and 23 somatic cell types and subtypes. An unanticipated diversity was uncovered of escort cells, the somatic cells that directly interact with differentiating germline cysts. Three escort cell subtypes reside in discrete anatomical positions, and they express distinct sets of secreted and transmembrane proteins, suggesting that diverse micro-environments support the progressive differentiation of germ cells. Finally, 17 follicle cell subtypes were uncovered, and their transcriptional profiles were characterized. Altogether, this study provides a comprehensive resource of gene expression, cell type-specific markers, spatial coordinates and functional predictions for 34 ovarian cell types and subtypes.

Tuesday, October 5th - Adult neural development and function

Cheriyamkunnel, S. J., Rose, S., Jacob, P. F., Blackburn, L. A., Glasgow, S., Moorse, J., Winstanley, M., Moynihan, P. J., Waddell, S. and Rezaval, C. (2021). A neuronal mechanism controlling the choice between feeding and sexual behaviors in Drosophila. Curr Biol. PubMed ID: 34358444
Summary:
Animals must express the appropriate behavior that meets their most pressing physiological needs and their environmental context. However, it is currently unclear how alternative behavioral options are evaluated and appropriate actions are prioritized. This study describes how fruit flies choose between feeding and courtship; two behaviors necessary for survival and reproduction. Sex- and food-deprived male flies were shown to prioritize feeding over courtship initiation, and manipulation of food quality or the animal's internal state fine-tunes this decision. The tyramine signaling pathway was identified as an essential mediator of this decision. Tyramine biosynthesis is regulated by the fly's nutritional state and acts as a satiety signal, favoring courtship over feeding. Tyramine inhibits a subset of feeding-promoting tyramine receptor (TyrR)-expressing neurons and activates P1 neurons, a known command center for courtship. Conversely, the perception of a nutritious food source activates TyrR neurons and inhibits P1 neurons. Therefore, TyrR and P1 neurons are oppositely modulated by starvation, via tyramine levels, and food availability. It is proposed that antagonistic co-regulation of neurons controlling alternative actions is key to prioritizing competing drives in a context- dependent manner.
Vaze, K. M. and Helfrich-Forster, C. (2021). The Neuropeptide PDF Is Crucial for Delaying the Phase of Drosophila's Evening Neurons Under Long Zeitgeber Periods. J Biol Rhythms: 7487304211032336. PubMed ID: 34428956
Summary:
Full comprehension of circadian clocks function requires precise understanding of their entrainment to the environment. The phase of entrained clock is plastic, which depends on different factors such as the period of endogenous oscillator, the period of the zeitgeber cycle (T), and the proportion of light and darkness (day length). This study investigated the importance of the neuropeptide Pigment-Dispersing Factor (PDF) for entrainment by systematically studying locomotor activity rhythms of Pdf mutants and wild-type flies under different T-cycles (T22 to T32) and different day lengths (8, 12, and 16 hour [h]). Furthermore, this study analysed Period protein oscillations in selected groups of clock neurons in both genotypes under T24 and T32 at a day length of 16 h. As expected, it was found that the phase of Drosophila's evening activity and evening neurons advanced with increasing T in all the day lengths. This advance was much larger in Pdf mutants (~7 h) than in wild-type flies causing (1) pronounced desynchrony between morning and evening neurons and (2) evening activity to move in the morning instead of the evening. Most interestingly, it was found that the lights-off transition determines the phase of evening neurons in both genotypes and that PDF appears necessary to delay the evening neurons by ~3 h to their wild-type phase. Thus, in T32, PDF first delays the molecular cycling in the evening neurons, and then, as shown in previous studies, delays their neuronal firing rhythms to produce a total delay of ~7 h necessary for a wild-type evening activity phase. It is concluded that PDF is crucial for appropriate phasing of Drosophila activity rhythm.
Polcownuk, S., Yoshii, T. and Ceriani, M. F. (2021). DPP acutely defines the connectivity of central pacemaker neurons in Drosophila. J Neurosci. PubMed ID: 34429376
Summary:
Rhythmic rest-activity cycles are controlled by an endogenous clock. In Drosophila, this clock resides in ∼150 neurons organized in clusters whose hierarchy changes in response to environmental conditions. The concerted activity of the circadian network is necessary for the adaptive responses to synchronizing environmental stimuli. Thus far work was devoted to unravel the logic of the coordination of different clusters focusing on neurotransmitters and neuropeptides. This study further explored communication in the adult male brain through ligands belonging to the Bone Morphogenetic Protein (BMP) pathway. This study shows that the Lateral Neurons ventral (LNvs) express the small morphogen Decapentaplegic (DPP). DPP expression in the large LNvs triggered a period lengthening phenotype, while its downregulation caused reduced rhythmicity and affected anticipation at dawn and dusk, underscoring DPP per se conveys time-of-day relevant information. Surprisingly, its expression in the large LNvs impaired circadian remodeling of the small LNv axonal terminals, likely through local modulation of the guanine nucleotide exchange factor (GEF) Trio. These findings open the provocative possibility that the BMP pathway is recruited to strengthen/reduce the connectivity among specific clusters along the day and thus modulate their contribution to the circadian network.
Sterne, G. R., Otsuna, H., Dickson, B. J. and Scott, K. (2021). Classification and genetic targeting of cell types in the primary taste and premotor center of the adult Drosophila brain. Elife 10. PubMed ID: 34473057
Summary:
Neural circuits carry out complex computations that allow animals to evaluate food, select mates, move toward attractive stimuli, and move away from threats. In insects, the subesophageal zone (SEZ) is a brain region that receives gustatory, pheromonal, and mechanosensory inputs and contributes to the control of diverse behaviors, including feeding, grooming, and locomotion. Despite its importance in sensorimotor transformations, the study of SEZ circuits has been hindered by limited knowledge of the underlying diversity of SEZ neurons. This study generate a collection of split-GAL4 lines that provides precise genetic targeting of 138 different SEZ cell types in adult D. melanogaster, comprising approximately one third of all SEZ neurons. The single cell anatomy of these neurons was characterized, and they were found to cluster by morphology into six supergroups that organize the SEZ into discrete anatomical domains. The majority of local SEZ interneurons are not classically polarized, suggesting rich local processing, whereas SEZ projection neurons tend to be classically polarized, conveying information to a limited number of higher brain regions. This study provides insight into the anatomical organization of the SEZ and generates resources that will facilitate further study of SEZ neurons and their contributions to sensory processing and behavior.
Wang, Y., Guo, Y., Li, G., Liu, C., Wang, L., Zhang, A., Yan, Z. and Song, C. (2021). The push-to-open mechanism of the tethered mechanosensitive ion channel NompC. Elife 10. PubMed ID: 34101577
Summary:
NompC is a mechanosensitive ion channel responsible for the sensation of touch and balance in Drosophila melanogaster. Based on a resolved cryo-EM structure, all-atom molecular dynamics simulations and electrophysiological experiments were performed to study the atomistic details of NompC gating. The results showed that NompC could be opened by compression of the intracellular ankyrin repeat domain but not by a stretch, and a number of hydrogen bonds along the force convey pathway are important for the mechanosensitivity. Under intracellular compression, the bundled ankyrin repeat region acts like a spring with a spring constant of ~13 pN nm-1 by transferring forces at a rate of ~1.8 nm ps-1. The linker helix region acts as a bridge between the ankyrin repeats and the transient receptor potential (TRP) domain, which passes on the pushing force to the TRP domain to undergo a clockwise rotation, resulting in the opening of the channel. This could be the universal gating mechanism of similar tethered mechanosensitive TRP channels, which enable cells to feel compression and shrinkage.
Park, Y. J., Kim, S., Shim, H. P., Park, J. H., Lee, G., Kim, T. Y., Jo, M. C., Kwon, A. Y., Lee, M., Lee, S., Yeo, J., Chung, H. L., Bellen, H. J., Kwon, S. H. and Jeon, S. H. (2021). Phosphatidylserine synthase plays an essential role in glia and affects development, as well as the maintenance of neuronal function. iScience 24(8): 102899. PubMed ID: 34401677
Summary:
Phosphatidylserine (PS) is an integral component of eukaryotic cell membranes and organelles. The Drosophila genome contains a single PS synthase (PSS)-encoding gene (Pss) homologous to mammalian PSSs. Flies with Pss loss-of-function alleles show a reduced life span, increased bang sensitivity, locomotor defects, and vacuolated brain, which are the signs associated with neurodegeneration. Defective mitochondria were observed in mutant adult brain, as well as elevated production of reactive oxygen species, and an increase in autophagy and apoptotic cell death. Intriguingly, glial-specific knockdown or overexpression of Pss alters synaptogenesis and axonal growth in the larval stage, causes developmental arrest in pupal stages, and neurodegeneration in adults. This is not observed with pan-neuronal up- or down-regulation. These findings suggest that precisely regulated expression of Pss in glia is essential for the development and maintenance of brain function. A mechanism is proposed that underlies these neurodegenerative phenotypes triggered by defective PS metabolism.

Monday, October 4th - Behavior

van Breugel, F. (2021). Correlated decision making across multiple phases of olfactory-guided search in Drosophila improves search efficiency. J Exp Biol 224(16). PubMed ID: 34415028
Summary:
Nearly all motile organisms must search for food, often requiring multiple phases of exploration across heterogeneous environments. The fruit fly, Drosophila, has emerged as an effective model system for studying this behavior; however, little is known about the extent to which experiences at one point in their search might influence decisions in another. To investigate whether prior experiences impact flies' search behavior after landing, Individually labelled fruit flies were tracked as they explored three odor-emitting but food-barren objects. Two features of their behavior were found that are correlated with the distance they travel on foot. First, flies walked larger distances when they approached the odor source, which they were almost twice as likely to do when landing on the patch farthest downwind. Computational fluid dynamics simulations suggest this patch may have had a stronger baseline odor, but only ∼15% higher than the other two patches. This small increase, together with flies' high olfactory sensitivity, suggests that their flight trajectory used to approach the patches plays a role. Second, flies also walked larger distances when the time elapsed since their last visit was longer. However, the correlation is subtle and subject to a large degree of variability. Using agent-based models, it was shown that this small correlation can increase search efficiency by 25-50% across many scenarios. Furthermore, models provide mechanistic hypotheses explaining the variability through either a noisy or stochastic decision-making process. Surprisingly, these stochastic decision-making algorithms enhance search efficiency in challenging but realistic search scenarios compared with deterministic strategies.
Girardeau, A. R., Foley, B. R. and Saltz, J. B. (2021). Comparing single- and mixed-species groups in fruit flies: differences in group dynamics, but not group formation. J Hered. PubMed ID: 34453172
Summary:
Mixed-species groups describe active associations among individuals of 2 or more species at the same trophic level. Mixed-species groups are important to key ecological and evolutionary processes such as competition and predation, and research that ignores the presence of other species risks ignoring a key aspect of the environment in which social behavior is expressed and selected. Despite the defining emphasis of active formation for mixed-species groups, surprisingly little is known about the mechanisms by which mixed-species groups form. Furthermore, insects have been almost completely ignored in the study of mixed-species groups, despite their taxonomic importance and relative prominence in the study of single-species groups. In this study group formation processes were measured in Drosophila melanogaster and its sister species, Drosophila simulans. Each species was studied alone, and together, and one population of D. melanogaster was also studied both alone and with another, phenotypically distinct D. melanogaster population, in a nested-factorial design. This approach differs from typical methods of studying mixed-species groups in that group formation could be quantitatively compared between single-population, mixed-population, and mixed-species treatments. Surprisingly, no differences were found between treatments in the number, size, or composition of groups that formed, suggesting that single- and mixed-species groups form through similar mechanisms of active attraction. However, it was found that mixed-species groups showed elevated interspecies male-male interactions, relative to interpopulation or intergenotype interactions in single-species groups. These findings expand the conceptual and taxonomic study of mixed-species groups while raising new questions about the mechanisms of group formation broadly.
Steymans, I., Pujol-Lereis, L. M., Brembs, B. and Gorostiza, E. A. (2021). Collective action or individual choice: Spontaneity and individuality contribute to decision-making in Drosophila. PLoS One 16(8): e0256560. PubMed ID: 34437617
Summary:
Human's unique character traits make their behavior consistent and define our individuality. Yet, this consistency does not entail that people behave repetitively like machines. Like humans, animals also combine personality traits with spontaneity to produce adaptive behavior: consistent, but not fully predictable. This study examined an iconically rigid behavioral trait, insect phototaxis, that nevertheless also contains both components of individuality and spontaneity. In a light/dark T-maze, approximately 70% of a group of Drosophila fruit flies choose the bright arm of the T-Maze, while the remaining 30% walk into the dark. Taking the photopositive and the photonegative subgroups and re-testing them reveals the spontaneous component: a similar 70-30 distribution emerges in each of the two subgroups. Increasing the number of choices to ten choices, reveals the individuality component: flies with an extremely negative series of first choices were more likely to show photonegative behavior in subsequent choices and vice versa. General behavioral traits, independent of light/dark preference, contributed to the development of this individuality. The interaction of individuality and spontaneity together explains why group averages, even for such seemingly stereotypical behaviors, are poor predictors of individual choices.
Cellini, B., Salem, W. and Mongeau, J. M. (2021). Mechanisms of punctuated vision in fly flight. Curr Biol. PubMed ID: 34329590
Summary:
To guide locomotion, animals control gaze via movements of their eyes, head, and/or body, but how the nervous system controls gaze during complex motor tasks remains elusive. In many animals, shifts in gaze consist of periods of smooth movement punctuated by rapid eye saccades. Notably, eye movements are constrained by anatomical limits, which requires resetting eye position. By studying tethered, flying fruit flies (Drosophila), this study showed that flies perform stereotyped head saccades to reset gaze, analogous to optokinetic nystagmus in primates. Head-reset saccades interrupted head smooth movement for as little as 50 ms-representing less than 5% of the total flight time-thereby enabling punctuated gaze stabilization. By revealing the passive mechanics of the neck joint, it was shown that head-reset saccades leverage the neck's natural elastic recoil, enabling mechanically assisted redirection of gaze. The consistent head orientation at saccade initiation, the influence of the head's angular position on saccade rate, the decrease in wing saccade frequency in head-fixed flies, and the decrease in head-reset saccade rate in flies with their head range of motion restricted together implicate proprioception as the primary trigger of head-reset saccades. Wing-reset saccades were influenced by head orientation, establishing a causal link between neck sensory signals and the execution of body saccades. Head-reset saccades were abolished when flies switched to a landing state, demonstrating that head movements are gated by behavioral state. A control architecture is proposed for active vision systems with limits in sensor range of motion.
Eddison, M. (2021). A genetic screen for Drosophila social isolation mutants and analysis of sex pistol. Sci Rep 11(1): 17395. PubMed ID: 34462500
Summary:
Prolonged periods of forced social isolation is detrimental to well-being, yet little is known about which genes regulate susceptibility to its effects. In the fruit fly, Drosophila melanogaster, social isolation induces stark changes in behavior including increased aggression, locomotor activity, and resistance to ethanol sedation. To identify genes regulating sensitivity to isolation, A collection of sixteen hundred P-element insertion lines was screened for mutants with abnormal levels of all three isolation-induced behaviors. The screen identified three mutants whose affected genes are likely central to regulating the effects of isolation in flies. One mutant, sex pistol (sxp), became extremely aggressive and resistant to ethanol sedation when socially isolated. sxp also had a high level of male-male courtship. The mutation in sxp reduced the expression of two minor isoforms of the actin regulator hts (adducin), as well as mildly reducing expression of CalpA, a calcium-dependent protease. As a consequence, sxp also had increased expression of the insulin-like peptide, dILP5. Analysis of the social behavior of sxp suggests that these minor hts isoforms function to limit isolation-induced aggression, while chronically high levels of dILP5 increase male-male courtship.
Yost, R. T., Liang, E., Stewart, M. P., Chui, S., Greco, A. F., Long, S. Q., McDonald, I. S., McDowell, T., McNeil, J. N. and Simon, A. F. (2021). Drosophila melanogaster Stress Odorant (dSO) Displays the Characteristics of an Interspecific Alarm Cue. J Chem Ecol. PubMed ID: 34402994
Summary:
Organisms depend on visual, auditory, and olfactory cues to signal the presence of danger that could impact survival and reproduction. Drosophila melanogaster emits a volatile olfactory alarm signal, termed the Drosophila stress odorant (dSO), in response to mechanical agitation or electric shock. While it has been shown that conspecifics avoid areas previously occupied by stressed individuals, the contextual underpinnings of the emission of, and response to dSO, have received little attention. Using a binary choice assay, it was determined that neither age and sex of emitters, nor the time of the day, affected the emission or avoidance of dSO. However, both sex and mating status affected the response to dSO. It was also demonstrated that while D. melanogaster, D. simulans, and D. suzukii, have different dSO profiles, its avoidance was not species-specific. Thus, dSO should not be considered a pheromone but a general alarm signal for Drosophila. However, the response levels to both intra- and inter-specific cues differed between Drosophila species and possible reasons for these differences are discussed.

Friday, October 1st - Methods

Walters, J. D., Hatfield, J. S., Baker, B. B., Mackay, T. F. C. and Anholt, R. R. H. (2021). A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila. J Vis Exp(172). PubMed ID: 34180893
Summary:
Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.
Zhang, Y., Liu, Y., Xu, J., Wang, X., Peng, X., Song, J. and Yu, D. J. (2021). Leveraging the attention mechanism to improve the identification of DNA N6-methyladenine sites. Brief Bioinform. PubMed ID: 34459479
Summary:
DNA N6-methyladenine is an important type of DNA modification that plays important roles in multiple biological processes. Despite the recent progress in developing DNA 6mA site prediction methods, several challenges remain to be addressed. For example, although the hand-crafted features are interpretable, they contain redundant information that may bias the model training and have a negative impact on the trained model. Furthermore, although deep learning (DL)-based models can perform feature extraction and classification automatically, they lack the interpretability of the crucial features learned by those models. In this study, two new DL-based models were developed for improving the prediction of N6-methyladenine sites, termed LA6mA and AL6mA, which use bidirectional long short-term memory to respectively capture the long-range information and self-attention mechanism to extract the key position information from DNA sequences. The performance of the two proposed methods is benchmarked and evaluated on the two model organisms Arabidopsis thaliana and Drosophila melanogaster. On the two benchmark datasets, LA6mA achieves an area under the receiver operating characteristic curve (AUROC) value of 0.962 and 0.966, whereas AL6mA achieves an AUROC value of 0.945 and 0.941, respectively. Moreover, an in-depth analysis of the attention matrix is conducted to interpret the important information, which is hidden in the sequence and relevant for 6mA site prediction. The two novel pipelines developed for DNA 6mA site prediction in this work will facilitate a better understanding of the underlying principle of DL-based DNA methylation site prediction and its future applications.
Thackray, A. M., Andreoletti, O., Spiropoulos, J. and Bujdoso, R. (2021). A new model for sensitive detection of zoonotic prions by PrP transgenic Drosophila. J Biol Chem: 100878. PubMed ID: 34270959
Summary:
Prions are transmissible protein pathogens most reliably detected by bioassay in a suitable host, typically mice. However, the mouse bioassay is slow and cumbersome, and relatively insensitive to low titres of prion infectivity. Prions can be detected biochemically in vitro by the protein misfolding cyclic amplification (PMCA) technique, which amplifies disease-associated prion protein but does not detect bona fide prion infectivity. This study demonstrates that Drosophila transgenic for bovine PrP expression can serve as a model system for the detection of bovine prions significantly more efficiently than either the mouse prion bioassay or PMCA. Strikingly, bovine PrP transgenic Drosophila could detect bovine prion infectivity in the region of a 10-12 dilution of classical bovine spongiform encephalopathy (BSE) inoculum, which is 106-fold more sensitive than that achieved by the bovine PrP mouse bioassay. A similar level of sensitivity was observed in the detection of H-type and L-type atypical BSE and sheep-passaged BSE by bovine PrP transgenic Drosophila. Bioassays of bovine prions in Drosophila were performed within 7 weeks, whereas the mouse prion bioassay required at least a year to assess the same inoculum. In addition, bovine PrP transgenic Drosophila could detect classical BSE at a level 105-fold lower than that achieved by PMCA. These data show that PrP transgenic Drosophila represent a new tractable prion bioassay for the efficient and sensitive detection of mammalian prions, including those of known zoonotic potential.
Kapun, M., Nunez, J. C. B., ..., Flatt, T. and Bergland, A. O. (2021). Drosophila Evolution over Space and Time (DEST) - A New Population Genomics Resource. Mol Biol Evol. PubMed ID: 34469576
Summary:
Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers an ability to address questions about the evolution of this species. These issues were addressed by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). This pipeline was used to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in > 20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This dataset, which is called Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. This paper further provides guidelines on how to use Pool-Seq data for model-based demographic inference. The aim of this pipeline is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan dataset. This resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.
Okochi, Y., Sakaguchi, S., Nakae, K., Kondo, T. and Naoki, H. (2021). Model-based prediction of spatial gene expression via generative linear mapping. Nat Commun 12(1): 3731. PubMed ID: 34140477
Summary:
Decoding spatial transcriptomes from single-cell RNA sequencing (scRNA-seq) data has become a fundamental technique for understanding multicellular systems; however, existing computational methods lack both accuracy and biological interpretability due to their model-free frameworks. This paper introduces Perler, a model-based method to integrate scRNA-seq data with reference in situ hybridization (ISH) data. To calibrate differences between these datasets, a biologically interpretable model was developed that uses generative linear mapping based on a Gaussian mixture model using the Expectation-Maximization algorithm. Perler accurately predicts the spatial gene expression of Drosophila embryos, zebrafish embryos, mammalian liver, and mouse visual cortex from scRNA-seq data. Furthermore, the reconstructed transcriptomes do not over-fit the ISH data and preserved the timing information of the scRNA-seq data. These results demonstrate the generalizability of Perler for dataset integration, thereby providing a biologically interpretable framework for accurate reconstruction of spatial transcriptomes in any multicellular system.
Li, T., Weng, Y. and Yang, D. (2021). An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults. J Vis Exp(172). PubMed ID: 34180892
Summary:
Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but effective method to observe and document the behavior or morphology of flies. Flies were placed in a translucent observation chamber c.a. Ø15 x 5mm (no food inside) or Ø15 x 12 mm (with an 8 mm-high piece of food inside). After covering with an ultraviolet (UV)/clear filter with high light transmittance, the chamber was placed under a 5-50x zoom stereo microscope, and mini light-emitting diode (LED) video lights were placed on both sides of the microscope to illuminate the chamber to obtain uniform, soft, bright, and nearly shadow-free light. Then, a compact digital camera with 3-5x optical zoom, which can record 1080 P high-definition or higher resolution video (at a frame rate of ≥30 fps), was connected to the eyepiece of microscope through a bracket, and photographs or videos were taken through the eyepiece. By adjusting the zoom knob of the zoom stereo microscope, it was very easy to track the flies and take panoramic or detailed close-up images as needed, while the camera recorded everything under the microscope. Because the flies can stay at any position in the chamber, they can be observed and recorded from all directions. The photographs or videos taken are of good image quality. This method can be used both for scientific research and teaching.
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