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Wednesday, August 31st, 2022 - Autophagy and Apoptosis

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Macabenta, F., Sun, H. T. and Stathopoulos, A. (2022). BMP-gated cell-cycle progression drives anoikis during mesenchymal collective migration. Dev Cell. PubMed ID: 35709766
Tissue homeostasis involves the elimination of abnormal cells to avoid compromised patterning and function. Although quality control through cell competition is well studied in epithelial tissues, it is unknown if and how homeostasis is regulated in mesenchymal collectives. This study demonstrates that collectively migrating Drosophila muscle precursors utilize both fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling to promote homeostasis via anoikis, a form of cell death in response to substrate de-adhesion. Cell-cycle-regulated expression of the cell death gene head involution defective is responsible for caudal visceral mesoderm (CVM) anoikis. The secreted BMP ligand drives cell-cycle progression via a visceral mesoderm-specific cdc25/string enhancer to synchronize collective proliferation, as well as apoptosis of cells that have lost access to substrate-derived FGF. Perturbation of BMP-dependent cell-cycle progression is sufficient to confer anoikis resistance to mismigrating cells and thus facilitate invasion of other tissues. This BMP-gated cell-cycle checkpoint defines a quality control mechanism during mesenchymal collective migration.
Nagata, R., Akai, N., Kondo, S., Saito, K., Ohsawa, S. and Igaki, T. (2022). Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila. Curr Biol 32(5): 1064-1076.e1064. PubMed ID: 35134324
Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. The mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. This study shows that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. This study found that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. These data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells.
Shen, J. L., Doherty, J., Allen, E., Fortier, T. M. and Baehrecke, E. H. (2022). Atg6 promotes organismal health by suppression of cell stress and inflammation. Cell Death Differ. PubMed ID: 35523956
Autophagy targets cytoplasmic materials for degradation, and influences cell health. Alterations in Atg6/Beclin-1, a key regulator of autophagy, are associated with multiple diseases. While the role of Atg6 in autophagy regulation is heavily studied, the role of Atg6 in organism health and disease progression remains poorly understood. This study discovered that loss of Atg6 in Drosophila results in various alterations to stress, metabolic and immune signaling pathways. The increased levels of circulating blood cells and tumor-like masses in atg6 mutants vary depending on tissue-specific function of Atg6, with contributions from intestine and hematopoietic cells. These phenotypes are suppressed by decreased function of macrophage and inflammatory response receptors crq and drpr. Thus, these findings provide a basis for understanding how Atg6 systemically regulates cell health within multiple organs, and highlight the importance of Atg6 in inflammation to organismal health.
Shields, A., Amcheslavsky, A., Brown, E., Lee, T. V., Nie, Y., Tanji, T., Ip, Y. T. and Bergmann, A. (2022). Toll-9 interacts with Toll-1 to mediate a feedback loop during apoptosis-induced proliferation in Drosophila. Cell Rep 39(7): 110817. PubMed ID: 35584678
Drosophila Toll-1 and all mammalian Toll-like receptors regulate innate immunity. However, the functions of the remaining eight Toll-related proteins in Drosophila are not fully understood. This study shows that Drosophila Toll-9 is necessary and sufficient for a special form of compensatory proliferation after apoptotic cell loss (undead apoptosis-induced proliferation [AiP]). Mechanistically, for AiP, Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway for nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces expression of the pro-apoptotic genes reaper and hid. This activity contributes to the feedback amplification loop that operates in undead cells. Given that Toll-9 also functions in loser cells during cell competition, this study defines a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as AiP. This work identifies conceptual similarities between cell competition and AiP.
Wang, L., Bukhari, H., Kong, L., Hagemann, T. L., Zhang, S. C., Messing, A. and Feany, M. B. (2022). Anastasis Drives Senescence and Non-Cell Autonomous Neurodegeneration in the Astrogliopathy Alexander Disease. J Neurosci 42(12): 2584-2597. PubMed ID: 35105675
Anastasis is a recently described process in which cells recover after late-stage apoptosis activation. The functional consequences of anastasis for cells and tissues are not clearly understood. Using Drosophila, rat and human cells and tissues, including analyses of both males and females, this study presents evidence that glia undergoing anastasis in the primary astrogliopathy Alexander disease subsequently express hallmarks of senescence. These senescent glia promote non-cell autonomous death of neurons by secreting interleukin family cytokines. These findings demonstrate that anastasis can be dysfunctional in neurologic disease by inducing a toxic senescent population of astroglia.
Rahman, A., Lorincz, P., Gohel, R., Nagy, A., Csordas, G., Zhang, Y., Juhasz, G. and Nezis, I. P. (2022). GMAP is an Atg8a-interacting protein that regulates Golgi turnover in Drosophila. Cell Rep 39(9): 110903. PubMed ID: 35649355
Selective autophagy receptors and adapters contain short linear motifs called LIR motifs (LC3-interacting region), which are required for the interaction with the Atg8-family proteins. LIR motifs bind to the hydrophobic pockets of the LIR motif docking site (LDS) of the respective Atg8-family proteins. The physiological significance of LDS docking sites has not been clarified in vivo. This study shows that Atg8a-LDS mutant Drosophila flies accumulate autophagy substrates and have reduced lifespan. Using quantitative proteomics to identify the proteins that accumulate in Atg8a-LDS mutants, this study identified the cis-Golgi protein GMAP (Golgi microtubule-associated protein) as a LIR motif-containing protein that interacts with Atg8a. GMAP LIR mutant flies exhibit accumulation of Golgi markers and elongated Golgi morphology. These data suggest that GMAP mediates the turnover of Golgi by selective autophagy to regulate its morphology and size via its LIR motif-mediated interaction with Atg8a.

Tuesday, August 30th - Signaling

Nichols, R., Bass, C. and Katanski, C. (2022). Structure-activity relationship data and ligand-receptor interactions identify novel agonists consistent with sulfakinin tissue-specific signaling in Drosophila melanogaster heart. Front Biosci (Landmark Ed) 27(5): 150. PubMed ID: 35638417
The structures and activities of invertebrate sulfakinins that influence gut motility and heart rate are like the vertebrate cholecystokinin (CCK) peptides. Typical of sulfakinin precursors Drosophila melanogaster encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2) that bind DSK-R1 and DSK-R2. To explore the role of the nsDSK II N-terminal extension (GGDDQ) in gut its structure-activity relationship (SAR) were identified, and novel agonists were identified. Then the nsDSK II extension SAR was predicted to be tissue specific, consistent with cardiac CCK structure activity and signaling being different from gut. To evaluate this hypothesis, single-substituted alanine and asparagine analogs in heart were tested. RESULTS: Alanyl-substituted analogs were less active in heart than nsDSK II; in gut they include a super agonist and a protean agonist. Additionally, it was discovered that ns[N4]DSK II was more active than nsDSK II in pupal heart, while ns[N3]DSK II was inactive. In contrast, ns[N3]DSK II and ns[N4]DSK II were super agonists in adult heart, yet inactive in larva. Although this study reported nsDSK II acts through DSK-R2 in gut, its identity in heart was unknown. CONCLUSIONS: This study reviewed ligand-receptor interactions in conjunction with SAR data to suggest nsDSK II acts through DSK-R1 in heart consistent with sulfakinin tissue-specific signaling.
Li, W., Trigg, J. S. and Taghert, P. H. (2022). Regulation of PDF receptor signaling controlling daily locomotor rhythms in Drosophila. PLoS Genet 18(5): e1010013. PubMed ID: 35605015
Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in Drosophila through its receptor, PDFR, a Family B G protein-coupled receptor (GPCR). The in vivo process was studied by which PDFR signaling turns off, by converting as many as half of the 28 potential sites of phosphorylation in its C terminal tail to a non-phosphorylatable residue (alanine). Many such sites are conserved evolutionarily, and their conversion creates a specific behavioral syndrome opposite to loss-of-function phenotypes previously described for pdfr. That syndrome includes increases in the amplitudes of both Morning and Evening behavioral peaks, as well as multi-hour delays of the Evening phase. The precise behavioral effects were dependent on day-length, and most effects mapped to conversion of only a few, specific serine residues near the very end of the protein and specific to its A isoform. Behavioral phase delays of the Evening activity under entraining conditions predicted the phase of activity cycles under constant darkness. The behavioral phenotypes produced by the most severe PDFR variant were ligand-dependent in vivo, and not a consequence of changes to their pharmacological properties, nor of changes in their surface expression, as measured in vitro. The mechanisms underlying termination of PDFR signaling are complex, subject to regulation that is modified by season, and central to a better understanding of the peptidergic modulation of behavior.
Morioka, E., Kasuga, Y., Kanda, Y., Moritama, S., Koizumi, H., Yoshikawa, T., Miura, N., Ikeda, M., Higashida, H., Holmes, T. C. and Ikeda, M. (2022). Mitochondrial LETM1 drives ionic and molecular clock rhythms in circadian pacemaker neurons. Cell Rep 39(6): 110787. PubMed ID: 35545046
The mechanisms that generate robust ionic oscillation in circadian pacemaker neurons are under investigation. This study demonstrates critical functions of the mitochondrial cation antiporter leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1), which exchanges K(+)/H(+) in Drosophila and Ca(2+)/H(+) in mammals, in circadian pacemaker neurons. Letm1 knockdown in Drosophila pacemaker neurons reduced circadian cytosolic H(+) rhythms and prolonged nuclear PERIOD/TIMELESS expression rhythms and locomotor activity rhythms. In rat pacemaker neurons in the hypothalamic suprachiasmatic nucleus (SCN), circadian rhythms in cytosolic Ca(2+) and Bmal1 transcription were dampened by Letm1 knockdown. Mitochondrial Ca(2+) uptake peaks late during the day were also observed in rat SCN neurons following photolytic elevation of cytosolic Ca(2+). Since cation transport by LETM1 is coupled to mitochondrial energy synthesis, it is proposed that LETM1 integrates metabolic, ionic, and molecular clock rhythms in the central clock system in both invertebrates and vertebrates.
Langridge, P. D., Garcia Diaz, A., Chan, J. Y., Greenwald, I. and Struhl, G. (2022). Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins. Curr Biol 32(10): 2263-2271. PubMed ID: 35349791
The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. In the absence of ligand, a negative regulatory region (NRR) in the Notch ectodomain adopts an autoinhibited confirmation, masking an ADAM protease cleavage site; ligand binding induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction. In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a "pulling force" on Notch that is essential to expose the cleavage site in the NRR. This study shows that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila, evidence is presented that (1) the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a "leucine plug" that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs. These results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant.
Li, Y., Dong, P., Yang, Y., Guo, T., Zhao, Q., Miao, D., Li, H., Lu, T., Xia, F., Lyu, J., Ma, J., Kornberg, T. B., Zhang, Q. and Huang, H. (2022). Metabolic control of progenitor cell propagation during Drosophila tracheal remodeling. Nat Commun 13(1): 2817. PubMed ID: 35595807
Adult progenitor cells in the trachea of Drosophila larvae are activated and migrate out of niches when metamorphosis induces tracheal remodeling. In response to metabolic deficiency in decaying tracheal branches, signaling by the insulin pathway controls the progenitor cells by regulating Yorkie (Yki)-dependent proliferation and migration. Yki, a transcription coactivator that is regulated by Hippo signaling, promotes transcriptional activation of cell cycle regulators and components of the extracellular matrix in tracheal progenitor cells. These findings reveal that regulation of Yki signaling by the insulin pathway governs proliferation and migration of tracheal progenitor cells, thereby identifying the regulatory mechanism by which metabolic depression drives progenitor cell activation and cell division that underlies tracheal remodeling.
Mathieu, J., Michel-Hissier, P., Boucherit, V. and Huynh, J. R. (2022). The deubiquitinase USP8 targets ESCRT-III to promote incomplete cell division. Science 376(6595): 818-823. PubMed ID: 35587967
In many vertebrate and invertebrate organisms, gametes develop within groups of interconnected cells called germline cysts formed by several rounds of incomplete divisions. This study found that loss of the deubiquitinase USP8 gene in Drosophila can transform incomplete divisions of germline cells into complete divisions. Conversely, overexpression of USP8 in germline stem cells is sufficient for the reverse transformation from complete to incomplete cytokinesis. The ESCRT-III proteins CHMP2B and Shrub/CHMP4 are targets of USP8 deubiquitinating activity. In Usp8 mutant sister cells, ectopic recruitment of ESCRT proteins at intercellular bridges causes cysts to break apart. A Shrub/CHMP4 variant that cannot be ubiquitinated does not localize at abscission bridges and cannot complete abscission. These results uncover ubiquitination of ESCRT-III as a major switch between two types of cell division.

Monday September 29th, Adult Physiology and Metabolism

Levy, K. A., Weisz, E. D. and Jongens, T. A. (2022). Loss of neurexin-1 in Drosophila melanogaster results in altered energy metabolism and increased seizure susceptibility. Hum Mol Genet. PubMed ID: 35617143
While autism is typically characterized by differences in language, social interaction, and restrictive, repetitive behaviors, it is becoming more well known in the field that alterations in energy metabolism and mitochondrial function are a comorbid disorder in autism. The synaptic cell adhesion molecule, neurexin-1 (NRXN1), has previously been implicated in autism, and this study shows that in Drosophila melanogaster, the homologue of NRXN1, called Nrx-1, regulates energy metabolism and nutrient homeostasis. First, it was shown that Nrx-1-null flies exhibit decreased resistance to nutrient deprivation and heat stress compared to wildtype controls. Additionally, Nrx-1 mutants exhibit a significantly altered metabolic profile characterized by decreased lipid and carbohydrate stores. Nrx-1-null Drosophila also exhibit diminished levels of nicotinamide adenine dinucleotide (NAD+), an important coenzyme in major energy metabolism pathways. Moreover, loss of Nrx-1 resulted in striking abnormalities in mitochondrial morphology in the flight muscle of Nrx-1-null Drosophila, as well as impaired flight ability in these flies. Further, following a mechanical shock Nrx-1-null flies exhibited seizure-like activity, a phenotype previously linked to defects in mitochondrial metabolism and a common symptom of patients with NRXN1 deletions. The current studies indicate a novel role for neurexin-1 in the regulation of energy metabolism as well as uncover a clinically relevant seizure phenotype in Drosophila lacking Nrx-1.
Mlih, M. and Karpac, J. (2022). Integrin-ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium. PLoS Biol 20(5): e3001635. PubMed ID: 35522719
Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. Diedel, a secreted protein/cytokine, has been identified as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. This study shows that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, a resilience mechanism was uncovered of epithelial tissues, mediated by Integrin-ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, It was found that resilient epithelial cells, enriched for Diedel-Integrin-ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin-ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin-ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.
Zhang, J. M., Chen, M. J., He, J. H., Li, Y. P., Li, Z. C., Ye, Z. J., Bao, Y. H., Huang, B. J., Zhang, W. J., Kwan, P., Mao, Y. L. and Qiao, J. D. (2022). Ketone Body Rescued Seizure Behavior of LRP1 Deficiency in Drosophila by Modulating Glutamate Transport. J Mol Neurosci. PubMed ID: 35668313
LRP1, the low-density lipoprotein receptor 1, would be a novel candidate gene of epilepsy according to bioinformatic results and the animal study. This study explored the role of LRP1 in epilepsy and whether beta-hydroxybutyrate, the principal ketone body of the ketogenic diet, can treat epilepsy caused by LRP1 deficiency in drosophila. UAS/GAL4 system was used to establish different genotype models. Flies were given standard, high-sucrose, and ketone body food randomly. The bang-sensitive test was performed on flies and seizure-like behavior was assessed. In morphologic experiments, it was found that LRP1 deficiency caused partial loss of the ellipsoidal body and partial destruction of the fan-shaped body. Whole-body and glia LRP1 defect flies had a higher seizure rate compared to the control group. Ketone body decreased the seizure rate in behavior test in all LRP1 defect flies, compared to standard and high sucrose diet. Overexpression of glutamate transporter gene Eaat1 could mimic the ketone body effect on LRP1 deficiency flies. This study demonstrated that LRP1 defect globally or in glial cells or neurons could induce epilepsy in drosophila. The ketone body efficaciously rescued epilepsy caused by LRP1 knockdown. The results support screening for LRP1 mutations as discriminating conduct for individuals who require clinical attention and further clarify the mechanism of the ketogenic diet in epilepsy, which could help epilepsy patients make a precise treatment case by case.
Brischigliaro, M., Badocco, D., Costa, R., Viscomi, C., Zeviani, M., Pastore, P. and Fernandez-Vizarra, E. (2022). Mitochondrial Cytochrome c Oxidase Defects Alter Cellular Homeostasis of Transition Metals. Front Cell Dev Biol 10: 892069. PubMed ID: 35663391
The redox activity of cytochrome c oxidase (COX), the terminal oxidase of the mitochondrial respiratory chain (MRC), depends on the incorporation of iron and copper into its catalytic centers. Many mitochondrial proteins have specific roles for the synthesis and delivery of metal-containing cofactors during COX biogenesis. In addition, a large set of different factors possess other molecular functions as chaperones or translocators that are also necessary for the correct maturation of these complexes. Pathological variants in genes encoding structural MRC subunits and these different assembly factors produce respiratory chain deficiency and lead to mitochondrial disease. COX deficiency in Drosophila melanogaster, induced by downregulated expression of three different assembly factors and one structural subunit, resulted in decreased copper content in the mitochondria accompanied by different degrees of increase in the cytosol. The disturbances in metal homeostasis were not limited only to copper, as some changes in the levels of cytosolic and/or mitochondrial iron, manganase and, especially, zinc were observed in several of the COX-deficient groups. The altered copper and zinc handling in the COX defective models resulted in a transcriptional response decreasing the expression of copper transporters and increasing the expression of metallothioneins. It is concluded that COX deficiency is generally responsible for an altered mitochondrial and cellular homeostasis of transition metals, with variations depending on the origin of COX assembly defect.
Flores, C. C., Loschky, S. S., Marshall, W., Spano, G. M., Massaro Cenere, M., Tononi, G. and Cirelli, C. (2022). Identification of ultrastructural signatures of sleep and wake in the fly brain. Sleep 45(5). PubMed ID: 35554595
The cellular consequences of sleep loss are poorly characterized. In the pyramidal neurons of mouse frontal cortex, this study found that mitochondria and secondary lysosomes occupy a larger proportion of the cytoplasm after chronic sleep restriction compared to sleep, consistent with increased cellular burden due to extended wake. For each morphological parameter, the within-animal variance was high, suggesting that the effects of sleep and sleep loss vary greatly among neurons. However, the analysis was based on 4-5 mice/group and a single section/cell. This study applied serial block-face scanning electron microscopy to identify signatures of sleep and sleep loss in the Drosophila brain. Stacks of images were acquired and used to obtain full 3D reconstructions of the cytoplasm and nucleus of 263 Kenyon cells from adult flies collected after a night of sleep (S) or after 11 h (SD11) or 35 h (SD35) of sleep deprivation (9 flies/group). Relative to S flies, SD35 flies showed increased density of dark clusters of chromatin and Golgi apparata and a trend increase in the percent of cell volume occupied by mitochondria, consistent with increased need for energy and protein supply during extended wake. Logistic regression models could assign each neuron to the correct experimental group with good accuracy, but in each cell, nuclear and cytoplasmic changes were poorly correlated, and within-fly variance was substantial in all experimental groups. Together, these results support the presence of ultrastructural signatures of sleep and sleep loss but underscore the complexity of their effects at the single-cell level.
Gnocchini, E., Pilesi, E., Schiano, L. and Verni, F. (2022). Vitamin B6 Deficiency Promotes Loss of Heterozygosity (LOH) at the Drosophila warts (wts) Locus. Int J Mol Sci 23(11). PubMed ID: 35682766
The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is a cofactor for more than 200 enzymes involved in many metabolic pathways. Moreover, PLP has antioxidant properties and quenches the reactive oxygen species (ROS). Accordingly, PLP deficiency causes chromosome aberrations in Drosophila, yeast, and human cells. This work investigated whether PLP depletion can also cause loss of heterozygosity (LOH) of the tumor suppressor warts (wts) in Drosophila. LOH is usually initiated by DNA breakage in heterozygous cells for a tumor suppressor mutation and can contribute to oncogenesis inducing the loss of the wild-type allele. LOH at the wts locus results in epithelial wts homozygous tumors easily detectable on adult fly cuticle. This study found that PLP depletion, induced by two PLP inhibitors, promotes LOH of wts locus producing significant frequencies of wts tumors (~7% vs. 2.3%). In addition, mitotic recombination was identified as a possible mechanism through which PLP deficiency induces LOH. Moreover, LOH of wts locus, induced by PLP inhibitors, was rescued by PLP supplementation. These data further confirm the role of PLP in genome integrity maintenance and indicate that vitamin B6 deficiency may impact on cancer also by promoting LOH.

Friday August 26th - Evolution

Veltsos, P., Porcelli, D., Fang, Y., Cossins, A. R., Ritchie, M. G. and Snook, R. R. (2022). Experimental sexual selection reveals rapid evolutionary divergence in sex-specific transcriptomes and their interactions following mating. Mol Ecol 31(12): 3374-3388. PubMed ID: 35437824
Post copulatory interactions between the sexes in internally fertilizing species elicits both sexual conflict and sexual selection. Macroevolutionary and comparative studies have linked these processes to rapid transcriptomic evolution in sex-specific tissues and substantial transcriptomic post mating responses in females, patterns of which are altered when mating between reproductively isolated species. This study tested multiple predictions arising from sexual selection and conflict theory about the evolution of sex-specific and tissue-specific gene expression and the post mating response at the microevolutionary level. Following over 150 generations of experimental evolution under either reduced (enforced monogamy) or elevated (polyandry) sexual selection in Drosophila pseudoobscura, this study found a substantial effect of sexual selection treatment on transcriptomic divergence in virgin male and female reproductive tissues (testes, male accessory glands, the female reproductive tract and ovaries). Sexual selection treatment also had a dominant effect on the post mating response, particularly in the female reproductive tract - the main arena for sexual conflict - compared to ovaries. This effect was asymmetric with monandry females typically showing more post mating responses than polyandry females, with enriched gene functions varying across treatments. The evolutionary history of the male partner had a larger effect on the post mating response of monandry females, but females from both sexual selection treatments showed unique patterns of gene expression and gene function when mating with males from the alternate treatment. These microevolutionary results mostly confirm comparative macroevolutionary predictions on the role of sexual selection on transcriptomic divergence and altered gene regulation arising from divergent coevolutionary trajectories between sexual selection treatments.
Meshrif, W. S., Elkayal, S. H., Soliman, M. A., Seif, A. I. and Roeder, T. (2022). Metabolic and immunological responses of Drosophila melanogaster to dietary restriction and bacterial infection differ substantially between genotypes in a population. Ecol Evol 12(5): e8960. PubMed ID: 35646322
To respond to changing environmental conditions, a population may either shift toward better-adapted genotypes or adapt on an individual level. The present work aimed to quantify the relevance of these two processes by comparing the responses of defined Drosophila melanogaster populations to different stressors. To do this, two homogeneous populations (isofemale lines), which differ significantly in fitness, and a synthetic heterogeneous population were infected with a specific pathogen and/or exposed to food restriction. Pectobacterium carotovorum was used to infect Drosophila larvae either fed standard or protein-restricted diet. In particular, the two homogeneous groups, which diverged in their fitness, showed considerable differences in all parameters assessed (survivorship, protein and lipid contents, Phenol oxidase (PO) activity, and antibacterial rate). Under fully nutritious conditions, larvae of the homogeneous population with low fitness exhibited lower survivorship and protein levels, as well as higher PO activity and antibacterial rate compared with the fitter population. A protein-restricted diet and bacterial infection provoked a decrease in survivorship, and antibacterial rate in most populations. Bacterial infection elicited an opposite response in protein and lipid content in both isofemale lines tested. Interestingly, the heterogeneous population showed a complex response pattern. The response of the heterogeneous population followed the fit genotype in terms of survival and antibacterial activity but followed the unfit genotype in terms of PO activity. In conclusion, these results show that defined genotypes exhibit highly divergent responses to varying stressors that are difficult to predict. Furthermore, the responses of heterogeneous populations do not follow a fixed pattern showing a very high degree of plasticity and differences between different genotypes.
Ahlawat, N., Maggu, K., Jigisha, Arun, M. G., Meena, A., Agarwala, A. and Prasad, N. G. (2022). No major cost of evolved survivorship in Drosophila melanogaster populations coevolving with Pseudomonas entomophila. Proc Biol Sci 289(1974): 20220532. PubMed ID: 35506222
Rapid exaggeration of host and pathogen traits via arms race dynamics is one possible outcome of host-pathogen coevolution. However, the exaggerated traits are expected to incur costs in terms of resource investment in other life-history traits. The current study investigated the costs associated with evolved traits in a host-pathogen coevolution system. The Drosophila melanogaster (host)-Pseudomonas entomophila (pathogen) system was used to experimentally derive two selection regimes, one where the host and pathogen both coevolved, and the other, where only the host evolved against a non-evolving pathogen. After 17 generations of selection, it was found that hosts from both selected populations had better post-infection survivorship than controls. Even though the coevolving populations tended to have better survivorship post-infection, no clear evidence that the two selection regimes were significantly different from each other. There was weak evidence for the coevolving pathogens being more virulent than the ancestral pathogen. No major cost was found of increased post-infection survivorship. The costs were not different between the coevolving hosts and the hosts evolving against a non-evolving pathogen. No evolved costs were found in the coevolving pathogens. Thus, these results suggest that increased host immunity and pathogen virulence may not be costly.
Brand, C. L. and Levine, M. T. (2022). Cross-species incompatibility between a DNA satellite and the Drosophila Spartan homolog poisons germline genome integrity. Curr Biol. PubMed ID: 35643081
Satellite-rich genomic regions mediate strictly conserved, essential processes such as chromosome segregation and nuclear structure. A leading resolution to this paradox posits that satellite DNA and satellite-associated chromosomal proteins coevolve to preserve these essential functions. This study experimentally test this model of intragenomic coevolution by conducting the first evolution-guided manipulation of both chromosomal protein and DNA satellite. The 359bp satellite spans an 11 Mb array in Drosophila melanogaster that is absent from its sister species, Drosophila simulans. This species-specific DNA satellite colocalizes with the adaptively evolving, ovary-enriched protein, Maternal haploid (MH), the Drosophila homolog of Spartan. To determine if MH and 359bp coevolve, the D.simulans version of MH ("MH[sim]") was swapped into D. melanogaster. MH[sim] triggers ovarian cell death, reduced ovary size, and loss of mature eggs. Surprisingly, the D. melanogaster mh-null mutant has no such ovary phenotypes, suggesting that MH[sim] is toxic in a D. melanogaster background. Using both cell biology and genetics, it was discovered that MH[sim] poisons oogenesis through a DNA-damage pathway. Remarkably, deleting the D. melanogaster-specific 359bp satellite array completely restores mh[sim] germline genome integrity and fertility, consistent with a history of coevolution between these two fast-evolving loci. Germline genome integrity and fertility are also restored by overexpressing topoisomerase II (Top2), suggesting that MH[sim] interferes with Top2-mediated processing of 359bp. The observed 359bp-MH[sim] cross-species incompatibility supports a model under which seemingly inert repetitive DNA and essential chromosomal proteins must coevolve to preserve germline genome integrity.
Christodoulaki, E., Nolte, V., Lai, W. Y. and Schlotterer, C. (2022). Natural variation in Drosophila shows weak pleiotropic effects. Genome Biol 23(1): 116. PubMed ID: 35578368
Pleiotropy describes the phenomenon in which a gene affects multiple phenotypes. The extent of pleiotropy is still disputed, mainly because of issues of inadequate power of analyses. A further challenge is that empirical tests of pleiotropy are restricted to a small subset of all possible phenotypes. To overcome these limitations, this paper proposes a new measurement of pleiotropy that integrates across many phenotypes and multiple generations to improve power. Pleiotropy is infered from the fitness cost imposed by frequency changes of pleiotropic loci. Mixing Drosophila simulans populations, which adapted independently to the same new environment using different sets of genes, this study shows that the adaptive frequency changes have been accompanied by measurable fitness costs. Unlike previous studies characterizing the molecular basis of pleiotropy, this study shows that many loci, each of weak effect, contribute to genome-wide pleiotropy. It is proposed that the costs of pleiotropy are reduced by the modular architecture of gene expression, which facilitates adaptive gene expression changes with low impact on other functions.
Kopp, A. and Barmina, O. (2022). Interspecific variation in sex-specific gustatory organs in Drosophila. J Comp Neurol. PubMed ID: 35603778
Drosophila males use leg gustatory bristles to discriminate between male and female cuticular pheromones as an important part of courtship behavior. In Drosophila melanogaster, several male-specific gustatory bristles are present on the anterior surface of the first tarsal segment of the prothoracic leg, in addition to a larger set of gustatory bristles found in both sexes. These bristles are thought to be specialized for pheromone detection. This study reports the number and location of sex-specific gustatory bristles in 27 other Drosophila species. Although some species have a pattern similar to D. melanogaster, others lack anterior male-specific bristles but have many dorsal male-specific gustatory bristles instead. Some species have both anterior and dorsal male-specific bristles, while others lack sexual dimorphism entirely. In several distantly related species, the number of gustatory bristles is much greater in males than in females due to a male-specific transformation of ancestrally mechanosensory bristles to a chemosensory identity. This variation in the extent and pattern of sexual dimorphism may affect the formation and function of neuronal circuits that control Drosophila courtship and contribute to the evolution of mating behavior.

Thursday, August 25th - Adult Neural Development and Function

Matsushita, A., Stewart, F., Ilic, M., Chen, P. J., Wakita, D., Miyazaki, N., Murata, K., Kinoshita, M., Belusic, G. and Arikawa, K. (2022). Connectome of the lamina reveals the circuit for early color processing in the visual pathway of a butterfly. Curr Biol 32(10): 2291-2299.e2293. PubMed ID: 35439432
Connectomics has become a standard neuroscience methodology in a few model animals, with the visual system being a popular target of study. Combining connectomics with circuit and behavioral physiology, recent studies on the color vision of the fruit fly Drosophila melanogaster have focused on the mechanisms underlying early wavelength processing in the optic ganglia. However, the color vision capabilities of D. melanogaster are limited, compared with many flower-visiting insects. For example, a butterfly Papilio xuthus has six spectral classes of photoreceptors. Each ommatidium contains nine photoreceptors in one of three fixed combinations, making the eye an array of three spectrally distinct ommatidia types. Behaviorally, P. xuthus can detect 1 nm differences in light wavelength across the spectrum from ultraviolet to red, outperforming humans. What is the neuronal basis of such precise color vision? How does such a system evolve? Addressing these questions requires comparative studies at the circuit level. This study performed a connectome analysis in the first optic ganglion, the lamina, of P. xuthus. The lamina comprises cartridges, each typically containing nine photoreceptor axons from a single ommatidium and four second-order neurons. Abundant inter-photoreceptor connections were found that are absent in the lamina of D. melanogaster. Connections were identified between neighboring cartridges, particularly those receiving inputs from spectrally distinct ommatidia. The linear summation of synaptic connections well explains the spectral sensitivity of photoreceptors and second-order neurons in the lamina.
Farnworth, M. S., Bucher, G. and Hartenstein, V. (2022). An atlas of the developing Tribolium castaneum brain reveals conservation in anatomy and divergence in timing to Drosophila melanogaster. J Comp Neurol. PubMed ID: 35535818
Insect brains are formed by conserved sets of neural lineages whose fibers form cohesive bundles with characteristic projection patterns. Within the brain neuropil, these bundles establish a system of fascicles constituting the macrocircuitry of the brain. The overall architecture of the neuropils and the macrocircuitry appear to be conserved. However, variation is observed, for example, in size, shape, and timing of development. Unfortunately, the developmental and genetic basis of this variation is poorly understood, although the rise of new genetically tractable model organisms such as the red flour beetle Tribolium castaneum allows the possibility to gain mechanistic insights. To facilitate such work, this paper presents an atlas of the developing brain of T. castaneum, covering the first larval instar, the prepupal stage, and the adult, by combining wholemount immunohistochemical labeling of fiber bundles (acetylated tubulin) and neuropils (synapsin) with digital 3D reconstruction using the TrakEM2 software package. Upon comparing this anatomical dataset with the published work in Drosophila melanogaster, an overall high degree of conservation was confirmed. Fiber tracts and neuropil fascicles, which can be visualized by global neuronal antibodies like antiacetylated tubulin in all invertebrate brains, create a rich anatomical framework to which individual neurons or other regions of interest can be referred to. The framework of a largely conserved pattern allowed differences to be described between the two species with respect to parameters such as timing of neuron proliferation and maturation. These features likely reflect adaptive changes in developmental timing that govern the change from larval to adult brain.
Korona, D., Dirnberger, B., Giachello, C. N. G., Queiroz, R. M. L., Popovic, R., Müller, K. H., Minde, D. P., Deery, M. J., Johnson, G., Firth, L. C., Earley, F. G., Russell, S. and Lilley, K. S. (2022). Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins. Elife 11. PubMed ID: 35575460
Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs, a CRISPR/Cas9 strategy was used to generate null alleles for all ten nAChR subunit genes in a common genetic background. Interactions of nAChR subunits with peptide neurotoxins were studied by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, the effects were determined of α-Bungarotoxin (α -Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. Pull-down assays were employed to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, the localisation is reported of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.
Das Chakraborty, S., Chang, H., Hansson, B. S. and Sachse, S. (2022). Higher-order olfactory neurons in the lateral horn support odor valence and odor identity coding in Drosophila. Elife 11. PubMed ID: 35621267
Understanding neuronal representations of odor-evoked activities and their progressive transformation from the sensory level to higher brain centers features one of the major aims in olfactory neuroscience. This study investigated how odor information is transformed and represented in higher-order neurons of the lateral horn, one of the higher olfactory centers implicated in determining innate behavior, using Drosophila melanogaster. This study focused on a subset of third-order glutamatergic lateral horn neurons (LHNs) and characterized their odor coding properties in relation to their presynaptic partner neurons, the projection neurons (PNs), by two-photon functional imaging. Odors evoke reproducible, stereotypic, and odor-specific response patterns in LHNs. Notably, odor-evoked responses in these neurons are valence-specific in a way that their response amplitude is positively correlated with innate odor preferences. It is postulated that this valence-specific activity is the result of integrating inputs from multiple olfactory channels through second-order neurons. GRASP and micro-lesioning experiments provide evidence that glutamatergic LHNs obtain their major excitatory input from uniglomerular PNs, while they receive an odor-specific inhibition through inhibitory multiglomerular PNs. In summary, this study indicates that odor representations in glutamatergic LHNs encode hedonic valence and odor identity and primarily retain the odor coding properties of second-order neurons.
Dong, H., Guo, P., Zhang, J., Wu, L., Fu, Y., Li, L., Zhu, Y., Du, Y., Shi, J., Zhang, S., Li, G., Xu, B., Bian, L., Zhu, X., You, W., Shi, F., Yang, X., Huang, J. and Jin, Y. (2022). Self-avoidance alone does not explain the function of Dscam1 in mushroom body axonal wiring. Curr Biol. PubMed ID: 35659864
Alternative splicing of Drosophila Dscam1 into 38,016 isoforms provides neurons with a unique molecular code for self-recognition and self-avoidance. A canonical model suggests that the homophilic binding of identical Dscam1 isoforms on the sister branches of mushroom body (MB) axons supports segregation with high fidelity, even when only a single isoform is expressed. This study generated a series of mutant flies with a single exon 4, 6, or 9 variant, encoding 1,584, 396, or 576 potential isoforms, respectively. Surprisingly, most of the mutants in the latter two groups exhibited obvious defects in the growth, branching, and segregation of MB axonal sister branches. This demonstrates that the repertoires of 396 and 576 Dscam1 isoforms were not sufficient for the normal patterning of axonal branches. Moreover, reducing Dscam1 levels largely reversed the defects caused by reduced isoform diversity, suggesting a functional link between Dscam1 expression levels and isoform diversity. Taken together, these results indicate that canonical self-avoidance alone does not explain the function of Dscam1 in MB axonal wiring.
Engert, S., Sterne, G. R., Bock, D. D. and Scott, K. (2022). Drosophila gustatory projections are segregated by taste modality and connectivity. Elife 11. PubMed ID: 35611959
Gustatory sensory neurons detect caloric and harmful compounds in potential food and convey this information to the brain to inform feeding decisions. To examine the signals that gustatory neurons transmit and receive, this study reconstructed gustatory axons and their synaptic sites in the adult Drosophila melanogaster brain, utilizing a whole-brain electron microscopy volume. 87 gustatory projections were reconstructed from the proboscis labellum in the right hemisphere and 57 from the left, representing the majority of labellar gustatory axons. Gustatory neurons contain a nearly equal number of interspersed pre- and postsynaptic sites, with extensive synaptic connectivity among gustatory axons. Morphology- and connectivity-based clustering revealed six distinct groups, likely representing neurons recognizing different taste modalities. The vast majority of synaptic connections are between neurons of the same group. This study resolves the anatomy of labellar gustatory projections, reveals that gustatory projections are segregated based on taste modality, and uncovers synaptic connections that may alter the transmission of gustatory signals.

Wednesday, August 24th - Signaling

Taylor, S. E. and Dearden, P. K. (2022). The Nasonia pair-rule gene regulatory network retains its function over 300 million years of evolution. Development 149(5). PubMed ID: 35142336
Insect segmentation is a well-studied and tractable system with which to investigate the genetic regulation of development. Though insects segment their germband using a variety of methods, modelling work implies that a single gene regulatory network can underpin the two main types of insect segmentation. This means limited genetic changes are required to explain significant differences in segmentation mode between different insects. This idea needs to be tested in a wider variety of species, and the nature of the gene regulatory network (GRN) underlying this model has not been tested. Some insects, e.g. Nasonia vitripennis and Apis mellifera segment progressively, a pattern not examined in previous studies of this segmentation model, producing stripes at different times progressively through the embryo, but not from a segment addition zone. This study aimed to understand the GRNs patterning Nasonia using a simulation-based approach. An existing model of Drosophila segmentation= can be used to recapitulate the progressive segmentation of Nasonia, if provided with altered inputs in the form of expression of the timer genes Nv-caudal and Nv-odd paired. Limited topological changes to the pair-rule network are predicted, and by RNAi knockdown, that Nv-odd paired is required for morphological segmentation. Together this implies that very limited changes to the Drosophila network are required to simulate Nasonia segmentation, despite significant differences in segmentation modes, implying that Nasonia use a very similar version of an ancestral GRN used by Drosophila, which must therefore have been conserved for at least 300 million years.
Jimenez-Mejía #, G., Montalvo-Mendez, R., Hernandez-Bautista, C., Altamirano-Torres, C., Vazquez, M., Zurita, M. and Resendez-Perez, D. (2022). Trimeric complexes of Antp-TBP with TFIIEbeta or Exd modulate transcriptional activity. Hereditas 159(1): 23. PubMed ID: 35637493
Hox proteins finely coordinate antero-posterior axis during embryonic development and through their action specific target genes are expressed at the right time and space to determine the embryo body plan. This study reports Antennapedia (Antp) Hox protein-protein interaction with the TATA-binding protein (TBP) and the formation of novel trimeric complexes with TFIIEβ and Extradenticle (Exd), as well as its participation in transcriptional regulation. Using Bimolecular Fluorescence Complementation (BiFC), this study detected the interaction of Antp-TBP and, in combination with Forster Resonance Energy Transfer (BiFC-FRET), the formation of the trimeric complex with TFIIEβ and Exd in living cells. Mutational analysis showed that Antp interacts with TBP through their N-terminal polyglutamine-stretches. The trimeric complexes of Antp-TBP with TFIIEβ and Exd were validated using different Antp mutations to disrupt the trimeric complexes. Interestingly, the trimeric complex Antp-TBP-TFIIEβ significantly increased the transcriptional activity of Antp, whereas Exd diminished its transactivation. These findings provide important insights into the Antp interactome with the direct interaction of Antp with TBP and the two new trimeric complexes with TFIIEβ and Exd. These novel interactions open the possibility to analyze promoter function and gene expression to measure transcription factor binding dynamics at target sites throughout the genome.
Perez-Moreno, J. J., Santa-Cruz Mateos, C., Martin-Bermudo, M. D. and Estrada, B. (2021). LanB1 Cooperates With Kon-Tiki During Embryonic Muscle Migration in Drosophila. Front Cell Dev Biol 9: 749723. PubMed ID: 35047493
Muscle development is a multistep process that involves cell specification, myoblast fusion, myotube migration, and attachment to the tendons. In spite of great efforts trying to understand the basis of these events, little is known about the molecular mechanisms underlying myotube migration. Knowledge of the few molecular cues that guide this migration comes mainly from studies in Drosophila. The migratory process of Drosophila embryonic muscles involves a first phase of migration, where muscle progenitors migrate relative to each other, and a second phase, where myotubes migrate searching for their future attachment sites. During this phase, myotubes form extensive filopodia at their ends oriented preferentially toward their attachment sites. This myotube migration and the subsequent muscle attachment establishment are regulated by cell adhesion receptors, such as the conserved proteoglycan Kon-tiki/Perdido. Laminins have been shown to regulate the migratory behavior of many cell populations, but their role in myotube migration remains largely unexplored. This study showed that laminins, previously implicated in muscle attachment, are indeed required for muscle migration to tendon cells. Furthermore, it was fond that laminins genetically interact with kon-tiki/perdido to control both myotube migration and attachment. All together, these results uncover a new role for the interaction between laminins and Kon-tiki/Perdido during Drosophila myogenesis. The identification of new players and molecular interactions underlying myotube migration broadens understanding of muscle development and disease.
Hayden, L., Chao, A., Deneke, V. E., Vergassola, M., Puliafito, A. and Di Talia, S. (2022). Cullin-5 mutants reveal collective sensing of the nucleocytoplasmic ratio in Drosophila embryogenesis. Curr Biol 32(9): 2084-2092.e2084. PubMed ID: 35334230
In most metazoans, early embryonic development is characterized by rapid division cycles that pause before gastrulation at the midblastula transition (MBT). These cleavage divisions are accompanied by cytoskeletal rearrangements that ensure proper nuclear positioning. However, the molecular mechanisms controlling nuclear positioning are not fully elucidated. In Drosophila, early embryogenesis unfolds in a multinucleated syncytium. Nuclei rapidly move across the anterior-posterior (AP) axis at cell cycles 4-6 in a process driven by actomyosin contractility and cytoplasmic flows. In shackleton (shkl) mutants, this axial spreading is impaired. This study shows that shkl mutants carry mutations in the cullin-5 (cul-5) gene. Live imaging experiments show that Cul-5 is downstream of the cell cycle but is required for cortical actomyosin contractility. The nuclear spreading phenotype of cul-5 mutants can be rescued by reducing Src activity, suggesting that a major target of cul-5 is Src kinase. cul-5 mutants display gradients of nuclear density across the AP axis that were exploited to study cell-cycle control as a function of the N/C ratio. The N/C ratio is sensed collectively in neighborhoods of about 100 μm, and such collective sensing is required for a precise MBT, in which all the nuclei in the embryo pause their division cycle. Moreover, it was found that the response to the N/C ratio is slightly graded along the AP axis. These two features can be linked to Cdk1 dynamics. Collectively, this study revealed a new pathway controlling nuclear positioning and provide a dissection of how nuclear cycles respond to the N/C ratio.
Chen, S. L., Liu, B. T., Lee, W. P., Liao, S. B., Deng, Y. B., Wu, C. L., Ho, S. M., Shen, B. X., Khoo, G. H., Shiu, W. C., Chang, C. H., Shih, H. W., Wen, J. K., Lan, T. H., Lin, C. C., Tsai, Y. C., Tzeng, H. F. and Fu, T. F. (2022). WAKE-mediated modulation of cVA perception via a hierarchical neuro-endocrine axis in Drosophila male-male courtship behaviour. Nat Commun 13(1): 2518. PubMed ID: 35523813
The nervous and endocrine systems coordinate with each other to closely influence physiological and behavioural responses in animals. This study shows that Wake (encoded by wide awake) modulates membrane levels of GABA(A) receptor Resistance to Dieldrin (Rdl), in insulin-producing cells of adult male Drosophila melanogaster. This results in changes to secretion of insulin-like peptides which is associated with changes in juvenile hormone biosynthesis in the corpus allatum, which in turn leads to a decrease in 20-hydroxyecdysone levels. A reduction in ecdysone signalling changes neural architecture and lowers the perception of the male-specific sex pheromone 11-cis-vaccenyl acetate by odorant receptor 67d olfactory neurons. These finding explain why WAKE-deficient in Drosophila elicits significant male-male courtship behaviour.
de Torres-Jurado, A., Manzanero-Ortiz, S. and Carmena, A. (2022). Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division. Curr Biol 32(10): 2174-2188.e2173. PubMed ID: 35472309
Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. This study shows that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex.

Tuesday, August 23rd - Embryonic Development

Falo-Sanjuan, J. and Bray, S. (2022). Notch-dependent and -independent transcription are modulated by tissue movements at gastrulation.. Elife 11. PubMed ID: 35583918
Cells sense and integrate external information from diverse sources that include mechanical cues. Shaping of tissues during development may thus require coordination between mechanical forces from morphogenesis and cell-cell signalling to confer appropriate changes in gene expression. By live-imaging Notch-induced transcription in real time, it was discovered that morphogenetic movements during Drosophila gastrulation bring about an increase in activity-levels of a Notch-responsive enhancer. Mutations that disrupt the timing of gastrulation resulted in concomitant delays in transcription up-regulation that correlated with the start of mesoderm invagination. As a similar gastrulation-induced effect was detected when transcription was elicited by the intracellular domain NICD, it cannot be attributed to forces exerted on Notch receptor activation. A Notch-independent vnd enhancer also exhibited a modest gastrulation-induced activity increase in the same stripe of cells. Together, these observations argue that gastrulation-associated forces act on the nucleus to modulate transcription levels. This regulation was uncoupled when the complex linking the nucleoskeleton and cytoskeleton (LINC) was disrupted, indicating a likely conduit. It is proposed that the coupling between tissue-level mechanics, arising from gastrulation, and enhancer activity represents a general mechanism for ensuring correct tissue specification during development and that Notch-dependent enhancers are highly sensitive to this regulation.
Thukral, S., Kaity, B., Mitra, D., Dey, B., Dey, P., Uttekar, B., Mitra, M. K., Nandi, A. and Rikhy, R. (2022). Pseudocleavage furrows restrict plasma membrane-associated PH domain in syncytial Drosophila embryos. Biophys J 121(12): 2419-2435. PubMed ID: 35591789
Syncytial cells contain multiple nuclei and have local distribution and function of cellular components despite being synthesized in a common cytoplasm. The syncytial Drosophila blastoderm embryo shows reduced spread of organelle and plasma membrane-associated proteins between adjacent nucleo-cytoplasmic domains. Anchoring to the cytoarchitecture within a nucleo-cytoplasmic domain is likely to decrease the spread of molecules; however, its role in restricting this spread has not been assessed. In order to analyze the cellular mechanisms that regulate the rate of spread of plasma membrane-associated molecules in the syncytial Drosophila embryos, a pleckstrin homology (PH) domain was expressed in a localized manner at the anterior of the embryo by tagging it with the bicoid mRNA localization signal. Anteriorly expressed PH domain forms an exponential gradient in the anteroposterior axis with a longer length scale compared with Bicoid. Using a combination of experiments and theoretical modeling, it was found that the characteristic distribution and length scale emerge due to plasma membrane sequestration and restriction within an energid. Loss of plasma membrane remodeling to form pseudocleavage furrows shows an enhanced spread of PH domain but not Bicoid. Modeling analysis suggests that the enhanced spread of the PH domain occurs due to the increased spread of the cytoplasmic population of the PH domain in pseudocleavage furrow mutants. This analysis of cytoarchitecture interaction in regulating plasma membrane protein distribution and constraining its spread has implications on the mechanisms of spread of various molecules, such as morphogens in syncytial cells.
Simoes, S., Lerchbaumer, G., Pellikka, M., Giannatou, P., Lam, T., Kim, D., Yu, J., Ter Stal, D., Al Kakouni, K., Fernandez-Gonzalez, R. and Tepass, U. (2022). Crumbs complex-directed apical membrane dynamics in epithelial cell ingression. J Cell Biol 221(7). PubMed ID: 35588693
Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cells lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. This study shows that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. These findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer.
Fierling, J., John, A., Delorme, B., Torzynski, A., Blanchard, G. B., Lye, C. M., Popkova, A., Malandain, G., Sanson, B., Etienne, J., Marmottant, P., Quilliet, C. and Rauzi, M. (2022). Embryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation. Nat Commun 13(1): 3348. PubMed ID: 35688832
Cell apical constriction driven by actomyosin contraction forces is a conserved mechanism during tissue folding in embryo development. While much is now understood of the molecular mechanism responsible for apical constriction and of the tissue-scale integration of the ensuing in-plane deformations, it is still not clear if apical actomyosin contraction forces are necessary or sufficient per se to drive tissue folding. To tackle this question, this study used the Drosophila embryo model system that forms a furrow on the ventral side, initiating mesoderm internalization. Past computational models support the idea that cell apical contraction forces may not be sufficient and that active or passive cell apico-basal forces may be necessary to drive cell wedging leading to tissue furrowing. By using 3D computational modelling and in toto embryo image analysis and manipulation, this idea is now challenged, and it is shown that embryo-scale force balance at the tissue surface, rather than cell-autonomous shape changes, is necessary and sufficient to drive a buckling of the epithelial surface forming a furrow which propagates and initiates embryo gastrulation.

Monday, August 22 - RNA and Transposons

Corgiat, E. B., List, S. M., Rounds, J. C., Yu, D., Chen, P., Corbett, A. H. and Moberg, K. H. (2022). The Nab2 RNA-binding protein patterns dendritic and axonal projections through a planar cell polarity-sensitive mechanism. G3 (Bethesda) 12(6). PubMed ID: 35471546
RNA-binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RNA-binding protein is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1-interacting RNA-binding protein Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. This study presents an analysis of a brain proteomic dataset that indicates that multiple planar cell polarity proteins are affected by Nab2 loss, and couple this with genetic data that demonstrate that Nab2 has a previously unappreciated role in restricting the growth and branching of dendrites that elaborate from larval body-wall sensory neurons. Further analysis confirms that Nab2 loss sensitizes sensory dendrites to the genetic dose of planar cell polarity components and that Nab2-planar cell polarity genetic interactions are also observed during Nab2-dependent control of axon projection in the central nervous system mushroom bodies. Collectively, these data identify the conserved Nab2 RNA-binding protein as a likely component of post-transcriptional mechanisms that limit dendrite growth and branching in Drosophila sensory neurons and genetically link this role to the planar cell polarity pathway. Given that mammalian ZC3H14 localizes to dendritic spines and controls spine density in hippocampal neurons, these Nab2-planar cell polarity genetic data may highlight a conserved path through which Nab2/ZC3H14 loss affects morphogenesis of both axons and dendrites in diverse species.
Stanek, T. J., Cao, W., Mehra, R. M. and Ellison, C. E. (2022). Sex-specific variation in R-loop formation in Drosophila melanogaster. PLoS Genet 18(6): e1010268. PubMed ID: 35687614
R-loops are three-stranded nucleotide structures consisting of a DNA:RNA hybrid and a displaced ssDNA non-template strand. Previous work suggests that R-loop formation is primarily determined by the thermodynamics of DNA:RNA binding, which are governed by base composition (e.g., GC skew) and transcription-induced DNA superhelicity. However, R-loops have been described at genomic locations that lack these properties, suggesting that they may serve other context-specific roles. To better understand the genetic determinants of R-loop formation, this study has characterized the Drosophila melanogaster R-loop landscape across strains and between sexes using DNA:RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq). R-loops were found to be associated with sequence motifs that are G-rich or exhibit G/C skew, as well as highly expressed genes, tRNAs, and small nuclear RNAs, consistent with a role for DNA sequence and torsion in R-loop specification. However, motifs associated with R-loops that are A/T-rich and lack G/C skew were found, as well as a subset of R-loops that are enriched in polycomb-repressed chromatin. Differential enrichment analysis reveals a small number of sex-biased R-loops: while non-differentially enriched and male-enriched R-loops form at similar genetic features and chromatin states and contain similar sequence motifs, female-enriched R-loops form at unique genetic features, chromatin states, and sequence motifs and are associated with genes that show ovary-biased expression. Male-enriched R-loops are most abundant on the dosage-compensated X chromosome, where R-loops appear stronger compared to autosomal R-loops. R-loop-containing genes on the X chromosome are dosage-compensated yet show lower MOF binding and reduced H4K16ac compared to R-loop-absent genes, suggesting that H4K16ac or MOF may attenuate R-loop formation. Collectively, these results suggest that R-loop formation in vivo is not fully explained by DNA sequence and topology and raise the possibility that a distinct subset of these hybrid structures plays an important role in the establishment and maintenance of epigenetic differences between sexes.
Pandey, M., Luhur, A., Sokol, N. S. and Chawla, G. (2022). Molecular Dissection of a Conserved Cluster of miRNAs Identifies Critical Structural Determinants That Mediate Differential Processing. Front Cell Dev Biol 10: 909212. PubMed ID: 35784477
Differential processing is a hallmark of clustered microRNAs (miRNAs) and the role of position and order of miRNAs in a cluster together with the contribution of stem-base and terminal loops has not been explored extensively within the context of a polycistronic transcript. To elucidate the structural attributes of a polycistronic transcript that contribute towards the differences in efficiencies of processing of the co-transcribed miRNAs, a series of chimeric variants was constructed of Drosophila let-7-Complex that encodes three evolutionary conserved and differentially expressed miRNAs (miR-100, let-7 and miR-125) and examined the expression and biological activity of the encoded miRNAs. The kinetic effects of Drosha and Dicer processing on the chimeric precursors were examined by in vitro processing assays. The results highlight the importance of stem-base and terminal loop sequences in differential expression of polycistronic miRNAs and provide evidence that processing of a particular miRNA in a polycistronic transcript is in part determined by the kinetics of processing of adjacent miRNAs in the same cluster. Overall, this analysis provides specific guidelines for achieving differential expression of a particular miRNA in a cluster by structurally induced changes in primary miRNA (pri-miRNA) sequences.
Yushkova, E. (2022). Contribution of transposable elements to transgenerational effects of chronic radioactive exposure of natural populations of Drosophila melanogaster living for a long time in the zone of the Chernobyl nuclear disaster. J Environ Radioact 251-252: 106945. PubMed ID: 35696883
The accident at the Chernobyl Nuclear Power Plant (ChNPP) led to the negative impact of chronic radioactive contamination on populations of organisms associated with the transgenerational transmission of genome instability. In this study, for the first time, the features of the influence of transposable elements (TEs) on the long-term biological consequences of the ChNPP are considered. In this work, specimens of D. melanogaster obtained from natural populations in 2007 in the areas of the ChNPP with heterogeneous radioactive contamination were studied. The descendants from these populations were maintained in laboratory (inbred) conditions for 160 generations. A stable transgenerational transmission of dominant lethal mutations (DLMs) to the offspring of all studied populations was shown. The DLM frequencies strongly were correlated with the level of survival of offspring. The mean frequencies of recessive sex-linked lethal mutations varied at the level of spontaneous point mutations. The simultaneous presence of P, hobo and I elements indicates that the studied populations do not have a definite cytotype, their phenotypic status is unstable. The behavior of TEs in the genomes of offspring depends not only on parental exposure, but also on origin of population, distance to the ChNPP, and inbred conditions. The obtained results confirm the hypothesis that TEs are involved in transgenerational transmission and accumulation of mutations by the offspring of irradiated parents. The TEs pattern present in the Chernobyl genomes of D. melanogaster is a peculiar of epigenetic mechanism for the regulation of plasticity and adaptation of populations living for many generations under conditions of a technogenically caused radiation background.
Nguyen, A. H., Wang, W., Chong, E., Chatla, K. and Bachtrog, D. (2022). Transposable element accumulation drives size differences among polymorphic Y Chromosomes in Drosophila. Genome Res 32(6): 1074-1088. PubMed ID: 35501131
Y Chromosomes of many species are gene poor and show low levels of nucleotide variation, yet they often display high amounts of structural diversity. Dobzhansky cataloged several morphologically distinct Y Chromosomes in Drosophila pseudoobscura that differ in size and shape, but the molecular causes of their large size differences are unclear. This study used cytogenetics and long-read sequencing to study the sequence content of polymorphic Y Chromosomes in D. pseudoobscura. The Y Chromosomes were shown to differ almost twofold in size, ranging from 30 to 60 Mb. Most of this size difference is caused by a handful of active transposable elements (TEs) that have recently expanded on the largest Y Chromosome, with different elements being responsible for Y expansion on differently sized D. pseudoobscura Y's. Y Chromosomes differ in their heterochromatin enrichment and expression of Y-enriched TEs, and also influence expression of dozens of autosomal and X-linked genes. The same helitron element that showed the most drastic amplification on the largest Y in D. pseudoobscura independently amplified on a polymorphic large Y Chromosome in Drosophila affinis, suggesting that some TEs are inherently more prone to become deregulated on Y Chromosomes.
Chen, J., Huang, Y. and Zhang, K. (2022). The DEAD-Box Protein Rok1 Coordinates Ribosomal RNA Processing in Association with Rrp5 in Drosophila. Int J Mol Sci 23(10). PubMed ID: 35628496
Ribosome biogenesis and processing involve the coordinated action of many components. The DEAD-box RNA helicase (Rok1) is essential for cell viability, and the depletion of Rok1 inhibits pre-rRNA processing. Previous research on Rok1 and its cofactor Rrp5 has been performed primarily in yeast. Few functional studies have been performed in complex multicellular eukaryotes. This study used a combination of genetics and developmental experiments to show that Rok1 and Rrp5, which localize to the nucleolus, play key roles in the pre-rRNA processing and ribosome assembly in D. melanogaster. The accumulation of pre-rRNAs caused by Rok1 depletion can result in developmental defects. The loss of Rok1 enlarged the nucleolus and led to stalled ribosome assembly and pre-rRNA processing in the nucleolus, thereby blocking rRNA maturation and exacerbating the inhibition of mitosis in the brain. This study also discovered that rrp5(4-2/4-2) displayed significantly increased ITS1 signaling by fluorescence in situ hybridization, and a reduction in ITS2. Rrp5 signal was highly enriched in the core of the nucleolus in the rok1(167/167) mutant, suggesting that Rok1 is required for the accurate cellular localization of Rrp5 in the nucleolus. This study has thus uncovered functions of Rok1 that reveal important implications for ribosome processing in eukaryotes.

Friday, August 19th - Cytoskeleton

Molina, E., Cataldo, V. F., Eggers, C., Munoz-Madrid, V. and Glavic, A. (2022). p53 Related Protein Kinase is Required for Arp2/3-Dependent Actin Dynamics of Hemocytes in Drosophila melanogaster. Front Cell Dev Biol 10: 859105. PubMed ID: 35721516
Cells extend membrane protrusions like lamellipodia and filopodia from the leading edge to sense, to move and to form new contacts. The Arp2/3 complex sustains lamellipodia formation, and in conjunction with the actomyosin contractile system, provides mechanical strength to the cell. Drosophila p53-related protein kinase (Prpk), a Tsc5p ortholog, has been described as essential for cell growth and proliferation. In addition, Prpk interacts with proteins associated to actin filament dynamics such as α-spectrin and the Arp2/3 complex subunit Arpc4. This study investigated the role of Prpk in cell shape changes, specifically regarding actin filament dynamics and membrane protrusion formation. Reductions in Prpk alter cell shape and the structure of lamellipodia, mimicking the phenotypes evoked by Arp2/3 complex deficiencies. Prpk co-localize and co-immunoprecipitates with the Arp2/3 complex subunit Arpc1 and with the small GTPase Rab35. Importantly, expression of Rab35, known by its ability to recruit upstream regulators of the Arp2/3 complex, could rescue the Prpk knockdown phenotypes. Finally, the requirement of Prpk was evaluated in different developmental contexts, where it was shown to be essential for correct Arp2/3 complex distribution and actin dynamics required for hemocytes migration, recruitment, and phagocytosis during immune response.
Thyagarajan, P., Feng, C., Lee, D., Shorey, M. and Rolls, M. M. (2022). Microtubule polarity is instructive for many aspects of neuronal polarity. Dev Biol 486: 56-70. PubMed ID: 35341730
Many neurons in bilaterian animals are polarized with functionally distinct axons and dendrites. Microtubule polarity, microtubule stability, and the axon initial segment (AIS) have all been shown to influence polarized transport in neurons. Each of these cytoskeletal cues could act independently to control axon and dendrite identity, or there could be a hierarchy in which one acts upstream of the others. This study tested the hypothesis that microtubule polarity acts as a master regulator of neuronal polarity by using a Drosophila genetic background in which some dendrites have normal minus-end-out microtubule polarity and others have the axonal plus-end-out polarity. In these mosaic dendrite arbors, this study found that ribosomes, which are more abundant in dendrites than axons, were reduced in plus-end-out dendrites, while an axonal cargo was increased. In addition, it was determined that microtubule stability was different in plus-end-out and minus-end-out dendrites, with plus-end-out ones having more stable microtubules like axons. Similarly, it was found that ectopic diffusion barriers, like those at the AIS, formed at the base of dendrites with plus-end-out regions. Thus, changes in microtubule polarity were sufficient to rearrange other cytoskeletal features associated with neuronal polarization. However, overall neuron shape was maintained with only subtle changes in branching in mosaic arbors. It is concluded that microtubule polarity can act upstream of many aspects of intracellular neuronal polarization, but shape is relatively resilient to changes in microtubule polarity in vivo.
Bu, S., Tang, Q., Wang, Y., Lau, S. S. Y., Yong, W. L. and Yu, F. (2022). Drosophila CLASP regulates microtubule orientation and dendrite pruning by suppressing Par-1 kinase. Cell Rep 39(9): 110887. PubMed ID: 35649352
The evolutionarily conserved CLASPs (cytoplasmic linker-associated proteins) are microtubule-associated proteins that inhibit microtubule catastrophe and promote rescue. CLASPs can regulate axonal elongation and dendrite branching in growing neurons. However, their roles in microtubule orientation and neurite pruning in remodeling neurons remain unknown. This study identified the Drosophila CLASP homolog Orbit/MAST, which is required for dendrite pruning in ddaC sensory neurons during metamorphosis. Orbit is important for maintenance of the minus-end-out microtubule orientation in ddaC dendrites. Structural analysis reveals that the microtubule lattice-binding TOG2 domain is required for Orbit to regulate dendritic microtubule orientation and dendrite pruning. In a genetic modifier screen, the conserved Par-1 kinase was further identified as a suppressor of Orbit in dendritic microtubule orientation. Moreover, elevated Par-1 function impairs dendritic microtubule orientation and dendrite pruning, phenocopying orbit mutants. Overall, this study demonstrates that Drosophila CLASP governs dendritic microtubule orientation and dendrite pruning at least partly via suppressing Par-1 kinase.
Repton, C., Cullen, C. F., Costa, M. F. A., Spanos, C., Rappsilber, J. and Ohkura, H. (2022). The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin. PLoS Genet 18(6): e1009995. PubMed ID: 35666772
Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. Previous work has uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. This paper reports systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, proteins or complexes were identified whose ability to bind microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function.
Villars, A., Matamoro-Vidal, A., Levillayer, F. and Levayer, R. (2022). Microtubule disassembly by caspases is an important rate-limiting step of cell extrusion. Nat Commun 13(1): 3632. PubMed ID: 35752632
The expulsion of dying epithelial cells requires well-orchestrated remodelling steps to maintain tissue sealing. This process, named cell extrusion, has been mostly analysed through the study of actomyosin regulation. Yet, the mechanistic relationship between caspase activation and cell extrusion is still poorly understood. Using the Drosophila pupal notum, a single layer epithelium where extrusions are caspase-dependent, this study showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration and dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion, while microtubule stabilisation strongly impairs cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting step of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation.
Aydogan, M. G., Hankins, L. E., Steinacker, T. L., Mofatteh, M., Saurya, S., Wainman, A., Wong, S. S., Lu, X., Zhou, F. Y. and Raff, J. W. (2022). Centriole distal-end proteins CP110 and Cep97 influence centriole cartwheel growth at the proximal-end. J Cell Sci. PubMed ID: 35707992
Centrioles are composed of a central cartwheel tethered to nine-fold symmetric microtubule (MT) blades. The centriole cartwheel and MTs are thought to grow from opposite ends of these organelles, so it is unclear how they coordinate their assembly. Previous work showed that an oscillation of Polo-like kinase 4 (Plk4) helps to initiate and time the growth of the cartwheel at the proximal end. This study showed that CP110 and Cep97 form a complex close to the distal-end of the centriole MTs whose levels rise and fall as the new centriole MTs grow, in a manner that appears to be entrained by the core Cdk/Cyclin oscillator that drives the nuclear divisions in these embryos. These CP110/Cep97 dynamics, however, do not appear to time the period of centriole MT growth directly. Instead, changing the levels of CP110/Cep97 appears to alter the Plk4 oscillation and the growth of the cartwheel at the proximal end. These findings reveal an unexpected potential crosstalk between factors normally concentrated at opposite ends of the growing centrioles, which may help to coordinate centriole growth.

Thursday, August 18th - Larval and Adult Neural Function

Kohatsu, S., Tanabe, N., Yamamoto, D. and Isono, K. (2022). Which Sugar to Take and How Much to Take? Two Distinct Decisions Mediated by Separate Sensory Channels. Front Mol Neurosci 15: 895395. PubMed ID: 35726300
In Drosophila melanogaster, gustatory receptor neurons (GRNs) for sugar taste coexpress various combinations of gustatory receptor (Gr) genes and are found in multiple sites in the body. To determine whether diverse sugar GRNs expressing different combinations of Grs have distinct behavioral roles, the effects on feeding behavior were examined of genetic manipulations which promote or suppress functions of GRNs that express either or both of the sugar receptor genes Gr5a (Gr5a+ GRNs) and Gr61a (Gr61a+ GRNs). Cell-population-specific overexpression of the wild-type form of Gr5a (Gr5a(+)) in the Gr5a mutant background revealed that Gr61a+ GRNs localized on the legs and internal mouthpart critically contribute to food choice but not to meal size decisions, while Gr5a+ GRNs, which are broadly expressed in many sugar-responsive cells across the body with an enrichment in the labella, are involved in both food choice and meal size decisions. The legs harbor two classes of Gr61a expressing GRNs, one with Gr5a expression (Gr5a+/Gr61a+ GRNs) and the other without Gr5a expression (Gr5a-/Gr61a+ GRNs). Blocking the Gr5a+ class in the entire body reduced the preference for trehalose and blocking the Gr5a- class reduced the preference for fructose. These two subsets of GRNs are also different in their central projections: axons of tarsal Gr5a+/Gr61a+ GRNs terminate exclusively in the ventral nerve cord, while some axons of tarsal Gr5a-/Gr61a+ GRNs ascend through the cervical connectives to terminate in the subesophageal ganglion. It is proposed that tarsal Gr5a+/Gr61a+ GRNs and Gr5a-/Gr61a+ GRNs represent functionally distinct sensory pathways that function differently in food preference and meal-size decisions.
Adel, M., Chen, N., Zhang, Y., Reed, M. L., Quasney, C. and Griffith, L. C. (2022). Pairing-Dependent Plasticity in a Dissected Fly Brain Is Input-Specific and Requires Synaptic CaMKII Enrichment and Nighttime Sleep. J Neurosci 42(21): 4297-4310. PubMed ID: 35474278
In Drosophila, in vivo functional imaging studies revealed that associative memory formation is coupled to a cascade of neural plasticity events in distinct compartments of the mushroom body (MB). In-depth investigation of the circuit dynamics, however, will require an ex vivo model that faithfully mirrors these events to allow direct manipulations of circuit elements that are inaccessible in the intact fly. The current ex vivo models have been able to reproduce the fundamental plasticity of aversive short-term memory, a potentiation of the MB intrinsic neuron (Kenyon cells [KCs]) responses after artificial learning ex vivo However, this potentiation showed different localization and encoding properties from those reported in vivo and failed to generate the previously reported suppression plasticity in the MB output neurons (MBONs). This study developed an ex vivo model using the female Drosophila brain that recapitulates behaviorally evoked plasticity in the KCs and MBONs. This plasticity accurately localizes to the MB α'3 compartment and is encoded by a coincidence between KC activation and dopaminergic input. The formed plasticity is input-specific, requiring pairing of the conditioned stimulus and unconditioned stimulus pathways; hence, it was named pairing-dependent plasticity. Pairing-dependent plasticity formation requires an intact CaMKII gene and is blocked by previous-night sleep deprivation but is rescued by rebound sleep. In conclusion, this study showed that the ex vivo preparation recapitulates behavioral and imaging results from intact animals and can provide new insights into mechanisms of memory formation at the level of molecules, circuits, and brain state.
Hanson, M. A. and Lemaitre, B. (2022). Repeated truncation of a modular antimicrobial peptide gene for neural context. PLoS Genet 18(6): e1010259. PubMed ID: 35714143
Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading pathogens. These genes commonly encode multiple products as post-translationally cleaved polypeptides. Recent studies have highlighted roles for AMPs in neurological contexts suggesting functions for these defence molecules beyond infection. During an immune study characterizing the antimicrobial peptide gene Baramicin, multiple Baramicin paralogs were uncovered in Drosophila melanogaster and other species, united by their N-terminal IM24 domain. Not all paralogs were immune-induced. In this study, through careful dissection of the Baramicin family's evolutionary history, it was found that paralogs lacking immune induction result from repeated events of duplication and subsequent truncation of the coding sequence from an immune-inducible ancestor. These truncations leave only the IM24 domain as the prominent gene product. Surprisingly, using mutation and targeted gene silencing it was demonstrated that two such genes are adapted for function in neural contexts in D. melanogaster. Enrichment in the head was found for independent Baramicin genes in other species. The Baramicin evolutionary history reveals that the IM24 Baramicin domain is not strictly useful in an immune context. This study thus provides a case study for how an AMP-encoding gene might play dual roles in both immune and non-immune processes via its multiple peptide products. As many AMP genes encode polypeptides, a full understanding of how immune effectors interact with the nervous system will require consideration of all their peptide products.
Li, Q., Wang, M., Zhang, P., Liu, Y., Guo, Q., Zhu, Y., Wen, T., Dai, X., Zhang, X., Nagel, M., Dethlefsen, B. H., Xie, N., Zhao, J., Jiang, W., Han, L., Wu, L., Zhong, W., Wang, Z., Wei, X., Dai, W., Liu, L., Xu, X., Lu, H., Yang, H., Wang, J., Boomsma, J. J., Liu, C., Zhang, G. and Liu, W. (2022). A single-cell transcriptomic atlas tracking the neural basis of division of labour in an ant superorganism. Nat Ecol Evol. PubMed ID: 35711063
Ant colonies with permanent division of labour between castes and highly distinct roles of the sexes have been conceptualized to be superorganisms, but the cellular and molecular mechanisms that mediate caste/sex-specific behavioural specialization have remained obscure. This study characterized the brain cell repertoire of queens, gynes (virgin queens), workers and males of Monomorium pharaonis by obtaining 206,367 single-nucleus transcriptomes. In contrast to Drosophila, the mushroom body Kenyon cells are abundant in ants and display a high diversity with most subtypes being enriched in worker brains, the evolutionarily derived caste. Male brains are as specialized as worker brains but with opposite trends in cell composition with higher abundances of all optic lobe neuronal subtypes, while the composition of gyne and queen brains remained generalized, reminiscent of solitary ancestors. Role differentiation from virgin gynes to inseminated queens induces abundance changes in roughly 35% of cell types, indicating active neurogenesis and/or programmed cell death during this transition. Insemination-induced cell changes were also identified probably associated with the longevity and fecundity of the reproductive caste, including increases of ensheathing glia and a population of dopamine-regulated Dh31-expressing neurons. It is concluded that permanent caste differentiation and extreme sex-differentiation induced major changes in the neural circuitry of ants.
Richard, M., Doubkova, K., Nitta, Y., Kawai, H., Sugie, A. and Tavosanis, G. (2022). A Quantitative Model of Sporadic Axonal Degeneration in the Drosophila Visual System. J Neurosci 42(24): 4937-4952. PubMed ID: 35534228
In human neurodegenerative diseases, neurons undergo axonal degeneration months to years before they die. This study developed a system modeling early degenerative events in Drosophila adult photoreceptor cells. Thanks to the stereotypy of their axonal projections, this system delivers quantitative data on sporadic and progressive axonal degeneration of photoreceptor cells. Using this method, it was shown that exposure of adult female flies to a constant light stimulation for several days overcomes the intrinsic resilience of R7 photoreceptors and leads to progressive axonal degeneration. This was not associated with apoptosis. Evidence is provided that loss of synaptic integrity between R7 and a postsynaptic partner preceded axonal degeneration, thus recapitulating features of human neurodegenerative diseases. Finally, these experiments uncovered a role of postsynaptic partners of R7 to initiate degeneration, suggesting that postsynaptic cells signal back to the photoreceptor to maintain axonal structure. This model can be used to dissect cellular and circuit mechanisms involved in the early events of axonal degeneration, allowing for a better understanding of how neurons cope with stress and lose their resilience capacities.
Simoes, A. R., Neto, M., Alves, C. S., Santos, M. B., Fernandez-Hernandez, I., Veiga-Fernandes, H., Brea, D., Dur, I., Encinas, J. M. and Rhiner, C. (2022). Damage-responsive neuro-glial clusters coordinate the recruitment of dormant neural stem cells in Drosophila. Dev Cell. PubMed ID: 35716661
Recruitment of stem cells is crucial for tissue repair. Although stem cell niches can provide important signals, little is known about mechanisms that coordinate the engagement of disseminated stem cells across an injured tissue. In Drosophila, adult brain lesions trigger local recruitment of scattered dormant neural stem cells suggesting a mechanism for creating a transient stem cell activation zone. This study found that injury triggers a coordinated response in neuro-glial clusters that promotes the spread of a neuron-derived stem cell factor via glial secretion of the lipocalin-like transporter Swim. Strikingly, swim is induced in a Hif1-α-dependent manner in response to brain hypoxia. Mammalian Swim (Lcn7) is also upregulated in glia of the mouse hippocampus upon brain injury. These results identify a central role of neuro-glial clusters in promoting neural stem cell activation at a distance, suggesting a conserved function of the HIF1-α/Swim/Wnt module in connecting injury-sensing and regenerative outcomes.

Wednesday, August 18th - Behavior

Zhong, L., Yang, Z., Tang, H., Xu, Y., Liu, X. and Shen, J. (2022). Differential analysis of negative geotaxis climbing trajectories in Drosophila under different conditions. Arch Insect Biochem Physiol: e21922. PubMed ID: 35666567
The decline of Drosophila climbing behavior is one of the common phenomena of Drosophila aging. The so-called negative geotaxis refers to the natural upward climbing behavior of Drosophila melanogaster after it oscillates to the bottom of the test tube. The strength of climbing ability is regarded as the index of aging change of D. melanogaster. At present, many laboratories use the percentage of 10 fruit flies climbing a specific height in 5 s as a general indicator of the climbing ability of fruit flies. This group research index ignores the climbing performance of a single fruit fly, and the climbing height belongs to the concept of vertical distance in physics, which cannot truly and effectively reflect the concept of curve distance in the actual climbing process of fruit flies. Therefore, based on the image processing algorithm, an experimental method was added to draw the climbing trajectory of a single fruit fly. By comparing the differences in climbing behavior of fruit flies under different sex, group or single, oscillation condition or rotation inversion condition, it was possible to find that the K-Nearest Neighbor target detection algorithm has good applicability in fruit fly climbing experiment, and the climbing ability of fruit flies decreases with age. Under the same experimental conditions, the climbing ability of female fruit flies was greater than that of male fruit flies. The climbing track length of a single fruit fly can better reflect the climbing process of a fruit fly.
Sun, X., Liu, Y., Liu, C., Mayumi, K., Ito, K., Nose, A. and Kohsaka, H. (2022). A neuromechanical model for Drosophila larval crawling based on physical measurements. BMC Biol 20(1): 130. PubMed ID: 35701821
Animal locomotion requires dynamic interactions between neural circuits, the body (typically muscles), and surrounding environments. How these outputs are integrated with body mechanics (neuromechanics) is less clear, in part due to the lack of understanding of the biomechanical properties of animal bodies. This paper proposes an integrated neuromechanical model of movement based on physical measurements by taking Drosophila larvae as a model of soft-bodied animals. First, the kinematics of forward crawling in Drosophila larvae was characterized at a segmental and whole-body level. Then the biomechanical parameters of fly larvae were characterized, namely the contraction forces generated by neural activity, and passive elastic and viscosity of the larval body was characterized using a stress-relaxation test. A mathematical neuromechanical model was established based on the physical measurements described above, obtaining seven kinematic values characterizing crawling locomotion. By optimizing the parameters in the neural circuit, the neuromechanical model succeeded in quantitatively reproducing the kinematics of larval locomotion that were obtained experimentally. This model could reproduce the observation of optogenetic studies reported previously. The model predicted that peristaltic locomotion could be exhibited in a low-friction condition. Analysis of floating larvae provided results consistent with this prediction. Furthermore, the model predicted a significant contribution of intersegmental connections in the central nervous system, which contrasts with a previous study. This study generated a neurochemical model based on physical measurement to provide a new foundation to study locomotion in soft-bodied animals and soft robot engineering.
Wu, X., Wang, S., Zhao, X., Wen, J., Li, Y., Zhao, Z. and Du, J. (2022). Analysis of sleep in individual Drosophila melanogaster reveals a self-regulatory role for cuticular hydrocarbons pheromones Insect Sci. PubMed ID: 35199930
It is well established that pheromones are used by insects to transmit information between individuals. However, research has revealed that individual insects can be both the sender and the receiver of some pheromonal signals. It is therefore interesting to consider whether the pheromonal state of an individual insect can exert an effect on itself. This study monitored the sleep activity of single flies exhibiting a mutation that leads to pheromonal deficiency and found that cuticular hydrocarbons (CHs) exerted self-regulatory effects on the amount of sleep experienced by these flies. To identify the physiological significance of this mechanism, the amounts of sleep were compared in individual young flies and individual old flies (flies are known to sleep less as they get older), and this data was compared with young and old flies exhibiting mutations that lead to CH reception defects. The differences in the amount of sleep experienced by young and old mutant flies were significantly lower than those of the control flies. These data show that hydrocarbon signals produced by the cuticle in Drosophila can be self-perceived and regulate the amount of sleep acquired in a maturation-dependent manner.
Wang, Y., Sun, W., Fleischmann, S., Millar, J. G., Ruther, J. and Verhulst, E. C. (2022). Silencing Doublesex expression triggers three-level pheromonal feminization in Nasonia vitripennis males. Proc Biol Sci 289(1967): 20212002. PubMed ID: 35078369
Doublesex (Dsx) has a conserved function in controlling sexual morphological differences in insects, but knowledge of its role in regulating sexual behaviour is primarily limited to Drosophila. This study shows with the parasitoid wasp Nasonia vitripennis that males whose Dsx gene had been silenced (NvDsx-i) underwent a three-level pheromonal feminization: (1) NvDsx-i males were no longer able to attract females from a distance, owing to drastically reduced titres of the long-range sex pheromone; (2) NvDsx-i males were courted by wild-type males as though they were females, which correlated with a lower abundance of alkenes in their cuticular hydrocarbon (CHC) profiles. Supplementation with realistic amounts of synthetic (Z)-9-hentriacontene (Z9C31), the most significantly reduced alkene in NvDsx-i males, to NvDsx-i males interrupted courtship by wild-type conspecific males. Supplementation of female CHC profiles with Z9C31 reduced courtship and mating attempts by wild-type males. These results prove that Z9C31 is crucial for sex discrimination in N. vitripennis; and (3) Nvdsx-i males were hampered in eliciting female receptivity and thus experienced severely reduced mating success, suggesting that they are unable to produce the to-date unidentified oral aphrodisiac pheromone reported in N. vitripennis males. It is concluded that Dsx is a multi-level key regulator of pheromone-mediated sexual communication in N. vitripennis.
Tanaka, R. and Clark, D. A. (2022). Neural mechanisms to exploit positional geometry for collision avoidance. Curr Biol 32(11): 2357-2374. PubMed ID: 35508172
Visual motion provides rich geometrical cues about the three-dimensional configuration of the world. However, how brains decode the spatial information carried by motion signals remains poorly understood. This paper studied a collision-avoidance behavior in Drosophila as a simple model of motion-based spatial vision. With simulations and psychophysics, it was demonstrated that walking Drosophila exhibit a pattern of slowing to avoid collisions by exploiting the geometry of positional changes of objects on near-collision courses. This behavior requires the visual neuron LPLC1, whose tuning mirrors the behavior and whose activity drives slowing. LPLC1 pools inputs from object and motion detectors, and spatially biased inhibition tunes it to the geometry of collisions. Connectomic analyses identified circuitry downstream of LPLC1 that faithfully inherits its response properties. Overall, these results reveal how a small neural circuit solves a specific spatial vision task by combining distinct visual features to exploit universal geometrical constraints of the visual world.
Grinberg, M., Levin, R., Neuman, H., Ziv, O., Turjeman, S., Gamliel, G., Nosenko, R. and Koren, O. (2022). Antibiotics increase aggression behavior and aggression-related pheromones and receptors in Drosophila melanogaster iScience 25(6): 104371. PubMed ID: 35620429
Aggression is a behavior common in most species; it is controlled by internal and external drivers, including hormones, environmental cues, and social interactions, and underlying pathways are understood in a broad range of species. To date, though, effects of gut microbiota on aggression in the context of gut-brain communication and social behavior have not been completely elucidated. This study examined how manipulation of Drosophila melanogaster microbiota affects aggression as well as the pathways that underlie the behavior in this species. Male flies treated with antibiotics exhibited significantly more aggressive behaviors. Furthermore, they had higher levels of cVA and (Z)-9 Tricosene, pheromones associated with aggression in flies, as well as higher expression of the relevant pheromone receptors and transporters OR67d, OR83b, GR32a, and LUSH. These findings suggest that aggressive behavior is, at least in part, mediated by bacterial species in flies.

Tuesday, August 16th - Larval and Adult Physiology and Metabolism

Tamang, A. M., Parkash, R., Srivastava, R. K. and Singh, P. (2022). Adaptive changes in energy reserves and effects of body melanization on thermal tolerance in Drosophila simulans Comp Biochem Physiol A Mol Integr Physiol 271: 111258. PubMed ID: 35705113
Seasonally polyphenic types have been documented in many Drosophilids, which differ significantly during thermal stress. Although Drosophila simulans is a sibling species to Drosophila melanogaster, both thrive in the temperate and tropical climates, but various climatic factors are expected to impact their distribution and abundance. As a result, D. simulans may use phenotypic plasticity to adapt to colder and drier circumstances in temperate zones, although such studies are less known. The main aim of this study was to find a link between adaptive plasticity and thermal tolerance in D. simulans. Two morphs in D. simulans flies were characterized based on the abdominal melanization collected from the same locality and season, as this trait is highly associated with the larval developmental conditions. The results suggested that flies reared from dark and light morph showed significant differences in the basal level of proline, carbohydrates (trehalose, glycogen), and lipids (cuticular lipids and total body lipids) within simulated seasons and morph lineages in D. simulans flies. It was further shown that D. simulans reared from dark morph are better adapted to cold conditions, whereas light flies are more adapted to warm conditions. The flies, both from light and dark morph lineages, when reared at 15 °C, showed an increase in the level of total body lipids after acclimation at 0 °C but a decrease in the level of proline and carbohydrates (trehalose, glycogen). Heat acclimation increases glycogen levels in the flies from light morph lineage while decreases trehalose and proline.
Gupta, V., Frank, A. M., Matolka, N. and Lazzaro, B. P. (2022). Inherent constraints on a polyfunctional tissue lead to a reproduction-immunity tradeoff BMC Biol 20(1): 127. PubMed ID: 35655304
Single tissues can have multiple functions, which can result in constraints, impaired function, and tradeoffs. The insect fat body performs remarkably diverse functions including metabolic control, reproductive provisioning, and systemic immune responses. How polyfunctional tissues simultaneously execute multiple distinct physiological functions is generally unknown. Immunity and reproduction are observed to trade off in many organisms but the mechanistic basis for this tradeoff is also typically not known. This study investigated constraints and trade-offs in the polyfunctional insect fat body. Using single-nucleus sequencing, it was determined that the Drosophila melanogaster fat body executes diverse basal functions with heterogenous cellular subpopulations. The size and identity of these subpopulations are remarkably stable between virgin and mated flies, as well as before and after infection. However, as an emergency function, the immune response engages the entire tissue and all cellular subpopulations produce induce expression of defense genes. Reproductively active females who were given bacterial infection exhibited signatures of ER stress and impaired capacity to synthesize new protein in response to infection, including decreased capacity to produce antimicrobial peptides. Transient provision of a reversible translation inhibitor to mated females prior to infection rescued general protein synthesis, specific production of antimicrobial peptides, and survival of infection. The commonly observed tradeoff between reproduction and immunity appears to be driven, in D. melanogaster, by a failure of the fat body to be able to handle simultaneous protein translation demands of reproductive provisioning and immune defense. It is suggested that inherent cellular limitations in tissues that perform multiple functions may provide a general explanation for the wide prevalence of physiological and evolutionary tradeoffs.
Sano, H., Nakamura, A., Yamane, M., Niwa, H., Nishimura, T., Araki, K., Takemoto, K., Ishiguro, K. I., Aoki, H., Kato, Y. and Kojima, M. (2022). The polyol pathway is an evolutionarily conserved system for sensing glucose uptake PLoS Biol 20(6): e3001678. PubMed ID: 35687590
Cells must adjust the expression levels of metabolic enzymes in response to fluctuating nutrient supply. For glucose, such metabolic remodeling is highly dependent on a master transcription factor ChREBP/MondoA. However, it remains elusive how glucose fluctuations are sensed by ChREBP/MondoA despite the stability of major glycolytic pathways. This study shows that in both flies and mice, ChREBP/MondoA activation in response to glucose ingestion involves an evolutionarily conserved glucose-metabolizing pathway: the polyol pathway. The polyol pathway converts glucose to fructose via sorbitol. It has been believed that this pathway is almost silent, and its activation in hyperglycemic conditions has deleterious effects on human health. This study shows that the polyol pathway regulates the glucose-responsive nuclear translocation of Mondo, a Drosophila homologue of ChREBP/MondoA, which directs gene expression for organismal growth and metabolism. Likewise, inhibition of the polyol pathway in mice impairs ChREBP's nuclear localization and reduces glucose tolerance. It is proposed that the polyol pathway is an evolutionarily conserved sensing system for glucose uptake that allows metabolic remodeling.
Hale, C., Moulton, J. K., Otis, Y. and Ganter, G. (2022). ARMADILLO REGULATES NOCICEPTIVE SENSITIVITY IN THE ABSENCE OF INJURY Mol Pain: 17448069221111155. PubMed ID: 35712882
Abnormal pain has recently been estimated to affect ~50 million adults each year within the United States. With many treatment options for abnormal pain, such as opioid analgesics, carrying numerous deleterious side effects, research into safer and more effective treatment options is crucial. To help elucidate the mechanisms controlling nociceptive sensitivity, the Drosophila melanogaster larval nociception model has been used to characterize well-conserved pathways through the use of genetic modification and/or injury to alter the sensitivity of experimental animals. Mammalian models have provided evidence of β-catenin signaling involvement in neuropathic pain development. By capitalizing on the conserved nature of β-catenin functions in the fruit fly, this study describes a role for Armadillo, the fly homolog to mammalian β-catenin, in regulating baseline sensitivity in the primary nociceptor of the fly, in the absence of injury, using under- and over-expression of Armadillo in a cell-specific manner. Underexpression of Armadillo resulted in hyposensitivity, while overexpression of wild-type Armadillo or expression of a degradation-resistant Armadillo resulted in hypersensitivity. Neither underexpression nor overexpression of Armadillo resulted in dendritic morphological changes that could contribute to behavioral phenotypes observed. These results showed that focused manipulation of Armadillo expression within the nociceptors is sufficient to modulate baseline response in the nociceptors to a noxious stimulus and that these changes are not shown to be associated with a morphogenetic effect.
Suito, T., Nagao, K., Juni, N., Hara, Y., Sokabe, T., Atomi, H. and Umeda, M. (2022). Regulation of thermoregulatory behavior by commensal bacteria in Drosophila Biosci Biotechnol Biochem. PubMed ID: 35671161
Commensal bacteria affect many aspects of host physiology. This study focused on the role of commensal bacteria in the thermoregulatory behavior of Drosophila melanogaster. The elimination of commensal bacteria caused an increase in the preferred temperature of Drosophila third-instar larvae without affecting the activity of transient receptor potential ankyrin 1 (TRPA1)-expressing thermosensitive neurons. Eight bacterial strains were isolated from the gut and culture medium of conventionally reared larvae, and it was found that the preferred temperature of the larvae was decreased by mono-association with Lactobacillus plantarum or Corynebacterium nuruki. Mono-association with these bacteria did not affect the indices of energy metabolism such as ATP and glucose levels of larvae, which are closely linked to thermoregulation in animals. Thus, this study shows a novel role for commensal bacteria in host thermoregulation and identifies two bacterial species that affect thermoregulatory behavior in Drosophila.
Yang, S. and Zhang, W. (2022). Systematic analysis of olfactory protein-protein interactions network of fruitfly, Drosophila melanogaster Arch Insect Biochem Physiol 110(2): e21882. PubMed ID: 35249240
Olfaction is one of the physiological traits of insect behavior. Insects have evolved a sophisticated olfactory system and use a combined coding strategy to process general odor. Drosophila melanogaster is a powerful model to reveal the molecular and cellular mechanisms of odor detection. Identifying new olfactory targets through complex interactions will contribute to a better understanding of the functions, interactions, and signaling pathways of olfactory proteins. However, the mechanism of D. melanogaster olfaction is still unclear, and more olfactory proteins are required to be discovered. This study explored essential proteins in the olfactory system of D. melanogaster and conducted protein-protein interactions (PPIs) analysis. The PPIs network was constructed of the olfactory system of D. melanogaster, consisting of 863 proteins and 18,959 interactions. Various methods were used to perform functional enrichment analysis, topological analysis and cluster analysis. The results confirmed that Class B scavenger receptors (SR-Bs), glutathione S-transferases (GSTs), and UDP-glycosyltransferases (UGTs) play an essential role in olfaction of D. melanogaster. The proteins obtained in this study can be used for subsequent functional identification in D. melanogaster olfactory.

Monday, August 15th - Enzyme and Protein Characterization

Wang, Z., Zeng, P. and Zhou, B. (2022). Identification and characterization of a heme exporter from the MRP family in Drosophila melanogaster BMC Biol 20(1): 126. PubMed ID: 35655259
The heme group constitutes a major functional form of iron, which plays vital roles in various biological processes including oxygen transport and mitochondrial respiration. Heme is an essential nutrient, but its pro-oxidant nature may have toxic cellular effects if present at high levels, and its synthesis is therefore tightly regulated. Deficiency and excess of heme both lead to pathological processes; however, current understanding of metazoan heme transport is largely limited to work in mammals and the worm Caenorhabditis elegans, while functional analyses of heme transport in the genetically amenable Drosophila melanogaster and other arthropods have not been explored. This study implemented a functional screening in Schneider 2 (S2) cells to identify putative heme transporters of D. melanogaster. A few multidrug resistance-associated protein (MRP) members were found to be induced by hemin and/or involved in heme export. Between the two plasma membrane-resident heme exporters CG4562 and CG7627, the former is responsible for heme transit across the intestinal epithelium. CG4562 knockdown resulted in heme accumulation in the intestine and lethality that could be alleviated by heme synthesis inhibition, human MRP5 (hMRP5) expression, heme oxygenase (HO) expression, or zinc supplement. CG4562 is mainly expressed in the gastric caeca and the anterior part of the midgut, suggesting this is the major site of heme absorption. It thus appears that CG4562 is the functional counterpart of mammalian MRP5. Mutation analyses in the transmembrane and nucleotide binding domains of CG4562 characterized some potential binding sites and conservative ATP binding pockets for the heme transport process. Furthermore, some homologs in Aedes aegypti, including that of CG4562, have also been characterized as heme exporters. Together, these findings suggest a conserved heme homeostasis mechanism within insects, and between insects and mammals. The fly model may be a good complement to the existing platforms of heme studies.
Weber, J. J., Brummett, L. M., Coca, M. E., Tabunoki, H., Kanost, M. R., Ragan, E. J., Park, Y. and Gorman, M. J. (2022). Phenotypic analyses, protein localization, and bacteriostatic activity of Drosophila melanogaster transferrin-1 Insect Biochem Mol Biol: 103811. PubMed ID: 35781032
Transferrin-1 (Tsf1) is an extracellular insect protein with a high affinity for iron. The functions of Tsf1 are still poorly understood; however, Drosophila melanogaster Tsf1 has been shown to influence iron distribution in the fly body and to protect flies against some infections. The goal of this study was to better understand the physiological functions of Tsf1 in D. melanogaster by 1) investigating Tsf1 null phenotypes, 2) determining tissue-specific localization of Tsf1, 3) measuring the concentration of Tsf1 in hemolymph, 4) testing Tsf1 for bacteriostatic activity, and 5) evaluating the effect of metal and paraquat treatments on Tsf1 abundance. Flies lacking Tsf1 had more iron than wild-type flies in specialized midgut cells that take up iron from the diet; however, the absence of Tsf1 had no effect on the iron content of whole midguts, fat body, hemolymph, or heads. Thus, as previous studies have suggested, Tsf1 appears to have a minor role in iron transport. Tsf1 was abundant in hemolymph from larvae (0.4 μM), pupae (1.4 μM), adult females (4.4 μM) and adult males (22 μM). Apo-Tsf1 at 1 μM had bacteriostatic activity whereas holo-Tsf1 did not, suggesting that Tsf1 can inhibit microbial growth by sequestering iron in hemolymph and other extracellular environments. This hypothesis was supported by detection of secreted Tsf1 in tracheae, testes and seminal vesicles. Colocalization of Tsf1 with an endosome marker in oocytes suggested that Tsf1 may provide iron to developing eggs; however, eggs from mothers lacking Tsf1 had the same amount of iron as control eggs, and they hatched at a wild-type rate. Thus, the primary function of Tsf1 uptake by oocytes may be to defend against infection rather than to provide eggs with iron. In beetles, Tsf1 plays a role in protection against oxidative stress. In contrast, this study found that flies lacking Tsf1 had a typical life span and greater resistance to paraquat-induced oxidative stress. In addition, Tsf1 abundance remained unchanged in response to ingestion of iron, cadmium or paraquat or to injection of iron. These results suggest that Tsf1 has a limited role in protection against oxidative stress in D. melanogaster.
Makamte, S., Thureau, A., Jabrani, A., Paquelin, A., Plessis, A., Sanial, M., Rudenko, O., Oteri, F., Baaden, M. and Biou, V. (2022). A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study J Struct Biol 214(2): 107853. PubMed ID: 35364288
Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in Drosophila. Previous publications showed that the crystal structures of human and Drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, full-length SuFu's structure in solution was studied, both with circular dichroism and small angle X-ray scattering, comparing Drosophila, zebrafish and human species, to better understand this considerable difference. These studies show that, in spite of similar crystal structures restricted to ordered domains, Drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, this study showed that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.
Markusson, S., Hallin, E. I., Bustad, H. J., Raasakka, A., Xu, J., Muruganandam, G., Loris, R., Martinez, A., Bramham, C. R. and Kursula, P. (2022). High-affinity anti-Arc nanobodies provide tools for structural and functional studies PLoS One 17(6): e0269281. PubMed ID: 35671319
Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is poorly understood. Arc has an N-terminal domain (NTD) involved in membrane binding and a C-terminal domain (CTD) that binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerisation, including assembly of retrovirus-like capsids involved in intercellular signalling. To obtain new tools for studies on Arc structure and function, six high-affinity anti-Arc nanobodies (Nb) were produced and characterised. The CTD of rat and human Arc were both crystallised in ternary complexes with two Nbs. One Nb bound deep into the stargazin-binding pocket of Arc CTD and suggested competitive binding with Arc ligand peptides. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. The collapsed conformation closely resembles Drosophila Arc in capsids, suggesting that this study has trapped a capsid-like conformation of the human Arc CTD. These data obtained with the help of anti-Arc Nbs suggest that structural dynamics of the CTD and dimerisation of the NTD may promote the formation of capsids. Taken together, the recombinant high-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function, as well as mechanisms of Arc capsid formation, both in vitro and in vivo. For example, the Nbs could serve as a genetically encoded tools for inhibition of endogenous Arc interactions in the study of neuronal function and plasticity.
Khoury, M. J. and Bilder, D. (2022). Minimal functional domains of the core polarity regulator Dlg Biol Open. PubMed ID: 35722710
The compartmentalized domains of polarized epithelial cells arise from mutually antagonistic actions between the apical Par complex and the basolateral Scrib module. In Drosophila, the Scrib module proteins Scribble (Scrib) and Discs-large (Dlg) are required to limit Lgl phosphorylation at the basolateral cortex, but how Scrib and Dlg could carry out such a 'protection' activity is not clear. This study tested Protein Phosphatase 1α (PP1) as a potential mediator of this activity but demonstrate that a significant component of Scrib and Dlg regulation of Lgl is PP1-independent, and found no evidence for a Scrib-Dlg-PP1 protein complex. However, the Dlg SH3 domain plays a role in Lgl protection and, in combination with the N-terminal region of the Dlg HOOK domain, in recruitment of Scrib to the membrane. A 'minimal Dlg' was identified, comprised of the SH3 and HOOK domains that is both necessary and sufficient for Scrib localization and epithelial polarity function in vivo.
Frampton, S. L., Sutcliffe, C., Baldock, C. and Ashe, H. L. (2022). Modelling the structure of Short Gastrulation and generation of a toolkit for studying its function in Drosophila Biol Open 11(6). PubMed ID: 35603711
A BMP gradient is essential for patterning the dorsal-ventral axis of invertebrate and vertebrate embryos. The extracellular BMP binding protein Short Gastrulation (Sog) in Drosophila plays a key role in BMP gradient formation. This study combined genome editing, structural and developmental approaches to study Sog function in Drosophila. A sog knockout fly stock was generated that allows simple reintegration of altered versions of the sog coding sequence. As proof-of-principle, the requirement was tested for two cysteine residues that were previously identified as targets for palmitoylation, which has been proposed to enhance Sog secretion. However, it was shown that the sogC27,28S mutant is viable with only very mild phenotypes, indicating that these residues and their potential modification are not critical for Sog secretion in vivo. Additionally, experimental negative stain EM imaging and hydrodynamic data were used to validate the AlphaFold structure prediction for Sog. The model suggests a more compact shape than the vertebrate ortholog Chordin and conformational flexibility between the C-terminal von Willebrand C domains. How this altered compactness may contribute to mechanistic differences in Sog and Chordin function during BMP gradient formation is discussed.

Friday, August 12th - Adult and Larval Physiology and Metabolism

Huisamen, E. J., Colinet, H., Karsten, M. and Terblanche, J. S. (2022). Dietary salt supplementation adversely affects thermal acclimation responses of flight ability in Drosophila melanogaster J Insect Physiol 140: 104403. PubMed ID: 35667397
Cold acclimation may enhance low temperature flight ability, and salt loading can alter an insects' cold tolerance by affecting their ability to maintain ion balance in the cold. Presently however, it remains unclear if dietary salt impacts thermal acclimation of flight ability in insects. This study examined the effect of a combination of dietary salt loading (either NaCl or KCl) and low temperature exposure on the flight ability of Drosophila melanogaster at low (15 °C) and benign (optimal, 22 °C) temperatures. Additionally, whether dietary salt supplementation translates into increased K(+) and Na(+) levels in the bodies of D. melanogaster was determined. Lastly, it was determined whether salt supplementation impacts body mass and wing morphology, to ascertain whether any changes in flight ability were potentially driven by flight-related morphometric variation. In control flies, it was found that cold acclimation enhances low temperature flight ability over non-acclimated flies confirming the beneficial acclimation hypothesis. By contrast, flies supplemented with KCl that were cold acclimated and tested at a cold temperature had the lowest flight ability, suggesting that excess dietary KCl during development negates the beneficial cold acclimation process that would have otherwise taken place. Overall, the NaCl-supplemented flies and the control group had the greatest flight ability, whilst those fed a KCl-supplemented diet had the lowest. Dietary salt supplementation translated into increased Na(+) and K(+) concentration in the body tissues of flies, confirming that dietary shifts are reflected in changes in body composition and are not simply regulated out of the body by homeostasis over the course of development. Flies fed with a KCl-supplemented diet tended to be larger with larger wings, whilst those reared on the control or NaCl-supplemented diet were smaller with smaller wings. Additionally, the flies with greater flight ability tended to be smaller and have lower wing loading. In conclusion, dietary salts affected wing morphology as well as ion balance, and dietary KCl seemed to have a detrimental effect on cold acclimation responses of flight ability in D. melanogaster.
Joshi, R., Cai, Y. D., Xia, Y., Chiu, J. C. and Emery, P. (2022). PERIOD Phosphoclusters Control Temperature Compensation of the Drosophila Circadian Clock Front Physiol 13: 888262. PubMed ID: 35721569
Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. This study uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, a series of mutations was introduced that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the pers phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. It was observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, these results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. This work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.
Jorgensen, D. B., Orsted, M. and Kristensen, T. N. (2022). Sustained positive consequences of genetic rescue of fitness and behavioural traits in inbred populations of Drosophila melanogaster J Evol Biol 35(6): 868-878. PubMed ID: 35532930
One solution to alleviate the detrimental genetic effects associated with reductions in population size and fragmentation is to introduce immigrants from other populations. While the effects of this genetic rescue on fitness traits are fairly well known, it is less clear to what extent inbreeding depression and subsequent genetic rescue affect behavioural traits. In this study, replicated crosses between inbred lines of Drosophila melanogaster were performed in order to investigate the effects of inbreeding and genetic rescue on egg-to-adult viability and negative geotaxis behaviour-a locomotor response used to measure, e.g. the effects of physiological ageing. Transgenerational effects of outcrossing were investigated by examining the fitness consequences in both the F(1) and F(4)  generation. The majority of inbred lines showed evidence for inbreeding depression for both egg-to-adult viability and behavioural performance (95% and 66% of lines, respectively), with inbreeding depression being more pronounced for viability compared with the locomotor response. Subsequent outcrossing with immigrants led to an alleviation of the negative effects for both viability and geotaxis response resulting in inbred lines being similar to the outbred controls, with beneficial effects persisting from F(1) to F(4). Overall, the results clearly show that genetic rescue can provide transgenerational rescue of small, inbred populations by rapidly improving population fitness components. Thus, this study showed that even the negative effects of inbreeding on behaviour, similar to that of neurodegeneration associated with physiological ageing, can be reversed by genetic rescue.
Lu, J., Dong, W., Hammond, G. R. and Hong, Y. (2022). Hypoxia controls plasma membrane targeting of polarity proteins by dynamic turnover of PI4P and PI(4,5)P2 Elife 11. PubMed ID: 35678383
Phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PIP2) are key phosphoinositides that determine the identity of the plasma membrane (PM) and regulate numerous key biological events there. To date, mechanisms regulating the homeostasis and dynamic turnover of PM PI4P and PIP2 in response to various physiological conditions and stresses remain to be fully elucidated. This study reports that hypoxia in Drosophila induces acute and reversible depletion of PM PI4P and PIP2 that severely disrupts the electrostatic PM targeting of multiple polybasic polarity proteins. Genetically encoded ATP sensors confirmed that hypoxia induces acute and reversible reduction of cellular ATP levels which showed a strong real-time correlation with the levels of PM PI4P and PIP2 in cultured cells. By combining genetic manipulations with quantitative imaging assays this study showed that PI4KIIIα, as well as Rbo/EFR3 and TTC7 that are essential for targeting PI4KIIIα to PM, are required for maintaining the homeostasis and dynamic turnover of PM PI4P and PIP2 under normoxia and hypoxia. These results revealed that in cells challenged by energetic stresses triggered by hypoxia, ATP inhibition and possibly ischemia, dramatic turnover of PM PI4P and PIP2 could have profound impact on many cellular processes including electrostatic PM targeting of numerous polybasic proteins.
Hong, S., Sun, Y., Sun, D. and Wang, C. (2022). Microbiome assembly on Drosophila body surfaces benefits the flies to combat fungal infections iScience 25(6): 104408. PubMed ID: 35663020
In contrast to the well-characterized gut microbiomes, the composition and function of the insect body-surface microbiotas are still elusive and highly underexplored. This study reports the dynamic features of the Drosophila melanogaster surface microbiomes. It was found that the microbiomes assembled on fly surfaces could defend insects against fungal parasitic infections. The substantial increase of bacterial loads occurred within 10 days of fly eclosion, especially the expansion of Gilliamella species. The culturable bacteria such as Lactiplantibacillus plantarum could effectively inhibit fungal spore germinations, and the gnotobiotic addition of the isolated bacteria could substantially delay fungal infection of axenic flies. The fly tarsal segments were found to be largely accumulated with bacterial cells, which could accelerate cell dispersal onto different body parts to deter fungal spore germinations. These findings will facilitate future investigations of the surface microbiotas affecting insect physiologies.
Hibicke, M. and Nichols, C. D. (2022). Validation of the forced swim test in Drosophila, and its use to demonstrate psilocybin has long-lasting antidepressant-like effects in flies. Sci Rep 12(1): 10019. PubMed ID: 35705666
Psilocybin has been shown to be a powerful, long-lasting antidepressant in human clinical trials and in rodent models. The forced swim test (FST), which has been used extensively to evaluate compounds for antidepressant efficacy, has recently been adapted for Drosophila. The fly FST has potential to be a cost-effective, high-throughput assay for evaluating potential antidepressants. This study pharmacologically validated the fly FST using methamphetamine, DL-α-methyltyrosine, and the antidepressant citalopram. While methamphetamine and DL-α-methyltyrosine altered overall locomotor activity in the Drosophila Activity Monitor System (DAMS), they had no significant impact on measures of immobility in the FST. Conversely, chronic citalopram decreased measures of immobility in the FST in both sexes without increasing DAMS activity. The validated FST was used to evaluate the antidepressant-like effects of high (3.5 mM) and low (0.03 mM) doses of psilocybin. Both doses of psilocybin significantly reduced measures of immobility in male flies, but not females. 0.03 mM had an effect size comparable to chronic citalopram, and 3.5 mM had an effect size approximately twice that of chronic citalopram.

Thursday, August 11th - Signaling

Singh, G., Chakraborty, S. and Lakhotia, S. C. (2022). Elevation of major constitutive heat shock proteins is heat shock factor independent and essential for establishment and growth of Lgl loss and Yorkie gain-mediated tumors in Drosophila. Cell Stress Chaperones PubMed ID: 35704239
Cancer cells generally overexpress heat shock proteins (Hsps), the major components of cellular stress response, to overcome and survive the diverse stresses. However, the specific roles of Hsps in initiation and establishment of cancers remain unclear. Using loss of Lgl-mediated epithelial tumorigenesis in Drosophila, tumorigenic somatic clones of different genetic backgrounds were induced to examine the temporal and spatial expression and roles of major heat shock proteins in tumor growth. The constitutively expressed Hsp83, Hsc70 (heat shock cognate), Hsp60 and Hsp27 show elevated levels in all cells of the tumorigenic clone from early stages that persists until their transformation. However, the stress-inducible Hsp70 is expressed only in a few cells at later stage of established tumorous clones that show high F-actin aggregation. Intriguingly, levels of Heat shock factor (HSF), the master regulator of Hsps, remain unaltered in these tumorous cells and its down-regulation does not affect tumorigenic growth of lgl- clones overexpressing Yorkie, although down-regulation of Hsp83 prevents their survival and growth. Interestingly, overexpression of HSF or Hsp83 in lgl- cells makes them competitively successful in establishing tumorous clones. These results show that the major constitutively expressed Hsps, but not the stress-inducible Hsp70, are involved in early as well as late stages of epithelial tumors and their elevated expression in lgl- clones co-overexpressing Yorkie is independent of HSF.
Valencia-Exposito, A., Gomez-Lamarca, M. J., Widmann, T. J. and Martin-Bermudo, M. D. (2022). Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis Front Cell Dev Biol 10: 892691. PubMed ID: 35769262
Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. This study used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. Reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, the results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, it was shown that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, these results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells.
Tang, X., Zhang, Y., Wang, G., Zhang, C., Wang, F., Shi, J., Zhang, T. and Ding, J. (2022). Molecular mechanism of S-adenosylmethionine sensing by SAMTOR in mTORC1 signaling Sci Adv 8(26): eabn3868. PubMed ID: 35776786
The mechanistic target of rapamycin-mLST8-raptor complex (mTORC1) functions as a central regulator of cell growth and metabolism in response to changes in nutrient signals such as amino acids. SAMTOR is an S-adenosylmethionine (SAM) sensor, which regulates the mTORC1 activity through its interaction with the GTPase-activating protein activity toward Rags-1 (GATOR1)-KPTN, ITFG2, C12orf66 and SZT2-containing regulator (KICSTOR) complex. This paper reports the crystal structures of Drosophila melanogaster SAMTOR in apo form and in complex with SAM. SAMTOR comprises an N-terminal helical domain and a C-terminal SAM-dependent methyltransferase (MTase) domain. The MTase domain contains the SAM-binding site and the potential GATOR1-KICSTOR-binding site. The helical domain functions as a molecular switch, which undergoes conformational change upon SAM binding and thereby modulates the interaction of SAMTOR with GATOR1-KICSTOR. The functional roles of the key residues and the helical domain are validated by functional assays. These structural and functional data together reveal the molecular mechanism of the SAM sensing of SAMTOR and its functional role in mTORC1 signaling.
Mieszczanek, J., Strutt, H., Rutherford, T. J., Strutt, D., Bienz, M. and Gammons, M. V. (2022). Selective function of the PDZ domain of Dishevelled in noncanonical Wnt signalling J Cell Sci 135(11). PubMed ID: 35542970
Dishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (β-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. This study used genome editing to delete the PDZ domain-encoding region from Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. This study used nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, changes were detected in the proximodistal distribution of the polarity protein Flamingo (also known as Starry night) in pupal wings that suggest a modulatory role of these motifs in polarity signalling. New genetic evidence is provided that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.
Zhou, Y., Liu, J. and Liu, J. L. (2022). Connecting Ras and CTP synthase in Drosophila Exp Cell Res 416(1): 113155. PubMed ID: 35427600
CTP synthase (CTPS), the enzyme responsible for the last step of de novo synthesis of CTP, forms filamentous structures termed cytoophidia in all three domains of life. This study reports that oncogenic Ras regulates cytoophidium formation in Drosophila intestines. Overexpressing active Ras induces elongate and abundant cytoophidia in intestinal stem cells (ISCs) and enteroblasts (EBs). Knocking-down CTPS in ISCs/EBs suppresses the over- proliferation phenotype induced by ectopic expression of active Ras. Moreover, disrupting cytoophidium formation increases the number of proliferating cells in the background of overexpressing active Ras. Therefore, these results demonstrate a link between Ras and CTPS.
Du, L., Sohr, A., Li, Y. and Roy, S. (2022). GPI-anchored FGF directs cytoneme-mediated bidirectional contacts to regulate its tissue-specific dispersion Nat Commun 13(1): 3482. PubMed ID: 35710780
How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). This study shows that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and promotes target-specific cytoneme contacts and contact-dependent FGF release. FGF-source and ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through cell-adhesion-molecule (CAM)-like FGF-FGFR binding. Contact-mediated FGF-FGFR interactions induce bidirectional responses in ASP and source cells that, in turn, polarize FGF-sending and FGF-receiving cytonemes toward each other to reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion.

Wednesday, August 10th - Larval and Adult Development

Nogueira Alves, A., Oliveira, M. M., Koyama, T., Shingleton, A. and Mirth, C. K. (2022). Ecdysone coordinates plastic growth with robust pattern in the developing wing. Elife 11. PubMed ID: 35261337
Animals develop in unpredictable, variable environments. In response to environmental change, some aspects of development adjust to generate plastic phenotypes. Other aspects of development, however, are buffered against environmental change to produce robust phenotypes. How organ development is coordinated to accommodate both plastic and robust developmental responses is poorly understood. This study demonstrates that the steroid hormone ecdysone coordinates both plasticity of organ size and robustness of organ pattern in the developing wings of the fruit fly Drosophila melanogaster. Using fed and starved larvae that lack prothoracic glands, which synthesize ecdysone, this study showed that nutrition regulates growth both via ecdysone and via an ecdysone-independent mechanism, while nutrition regulates patterning only via ecdysone. It was then demonstrated that growth shows a graded response to ecdysone concentration, while patterning shows a threshold response. Collectively, these data support a model where nutritionally regulated ecdysone fluctuations confer plasticity by regulating disc growth in response to basal ecdysone levels and confer robustness by initiating patterning only once ecdysone peaks exceed a threshold concentration. This could represent a generalizable mechanism through which hormones coordinate plastic growth with robust patterning in the face of environmental change.
Xu, J., Liu, Y., Li, H., Tarashansky, A. J., Kalicki, C. H., Hung, R. J., Hu, Y., Comjean, A., Kolluru, S. S., Wang, B., Quake, S. R., Luo, L., McMahon, A. P., Dow, J. A. T. and Perrimon, N. (2022). Transcriptional and functional motifs defining renal function revealed by single-nucleus RNA sequencing. Proc Natl Acad Sci U S A 119(25): e2203179119. PubMed ID: 35696569
Recent advances in single-cell sequencing provide a unique opportunity to gain novel insights into the diversity, lineage, and functions of cell types constituting a tissue/organ. A single-nucleus study of the adult Drosophila renal system was performed, consisting of Malpighian tubules and nephrocytes, which shares similarities with the mammalian kidney. Eleven distinct clusters representing renal stem cells, stellate cells, regionally specific principal cells, garland nephrocyte cells, and pericardial nephrocytes. Characterization of the transcription factors specific to each cluster identified fruitless (fru) as playing a role in stem cell regeneration and Hepatocyte nuclear factor 4 (Hnf4) in regulating glycogen and triglyceride metabolism. In addition, a number of genes, including Rho guanine nucleotide exchange factor at 64C (RhoGEF64c), Prip, and CG1093 were identified that are involved in regulating the unusual star shape of stellate cells. Importantly, the single-nucleus dataset allows visualization of the expression at the organ level of genes involved in ion transport and junctional permeability, providing a systems-level view of the organization and physiological roles of the tubules. Finally, a cross-species analysis allowed matching the fly kidney cell types to mouse kidney cell types and planarian protonephridia, knowledge that will help the generation of kidney disease models. Altogether, this study provides a comprehensive resource for studying the fly kidney.
Cortot, J., Farine, J. P., Cobb, M., Everaerts, C. and Ferveur, J. F. (2022). Factors affecting the biosynthesis and emission of a Drosophila pheromone. J Exp Biol 225(13). PubMed ID: 35678110
The most studied pheromone in Drosophila melanogaster, cis-vaccenyl acetate (cVA), is synthesized in the male ejaculatory bulb and transferred to the female during copulation. Combined with other chemicals, cVA can modulate fly aggregation, courtship, mating and fighting. This study explored the mechanisms underlying both cVA biosynthesis and emission in males of two wild types and a pheromonal mutant line. The effects of ageing, adult social interaction, and maternally transmitted cVA and microbes - both associated with the egg chorion - on cVA biosynthesis and emission were measured. While ageing and genotype changed both biosynthesis and emission in similar ways, early developmental exposure to maternally transmitted cVA and microbes strongly decreased cVA emission but not the biosynthesis of this molecule. This indicates that the release - but not the biosynthesis - of this sex pheromone strongly depends on early developmental context. The mechanism by which the preimaginal effects occur is unknown, but reinforces the significance of development in determining adult physiology and behaviour.
Tse, J., Li, T. H., Zhang, J., Lee, A. C. K., Lee, I., Qu, Z., Lin, X., Hui, J. and Chan, T. F. (2022). Single-Cell Atlas of the Drosophila Leg Disc Identifies a Long Non-Coding RNA in Late Development. Int J Mol Sci 23(12). PubMed ID: 35743238
The Drosophila imaginal disc has been an excellent model for the study of developmental gene regulation. In particular, long non-coding RNAs (lncRNAs) have gained widespread attention in recent years due to their important role in gene regulation. Their specific spatiotemporal expressions further support their role in developmental processes and diseases. This study explored the role of a novel lncRNA in Drosophila leg development by dissecting and dissociating w(1118) third-instar larval third leg (L3) discs into single cells and single nuclei, and performing single-cell RNA-sequencing (scRNA-seq) and single-cell assays for transposase-accessible chromatin (scATAC-seq). Single-cell transcriptomics analysis of the L3 discs across three developmental timepoints revealed different cell types and identified lncRNA:CR33938 as a distal specific gene with high expression in late development. This was further validated by fluorescence in-situ hybridization (FISH). The scATAC-seq results reproduced the single-cell transcriptomics landscape and elucidated the distal cell functions at different timepoints. Furthermore, overexpression of lncRNA:CR33938 in the S2 cell line increased the expression of leg development genes, further elucidating its potential role in development
Hanna, L., Lamouret, T., Poças, G. M., Mirth, C. K., Moczek, A. P., Nijhout, F. H. and Abouheif, E. (2022). Evaluating old truths: Final adult size in holometabolous insects is set by the end of larval development. J Exp Zool B Mol Dev Evol. PubMed ID: 35676886
For centuries, it has been understood that the final size of adult holometabolous insects is determined by the end of the larval stage, and that once they transform to adults, holometabolous insects do not grow. Despite this, no previous study has directly tested these 'old truths' across holometabolous insects. This study demonstrates that final adult size is set at the end of the last larval stage in species representing each of the four orders of holometabolous insects: the fruit fly Drosophila melanogaster (Diptera), the tobacco hornworm Manduca sexta (Lepidoptera), the dung beetle Onthophagus taurus (Coleoptera), and the Florida carpenter ant Camponotus floridanus (Hymenoptera). Furthermore, in both D. melanogaster and C. floridanus, this study showed that the size of adult individuals fluctuates but does not significantly change. Therefore, this study finally confirms these two basic assumptions in the biology of insects, which have for centuries served as the foundation for studies of insect growth, size, and allometry.
Hun, L. V., Okamoto, N., Imura, E., Maxson, R., Bittar, R. and Yamanaka, N. (2022). Essential functions of mosquito ecdysone importers in development and reproduction. Proc Natl Acad Sci U S A 119(25): e2202932119. PubMed ID: 35696563
The primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. This study reports three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, -3, and -4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, these results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes.

Tuesday, August 9th - Disease Models

Malacrida, S., De Lazzari, F., Mrakic-Sposta, S., Vezzoli, A., Zordan, M. A., Bisaglia, M., Menti, G. M., Meda, N., Frighetto, G., Bosco, G., Dal Cappello, T., Strapazzon, G., Reggiani, C., Gussoni, M. and Megighian, A. (2022). Lifespan and ROS levels in different Drosophila melanogaster strains after 24 h hypoxia exposure. Biol Open 11(6). PubMed ID: 35616023
During recent decades, model organisms such as Drosophila melanogaster have made it possible to study the effects of different environmental oxygen conditions on lifespan and oxidative stress. In this study, longevity and ROS levels were compared in young, unmated males of three laboratory wild-type lines (Canton-S, Oregon-R and Berlin-K) and one mutant line (Sod1n1) as a positive control of redox imbalance, under both normoxic and hypoxic (2% oxygen for 24 h) conditions. The genetic background was found to be a relevant factor involved in D. melanogaster longevity and ROS levels. Indeed, as expected, in normoxia Sod1n1 are the shortest-lived, while the wild-type strains, despite a longer lifespan, show some differences, with the Canton-S line displaying the lowest mortality rate. After hypoxic stress these variances are amplified, with Berlin-K flies showing the highest mortality rate and most evident reduction of lifespan. Moreover, this analysis highlighted differential effects of hypoxia on redox balance/unbalance. Canton-S flies had the lowest increase of ROS level compared to all the other strains, confirming it to be the less sensitive to hypoxic stress. Sod1n1 flies displayed the highest ROS levels in normoxia and after hypoxia. These results should be used to further standardize future Drosophila research models designed to investigate genes and pathways that may be involved in lifespan and/or ROS, as well as comparative studies on specific mutant strains.
Mascolo, E., Liguori, F., Merigliano, C., Schiano, L., Gnocchini, E., Pilesi, E., Volonts, C., Di Salvo, M. L., Contestabile, R., Tramonti, A. and Verni, F. (2022). Vitamin B6 rescues insulin resistance and glucose-induced DNA damage caused by reduced activity of Drosophila PI3K. J Cell Physiol. PubMed ID: 35678366
The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3-kinase (PI3K) contributes to the cascade of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. This study shows that RNAi-induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, hyperglycemia was found to produce chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end-products and reactive oxygen species. Rearing PI3K(RNAi) flies in a medium supplemented with pyridoxal 5'-phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3K(RNAi) larvae with the PLP inhibitor 4-deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, these results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, these findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications.
Palumbo, R. J., Belkevich, A. E., Pascual, H. G. and Knutson, B. A. (2022). A clinically-relevant residue of POLR1D is required for Drosophila development. Dev Dyn. PubMed ID: 35656583
POLR1D is a subunit of RNA Polymerases I and III, which synthesize ribosomal RNAs. Dysregulation of these polymerases cause several types of diseases, including ribosomopathies. The craniofacial disorder Treacher Collins Syndrome (TCS) is a ribosomopathy caused by mutations in several subunits of RNA Polymerase I, including POLR1D. This study characterized the effect of a missense mutation in POLR1D and RNAi knockdown of POLR1D on Drosophila development. RESULTS: A missense mutation in Drosophila POLR1D (G30R) reduced larval rRNA levels, slowed larval growth, and arrested larval development. Remarkably, the G30R substitution is at an orthologous glycine in POLR1D that is mutated in a TCS patient (G52E). The G52E mutation in human POLR1D, and the comparable substitution (G30E) in Drosophila POLR1D, reduced their ability to heterodimerize with POLR1C in vitro. It was also found that POLR1D is required early in the development of Drosophila neural cells. Furthermore, an RNAi screen revealed that POLR1D is also required for development of non-neural Drosophila cells, suggesting the possibility of defects in other cell types. These results establish a role for POLR1D in Drosophila development, and present Drosophila as an attractive model to evaluate the molecular defects of TCS mutations in POLR1D.
Prakash, P., Pradhan, A. K. and Sheeba, V. (2022). Hsp40 overexpression in pacemaker neurons delays circadian dysfunction in a Drosophila model of Huntington's disease. Dis Model Mech 15(6). PubMed ID: 35645202
Circadian disturbances are early features of neurodegenerative diseases, including Huntington's disease (HD). Emerging evidence suggests that circadian decline feeds into neurodegenerative symptoms, exacerbating them. Therefore, it was asked whether known neurotoxic modifiers can suppress circadian dysfunction. A screen was performed of neurotoxicity-modifier genes to suppress circadian behavioural arrhythmicity in a Drosophila circadian HD model. The molecular chaperones Hsp40 and HSP70 emerged as significant suppressors in the circadian context, with Hsp40 being the more potent mitigator. Upon Hsp40 overexpression in the Drosophila circadian ventrolateral neurons (LNv), the behavioural rescue was associated with neuronal rescue of loss of circadian proteins from small LNv soma. Specifically, there was a restoration of the molecular clock protein Period and its oscillations in young flies and a long-lasting rescue of the output neuropeptide Pigment dispersing factor. Significantly, there was a reduction in the expanded Huntingtin inclusion load, concomitant with the appearance of a spot-like Huntingtin form. Thus, this study provided evidence implicating the neuroprotective chaperone Hsp40 in circadian rehabilitation. The involvement of molecular chaperones in circadian maintenance has broader therapeutic implications for neurodegenerative diseases.
Shaposhnikov, M. V., Guvatova, Z. G., Zemskaya, N. V., Koval, L. A., Schegoleva, E. V., Gorbunova, A. A., Golubev, D. A., Pakshina, N. R., Ulyasheva, N. S., Solovev, I. A., Bobrovskikh, M. A., Gruntenko, N. E., Menshanov, P. N., Krasnov, G. S., Kudryavseva, A. V. and Moskalev, A. A. (2022). Molecular mechanisms of exceptional lifespan increase of Drosophila melanogaster with different genotypes after combinations of pro-longevity interventions. Commun Biol 5(1): 566. PubMed ID: 35681084
Aging is a global challenge. The search for new anti-aging interventions is also an issue of great actuality. This report describes the success of Drosophila melanogaster lifespan extension under the combined influence of dietary restriction, co-administration of berberine, fucoxanthin, and rapamycin, photodeprivation, and low-temperature conditions up to 185 days in w(1118) strain and up to 213 days in long-lived E(z)/w mutants. The trade-off was found between longevity and locomotion. The transcriptome analysis showed an impact of epigenetic alterations, lipid metabolism, cellular respiration, nutrient sensing, immune response, and autophagy in the registered effect.
Bertapelle, C., Carillo, M. R., Cacciola, N. A., Shidlovskii, Y. V., Peluso, G. and Digilio, F. A. (2022). The Reversible Carnitine Palmitoyltransferase 1 Inhibitor (Teglicar) Ameliorates the Neurodegenerative Phenotype in a Drosophila Huntington's Disease Model by Acting on the Expression of Carnitine-Related Genes. Molecules 27(10). PubMed ID: 35630602
Huntington's disease (HD) is a dramatic neurodegenerative disorder caused by the abnormal expansion of a CAG triplet in the huntingtin gene, producing an abnormal protein. As it leads to the death of neurons in the cerebral cortex, the patients primarily present with neurological symptoms, but recently metabolic changes resulting from mitochondrial dysfunction have been identified as novel pathological features. The carnitine shuttle is a complex consisting of three enzymes whose function is to transport the long-chain fatty acids into the mitochondria. In this study, its pharmacological modification was used to test the hypothesis that shifting metabolism to lipid oxidation exacerbates the HD symptoms. Behavioural and transcriptional analyses were carried out on HD Drosophila model, to evaluate the involvement of the carnitine cycle in this pathogenesis. Pharmacological inhibition of CPT1, the rate-limiting enzyme of the carnitine cycle, ameliorates the HD symptoms in Drosophila, likely acting on the expression of carnitine-related genes.

Monday, August 8th - Evolution

Ahlawat, N., Geeta Arun, M., Maggu, K., Jigisha, Singh, A. and Prasad, N. G. (2022). Drosophila melanogaster hosts coevolving with Pseudomonas entomophila pathogen show sex-specific patterns of local adaptation. BMC Ecol Evol 22(1): 77. PubMed ID: 35717176
In spatially structured populations, local adaptation improves organisms' fitness in their native environment. Hosts and pathogens can rapidly adapt to their local antagonist. Since males and females can differ in their immunocompetence, the patterns of local adaptation can be different between the sexes. However, there is little information about sex differences in local adaptation in host-pathogen systems. This study experimentally coevolved four different replicate populations of Drosophila melanogaster (host) and Pseudomonas entomophila (pathogen) along with appropriate controls. The four host-pathogen coevolution populations were used to investigate the occurrence of local adaptation separately in males and females of the coevolving hosts. Local adaptation was also assessed in pathogens. A reciprocal infection experiment was set up where each of the four coevolving hosts were infected with their local pathogen or non-local pathogens from the other three replicate populations. Overall, male and female hosts had better survivorship when infected with local pathogens, indicating that they were locally adapted. Interestingly, males were more susceptible to non-local pathogens compared to females. In addition, no fecundity cost was found in females infected with either local or non-local pathogens. No evidence was found of local adaptation among the pathogens. This study showed sex-specific adaptation in the coevolving hosts where female hosts had a broader response against allopatric coevolving pathogens with no cost in fecundity. Thus, these results might suggest a novel mechanism that can maintain variation in susceptibility in spatially structured populations.
Venkitachalam, S., Das, S., Deep, A. and Joshi, A. (2022). Density-dependent selection in Drosophila: evolution of egg size and hatching time. J Genet 101. PubMed ID: 35129132
Many different laboratory studies of adaptation to larval crowding in Drosophila spp. have all yielded the evolution of preadult competitive ability, even though the ecological context in which crowding was experienced varied across studies. However, the evolution of competitive ability was achieved through different suites of traits in studies wherein crowding was imposed in slightly different ways. This study reports results from a study in which egg size and hatching time were assayed on three sets of populations adapted to larval crowding experienced in slightly different ways, as well as their low density ancestral control populations. Egg size and hatching time are traits that may provide larvae with initial advantages under crowding through increased starting larval size and a temporal head-start, respectively. In each set of populations adapted to some form of larval crowding, the evolution of longer and wider eggs was seen, compared to controls, thus making egg size the first consistent correlate of the evolution of increased larval competitive ability across Drosophila populations experiencing crowding in slightly different ways. Among the crowding-adapted populations, those crowded at the lowest overall eggs/food density, but the highest density of larvae in the feeding band, showed the largest eggs, on an average. All three sets of crowding-adapted populations showed shorter average egg hatching time than controls, but the difference was significant only in the case of populations experiencing the highest feeding band density. These results underscore the importance of considering factors other than just eggs/food density when studying the evolution of competitive ability, as also the advantages of having multiple selection regimes within one experimental set up, allowing for a more nuanced understanding of the subtlety with which adaptive evolutionary trajectories can vary across even fairly similar selection regimes.
Duranton, M. and Pool, J. E. (2022). Interactions between natural selection and recombination shape the genomic landscape of introgression. Mol Biol Evol. PubMed ID: 35666817
Hybridization between lineages that have not reached complete reproductive isolation appears more and more like a common phenomenon. Indeed, speciation genomics studies have now extensively shown that many species' genomes have hybrid ancestry. However, genomic patterns of introgression are often heterogeneous across the genome. In many organisms, a positive correlation between introgression levels and recombination rate has been observed. It is usually explained by the purging of deleterious introgressed material due to incompatibilities. However, the opposite relationship was observed in a North American population of Drosophila melanogaster with admixed European and African ancestry. In order to explore how directional and epistatic selection can impact the relationship between introgression and recombination, forward simulations were performed of whole D. melanogaster genomes reflecting the North American population's history. The results revealed that the simplest models of positive selection often yield negative correlations between introgression and recombination such as the one observed in D. melanogaster. It was also confirmed that incompatibilities tend to produce positive introgression-recombination correlations. And yet, this study has identified parameter space under each model where the predicted correlation is reversed. These findings deepen understanding of the evolutionary forces that may shape patterns of ancestry across genomes, and they strengthen the foundation for future studies aimed at estimating genome-wide parameters of selection in admixed populations.
Rusuwa, B. B., Chung, H., Allen, S. L., Frentiu, F. D. and Chenoweth, S. F. (2022). Natural variation at a single gene generates sexual antagonism across fitness components in Drosophila. Curr Biol. PubMed ID: 35700732
Mutations with conflicting fitness effects in males and females accumulate in sexual populations, reducing their adaptive capacity. Although quantitative genetic studies indicate that sexually antagonistic polymorphisms are common, their molecular basis and population genetic properties remain poorly understood. This study shows in fruit flies how natural variation at a single gene generates sexual antagonism through phenotypic effects on cuticular hydrocarbon (CHC) traits that function as both mate signals and protectors against abiotic stress across a latitudinal gradient. Tropical populations of Drosophila serrata have polymorphic CHCs producing sexual antagonism through opposing but sex-limited effects on these two fitness-related functions. This study dissected this polymorphism to a single fatty-acyl CoA reductase gene, DsFAR2-B, that is expressed in oenocyte cells where CHCs are synthesized. RNAi-mediated disruption of the DsFAR2-B ortholog in D. melanogaster oenocytes affected CHCs in a similar way to that seen in D.serrata. Population genomic analysis revealed that balancing selection likely operates at the DsFAR2-B locus in the wild. This study provides insights into the genetic basis of sexual antagonism in nature and connects sexually varying antagonistic selection on phenotypes with balancing selection on genotypes that maintains molecular variation.
Bubnell, J. E., Ulbing, C. K. S., Fernandez Begne, P. and Aquadro, C. F. (2022). Functional divergence of the bag of marbles gene in the Drosophila melanogaster species group. Mol Biol Evol. PubMed ID: 35714266
In Drosophila melanogaster, a key germline stem cell (GSC) differentiation factor, bag of marbles (bam) shows rapid bursts of amino acid fixations between sibling species D. melanogaster and D. simulans, but not in the outgroup species D. ananassae. This study tested the null hypothesis that the bam differentiation function is conserved between D. melanogaster and four additional Drosophila species in the melanogaster species group spanning approximately 30 million years of divergence. Surprisingly, it was demonstrated that bam is not necessary for oogenesis or spermatogenesis in D. teissieri nor is bam necessary for spermatogenesis in D. ananassae. Remarkably bam function may change on a relatively short time scale. Neutral sequence evolution at bam was tested in additional species of Drosophila, and a positive, but not perfect, correlation was found between evidence for positive selection at bam and its essential role in GSC regulation and fertility for both males and females. Further characterization of bam function in more divergent lineages will be necessary to distinguish between the bam critical gametogenesis role being newly derived in D. melanogaster, D. simulans, D. yakuba, and D. ananassae females or it being basal to the genus and subsequently lost in numerous lineages.
Sepil, I., Perry, J. C., Dore, A., Chapman, T. and Wigby, S. (2022). Experimental evolution under varying sex ratio and nutrient availability modulates male mating success in Drosophila melanogaster. Biol Lett 18(6): 20210652. PubMed ID: 35642384
Biased population sex ratios can alter optimal male mating strategies, and allocation to reproductive traits depends on nutrient availability. However, there is little information on how nutrition interacts with sex ratio to influence the evolution of pre-copulatory and post-copulatory traits separately. To address this omission, this study tested how male mating success and reproductive investment evolve under varying sex ratios and adult diet in Drosophila melanogaster, using experimental evolution. Sex ratio and nutrient availability were found to interact to determine male pre-copulatory performance. Males from female-biased populations were slow to mate when they evolved under protein restriction. By contrast, direct and non-interacting effects of sex ratio and nutrient availability on post-copulatory success were found. Males that evolved under protein restriction were relatively poor at suppressing female remating. Males that evolved under equal sex ratios fathered more offspring and were better at supressing female remating, relative to males from male-biased or female-biased populations. These results support the idea that sex ratios and nutrition interact to determine the evolution of pre-copulatory mating traits, but independently influence the evolution of post-copulatory traits.

Friday, August 5th - Synapse and Vesicles

Lee, K. M., Linskens, A. M. and Doe, C. Q. (2022). Hunchback activates Bicoid in Pair1 neurons to regulate synapse number and locomotor circuit function. Curr Biol 32(11): 2430-2441.e2433. PubMed ID: 35512697
Neural circuit function underlies cognition, sensation, and behavior. Proper circuit assembly depends on the identity of the neurons in the circuit (gene expression, morphology, synapse targeting, and biophysical properties). Neuronal identity is established by spatial and temporal patterning mechanisms, but little is known about how these mechanisms drive circuit formation in postmitotic neurons. Temporal patterning involves the sequential expression of transcription factors (TFs) in neural progenitors to diversify neuronal identity, in part through the initial expression of homeodomain TF combinations. This study addresses the role of the Drosophila temporal TF Hunchback and the homeodomain TF Bicoid in the assembly of the Pair1 (SEZ_DN1) descending neuron locomotor circuit, which promotes larval pausing and head casting. Both Hunchback and Bicoid are expressed in larval Pair1 neurons, Hunchback activates Bicoid in Pair1 (opposite of their embryonic relationship), and the loss of Hunchback function or Bicoid function from Pair1 leads to ectopic presynapse numbers in Pair1 axons and an increase in Pair1-induced pausing behavior. These phenotypes are highly specific, as the loss of Bicoid or Hunchback has no effect on Pair1 neurotransmitter identity, dendrite morphology, or axonal morphology. Importantly, the loss of Hunchback or Bicoid in Pair1 leads to the addition of new circuit partners that may underlie the exaggerated locomotor pausing behavior. These data are the first to show a role for Bicoid outside of embryonic patterning and the first to demonstrate a cell-autonomous role for Hunchback and Bicoid in interneuron synapse targeting and locomotor behavior.
Ueda, A., O'Harrow, T., Xing, X., Ehaideb, S., Manak, J. R. and Wu, C. F. (2022). Abnormal larval neuromuscular junction morphology and physiology in Drosophila prickle isoform mutants with known axonal transport defects and adult seizure behavior. J Neurogenet: 1-9. PubMed ID: 35775303
Previous studies have demonstrated the striking mutational effects of the Drosophila planar cell polarity gene prickle (pk) on larval motor axon microtubule-mediated vesicular transport and on adult epileptic behavior associated with neuronal circuit hyperexcitability. Mutant alleles of the prickle-prickle (pk(pk)) and prickle-spiny-legs (pk(sple)) isoforms (hereafter referred to as pk and sple alleles, respectively) exhibit differential phenotypes. While both pk and sple affect larval motor axon transport, only sple confers motor circuit and behavior hyperexcitability. However, mutations in the two isoforms apparently counteract to ameliorate adult motor circuit and behavioral hyperexcitability in heteroallelic pk/pk/pk(sple) flies. The consequences of altered axonal transport was further investigated in the development and function of the larval neuromuscular junction (NMJ). Robust dominant phenotypes were uncovered in both pk and sple alleles, including synaptic terminal overgrowth (as revealed by anti-HRP and -Dlg immunostaining) and poor vesicle release synchronicity (as indicated by synaptic bouton focal recording). However, recessive alteration of synaptic transmission was observed only in pk/pk larvae, i.e. increased excitatory junctional potential (EJP) amplitude in pk/pk but not in pk/+ or sple/sple. Interestingly, for motor terminal excitability sustained by presynaptic Ca(2+) channels, both pk and sple exerted strong effects to produce prolonged depolarization. Notably, only sple acted dominantly whereas pk/+ appeared normal, but was able to suppress the sple phenotypes, i.e. pk/sple appeared normal. Thesd observations contrast the differential roles of the pk and sple isoforms and highlight their distinct, variable phenotypic expression in the various structural and functional aspects of the larval NMJ.
Hodgson, J. J., Buchon, N. and Blissard, G. W. (2022). Identification of Cellular Genes Involved in Baculovirus GP64 Trafficking to the Plasma Membrane. J Virol 96(12): e0021522. PubMed ID: 35608346
The baculovirus envelope protein GP64 is an essential component of the budded virus and is necessary for efficient virion assembly. Little is known regarding intracellular trafficking of GP64 to the plasma membrane, where it is incorporated into budding virions during egress. To identify host proteins and potential cellular trafficking pathways that are involved in delivery of GP64 to the plasma membrane, this study developed and characterized a stable Drosophila cell line that inducibly expresses the AcMNPV GP64 protein and used that cell line in combination with a targeted RNA interference (RNAi) screen of vesicular protein trafficking pathway genes. Of the 37 initial hits from the screen, six host genes were validated and examined that were important for trafficking of GP64 to the cell surface. Validated hits included Rab GTPases Rab1 and Rab4, Clathrin heavy chain, clathrin adaptor protein genes AP-1-2β and AP-2&my;, and Snap29. Two gene knockdowns (Rab5 and Exo84) caused substantial increases (up to 2.5-fold) of GP64 on the plasma membrane. A small amount of GP64 is released from cells in exosomes, and tsome portion of cell surface GP64 is endocytosed, suggesting that recycling helps to maintain GP64 at the cell surface.
Astacio, H., Vasin, A. and Bykhovskaia, M. (2022). Stochastic Properties of Spontaneous Synaptic Transmission at Individual Active Zones. J Neurosci 42(6): 1001-1019. PubMed ID: 34969867
Using postsynaptically tethered calcium sensor GCaMP, this study investigated spontaneous synaptic transmission at individual active zones (AZs) at the Drosophila (both sexes) neuromuscular junction. Optical monitoring of GCaMP events coupled with focal electrical recordings of synaptic currents revealed "hot spots" of spontaneous transmission, which corresponded to transient states of elevated activity at selected AZs. The elevated spontaneous activity had two temporal components, one at a timescale of minutes and the other at a subsecond timescale. To investigate the mechanisms of elevated activity, focused was first placed on the protein Complexin, which binds the SNARE protein complex and serves to clamp spontaneous fusion. Overexpression of Drosophila complexin largely abolished the high-activity states of AZs, while complexin deletion drastically promoted it. How presynaptic Ca(2+) transients affect the states of elevated activity was investiged at individual AZs. Ca(2+) influx was blocked or promoted pharmacologically, and also Ca(2+) release was promoted from internal stores. These experiments coupled with computations revealed that Ca(2+) transients can trigger bursts of spontaneous events from individual AZs or AZ clusters at a subsecond timescale. Together, these results demonstrated that spontaneous transmission is highly heterogeneous, with transient hot spots being regulated by the SNARE machinery and Ca(2+).
Ochi, Y., Yamashita, H., Yamada, Y., Satoh, T. and Satoh, A. K. (2022). Stratum is required for both apical and basolateral transport through stable expression of Rab10 and Rab35 in Drosophila photoreceptors. Mol Biol Cell: mbcE21120596. PubMed ID: 35767331
Post-Golgi transport for specific membrane domains, also termed polarized transport, is essential for the construction and maintenance of polarized cells. Highly polarized Drosophila photoreceptors serve as a good model system for studying the mechanisms underlying polarized transport. The Mss4 Drosophila ortholog, Stratum (Strat), controls basal restriction of basement membrane proteins in follicle cells, and Rab8 acts downstream of Strat. This study investigated the function of Strat in fly photoreceptors and found that polarized transport in both the basolateral and rhabdomere membrane domains was inhibited in Strat-deficient photoreceptors. 79% and 55% reductions in Rab10 and Rab35 levels, respectively, were also observed but no reduction in Rab11 levels in whole-eye homozygous clones of Strat(null). Moreover, Rab35 was localized in the rhabdomere, and loss of Rab35 resulted in impaired Rh1 transport to the rhabdomere. These results indicate that Strat is essential for the stable expression of Rab10 and Rab35, which regulate basolateral and rhabdomere transport, respectively, in fly photoreceptors.
Courtney, K. C., Wu, L., Mandal, T., Swift, M., Zhang, Z., Alaghemandi, M., Wu, Z., Bradberry, M. M., Deo, C., Lavis, L. D., Volkmann, N., Hanein, D., Cui, Q., Bao, H. and Chapman, E. R. (2022). The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes. Nat Struct Mol Biol 29(2): 97-107. PubMed ID: 35132256
Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. This paper reports that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx: see Drosophila Complexin), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, this study demonstrated that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. It is proposed that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.

Thursday, August 4th - Disease Models

Hope, K. A., Berman, A. R., Peterson, R. T. and Chow, C. Y. (2022). An in vivo drug repurposing screen and transcriptional analyses reveals the serotonin pathway and GSK3 as major therapeutic targets for NGLY1 deficiency. PLoS Genet 18(6): e1010228. PubMed ID: 35653343
NGLY1 deficiency, a rare disease with no effective treatment, is caused by autosomal recessive, loss-of-function mutations in the N-glycanase 1 (NGLY1) gene and is characterized by global developmental delay, hypotonia, alacrima, and seizures. This study used a Drosophila model of NGLY1 deficiency to conduct an in vivo, unbiased, small molecule, repurposing screen of FDA-approved drugs to identify therapeutic compounds. Seventeen molecules partially rescued lethality in a patient-specific NGLY1 deficiency model, including multiple serotonin and dopamine modulators. Exclusive dNGLY1 expression in serotonin and dopamine neurons, in an otherwise dNGLY1 deficient fly, was sufficient to partially rescue lethality. Further, genetic modifier and transcriptomic data supports the importance of serotonin signaling in NGLY1 deficiency. Connectivity Map analysis identified glycogen synthase kinase 3 (GSK3) inhibition as a potential therapeutic mechanism for NGLY1 deficiency, which this study experimentally validated with TWS119, lithium, and GSK3 knockdown. Strikingly, GSK3 inhibitors and a serotonin modulator rescued size defects in dNGLY1 deficient larvae upon proteasome inhibition, suggesting that these compounds act through NRF1, a transcription factor that is regulated by NGLY1 and regulates proteasome expression. This study reveals the importance of the serotonin pathway in NGLY1 deficiency, and serotonin modulators or GSK3 inhibitors may be effective therapeutics for this rare disease.
Huynh, T. K. T., Mai, T. T. T., Huynh, M. A., Yoshida, H., Yamaguchi, M. and Dang, T. T. P. (2022). Crucial Roles of Ubiquitin Carboxy-Terminal Hydrolase L1 in Motor Neuronal Health by Drosophila Model. Antioxid Redox Signal.37(4-6):257-273 PubMed ID: 35343238
Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) plays an important role in the ubiquitin-proteasome system and is distributed mostly in the brain. Previous studies have shown that mutated forms or reduction of UCH-L1 are related to neurodegenerative disorders, but the mechanisms of pathogenesis are still not well understood. To study its roles in motor neuronal health, the Drosophila model in which dUCH, a homolog of human UCH-L1, was specifically knocked down in motor neurons. RThe reduction of Drosophila ubiquitin carboxyl-terminal hydrolase (dUCH) in motor neurons induced excessive reactive oxygen species production and multiple aging-like phenotypes, including locomotive defects, muscle degeneration, enhanced apoptosis, and shortened longevity. In addition, there is a decrease in the density of the synaptic active zone and glutamate receptor area at the neuromuscular junction. Interestingly, all these defects were rescued by vitamin C treatment, suggesting a close association with oxidative stress. Strikingly, the knockdown of dUCH at motor neurons exhibited aberrant morphology and function of mitochondria, such as mitochondrial DNA (mtDNA) depletion, an increase in mitochondrial size, and overexpression of antioxidant enzymes. This research indicates a new, possible pathogenesis of dUCH deficiency in the ventral nerve cord and peripheral nervous systems, which starts with abnormal mitochondria, leading to oxidative stress and accumulation aging-like defects in general. Taken together, by using the Drosophila model, these findings strongly emphasize how the UCH-L1 shortage affects motor neurons and further demonstrate the crucial roles of UCH-L1 in neuronal health.
Luong, H. N. B., Kalogeridi, M., Vontas, J. and Denecke, S. (2022). Using tissue specific P450 expression in Drosophila melanogaster larvae to understand the spatial distribution of pesticide metabolism in feeding assays. Insect Mol Biol 31(3): 369-376. PubMed ID: 35118729
Drug metabolizing enzymes such as cytochrome P450s have often been implicated in influencing levels of pesticide toxicology and resistance. Consequently, a variety of different P450 genes and variants have been linked to pesticide metabolism. Substantially less is known in regards to which tissues these P450s contribute to pesticide metabolism. This study isolated the effect of different tissues in pesticide toxicology by driving the model P450 Cyp6g1 in specific tissues of Drosophila melanogaster. Fluorescent and luminescent assays were used to compare the strength of GAL4 lines specific to the midgut (Mex-GAL4), Malpighian tubules (UO-GAL4) and the fat body (LSP2-GAL4) with the widely used HR-GAL4 line which drives GAL4 expression in all three tissues simultaneously. These data suggested that GAL4 drivers specific for the midgut and fat body were of approximately equal strength to the HR-GAL4 line, while the Malpighian tubule specific line was significantly weaker. Multiple toxicology assays using the pesticides bendiocarb, imidacloprid and malathion were then performed to assess which tissues provide the most chemoprotection. In the long-term feeding assay, transgenic expression of Cyp6g1 specifically in the midgut accounted for the majority of the resistance caused by Cyp6g1 overexpression with the HR-GAL4 driver. Real-time toxicology assays on third instar larvae were also performed and showed variable contributions of tissues to acute toxicology response depending on which pesticide was used. These data suggest a strong influence of bioassay parameters such as life stage and dosing method on outcome but suggest a prominent role for the midgut in larval toxicology.
Larsson, J. N. K., Nystrom, S. and Hammarstrom, P. (2022). HSP10 as a Chaperone for Neurodegenerative Amyloid Fibrils. Front Neurosci 16: 902600. PubMed ID: 35769706
Neurodegenerative diseases (NDs) are associated with accumulated misfolded proteins (MPs). MPs oligomerize and form multiple forms of amyloid fibril polymorphs that dictate fibril propagation and cellular dysfunction. Protein misfolding processes that impair protein homeostasis are implicated in onset and progression of NDs. A wide variety of molecular chaperones safeguard the cell from MP accumulation. A rather overlooked molecular chaperone is HSP10, known as a co-chaperone for HSP60. Due to the ubiquitous presence in human tissues and protein overabundance compared with HSP60, this work shows how HSP10 alone influences fibril formation in vitro of Alzheimer's disease-associated Aβ1-42 (see Appl). At sub-stoichiometric concentrations, eukaryotic HSP10s (human and Drosophila) significantly influenced the fibril formation process and the fibril structure of Aβ1-42, more so than the prokaryotic HSP10 GroES. Similar effects were observed for prion disease-associated prion protein HuPrP90-231. Paradoxically, for a chaperone, low concentrations of HSP10 appeared to promote fibril nucleation by shortened lag-phases, which were chaperone and substrate dependent. Higher concentrations of chaperone while still sub-stoichiometric extended the nucleation and/or the elongation phase. It is hypothesized that HSP10 by means of its seven mobile loops provides the chaperone with high avidity binding to amyloid fibril ends. The preserved sequence of the edge of the mobile loop GGIM(V)L (29-33 human numbering) normally dock to the HSP60 apical domain. Interestingly, this segment shows sequence similarity to amyloidogenic core segments of Aβ1-42, GGVVI (37-41), and HuPrP90-231 GGYML (126-130) likely allowing efficient competitive binding to fibrillar conformations of these MPs. Thee results propose that HSP10 can function as an important molecular chaperone in human proteostasis in NDs.
Miller, H. A., Huang, S., Dean, E. S., Schaller, M. L., Tuckowski, A. M., Munneke, A. S., Beydoun, S., Pletcher, S. D. and Leiser, S. F. (2022). Serotonin and dopamine modulate aging in response to food odor and availability. Nat Commun 13(1): 3271. PubMed ID: 35672307
An organism's ability to perceive and respond to changes in its environment is crucial for its health and survival. This study reveals how the most well-studied longevity intervention, dietary restriction, acts in-part through a cell non-autonomous signaling pathway that is inhibited by the presence of attractive smells. Using an intestinal reporter for a key gene induced by dietary restriction but suppressed by attractive smells, this study identified three compounds that block food odor effects in C. elegans, thereby increasing longevity as dietary restriction mimetics. These compounds clearly implicate serotonin and dopamine in limiting lifespan in response to food odor. A chemosensory neuron that likely perceives food odor, an enteric neuron that signals through the serotonin receptor 5-HT1A/SER-4, and a dopaminergic neuron that signals through the dopamine receptor DRD2/DOP-3. Aspects of this pathway are conserved in D. melanogaster. Thus, blocking food odor signaling through antagonism of serotonin or dopamine receptors is a plausible approach to mimic the benefits of dietary restriction.
Hodge, B. A., Meyerhof, G. T., Katewa, S. D., Lian, T., Lau, C., Bar, S., Leung, N. Y., Li, M., Li-Kroeger, D., Melov, S., Schilling, B., Montell, C. and Kapahi, P. (2022). Dietary restriction and the transcription factor clock delay eye aging to extend lifespan in Drosophila Melanogaster. Nat Commun 13(1): 3156. PubMed ID: 35672419
Many vital processes in the eye are under circadian regulation, and circadian dysfunction has emerged as a potential driver of eye aging. Dietary restriction is one of the most robust lifespan-extending therapies and amplifies circadian rhythms with age. This study demonstrates that dietary restriction extends lifespan in Drosophila melanogaster by promoting circadian homeostatic processes that protect the visual system from age- and light-associated damage. Altering the positive limb core molecular clock transcription factor, CLOCK, or CLOCK-output genes, accelerates visual senescence, induces a systemic immune response, and shortens lifespan. Flies subjected to dietary restriction are protected from the lifespan-shortening effects of photoreceptor activation. Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in constant darkness, primarily extends the lifespan of flies reared on a high-nutrient diet. These findings establish the eye as a diet-sensitive modulator of lifespan and indicates that vision is an antagonistically pleiotropic process that contributes to organismal aging.

Wednesday, August 4th - RNA and transposons

Peng, Y. and Gavis, E. R. (2022). The Drosophila hnRNP F/H homolog Glorund recruits dFMRP to inhibit nanos translation elongation. Nucleic Acids Res. PubMed ID: 35699205
Translational control of maternal mRNAs generates spatial and temporal patterns of protein expression necessary to begin animal development. Translational repression of unlocalized nanos (nos) mRNA in late-stage Drosophila oocytes by the hnRNP F/H homolog, Glorund (Glo), is important for embryonic body patterning. While previous work has suggested that repression occurs at both the translation initiation and elongation phases, the molecular mechanism by which Glo regulates nos translation remains elusive. This study identified the Drosophila fragile X mental retardation protein, dFMRP, as a Glo interaction partner with links to the translational machinery. Using an oocyte-based in vitro translation system, it was confirmed that Glo regulates both initiation and elongation of a nos translational reporter and showed that dFMRP specifically represses translation elongation and promotes ribosome stalling. Furthermore, mutational analysis and in vivo and in vitro binding assays were combined to show that Glo's qRRM2 domain specifically and directly interacts with dFMRP. These findings suggest that Glo regulates nos translation elongation by recruiting dFMRP and that Glo's RNA-binding domains can also function as protein-protein interaction interfaces critical for its regulatory functions. Additionally, they reveal a mechanism for targeting dFMRP to specific transcripts.
Weidman, T., Nagengast, A. A. and DiAngelo, J. R. (2022). The splicing factor 9G8 regulates the expression of NADPH-producing enzyme genes in Drosophila. Biochem Biophys Res Commun 620: 92-97. PubMed ID: 35780586
Excess nutrients are stored as triglycerides, mostly as lipid droplets found in adipose tissue. Previous studies have characterized a group of splicing factors called serine/arginine rich (SR) proteins that function to identify intron/exon borders in regulating metabolic homeostasis in the Drosophila fat body. Decreasing the function of one SR protein, 9G8, causes an increase in triglyceride storage; however, the full complement of genes regulated by 9G8 to control metabolism is unknown. To address this question, RNA sequencing was performed on Drosophila fat bodies with 9G8 levels reduced by RNAi. Differential expression and differential exon usage analyses revealed several genes involved in the immune response, xenobiotic biology, protein translation, sleep, and lipid and carbohydrate metabolism whose expression or splicing is altered in 9G8-RNAi fat bodies. One gene that was both downregulated and had altered splicing in 9G8-RNAi fat bodies was Zwischenferment (Zw), the Drosophila homolog of human glucose 6-phosphate dehydrogenase (G6PD). G6PD regulates flux of glucose 6-phosphate (G6P) into the pentose phosphate pathway, which generates NADPH, a coenzyme for lipid synthesis. Interestingly, the other NADPH-producing enzyme genes in Drosophila (phosphogluconate dehydrogenase, isocitrate dehydrogenase and malic enzyme) were also decreased in 9G8-RNAi flies. Together, these findings suggest that 9G8 regulates several classes of genes and may regulate NADPH-producing enzyme genes to maintain metabolic homeostasis.
van Leeuwen, W., VanInsberghe, M., Battich, N., Salmen, F., van Oudenaarden, A. and Rabouille, C. (2022). Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo. Cell Rep Methods 2(6): 100235. PubMed ID: 35784648
Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. This study used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. An RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, this purification-free method was found to reliably identify stress granule RNAs in bulk and single S2 cells and in Drosophila neurons. Similar to mammalian cells, this study found that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues.
Singh, M., Ye, B. and Kim, J. H. (2022). Dual leucine zipper kinase regulates Dscam expression through a non-canonical function of the cytoplasmic poly(A)-binding protein. J Neurosci. PubMed ID: 35764381
Dual leucine zipper kinase (DLK) plays a pivotal role in the development, degeneration, and regeneration of neurons. DLK can regulate gene expression post-transcriptionally, but the underlying mechanism remains poorly understood. The Drosophila DLK, Wallenda (Wnd) regulates the expression of Down syndrome cell adhesion molecule (Dscam) to control presynaptic arbor growth. This regulation is mediated by the 3' untranslated region (3'UTR) of Dscam mRNA, which suggests that RNA binding proteins (RBPs) mediate DLK function. This study performed a genome-wide cell-based RNAi screen of RBPs and identified the cytoplasmic poly(A)-binding protein, pAbp, as an RBP that mediates Wnd-induced increase in Dscam expression. Genetic analysis shows that Wnd requires pAbp for promoting presynaptic arbor growth and for enhancing Dscam expression. This analysis revealed that Dscam mRNAs harbor short poly(A) tails. A region was identified in Dscam 3'UTR that specifically interacts with pAbp. Removing this region significantly reduced Wnd-induced increase in Dscam expression. These suggest that a non-canonical interaction of PABP with the 3'UTR of target transcripts is essential for DLK functions.
Su, S., Wang, J., Deng, T., Yuan, X., He, J., Liu, N., Li, X., Huang, Y., Wang, H. W. and Ma, J. (2022). Structural insights into dsRNA processing by Drosophila Dicer-2-Loqs-PD. Nature. PubMed ID: 35768513
Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs). ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes. This study reports the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5'-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2-Loqs-PD.
Tng, P. Y. L., Carabajal Paladino, L. Z., Anderson, M. A. E., Adelman, Z. N., Fragkoudis, R., Noad, R. and Alphey, L. (2022). Intron-derived small RNAs for silencing viral RNAs in mosquito cells. PLoS Negl Trop Dis 16(6): e0010548. PubMed ID: 35737714
Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. This study tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3' UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.

Tuesday, August 2nd - Adult and larval physiology and Metabolism

Sirocko, K. T., Angstmann, H., Papenmeier, S., Wagner, C., Spohn, M., Indenbirken, D., Ehrhardt, B., Kovacevic, D., Hammer, B., Svanes, C., Rabe, K. F., Roeder, T., Uliczka, K. and Krauss-Etschmann, S. (2022). Early-life exposure to tobacco smoke alters airway signaling pathways and later mortality in D. melanogaster. Environ Pollut: 119696. PubMed ID: 35780997
Early life environmental influences such as exposure to cigarette smoke (CS) can disturb molecular processes of lung development and thereby increase the risk for later development of chronic respiratory diseases. Among the latter, asthma and chronic obstructive pulmonary disease (COPD) are the most common. The airway epithelium plays a key role in their disease pathophysiology but how CS exposure in early life influences airway developmental pathways and epithelial stress responses or survival is poorly understood. Using Drosophila melanogaster larvae as a model for early life, this study demonstrates that CS enters the entire larval airway system, where it activates cyp18a1 which is homologues to human CYP1A1 to metabolize CS-derived polycyclic aromatic hydrocarbons and further induces heat shock protein 70. RNASeq studies of isolated airways showed that CS dysregulates pathways involved in oxidative stress response, innate immune response, xenobiotic and glutathione metabolic processes as well as developmental processes (BMP, FGF signaling) in both sexes, while other pathways were exclusive to females or males. Glutathione S-transferase genes were further validated by qPCR showing upregulation of gstD4, gstD5 and gstD8 in respiratory tracts of females, while gstD8 was downregulated and gstD5 unchanged in males. ROS levels were increased in airways after CS. Exposure to CS further resulted in higher larval mortality, lower larval-pupal transition, and hatching rates in males only as compared to air-exposed controls. Taken together, early life CS induces airway epithelial stress responses and dysregulates pathways involved in the fly's branching morphogenesis as well as in mammalian lung development. CS further affected fitness and development in a highly sex-specific manner.
Freda, P. J., Toxopeus, J., Dowle, E. J., Ali, Z. M., Heter, N., Collier, R. L., Sower, I., Tucker, J. C., Morgan, T. J. and Ragland, G. J. (2022). Transcriptomic and functional genetic evidence for distinct ecophysiological responses across complex life cycle stages. J Exp Biol 225(11). PubMed ID: 35578907
Organisms with complex life cycles demonstrate a remarkable ability to change their phenotypes across development, presumably as an evolutionary adaptation to developmentally variable environments. Developmental variation in environmentally sensitive performance, and thermal sensitivity in particular, has been well documented in holometabolous insects. For example, thermal performance in adults and juvenile stages exhibit little genetic correlation (genetic decoupling) and can evolve independently, resulting in divergent thermal responses. Yet, very little is understood about how this genetic decoupling occurs. This study tested the hypothesis that genetic decoupling of thermal physiology is driven by fundamental differences in physiology between life stages, despite a potentially conserved cellular stress response. RNAseq was used to compare transcript expression in response to a cold stressor in Drosophila melanogaster larvae and adults and RNA interference (RNAi) was used to test whether knocking down nine target genes differentially affected larval and adult cold tolerance. Transcriptomic responses of whole larvae and adults during and following exposure to -5°C were largely unique both in identity of responding transcripts and in temporal dynamics. Further, the tissue-specificity of differentially expressed transcripts were analyzed from FlyAtlas 2 data, and it was concluded that stage-specific differences in transcription were not simply driven by differences in tissue composition. In addition, RNAi of target genes resulted in largely stage-specific and sometimes sex-specific effects on cold tolerance. The combined evidence suggests that thermal physiology is largely stage-specific at the level of gene expression, and thus natural selection may be acting on different loci during the independent thermal adaptation of different life stages.
Brischigliaro, M., Cabrera-Orefice, A., Sturlese, M., Elurbe, D. M., Frigo, E., Fernandez-Vizarra, E., Moro, S., Huynen, M. A., Arnold, S., Viscomi, C. and Zeviani, M. (2022). CG7630 is the Drosophila melanogaster homolog of the cytochrome c oxidase subunit COX7B. EMBO Rep: e54825. PubMed ID: 35699132
The mitochondrial respiratory chain (MRC) is composed of four multiheteromeric enzyme complexes. According to the endosymbiotic origin of mitochondria, eukaryotic MRC derives from ancestral proteobacterial respiratory structures consisting of a minimal set of complexes formed by a few subunits associated with redox prosthetic groups. These enzymes, which are the "core" redox centers of respiration, acquired additional subunits, and increased their complexity throughout evolution. Cytochrome c oxidase (COX), the terminal component of MRC, has a highly interspecific heterogeneous composition. Mammalian COX consists of 14 different polypeptides, of which COX7B is considered the evolutionarily youngest subunit. This study applied proteomic, biochemical, and genetic approaches to investigate the COX composition in the invertebrate model Drosophila melanogaster. A novel subunit was identified and characterized that is widely different in amino acid sequence, but similar in secondary and tertiary structures to COX7B, and evidence is provided that this object is in fact replacing the latter subunit in virtually all protostome invertebrates. These results demonstrate that although individual structures may differ the composition of COX is functionally conserved between vertebrate and invertebrate species.
Ding, M., Li, Q. F., Yin, G., Liu, J. L., Jan, X. Y., Huang, T., Li, A. C. and Zheng, L. (2022). Effects of Drosophila melanogaster regular exercise and apolipoprotein B knockdown on abnormal heart rhythm induced by a high-fat diet. PLoS One 17(6): e0262471. PubMed ID: 35657779
Abnormal heart rhythm is a common cardiac dysfunction in obese patients, and its pathogenesis is related to systemic lipid accumulation. The cardiomyocyte-derived apoLpp (homologous gene in Drosophila of the human apolipoprotein B) plays an important role in whole-body lipid metabolism of Drosophila under a high-fat diet (HFD). Knockdown of apoLpp derived from cardiomyocytes can reduce HFD-induced weight gain and abdominal lipid accumulation. In addition, exercise can reduce the total amount of apoLpp in circulation. However, the relationship between regular exercise, cardiomyocyte-derived apoLpp and abnormal heart rhythm is unclear. This study found that an HFD increased the level of triglyceride (TG) in the whole-body, lipid accumulation and obesity in Drosophila. Moreover, the expression of apoLpp in the heart increased sharply, the heart rate and arrhythmia index increased and fibrillation occurred. Conversely, regular exercise or cardiomyocyte-derived apoLpp knockdown reduced the TG level in the whole-body of Drosophila. This significantly reduced the arrhythmia induced by obesity, including the reduction of heart rate, arrhythmia index, and fibrillation. Under HFD conditions, flies with apoLpp knockdown in the heart could resist the abnormal cardiac rhythm caused by obesity after receiving regular exercise. HFD-induced obesity and abnormal cardiac rhythm may be related to the acute increase of cardiomyocyte-derived apoLpp. Regular exercise and inhibition of cardiomyocyte-derived apoLpp can reduce the HFD-induced abnormal cardiac rhythm.
Das, P. N., Basu, A. K. and Prasad, N. G. (2022). Increasing adult density compromises survival following bacterial infections in Drosophila melanogaster. J Insect Physiol: 104415. PubMed ID: 35753428
The density-dependent prophylaxis hypothesis predicts that risk of pathogen transmission increases with increase in population density, and in response to this, organisms mount a prophylactic immune response when exposed to high density. This prophylactic response is expected to help organisms improve their chances of survival when exposed to pathogens. Alternatively, organisms living at high densities can exhibit compromised defense against pathogens due to lack of resources and density associated physiological stress; the crowding stress hypothesis. This study housed adult Drosophila melanogaster flies at different densities and measured the effect this has on their post-infection survival and resistance to starvation. Flies housed at higher densities show greater mortality after being infected with bacterial pathogens, while also exhibiting increased resistance to starvation. These results are more in line with the density-stress hypothesis that postulates a compromised immune system when hosts are subjected to high densities.
Liu, Z., Jiang, L., Li, C., Li, C., Yang, J., Yu, J., Mao, R. and Rao, Y. (2022). LKB1 is physiologically required for sleep from Drosophila melanogaster to the Mus musculus. Genetics 221(3). PubMed ID: 35579349
Liver Kinase B1 (LKB1) is known as a master kinase for 14 kinases related to the adenosine monophosphate-activated protein kinase. Two of them salt inducible kinase 3 and adenosine monophosphate-activated protein kinase α have previously been implicated in sleep regulation. This study generated loss-of-function mutants for Lkb1 in both Drosophila and mice. Sleep, but not circadian rhythms, was reduced in Lkb1-mutant flies and in flies with neuronal deletion of Lkb1. Genetic interactions between Lkb1 and threonine to alanine mutation at residue 184 of adenosine monophosphate-activated protein kinase in Drosophila sleep or those between Lkb1 and Threonine to Glutamic Acid mutation at residue 196 of salt inducible kinase 3 in Drosophila viability have been observed. Sleep was reduced in mice after virally mediated reduction of Lkb1 in the brain. Electroencephalography analysis showed that nonrapid eye movement sleep and sleep need were both reduced in Lkb1-mutant mice. These results indicate that liver kinase B1 plays a physiological role in sleep regulation conserved from flies to mice.

Monday, August 1st - Disease Models

Delventhal, R., Wooder, E. R., Basturk, M., Sattar, M., Lai, J., Bolton, D., Muthukumar, G., Ulgherait, M. and Shirasu-Hiza, M. M.. (2022). Dietary restriction ameliorates TBI-induced phenotypes in Drosophila melanogaster. Sci Rep 12(1): 9523. PubMed ID: 35681073
Traumatic brain injury (TBI) affects millions annually and is associated with long-term health decline. TBI also shares molecular and cellular hallmarks with neurodegenerative diseases (NDs), typically increasing in prevalence with age, and is a major risk factor for developing neurodegeneration later in life. While understanding of genes and pathways that underlie neurotoxicity in specific NDs has advanced, a complete understanding of early molecular and physiological changes that drive neurodegeneration, particularly as an individual ages following a TBI, is lacking. Recently Drosophila has been introduced as a model organism for studying closed-head TBI. In this paper, a TBI is delivered to flies early in adult life, and then molecular and physiological phenotypes were measured at short-, mid-, and long-term timepoints following the injury. The aim was to identify the timing of changes that contribute to neurodegeneration. Prior work is confirmed demonstrating a TBI-induced decline in lifespan, and evidence is presented of a progressive decline in locomotor function, robust acute and modest chronic neuroinflammation, and a late-onset increase in protein aggregation. Evidence is also presented of metabolic dysfunction, in the form of starvation sensitivity and decreased lipids, that persists beyond the immediate injury response, but does not differ long-term. An intervention of dietary restriction (DR) partially ameliorates some TBI-induced phenotypes, including lifespan and locomotor function, though it does not alter the pattern of starvation sensitivity of injured flies. In the future, molecular pathways identified as altered following TBI-particularly in the short-, or mid-term-could present potential therapeutic targets.
Sanz, F. J., Solana-Manrique, C., Lilao-Garzon, J., Brito-Casillas, Y., Muñoz-Descalzo, S. and Paricio, N. (2022). Exploring the link between Parkinson's disease and type 2 diabetes mellitus in Drosophila. Faseb j 36(8): e22432. PubMed ID: 35766235
Parkinson's disease (PD) is the second most common neurodegenerative disease. Diabetes mellitus (DM) is a metabolic disease characterized by high levels of glucose in blood. Recent epidemiological studies have highlighted the link between both diseases; it is even considered that DM might be a risk factor for PD. To further investigate the likely relation of these diseases, a Drosophila PD model was used based on inactivation of the DJ-1β gene (ortholog of human DJ-1), and diet-induced Drosophila and mouse type 2 DM (T2DM) models, together with human neuron-like cells. T2DM models were obtained by feeding flies with a high sugar-containing medium, and mice with a high fat diet. These results showed that both fly models exhibit common phenotypes such as alterations in carbohydrate homeostasis, mitochondrial dysfunction or motor defects, among others. In addition, it was demonstrated that T2DM might be a risk factor of developing PD since the diet-induced fly and mouse T2DM models present DA neuron dysfunction, a hallmark of PD. It was also confirmed that neurodegeneration is caused by increased glucose levels, which has detrimental effects in human neuron-like cells by triggering apoptosis and leading to cell death. Besides, the observed phenotypes were exacerbated in DJ-1β mutants cultured in the high sugar medium, indicating that DJ-1 might have a role in carbohydrate homeostasis. Finally, it was confirmed that metformin, an antidiabetic drug, is a potential candidate for PD treatment and that it could prevent PD onset in T2DM model flies. This result supports antidiabetic compounds as promising PD therapeutics.
Chocron, E. S., Munkácsy, E., Kim, H. S., Karpowicz, P., Jiang, N., Van Skike, C. E., DeRosa, N., Banh, A. Q., Palavicini, J. P., Wityk, P., Kalinowski, L., Galvan, V., Osmulski, P. A., Jankowska, E., Gaczynska, M. and Pickering, A. M. (2022). Genetic and pharmacologic proteasome augmentation ameliorates Alzheimer's-like pathology in mouse and fly APP overexpression models. Sci Adv 8(23): eabk2252. PubMed ID: 35675410
The proteasome has key roles in neuronal proteostasis, including the removal of misfolded and oxidized proteins, presynaptic protein turnover, and synaptic efficacy and plasticity. Proteasome dysfunction is a prominent feature of Alzheimer's disease (AD). Prevention of proteasome dysfunction by genetic manipulation delays mortality, cell death, and cognitive deficits in fly and cell culture AD models. This study developed a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an AD mouse model, showed reduced mortality and cognitive deficits. To establish translational relevance, a set of TAT-based proteasome-activating peptidomimetics was developed that stably penetrated the blood-brain barrier and enhanced 20S/26S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. The protective effects of proteasome overexpression appear to be driven, at least in part, by the proteasome's increased turnover of the amyloid precursor protein along with the prevention of overall proteostatic dysfunction.
Xue, J., Zhu, Y., Wei, L., Huang, H., Li, G., Huang, W., Zhu, H. and Duan, R. (2022). Loss of Drosophila NUS1 results in cholesterol accumulation and Parkinson's disease-related neurodegeneration. Faseb j 36(7): e22411. PubMed ID: 35695805
=NgBR is the Nogo-B receptor, encoded by NUS1 gene. As NgBR contains a C-terminal domain that is similar to cis-isoprenyltransferase (cis-IPTase), NgBR was speculated to stabilize nascent Niemann-Pick type C 2 (NPC2) to facilitate cholesterol transport out of lysosomes. Mutations in the NUS1 were known as risk factors for Parkinson's disease (PD). In a previous study, it was shown that knockdown of Drosophila NUS1 orthologous gene tango14 causes decreased climbing ability, loss of dopaminergic neurons, and decreased dopamine contents. In this study, tango14 mutant flies were generated with a mutation in the C-terminal enzyme activity region using CRISPR/Cas9. tango14 mutant showed a reduced lifespan with locomotive defects and cholesterol accumulation in Malpighian tubules and brains, especially in dopaminergic neurons. Multilamellar bodies were found in tango14 mutants using electron microscopy. Neurodegenerative-related brain vacuolization was also detected in tango14 knockdown flies in an age-dependent manner. In addition, tango14 knockdown increased α-synuclein (α-syn) neurotoxicity in α-syn-overexpressing flies, with decreased locomotive activities, dopamine contents, and the numbers of dopaminergic neurons in aging flies. Thus, these observations suggest a role of NUS1, the ortholog of tango14, in PD-related pathogenesis.
Pant, D. C., Parameswaran, J., Rao, L., Loss, I., Chilukuri, G., Parlato, R., Shi, L., Glass, J. D., Bassell, G. J., Koch, P., Yilmaz, R., Weishaupt, J. H., Gennerich, A. and Jiang, J. (2022). ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain-of-function. EMBO Rep: e54234. PubMed ID: 35735139
Mutations in the human kinesin family member 5A (KIF5A; see Drosophila Khc) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. This study confirms the expression of KIF5A mutant proteins in patient iPSC-derived motor neurons. A comprehensive analysis was performed of ΔExon27 at the single-molecule, cellular, and organism levels. The results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. These results suggest gain-of-function as an underlying disease mechanism in KIF5A-associated ALS.
Chongtham, A., Yoo, J. H., Chin, T. M., Akingbesote, N. D., Huda, A., Marsh, J. L. and Khoshnan, A. (2022). Gut Bacteria Regulate the Pathogenesis of Huntington's Disease in Drosophila Model. Front Neurosci 16: 902205. PubMed ID: 35757549
Changes in the composition of gut microbiota are implicated in the pathogenesis of several neurodegenerative disorders. This study investigated whether gut bacteria affect the progression of Huntington's disease (HD) in transgenic Drosophila melanogaster models expressing full-length or N-terminal fragments of human mutant huntingtin (HTT) protein. Elimination of commensal gut bacteria by antibiotics was found to reduce the aggregation of amyloidogenic N-terminal fragments of HTT and delays the development of motor defects. Conversely, colonization of HD flies with Escherichia coli (E. coli), a known pathobiont of human gut with links to neurodegeneration and other morbidities, accelerates HTT aggregation, aggravates immobility, and shortens lifespan. Similar to antibiotics, treatment of HD flies with small compounds such as luteolin, a flavone, or crocin a beta-carotenoid, ameliorates disease phenotypes, and promotes survival. Crocin prevents colonization of E. coli in the gut and alters the levels of commensal bacteria, which may be linked to its protective effects. The opposing effects of E. coli and crocin on HTT aggregation, motor defects, and survival in transgenic Drosophila models support the involvement of gut-brain networks in the pathogenesis of HD.

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