What's hot today:
Current papers in developmental biology and gene function


Tuesday, October 31st, 2023 - Behavior

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Goncharova, A. A., Besedina, N. G., Bragina, J. V., Danilenkova, L. V., Kamysheva, E. A. and Fedotov, S. A. (2023). Courtship suppression in Drosophila melanogaster: The role of mating failure. PLoS One 18(8): e0290048. PubMed ID: 37561803
Drosophila melanogaster is a popular model organism in the study of memory due to a wide arsenal of methods used to analyze neuronal activity. The most commonly used tests in research of behavioral plasticity are shock avoidance associated with chemosensory cues and courtship suppression after mating failure. Many authors emphasize the value of courtship suppression as a model of behavior most appropriate to natural conditions. However, researchers often investigate courtship suppression using immobilized and decapitated females as targets of courtship by males, which makes the data obtained from such flies less valuable. In this study, courtship suppression towards immature mobile non-receptive females was evaluated after training with mated or immature females combined with an aversive stimulus (quinine). The previously described mechanisms of courtship suppression, as a result of the association of the courtship object with the repellent, as well as due to increased sensitivity to the anti-aphrodisiac cVA after mating failure, were not confirmed when immature mobile females were used. The reasons for the discrepancies between the current results and literature data, define the conditions to be met in the courtship suppression test if the aim is to analyze the natural forms of behavioral plasticity, and present data on the test modifications to approximate conditions to natural ones.
Krama, T., Munkevics, M., Krams, R., Grigorjeva, T., Trakimas, G., Jõers, P., Popovs, S., Zants, K., Elferts, D., Rantala, M. J., Sledevskis, E., Contreras-Garduño, J., de Bivort, B. L. and Krams, I. A. (2023). Development under predation risk increases serotonin-signaling, variability of turning behavior and survival in adult fruit flies Drosophila melanogaster. Front Behav Neurosci 17: 1189301. PubMed ID: 37304760
The development of high-throughput behavioral assays, where numerous individual animals can be analyzed in various experimental conditions, has facilitated the study of animal personality. Previous research showed that isogenic Drosophila melanogaster flies exhibit striking individual non-heritable locomotor handedness. The variability of this trait, i.e., the predictability of left-right turn biases, varies across genotypes and under the influence of neural activity in specific circuits. This suggests that the brain can dynamically regulate the extent of animal personality. It has been recently shown that predators can induce changes in prey phenotypes via lethal or non-lethal effects affecting the serotonergic signaling system. In this study, we tested whether fruit flies grown with predators exhibit higher variability/lower predictability in their turning behavior and higher survival than those grown with no predators in their environment. These predictions were confirmed, and it was found that both effects were blocked when flies were fed an inhibitor (αMW) of serotonin synthesis. The results of this study demonstrate a negative association between the unpredictability of turning behavior of fruit flies and the hunting success of their predators. It was also shown that the neurotransmitter serotonin controls predator-induced changes in the turning variability of fruit flies, regulating the dynamic control of behavioral predictability.
Ji, X., Li, X., Wang, L., Liu, S., Jiang, X. and Pan, Y. (2023). Asexuality in Drosophila juvenile males is organizational and independent of juvenile hormone. EMBO Rep: e56898. PubMed ID: 37530648
Sexuality is generally prevented in newborns and arises with organizational rewiring of neural circuitry and optimization of fitness for reproduction competition. Recent studies reported that sex circuitry in Drosophila melanogaster is developed in juvenile males but functionally inhibited by juvenile hormone (JH). This study found that the fly sex circuitry, mainly expressing the male-specific fruitless (fruM) and/or doublesex (dsx), is organizationally undeveloped and functionally inoperative in juvenile males. Artificially activating all fruM neurons induces substantial courtship in solitary adult males but not in juvenile males. Synaptic transmissions between major courtship regulators and all dsx neurons are strong in adult males but either weak or undetectable in juvenile males. It was further found that JH does not inhibit male courtship in juvenile males but instead promotes courtship robustness in adult males. These results indicate that the transition to sexuality from juvenile to adult flies requires organizational rewiring of neural circuitry.
Kim, G., An, J., Ha, S. and Kim, A. J. (2023).. A deep learning analysis of Drosophila body kinematics during magnetically tethered flight. J Neurogenet 37(1-2): 47-56. PubMed ID: 37200153
Flying Drosophila rely on their vision to detect visual objects and adjust their flight course. Despite their robust fixation on a dark, vertical bar, understanding of the underlying visuomotor neural circuits remains limited, in part due to difficulties in analyzing detailed body kinematics in a sensitive behavioral assay. This study observed the body kinematics of flying Drosophila using a magnetically tethered flight assay, in which flies are free to rotate around their yaw axis, enabling naturalistic visual and proprioceptive feedback. Additionally, deep learning-based video analyses was used to characterize the kinematics of multiple body parts in flying animals. By applying this pipeline of behavioral experiments and analyses, this study characterized the detailed body kinematics during rapid flight turns (or saccades) in two different visual conditions: spontaneous flight saccades under static screen and bar-fixating saccades while tracking a rotating bar. Both types of saccades involved movements of multiple body parts, and the overall dynamics were comparable. This study highlights the importance of sensitive behavioral assays and analysis tools for characterizing complex visual behaviors.
Durkin, J., Poe, A. R., Belfer, S. J., Rodriguez, A., Tang, S. H., Walker, J. A. and Kayser, M. S. (2023). Neurofibromin 1 regulates early developmental sleep in Drosophila. Neurobiol Sleep Circadian Rhythms 15: 100101. PubMed ID: 37593040
Sleep disturbances are common in neurodevelopmental disorders, but knowledge of molecular factors that govern sleep in young animals is lacking. Evidence across species, including Drosophila, suggests that juvenile sleep has distinct functions and regulatory mechanisms in comparison to sleep in maturity. In flies, manipulation of most known adult sleep regulatory genes is not associated with sleep phenotypes during early developmental (larval) stages. This study examined the role of the neurodevelopmental disorder-associated gene Neurofibromin 1 (Nf1) in sleep during numerous developmental periods. Mutations in Neurofibromin 1 (Nf1) are associated with sleep and circadian disorders in humans and adult flies. It was found in flies that Nf1 acts to regulate sleep across the lifespan, beginning during larval stages. Nf1 is required in neurons for this function, as is signaling via the Alk pathway. These findings identify Nf1 as one of a small number of genes positioned to regulate sleep across developmental periods.
He, L., Wu, B., Shi, J., Du, J. and Zhao, Z. (2023). Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila. Cell Rep 42(8): 112912. PubMed ID: 37531254
Feeding behavior is essential for growth and survival of animals; however, relatively little is known about its intrinsic mechanisms. This study demonstrates that Gart is expressed in the glia, fat body, and gut and positively regulates feeding behavior via cooperation and coordination. Gart in the gut is crucial for maintaining endogenous feeding rhythms and food intake, while Gart in the glia and fat body regulates energy homeostasis between synthesis and metabolism. These roles of Gart further impact Drosophila lifespan. Importantly, Gart expression is directly regulated by the CLOCK/CYCLE heterodimer via canonical E-box, in which the CLOCKs (CLKs) in the glia, fat body, and gut positively regulate Gart of peripheral tissues, while the core CLK in brain negatively controls Gart of peripheral tissues. This study provides insight into the complex and subtle regulatory mechanisms of feeding and lifespan extension in animals.

Monday, October 30th - Immune Response

Imrie, R. M., Walsh, S. K., Roberts, K. E., Lello, J. and Longdon, B. (2023). Investigating the outcomes of virus coinfection within and across host species. PLoS Pathog 19(5): e1011044. PubMed ID: 37216391
Interactions between coinfecting pathogens have the potential to alter the course of infection and can act as a source of phenotypic variation in susceptibility between hosts. This phenotypic variation may influence the evolution of host-pathogen interactions within host species and interfere with patterns in the outcomes of infection across host species. This study examine experimental coinfections of two Cripaviruses-Cricket Paralysis Virus (CrPV), and Drosophila C Virus (DCV)-across a panel of 25 Drosophila melanogaster inbred lines and 47 Drosophilidae host species. Interactions between these viruses were found to alter viral loads across D. melanogaster genotypes, with a ~3 fold increase in the viral load of DCV and a ~2.5 fold decrease in CrPV in coinfection compared to single infection, but little evidence was found of a host genetic basis for these effects. Across host species, no evidence was found of systematic changes in susceptibility during coinfection, with no interaction between DCV and CrPV detected in the majority of host species. These results suggest that phenotypic variation in coinfection interactions within host species can occur independently of natural host genetic variation in susceptibility, and that patterns of susceptibility across host species to single infections can be robust to the added complexity of coinfection.
Hixson, B., Huot, L., Morejon, B., Yang, X., Nagy, P., Michel, K. and Buchon, N. (2023). The transcriptional response in mosquitoes distinguishes between fungi and bacteria but not Gram types. bioRxiv. PubMed ID: 37546902
Mosquitoes are prolific vectors of human pathogens; a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. To determine whether Drosophila pathway-specific discrimination between pathogens is shared by mosquitoes, RNAseq was used to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, Ae. aegypti and An. gambiae were both found to mount a core response to all categories of infection. When the transcriptomes of mosquitoes infected with different types of bacteria were compared, it was observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, transcriptional signatures specific to bacterial infection and to fungal infection were identified. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi.
Cabrera, K., Hoard, D. S., Gibson, O., Martinez, D. I. and Wunderlich, Z. (2023). Drosophila immune priming to Enterococcus faecalis relies on immune tolerance rather than resistance. PLoS Pathog 19(8): e1011567. PubMed ID: 37566589
Innate immune priming increases an organism's survival of a second infection after an initial, non-lethal infection. Drosophila melanogaster and an insect-derived strain of Enterococcus faecalis were used to study transcriptional control of priming. In contrast to other pathogens, the enhanced survival in primed animals does not correlate with decreased E. faecalis load. Further analysis shows that primed organisms tolerate, rather than resist infection. Using RNA-seq of immune tissues, many genes were found to be upregulated in only primed flies, suggesting a distinct transcriptional program in response to initial and secondary infections. In contrast, few genes continuously express throughout the experiment or more efficiently re-activate upon reinfection. Priming experiments in immune deficient mutants revealed Imd is largely dispensable for responding to a single infection but needed to fully prime. Together, this indicates the fly's innate immune response is plastic-differing in immune strategy, transcriptional program, and pathway use depending on infection history.
Kong, F., Qadeer, A., Xie, Y., Jin, Y., Li, Q., Xiao, Y., She, K., Zheng, X., Li, J., Ji, S. and Zhu, Y. (2023). Dietary Supplementation of Aspirin Promotes Drosophila Defense against Viral Infection. Molecules 28(14). PubMed ID: 37513173
Aspirin, also known as acetylsalicylic acid, is widely consumed as a pain reliever and an anti-inflammatory as well as anti-platelet agent. Recently, our studies using the animal model of Drosophila demonstrated that the dietary supplementation of aspirin renovates age-onset intestinal dysfunction and delays organismal aging. Nevertheless, it remains probable that aspirin plays functional roles in other biological activities, for instance antiviral defense reactions. Intriguingly, this study observed that the replications of several types of viruses were drastically antagonized in Drosophila macrophage-like S2 cells with the addition of aspirin. Further in vivo experimental approaches illustrate that adult flies consuming aspirin harbor higher resistances to viral infections with respect to flies without aspirin treatment. Mechanistically, aspirin positively contributes to the Drosophila antiviral defense largely through mediating the STING (stimulator of interferon genes) but not the IMD (immune deficiency) signaling pathway. Collectively, these studies uncover a novel biological function of aspirin in modulating Drosophila antiviral immunity and provide theoretical bases for exploring new antiviral treatments in clinical trials.
Joshi, M., Viallat-Lieutaud, A. and Royet, J. (2023). Role of Rab5 early endosomes in regulating Drosophila gut antibacterial response. iScience 26(8): 107335. PubMed ID: 37529104
Interactions between prokaryotes and eukaryotes require a dialogue between microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs). In Drosophila, bacterial peptidoglycan is detected by PGRP receptors. While the components of the signaling cascades activated upon PGN/PGRP interactions are well characterized, little is known about the subcellular events that translate these early signaling steps into target gene transcription. Using a Drosophila enteric infection model, this study showed that gut-associated bacteria can induce the formation of intracellular PGRP-LE aggregates which colocalized with the early endosome marker Rab5. Combining microscopic and RNA-seq analysis, it was demonstrated that RNAi inactivation of the endocytosis pathway in the Drosophila gut affects the expression of essential regulators of the NF-κB response leading not only to a disruption of the immune response locally in the gut but also at the systemic level. This work sheds new light on the involvement of the endocytosis pathway in the control of the gut response to intestinal bacterial infection.
Loudhaief, R., Jneid, R., Christensen, C. F., Mackay, D. J., Andersen, D. S. and Colombani, J. (2023). The Drosophila tumor necrosis factor receptor, Wengen, couples energy expenditure with gut immunity. Sci Adv 9(23): eadd4977. PubMed ID: 37294765
It is well established that tumor necrosis factor (TNF) plays an instrumental role in orchestrating the metabolic disorders associated with late stages of cancers. However, it is not clear whether TNF/TNF receptor (TNFR) signaling controls energy homeostasis in healthy individuals. This study shows that the highly conserved Drosophila TNFR, Wengen (Wgn), is required in the enterocytes (ECs) of the adult gut to restrict lipid catabolism, suppress immune activity, and maintain tissue homeostasis. Wgn limits autophagy-dependent lipolysis by restricting cytoplasmic levels of the TNFR effector, TNFR-associated factor 3 (dTRAF3), while it suppresses immune processes through inhibition of the dTAK1/TAK1-Relish/NF-κB pathway in a dTRAF2-dependent manner. Knocking down dTRAF3 or overexpressing dTRAF2 is sufficient to suppress infection-induced lipid depletion and immune activation, respectively, showing that Wgn/TNFR functions as an intersection between metabolism and immunity allowing pathogen-induced metabolic reprogramming to fuel the energetically costly task of combatting an infection.

Friday, October 27th - Transposons and RNAs

Eichler, C. E., Li, H., Grunberg, M. E. and Gavis, E. R. (2023). Localization of oskar mRNA by agglomeration in ribonucleoprotein granules. PLoS Genet 19(8): e1010877. PubMed ID: 37624861
Localization of oskar mRNA to the posterior of the Drosophila oocyte is essential for abdominal patterning and germline development. oskar localization is a multi-step process involving temporally and mechanistically distinct transport modes. Little is known about the requirements for the posterior localization phase, which depends on cytoplasmic flows and results in the accumulation of large oskar ribonucleoprotein granules, called founder granules, by the end of oogenesis. Using super-resolution microscopy, this study showed that founder granules are agglomerates of smaller oskar transport particles. In contrast to the earlier kinesin-dependent oskar transport, late-phase localization depends on the sequence as well as on the structure of the spliced oskar localization element (SOLE), but not on the adjacent exon junction complex deposition. Late-phase localization also requires the oskar 3' untranslated region (3' UTR), which targets oskar to founder granules. Together, these results show that 3' UTR-mediated targeting together with SOLE-dependent agglomeration leads to accumulation of oskar in large founder granules at the posterior of the oocyte during late stages of oogenesis. In light of previous work showing that oskar transport particles are solid-like condensates, these findings indicate that founder granules form by a process distinct from that of well-characterized ribonucleoprotein granules like germ granules, P bodies, and stress granules. Additionally, they illustrate how an individual mRNA can be adapted to exploit different localization mechanisms depending on the cellular context.
Iki, T., Kawaguchi, S. and Kai, T. (2023). miRNA/siRNA-directed pathway to produce noncoding piRNAs from endogenous protein-coding regions ensures Drosophila spermatogenesis. Sci Adv 9(29): eadh0397. PubMed ID: 37467338
PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. This study shows that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in Drosophila melanogaster testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. This study further demonstrates that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, these data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.
Ho, S., Rice, N. P., Yu, T., Weng, Z. and Theurkauf, W. E. (2023). Aub, Vasa and Armi localization to phase separated nuage is dispensable for piRNA biogenesis and transposon silencing in Drosophila. bioRxiv. PubMed ID: 37546958
From nematodes to placental mammals, key components of the germline transposon silencing piRNAs pathway localize to phase separated perinuclear granules. In Drosophila, the PIWI protein Aub, DEAD box protein Vasa and helicase Armi localize to nuage granules and are required for ping-pong piRNA amplification and phased piRNA processing. Drosophila piRNA mutants lead to genome instability and mnk double mutants, we show that Chk2 activation disrupts nuage localization of Aub and Vasa, and that the HP1 homolog Rhino, which drives piRNA precursor transcription, is required for Aub, Vasa, and Armi localization to nuage. However, these studies also show that ping-pong amplification and phased piRNA biogenesis are independent of nuage localization of Vasa, Aub and Armi. Dispersed cytoplasmic proteins thus appear to mediate these essential piRNA pathway functions.
Kim, C. J., Kim, H. H., Kim, H. K., Lee, S., Jang, D., Kim, C. and Lim, D. H. (2023). MicroRNA miR-263b-5p Regulates Developmental Growth and Cell Association by Suppressing Laminin A in Drosophila. Biology (Basel) 12(8). PubMed ID: 37626982
Basement membranes (BMs) play important roles under various physiological conditions in animals, including ecdysozoans. During development, BMs undergo alterations through diverse intrinsic and extrinsic regulatory mechanisms; however, the full complement of pathways controlling these changes remain unclear. This study found that fat body-overexpression of Drosophila miR-263b, which is highly expressed during the larval-to-pupal transition, resulted in a decrease in the overall size of the larval fat body, and ultimately, in a severe growth defect accompanied by a reduction in cell proliferation and cell size. Interestingly, it was further observed that a large proportion of the larval fat body cells were prematurely disassociated from each other. Moreover, evidence is presented that miR-263b-5p suppresses the main component of BMs, Laminin A (LanA). Through experiments using RNA interference (RNAi) of LanA, it was found that its depletion phenocopied the effects in miR-263b-overexpressing flies. Overall, these findings suggest a potential role for miR-263b in developmental growth and cell association by suppressing LanA expression in the Drosophila fat body.
Lin, Y., Suyama, R., Kawaguchi, S., Iki, T. and Kai, T. (2023). Tejas functions as a core component in nuage assembly and precursor processing in Drosophila piRNA biogenesis. J Cell Biol 222(10):e202303125. PubMed ID: 37555815
PIWI-interacting RNAs (piRNAs), which protect genome from the attack by transposons, are produced and amplified in membraneless granules called nuage. In Drosophila, PIWI family proteins, Tudor-domain-containing (Tdrd) proteins, and RNA helicases are assembled and form nuage to ensure piRNA production. However, the molecular functions of the Tdrd protein Tejas (Tej) in piRNA biogenesis remain unknown. This study conducted a detailed analysis of the subcellular localization of fluorescently tagged nuage proteins and behavior of piRNA precursors. The results demonstrate that Tej functions as a core component that recruits Vasa (Vas) and Spindle-E (Spn-E) into nuage granules through distinct motifs, thereby assembling nuage and engaging precursors for further processing. This study also reveals that the low-complexity region of Tej regulates the mobility of Vas. Based on these results, it is proposed that Tej plays a pivotal role in piRNA precursor processing by assembling Vas and Spn-E into nuage and modulating the mobility of nuage components.
Iyer, S. S., Sun, Y., Seyfferth, J., Manjunath, V., Samata, M., Alexiadis, A., Kulkarni, T., Gutierrez, N., Georgiev, P., Shvedunova, M. and Akhtar, A. (2023). The NSL complex is required for piRNA production from telomeric clusters. Life Sci Alliance 6(9). PubMed ID: 37399316
The NSL complex is a transcriptional activator. Germline-specific knockdown of NSL complex subunits NSL1, NSL2, and NSL3 results in reduced piRNA production from a subset of bidirectional piRNA clusters, accompanied by widespread transposon derepression. The piRNAs most transcriptionally affected by NSL2 and NSL1 RNAi map to telomeric piRNA clusters. At the chromatin level, these piRNA clusters also show decreased levels of H3K9me3, HP1a, and Rhino after NSL2 depletion. Using NSL2 ChIP-seq in ovaries, we found that this protein specifically binds promoters of telomeric transposons HeT-A, TAHRE, and TART Germline-specific depletion of NSL2 also led to a reduction in nuclear Piwi in nurse cells. These findings thereby support a role for the NSL complex in promoting the transcription of piRNA precursors from telomeric piRNA clusters and in regulating Piwi levels in the Drosophila female germline.

Thursday, October 26th - Larval and Adult Neural Development and Function

Guss, E. J., Akbergenova, Y., Cunningham, K. L. and Littleton, J. T. (2023). Loss of the extracellular matrix protein Perlecan disrupts axonal and synaptic stability during Drosophila development. Elife 12. PubMed ID: 37368474
Heparan sulfate proteoglycans (HSPGs) form essential components of the extracellular matrix (ECM) and basement membrane (BM) and have both structural and signaling roles. Perlecan is a secreted ECM-localized HSPG that contributes to tissue integrity and cell-cell communication. Although a core component of the ECM, the role of Perlecan in neuronal structure and function is less understood. This study identified a role for Drosophila Perlecan in the maintenance of larval motoneuron axonal and synaptic stability. Loss of Perlecan causes alterations in the axonal cytoskeleton, followed by axonal breakage and synaptic retraction of neuromuscular junctions. These phenotypes are not prevented by blocking Wallerian degeneration and are independent of Perlecan's role in Wingless signaling. Expression of Perlecan solely in motoneurons cannot rescue synaptic retraction phenotypes. Similarly, removing Perlecan specifically from neurons, glia, or muscle does not cause synaptic retraction, indicating the protein is secreted from multiple cell types and functions non-cell autonomously. Within the peripheral nervous system, Perlecan predominantly localizes to the neural lamella, a specialized ECM surrounding nerve bundles. Indeed, the neural lamella is disrupted in the absence of Perlecan, with axons occasionally exiting their usual boundary in the nerve bundle. In addition, entire nerve bundles degenerate in a temporally coordinated manner across individual hemi-segments throughout larval development. These observations indicate disruption of neural lamella ECM function triggers axonal destabilization and synaptic retraction of motoneurons, revealing a role for Perlecan in axonal and synaptic integrity during nervous system development.
Jindal, D. A., Leier, H. C., Salazar, G., Foden, A. J., Seitz, E. A., Wilkov, A. J., Coutinho-Budd, J. C. and Broihier, H. T. (2023). Early Draper-mediated glial refinement of neuropil architecture and synapse number in the Drosophila antennal lobe. Front Cell Neurosci 17: 1166199. PubMed ID: 37333889
Glial phagocytic activity refines connectivity, though molecular mechanisms regulating this exquisitely sensitive process are incompletely defined. This study developed the Drosophila antennal lobe as a model for identifying molecular mechanisms underlying glial refinement of neural circuits in the absence of injury. Antennal lobe organization is stereotyped and characterized by individual glomeruli comprised of unique olfactory receptor neuronal (ORN) populations. The antennal lobe interacts extensively with two glial subtypes: ensheathing glia wrap individual glomeruli, while astrocytes ramify considerably within them. Phagocytic roles for glia in the uninjured antennal lobe are largely unknown. Thus, this study tested whether Draper regulates ORN terminal arbor size, shape, or presynaptic content in two representative glomeruli: VC1 and VM7. Glial Draper was found to limits the size of individual glomeruli and restrains their presynaptic content. Moreover, glial refinement is apparent in young adults, a period of rapid terminal arbor and synapse growth, indicating that synapse addition and elimination occur simultaneously. Draper has been shown to be expressed in ensheathing glia; unexpectedly, it was found to be expressed at high levels in late pupal antennal lobe astrocytes. Surprisingly, Draper plays differential roles in ensheathing glia and astrocytes in VC1 and VM7. In VC1, ensheathing glial Draper plays a more significant role in shaping glomerular size and presynaptic content; while in VM7, astrocytic Draper plays the larger role. Together, these data indicate that astrocytes and ensheathing glia employ Draper to refine circuitry in the antennal lobe before the terminal arbors reach their mature form and argue for local heterogeneity of neuron-glia interactions.
Hamid, A., Gattuso, H., Caglar, A. N., Pillai, M., Steele, T., Gonzalez, A., Nagel, K. and Syed, M. H. (2023). The RNA-binding protein, Imp specifies olfactory navigation circuitry and behavior in Drosophila. bioRxiv. PubMed ID: 37398350
Complex behaviors depend on the precise developmental specification of neuronal circuits, but the relationship between genetic programs for neural development, circuit structure, and behavioral output is often unclear. The central complex (CX) is a conserved sensory-motor integration center in insects that governs many higher order behaviors and largely derives from a small number of Type II neural stem cells. This study shows that Imp, a conserved IGF-II mRNA-binding protein expressed in Type II neural stem cells, specifies components of CX olfactory navigation circuitry. This study shows: (1) that multiple components of olfactory navigation circuitry arise from Type II neural stem cells and manipulating Imp expression in Type II neural stem cells alters the number and morphology of many of these circuit elements, with the most potent effects on neurons targeting the ventral layers of the fan-shaped body. (2) Imp regulates the specification of Tachykinin expressing ventral fan-shaped body input neurons. (3) Imp in Type II neural stem cells alters the morphology of the CX neuropil structures. (4) Loss of Imp in Type II neural stem cells abolishes upwind orientation to attractive odor while leaving locomotion and odor-evoked regulation of movement intact. Taken together, this work establishes that a single temporally expressed gene can regulate the expression of a complex behavior through the developmental specification of multiple circuit components and provides a first step towards a developmental dissection of the CX and its roles in behavior.
Hurkey, S., Niemeyer, N., Schleimer, J. H., Ryglewski, S., Schreiber, S. and Duch, C. (2023). Gap junctions desynchronize a neural circuit to stabilize insect flight. Nature 618(7963): 118-125. PubMed ID: 37225999
Insect asynchronous flight is one of the most prevalent forms of animal locomotion used by more than 600,000 species. Despite profound insights into the motor patterns, biomechanics and aerodynamics underlying asynchronous flight, the architecture and function of the central-pattern-generating (CPG) neural network remain unclear. On the basis of an experiment-theory approach including electrophysiology, optophysiology, Drosophila genetics and mathematical modelling, this study identified a miniaturized circuit solution with unexpected properties. The CPG network consists of motoneurons interconnected by electrical synapses that, in contrast to doctrine, produce network activity splayed out in time instead of synchronized across neurons. Experimental and mathematical evidence support a generic mechanism for network desynchronization that relies on weak electrical synapses and specific excitability dynamics of the coupled neurons. In small networks, electrical synapses can synchronize or desynchronize network activity, depending on the neuron-intrinsic dynamics and ion channel composition. In the asynchronous flight CPG, this mechanism translates unpatterned premotor input into stereotyped neuronal firing with fixed sequences of cell activation that ensure stable wingbeat power and, as we show, is conserved across multiple species. These findings prove a wider functional versatility of electrical synapses in the dynamic control of neural circuits and highlight the relevance of detecting electrical synapses in connectomics.
Ji, H., Wang, B., Shen, Y., Labib, D., Lei, J., Chen, X., Sapar, M., Boulanger, A., Dura, J. M. and Han, C. (2023). The Drosophila chemokine-like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons. Proc Natl Acad Sci U S A 120(24): e2303392120. PubMed ID: 37276397
Phagocytic clearance of degenerating neurons is triggered by "eat-me" signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes in vivo remains poorly understood. Using multiple models of dendrite degeneration, this study shows that the Drosophila chemokine-like protein Orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of Orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the Orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between Orion and human immunomodulatory proteins are important for Orion function. Thus, these results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons.
Kasturacharya, N., Dhall, J. K. and Hasan, G. (2023). A STIM dependent dopamine-neuropeptide axis maintains the larval drive to feed and grow in Drosophila. PLoS Genet 19(6): e1010435. PubMed ID: 37363909
Appropriate nutritional intake is essential for organismal survival. In holometabolous insects such as Drosophila melanogaster, the quality and quantity of food ingested as larvae determines adult size and fecundity. This study has identified a subset of dopaminergic neurons (THD') that maintain the larval motivation to feed. Dopamine release from these neurons requires the ER Ca2+ sensor STIM. Larvae with loss of STIM stop feeding and growing, whereas expression of STIM in THD' neurons rescues feeding, growth and viability of STIM null mutants to a significant extent. Moreover STIM is essential for maintaining excitability and release of dopamine from THD' neurons. Optogenetic stimulation of THD' neurons activated neuropeptidergic cells, including median neuro secretory cells that secrete insulin-like peptides. Loss of STIM in THD' cells alters the developmental profile of specific insulin-like peptides including ilp3. Loss of ilp3 partially rescues STIM null mutants and inappropriate expression of ilp3 in larvae affects development and growth. In summary this study has identified a novel STIM-dependent function of dopamine neurons that modulates developmental changes in larval feeding behaviour and growth.

Wednesday, October 25th - Evolution

Homberg, U., Kirchner, M., Kowalewski, K., Pitz, V., Kinoshita, M., Kern, M. and Seyfarth, J. (2023). Comparative morphology of serotonin-immunoreactive neurons innervating the central complex in the brain of dicondylian insects. J Comp Neurol 531(14): 1482-1508. PubMed ID: 37478205
Serotonin (5-hydroxytryptamine) acts as a widespread neuromodulator in the nervous system of vertebrates and invertebrates. In insects, it promotes feeding, enhances olfactory sensitivity, modulates aggressive behavior, and, in the central complex of Drosophila, serves a role in sleep homeostasis. In addition to a role in sleep-wake regulation, the central complex has a prominent role in spatial orientation, goal-directed locomotion, and navigation vector memory. To further understand the role of serotonergic signaling in this brain area, this study analyzed the distribution and identity of serotonin-immunoreactive neurons across a wide range of insect species. While one bilateral pair of tangential neurons innervating the central body was present in all species studied, a second type was labeled in all neopterans but not in dragonflies and firebrats. Both cell types show conserved major fiber trajectories but taxon-specific differences in dendritic targets outside the central body and axonal terminals in the central body, noduli, and lateral accessory lobes. In addition, numerous tangential neurons of the protocerebral bridge were labeled in all studied polyneopteran species except for Phasmatodea, but not in Holometabola. Lepidoptera and Diptera showed additional labeling of two bilateral pairs of neurons of a third type. The presence of serotonin in systems of columnar neurons apparently evolved independently in dragonflies and desert locusts. The data suggest distinct evolutionary changes in the composition of serotonin-immunolabeled neurons of the central complex and provides a promising basis for a phylogenetic study in a wider range of arthropod species.
Kapun, M., Mitchell, E. D., Kawecki, T. J., Schmidt, P. and Flatt, T. (2023). An Ancestral Balanced Inversion Polymorphism Confers Global Adaptation. Mol Biol Evol 40(6). PubMed ID: 37220650
Since the pioneering work of Dobzhansky in the 1930s and 1940s, many chromosomal inversions have been identified, but how they contribute to adaptation remains poorly understood. In Drosophila melanogaster, the widespread inversion polymorphism In(3R)Payne underpins latitudinal clines in fitness traits on multiple continents. This study used single-individual whole-genome sequencing, transcriptomics, and published sequencing data to study the population genomics of this inversion on four continents: in its ancestral African range and in derived populations in Europe, North America, and Australia. The results confirm that this inversion originated in sub-Saharan Africa and subsequently became cosmopolitan; marked monophyletic divergence of inverted and noninverted karyotypes was observed, with some substructure among inverted chromosomes between continents. Despite divergent evolution of this inversion since its out-of-Africa migration, derived non-African populations exhibit similar patterns of long-range linkage disequilibrium between the inversion breakpoints and major peaks of divergence in its center, consistent with balancing selection and suggesting that the inversion harbors alleles that are maintained by selection on several continents. Using RNA-sequencing, this study identified overlap between inversion-linked single-nucleotide polymorphisms and loci that are differentially expressed between inverted and noninverted chromosomes. Expression levels are higher for inverted chromosomes at low temperature, suggesting loss of buffering or compensatory plasticity and consistent with higher inversion frequency in warm climates. These results suggest that this ancestrally tropical balanced polymorphism spread around the world and became latitudinally assorted along similar but independent climatic gradients, always being frequent in subtropical/tropical areas but rare or absent in temperate climates.
Fontana, P., Buch-Larsen, S. C., Suyari, O., Smith, R., Suskiewicz, M. J., Schützenhofer, K., Ariza, A., Rack, J. G. M., Nielsen, M. L. and Ahel, I. (2023). Serine ADP-ribosylation in Drosophila provides insights into the evolution of reversible ADP-ribosylation signalling. Nat Commun 14(1): 3200. PubMed ID: 37268618
In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. This study shows by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the dParp1:dHpf1 complex. Moreover, structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, these data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling.
Glaser-Schmitt, A., Ramnarine, T. J. S. and Parsch, J. (2023). Rapid evolutionary change, constraints and the maintenance of polymorphism in natural populations of Drosophila melanogaster. Mol Ecol. PubMed ID: 37222070
Allele frequencies can shift rapidly within natural populations. Under certain conditions, repeated rapid allele frequency shifts can lead to the long-term maintenance of polymorphism. In recent years, studies of the model insect Drosophila melanogaster have suggested that this phenomenon is more common than previously believed and is often driven by some form of balancing selection, such as temporally fluctuating or sexually antagonistic selection. This study discusses some of the general insights into rapid evolutionary change revealed by large-scale population genomic studies, as well as the functional and mechanistic causes of rapid adaptation uncovered by single-gene studies. As an example of the latter, a regulatory polymorphism of the D. melanogaster fezzik gene is considered. Polymorphism at this site has been maintained at intermediate frequency over an extended period of time. Regular observations from a single population over a period of 7 years revealed significant differences in the frequency of the derived allele and its variance across collections between the sexes. These patterns are highly unlikely to arise from genetic drift alone or from the action of sexually antagonistic or temporally fluctuating selection individually. Instead, the joint action of sexually antagonistic and temporally fluctuating selection can best explain the observed rapid and repeated allele frequency shifts. Temporal studies such as those reviewed here further understanding of how rapid changes in selection can lead to the long-term maintenance of polymorphism as well as improve our knowledge of the forces driving and limiting adaptation in nature.
Grandchamp, A., Kuhl, L., Lebherz, M., Brüggemann, K., Parsch, J. and Bornberg-Bauer, E. (2023). Population genomics reveals mechanisms and dynamics of de novo expressed open reading frame emergence in Drosophila melanogaster. Genome Res 33(6): 872-890. PubMed ID: 37442576
Novel genes are essential for evolutionary innovations and differ substantially even between closely related species. This study sought de novo expressed open reading frames (neORFs), the not-yet fixed precursors of de novo genes that emerged within a single species. Genomes were with long-read technology and the corresponding transcriptomes from inbred lines of Drosophila melanogaster, derived from seven geographically diverse populations. We found line-specific neORFs in abundance but few neORFs shared by lines, suggesting a rapid turnover. Gain and loss of transcription is more frequent than the creation of ORFs, for example, by forming new start and stop codons. Consequently, the gain of ORFs becomes rate limiting and is frequently the initial step in neORFs emergence. Furthermore, transposable elements (TEs) are major drivers for intragenomic duplications of neORFs, yet TE insertions are less important for the emergence of neORFs. However, highly mutable genomic regions around TEs provide new features that enable gene birth. In conclusion, neORFs have a high birth-death rate, are rapidly purged, but surviving neORFs spread neutrally through populations and within genomes.
Karasawa, T., Saito, N. and Koshikawa, S. (2023). Cis-regulatory evolution underlying the changes in wingless expression pattern associated with wing pigmentation of Drosophila. FEBS Lett 597(14): 1837-1847. PubMed ID: 37194970
The co-option of regulatory genes has the potential to play an important role in the evolutionary gain of new traits. However, the changes at the sequence level that underlie such a co-option event are still elusive. This study identified the changes in the cis-regulatory sequence of wingless that caused co-option of wingless and led to its expression in new places in Drosophila guttifera, which has unique pigmentation patterns on its wings. The newly gained function of gene expression activation was acquired evolutionarily via a combination of pre-existing sequences containing a putative binding site for SMAD transcription factors that exhibit an ancestral function in driving expression at crossveins, and a sequence that is specific to the lineage leading to D. guttifera.

Tuesday, October 24th - Enhancers and Transcriptional Regulation

Dollinger, R., Deng, E. B., Schultz, J., Wu, S., Deorio, H. R. and Gilmour, D. S. (2023). DSIF directly facilitates promoter proximal pausing of RNA Polymerase II. J Biol Chem: 105106. PubMed ID: 37517697
Promoter proximal pausing of RNA Polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires the transcription factor DSIF and the inhibitory factor NELF. DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. This study used a highly purified in vitro system and Drosophila nuclear extract to investigate the role of DSIF in promoter proximal pausing. Two domains of Spt5 were identified, the KOW4 and NGN domains, that directly facilitate Pol II pausing. The KOW4 domain promotes pausing through its interaction with the nascent RNA while the NGN domain does so through a short helical motif that is in close proximity to the non-transcribed DNA template strand. Removal of this sequence in Drosophila has a male-specific dominant negative effect. The alpha helical motif is also needed to support fly viability. It was also shown that the interaction between the Spt5 KOW1 domain and the upstream DNA helix is required for DSIF association with the Pol II elongation complex. Disruption of the KOW1-DNA interaction is dominant lethal in vivo. Finally, the KOW2-3 domain of Spt5 was shown to mediate the recruitment of NELF to the elongation complex. In summary, these results reveal additional roles for DSIF in transcription regulation and identify specific domains important for facilitating Pol II pausing.
Cheng, Y., Chan, F. and Kassis, J. A. (2023). The activity of engrailed imaginal disc enhancers is modulated epigenetically by chromatin and autoregulation. bioRxiv. PubMed ID: 37502849
engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal disks; small IDE-reporter transgenes are expressed in the anterior compartment, the opposite of what is expected. The data show that the En protein binds to en IDEs, and it is suggested that En directly represses IDE function. Two en IDEs, 'O' and 'S' were identified. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from the endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether these data show that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic regulators. Further, the endogenous locus imparts a stability to en function not seen even in a large transgene, reflecting the importance of both positive and negative epigenetic influences that act over relatively large distances in chromatin.
Cho, C. Y. and O'Farrell, P. H. (2023). Stepwise modifications of transcriptional hubs link pioneer factor activity to a burst of transcription. Nat Commun 14(1): 4848. PubMed ID: 37563108
Binding of transcription factors (TFs) promotes the subsequent recruitment of coactivators and preinitiation complexes to initiate eukaryotic transcription, but this time course is usually not visualized. It is commonly assumed that recruited factors eventually co-reside in a higher-order structure, allowing distantly bound TFs to activate transcription at core promoters. This study used live imaging of endogenously tagged proteins, including the pioneer TF Zelda, the coactivator dBrd4, and RNA polymerase II (RNAPII), to define a cascade of events upstream of transcriptional initiation in early Drosophila embryos. These factors are sequentially and transiently recruited to discrete clusters during activation of non-histone genes. Zelda and the acetyltransferase dCBP nucleate dBrd4 clusters, which then trigger pre-transcriptional clustering of RNAPII. Subsequent transcriptional elongation disperses clusters of dBrd4 and RNAPII. These results suggest that activation of transcription by eukaryotic TFs involves a succession of distinct biomolecular condensates that culminates in a self-limiting burst of transcription.
Eggers, N., Gkountromichos, F., Krause, S., Campos-Sparr, A. and Becker, P. B. (2023). Physical interaction between MSL2 and CLAMP assures direct cooperativity and prevents competition at composite binding sites. Nucleic Acids Res. PubMed ID: 37602401
MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. This study explored the nature of the cooperative binding to these GA-rich, composite sequence elements in reconstituted naive embryonic chromatin. It was found that the cooperativity requires physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. The cooperation between MSL2/CLAMP was not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in the reconstitution, such as roX2 lncRNA.
Dresch, J. M., Conrad, R. D., Klonaros, D. and Drewell, R. A. (2023). Investigating the sequence landscape in the Drosophila initiator core promoter element using an enhanced MARZ algorithm. PeerJ 11: e15597. PubMed ID: 37366427
The core promoter elements are important DNA sequences for the regulation of RNA polymerase II transcription in eukaryotic cells. Despite the broad evolutionary conservation of these elements, there is extensive variation in the nucleotide composition of the actual sequences. This study aimed to improve understanding of the complexity of this sequence variation in the TATA box and initiator core promoter elements in Drosophila melanogaster. Using computational approaches, including an enhanced version of the previously developed MARZ algorithm that utilizes gapped nucleotide matrices, several sequence landscape features are uncovered, including an interdependency between the nucleotides in position 2 and 5 in the initiator. Incorporating this information in an expanded MARZ algorithm improves predictive performance for the identification of the initiator element. Overall these results demonstrate the need to carefully consider detailed sequence composition features in core promoter elements in order to make more robust and accurate bioinformatic predictions.
Jacobs, J., Pagani, M., Wenzl, C. and Stark, A. (2023). Widespread regulatory specificities between transcriptional co-repressors and enhancers in Drosophila. Science 381(6654): 198-204. PubMed ID: 37440660
Gene expression is controlled by the precise activation and repression of transcription. Repression is mediated by specialized transcription factors (TFs) that recruit co-repressors (CoRs) to silence transcription, even in the presence of activating cues. However, whether CoRs can dominantly silence all enhancers or display distinct specificities is unclear. This work reports that most enhancers in Drosophila can be repressed by only a subset of CoRs, and enhancers classified by CoR sensitivity show distinct chromatin features, function, TF motifs, and binding. Distinct TF motifs render enhancers more resistant or sensitive to specific CoRs, as was demonstrated by motif mutagenesis and addition. These CoR-enhancer compatibilities constitute an additional layer of regulatory specificity that allows differential regulation at close genomic distances and is indicative of distinct mechanisms of transcriptional repression.

Monday, October 23rd - Signal Transduction

Gu, J., Ye, Y., Zheng, Z. W., Luo, W., Gong, Y. J., Feng, Q. L., Li, S. and Huang, L. H. (2023). Cytoplasmic Hsp70s promote EcR transport into the nucleus by responding to various stimuli. Insect Biochem Mol Biol 157: 103964. PubMed ID: 37230333
Metamorphosis is one of the most important physiological processes in insects, which is coordinated by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Ecdysone receptor (EcR) is a steroid receptor (SR), which usually presents in cytoplasm and transfers into nucleus after binding to 20E. Heat shock proteins (Hsps) are suggested to be important members of the SR complex. However, their role in nucleocytoplasmic shuttle of the EcR remains unclear. The present study found that apoptozole (Hsp70 inhibitor) suppressed the larval molting by decreasing the expression of ecdysone signaling genes. Two cytoplasmic (Cy) Hsp70s (Hsp72 and (Hsp73) interacted with both EcR and Ultraspiracle (USP, the heterodimer partner of EcR). By immunohistochemistry experiments, it was revealed that CyHsp70 co-localized with EcR in the cytoplasm, and that both apoptozole and interfering of CyHsp70 significantly inhibited the process of EcR entering the nucleus under 20E induction, while reducing the expression of ecdysone signaling genes. Interestingly, the nuclear localization of EcR was also promoted by two other stimuli, including JH and heat stress, and this promotion was inhibited by apoptozole. This implies that various stimuli can induce EcR entry into the nucleus, and that this process is mediated by CyHsp70. Curiously, neither JH nor heat stress activated the ecdysone signaling genes; instead, they have a significant inhibitory effect on them. Taken together, it seems that Cytoplasmic Hsp70s promote EcR transport into the nucleus by responding to various stimuli, and that the biological effects of various stimuli passing through the EcR are different. Thus, these data provide a new viewpoint to understand the mechanism of nucleocytoplasmic shuttle of EcR.
Guo, Y., Zeng, Q., Brooks, D. and Geisbrecht, E. R. (2023). A conserved STRIPAK complex is required for autophagy in muscle tissue. Mol Biol Cell 34(9): ar91. PubMed ID: 37379167
Autophagy is important for cellular homeostasis and to prevent the abnormal accumulation of proteins. While many proteins that comprise the canonical autophagy pathway have been characterized, the identification of new regulators may help understand tissue and/or stress-specific responses. Using an in-silico approach, Striatin interacting protein (Strip), MOB kinase activator 4, and fibroblast growth factor receptor 1 oncogene partner 2 were identified as conserved mediators of muscle tissue maintenance. Affinity purification-mass spectrometry (AP-MS) experiments with Drosophila melanogaster Strip was used as a bait protein and copurified additional Striatin-interacting phosphatase and kinase (STRIPAK) complex members from larval muscle tissue. NUAK family kinase 1 (NUAK) and Starvin (Stv) also emerged as Strip-binding proteins and these physical interactions were verified in vivo using proximity ligation assays. To understand the functional significance of the STRIPAK-NUAK-Stv complex, a sensitized genetic assay combined with RNA interference (RNAi) were used to demonstrate that both NUAK and stv function in the same biological process with genes that encode for STRIPAK complex proteins. RNAi-directed knockdown of Strip in muscle tissue led to the accumulation of ubiquitinated cargo, p62, and Autophagy-related 8a, consistent with a block in autophagy. Indeed, autophagic flux was decreased in Strip RNAi muscles, while lysosome biogenesis and activity were unaffected. These results support a model whereby the STRIPAK-NUAK-Stv complex coordinately regulates autophagy in muscle tissue.
Han, I., Nassar, L. S., Page-McCaw, A. and Hutson, M. S. (2023). After wounding, a G-protein coupled receptor restores tension to epithelial cells in a dynamic inward-traveling wave. bioRxiv. PubMed ID: 37398151 ID:
The maintenance of epithelial barrier function is due in part to cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension and wound-induced changes in tension may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension, a laser-recoil assay was used to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored as an inward traveling wave that reached the wound margin about 10 minutes after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. The wave of tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.
Humphries, A. C., Molina-Pelayo, C., Sil, P., Hazelett, C. C., Devenport, D. and Mlodzik, M. (2023). A Van Gogh/Vangl tyrosine phosphorylation switch regulates its interaction with core Planar Cell Polarity factors Prickle and Dishevelled. PLoS Genet 19(7): e1010849. PubMed ID: 37463168
Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). In this study, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3) was dissected. Pk and Dsh/Dvl were shown to bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment.
Erazo-Oliveras, A., Muñoz-Vega, M., Mlih, M., Thiriveedi, V., Salinas, M. L., Rivera-Rodríguez, J. M., Kim, E., Wright, R. C., Wang, X., Landrock, K. K., Goldsby, J. S., Mullens, D. A., Roper, J., Karpac, J. and Chapkin, R. S. (2023). Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin. Nat Commun 14(1): 4342. PubMed ID: 37468468
Although the role of the Wnt pathway in colon carcinogenesis has been described previously, it has been recently demonstrated that Wnt signaling originates from highly dynamic nano-assemblies at the plasma membrane. However, little is known regarding the role of oncogenic APC in reshaping Wnt nanodomains. This is noteworthy, because oncogenic APC does not act autonomously and requires activation of Wnt effectors upstream of APC to drive aberrant Wnt signaling. This study demonstrates the role of oncogenic APC in increasing plasma membrane free cholesterol and rigidity, thereby modulating Wnt signaling hubs. This results in an overactivation of Wnt signaling in the colon. Finally, using the Drosophila sterol auxotroph model, this study demonstrated the unique ability of exogenous free cholesterol to disrupt plasma membrane homeostasis and drive Wnt signaling in a wildtype APC background. Collectively, these findings provide a link between oncogenic APC, loss of plasma membrane homeostasis and CRC development.
Ghosh, A., Venugopal, A., Shinde, D., Sharma, S., Krishnan, M., Mathre, S., Krishnan, H., Saha, S. and Raghu, P. (2023). PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase. Life Sci Alliance 6(9). PubMed ID: 37316298
Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P in vitro. This study repors a role for PIP4K in regulating PI3P levels in Drosophila Loss-of-function mutants of the only Drosophila PIP4K gene show reduced cell size in salivary glands. PI3P levels are elevated in dPIP4K 29 and reverting PI3P levels back towards WT, without changes in PI5P levels, can rescue the reduced cell size. dPIP4K 29 mutants also show up-regulation in autophagy and the reduced cell size can be reverted by depleting Atg8a that is required for autophagy. Lastly, increasing PI3P levels in WT can phenocopy the reduction in cell size and associated autophagy up-regulation seen in dPIP4K 29 Thus, this study reports a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size.

Friday, October 20th - Larval and Adult Physiology and Metabolism

Huang, J., Wang, P., Wu, Y., Zeng, L., Ji, X., Zhang, X., Wu, M., Tong, H. and Yang, Y. (2023). Rapid determination of triglyceride and glucose levels in Drosophila melanogaster induced by high-sugar or high-fat diets based on near-infrared spectroscopy. Heliyon 9(6): e17389. PubMed ID: 37426790
Triglyceride and glucose levels are important indicators for determining metabolic syndrome, one of the leading public-health burdens worldwide. Drosophila melanogaster is an ideal model for investigating metabolic diseases because it has 70% homology to human genes and its regulatory mechanism of energy metabolism homeostasis is highly similar to that of mammals. However, traditional analytical methods of triglyceride and glucose are time-consuming, laborious, and costly. In this study, a simple, practical, and reliable near-infrared (NIR) spectroscopic analysis method was developed for the rapid determination of glucose and triglyceride levels in an in vivo model of metabolic disorders using Drosophila induced by high-sugar or high-fat diets. The partial least squares (PLS) model was constructed and optimized using different spectral regions and spectral pretreatment methods. The overall results had satisfactory prediction performance. For Drosophila induced by high-sugar diets, the correlation coefficient (R(P)) and root mean square error of prediction (RMSEP) were 0.919 and 0.228 mmoL gprot(-1) for triglyceride and 0.913 and 0.143 mmoL gprot(-1) for glucose respectively; for Drosophila induced by high-fat diets, the R(P) and RMSEP were 0.871 and 0.097 mmoL gprot(-1) for triglyceride and 0.853 and 0.154 mmoL gprot(-1) for glucose, respectively. This study demonstrated the potential of using NIR spectroscopy combined with PLS in the determination of triglyceride and glucose levels in Drosophila, providing a rapid and effective method for monitoring metabolite levels during disease development and a possibility for evaluating metabolic diseases in humans in clinical practice.
Dobson, A. J., Voigt, S., Kumpitsch, L., Langer, L., Voigt, E., Ibrahim, R., Dowling, D. K. and Reinhardt, K. (2023). Mitonuclear interactions shape both direct and parental effects of diet on fitness and involve a SNP in mitoribosomal 16s rRNA. PLoS Biol 21(8): e3002218. PubMed ID: 37603597
Nutrition is a primary determinant of health, but responses to nutrition vary with genotype. Epistasis between mitochondrial and nuclear genomes may cause some of this variation, but which mitochondrial loci and nutrients participate in complex gene-by-gene-by-diet interactions? Furthermore, it remains unknown whether mitonuclear epistasis is involved only in the immediate responses to changes in diet, or whether mitonuclear genotype might modulate sensitivity to variation in parental nutrition, to shape intergenerational fitness responses. This study has shown that in Drosophila melanogaster mitonuclear epistasis shapes fitness responses to variation in dietary lipids and amino acids. It was also shown that mitonuclear genotype modulates the parental effect of dietary lipid and amino acid variation on offspring fitness. Effect sizes for the interactions between diet, mitogenotype, and nucleogenotype were equal to or greater than the main effect of diet for some traits, suggesting that dietary impacts cannot be understood without first accounting for these interactions. Associating phenotype to mtDNA variation in a subset of populations implicated a C/T polymorphism in mt:lrRNA, which encodes the 16S rRNA of the mitochondrial ribosome. This association suggests that directionally different responses to dietary changes can result from variants on mtDNA that do not change protein coding sequence, dependent on epistatic interactions with variation in the nuclear genome.
Fujita, H., Kaneshiro, J., Takeda, M., Sasaki, K., Yamamoto, R., Umetsu, D., Kuranaga, E., Higo, S., Kondo, T., Asano, Y., Sakata, Y., Miyagawa, S. and Watanabe, T. M. (2023). Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation. Life Sci Alliance 6(7). PubMed ID: 37236659
Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. This study established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, this study has successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, this study would be of help in the risk assessment of future heart failure.
Gandara, A. C. P. and Drummond-Barbosa, D. (2023). Chronic exposure to warm temperature causes low sperm abundance and quality in Drosophila melanogaster. Sci Rep 13(1): 12331. PubMed ID: 37518578
Temperature influences male fertility across organisms; however, how suboptimal temperatures affect adult spermatogenesis remains understudied. In a recent study on Drosophila melanogaster oogenesis, a drastic reduction was observed in the fertility of adult males exposed to warm temperature (29s°C). This study showss that males become infertile at 29 °sC because of low sperm abundance and quality. The low sperm abundance at 29 °C does not stem from reduced germline stem cell or spermatid numbers, as those numbers remain comparable between 29 °C and control 25 °C. Notably, males at cold 18 °C and 29 °C had similarly increased frequencies of spermatid elongation and individualization defects which, considering the high sperm abundance and male fertility measured at 18 °C, indicate that spermatogenesis has a high tolerance for elongation and individualization defects. Interestingly, the abundance of sperm at 29 °C decreases abruptly and with no evidence of apoptosis as they transition into the seminal vesicle near the end of spermatogenesis, pointing to sperm elimination through an unknown mechanism. Finally, sperm from males at 29 °C fertilize eggs less efficiently and do not support embryos past the first stage of embryogenesis, indicating that poor sperm quality is an additional cause of male infertility at 29 °C.
Grenier, T., Consuegra, J., Ferrarini, M. G., Akherraz, H., Bai, L., Dusabyinema, Y., Rahioui, I., Da Silva, P., Gillet, B., Hughes, S., Ramos, C. I., Matos, R. C. and Leulier, F. (2023). Intestinal GCN2 controls Drosophila systemic growth in response to Lactiplantibacillus plantarum symbiotic cues encoded by r/tRNA operons. Elife 12. PubMed ID: 37294006
Symbiotic bacteria interact with their host through symbiotic cues. This study took advantage of the mutualism between Drosophila and Lactiplantibacillus plantarum (Lp) to investigate a novel mechanism of host-symbiont interaction. Using chemically defined diets, it was found that association with Lp improves the growth of larvae-fed amino acid-imbalanced diets, even though Lp cannot produce the limiting amino acid. In this context Lp supports its host's growth through a molecular dialogue that requires functional operons encoding ribosomal and transfer RNAs (r/tRNAs) in Lp and the general control nonderepressible 2 (GCN2) kinase in Drosophila's enterocytes. These data indicate that Lp's r/tRNAs are packaged in extracellular vesicles and activate GCN2 in a subset of larval enterocytes, a mechanism necessary to remodel the intestinal transcriptome and ultimately to support anabolic growth. Based on these findings, a novel beneficial molecular dialogue is proposed between host and microbes, which relies on a non-canonical role of GCN2 as a mediator of non-nutritional symbiotic cues encoded by r/tRNA operons.
Guo, L., Qiao, X., Haji, D., Zhou, T., Liu, Z., Whiteman, N. K. and Huang, J. (2023). Convergent resistance to GABA receptor neurotoxins through plant-insect coevolution. Nat Ecol Evol. PubMed ID: 37460839
The molecular mechanisms of coevolution between plants and insects remain elusive. GABA receptors are targets of many neurotoxic terpenoids, which represent the most diverse array of natural products known. Over deep evolutionary time, as plant terpene synthases diversified in plants, so did plant terpenoid defence repertoires. This study shows that herbivorous insects and their predators evolved convergent amino acid changing substitutions in duplicated copies of the Resistance to dieldrin (Rdl) gene that encodes the GABA receptor, and that the evolution of duplicated Rdl and terpenoid-resistant GABA receptors is associated with the diversification of moths and butterflies. These same substitutions also evolved in pests exposed to synthetic insecticides that target the GABA receptor. In vivo genome editing in Drosophila melanogaster was used to evaluate the fitness effects of each putative resistance mutation, and pleiotropy was found to both facilitate and constrain the evolution of GABA receptor resistance. The same genetic changes that confer resistance to terpenoids across 300 Myr of insect evolution have re-evolved in response to synthetic analogues over one human lifespan.

Thursday, October 19th - Tumors, Cancer and Growth

Hsi, T. C., Ong, K. L., Sepers, J. J., Kim, J. and Bilder, D. (2023). Systemic coagulopathy promotes host lethality in a new Drosophila tumor model. Curr Biol 33(14): 3002-3010. PubMed ID: 37354901
Malignant tumors trigger a complex network of inflammatory and wound repair responses, prompting Dvorak's characterization of tumors as "wounds that never heal." Some of these responses lead to profound defects in blood clotting, such as disseminated intravascular coagulopathy (DIC), which correlate with poor prognoses. This study demonstrates that a new tumor model in Drosophila provokes phenotypes that resemble coagulopathies observed in patients. Fly ovarian tumors overproduce multiple secreted components of the clotting cascade and trigger hypercoagulation of fly blood (hemolymph). Hypercoagulation occurs shortly after tumor induction and is transient; it is followed by a hypocoagulative state that is defective in wound healing. Cellular clotting regulators accumulate on the tumor over time and are depleted from the body, suggesting that hypocoagulation is caused by exhaustion of host clotting components. Rescuing coagulopathy by depleting a tumor-produced clotting factor improves survival of tumor-bearing flies, despite the fact that flies have an open (non-vascular) circulatory system. As clinical studies suggest that lethality in patients with high serum levels of clotting components can be independent of thrombotic events, this work establishes a platform for identifying alternative mechanisms by which tumor-driven coagulopathy triggers early mortality. Moreover, it opens up exploration of other conserved mechanisms of host responses to chronic wounds.
Datta, I. and Bangi, E. (2023). Senescent cells and macrophages cooperate through a multi-kinase signaling network to promote intestinal transformation in Drosophila. bioRxiv. PubMed ID: 37292988
Cellular senescence is a conserved biological process essential for embryonic development, tissue remodeling, repair, and a key regulator of aging. Senescence also plays a crucial role in cancer, though this role can be tumor-suppressive or tumor-promoting, depending on the genetic context and the microenvironment. The highly heterogeneous, dynamic, and context-dependent nature of senescence-associated features and the relatively small numbers of senescent cells in tissues makes in vivo mechanistic studies of senescence challenging. As a result, which senescence-associated features are observed in which disease contexts and how they contribute to disease phenotypes remain largely unknown. Similarly, the specific mechanisms by which various senescence-inducing signals are integrated in vivo to induce senescence and why some cells become senescent while their immediate neighbors do not are unclear. This study identified a small number of cells that exhibit multiple features of senescence in a genetically complex model of intestinal transformation established in the developing Drosophila larval hindgut epithelium. These cells emerge in response to concurrent activation of AKT, JNK, and DNA damage response pathways within transformed tissue. Eliminating senescent cells, genetically or by treatment with senolytic compounds, reduces overgrowth and improves survival. This tumor-promoting role is mediated by Drosophila macrophages recruited to the transformed tissue by senescent cells, which results in non-autonomous activation of JNK signaling within the transformed epithelium. These findings emphasize complex cell-cell interactions underlying epithelial transformation and identify senescent cell-macrophage interactions as a potential druggable node in cancer. It is concluded that interactions between transformed senescent cells and macrophages drive tumorigenesis.
Bosso, G., Cipressa, F., Tullo, L. and Cenci, G. (2023). Co-amplification of CBX3 with EGFR or RAC1 in human cancers corroborated by a conserved genetic interaction among the genes. Cell Death Discov 9(1): 317. PubMed ID: 37633946
Chromobox Protein 3 (CBX3) overexpression is a common event occurring in cancer, promotes cancer cell proliferation and represents a poor prognosis marker in a plethora of human cancers. This study describes that a wide spectrum of human cancers harbors a co-amplification of CBX3 (see Drosophila HP1b) gene with either EGFR or RAC1, which yields a statistically significant increase of both mRNA and protein levels of CBX3, EGFR and RAC1. It was also revealed that the simultaneous overexpression of CBX3, RAC1 and EGFR gene products correlates with a worse prognosis compared to the condition when CBX3, RAC1 and EGFR are singularly upregulated. Furthermore, this study also showed that a co-occurrence of low-grade amplification, in addition to high-grade amplification, between CBX3 and EGFR or RAC1 is associated with a reduced patient lifespan. Finally, it was found that CBX3 and RAC1/EGFR genetically interact in the model organism Drosophila melanogaster, suggesting that the simultaneous overexpression as well as well the co-occurrence of high- or low-grade copy number alterations in these genes is not accidental and could reflect evolutionarily conserved functional relationships.
Chen, D., Lan, X., Huang, X., Huang, J., Zhou, X., Liu, J. and Hoffmann, J. A. (2023). mthl1, a potential Drosophila homologue of mammalian adhesion GPCRs, is involved in antitumor reactions to injected oncogenic cells in flies. Proc Natl Acad Sci U S A 120(30): e2303462120. PubMed ID: 37459549
Injection of OCs into adult male flies induces a strong transcriptomic response in the host flies featuring in particular genes encoding bona fide G coupled proteins, among which the gene for methuselah like 1 is prominent. The injection is followed after a 3-d lag period, by the proliferation of the oncogenic cells. It was hypothesized that through the product of mthl1 the host might control, at least in part, this proliferation as a defense reaction. Through a combination of genetic manipulations of the mthl1 gene (loss of function and overexpression of mthl1), this study documented that indeed this gene has an antiproliferative effect. Parallel injections of primary embryonic Drosophila cells or of various microbes do not exhibit this effect. It was further shown that mthl1 controls the expression of a large number of genes coding for chemoreceptors and genes implicated in regulation of development. Of great potential interest is the observation that the expression of the mouse gene coding for the adhesion G-protein-coupled receptor E1 (Adgre1, also known as F4/80), a potential mammalian homologue of mthl1, is significantly induced by B16-F10 melanoma cell inoculation 3 d postinjection in both the bone marrow and spleen (nests of immature and mature myeloid-derived immune cells), respectively. This observation is compatible with a role of this GPCR in the early response to injected tumor cells in mice.
Gao, Y., Shan, Z., Jian, C., Wang, Y., Yao, X., Li, S., Ti, X., Zhao, G., Liu, C. and Zhang, Q. (2023). HIB/SPOP inhibits Ci/Gli-mediated tumorigenesis by modulating the RNA Polymerase II components stabilities. iScience 26(8): 107334. PubMed ID: 37554435
Hedgehog (Hh) signaling mediated by transcription factor Ci/Gli plays a vital role in embryonic development and adult tissue homeostasis in invertebrates and vertebrates, whose dysregulation leads to many human disorders, including cancer. However, till now, cofactors of Ci/Gli which can affect tumorigenesis are not well known. Through genetic screen, this study found overexpression of active Ci alone is not sufficient to generate tumor-like eye phenotype in Drosophila, however, its overexpression combined with knockdown of hib causes a striking tumor-like big eye phenotype. Mechanistically, HIB/SPOP inhibits Ci/Gli-mediated tumorigenesis by modulating the RNA polymerase II (RNAPII) components Rpb3/Rpb7 stabilities in E3 ligase dependent manner. In addition, Ci/Gli can promote HIB/SPOP-mediated Rpb7/Rpb3 degradation. Taken together, these results indicate Ci/Gli needs to hook up with suitable RNAPII together to achieve the tumor-like eye phenotype and HIB/SPOP plays dual roles through controlling Ci/Gli and Rpb3/Rpb7 protein stabilities to temper Ci/Gli/RNAPII-mediated tumorigenesis.
Garcia-Arias, J. M., Pinal, N., Cristobal-Vargas, S., Estella, C. and Morata, G. (2023). Lack of apoptosis leads to cellular senescence and tumorigenesis in Drosophila epithelial cells. Cell Death Discov 9(1): 281. PubMed ID: 37532716
Programmed cell death (apoptosis) is a homeostasis program of animal tissues designed to remove cells that are unwanted or are damaged by physiological insults. To assess the functional role of apoptosis, the consequences were studied of subjecting Drosophila epithelial cells defective in apoptosis to stress or genetic perturbations that normally cause massive cell death. Many of those cells acquire persistent activity of the JNK pathway, which drives them into senescent status, characterized by arrest of cell division, cell hypertrophy, Senescent Associated β-gal activity (SA-β-gal), reactive oxygen species (ROS) production, Senescent Associated Secretory Phenotype (SASP) and migratory behaviour. Two classes of senescent cells were identified in the wing disc: 1) those that localize to the appendage part of the disc, express the upd, wg and dpp signalling genes and generate tumour overgrowths, and 2) those located in the thoracic region do not express wg and dpp nor they induce tumour overgrowths. Whether to become tumorigenic or non-tumorigenic depends on the original identity of the cell prior to the transformation. The p53 gene was also found to contribute to senescence by enhancing the activity of JNK.

Wednesday, October 18th - Characterization of Protein Structure and Function

Gurley, N. J., Szymanski, R. A., Dowen, R. H., Butcher, T. A., Ishiyama, N. and Peifer, M. (2023). Exploring the evolution and function of Canoe's intrinsically disordered region in linking cell-cell junctions to the cytoskeleton during embryonic morphogenesis. PLoS One 18(8): e0289224. PubMed ID: 37535684
One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. This study focused on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. This study has combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. This bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When shorter evolutionary time scales were examined, multiple conserved motifs were identified. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. The lesions were identified in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including the conserved motifs in the IDR, are critical for protein function. These data provide the foundation for further analysis of IDR function.
Groppe, J. C., Lu, G., Tandang-Silvas, M. R., Pathi, A., Konda, S., Wu, J., Le, V. Q., Culbert, A. L., Shore, E. M., Wharton, K. A. and Kaplan, F. S. (2023). Polypeptide Substrate Accessibility Hypothesis: Gain-of-Function R206H Mutation Allosterically Affects Activin Receptor-like Protein Kinase Activity. Biomolecules 13(7). PubMed ID: 37509165
Although structurally similar to type II counterparts, type I or activin receptor-like kinases (ALKs; see Drosophila Babboon) are set apart by a metastable helix-loop-helix (HLH) element preceding the protein kinase domain that, according to a longstanding paradigm, serves passive albeit critical roles as an inhibitor-to-substrate-binding switch. A single recurrent mutation in the codon of the penultimate residue, directly adjacent the position of a constitutively activating substitution, causes milder activation of ACVR1/ALK2 leading to sporadic heterotopic bone deposition in patients presenting with fibrodysplasia ossificans progressiva, or FOP. To determine the protein structural-functional basis for the gain of function, R206H mutant, Q207D (aspartate-substituted caALK2) and HLH subdomain-truncated (208 Ntrunc) forms were compared to one another and the wild-type enzyme through in vitro kinase and protein-protein interaction analyses that were complemented by signaling read-out (p-Smad) in primary mouse embryonic fibroblasts and Drosophila S2 cells. Contrary to the paradigm, the HLH subdomain actively suppressed the phosphotransferase activity of the enzyme, even in the absence of FKBP12. Unexpectedly, perturbation of the HLH subdomain elevated kinase activity at a distance, i.e., allosterically, at the ATP-binding and polypeptide-interacting active site cleft. Accessibility to polypeptide substrate (BMP Smad C-terminal tails) due to allosterically altered conformations of type I active sites within heterohexameric cytoplasmic signaling complexes-assembled noncanonically by activin-type II receptors extracellularly-is hypothesized to produce a gain of function of the R206H mutant protein responsible for episodic heterotopic ossification in FOP.
Hamada-Kawaguchi, N., Nore, B. F., Zain, R., Engstrom, Y., Smith, C. I. E. and Yamamoto, D. (2023). Exposure to Therapeutic BTK Inhibitors Induces Phenocopying of Btk29A Mutants in the Fruit Fly Drosophila melanogaster. Front Biosci (Landmark Ed) 28(6): 124. PubMed ID: 37395037
Bruton's tyrosine kinase (BTK) is a non-receptor type tyrosine kinase originally identified as the genetic signature responsible for X-linked agammaglobulinemia (XLA) when mutated. Its functional form is required for B lymphocyte maturation in both humans and mice, whereas loss-of-function causes a different form of developmental defect in the fruit fly, Drosophila melanogaster. Ibrutinib and other therapeutic inhibitors of BTK have been extensively used to successfully treat various leukemias and lymphomas. Btk29A type 2 is the ortholog of BTK in the fruit fly. Feeding wild-type flies an ibrutinib-containing diet induces phenocopying of Btk29A mutants, i.e., failure in the fusion of left and right halves of the dorsal cuticles, partial loss of wing tissues and dysregulation of germ cell production. It has been previously reported that Btk29A phosphorylates Drosophila Arm (β-catenin), and ibrutinib reduces phosphorylation at Tyrosine142 of endogenously expressed β-catenin in Cos7 cells transfected with Btk29A type 2 cDNA. Thus, Drosophila is suitable for screens of novel BTK inhibitor candidates and offers a unique in vivo system in which the mode of action of BTK inhibitors can be examined at the molecular, cellular, and organismal levels.
He, Y., Cotten, M. L., Yin, J., Yuan, Q. and Tjandra, N. (2023). Expression and purification of Drosophila OBP44a with the aids of LC-MS and NMR. Protein Expr Purif 212: 106354. PubMed ID: 37597794
The production of highly purified native soluble proteins in large quantities is crucial for studying protein structure and function. Odorant binding proteins (OBPs) are small, soluble, extracellular proteins with multiple disulfide bonds, whose functions include, but are not limited to, binding hydrophobic molecules and delivering them to their corresponding receptors expressed on insect olfactory receptor neurons. Expression of proteins with multiple disulfide bonds like OBPs usually results in insolubility and low yield, which has been a significant barrier to understanding their biological roles and physiological functions. In the E. coli system, expression of OBPs often results in insoluble inclusion bodies or a limited amount of periplasmic soluble proteins. Although expression of OBPs in eukaryotic systems such as Sf9 insect cells or yeast Pichia pastoris can increase the solubility of the protein, the process remains insufficient. Additionally, monitoring the purity and native apo state of the protein is critical for establishing the correct conformation of the protein. This study employed an E. coli host with an altered intracellular environment to produce cytosolic soluble OBP44a protein, which yielded over 100 mg/L. The integrity of disulfide bonds was monitored throughout the purification process using LC-MS and used NMR to ensure the final product adopted a single conformation. This study presents an efficient method for obtaining large quantities of soluble proteins in a single conformation, which enables extensive in vitro studies of secreted proteins like OBPs.
Emond-Fraser, V., Larouche, M., Kubiniok, P., Bonneil, É., Li, J., Bourouh, M., Frizzi, L., Thibault, P. and Archambault, V. (2023). Identification of PP2A-B55 targets uncovers regulation of emerin during nuclear envelope reassembly in Drosophila. Open Biol 13(7): 230104. PubMed ID: 37463656
Mitotic exit requires the dephosphorylation of many proteins whose phosphorylation was needed for mitosis. Protein phosphatase 2A (Microtubule star in Drosophila) with its B55 regulatory subunit (PP2A-B55: Twins in Drosophila) promotes this transition. However, the events and substrates that it regulates are incompletely understood. This study used proteomic approaches in Drosophila to identify proteins that interact with and are dephosphorylated by PP2A-B55. Among several candidates, emerin (otefin in Drosophila) was identified. Emerin resides in the inner nuclear membrane and interacts with the DNA-binding protein barrier-to-autointegration factor (BAF) via a LEM domain. The phosphorylation of emerin at Ser50 and Ser54 near its LEM domain negatively regulates its association with BAF, lamin and additional emerin in mitosis. Dephosphorylation of emerin at these sites by PP2A-B55 determines the timing of nuclear envelope reformation. Genetic experiments indicate that this regulation is required during embryonic development. Phosphoregulation of the emerin-BAF complex formation by PP2A-B55 appears as a key event of mitotic exit that is likely conserved across species.
Fedotova, A. A., Georgiev, P. G. and Bonchuk, A. N. (2023). Study of the in Vivo Functional Role of Mutations in the BTB Domain of the CP190 Protein of Drosophila melanogaster. Dokl Biochem Biophys 509(1): 47-50. PubMed ID: 37340291
The Drosophila transcription factor СР190 is one of the key proteins that determine the activity of housekeeping gene promoters and insulators. CP190 has an N-terminal BTB domain that allows for dimerization. Many of known Drosophila architectural proteins interact with the hydrophobic peptide-binding groove in the BTB domain, which is presumably a mechanisms for recruiting CP190 to regulatory elements. To study the role of the BTB domain in the interaction with architectural proteins, transgenic flies were obtained expressing CP190 variants with mutations in the peptide-binding groove, which disrupts their interaction with architectural proteins. As a result of the studies, it was found that mutations in the BTB domain do not affect binding of the CP190 protein to polytene chromosomes. Thus, these studies confirm the previously obtained data that CP190 is recruited to regulatory elements by several transcription factors interacting, in addition to BTB, with other CP190 domains.

Tuesday, October 17th - Synapse and Vesicles

He, K., Han, Y., Li, X., Hernandez, R. X., Riboul, D. V., Feghhi, T., Justs, K. A., Mahneva, O., Perry, S., Macleod, G. T. and Dickman, D. (2023). Physiologic and Nanoscale Distinctions Define Glutamatergic Synapses in Tonic vs Phasic Neurons. J Neurosci 43(25): 4598-4611. PubMed ID: 37221096
Neurons exhibit a striking degree of functional diversity, each one tuned to the needs of the circuitry in which it is embedded. A fundamental functional dichotomy occurs in activity patterns, with some neurons firing at a relatively constant "tonic" rate, while others fire in bursts, a "phasic" pattern. Synapses formed by tonic versus phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic. A major challenge toward illuminating the synaptic differences between tonic and phasic neurons is the difficulty in isolating their physiological properties. At the Drosophila neuromuscular junction, most muscle fibers are coinnervated by two motor neurons: the tonic "MN-Ib" and phasic "MN-Is." This study used selective expression of a newly developed botulinum neurotoxin transgene to silence tonic or phasic motor neurons in Drosophila larvae of either sex. This approach highlighted major differences in their neurotransmitter release properties, including probability, short-term plasticity, and vesicle pools. Furthermore, Ca(2+) imaging demonstrated ∼2-fold greater Ca(2+) influx at phasic neuron release sites relative to tonic, along with an enhanced synaptic vesicle coupling. Finally, confocal and super-resolution imaging revealed that phasic neuron release sites are organized in a more compact arrangement, with enhanced stoichiometry of voltage-gated Ca(2+) channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca(2+) influx collaborate to differentially tune glutamate release at tonic versus phasic synaptic subtypes.
Greaney, M. R., Wreden, C. C. and Heckscher, E. S. (2023). Distinctive features of the central synaptic organization of Drosophila larval proprioceptors. Front Neural Circuits 17: 1223334. PubMed ID: 37564629
Proprioceptive feedback is critically needed for locomotor control, but how this information is incorporated into central proprioceptive processing circuits remains poorly understood. Circuit organization emerges from the spatial distribution of synaptic connections between neurons. This distribution is difficult to discern in model systems where only a few cells can be probed simultaneously. Therefore, this study turned to a relatively simple and accessible nervous system to ask: how are proprioceptors' input and output synapses organized in space, and what principles underlie this organization? Using the Drosophila larval connectome, a map was generated of the input and output synapses of 34 proprioceptors in several adjacent body segments (5-6 left-right pairs per segment). The spatial organization of these synapses was characterized, and this organization was compared to that of other somatosensory neurons' synapses. Three distinguishing features of larval proprioceptor synapses were found: (1) Generally, individual proprioceptor types display segmental somatotopy. (2) Proprioceptor output synapses both converge and diverge in space; they are organized into six spatial domains, each containing a unique set of one or more proprioceptors. Proprioceptors form output synapses along the proximal axonal entry pathway into the neuropil. (3) Proprioceptors receive few inhibitory input synapses. Further, it was found that these three features do not apply to other larval somatosensory neurons. Thus, this study has generated the most comprehensive map to date of how proprioceptor synapses are centrally organized. This map documents previously undescribed features of proprioceptors, raises questions about underlying developmental mechanisms, and has implications for downstream proprioceptive processing circuits.
Gujar, M. R., Gao, Y., Teng, X., Deng, Q., Lin, K. Y., Tan, Y. S., Toyama, Y. and Wang, H. (2023). Golgi-dependent reactivation and regeneration of Drosophila quiescent neural stem cells. Dev Cell. PubMed ID: 37567172
The ability of stem cells to switch between quiescent and proliferative states is crucial for maintaining tissue homeostasis and regeneration. In Drosophila, quiescent neural stem cells (qNSCs) extend a primary protrusion, a hallmark of qNSCs. This study found that qNSC protrusions can be regenerated upon injury. This regeneration process relies on the Golgi apparatus that acts as the major acentrosomal microtubule-organizing center in qNSCs. A Golgi-resident GTPase Arf1 and its guanine nucleotide exchange factor Sec71 promote NSC reactivation and regeneration via the regulation of microtubule growth. Arf1 physically associates with its new effector mini spindles (Msps)/XMAP215, a microtubule polymerase. Finally, Arf1 functions upstream of Msps to target the cell adhesion molecule E-cadherin to NSC-neuropil contact sites during NSC reactivation. Thses findings have established Drosophila qNSCs as a regeneration model and identified Arf1/Sec71-Msps pathway in the regulation of microtubule growth and NSC reactivation.
Hossain, M. S., Yao, A., Qiao, X., Shi, W., Xie, T., Chen, C. and Zhang, Y. Q. (2023). Gbb glutathionylation promotes its proteasome-mediated degradation to inhibit synapse growth. J Cell Biol 222(9). PubMed ID: 37389657
Glutathionylation is a posttranslational modification involved in various molecular and cellular processes. However, it remains unknown whether and how glutathionylation regulates nervous system development. To identify critical regulators of synapse growth and development, this study performed an RNAi screen and found that postsynaptic knockdown of glutathione transferase omega 1 (GstO1) caused significantly more synaptic boutons at the Drosophila neuromuscular junctions. Genetic and biochemical analysis revealed an increased level of glass boat bottom (Gbb), the Drosophila homolog of mammalian bone morphogenetic protein (BMP), in GstO1 mutants. Further experiments showed that GstO1 is a critical regulator of Gbb glutathionylation at cysteines 354 and 420, which promoted its degradation via the proteasome pathway. Moreover, the E3 ligase Ctrip negatively regulated the Gbb protein level by preferentially binding to glutathionylated Gbb. These results unveil a novel regulatory mechanism in which glutathionylation of Gbb facilitates its ubiquitin-mediated degradation. Taken together, these findings shed new light on the crosstalk between glutathionylation and ubiquitination of Gbb in synapse development.
Fernandes, A. R., Martins, J. P., Gomes, E. R., Mendes, C. S. and Teodoro, R. O. (2023). Drosophila motor neuron boutons remodel through membrane blebbing coupled with muscle contraction. Nat Commun 14(1): 3352. PubMed ID: 37291089
Wired neurons form new presynaptic boutons in response to increased synaptic activity, however the mechanism(s) by which this occurs remains uncertain. Drosophila motor neurons (MNs) have clearly discernible boutons that display robust structural plasticity, being therefore an ideal system in which to study activity-dependent bouton genesis. This study showed that in response to depolarization and in resting conditions, MNs form new boutons by membrane blebbing, a pressure-driven mechanism that occurs in 3-D cell migration, but not previously described to occur in neurons. Accordingly, F-actin is decreased in boutons during outgrowth, and non-muscle myosin-II is dynamically recruited to newly formed boutons. Furthermore, muscle contraction plays a mechanical role, which is hypothesized to promotes bouton addition by increasing MN confinement. Overall, this study identified a mechanism by which established circuits form new boutons allowing their structural expansion and plasticity, using trans-synaptic physical forces as the main driving force.
Han, Y., Goel, P., Chen, J., Perry, S., Tran, N., Nishimura, S., Sanjani, M., Chien, C. and Dickman, D. (2023). Excess glutamate release triggers subunit-specific homeostatic receptor scaling. Cell Rep 42(7): 112775. PubMed ID: 37436892
Ionotropic glutamate receptors (GluRs) are targets for modulation in Hebbian and homeostatic synaptic plasticity and are remodeled by development, experience, and disease. This study has probed the impact of synaptic glutamate levels on the two postsynaptic GluR subtypes at the Drosophila neuromuscular junction, GluRA and GluRB. It was demonstrated that GluRA and GluRB compete to establish postsynaptic receptive fields, and that proper GluR abundance and composition can be orchestrated in the absence of any synaptic glutamate release. However, excess glutamate adaptively tunes postsynaptic GluR abundance, echoing GluR scaling observed in mammalian systems. Furthermore, when GluRA vs. GluRB competition is eliminated, GluRB becomes insensitive to glutamate modulation. In contrast, GluRA is now homeostatically regulated by excess glutamate to maintain stable miniature activity, where Ca(2+) permeability through GluRA receptors is required. Thus, excess glutamate, GluR competition, and Ca(2+) signaling collaborate to selectively target GluR subtypes for homeostatic regulation at postsynaptic compartments.

Monday, October 16th - Disease Models

Geier, B., Neely, L., Coronado, E. and Reiter, L. T. (2023). Drosophila UBE3A regulates satiety signaling through the Piezo mechanosensitive ion channel. Res Sq. PubMed ID: 37461494
Angelman syndrome (AS) is a rare neurogenetic disorder characterized by developmental delays, speech impairments, ataxic movements, and in some cases, hyperphagic feeding behavior. Loss of function mutations, loss of expression from the maternal allele or absence of maternal UBE3A result in AS. Recent studies have established a connection between UBE3A and the mechanosensitive ion channel PIEZO2, suggesting the potential role of UBE3A in the regulation of PIEZO channels. This study investigated the role of Drosophila UBE3A (Dube3a) in Piezo associated hyperphagic feeding behavior. A novel assay was developed using green fluorescent protein (GFP) expressing yeast to quantify gut distention in flies with Piezo and Dube3a mutations. It was confirmed that Dube3a (15b) loss of function flies displayed gut distention to almost identical levels as Piezo (KO) flies. Further analysis using deficiency (Df) lines encompassing the Dube3a locus provided proof for a role of Dube3a in satiety signaling. Endogenous Piezo expression was examined across the fly midgut and tracheal system. Piezo protein could be detected in both neurons and trachea of the midgut. Overexpression of Dube3a driven by the Piezo promoter resulted in distinct tracheal remodeling within the midgut. These findings suggest that Dube3a plays a key role in the regulation of Piezo and that subsequent dysregulation of these ion channels may explain the hyperphagic behavior observed in 32% of cases of AS. Further investigation will be needed to identify the intermediate protein(s) interacting between the Dube3a ubiquitin ligase and Piezo channels, as Piezo does not appear to be a direct ubiquitin substrate for UBE3A in mice and humans.
Hardy, R. E., Chung, I., Yu, Y., Loh, S. H. Y., Morone, N., Soleilhavoup, C., Travaglio, M., Serreli, R., Panman, L., Cain, K., Hirst, J., Martins, L. M., MacFarlane, M. and Pryde, K. R. (2023). The antipsychotic medications aripiprazole, brexpiprazole and cariprazine are off-target respiratory chain complex I inhibitors. Biol Direct 18(1): 43. PubMed ID: 37528429
Antipsychotic drugs are the mainstay of treatment for schizophrenia and provide adjunct therapies for other prevalent psychiatric conditions, including bipolar disorder and major depressive disorder. However, they also induce debilitating extrapyramidal syndromes (EPS), such as Parkinsonism, in a significant minority of patients. The majority of antipsychotic drugs function as dopamine receptor antagonists in the brain while the most recent 'third'-generation, such as aripiprazole, act as partial agonists. Despite showing good clinical efficacy, these newer agents are still associated with EPS in ~ 5 to 15% of patients. However, it is not fully understood how these movement disorders develop. This study combined clinically-relevant drug concentrations with mutliscale model systems to show that aripiprazole and its primary active metabolite induce mitochondrial toxicity inducing robust declines in cellular ATP and viability. Aripiprazole, brexpiprazole and cariprazine were shown to directly inhibit respiratory complex I through its ubiquinone-binding channel. Importantly, all three drugs induced mitochondrial toxicity in primary embryonic mouse neurons, with greater bioenergetic inhibition in ventral midbrain neurons than forebrain neurons. Finally, chronic feeding with aripiprazole resulted in structural damage to mitochondria in the brain and thoracic muscle of adult Drosophila melanogaster consistent with locomotor dysfunction. Taken together, this study showed show that antipsychotic drugs acting as partial dopamine receptor agonists exhibit off-target mitochondrial liabilities targeting complex I.
Hao, H., Song, L. and Zhang, L. (2023). Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis. PLoS Genet 19(7): e1010827. PubMed ID: 37399203
Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, this study aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. It was found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, a role is proposed for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations.
Ham, S. J., Yoo, H., Woo, D., Lee, D. H., Park, K. S. and Chung, J. (2023). PINK1 and Parkin regulate IP(3)R-mediated ER calcium release. Nat Commun 14(1): 5202. PubMed ID: 37626046
Although defects in intracellular calcium homeostasis are known to play a role in the pathogenesis of Parkinson's disease (PD), the underlying molecular mechanisms remain unclear. This study shows that loss of PTEN-induced kinase 1 (PINK1) and Parkin leads to dysregulation of inositol 1,4,5-trisphosphate receptor (IP(3)R) activity, robustly increasing ER calcium release. In addition, CDGSH iron sulfur domain 1 (CISD1, also known as mitoNEET) functions were identifed downstream of Parkin to directly control IP(3)R. Both genetic and pharmacologic suppression of CISD1 and its Drosophila homolog CISD (also known as Dosmit) restore the increased ER calcium release in PINK1 and Parkin null mammalian cells and flies, respectively, demonstrating the evolutionarily conserved regulatory mechanism of intracellular calcium homeostasis by the PINK1-Parkin pathway. More importantly, suppression of CISD in PINK1 and Parkin null flies rescues PD-related phenotypes including defective locomotor activity and dopaminergic neuronal degeneration. Based on these data, it is proposed that the regulation of ER calcium release by PINK1 and Parkin through CISD1 and IP(3)R is a feasible target for treating PD pathogenesis.
Guichard, A., Lu, S., Kanca, O., Bressan, D., Huang, Y., Ma, M., Sanz Juste, S., Andrews, J. C., Jay, K. L., Sneider, M., Schwartz, R., Huang, M. C., Bei, D., Pan, H., Ma, L., Lin, W. W., Auradkar, A., Bhagwat, P., Park, S., Wan, K. H., Ohsako, T., Takano-Shimizu, T., Celniker, S. E., Wangler, M. F., Yamamoto, S., Bellen, H. J. and Bier, E. (2023). A comprehensive Drosophila resource to identify key functional interactions between SARS-CoV-2 factors and host proteins. Cell Rep 42(8): 112842. PubMed ID: 37480566
Development of effective therapies against SARS-CoV-2 infections relies on mechanistic knowledge of virus-host interface. Abundant physical interactions between viral and host proteins have been identified, but few have been functionally characterized. Harnessing the power of fly genetics, this study developed a comprehensive Drosophila COVID-19 resource (DCR) consisting of publicly available strains for conditional tissue-specific expression of all SARS-CoV-2 encoded proteins, UAS-human cDNA transgenic lines encoding established host-viral interacting factors, and GAL4 insertion lines disrupting fly homologs of SARS-CoV-2 human interacting proteins. The utility was demonstrated of the DCR to functionally assess SARS-CoV-2 genes and candidate human binding partners. NSP8 was shown to engage in strong genetic interactions with several human candidates, most prominently with the ATE1 arginyltransferase to induce actin arginylation and cytoskeletal disorganization, and that two ATE1 inhibitors can reverse NSP8 phenotypes. The DCR enables parallel global-scale functional analysis of SARS-CoV-2 components in a prime genetic model system.
Gomes, K. K., Dos Santos, A. B., Dos Anjos, J. S., Leandro, L. P., Mariano, M. T., Pinheiro, F. L., Farina, M., Franco, J. L. and Posser, T. (2023). Increased Iron Levels and Oxidative Stress Mediate Age-Related Impairments in Male and Female Drosophila melanogaster. Oxid Med Cell Longev 2023: 7222462. PubMed ID: 37333463
Aging is characterized by a functional decline in the physiological functions and organic systems, causing frailty, illness, and death. Ferroptosis is an iron- (Fe-) dependent regulated cell death, which has been implicated in the pathogenesis of several disorders, such as cardiovascular and neurological diseases. The present study investigated behavioral and oxidative stress parameters over the aging of Drosophila melanogaster that, together with augmented Fe levels, indicate the occurrence of ferroptosis. This work demonstrated that older flies (30-day-old) of both sexes presented impaired locomotion and balance when compared with younger flies (5-day-old). Older flies also produced higher reactive oxygen species (ROS) levels, decreased glutathione levels (GSH), and increased lipid peroxidation. In parallel, Fe levels were augmented in the fly's hemolymph. The GSH depletion with diethyl maleate potentiated the behavioral damage associated with age. These data demonstrated biochemical effects that characterize the occurrence of ferroptosis over the age of D. melanogaster and reports the involvement of GSH in the age-associated damages, which could be in part attributed to the augmented levels of Fe.

Friday, October 13th - Immune Response

Cai, H., Li, L., Slavik, K., Huang, J., Yin, T., Hedelin, L., Xiang, Z., Yang, Y., Li, X., Chen, Y., Wei, Z., Deng, H., Chen, D., Jiao, R., Martins, N., Meignin, C., Kranzusch, P. and Imler, J. L. (2023). A novel virus-induced cyclic dinucleotide, 2'3'-c-di-GMP, mediates STING-dependent antiviral immunity in Drosophila. bioRxiv. PubMed ID: 37214844
In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP. This CDN binds to and activates the protein STING to trigger immunity. Two cGAS-like receptors (cGLRs) have been discovered that activate STING-dependent antiviral immunity and can produce 3'2'-cGAMP, in addition to 2'3'-cGAMP. This study explore dCDN-mediated immunity in 14 different Drosophila species covering 50 million years of evolution and reports that 2'3'-cGAMP and 3'2'-cGAMP fail to control infection by Drosophila C virus in D. serrata, D. sechellia and D. mojavensis. Using an accurate and sensitive mass spectrometry method, an unexpected diversity was discovered of CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including a novel CDN, 2'3'-c-di-GMP. 2'3'-c-di-GMP was shown to be the most potent STING agonist identified so far in D. melanogaster, and this molecule also activates a strong antiviral transcriptional response in D. serrata. These results shed light on the evolution of cGLRs in flies and provide a basis for the understanding of the function and regulation of this emerging family of PRRs in animal innate immunity.
Aida, A., Yuswan, K., Kawai, Y., Hasegawa, K., Nakajima, Y. I. and Kuranaga, E. (2023). Drosophila innate immunity suppresses the survival of xenografted mammalian tumor cells. Sci Rep 13(1): 12334. PubMed ID: 37518191
Patient-derived xenograft (PDX) is an emerging tool established in immunodeficient vertebrate models to assess individualized treatments for cancer patients. Current xenograft models are deficient in adaptive immune systems. However, the precise role of the innate immunity in the xenograft models is unknown. With conserved signaling pathways and established genetic tools, Drosophila has contributed to the understanding of the mechanism of tumor growth as well as tumor-host interactions for decades, making it a promising candidate model for studying whether or not the hosts' innate immunity can accommodate transplanted human tumor cells. This study shows initial observations that assess the behavior and impact of several human tumor cell lines when transplanted into Drosophila. Some injected cell lines persisted for a longer duration and reduced hosts' lifespan. In particular, the human lung cancer cell line A549 were observed adjacent to the fly host tissues. Two factors were examined that affect the survivability of cancer cells: (1) the optimal temperature of each cell line and (2) the innate immunity of Drosophila hosts. Especially, transplanted human tumor cells survived longer in immunodeficient flies, suggesting that the host innate immune system impedes the growth of xenografted cells. These attempts for xenografting fly models thus provide necessary steps to overcome for establishing PDX cancer models using invertebrates.
Arunkumar, R., Zhou, S. O., Day, J. P., Bakare, S., Pitton, S., Zhang, Y., Hsing, C. Y., O'Boyle, S., Pascual-Gil, J., Clark, B., Chandler, R. J., Leitao, A. B. and Jiggins, F. M. (2023). Natural selection has driven the recurrent loss of an immunity gene that protects Drosophila against a major natural parasite. Proc Natl Acad Sci U S A 120(33): e2211019120. PubMed ID: 37552757
Polymorphisms in immunity genes can have large effects on susceptibility to infection. To understand the origins of this variation, this study has investigated the genetic basis of resistance to the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster. Increased expression of the gene lectin-24A after infection by parasitic wasps was associated with a faster cellular immune response and greatly increased rates of killing the parasite. lectin-24A encodes a protein that is strongly up-regulated in the fat body after infection and localizes to the surface of the parasite egg. In certain susceptible lines, a deletion upstream of the lectin-24A has largely abolished expression. Other mutations predicted to abolish the function of this gene have arisen recurrently in this gene, with multiple loss-of-expression alleles and premature stop codons segregating in natural populations. The frequency of these alleles varies greatly geographically, and in some southern African populations, natural selection has driven them near to fixation. It is concluded that natural selection has favored the repeated loss of an important component of the immune system, suggesting that in some populations, a pleiotropic cost to lectin-24A expression outweighs the benefits of resistance.
Li, Y., Slavik, K. M., Morehouse, B. R., de Oliveira Mann, C. C., Mears, K., Liu, J., Kashin, D., Schwede, F. and Kranzusch, P. J. (2023). cGLRs are a diverse family of pattern recognition receptors in animal innate immunity. bioRxiv. PubMed ID: 36865129
cGAS (cyclic GMP-AMP synthase; CG7194 in Drosophila) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates the protein STING and downstream immunity. This study discovered cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in animal innate immunity. Building on recent analysis in Drosophila, a bioinformatic approach was used to identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screen of 140 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of alternative nucleotide signals including isomers of cGAMP and cUMP-AMP. Using structural biology, this study explains how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Together these results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.
Li, Q., Zhang, C., Zhang, C., Duan, R. and Hua, Y. (2023). CG4968 positively regulates the immune deficiency pathway by targeting Imd protein in Drosophila. PeerJ 11: e14870. PubMed ID: 36778143
Drosophila melanogaster relies solely on innate immunity to defend against various microbial pathogens. Although it is well-known that the adaptor protein Imd undergoes K63-linked ubiquitination to activate the downstream signaling cascades, its involvement with K48-linked ubiquitination and what is responsible for controlling this modification remain largely unknown. This study explored the immunological function of CG4968, which encodes a typical ovarian tumour-associated protease (OTU)-type deubiquitinase (Dub) in flies. In vitro and vivo evidence demonstrated that CG4968 plays a positive role in governing the immune deficiency (IMD), but not the Toll innate immune response in an OTU domain-dependent manner. Mechanistically, it was found that CG4968 is associated with Imd to restrict its K48-linked ubiquitination, thereby contributing to its turnover. Collectively, this study uncovered a novel regulatory mechanism involving the K48-linked ubiquitination of Imd in Drosophila innate immunity.
Hanson, M. A., Grollmus, L. and Lemaitre, B. (2023). Ecology-relevant bacteria drive the evolution of host antimicrobial peptides in Drosophila. Science 381(6655): eadg5725. PubMed ID: 37471548
Antimicrobial peptides are host-encoded immune effectors that combat pathogens and shape the microbiome in plants and animals. However, little is known about how the host antimicrobial peptide repertoire is adapted to its microbiome. This study characterized the function and evolution of the Diptericin antimicrobial peptide family of Diptera. Using mutations affecting the two Diptericins (Dpt) of Drosophila melanogaster, the specific role of DptA for the pathogen Providencia rettgeri and DptB for the gut mutualist Acetobacter. The presence of DptA- or DptB-like genes across Diptera correlates with the presence of Providencia and Acetobacter in their environment. Moreover, DptA- and DptB-like sequences predict host resistance against infection by these bacteria across the genus Drosophila. This study explains the evolutionary logic behind the bursts of rapid evolution of an antimicrobial peptide family and reveals how the host immune repertoire adapts to changing microbial environments.

Thursday, October 12th - Stress Response

Weingartner, K. A., Tran, T., Tripp, K. W. and Kavran, J. M. (2023). Dimerization and autophosphorylation of the MST family of kinases are controlled by the same set of residues. bioRxiv. PubMed ID: 36945437
The Hippo pathway controls tissue growth and regulates stem cell fate through the activities of core kinase cassette that begins with the Sterile 20-like kinase MST1/2. Activation of MST1/2 relies on trans -autophosphorylation but the details of the mechanisms regulating that reaction are not fully elucidated. Proposals include dimerization as a first step and include multiple models for potential kinase-domain dimers. Efforts to verify and link these dimers to trans -autophosphorylation were unsuccessful. This study explored the link between dimerization and trans -autophosphorylation for MST2 and the entire family of MST kinases. Crystal lattice contacts were analyzed of structures of MST kinases and identified an ensemble of kinase-domain dimers compatible with trans -autophosphorylation. These dimers share a common dimerization interface comprised of the activation loop and αG-helix while the arrangements of the kinase-domains within the dimer varied depending on their activation state. Then the dimerization interface was verified, and its function was determined using MST2. Variants bearing alanine substitutions of the αG-helix prevented dimerization of the MST2 kinase domain both in solution and in cells. These substitutions also blocked autophosphorylation of full-length MST2 and its Drosophila homolog Hippo in cells. These variants retain the same secondary structure as wild-type and capacity to phosphorylate a protein substrate, indicating the loss of MST2 activation can be directly attributed to a loss of dimerization rather than loss of either fold or catalytic function. Together this data functionally links dimerization and autophosphorylation for MST2 and suggests this activation mechanism is conserved across both species and the entire MST family.
Bii, V. M., Rudoy, D., Klezovitch, O. and Vasioukhin, V. (2023P. Lethal giant larvae gene family (Llgl1 and Llgl2) functions as a tumor suppressor in mouse skin epidermis. bioRxiv. PubMed ID: 36945368
Loss of cell polarity and tissue disorganization occurs in majority of epithelial cancers. Studies in simple model organisms identified molecular mechanisms responsible for the establishment and maintenance of cellular polarity, which play a pivotal role in establishing proper tissue architecture. The exact role of these cell polarity pathways in mammalian cancer is not completely understood. This study analyzed the mammalian orthologs of drosophila apical-basal polarity gene lethal giant larvae (lgl), which regulates asymmetric stem cell division and functions as a tumor suppressor in flies. There are two mammalian orthologs of lgl (Llgl1 and Llgl2). To determine the role of the entire lgl signaling pathway in mammals this study generated mice with ablation of both Llgl1 and Llgl2 in skin epidermis using K14-Cre (Llgl1/2 (-/-) cKO mice). Surprisingly, it was found that ablation of Llgl1/2 genes does not impact epidermal polarity in adult mice. However, old Llgl1/2 cKO mice present with focal skin lesions which are missing epidermal layer and ripe with inflammation. To determine the role of lgl signaling pathway in cancer Trp53 (-/-)/Llgl1/2 (-/-) cKO and Trp53 (-/+) /Llgl1/2 (-/-) cKO mice were generated. Loss of Llgl1/2 promoted squamous cell carcinoma (SCC) development in Trp53 (-/-) cKO and caused SCC in Trp53 (-/+) cKO mice, while no cancer was observed in Trp53 (-/+) cKO controls. Mechanistically, it was shown that ablation of Llgl1/2 causes activation of aPKC and upregulation of NF-kB signaling pathway, which may be necessary for SCC in Trp53 (-/+) /Llgl1/2 (-/-) cKO mice. It is concluded that Lgl signaling pathway functions as a tumor suppressor in mammalian skin epidermis.
Maschi, D., Fernandez-Alvarez, A. J. and Boccaccio, G. L. (2023). The RNA-binding protein NANOS1 controls hippocampal synaptogenesis. PLoS One 18(4): e0284589. PubMed ID: 37058523
Proteins from the NANOS family are conserved translational repressors with a well-known role in gonad development in both vertebrates and invertebrates. In addition, Drosophila Nanos controls neuron maturation and function, and rodent Nanos1 affects cortical neuron differentiation. This study showed that rat Nanos1 is expressed in hippocampal neurons and that the siRNA-mediated knockdown of Nanos1 impairs synaptogenesis. This study found that both dendritic spine size and number were affected by Nanos1 KD. Dendritic spines were smaller and more numerous. Moreover, whereas in control neurons most dendritic PSD95 clusters contact pre-synaptic structures, a larger proportion of PSD95 clusters lacked a synapsin counterpart upon Nanos1 loss-of-function. Finally, Nanos1 KD impaired the induction of ARC typically triggered by neuron depolarization. These results expand knowledge on the role of NANOS1 in CNS development and suggest that RNA regulation by NANOS1 governs hippocampal synaptogenesis.
Thoma, V., Sakai, S., Nagata, K., Ishii, Y., Maruyama, S., Abe, A., Kondo, S., Kawata, M., Hamada, S., Deguchi, R. and Tanimoto, H. (2023). On the origin of appetite: GLWamide in jellyfish represents an ancestral satiety neuropeptide. Proc Natl Acad Sci U S A 120(15): e2221493120.. PubMed ID: 37011192
Food intake is regulated by internal state. This function is mediated by hormones and neuropeptides, which are best characterized in popular model species. However, the evolutionary origins of such feeding-regulating neuropeptides are poorly understood. The jellyfish Cladonema was used to address this question. Combined transcriptomic, behavioral, and anatomical approaches identified GLWamide as a feeding-suppressing peptide that selectively inhibits tentacle contraction in this jellyfish. In the fruit fly Drosophila, myoinhibitory peptide (MIP) is a related satiety peptide. Surprisingly, it was found that GLWamide and MIP were fully interchangeable in these evolutionarily distant species for feeding suppression. These results suggest that the satiety signaling systems of diverse animals share an ancient origin.
Jin, Z., Yu, B. and Huang, Y. (2023). Structural insights into the chromodomain of Oxpecker in complex with histone H3 lysine 9 trimethylation reveal a transposon silencing mechanism by heterodimerization. Biochem Biophys Res Commun 652: 95-102. PubMed ID: 36841100
Oxpecker, the homolog of Rhino/HP1D, exclusively expressed in Drosophila ovaries, belongs to the Heterochromatin Protein 1 family, as does Rhino. Rhi recognizes piRNA clusters enriched with the heterochromatin marker H3K9me3 via its N-terminal chromodomain and recruits Deadlock via its C-terminal chromoshadow domain, further recruits Moonshiner, a paralog of the TATA box-binding protein-related factor 2 large subunits, to promote transcription of piRNA precursors, thereby protecting the genome. Despite Oxp possessing only the chromodomain, its loss leads to the upregulation of transposons in the female germline. This study ∂solved the crystal structure of the Oxp chromodomain in complex with the histone H3K9me3 peptide. As the Oxp chromodomain dimerizes, two H3K9me3 peptides bind to the Oxp chromodomain in an antiparallel manner. ITC experiments and site-directed mutagenesis experiments showed that E44 determines Oxp's five-fold stronger binding ability to H3K9me3 than that of Rhi. In addition, it was found that Oxp and Rhi can form a heterodimer, which may shed light on the molecular mechanism by which Oxp regulates transposon silencing in the absence of CSD.
Fixsen, B. R., Han, C. Z., Zhou, Y., Spann, N. J., Saisan, P., Shen, Z., Balak, C., Sakai, M., Cobo, I., Holtman, I. R., Warden, A. S., Ramirez, G., Collier, J. G., Pasillas, M. P., Yu, M., Hu, R., Li, B., Belhocine, S., Gosselin, D., Coufal, N. G., Ren, B. and Glass, C. K. (2023). SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity. Nat Immunol 24(7): 1188-1199. PubMed ID: 37322178
Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. This study demonstrates that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, evidence is provided for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFβ and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFβ-SMAD signaling axis.

Monday, October 9th - Larval and Adult Development

Casal, J., Storer, F. and Lawrence, P. A. (2023). . Planar cell polarity: intracellular asymmetry and supracellular gradients of Frizzled, Open Biol 13(6): 230105. PubMed ID: 37311537

Planar cell polarity (PCP), the coordinated orientation of structures such as cilia, mammalian hairs or insect bristles, depends on at least two molecular systems. It has been argued that these two systems use similar mechanisms; each depending on a supracellular gradient of concentration that spans a field of cells. In a linked paper, the Dachsous/Fat system was analyzed. A graded distribution of Dachsous was found in vivo in a segment of the pupal epidermis in the abdomen of Drosophila. This study report a similar study of the key molecule for the Starry Night/Frizzled or 'core' system. The distribution was measured of the receptor Frizzled on the cell membranes of all cells of one segment in the living pupal abdomen of Drosophila. A supracellular gradient was found that falls about 17% in concentration from the front to the rear of the segment. Some evidence is presented that the gradient then resets in the most anterior cells of the next segment back. An intracellular asymmetry was found in all the cells, the posterior membrane of each cell carrying about 22% more Frizzled than the anterior membrane. These direct molecular measurements add to earlier evidence that the two systems of PCP act independently.

Olenik, M., Turley, J., Cross, S., Weavers, H., Martin, P., Chenchiah, I. V. and Liverpool, T. B. (2023). Fluctuations of cell geometry and their nonequilibrium thermodynamics in living epithelial tissue. Phys Rev E 107(1-1): 014403. PubMed ID: 36797912
This study measured different contributions to entropy production in a living functional epithelial tissue. This was done by extracting the functional dynamics of development while at the same time quantifying fluctuations. Using the translucent Drosophila melanogaster pupal epithelium as an ideal tissue for high-resolution live imaging, the entropy was measured associated with the stochastic geometry of cells in the epithelium. This was done using a detailed analysis of the dynamics of the shape and orientation of individual cells which enables separation of local and global aspects of the tissue behavior. Intriguingly, it was possible to observe irreversible dynamics in the cell geometries but without a change in the entropy associated with those degrees of freedom, showing that there is a flow of energy into those degrees of freedom. Hence, the living system is controlling how the entropy is being produced and partitioned into its different parts.
Palmer, M. J. and Fitzsimons, H. L. (2023). Herzog is not required for mushroom body development or courtship learning & memory but is required for eye development in Drosophila melanogaster MicroPubl Biol 2023. PubMed ID: 36798589
Herzog (Hzg, CG5830) shares similarity to members of the haloacid dehalogenase subfamily of small CTD phosphatases. In Drosophila it is a maternal gene essential for establishment of embryonic segment polarity, and oligomerization is required for activation of phosphatase activity. While Hzg is expressed in the brain, its role has not been investigated. To that end, this study further characterized Hzg expression in the brain, and it was found to be highly expressed in neurons of the mushroom body where it localises to axons and is also expressed in cortical glia. This study investigated its role in mushroom body development as well as courtship learning and memory, but found that knockdown of Hzg had no impact on these processes. In contrast, knockdown in post-mitotic neurons in the eye resulted in disruption to ommatidial patterning and pigmentation, indicating it plays an important role in eye development.
Shimell, M. and O'Connor, M. B. (2023). Endoreplication in the Drosophila melanogaster prothoracic gland is dispensable for the critical weight checkpoint. MicroPubl Biol 2023. PubMed ID: 36908310
Critical weight (CW) attainment is a key life event in the development of holometabolous insects including Drosophila melanogaster. It indicates that sufficient growth has occurred to initiate the juvenile-to-adult transition. The prothoracic gland (PG), the major insect larval endocrine organ, is a polyploid tissue that plays a key role in the determination of CW via release of the steroid hormone ecdysone. This study showed that when the cells of the PG fail to make the mitotic-to-endocycle switch, but instead remain mitotic, the result is more but smaller cells. Nevertheless, they reach the same CW and produce healthy adults after only a moderate developmental delay. It is proposed that the CW checkpoint can be reached by either an endocycling or mitotic PG and may simply reflect the attainment of sufficient ecdysone biosynthetic capacity to initiate metamorphosis.
Adelmann, J. A., Vetter, R. and Iber, D. (2023). The impact of cell size on morphogen gradient precision. Development 150(10). PubMed ID: 37249125

Tissue patterning during embryonic development is remarkably precise. This study numerically determine the impact of the cell diameter, gradient length and the morphogen source on the variability of morphogen gradients. It was shown that the positional error increases with the gradient length relative to the size of the morphogen source, and with the square root of the cell diameter and the readout position. Theoretical explanations are provided for these relationships, and it was shown that they enable high patterning precision over developmental time for readouts that scale with expanding tissue domains, as observed in the Drosophila wing disc. This analysis suggests that epithelial tissues generally achieve higher patterning precision with small cross-sectional cell areas. An extensive survey of measured apical cell areas shows that they are indeed small in developing tissues that are patterned by morphogen gradients. Enhanced precision may thus have led to the emergence of pseudostratification in epithelia, a phenomenon for which the evolutionary benefit had so far remained elusive.

Dong, W., Li, T., Wu, W. J. and Zhang, X. B. (2023). Cell lineage analysis reveals signal tracing and compartment characterisation in Drosophila haltere. Insect Mol Biol. PubMed ID: 37212394

Insect halteres, as specialised hind wings, play an important role during aerial manoeuvres. In Drosophila, halteres and wings are homologous appendages with different morphology. Previous studies have focused on the metamorphosis of halteres, while current knowledge about its cell lineage and regional compartmentalization is still limited. In this study cell-lineage tracing was performed of canonical landmark signals in halteres and present a simple model for haltere development. Cell lineage tracing in wings was used as a reference. nub showed wing-like expressions in halteres, whereas hth and pnr exhibited different expressions in adult wings and halteres. The lineage tracing revealed that the pouch region gives rise to end-bulb, and hinge cells contribute to proximal haltere formation. Moreover, it was demonstrated that twi-expressing cells participate in the cell population of the distal end-bulb. Haematoxylin and eosin staining indicated that muscle cells were present at the distal end-bulb. These results indicated that adult halteres displayed unique cell lineage patterns and the muscle cells are important components of end-bulbs.

Friday, October 6th- Neural function adult

Ferreira, A. A. G. and Desplan, C. (2023). An Atlas of the Developing Drosophila Visual System Glia and Subcellular mRNA Localization of Transcripts in Single Cells. bioRxiv. PubMed ID: 37609218
Glial cells are essential for proper nervous system development and function. To understand glial development and function, glial cells were comprehensively annotated in a single-cell mRNA-sequencing (scRNAseq) atlas of the developing Drosophila visual system. This allowed a study of their developmental trajectories, from larval to adult stages, and to understand how specific types of glia diversify during development. For example, neuropil glia that are initially transcriptionally similar in larvae, split into ensheathing and astrocyte-like glia during pupal stages. Other glial types, such as chiasm glia change gradually during development without splitting into two cell types. The analysis of scRNA-seq led to the discovery that the transcriptome of glial cell bodies can be distinguished from that of their broken processes. The processes contain distinct enriched mRNAs that were validated in vivo. Therefore, this study has identified most glial types in the developing optic lobe and devised a computational approach to identify mRNA species that are localized to cell bodies or cellular processes.
Dong, H., Yang, X., Wu, L., Zhang, S., Zhang, J., Guo, P., Du, Y., Pan, C., Fu, Y., Li, L., Shi, J., Zhu, Y., Ma, H., Bian, L., Xu, B., Li, G., Shi, F., Huang, J., He, H. and Jin, Y. (2023). A systematic CRISPR screen reveals redundant and specific roles for Dscam1 isoform diversity in neuronal wiring. PLoS Biol 21(7): e3002197. PubMed ID: 37410725
Drosophila melanogaster Down syndrome cell adhesion molecule 1 (Dscam1) encodes 19,008 diverse ectodomain isoforms via the alternative splicing of exon 4, 6, and 9 clusters. However, whether individual isoforms or exon clusters have specific significance is unclear. Using phenotype-diversity correlation analysis, this study reveal the redundant and specific roles of Dscam1 diversity in neuronal wiring. A series of deletion mutations were performed from the endogenous locus harboring exon 4, 6, or 9 clusters, reducing to 396 to 18,612 potential ectodomain isoforms. Of the 3 types of neurons assessed, dendrite self/non-self discrimination required a minimum number of isoforms (approximately 2,000), independent of exon clusters or isoforms. In contrast, normal axon patterning in the mushroom body and mechanosensory neurons requires many more isoforms that tend to associate with specific exon clusters or isoforms. It is concluded that the role of the Dscam1 diversity in dendrite self/non-self discrimination is nonspecifically mediated by its isoform diversity. In contrast, a separate role requires variable domain- or isoform-related functions and is essential for other neurodevelopmental contexts, such as axonal growth and branching. These findings shed new light on a general principle for the role of Dscam1 diversity in neuronal wiring.
Duan, W., Zhang, Y., Zhang, X., Yang, J., Shan, H., Liu, L. and Wei, H. (2023). A Visual Pathway into Central Complex for High-Frequency Motion-Defined Bars in Drosophila. J Neurosci 43(26): 4821-4836. PubMed ID: 37290936
Relative motion breaks a camouflaged target from a same-textured background, thus eliciting discrimination of a motion-defined object. Ring (R) neurons are critical components in the Drosophila central complex, which has been implicated in multiple visually guided behaviors. Using two-photon calcium imaging with female flies, this study demonstrated that a specific population of R neurons that innervate the superior domain of bulb neuropil, termed superior R neurons, encoded a motion-defined bar with high spatial frequency contents. Upstream superior tuberculo-bulbar (TuBu) neurons transmitted visual signals by releasing acetylcholine within synapses connected with superior R neurons. Blocking TuBu or R neurons impaired tracking performance of the bar, which reveals their importance in motion-defined feature encoding. Additionally, the presentation of a low spatial frequency luminance-defined bar evoked consistent excitation in R neurons of the superior bulb, whereas either excited or inhibited responses were evoked in the inferior bulb. The distinct properties of the responses to the two bar stimuli indicate there is a functional division between the bulb subdomains. Moreover, physiological and behavioral tests with restricted lines suggest that R4d neurons play a vital role in tracking motion-defined bars. It is concluded that the central complex receives the motion-defined features via a visual pathway from superior TuBu to R neurons and might encode different visual features via distinct response patterns at the population level, thereby driving visually guided behaviors.
Ehrhardt, E., Whitehead, S. C., Namiki, S., Minegishi, R., Siwanowicz, I., Feng, K., Otsuna, H., Meissner, G. W., Stern, D., Truman, J., Shepherd, D., Dickinson, M. H., Ito, K., Dickson, B. J., Cohen, I., Card, G. M. and Korff, W. (2023). Single-cell type analysis of wing premotor circuits in the ventral nerve cord of Drosophila melanogaster. bioRxiv. PubMed ID: 37398009
To perform most behaviors, animals must send commands from higher-order processing centers in the brain to premotor circuits that reside in ganglia distinct from the brain, such as the mammalian spinal cord or insect ventral nerve cord. How these circuits are functionally organized to generate the great diversity of animal behavior remains unclear. An important first step in unraveling the organization of premotor circuits is to identify their constituent cell types and create tools to monitor and manipulate these with high specificity to assess their function. This is possible in the tractable ventral nerve cord of the fly. To generate such a toolkit, a combinatorial genetic technique (split-GAL4) was used to create 195 sparse driver lines targeting 198 individual cell types in the ventral nerve cord. These included wing and haltere motoneurons, modulatory neurons, and interneurons. Using a combination of behavioral, developmental, and anatomical analyses, the cell types targeted in this collection was systematically characterized. Taken together, the resources and results presented in this study form a powerful toolkit for future investigations of neural circuits and connectivity of premotor circuits while linking them to behavioral outputs.
Gendron, C. M., Chakraborty, T. S., Duran, C., Dono, T. and Pletcher, S. D. (2023). Ring neurons in the Drosophila central complex act as a rheostat for sensory modulation of aging. PLoS Biol 21(6): e3002149. PubMed ID: 37310911
Sensory perception modulates aging, yet little is known about how. An understanding of the neuronal mechanisms through which animals orchestrate biological responses to relevant sensory inputs would provide insight into the control systems that may be important for modulating lifespan. This study provides new awareness into how the perception of dead conspecifics, or death perception, which elicits behavioral and physiological effects in many different species, affects lifespan in the fruit fly, Drosophila melanogaster. Previous work demonstrated that cohousing Drosophila with dead conspecifics decreases fat stores, reduces starvation resistance, and accelerates aging in a manner that requires both sight and the serotonin receptor 5-HT2A. This study demonstrated that a discrete, 5-HT2A-expressing neural population in the ellipsoid body (EB) of the Drosophila central complex, identified as R2/R4 neurons, acts as a rheostat and plays an important role in transducing sensory information about the presence of dead individuals to modulate lifespan. Expression of the insulin-responsive transcription factor foxo in R2/R4 neurons and insulin-like peptides dilp3 and dilp5, but not dilp2, are required, with the latter likely altered in median neurosecretory cells (MNCs) after R2/R4 neuronal activation. These data generate new insights into the neural underpinnings of how perceptive events may impact aging and physiology across taxa.

Gugel, Z. V., Maurais, E. G. and Hong, E. J. (2023). Chronic exposure to odors at naturally occurring concentrations triggers limited plasticity in early stages of Drosophila olfactory processing. Elife 12. PubMed ID: 37195027
In insects and mammals, olfactory experience in early life alters olfactory behavior and function in later life. In the vinegar fly Drosophila, flies chronically exposed to a high concentration of a monomolecular odor exhibit reduced behavioral aversion to the familiar odor when it is reencountered. This change in olfactory behavior has been attributed to selective decreases in the sensitivity of second-order olfactory projection neurons (PNs) in the antennal lobe that respond to the overrepresented odor. However, since odorant compounds do not occur at similarly high concentrations in natural sources, the role of odor experience-dependent plasticity in natural environments is unclear. This study investigated olfactory plasticity in the antennal lobe of flies chronically exposed to odors at concentrations that are typically encountered in natural odor sources. These stimuli were chosen to each strongly and selectively excite a single class of primary olfactory receptor neuron (ORN), thus facilitating a rigorous assessment of the selectivity of olfactory plasticity for PNs directly excited by overrepresented stimuli. Unexpectedly, it was found that chronic exposure to three such odors did not result in decreased PN sensitivity but rather mildly increased responses to weak stimuli in most PN types. Odor-evoked PN activity in response to stronger stimuli was mostly unaffected by odor experience. When present, plasticity was observed broadly in multiple PN types and thus was not selective for PNs receiving direct input from the chronically active ORNs. The DL5 olfactory coding channel was further investigated and it was found that chronic odor-mediated excitation of its input ORNs did not affect PN intrinsic properties, local inhibitory innervation, ORN responses or ORN-PN synaptic strength; however, broad-acting lateral excitation evoked by some odors was increased. These results show that PN odor coding is only mildly affected by strong persistent activation of a single olfactory input, highlighting the stability of early stages of insect olfactory processing to significant perturbations in the sensory environment.

Thursday October 5th - Behavior

Cho, H. and Rohlfs, M. (2023). Transmission of beneficial yeasts accompanies offspring production in Drosophila-An initial evolutionary stage of insect maternal care through manipulation of microbial load? Ecol Evol 13(6): e10184. PubMed ID: 37332518
Parent-to-offspring transmission of beneficial microorganisms is intimately interwoven with the evolution of social behaviors. Ancestral stages of complex sociality-microbe vectoring interrelationships may be characterized by high costs of intensive parental care and hence only a weak link between the transmission of microbial symbionts and offspring production. This study investigate the relationship between yeast symbiont transmission and egg-laying, as well as some general factors thought to drive the "farming" of microscopic fungi by the fruit fly Drosophila melanogaster, an insect with no obvious parental care but which is highly dependent on dietary microbes during offspring development. The process of transmitting microbes involves flies ingesting microbes from their previous environment, storing and vectoring them, and finally depositing them to a new environment. This study revealed that fecal materials of adult flies play a significant role in this process, as they contain viable yeast cells that support larval development. During single patch visits, egg-laying female flies transmitted more yeast cells than non-egg-laying females, suggesting that dietary symbiont transmission is not random, but linked to offspring production. The crop, an extension of the foregut, was identified as an organ capable of storing viable yeast cells during travel between egg-laying sites. However, the amount of yeast in the crop reduced rapidly during periods of starvation. Although females starved for 24 h deposited a smaller amount of yeast than those starved for 6 h, the yeast inoculum produced still promoted the development of larval offspring. The results of these experiments suggest that female Drosophila fruit flies have the ability to store and regulate the transfer of microorganisms beneficial to their offspring via the shedding of fecal material. It is argue that this observation may represent an initial evolutionary stage of maternal care through the manipulation of microbial load, from which more specialized feedbacks of sociality and microbe management may evolve.
Belkina, E. G., Seleznev, D. G., Sorokina, S. Y., Kulikov, A. M. and Lazebny, O. E. (2023). The Effect of Chromosomes on Courtship Behavior in Sibling Species of the Drosophila virilis Group. Insects 14(7). PubMed ID: 37504615
courtship behavior is not a typical quantitative trait that can be easily measured or quantified in both females and males, Each courtship element involves the participation of both female and male partners, making the genetic analysis of this behavior complex. This study employed a modified design to analysis of courtship behavior by introducing what is referred to as 'reference partners' during the testing of hybrid individuals from F1, F2, and backcrosses. These reference partners represented one of the parental species. This approach allowed categorization of all possible test combinations into four groups based on the reference partner's sex (female or male) and their constant genotype towards one of the parental species (D. virilis or D. americana). The genotype of the second partner in the within-group test combinations varied from completely conspecific to completely heterospecific, based on the parental chromosomal sets. To assess the contribution of partner genotypes to the variability of courtship-element parameters, structural equation modeling (SEM) was employed. SEM enabled estimation of the regression of the proportion of chromosomes of a specific species type on the value of each courtship-element parameter in partners with varying genotypes across different test combinations. The aim of the current study was to analyze the involvement of sex chromosomes and autosomes in the formation of courtship structure in D. virilis and D. americana. The genetic analysis was complemented by video recording and formalization of courtship-ritual elements. D. virilis was found to be more sensitive to mate stimuli compared to D. americana. The majority of species-specific parameters, such as latency and duration of courtship elements (e.g., male and female song, following, licking, and circling), were shown to be influenced by the D. virilis genotype. However, not all of these parameters significantly impact copulation success, with the male song, licking, and following being the most significant. In females, the female song was found to have a significant relationship only with copulation duration. The influence of the female genotype on the species-specific parameters of courtship elements is primarily related to autosomes, while the male genotype is associated with the X chromosomes. The study suggests that sexual selection primarily occurs through acoustic and chemoreceptor channels.
Brisevac, D., Prakash, C. and Kaiser, T. S. (2023). Genetic analysis of a phenotypic loss in the mechanosensory entrainment of a circalunar clock. PLoS Genet 19(6): e1010763. PubMed ID: 37347791
Genetic variants underlying traits that become either non-adaptive or selectively neutral are expected to have altered evolutionary trajectories. Uncovering genetic signatures associated with phenotypic loss presents the opportunity to discover the molecular basis for the phenotype in populations where it persists. Circalunar clocks were studied in populations of the marine midge Clunio marinus. The circalunar clock synchronizes development to the lunar phase, and it is set by moonlight and tidal cycles of mechanical agitation. Two out of ten studied populations have lost their sensitivity to mechanical agitation while preserving sensitivity to moonlight. Intriguingly, the F1 offspring of the two insensitive populations regained the sensitivity to mechanical entrainment, implying a genetically independent loss of the phenotype. By combining quantitative trait locus mapping and genome-wide screens, this study explored the genetics of this phenotypic loss. QTL analysis suggested an oligogenic origin with one prevalent additive locus in one of the strains. In addition, it confirmed a distinct genetic architecture in the two insensitive populations. Genomic screens further uncovered several candidate genes underlying QTL regions. The strongest signal under the most prominent QTL contains a duplicated STAT1 gene, which has a well-established role in development, and CG022363, an ortholog of the Drosophila melanogaster CG32100 gene, which plays a role in gravitaxis. These results support the notion that adaptive phenotypes have a complex genetic basis with mutations occurring at several loci. By dissecting the most prevalent signals, this study started to reveal the molecular machinery responsible for the entrainment of the circalunar clock.
Bassetto, M., Reichl, T., Kobylkov, D., Kattnig, D. R., Winklhofer, M., Hore, P. J. and Mouritsen, H. (2023). No evidence for magnetic field effects on the behaviour of Drosophila. Nature 620(7974): 595-599. PubMed ID: 37558871
Migratory songbirds have the remarkable ability to extract directional information from the Earth's magnetic field. The exact mechanism of this light-dependent magnetic compass sense, however, is not fully understood. The most promising hypothesis focuses on the quantum spin dynamics of transient radical pairs formed in cryptochrome proteins in the retina. Frustratingly, much of the supporting evidence for this theory is circumstantial, largely because of the extreme challenges posed by genetic modification of wild birds. Drosophila has therefore been recruited as a model organism, and several influential reports of cryptochrome-mediated magnetic field effects on fly behaviour have been widely interpreted as support for a radical pair-based mechanism in birds. This study reportd the results of an extensive study testing magnetic field effects on 97,658 flies moving in a two-arm maze and on 10,960 flies performing the spontaneous escape behaviour known as negative geotaxis. Under meticulously controlled conditions and with vast sample sizes, it was not possible to find evidence for magnetically sensitive behaviour in Drosophila. Moreover, after reassessment of the statistical approaches and sample sizes used in the studies that this study tried to replicate, it is suggested that many-if not all-of the original results were false positives. These findings therefore cast considerable doubt on the existence of magnetic sensing in Drosophila and thus strongly suggest that night-migratory songbirds remain the organism of choice for elucidating the mechanism of light-dependent magnetoreception.
Bi, Y., Zhang, X., Chang, X., Li, J., Xiao, S., Zhang, B., Dang, C., Sun, L., Yao, H., Fang, Q., Wang, F. and Ye, G. (2023). Olfactory behavioral responses of two Drosophila species and their pupal parasitoid to volatiles from bananas. Pest Manag Sci. PubMed ID: 37357260
Behavior-based manipulation is an essential part of Drosophila integrated pest management (IPM). This study investigated the olfactory behavior of two Drosophila species, as well as their pupal parasitoid, to volatiles from bananas, for the identification of effective compounds to control fly populations. The results showed that overripe bananas were most attractive to both flies, with a higher attraction index (AI) in D. melanogaster than in D. suzukii. The profiles of volatile organic compounds (VOCs) from bananas with three different ripening stages were compared, and six VOCs were selected for behavioral tests. D. suzukii showed significantly different responses to isoamyl alcohol with D. melanogaster. The effects of ethyl butyrate and ethyl isovalerate on the two flies were dose-dependent, with lower concentrations acting as repellent and higher concentrations acting as attractant. Isoamyl acetate, isoamyl butyrate and isoamyl isovalerate were attractive to both flies. The parasitoid wasp Pachycrepoidus vindemmiae was repelled by isoamyl alcohol and ethyl butyrate, and attracted by ethyl isovalerate, but showed no significant response to isoamyl acetate, isoamyl butyrate and isoamyl isovalerate at the tested concentration. This study indicated that the behavioral response of Drosophila to the odor of chemical compounds is dose-dependent. Isoamyl alcohol and isoamyl acetate at certain concentrations could be used as repellents, whereas ethyl isovalerate as an attractant in control strategy design for D. suzukii.
Duhart, J. M., Buchler, J. R., Inami, S., Kennedy, K. J., Jenny, B. P., Afonso, D. J. S. and Koh, K. (2023). Modulation and neural correlates of postmating sleep plasticity in Drosophila females. Curr Biol 33(13): 2702-2716.e2703. PubMed ID: 37352854
Sleep is essential, but animals may forgo sleep to engage in other critical behaviors, such as feeding and reproduction. Previous studies have shown that female flies exhibit decreased sleep after mating, but understanding of the process is limited. This study reports that postmating nighttime sleep loss is modulated by diet and sleep deprivation, demonstrating a complex interaction among sleep, reproduction, and diet. It was also found that female-specific pC1 neurons and sleep-promoting dorsal fan-shaped body (dFB) neurons are required for postmating sleep plasticity. Activating pC1 neurons leads to sleep suppression on standard fly culture media but has little sleep effect on sucrose-only food. Published connectome data suggest indirect, inhibitory connections among pC1 subtypes. Using calcium imaging, it was shown that activating the pC1e subtype inhibits dFB neurons. It is proposed that pC1 and dFB neurons integrate the mating status, food context, and sleep drive to modulate postmating sleep plasticity.

Wednesday, October 4th - Evolution

Doyle, D. A., Burian, F. N., Aharoni, B., Klinder, A. J., Menzel, M. M., Nifras, G. C. C., Shabazz-Henry, A. L., Palma, B. U., Hidalgo, G. A., Sottolano, C. J., Ortega, B. M. and Niepielko, M. G. (2023). Germ Granule Evolution Provides Mechanistic Insight into Drosophila Germline Development. Mol Biol Evol 40(8). PubMed ID: 37527522

The copackaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to posttranscriptionally regulate germline mRNAs. In Drosophila melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates containing multiple transcripts from the same gene. Nucleated by Oskar (Osk), homotypic clusters are generated through a stochastic seeding and self-recruitment process that requires the 3' untranslated region (UTR) of germ granule mRNAs. Interestingly, the 3' UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species and it was hypothesized that this diversity influences homotypic clustering. To test this hypothesis, the homotypic clustering of nos and polar granule component (pgc) was investigated in four Drosophila species and it was concluded that clustering is a conserved process used to enrich germ granule mRNAs. However, it was discovered germ granule phenotypes that included significant changes in the abundance of transcripts present in species' homotypic clusters, which also reflected diversity in the number of coalesced primordial germ cells within their embryonic gonads. By integrating biological data with computational modeling, it was found that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels and/or homotypic clustering efficacy. Furthermore, it was demonstrated how the nos 3' UTR from different species influences nos clustering, causing granules to have ∼70% less nos and increasing the presence of defective primordial germ cells. These results highlight the impact that evolution has on germ granules, which should provide broader insight into processes that modify compositions and activities of other classes of biomolecular condensate.

Dagilis, A. J. and Matute, D. R. (2023). The fitness of an introgressing haplotype changes over the course of divergence and depends on its size and genomic location. PLoS Biol 21(7): e3002185. PubMed ID: 37459351
The genomic era has made clear that introgression, or the movement of genetic material between species, is a common feature of evolution. Examples of both adaptive and deleterious introgression exist in a variety of systems. What is unclear is how the fitness of an introgressing haplotype changes as species diverge or as the size of the introgressing haplotype changes. In a simple model, this study showed that introgression may more easily occur into parts of the genome which have not diverged heavily from a common ancestor. The key insight is that alleles from a shared genetic background are likely to have positive epistatic interactions, increasing the fitness of a larger introgressing block. In regions of the genome where few existing substitutions are disrupted, this positive epistasis can be larger than incompatibilities with the recipient genome. Further, this study showed that early in the process of divergence, introgression of large haplotypes can be favored more than introgression of individual alleles. This model is consistent with observations of a positive relationship between recombination rate and introgression frequency across the genome; however, it generates several novel predictions. First, the model suggests that the relationship between recombination rate and introgression may not exist, or may be negative, in recently diverged species pairs. Furthermore, the model suggests that introgression that replaces existing derived variation will be more deleterious than introgression at sites carrying ancestral variants. These predictions are tested in an example of introgression in Drosophila melanogaster, with some support for both. Finally, the model provides a potential alternative explanation to asymmetry in the direction of introgression, with expectations of higher introgression from rapidly diverged populations into slowly evolving ones.

Charlesworth, B. (2023). The effects of inversion polymorphisms on patterns of neutral genetic diversity. Genetics 224(4). PubMed ID: 37348059
The strong reduction in the frequency of recombination in heterozygotes for an inversion and a standard gene arrangement causes the arrangements to become partially isolated genetically, resulting in sequence divergence between them and changes in the levels of neutral variability at nucleotide sites within each arrangement class. Previous theoretical studies on the effects of inversions on neutral variability have assumed either that the population is panmictic or that it is divided into 2 populations subject to divergent selection. In this study, the theory is extended to a model of an arbitrary number of demes connected by migration, using a finite island model with the inversion present at the same frequency in all demes. Recursion relations for mean pairwise coalescent times are used to obtain simple approximate expressions for diversity and divergence statistics for an inversion polymorphism at equilibrium under recombination and drift, and for the approach to equilibrium following the sweep of an inversion to a stable intermediate frequency. The effects of an inversion polymorphism on patterns of linkage disequilibrium are also examined. The reduction in effective recombination rate caused by population subdivision can have significant effects on these statistics. The theoretical results are discussed in relation to population genomic data on inversion polymorphisms, with an emphasis on Drosophila melanogaster. Methods are proposed for testing whether or not inversions are close to recombination-drift equilibrium, and for estimating the rate of recombinational exchange in heterozygotes for inversions; difficulties involved in estimating the ages of inversions are also discussed.

Dobbelaere, J., Su, T. Y., Erdi, B., Schleiffer, A. and Dammermann, A. (2023). A phylogenetic profiling approach identifies novel ciliogenesis genes in Drosophila and C. elegans. Embo j 42(16): e113616. PubMed ID: 37317646

Cilia are cellular projections that perform sensory and motile functions in eukaryotic cells. A defining feature of cilia is that they are evolutionarily ancient, yet not universally conserved. In this study, This study used the resulting presence and absence pattern in the genomes of diverse eukaryotes to identify a set of 386 human genes associated with cilium assembly or motility. Comprehensive tissue-specific RNAi in Drosophila and mutant analysis in C. elegans revealed signature ciliary defects for 70-80% of novel genes, a percentage similar to that for known genes within the cluster. Further characterization identified different phenotypic classes, including a set of genes related to the cartwheel component Bld10/CEP135 and two highly conserved regulators of cilium biogenesis. It is proposed that this dataset defines the core set of genes required for cilium assembly and motility across eukaryotes and presents a valuable resource for future studies of cilium biology and associated disorders.

Davies, N., Janicke, T. and Morrow, E. H. (2023). Evidence for stronger sexual selection in males than females using an adapted method of Bateman's classic study of Drosophila melanogaster. Evolution. PubMed ID: 37624087
Bateman's principles, originally a test of Darwin's theoretical ideas, has since become fundamental to sexual selection theory and vital to contextualising the role of anisogamy in sex differences of precopulatory sexual selection. Despite this, Bateman's principles have received substantial criticism, and researchers have highlighted both statistical and methodological errors, suggesting that Bateman's original experiment contains too much sampling bias for there to be any evidence of sexual selection. This study uses Bateman's original method as a template, accounting for two fundamental flaws in his original experiments, (i) viability effects and (ii) a lack of mating behaviour observation. Experimental populations of Drosophila melanogaster consisted of wild-type focal individuals and non-focal individuals established by backcrossing the brown eye (bw  -) eye-colour marker - thereby avoiding viability effects. Mating assays included direct observation of mating behaviour and total number of offspring, to obtain measures of mating success, reproductive success, and standardised variance measures based on Bateman's principles. The results provide observational support for Bateman's principles, particularly that (i) males had significantly more variation in number of mates compared to females and (ii) males had significantly more individual variation in total number of offspring. Significantly steeper Bateman gradient was found for males compared to females, suggesting that sexual selection is operating more intensely in males. However, female remating was limited, providing the opportunity for future study to further explore female reproductive success in correlation with higher levels of remating.
Erlenbach, T., Haynes, L., Fish, O., Beveridge, J., Bingolo, E., Giambrone, S. A., Kropelin, G., Rudisill, S., Chialvo, P., Reed, L. K., Dyer, K. A. and Chialvo, C. S. (2023). Investigating the phylogenetic history of toxin tolerance in mushroom-feeding Drosophila. bioRxiv. PubMed ID: 37577671
Understanding how and when key novel adaptations evolved is a central goal of evolutionary biology. Within the immigrans-tripunctata radiation of Drosophila , many mushroom-feeding species are tolerant of host toxins, such as cyclopeptides, that are lethal to nearly all other eukaryotes. In this study, phylogenetic and functional approaches were used to investigate the evolution of cyclopeptide tolerance in the immigrans-tripunctata radiation of Drosophila. First, the evolutionary relationships among 48 species in this radiation was inferred using 978 single copy orthologs. These results resolved previous incongruities within species groups across the phylogeny. Second, this study expanded on previous studies of toxin tolerance by assaying 16 of these species for tolerance to α-amanitin and found that six of these species could develop on diet with toxin. Third, fly development was examined on a diet containing a natural mix of toxins extracted from the Death Cap Amanita phalloides mushroom. Both tolerant and susceptible species developed on diet with this mix, though tolerant species survived at significantly higher concentrations. Finally, it was asked how cyclopeptide tolerance might have evolved across the immigrans-tripunctata radiation and inferred that toxin tolerance was ancestral and subsequently lost multiple times. These results suggest the evolutionary history of cyclopeptide tolerance is complex, and simply describing this trait as present or absent does not fully capture the occurrence or impact on this adaptive radiation. More broadly, the evolution of novelty can be more complex than previously thought, and that accurate descriptions of such novelties are critical in studies examining their evolution.

Tuesday October 3rd - Cytoskeleton and Junction

Carim, S. C. and Hickson, G. R. X. (2023). The Rho1 GTPase controls anillo-septin assembly to facilitate contractile ring closure during cytokinesis. iScience 26(6): 106903. PubMed ID: 37378349
Animal cell cytokinesis requires activation of the GTPase RhoA (Rho1 in Drosophila), which assembles an F-actin- and myosin II-dependent contractile ring (CR) at the equatorial plasma membrane. CR closure is poorly understood, but involves the multidomain scaffold protein, Anillin. Anillin binds many CR components including F-actin and myosin II (collectively actomyosin), RhoA and the septins. Anillin recruits septins to the CR but the mechanism is unclear. Live imaging of Drosophila S2 cells and HeLa cells revealed that the Anillin N-terminus, which scaffolds actomyosin, cannot recruit septins to the CR. Rather, septin recruitment required the ability of the Anillin C-terminus to bind Rho1-GTP and the presence of the Anillin PH domain, in a sequential mechanism occurring at the plasma membrane, independently of F-actin. Anillin mutations that blocked septin recruitment, but not actomyosin scaffolding, slowed CR closure and disrupted cytokinesis. Thus, CR closure requires coordination of two Rho1-dependent networks: actomyosin and anillo-septin.
Chen, J., Verissimo, A. F., Kull, A. R. and He, B. (2023). Early zygotic gene product Dunk interacts with anillin to regulate Myosin II during Drosophila cleavage. PubMed ID: 37494082
Drosophila melanogaster cellularization is a special form of cleavage that converts syncytial embryos into cellular blastoderms by partitioning the peripherally localized nuclei into individual cells. An early event in cellularization is the recruitment of nonmuscle myosin II ("myosin") to the leading edge of cleavage furrows, where myosin forms an interconnected basal array before reorganizing into individual cytokinetic rings. The initial recruitment and organization of basal myosin are regulated by a cellularization-specific gene, dunk, but the underlying mechanism is unclear. Through a genome-wide yeast two-hybrid screen, this study identified anillin (Scraps in Drosophila), a conserved scaffolding protein in cytokinesis, as the primary binding partner of Dunk. Dunk colocalizes with anillin and regulates its cortical localization during the formation of cleavage furrows, while the localization of Dunk is independent of anillin. Furthermore, Dunk genetically interacts with anillin to regulate the basal myosin array during cellularization. Similar to Dunk, anillin colocalizes with myosin since the very early stage of cellularization and is required for myosin retention at the basal array, before the well-documented function of anillin in regulating cytokinetic ring assembly. Based on these results, it is proposed that Dunk regulates myosin recruitment and spatial organization during early cellularization by interacting with and regulating anillin.
Fang, H. Y., Forghani, R., Clarke, A., McQueen, P. G., Chandrasekaran, A., O'Neill, K. M., Losert, W., Papoian, G. A. and Giniger, E. (2023). Enabled primarily controls filopodial morphology, not actin organization, in the TSM1 growth cone in Drosophila. Mol Biol Cell 34(8): ar83. PubMed ID: 37223966
Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. This study used in vivo live imaging of morphology and actin distribution to determine the role of Ena in promoting the growth of the TSM1 axon of the Drosophila wing. Altering Ena activity causes stalling and misrouting of TSM1. These data show that Ena has a substantial impact on filopodial morphology in this growth cone but exerts only modest effects on actin distribution. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which was shown previously to have profound effects on actin and only mild effects on TSM1 growth cone morphology. These data are interpreted as suggesting that the primary role of Ena in this axon may be to link actin to the morphogenetic processes of the plasma membrane, rather than to regulate actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain consistent organization and reliable evolution of growth cone structure, even as Abl activity varies in response to guidance cues in the environment.
Gaspar, I., Phea, L. J., McClintock, M. A., Heber, S., Bullock, S. L. and Ephrussi, A. (2023). An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport. J Cell Biol 222(7). PubMed ID: 37213090
Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). This study shows that orchestration of oskar RNP transport in the Drosophila germline relies on interplay between two double-stranded RNA-binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). It was found that Staufen antagonizes Egl-mediated transport of oskar mRNA by dynein both in vitro and in vivo. Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. It was additionally shown that Egl associates with staufen (stau) mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. These observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation of stau mRNA, and thus protein, in the oocyte enables motor switching on oskar RNPs by downregulating dynein activity.
Goldner, A. N., Fessehaye, S. M., Rodriguez, N., Mapes, K. A., Osterfield, M. and Doubrovinski, K. (2023). Evidence that tissue recoil in the early Drosophila embryo is a passive not active process. Mol Biol Cell 34(10): br16. PubMed ID: 37405768
Understanding tissue morphogenesis is impossible without knowing the mechanical properties of the tissue being shaped. Although techniques for measuring tissue material properties are continually being developed, methods for determining how individual proteins contribute to mechanical properties are very limited. This study developed two complementary techniques for the acute inactivation of spaghetti squash (the Drosophila myosin regulatory light chain), one based on the recently introduced (auxin-inducible degron 2 (AID2) system, and the other based on a novel method for conditional protein aggregation that results in nearly instantaneous protein inactivation. Combining these techniques with rheological measurements, it was shown that passive material properties of the cellularization-stage Drosophila embryo are essentially unaffected by myosin activity. These results suggest that this tissue is elastic, not predominantly viscous, on the developmentally relevant timescale.

Gonzalez Morales, N., Marescal, O., Szikora, S., Katzemich, A., Correia-Mesquita, T., Bíro, P., Erdelyi, M., Mihaly, J. and Schock, F. (2023). The oxoglutarate dehydrogenase complex is involved in myofibril growth and Z-disc assembly in Drosophila. J Cell Sci 136(13). PubMed ID: 37272588
Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. It has been shown shown that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. This study looked for Zasp-binding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. This study found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, it was revealed the overall organization of the complex at the Z-disc. Metabolomics identified an amino acid imbalance affecting protein synthesis as a possible cause of myofibril defects, which is supported by OGDH-dependent localization of ribosomes at the Z-disc.
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