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Current papers in developmental biology and gene function
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| ARCHIVE | Friday February 26th, 2021 - Signal Transduction |
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What's hot today
February 2024 January 2024 December 2023 November 2023 October 2023 September 2023 August 2023 July 2023 June 2023 May 2023 April 2023 March 2023 February 2023 January 2023 December 2022 November 2022 October 2022 September 2022 August 2022 July 2022 June 2022 May 2022 April 2022 March 2022 February 2022 Januaru 2022 December 2021 November 2021 October 2021 September 2021 August 2021 July 2021 June 2021 May 2021 April 2021 March 2021 February 2021 January 2021 May 2019 April 2019 | Medina-Yanez, I., Olivares, G. H., Vega-Macaya, F., Mlodzik, M. and Olguin, P. (2020). Phosphatidic acid increases Notch signalling by affecting Sanpodo trafficking during Drosophila sensory organ development. Sci Rep 10(1): 21731. PubMed ID: 33303974
Summary: Organ cell diversity depends on binary cell-fate decisions mediated by the Notch signalling pathway during development and tissue homeostasis. A clear example is the series of binary cell-fate decisions that take place during asymmetric cell divisions that give rise to the sensory organs of Drosophila melanogaster. The regulated trafficking of Sanpodo, a transmembrane protein that potentiates receptor activity, plays a pivotal role in this process. Membrane lipids can regulate many signalling pathways by affecting receptor and ligand trafficking. It remains unknown, however, whether phosphatidic acid regulates Notch-mediated binary cell-fate decisions during asymmetric cell divisions, and what are the cellular mechanisms involved. This study shows that increased phosphatidic acid derived from Phospholipase D leads to defects in binary cell-fate decisions that are compatible with ectopic Notch activation in precursor cells, where it is normally inactive. Null mutants of numb or the α-subunit of Adaptor Protein complex-2 enhance dominantly this phenotype while removing a copy of Notch or sanpodo suppresses it. In vivo analyses show that Sanpodo localization decreases at acidic compartments, associated with increased internalization of Notch. It is proposed that Phospholipase D-derived phosphatidic acid promotes ectopic Notch signalling by increasing receptor endocytosis and inhibiting Sanpodo trafficking towards acidic endosomes. | Ogawa, M., Kawarazaki, Y., Fujita, Y., Naguro, I. and Ichijo, H. (2020). FGF21 Induced by the ASK1-p38 Pathway Promotes Mechanical Cell Competition by Attracting Cells. Curr Biol. PubMed ID: 33357449
Summary: Cell competition is a social cellular phenomenon in which unfit cells are selectively eliminated to maintain tissue homeostasis. Recent studies have revealed that mechanical forces induce competitive cell-cell interactions in Drosophila. This mechanical cell competition has also been reported to play an important role in mammalian cells, using Madin-Darby canine kidney (MDCK) cells depleted of a polarity regulator Scribble in a tetracycline-inducible manner (scrib(KD) cells). scrib(KD) cells are hypersensitive to crowding due to the lower homeostatic density than wild-type (WT) cells, and in the context of cell competition, scrib(KD) cells are compacted and eliminated by WT cells. Although p38 and p53 are involved in this process, the molecular mechanism by which WT cells recognize and mechanically eliminate scrib(KD) cells remains unclear. This study reports that scrib(KD) cells secrete fibroblast growth factor 21 (FGF21) to drive cell competition. Knockdown of FGF21 in scrib(KD) cells or loss of FGFR1 in WT cells suppresses cell competition, suggesting that WT cells recognize scrib(KD) cells through FGF21. FGF21-containing culture medium of scrib(KD) cells activates cell motility. Moreover, FGF21 promotes the compression and elimination of scrib(KD)) cells by attracting surrounding WT cells. It was also demonstrated that activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway in scrib(KD) cells induces FGF21 to drive cell competition. These findings reveal a mechanism whereby WT cells mechanically eliminate scrib(KD) cells and propose a new function for FGF21 in cell-cell communication. |
| Lee, H. S., Mo, Y., Shin, H. C., Kim, S. J. and Ku, B. (2020). Structural and Biochemical Characterization of the Two Drosophila Low Molecular Weight-Protein Tyrosine Phosphatases DARP and Primo-1. Mol Cells 43(12): 1035-1045. PubMed ID: 33372666
Summary: The Drosophila genome contains four low molecular weight protein tyrosine phosphatase (LMW-PTP) members: Primo-1, Primo-2, CG14297, and CG31469. The lack of intensive biochemical analysis has limited understanding of these proteins. Primo-1 and CG31469 were previously classified as pseudophosphatases, but CG31469 was also suggested to be a putative protein arginine phosphatase. This study presents the crystal structures of CG31469 and Primo-1, which are the first Drosophila LMW-PTP structures. Structural analysis showed that the two proteins adopt the typical LMW-PTP fold and have a canonically arranged P-loop. Intriguingly, while Primo-1 is presumed to be a canonical LMW-PTP, CG31469 is unique as it contains a threonine residue at the fifth position of the P-loop motif instead of highly conserved isoleucine and a characteristically narrow active site pocket, which should facilitate the accommodation of phosphoarginine. Subsequent biochemical analysis revealed that Primo-1 and CG31469 are enzymatically active on phosphotyrosine and phosphoarginine, respectively, refuting their classification as pseudophosphatases. Collectively, this study provides structural and biochemical data on two Drosophila proteins: Primo-1, the canonical LMW-PTP protein, and CG31469, the first investigated eukaryotic protein arginine phosphatase. CG31469 was named DARP, which stands for Drosophila ARginine Phosphatase. | Nakamura, M., Verboon, J. M., Allen, T. E., Abreu-Blanco, M. T., Liu, R., Dominguez, A. N. M., Delrow, J. J. and Parkhurst, S. M. (2020). Autocrine insulin pathway signaling regulates actin dynamics in cell wound repair. PLoS Genet 16(12): e1009186. PubMed ID: 33306674
Summary: Cells are exposed to frequent mechanical and/or chemical stressors that can compromise the integrity of the plasma membrane and underlying cortical cytoskeleton. The molecular mechanisms driving the immediate repair response launched to restore the cell cortex and circumvent cell death are largely unknown. Using microarrays and drug-inhibition studies to assess gene expression, this study found that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. 253 genes were identified whose expression is up-regulated (80) or down-regulated (173) in response to laser wounding. A subset of these genes were validated using RNAi knockdowns and exhibit aberrant actomyosin ring assembly and/or actin remodeling defects. Strikingly, it was found that the canonical insulin signaling pathway controls actin dynamics through the actin regulators Girdin and Chickadee (profilin), and its disruption leads to abnormal wound repair. These results provide new insight for understanding how cell wound repair proceeds in healthy individuals and those with diseases involving wound healing deficiencies. |
| Park, J., Jun, K., Choi, Y., Yoon, E., Kim, W., Jang, Y. G. and Chung, J. (2020). CORO7 functions as a scaffold protein for the core kinase complex assembly of the Hippo pathway. J Biol Chem 296: 100040. PubMed ID: 33410403
Summary: The Hippo pathway controls organ size and tissue homeostasis through the regulation of cell proliferation and apoptosis. However, the exact molecular mechanisms underpinning Hippo pathway regulation are not fully understood. This study identified a new component of the Hippo pathway: coronin 7 (CORO7), a coronin protein family member that is involved in organization of the actin cytoskeleton. pod1, the Drosophila ortholog of CORO7, genetically interacts with key Hippo pathway genes in Drosophila. In mammalian cells, CORO7 is required for the activation of the Hippo pathway in response to cell-cell contact, serum deprivation, and cytoskeleton damage. CORO7 forms a complex with the core components of the pathway and functions as a scaffold for the Hippo core kinase complex. Collectively, these results demonstrate that CORO7 is a key scaffold controlling the Hippo pathway via modulating protein-protein interactions. | Krautz, R., Khalili, D. and Theopold, U. (2020). Tissue-autonomous immune response regulates stress signalling during hypertrophy. Elife 9. PubMed ID: 33377870
Summary: Postmitotic tissues are incapable of replacing damaged cells through proliferation, but need to rely on buffering mechanisms to prevent tissue disintegration. By constitutively activating the Ras/MAPK-pathway via Ras(V12)-overexpression in the postmitotic salivary glands of Drosophila larvae, the glands adaptability to growth signals and induced hypertrophy was overridden. The accompanied loss of tissue integrity, recognition by cellular immunity and cell death are all buffered by blocking stress signalling through a genuine tissue-autonomous immune response. This novel, spatio-temporally tightly regulated mechanism relies on the inhibition of a feedback-loop in the JNK-pathway by the immune effector and antimicrobial peptide Drosomycin. While this interaction might allow growing salivary glands to cope with temporary stress, continuous Drosomycin expression in Ras(V12)-glands favors unrestricted hypertrophy. These findings indicate the necessity to refine therapeutic approaches that stimulate immune responses by acknowledging their possible, detrimental effects in damaged or stressed tissues. |
Thursday, February 25th - Disease Models |
| Padash Barmchi, M., Thomas, M., Thatte, J. V., Vats, A., Zhang, B., Cagan, R. L. and Banks, L. (2021). Inhibition of kinase IKKbeta suppresses cellular abnormalities induced by the human papillomavirus oncoprotein HPV 18E6. Sci Rep 11(1): 1111. PubMed ID: 33441820
Summary: Human papillomavirus (HPV) is the leading cause of cervical cancer and has been implicated in several other cancer types including vaginal, vulvar, penile, and oropharyngeal cancers. Despite the recent availability of a vaccine, there are still over 310,000 deaths each year worldwide. Current treatments for HPV-mediated cancers show limited efficacy, and would benefit from improved understanding of disease mechanisms. Recently, a Drosophila 'HPV 18 E6' model was developed that displayed loss of cellular morphology and polarity, junctional disorganization, and degradation of the major E6 target Magi; this study further provides evidence that mechanisms underlying HPV E6-induced cellular abnormalities are conserved between humans and flies. A functional genetic screen of the Drosophila kinome identified IKK-a regulator of NF-κB-as an enhancer of E6-induced cellular defects. Inhibition of IKK reduces Magi degradation, and this effect correlates with hyperphosphorylation of E6. Further, the reduction in IKKβ suppressed the cellular transformation caused by the cooperative action of HPVE6 and the oncogenic Ras. Finally, we demonstrate that the interaction between IKKβ and E6 is conserved in human cells: inhibition of IKKβ blocked the growth of cervical cancer cells, suggesting that IKKβ may serve as a novel therapeutic target for HPV-mediated cancers. | Neuman, S. D., Terry, E. L., Selegue, J. E., Cavanagh, A. T. and Bashirullah, A. (2020). Mistargeting of secretory cargo in retromer-deficient cells. Dis Model Mech. 14(1):dmm046417 PubMed ID: 33380435
Summary: Intracellular trafficking is a basic and essential cellular function required for delivery of proteins to the appropriate subcellular destination; this process is especially demanding in professional secretory cells, which synthesize and secrete massive quantities of cargo proteins via regulated exocytosis. The Drosophila larval salivary glands are professional secretory cells that synthesize and secrete mucin proteins at the onset of metamorphosis. Using the larval salivary glands as a model system, a role was identified for the highly conserved retromer complex in trafficking of secretory granule membrane proteins. This study demonstrates that retromer-dependent trafficking via endosomal tubules is induced at the onset of secretory granule biogenesis, and that recycling via endosomal tubules is required for delivery of essential secretory granule membrane proteins to nascent granules. Without retromer function, nascent granules do not contain the proper membrane proteins; as a result, cargo from these defective granules is mistargeted to Rab7-positive endosomes, where it progressively accumulates to generate dramatically enlarged endosomes. Retromer complex dysfunction is strongly associated with neurodegenerative diseases, including Alzheimer's disease, characterized by accumulation of amyloid β (Aβ). Ectopically expressed amyloid precursor protein (APP) undergoes regulated exocytosis in salivary glands and accumulates within enlarged endosomes in retromer-deficient cells. These results highlight recycling of secretory granule membrane proteins as a critical step during secretory granule maturation and provide new insights into our understanding of retromer complex function in secretory cells. These findings also suggest that missorting of secretory cargo, including APP, may contribute to the progressive nature of neurodegenerative disease (Neuman, 2020). |
| Matsukawa, K., Kukharsky, M. S., Park, S. K., Park, S., Watanabe, N., Iwatsubo, T., Hashimoto, T., Liebman, S. W. and Shelkovnikova, T. A. (2021). Long non-coding RNA NEAT1_1 ameliorates TDP-43 toxicity in in vivo models of TDP-43 proteinopathy. RNA Biol: 1-9. PubMed ID: 33427561
Summary: Pathological changes involving TDP-43 protein ('TDP-43 proteinopathy') are typical for several neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). FTLD-TDP cases are characterized by increased binding of TDP-43 to an abundant lncRNA, NEAT1, in the cortex. However it is unclear whether enhanced TDP-43-NEAT1 interaction represents a protective mechanism. This study shows that accumulation of human TDP-43 leads to upregulation of the constitutive NEAT1 isoform, NEAT1_1, in cultured cells and in the brains of transgenic mice. Further, it was demonstrated that overexpression of NEAT1_1 ameliorates TDP-43 toxicity in Drosophila and yeast models of TDP-43 proteinopathy. Thus, NEAT1_1 upregulation may be protective in TDP-43 proteinopathies affecting the brain. Approaches to boost NEAT1_1 expression in the CNS may prove useful in the treatment of these conditions. | Millet-Boureima, C., Rozencwaig, R., Polyak, F. and Gamberi, C. (2020). Cyst Reduction by Melatonin in a Novel Drosophila Model of Polycystic Kidney Disease. Molecules 25(22). PubMed ID: 33238462
Summary: Autosomal dominant polycystic kidney disease (ADPKD) causes progressive cystic degeneration of the renal tubules, the nephrons, eventually severely compromising kidney function. ADPKD is incurable, with half of the patients eventually needing renal replacement. Treatments for ADPKD patients are limited and new effective therapeutics are needed. Melatonin, a central metabolic regulator conserved across all life kingdoms, exhibits oncostatic and oncoprotective activity and no detected toxicity. This study used the Bicaudal C (BicC) Drosophila model of polycystic kidney disease to test the cyst-reducing potential of melatonin. Significant cyst reduction was found in the renal (Malpighian) tubules upon melatonin administration and suggest mechanistic sophistication. Similar to vertebrate PKD, the BicC fly PKD model responds to the antiproliferative drugs rapamycin and mimics of the second mitochondria-derived activator of caspases (Smac). Melatonin appears to be a new cyst-reducing molecule with attractive properties as a potential candidate for PKD treatment. |
| Lohr, K. M., Frost, B., Scherzer, C. and Feany, M. B. (2020). Biotin rescues mitochondrial dysfunction and neurotoxicity in a tauopathy model. Proc Natl Acad Sci U S A 117(52): 33608-33618. PubMed ID: 33318181
Summary: Mitochondrial and metabolic dysfunction are often implicated in neurological disease, but effective mechanism-based therapies remain elusive. A genome-scale forward genetic screen was performed in a Drosophila model of tauopathy, a class of neurodegenerative disorders characterized by the accumulation of the protein tau, and identified manipulation of the B-vitamin biotin as a potential therapeutic approach in tauopathy. Tau transgenic flies have an innate biotin deficiency due to tau-mediated relaxation of chromatin and consequent aberrant expression of multiple biotin-related genes, disrupting both carboxylase and mitochondrial function. Biotin depletion alone causes mitochondrial pathology and neurodegeneration in both flies and human neurons, implicating mitochondrial dysfunction as a mechanism in biotin deficiency. Finally, carboxylase biotin levels are reduced in mammalian tauopathies, including brains of human Alzheimer's disease patients. These results provide insight into pathogenic mechanisms of human biotin deficiency, the resulting effects on neuronal health, and a potential therapeutic pathway in the treatment of tau-mediated neurotoxicity. | Liu, J., Wang, X., Ma, R., Li, T., Guo, G., Ning, B., Moran, T. H. and Smith, W. W. (2020). AMPK signaling mediates synphilin-1-induced hyperphagia and obesity in Drosophila. J Cell Sci. PubMed ID: 33443093
Summary: Expression of synphilin-1 in neurons induces hyperphagia and obesity in a Drosophila model. However, the molecular pathways underlying synphilin-1-linked obesity remain unclear. This study used the Drosophila model, and genetic tools were used to study the synphilin-1-linked pathways in energy balance by combining molecular biology and pharmacological approaches. Expression of human synphilin-1 in flies increased AMPK phosphorylation at Thr172 compared with non-transgenic flies. Knockdown of AMPK reduced AMPK phosphorylation and food intake in non-transgenic flies, and further suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia, fat storage, and body weight gain in transgenic flies. Expression of constitutively activated AMPK significantly increased food intake and body weight gain in non-transgenic flies, but it did not alter food intake in the synphilin-1 transgenic flies. In contrast, expression of dominant-negative AMPK reduced food intake in both non-transgenic and synphilin-1 transgenic flies. Treatment with STO609 also suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia and body weight gain. These results demonstrated that the AMPKsignaling pathway plays a critical role in synphilin-1-induced hyperphagia and obesity. These findings provide new insights into the mechanisms of synphilin-1 controlled energy homeostasis. |
Wednesday, February 24th - Immune Response |
| Campbell, J. S., Davidson, A. J., Todd, H., Rodrigues, F., Elliot, A. M., Early, J. J., Lyons, D. A., Feng, Y. and Wood, W. (2020). PTPN21/Pez Is a Novel and Evolutionarily Conserved Key Regulator of Inflammation In Vivo. Curr Biol. PubMed ID: 33296680
Summary: Drosophila provides a powerful model in which to study inflammation in vivo, and previous studies have revealed many of the key signaling events critical for recruitment of immune cells to tissue damage. In the fly, wounding stimulates the rapid production of hydrogen peroxide (H(2)O(2)). This then acts as an activation signal by triggering a signaling pathway within responding macrophages by directly activating the Src family kinase (SFK) Src42A, which in turn phosphorylates the damage receptor Draper. Activated Draper then guides macrophages to the wound through the detection of an as-yet unidentified chemoattractant. Similar H(2)O(2)-activated signaling pathways are also critical for leukocyte recruitment following wounding in larval zebrafish, where H(2)O(2) activates the SFK Lyn to drive neutrophil chemotaxis. This study combined proteomics, live imaging, and genetics in the fly to identify a novel regulator of inflammation in vivo; the PTP-type phosphatase Pez. Pez is expressed in macrophages and is critical for their efficient migration to wounds. Pez functions within activated macrophages downstream of damage-induced H(2)O(2) and operates, via its band 4.1 ezrin, radixin, and moesin (FERM) domain, together with Src42A and Draper to ensure effective inflammatory cell recruitment to wounds. This key role is conserved in vertebrates, because "crispant" zebrafish larvae of the Draper ortholog (MEGF10) or the Pez ortholog (PTPN21) exhibit a failure in leukocyte recruitment to wounds. This study demonstrates evolutionary conservation of inflammatory signaling and identifies MEGF10 and PTPN21 as potential therapeutic targets for the treatment of inflammatory disorders. | Martonez, B. A., Hoyle, R. G., Yeudall, S., Granade, M. E., Harris, T. E., Castle, J. D., Leitinger, N. and Bland, M. L. (2020). Innate immune signaling in Drosophila shifts anabolic lipid metabolism from triglyceride storage to phospholipid synthesis to support immune function. PLoS Genet 16(11): e1009192. PubMed ID: 33227003
Summary: During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. This study asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. Genetic or physiological activation of fat body Toll signaling was found to lead to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy phospholipid synthesis pathway induction is an impaired immune response to bacterial infection. These results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage. |
| Cai, H., Holleufer, A., Simonsen, B., Schneider, J., Lemoine, A., Gad, H. H., Huang, J., Huang, J., Chen, D., Peng, T., Marques, J. T., Hartmann, R., Martins, N. E. and Imler, J. L. (2020). 2'3'-cGAMP triggers a STING- and NF-kappaB-dependent broad antiviral response in Drosophila. Sci Signal 13(660). PubMed ID: 33262294
Summary: An ortholog of STING has been reported to regulate infection by picorna-like viruses in Drosophila In mammals, STING is activated by the cyclic dinucleotide 2'3'-cGAMP produced by cGAS, which acts as a receptor for cytosolic DNA. This study showed that injection of flies with 2'3'-cGAMP induced the expression of dSTING-regulated genes. Coinjection of 2'3'-cGAMP with a panel of RNA or DNA viruses resulted in substantially reduced viral replication. This 2'3'-cGAMP-mediated protection was still observed in flies with mutations in Atg7 and AGO2, genes that encode key components of the autophagy and small interfering RNA pathways, respectively. By contrast, this protection was abrogated in flies with mutations in the gene encoding the NF-κB transcription factor Relish. Transcriptomic analysis of 2'3'-cGAMP-injected flies revealed a complex response pattern in which genes were rapidly induced, induced after a delay, or induced in a sustained manner. These results reveal that dSTING regulates an NF-κB-dependent antiviral program that predates the emergence of interferons in vertebrates. | Madhwal, S., Shin, M., Kapoor, A., Goyal, M., Joshi, M. K., Ur Rehman, P. M., Gor, K., Shim, J. and Mukherjee, T. (2020). Metabolic control of cellular immune-competency by odors in Drosophila. Elife 9. PubMed ID: 33372660
Summary: Studies in different animal model systems have revealed the impact of odors on immune cells; however, any understanding on why and how odors control cellular immunity remained unclear. This study found that Drosophila employ an olfactory-immune cross-talk to tune a specific cell type, the lamellocytes, from hematopoietic-progenitor cells. Neuronally released GABA derived upon olfactory stimulation is utilized by blood-progenitor cells as a metabolite and through its catabolism, these cells stabilize Sima/HIFα protein. Sima capacitates blood-progenitor cells with the ability to initiate lamellocyte differentiation. This systemic axis becomes relevant for larvae dwelling in wasp-infested environments where chances of infection are high. By co-opting the olfactory route, the preconditioned animals elevate their systemic GABA levels leading to the upregulation of blood-progenitor cell Sima expression. This elevates their immune-potential and primes them to respond rapidly when infected with parasitic wasps. The present work highlights the importance of the olfaction in immunity and shows how odor detection during animal development is utilized to establish a long-range axis in the control of blood-progenitor competency and immune-priming. |
| Leitao, A. B., Arunkumar, R., Day, J. P., Geldman, E. M., Morin-Poulard, I., Crozatier, M. and Jiggins, F. M. (2020). Constitutive activation of cellular immunity underlies the evolution of resistance to infection in Drosophila. Elife 9. PubMed ID: 33357377
Summary: Organisms rely on inducible and constitutive immune defences to combat infection. Constitutive immunity enables a rapid response to infection but may carry a cost for uninfected individuals, leading to the prediction that it will be favoured when infection rates are high. When populations of Drosophila melanogaster were exposed to intense parasitism by the parasitoid wasp Leptopilina boulardi, they evolved resistance by developing a more reactive cellular immune response. Using single-cell RNA sequencing, this study found that immune-inducible genes had become constitutively upregulated. This was the result of resistant larvae differentiating precursors of specialized immune cells called lamellocytes that were previously only produced after infection. Therefore, populations evolved resistance by genetically hard-wiring the first steps of an induced immune response to become constitutive. | Mondotte, J. A., Gausson, V., Frangeul, L., Suzuki, Y., Vazeille, M., Mongelli, V., Blanc, H., Failloux, A. B. and Saleh, M. C. (2020). Evidence For Long-Lasting Transgenerational Antiviral Immunity in Insects. Cell Rep 33(11): 108506. PubMed ID: 33326778
Summary: Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. This study shows that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline. |
Tuesday, February 23rd - Methods |
| Gupta, S., Verma, A. K. and Ahmad, S. (2020). Feature Selection for Topological Proximity Prediction of Single-Cell Transcriptomic Profiles in Drosophila Embryo Using Genetic Algorithm. Genes (Basel) 12(1). PubMed ID: 33379262
Summary: Single-cell transcriptomics data, when combined with in situ hybridization patterns of specific genes, can help in recovering the spatial information lost during cell isolation. Dialogue for Reverse Engineering Assessments and Methods (DREAM) consortium conducted a crowd-sourced competition known as DREAM Single Cell Transcriptomics Challenge (SCTC) to predict the masked locations of single cells from a set of 60, 40 and 20 genes out of 84 in situ gene patterns known in Drosophila embryo. A genetic algorithm (GA) was applied to predict the most important genes that carry positional and proximity information of the single-cell origins, in combination with the base distance mapping algorithm DistMap. Resulting gene selection was found to perform well and was ranked among top 10 in two of the three sub-challenges. However, the details of the method did not make it to the main challenge publication, due to an intricate aggregation ranking. This work discusses the detailed implementation of GA and its post-challenge parameterization, with a view to identify potential areas where GA-based approaches of gene-set selection for topological association prediction may be improved, to be more effective. It is believed this work provides additional insights into the feature-selection strategies and their relevance to single-cell similarity prediction and will form a strong addendum to the recently published work from the consortium. | Melnattur, K., Morgan, E., Duong, V., Kalra, A. and Shaw, P. J. (2020). The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila. J Vis Exp(166). PubMed ID: 33369606
Summary: Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal kingdom. As a consequence, sleep deprivation and sleep restriction are powerful tools that are commonly used to provide insight into sleep function. Nonetheless, sleep deprivation experiments are inherently problematic in that the deprivation stimulus itself may be the cause of observed changes in physiology and behavior. Accordingly, successful sleep deprivation techniques should keep animals awake and, ideally, result in a robust sleep rebound without also inducing a large number of unintended consequences. This study describe a sleep deprivation technique for Drosophila melanogaster. The Sleep Nullifying Apparatus (SNAP) administers a stimulus every 10s to induce negative geotaxis. Although the stimulus is predictable, the SNAP effectively prevents >95% of nighttime sleep even in flies with high sleep drive. Importantly, the subsequent homeostatic response is very similar to that achieved using hand-deprivation. The timing and spacing of the stimuli can be modified to minimize sleep loss and thus examine non-specific effects of the stimulus on physiology and behavior. The SNAP can also be used for sleep restriction and to assess arousal thresholds. The SNAP is a powerful sleep disruption technique that can be used to better understand sleep function. |
| Januschke, J. and Loyer, N. (2020). Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging. J Vis Exp(166). PubMed ID: 33427234
Summary: Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of the fly such as imaginal discs, the larval brain or egg chambers of adult females or the adult intestine can be extracted and kept in culture for imaging with time-lapse microscopy, providing valuable insights into cell and developmental biology. This study describes in detail a current protocol for the dissection of Drosophila larval brains and then presents the current approach for immobilizing and orienting larval brains and other tissues on a glass coverslip using Fibrin clots. This immobilization method only requires the addition of Fibrinogen and Thrombin to the culture medium. It is suitable for high-resolution time lapse imaging on inverted microscopes of multiple samples in the same culture dish, minimizes the lateral drifting frequently caused by movements of the microscope stage in multi-point visiting microscopy and allows for the addition and removal of reagents during the course of imaging. Custom-made macros are presented that are routinely used to correct for drifting and to extract and process specific quantitative information from time-lapse analysis | Buddika, K., Xu, J., Ariyapala, I. S. and Sokol, N. S. (2021). I-KCKT allows dissection-free RNA profiling of adult Drosophila intestinal progenitor cells. Development 148(1). PubMed ID: 33246929
Summary: The adult Drosophila intestinal epithelium is a model system for stem cell biology, but its utility is limited by current biochemical methods that lack cell type resolution. This paper describes a new proximity-based profiling method that relies upon a GAL4 driver, termed intestinal-kickout-GAL4 (I-KCKT-GAL4), that is exclusively expressed in intestinal progenitor cells. This method uses UV crosslinked whole animal frozen powder as its starting material to immunoprecipitate the RNA cargoes of transgenic epitope-tagged RNA binding proteins driven by I-KCKT-GAL4 When applied to the general mRNA-binder, poly(A)-binding protein, the RNA profile obtained by this method identifies 98.8% of transcripts found after progenitor cell sorting, and has low background noise despite being derived from whole animal lysate. The targets of the more selective RNA binder, Fragile X mental retardation protein (FMRP), were mapped using enhanced crosslinking and immunoprecipitation (eCLIP), and report for the first time its binding motif in Drosophila cells. This method will therefore enable the RNA profiling of wild-type and mutant intestinal progenitor cells from intact flies exposed to normal and altered environments, as well as the identification of RNA-protein interactions crucial for stem cell function. |
| Hancock, C. E., Geurten, B. R. H. and Fiala, A. (2020). Visualization of naive and learned odor representations using in vivo calcium imaging and immunohistochemical bouton mapping of single Drosophila mushroom body neurons. STAR Protoc 1(3): 100210. PubMed ID: 33377104
Summary: This protocol enables the quantification of odor-evoked calcium activity in mushroom body Kenyon cells of the Drosophila melanogaster brain at the single bouton level. Subsequent characterization is presented of naive and learned odor representations in the context of olfactory coding. This approach to analyzing the neuronal basis of associative learning provides a substrate for similar studies, perhaps in other animals, to probe the attributes of a neuronal memory trace at the level of synapses distributed across neurons. For complete details on the use and execution of this protocol, please refer to Bilz (2020). | He, L., Wang, L., Zeng, H., Tan, P., Ma, G., Zheng, S., Li, Y., Sun, L., Dou, F., Siwko, S., Huang, Y., Wang, Y. and Zhou, Y. (2021). Engineering of a bona fide light-operated calcium channel. Nat Commun 12(1): 164. PubMed ID: 33431868
Summary: The current optogenetic toolkit lacks a robust single-component Ca(2+)-selective ion channel tailored for remote control of Ca(2+) signaling in mammals. Existing tools are either derived from engineered channelrhodopsin variants without strict Ca(2+) selectivity or based on the stromal interaction molecule 1 (STIM1) that might crosstalk with other targets. This paper describes the design of a light-operated Ca(2+) channel (designated LOCa) by inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel. LOCa displays biophysical features reminiscent of the ORAI1 channel (see Drosophila Orai), which enables precise optical control over Ca(2+) signals and hallmark Ca(2+)-dependent physiological responses. Furthermore, this study demonstrates the use of LOCa to modulate aberrant hematopoietic stem cell self-renewal, transcriptional programming, cell suicide, as well as neurodegeneration in a Drosophila model of amyloidosis. |
Monday, February 22nd - Adult Development |
| Bonche, R., Chessel, A., Boisivon, S., Smolen, P., Therond, P. and Pizette, S. (2020). Two different sources of Perlecan cooperate for its function in the basement membrane of the Drosophila wing imaginal disc. Dev Dyn. PubMed ID: 33269518
Summary: The basement membrane (BM) provides mechanical shaping of tissues during morphogenesis. The Drosophila BM proteoglycan Perlecan (Pcan) is vital for this process in the wing imaginal disc. This function is thought to be fostered by the heparan sulfate chains attached to the domain I of vertebrate Pcan. However, this domain is not present in Drosophila, and the source of Pcan for the wing imaginal disc BM remains unclear. These two issues were tackled. In silico analysis shows that Drosophila Pcan holds a domain I. Moreover, by combining in situ hybridization of Pcan mRNA and protein staining, together with tissue-specific Pcan depletion, this study found that there is an autonomous and a non-autonomous source for Pcan deposition in the wing imaginal disc BM. It was further shown that both sources cooperate for correct distribution of Pcan in the wing imaginal disc and morphogenesis of this tissue. These results show that Pcan is fully conserved in Drosophila, providing a valuable in vivo model system to study its role in BM function. The existence of two different sources for Pcan incorporation in the wing imaginal disc BM raises the possibility that inter-organ communication mediated at the level of the BM is involved in organogenesis. | Fan, Z., Zhang, J., Wang, D. and Shen, J. (2021). T-box transcription factors Dorsocross and optomotor-blind control Drosophila leg patterning in a functionally redundant manner. Insect Biochem Mol Biol 129: 103516. PubMed ID: 33412239
Summary: The T-box genes are essential transcription factors during limb development. In Drosophila, Dorsocross (Doc) and optomotor-blind (omb), members of the Tbx2 and Tbx6 families, are best studied in the Drosophila wing development. Despite prominently expressed in leg discs, the specific function of these genes in leg growth is still not revealed. This study demonstrated that Doc and omb regulated the morphogenesis of leg intermediate regions in a functionally redundant manner. Loss of Doc ir omb individually did not result in any developmental defects of the legs, but loss of both genes induced significant defects in femur and proximal tibia of the adult legs. These genes located in the dorsal domain, where the Doc region expanded and cross-overlapped with the omb region corresponding to the presumptive leg intermediate region. The normal epithelial folds in the leg discs were disrupted along with dorsal repression of cell proliferation and activation of cell apoptosis when Doc and omb were both reduced. Furthermore, the dorsal expression of dachshund (dac), a canonical leg developmental gene specifying the leg intermediate region, was maintained by Doc and omb. Meanwhile, the Notch pathway was compromised in the dorsal domain when these genes were reduced, which might contribute to the joint defect of the adult leg intermediate regions. This study provides cytological and genetic evidence for understanding the redundant function of Doc and omb in leg morphogenesis. |
| Iijima, N., Sato, K., Kuranaga, E. and Umetsu, D. (2020). Differential cell adhesion implemented by Drosophila Toll corrects local distortions of the anterior-posterior compartment boundary. Nat Commun 11(1): 6320. PubMed ID: 33303753
Summary: Maintaining lineage restriction boundaries in proliferating tissues is vital to animal development. A long-standing thermodynamics theory, the differential adhesion hypothesis, attributes cell sorting phenomena to differentially expressed adhesion molecules. However, the contribution of the differential adhesion system during tissue morphogenesis has been unsubstantiated despite substantial theoretical support. This study reports that Toll-1, a transmembrane receptor protein, acts as a differentially expressed adhesion molecule that straightens the fluctuating anteroposterior compartment boundary in the abdominal epidermal epithelium of the Drosophila pupa. Toll-1 is expressed across the entire posterior compartment under the control of the selector gene engrailed and displays a sharp expression boundary that coincides with the compartment boundary. Toll-1 corrects local distortions of the boundary in the absence of cable-like Myosin II enrichment along the boundary. The reinforced adhesion of homotypic cell contacts, together with pulsed cell contraction, achieves a biased vertex sliding action by resisting the separation of homotypic cell contacts in boundary cells. This work reveals a self-organizing system that integrates a differential adhesion system with pulsed contraction of cells to maintain lineage restriction boundaries. | Millington, J. W., Brownrigg, G. P., Chao, C., Sun, Z., Basner-Collins, P. J., Wat, L. W., Hudry, B., Miguel-Aliaga, I. and Rideout, E. J. (2021). Female-biased upregulation of insulin pathway activity mediates the sex difference in Drosophila body size plasticity. Elife 10. PubMed ID: 33448263
Summary: Nutrient-dependent body size plasticity differs between the sexes in most species, including mammals. Previous work in Drosophila showed that body size plasticity was higher in females, yet the mechanisms underlying increased female body size plasticity remain unclear. This study discovered that a protein-rich diet augments body size in females and not males because of a female-biased increase in activity of the conserved insulin/insulin-like growth factor signaling pathway (IIS). This sex-biased upregulation of IIS activity was triggered by a diet-induced increase in stunted mRNA in females, and required Drosophila insulin-like peptide 2, illuminating new sex-specific roles for these genes. Importantly, this study shows that sex determination gene transformer promotes the diet-induced increase in stunted mRNA via transcriptional coactivator Spargel to regulate the male-female difference in body size plasticity. Together, these findings provide vital insight into conserved mechanisms underlying the sex difference in nutrient-dependent body size plasticity. |
| Katti, P., Rai, M., Srivastava, S., Silva, P. and Nongthomba, U. (2021). Marf-mediated mitochondrial fusion is imperative for the development and functioning of indirect flight muscles (IFMs) in Drosophila. Exp Cell Res: 112486. PubMed ID: 33450208
Summary: Dynamic changes in mitochondrial shape and size are vital for mitochondrial health and for tissue development and function. Adult Drosophila indirect flight muscles contain densely packed mitochondria. This study shows here that mitochondrial fusion is critical during early muscle development (in pupa) and that silencing of the outer mitochondrial membrane fusion gene, Marf, in muscles results in smaller mitochondria that are functionally defective. This leads to abnormal muscle development resulting in muscle dysfunction in adult flies. However, post-developmental silencing of Marf has no obvious effects on mitochondrial and muscle phenotype in adult flies, indicating the importance of mitochondrial fusion during early muscle development. | Miller, A. C., Urban, E. A., Lyons, E. L., Herman, T. G. and Johnston, R. J., Jr. (2020). Interdependent regulation of stereotyped and stochastic photoreceptor fates in the fly eye. Dev Biol 471: 89-96. PubMed ID: 33333066
Summary: Diversification of neuronal subtypes often requires stochastic gene regulatory mechanisms. How stochastically expressed transcription factors interact with other regulators in gene networks to specify cell fates is poorly understood. The random mosaic of color-detecting R7 photoreceptor subtypes in Drosophila is controlled by the stochastic on/off expression of the transcription factor Spineless (Ss). In Ss(ON) R7s, Ss induces expression of Rhodopsin 4 (Rh4), whereas in Ss(OFF) R7s, the absence of Ss allows expression of Rhodopsin 3 (Rh3). This study finds that the transcription factor Runt, which is initially expressed in all R7s, is sufficient to promote stochastic Ss expression. Later, as R7s develop, Ss negatively feeds back onto Runt to prevent repression of Rh4 and ensure proper fate specification. Together, stereotyped and stochastic regulatory inputs are integrated into feedforward and feedback mechanisms to control cell fate. |
Friday, February 19th - Gonads |
| Li, W., Liang, J., Outeda, P., Turner, S., Wakimoto, B. T. and Watnick, T. (2020). A genetic screen in Drosophila reveals an unexpected role for the KIP1 ubiquitination-promoting complex in male fertility. PLoS Genet 16(12): e1009217. PubMed ID: 33378371
Summary: A unifying feature of polycystin-2 channels is their localization to both primary and motile cilia/flagella. In Drosophila melanogaster, the fly polycystin-2 homologue, Amo, is an ER protein early in sperm development but the protein must ultimately cluster at the flagellar tip in mature sperm to be fully functional. Male flies lacking appropriate Amo localization are sterile due to abnormal sperm motility and failure of sperm storage. A forward genetic screen was performed to identify additional proteins that mediate ciliary trafficking of Amo. Drosophila homologues of KPC1 and KPC2, which comprise the mammalian KIP1 ubiquitination-promoting complex (KPC), form a conserved unit that is required for the sperm tail tip localization of Amo. Male flies lacking either KPC1 or KPC2 phenocopy amo mutants and are sterile due to a failure of sperm storage. KPC is a heterodimer composed of KPC1, an E3 ligase, and KPC2 (or UBAC1), an adaptor protein. Like their mammalian counterparts Drosophila KPC1 and KPC2 physically interact and they stabilize one another at the protein level. In flies, KPC2 is monoubiquitinated and phosphorylated and this modified form of the protein is located in mature sperm. Neither KPC1 nor KPC2 directly interact with Amo but they are detected in proximity to Amo at the tip of the sperm flagellum. In summary this study has identified a new complex that is involved in male fertility in Drosophila melanogaster. | Ladyzhets, S., Antel, M., Simao, T., Gasek, N., Cowan, A. E. and Inaba, M. (2020). Self-limiting stem-cell niche signaling through degradation of a stem-cell receptor. PLoS Biol 18(12): e3001003. PubMed ID: 33315855
Summary: Stem-cell niche signaling is short-range in nature, such that only stem cells but not their differentiating progeny receive self-renewing signals. At the apical tip of the Drosophila testis, 8 to 10 germline stem cells (GSCs) surround the hub, a cluster of somatic cells that organize the stem-cell niche. Previous work has shown that GSCs form microtubule-based nanotubes (MT-nanotubes) that project into the hub cells, serving as the platform for niche signal reception; this spatial arrangement ensures the reception of the niche signal specifically by stem cells but not by differentiating cells. The receptor Thickveins (Tkv) is expressed by GSCs and localizes to the surface of MT-nanotubes, where it receives the hub-derived ligand Decapentaplegic (Dpp). The fate of Tkv receptor after engaging in signaling on the MT-nanotubes has been unclear. This study demonstrates that the Tkv receptor is internalized into hub cells from the MT-nanotube surface and subsequently degraded in the hub cell lysosomes. Perturbation of MT-nanotube formation and Tkv internalization from MT-nanotubes into hub cells both resulted in an overabundance of Tkv protein in GSCs and hyperactivation of a downstream signal, suggesting that the MT-nanotubes also serve a second purpose to dampen the niche signaling. Together, these results demonstrate that MT-nanotubes play dual roles to ensure the short-range nature of niche signaling by (1) providing an exclusive interface for the niche ligand-receptor interaction; and (2) limiting the amount of stem cell receptors available for niche signal reception. |
| Ma, T., Matsuoka, S. and Drummond-Barbosa, D. (2020). RNAi-based screens uncover a potential new role for the orphan neuropeptide receptor Moody in Drosophila female germline stem cell maintenance. PLoS One 15(12): e0243756. PubMed ID: 33307547
Summary: Reproduction is highly sensitive to changes in physiology and the external environment. Neuropeptides are evolutionarily conserved signaling molecules that regulate multiple physiological processes. However, the potential reproductive roles of many neuropeptide signaling pathways remain underexplored. This study describes the results of RNAi-based screens in Drosophila melanogaster to identify neuropeptides/neuropeptide receptors with potential roles in oogenesis. The screen read-outs were either the number of eggs laid per female per day over time or fluorescence microscopy analysis of dissected ovaries. The orphan neuropeptide receptor encoded by moody (homologous to mammalian melatonin receptors) is likely required in somatic cells for normal egg production and proper germline stem cell maintenance. However, the egg laying screens had low signal-to-noise ratio and did not lead to the identification of additional candidates. Thus, although egg count assays might be useful for large-scale screens to identify oogenesis regulators that result in dramatic changes in oogenesis, more labor-intensive microscopy-based screen are better applicable for identifying new physiological regulators of oogenesis with more subtle phenotypes. | Potter-Birriel, J. M., Gonsalvez, G. B. and Marzluff, W. F. (2021). A region of SLBP outside the mRNA processing domain is essential for deposition of histone mRNA into the Drosophila egg. J Cell Sci. PubMed ID: 33408246
Summary: Replication-dependent histone mRNAs are the only cellular mRNAs that are not polyadenylated, ending in a stemloop instead of a polyA tail, and are normally regulated coordinately with DNA replication. SLBP binds the 3' end of histone mRNA, and is required for processing and translation. During Drosophila oogenesis, large amounts of histone mRNAs and proteins are deposited in the developing oocyte. The maternally deposited histone mRNA is synthesized in stage 10B oocytes after the nurse cells complete endoreduplication. This study reports that in WT stage 10B oocytes, the Histone Locus Bodies (HLBs), formed on the histone genes, produce histone mRNAs in the absence of phosphorylation of Mxc, normally required for histone gene expression in S-phase cells. Two mutants of SLBP, one with reduced expression and another with a 10 aa deletion, fail to deposit sufficient histone mRNA in the oocyte, and don't transcribe the histone genes in stage 10B. Mutations in a putative SLBP nuclear localization sequence overlapping the deletion, phenocopy the deletion. It is concluded that a high concentration of SLBP in the nucleus of stage 10B oocytes is essential for histone gene transcription. |
| Dorogova, N. V., Galimova, Y. A., Bolobolova, E. U., Baricheva, E. M. and Fedorova, S. A. (2020). Loss of Drosophila E3 Ubiquitin Ligase Hyd Promotes Extra Mitosis in Germline Cysts and Massive Cell Death During Oogenesis. Front Cell Dev Biol 8: 600868. PubMed ID: 33240894
Summary: The Drosophila hyperplastic disc (hyd) gene is the ortholog of mammalian tumor suppressor EDD, which is implicated in a wide variety of cellular processes, and its regulation is impaired in various tumors. It is a member of the highly conserved HECT family of E3 ubiquitin ligases, which directly attach ubiquitin to targeted substrates. In early works, it was shown that Drosophila Hyd may be a tumor suppressor because it is involved in the control of imaginal-disc cell proliferation and growth. This study demonstrated that Hyd is also important for the regulation of female germ cell proliferation and that its depletion leads to additional germline cell mitoses. Furthermore, a previously unknown Hyd function was revealed, associated with the maintenance of germ cells' viability. A reduction in hyd expression by either mutations or RNA interference resulted in large-scale germ cell death at different stages of oogenesis. Thus, the analysis of phenotypes arising from the hyd deficiency points to Hyd's role in the regulation of germline metabolic processes during oogenesis. | Lamire, L. A., Milani, P., Runel, G., Kiss, A., Arias, L., Vergier, B., de Bossoreille, S., Das, P., Cluet, D., Boudaoud, A. and Grammont, M. (2020). Gradient in cytoplasmic pressure in germline cells controls overlying epithelial cell morphogenesis. PLoS Biol 18(11): e3000940. PubMed ID: 33253165
Summary: It is unknown how growth in one tissue impacts morphogenesis in a neighboring tissue. To address this, the Drosophila ovarian follicle, in which a cluster of 15 nurse cells and a posteriorly located oocyte are surrounded by a layer of epithelial cells, was used. It is known that as the nurse cells grow, the overlying epithelial cells flatten in a wave that begins in the anterior. This study demonstrates that an anterior to posterior gradient of decreasing cytoplasmic pressure is present across the nurse cells and that this gradient acts through TGFβ to control both the triggering and the progression of the wave of epithelial cell flattening. These data indicate that intrinsic nurse cell growth is important to control proper nurse cell pressure. Finally, it was revealed that nurse cell pressure and subsequent TGFβ activity in the stretched cells combine to increase follicle elongation in the anterior, which is crucial for allowing nurse cell growth and pressure control. More generally, the results reveal that during development, inner cytoplasmic pressure in individual cells has an important role in shaping their neighbors. |
Thursday, February 18th - Enhancers and gene regulation |
| Floc'hlay, S., Wong, E., Zhao, B., Viales, R. R., Thomas-Chollier, M., Thieffry, D., Garfield, D. A. and Furlong, E. E. M. (2020). Cis-acting variation is common across regulatory layers but is often buffered during embryonic development. Genome Res. PubMed ID: 33310749
Summary: Precise patterns of gene expression are driven by interactions between transcription factors, regulatory DNA sequence, and chromatin. How DNA mutations affecting any one of these regulatory 'layers' is buffered or propagated to gene expression remains unclear. To address this, allele-specific changes were quantified in chromatin accessibility, histone modifications, and gene expression in F1 embryos generated from eight Drosophila crosses at three embryonic stages, yielding a comprehensive dataset of 240 samples spanning multiple regulatory layers. Genetic variation (allelic imbalance) impacts gene expression more frequently than chromatin features, with metabolic and environmental response genes being most often affected. Allelic imbalance in cis-regulatory elements (enhancers) is common and highly heritable, yet its functional impact doesn't generally propagate to gene expression. When it does, genetic variation impacts RNA levels through H3K4me3 or independently through chromatin accessibility and H3K27ac. Changes in RNA are more predictive of variation in H3K4me3 than vice versa, suggesting a role for H3K4me3 downstream of transcription. The impact of a substantial proportion of genetic variation is consistent across embryonic stages, with 50% of allelic imbalanced features at one stage being also imbalanced at subsequent developmental stages. Crucially, buffering, as well as the magnitude and evolutionary impact of genetic variants, are influenced by regulatory complexity (i.e., number of enhancers regulating a gene), with transcription factors being most robust to cis-acting, but most influenced by trans-acting variation. | Lim, S. Y., You, H., Lee, J., Lee, J., Lee, Y., Lee, K. A., Kim, B., Lee, J. H., Jeong, J., Jang, S., Kim, B., Choi, H., Hwang, G., Choi, M. S., Yoon, S. E., Kwon, J. Y., Lee, W. J., Kim, Y. J. and Suh, G. S. B. (2020). Identification and characterization of GAL4 drivers that mark distinct cell types and regions in the Drosophila adult gut. J Neurogenet: 1-11. PubMed ID: 33326321
Summary: The gastrointestinal tract in the adult Drosophila serves as a model system for exploring the mechanisms underlying digestion, absorption and excretion, stem cell plasticity, and inter-organ communication, particularly through the gut-brain axis. It is also useful for studying the cellular and adaptive responses to dietary changes, alterations in microbiota and immunity, and systematic and endocrine signals. Despite the various cell types and distinct regions in the gastrointestinal tract, few tools are available to target and manipulate the activity of each cell type and region, and their gene expression. This study reports 353 GAL4 lines and several split-GAL4 lines that are expressed in enteric neurons (ENs), progenitors (ISCs and EBs), enterocytes (ECs), enteroendocrine cells (EEs), or/and other cell types that are yet to be identified in distinct regions of the gut. Initially approximately 600 GAL4 lines were collected that may be expressed in the gut based on RNA sequencing data, and then they were crossed to UAS-GFP to perform immunohistochemistry to identify those that are expressed selectively in the gut. The cell types and regional expression patterns that are associated with the entire set of GAL4 drivers and split-GAL4 combinations are annotated online (K-Gut Project). This GAL4 resource can be used to target specific populations of distinct cell types in the fly gut, and therefore, should permit a more precise investigation of gut cells that regulate important biological processes. |
| Judd, J., Duarte, F. M. and Lis, J. T. (2021). Pioneer-like factor GAF cooperates with PBAP (SWI/SNF) and NURF (ISWI) to regulate transcription. Genes Dev 35(1-2): 147-156. PubMed ID: 33303640
Summary: Transcriptionally silent genes must be activated throughout development. This requires nucleosomes be removed from promoters and enhancers to allow transcription factor (TF) binding and recruitment of coactivators and RNA polymerase II (Pol II). Specialized pioneer TFs bind nucleosome-wrapped DNA to perform this chromatin opening by mechanisms that remain incompletely understood. This study shows that GAGA factor (GAF), a Drosophila pioneer-like factor, functions with both SWI/SNF and ISWI family chromatin remodelers to allow recruitment of Pol II and entry to a promoter-proximal paused state, and also to promote Pol II's transition to productive elongation. GAF interacts with PBAP (SWI/SNF) to open chromatin and allow Pol II to be recruited. Importantly, this activity is not dependent on NURF as previously proposed; however, GAF also synergizes with NURF downstream from this process to ensure efficient Pol II pause release and transition to productive elongation, apparently through its role in precisely positioning the +1 nucleosome. These results demonstrate how a single sequence-specific pioneer TF can synergize with remodelers to activate sets of genes. Furthermore, this behavior of remodelers is consistent with findings in yeast and mice, and likely represents general, conserved mechanisms found throughout eukarya. | Bordet, G., Lodhi, N., Guo, D., Kossenkov, A. and Tulin, A. V. (2020). Poly(ADP-ribose) polymerase 1 in genome-wide expression control in Drosophila. Sci Rep 10(1): 21151. PubMed ID: 33273587
Summary: Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme involved in DNA repair and transcription regulation, among other processes. Malignant transformations, tumor progression, the onset of some neuropathies and other disorders have been linked to misregulation of PARP-1 activity. Despite intensive studies during the last few decades, the role of PARP-1 in transcription regulation is still not well understood. In this study, a transcriptomic analysis in Drosophila melanogaster third instar larvae was carried out. A total of 602 genes were identified, showing large-scale changes in their expression levels in the absence of PARP-1 in vivo. Among these genes, several functional gene groups were present, including transcription factors and cytochrome family members. The transcription levels of genes from the same functional group were affected by the absence of PARP-1 in a similar manner. In the absence of PARP-1, all misregulated genes coding for transcription factors were downregulated, whereas all genes coding for members of the cytochrome P450 family were upregulated. The cytochrome P450 proteins contain heme as a cofactor and are involved in oxidoreduction. Significant changes were also observed in the expression of several mobile elements in the absence of PARP-1, suggesting that PARP-1 may be involved in regulating the expression of mobile elements. |
| Domsch, K., Schroder, J., Janeschik, M., Schaub, C. and Lohmann, I. (2021). The Hox Transcription Factor Ubx Ensures Somatic Myogenesis by Suppressing the Mesodermal Master Regulator Twist. Cell Rep 34(1): 108577. PubMed ID: 33406430
Summary: Early lineage-specific master regulators are essential for the specification of cell types. However, once cells are committed to a specific fate, it is critical to restrict the activity of such factors to enable differentiation. To date, it remains unclear how these factors are silenced. Using the Drosophila mesoderm as a model and a comparative genomic approach, the Hox transcription factor Ultrabithorax (Ubx) was shown to be critical for the repression of the master regulator Twist. Mesoderm-specific Ubx loss-of-function experiments using CRISPR-Cas9 and overexpression studies demonstrate that Ubx majorly impacts twist transcription. A mechanistic analysis reveals that Ubx requires the NK-homeodomain protein Tinman to bind to the twist promoter. Furthermore, these factor interactions were found to be critical for silencing by recruiting the Polycomb DNA binding protein Pleiohomeotic. Altogether, these data reveal that Ubx is a critical player in mediating the silencing of Twist, which is crucial for coordinated muscle differentiation | Le Poul, Y., Xin, Y., Ling, L., Muhling, B., Jaenichen, R., Horl, D., Bunk, D., Harz, H., Leonhardt, H., Wang, Y., Osipova, E., Museridze, M., Dharmadhikari, D., Murphy, E., Rohs, R., Preibisch, S., Prud'homme, B. and Gompel, N. (2020). Regulatory encoding of quantitative variation in spatial activity of a Drosophila enhancer. Sci Adv 6(49). PubMed ID: 33268361
Summary: Developmental enhancers control the expression of genes prefiguring morphological patterns. The activity of an enhancer varies among cells of a tissue, but collectively, expression levels in individual cells constitute a spatial pattern of gene expression. How the spatial and quantitative regulatory information is encoded in an enhancer sequence is elusive. To link spatial pattern and activity levels of an enhancer, systematic mutations were used of the yellow spot enhancer, active in developing Drosophila wings; their effect was tested in a reporter assay. Moreover, an analytic framework was developed based on the comprehensive quantification of spatial reporter activity. The quantitative enhancer activity results from densely packed regulatory information along the sequence, and a complex interplay between activators and multiple tiers of repressors carves the spatial pattern. These results shed light on how an enhancer reads and integrates trans-regulatory landscape information to encode a spatial quantitative pattern. |
Wednesday February 17th - Disease Models |
| Katanaev, V. L., Kryuchkov, M., Averkov, V., Savitsky, M., Nikolaeva, K., Klimova, N., Khaustov, S. and Solis, G. P. (2020). HumanaFly: high-throughput transgenesis and expression of breast cancer transcripts in Drosophila eye discovers the RPS12-Wingless signaling axis. Sci Rep 10(1): 21013. PubMed ID: 33273532
Summary: Drosophila melanogaster has been a model for multiple human disease conditions, including cancer. Among Drosophila tissues, the eye development is particularly sensitive to perturbations of the embryonic signaling pathways, whose improper activation in humans underlies various forms of cancer. The HumanaFly project as been launched; human genes expressed in breast cancer patients are screened for their ability to aberrate development of the Drosophila eye, hoping to thus identify novel oncogenes. This study reports identification of a breast cancer transgene, which upon expression in Drosophila produces eye malformation similar to the famous Glazed phenotype discovered by Thomas Morgan and decades later dissected to originate from mis-expression of Wingless (Wg). Wg is the ortholog of human Wnt proteins serving as ligands to initiate the developmental/oncogenic Wnt signaling pathway. Through genetic experiments this transgene was shown to interact with the Wg production machinery, rather than with Wg signal transduction. In Drosophila imaginal discs, the transgene was shown to promote long-range diffusion of Wg, affecting expression of the Wg target genes. The transgene emerged to encode RPS12-a protein of the small ribosomal subunit overexpressed in several cancer types and known to also possess extra-ribosomal functions. This work identifies RPS12 as an unexpected regulator of secretion and activity of Wnts. As Wnt signaling is particularly important in the context of breast cancer initiation and progression, RPS12 might be implicated in tumorigenesis in this and other Wnt-dependent cancers. Continuation of the HumanaFly project may bring further discoveries on oncogenic mechanisms. | Khyati, Malik, I., Agrawal, N. and Kumar, V. (2020). Melatonin and curcumin reestablish disturbed circadian gene expressions and restore locomotion ability and eclosion behavior in Drosophila model of Huntington's disease. Chronobiol Int: 1-18. PubMed ID: 33334207
Summary: Deficit in locomotion (motor) ability and disturbance of the circadian behavior and sleep-wake pattern characterize Huntington's disease (HD). This study examined the disturbance of circadian timing with the progression of HD pathogenesis and tested the efficacy of melatonin and curcumin in preventing the motor deficit and disturbed eclosion behavior in the Drosophila model of HD. To examine circadian timing, mRNA expression was examined of genes of the transcriptional feedback (TF) loop that generates the near 24-h rhythmicity. qPCR was performed of the Period, Timeless, Clock, Cycle, Clockwork, and Cryptochrome genes in transgenic fly heads from elav-Gal4 (pan neuronal) and PDF-Gal4 (PDF-specific neurons) driver lines through the progression of HD disease post-eclosion, from day 1 to its terminal stage on day 13. Cycle was arrhythmic from day 1, but Period and Timeless became arrhythmic on day 13 of the HD pathogenesis in elav, but not PDF, neurons. Twenty-four-hour mRNA rhythms showed alteration in the waveform properties (mesor and amplitude, not acrophase), but not in the persistence, in both elav-Gal4 and PDF-Gal4 HD flies; however, disturbance of the clock gene rhythm was delayed in PDF-Gal4 flies. To assess the preventive effects on HD pathogenesis, flies of both driver lines were provided with melatonin (50, 100, or 150 μg) or curcumin (10 μM) in the diet commencing from the larval stage. Both melatonin (100 μg) and curcumin reestablished the 24-h pattern in mRNA expression of Period and Timeless to normal (control) levels, and significantly improved both locomotion ability and eclosion behavior of HD flies. It is suggested that the disturbance of circadian timekeeping progressively accelerated HD pathogenesis, possibly via modulation of the transcriptional state that resulted in the modification of the Huntington gene. These findings suggest melatonin and curcumin might be potential therapeutic agents for the treatment of HD in humans, although this needs specific investigation. |
| Gunaseelan, S., Wang, Z., Tong, V. K. J., Ming, S. W. S., Razar, R., Srimasorn, S., Ong, W. Y., Lim, K. L. and Chua, J. J. E. (2021). Loss of FEZ1, a gene deleted in Jacobsen syndrome, causes locomotion defects and early mortality by impairing motor neuron development. Hum Mol Genet. PubMed ID: 33395696
Summary: FEZ1-mediated axonal transport plays important roles in central nervous system development but its involvement in the peripheral nervous system is not well-characterised. FEZ1 is deleted in Jacobsen syndrome (JS), an 11q terminal deletion developmental disorder. JS patients display impaired psychomotor skills, including gross and fine motor delay, suggesting that FEZ1 deletion may be responsible for these phenotypes, given its association with the development of motor-related circuits. Supporting this hypothesis, the data shows that FEZ1 is selectively expressed in the rat brain and spinal cord. Its levels progressively increase over the developmental course of human motor neurons derived from embryonic stem cells. Deletion of FEZ1 strongly impaired axon and dendrite development, and significantly delayed the transport of synaptic proteins into developing neurites. Concurring with these observations, Drosophila unc-76 mutants showed severe locomotion impairments, accompanied by a strong reduction of synaptic boutons at neuromuscular junctions. These abnormalities were ameliorated by pharmacological activation of UNC-51/ATG1, a FEZ1-activating kinase, with rapamycin and metformin. Collectively, the results highlight a role for FEZ1 in motor neuron development and implicate its deletion as an underlying cause of motor impairments in JS patients. | Kratschmer, P., Lowe, S. A., Buhl, E., Chen, K. F., Kullmann, D. M., Pittman, A., Hodge, J. J. L. and Jepson, J. E. C. (2021). Impaired Pre-Motor Circuit Activity and Movement in a Drosophila Model of KCNMA1-Linked Dyskinesia. Mov Disord. PubMed ID: 33449381
Summary: Paroxysmal dyskinesias (PxDs) are characterized by involuntary movements and altered pre-motor circuit activity. This study utilized the fruit fly, Drosophila, to study a PxD linked to a gain-of-function (GOF) mutation in the KCNMA1/hSlo1 BK potassium channel (Drosophila homolog: Slowpoke). This study aimed to recreate the equivalent BK (big potassium) channel mutation in Drosophila. Attempts were made to determine how this mutation altered action potentials (APs) and synaptic release in vivo; to test whether this mutation disrupted pre-motor circuit function and locomotion; and to define neural circuits involved in locomotor disruption. A knock-in Drosophila model was generated using homologous recombination. Electrophysiological recordings and calcium-imaging were used to assess AP shape, neurotransmission, and the activity of the larval pre-motor central pattern generator (CPG). Video-tracking and automated systems were used to measure movement, and developed a genetic method to limit BK channel expression to defined circuits. Neuronal APs exhibited reduced width and an enhanced afterhyperpolarization in the PxD model. Calcium-dependent reductions were identified in neurotransmitter release, dysfunction of the CPG, and corresponding alterations in movement, in model larvae. Finally, aberrant locomotion and dyskinesia-like movements were identified in adult model flies, and the impact of GOF BK channels on movement was partially mapped to cholinergic neurons. This model supports a link between BK channel GOF and hyperkinetic movements, and provides a platform to dissect the mechanistic basis of PxDs. |
| Liang, Z., Chan, H. Y. E., Lee, M. M. and Chan, M. K. (2021). A SUMO1-derived peptide targeting SUMO-interacting motif inhibits α-synuclein aggregation. Cell Chem Biol. PubMed ID: 33444530
Summary: The accumulation of α-synuclein amyloid fibrils in the brain is linked to Parkinson's disease and other synucleinopathies. The intermediate species in the early aggregation phase of α-synuclein are involved in the emergence of amyloid toxicity and considered to be the most neurotoxic. The N-terminal region flanking the non-amyloid-β component domain of α-synuclein has been implicated in modulating its aggregation. This study reports the development of a SUMO1-derived peptide inhibitor (SUMO1(15-55); see Drosophila Sumo), which targets two SUMO-interacting motifs (SIMs) within this aggregation-regulating region and suppresses α-synuclein aggregation. Molecular modeling, site-directed mutagenesis, and binding studies are used to elucidate the mode of interaction, namely, via the binding of either of the two SIM sequences on α-synuclein to a putative hydrophobic binding groove on SUMO1(15-55). Subsequent studies show that SUMO1(15-55) also reduces α-synuclein-induced cytotoxicity in cell-based and Drosophila disease models. | Konar, A., Kalra, R. S., Chaudhary, A., Nayak, A., Guruprasad, K. P., Satyamoorthy, K., Ishida, Y., Terao, K., Kaul, S. C. and Wadhwa, R. (2020). Identification of Caffeic Acid Phenethyl Ester (CAPE) as a Potent Neurodifferentiating Natural Compound That Improves Cognitive and Physiological Functions in Animal Models of Neurodegenerative Diseases. Front Aging Neurosci 12: 561925. PubMed ID: 33244299
Summary: Using human neuroblastoma cells, this study identified caffeic acid phenethyl ester (CAPE) as a potent neurodifferentiating natural compound. Analyses of control and CAPE-induced neurodifferentiated cells revealed: (i) modulation of several key proteins (NF200, MAP-2, NeuN, PSD95, Tuj1, GAP43, and GFAP) involved in neurodifferentiation process; and (ii) attenuation of neuronal stemness (HOXD13, WNT3, and Msh-2) and proliferation-promoting (CDC-20, CDK-7, and BubR1) proteins. It is anticipated that the neurodifferentiation potential of CAPE was tested using the Drosophila model of Alzheimer's disease (AD) and mice model of amnesia/loss of memory. In both models, CAPE exhibited improved disease symptoms and activation of physiological functions. Remarkably, CAPE-treated mice showed increased levels of neurotrophin-BDNF, neural progenitor marker-Nestin, and differentiation marker-NeuN, both in the cerebral cortex and hippocampus. Taken together, this study demonstrated the differentiation-inducing and therapeutic potential of CAPE for neurodegenerative diseases. |
Tuesday, February 17th - Synapse and Vesicles |
| Kawamura, H., Hakeda-Suzuki, S. and Suzuki, T. (2020). Activity-dependent endocytosis of Wingless regulates synaptic plasticity in the Drosophila visual system. Genes Genet Syst. PubMed ID: 33298662
Summary: Neural activity contributes to synaptic regulation in sensory systems, which allows organisms to adjust to changing environments. However, little is known about how synaptic molecular components are regulated to achieve activity-dependent plasticity at central synapses. Previous studies have shown that following prolonged exposure to natural ambient light, the presynaptic active zone (AZ), an area associated with presynaptic neurotransmitter release in Drosophila photoreceptors, undergoes reversible remodeling. Other studies suggest that the secretory protein Wingless (Wg; an ortholog of Wnt-1) can mediate communication between synaptic cells to achieve synaptic remodeling. However, the source of Wg and the mechanism of Wg signal modulation by neuronal activity remained unclear. This study found that Wg secreted from glial cells regulates synaptic remodeling in photoreceptors. In addition, antibody staining revealed that Wg changes its localization depending on light conditions. Although Wg is secreted from glial cells, Wg appeared inside photoreceptor axons when flies were kept under light conditions, suggesting that an increase in neuronal activity causes Wg internalization into photoreceptors by endocytosis. Indeed, by blocking endocytosis in photoreceptors, the localization of Wg in photoreceptors disappeared. Interestingly, Wg accumulation was higher in axons with disassembled AZ structure than in axons whose AZ structure was stabilized at the single-cell level, indicating that Wg endocytosis may trigger AZ disassembly. Furthermore, when Wg signaling was genetically activated, Wg accumulation in photoreceptors decreased. Conversely, when Wg signaling was suppressed there was an increase in Wg accumulation. Through RNAi screening of Ca(2+)-binding proteins in photoreceptors, it was found that Calcineurin is a key molecule that triggers Wg endocytosis. Overall, it is proposed that Wg signaling is regulated by a negative feedback loop driven by Wg endocytosis. The increase in neuronal activity is transmitted via calcium signaling, which leads to a decrease in Wg signaling and thereby promotes presynaptic remodeling. | Baeumers, M., Ruhnau, K., Breuer, T., Pannen, H., Goerlich, B., Kniebel, A., Haensch, S., Weidtkamp-Peters, S., Schmitt, L. and Klein, T. (2020). Lethal (2) giant discs (Lgd)/CC2D1 is required for the full activity of the ESCRT machinery. BMC Biol 18(1): 200. PubMed ID: 33349255
Summary: A major task of the endosomal sorting complex required for transport (ESCRT) machinery is the pinching off of cargo-loaded intraluminal vesicles (ILVs) into the lumen of maturing endosomes (MEs), which is essential for the complete degradation of transmembrane proteins in the lysosome. The ESCRT machinery is also required for the termination of signalling through activated signalling receptors, as it separates their intracellular domains from the cytosol. The ESCRT-III complex is required for an increasing number of processes where membrane regions are abscised away from the cytosol. The core of ESCRT-III, comprising four members of the CHMP protein family, organises the assembly of a homopolymer of CHMP4, Shrub in Drosophila, that is essential for abscission. The tumour-suppressor lethal (2) giant discs (Lgd)/CC2D1 is a physical interactor of Shrub/CHMP4 in Drosophila and mammals, respectively. This study shows that the loss of function of lgd constitutes a state of reduced activity of Shrub/CHMP4/ESCRT-III. The forced incorporation in ILVs of lgd mutant MEs suppresses the uncontrolled and ligand-independent activation of Notch. Moreover, the analysis of Su(dx) lgd double mutants clarifies their relationship and suggests that they are not operating in a linear pathway. Since lgd mutants can be rescued to normal adult flies if extra copies of shrub (or its mammalian ortholog CHMP4B) are added into the genome, it is concluded that the net activity of Shrub is reduced upon loss of lgd function. In solution, CHMP4B/Shrub exists in two conformations. LGD1/Lgd binding does not affect the conformational state of Shrub, suggesting that Lgd is not a chaperone for Shrub/CHMP4B. These results suggest that Lgd is required for the full activity of Shrub/ESCRT-III. In its absence, the activity of the ESCRT machinery is reduced. This reduction causes the escape of a fraction of cargo, among it Notch, from incorporation into ILVs, which in turn leads to an activation of this fraction of Notch after fusion of the ME with the lysosome. These results highlight the importance of the incorporation of Notch into ILV not only to assure complete degradation, but also to avoid uncontrolled activation of the pathway. |
| Han, K. A., Kim, Y. J., Yoon, T. H., Kim, H., Bae, S., Um, J. W., Choi, S. Y. and Ko, J. (2020). LAR-RPTPs Directly interact with Neurexins to coordinate bidirectional assembly of molecular machineries. J Neurosci 40(44): 8438-8462. PubMed ID: 33037075
Summary: Neurexins (Nrxns) and LAR-RPTPs (leukocyte common antigen-related protein tyrosine phosphatases) are presynaptic adhesion proteins responsible for organizing presynaptic machineries through interactions with nonoverlapping extracellular ligands. This study reports that two members of the LAR-RPTP family, PTPσ and PTPδ, are required for the presynaptogenic activity of Nrxns. Intriguingly, Nrxn1 and PTPσ require distinct sets of intracellular proteins for the assembly of specific presynaptic terminals. In addition, Nrxn1α showed robust heparan sulfate (HS)-dependent, high-affinity interactions with Ig domains of PTPσ that were regulated by the splicing status of PTPσ. Furthermore, Nrxn1α WT, but not a Nrxn1α mutant lacking HS moieties (Nrxn1α ΔHS), inhibited postsynapse-inducing activity of PTPσ at excitatory, but not inhibitory, synapses. Similarly, cis expression of Nrxn1α WT, but not Nrxn1α ΔHS, suppressed the PTPσ-mediated maintenance of excitatory postsynaptic specializations in mouse cultured hippocampal neurons. Lastly, genetics analyses using male or female Drosophila Dlar and Dnrx mutant larvae identified epistatic interactions that control synapse formation and synaptic transmission at neuromuscular junctions. These results suggest a novel synaptogenesis model whereby different presynaptic adhesion molecules combine with distinct regulatory codes to orchestrate specific synaptic adhesion pathways. | Li, T. N., Chen, Y. J., Lu, T. Y., Wang, Y. T., Lin, H. C. and Yao, C. K. (2020). A positive feedback loop between Flower and PI(4,5)P(2) at periactive zones controls bulk endocytosis in Drosophila. Elife 9. PubMed ID: 33300871
Summary: Synaptic vesicle (SV) endocytosis is coupled to exocytosis to maintain SV pool size and thus neurotransmitter release. Intense stimulation induces activity-dependent bulk endocytosis (ADBE) to recapture large quantities of SV constituents in large endosomes from which SVs reform. How these consecutive processes are spatiotemporally coordinated remains unknown. This study shows that Flower Ca(2+) channel-dependent phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) compartmentalization governs control of these processes in Drosophila. Strong stimuli trigger PI(4,5)P(2) microdomain formation at larval neuromuscular junction periactive zones. Upon exocytosis, Flower translocates from SVs to periactive zones, where it increases PI(4,5)P(2) levels via Ca(2+) influxes. Remarkably, PI(4,5)P(2) directly enhances Flower channel activity, thereby establishing a positive feedback loop for PI(4,5)P(2) microdomain compartmentalization. PI(4,5)P(2) microdomains drive ADBE and SV reformation from bulk endosomes. PI(4,5)P(2) further retrieves Flower to bulk endosomes, terminating endocytosis. It is proposed that the interplay between Flower and PI(4,5)P(2) is the crucial spatiotemporal cue that couples exocytosis to ADBE and subsequent SV reformation. |
| de la Riva Carrasco, R., Perez Pandolfo, S., Freire, S. S., Romero, N. M., Bhujabal, Z., Johansen, T., Wappner, P. and Melani, M. (2020). The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues. Traffic. PubMed ID: 33336828
Summary: Exocytosis is a fundamental process in physiology, communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. This study reports a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, Zda was found to be required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen, the small GTPase RalA was identified as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane. | Fujii, S., Kurokawa, K., Tago, T., Inaba, R., Takiguchi, A., Nakano, A., Satoh, T. and Satoh, A. K. (2020). Sec71 separates Golgi stacks in Drosophila S2 cells. J Cell Sci 133(24). PubMed ID: 33262309
Summary: Golgi stacks are the basic structural units of the Golgi. Golgi stacks are separated from each other and scattered in the cytoplasm of Drosophila cells. This study reports that the ARF-GEF inhibitor Brefeldin A (BFA) induces the formation of BFA bodies, which are aggregates of Golgi stacks, trans-Golgi networks and recycling endosomes. Recycling endosomes are located in the centers of BFA bodies, while Golgi stacks surround them on their trans sides. Live imaging of S2 cells revealed that Golgi stacks repeatedly merged and separated on their trans sides, and BFA caused successive merger by inhibiting separation, forming BFA bodies. S2 cells carrying genome-edited BFA-resistant mutant Sec71(M717L) did not form BFA bodies at high concentrations of BFA; S2 cells carrying genome-edited BFA-hypersensitive mutant Sec71(F713Y) produced BFA bodies at low concentrations of BFA. These results indicate that Sec71 is the sole BFA target for BFA body formation and controls Golgi stack separation. Finally, this study showed that impairment of Sec71 in fly photoreceptors induces BFA body formation, with accumulation of both apical and basolateral cargoes, resulting in inhibition of polarized transport. |
Monday, February 15th - Adult physiology |
| Hofbauer, H. F., Heier, C., Sen Saji, A. K. and Kuhnlein, R. P. (2020). Lipidome remodeling in aging normal and genetically obese Drosophila males. Insect Biochem Mol Biol: 103498. PubMed ID: 33221388
Summary: Lipid homeostasis is essential for insects to maintain phospholipid (PL)-based membrane integrity and to provide on-demand energy supply throughout life. Triacylglycerol (TAG) is the major lipid class used for energy production and is stored in lipid droplets, the universal cellular fat storage organelles. Accumulation and mobilization of TAG are strictly regulated since excessive accumulation of TAG leads to obesity and has been correlated with adverse effects on health- and lifespan across phyla. Little is known, however, about when during adult life and why excessive storage lipid accumulation restricts lifespan. This study used genetically obese Drosophila mutant males, which were all shown to be short-lived compared to control males and applied single fly mass spectrometry-based lipidomics to profile TAG, diacylglycerol and major membrane lipid signatures throughout adult fly life from eclosion to death. This comparative approach revealed distinct phases of lipidome remodeling throughout aging. Quantitative and qualitative compositional changes of TAG and PL species, which are characterized by the length and saturation of their constituent fatty acids, were pronounced during young adult life. In contrast, lipid signatures of adult and senescent flies were remarkably stable. Genetically obese flies displayed both quantitative and qualitative changes in TAG species composition, while PL signatures were almost unaltered compared to normal flies at all ages. Collectively, this suggests a tight control of membrane composition throughout lifetime largely uncoupled from storage lipid metabolism. Finally, evidence is presented for a characteristic lipid signature of moribund flies, likely generated by a rapid and selective storage lipid depletion close to death. Of note, the analytical power to monitor lipid species profiles combined with high sensitivity of this single fly lipidomics approach is universally applicable to address developmental or behavioral lipid signature modulations of importance for insect life. | Idda, T., Bonas, C., Hoffmann, J., Bertram, J., Quinete, N., Schettgen, T., Fietkau, K., Esser, A., Stope, M. B., Leijs, M. M., Baron, J. M., Kraus, T., Voigt, A. and Ziegler, P. (2020). Metabolic activation and toxicological evaluation of polychlorinated biphenyls in Drosophila melanogaster. Sci Rep 10(1): 21587. PubMed ID: 33299007
Summary: Degradation of polychlorinated biphenyls (PCBs) is initiated by cytochrome P450 (CYP) enzymes and includes PCB oxidation to OH-metabolites, which often display a higher toxicity than their parental compounds. In search of an animal model reflecting PCB metabolism and toxicity, Drosophila melanogaster, a well-known model system for genetics and human disease, was tested. Feeding Drosophila with lower chlorinated (LC) PCB congeners 28, 52 or 101 resulted in the detection of a human-like pattern of respective OH-metabolites in fly lysates. Feeding flies high PCB 28 concentrations caused lethality. Thus selected CYPs were silenced via RNA interference, and the effect on PCB 28-derived metabolite formation by assaying 3-OH-2',4,4'-trichlorobiphenyl (3-OHCB 28) and 3'-OH-4',4,6'-trichlorobiphenyl (3'-OHCB 28) in fly lysates was analyzed. Several Drosophila CYPs (dCYPs) were identified whose knockdown reduced PCB 28-derived OH-metabolites and suppressed PCB 28 induced lethality including dCYP1A2. Following in vitro analysis using a liver-like CYP-cocktail, containing human orthologues of dCYP1A2, this study confirmed human CYP1A2 as a PCB 28 metabolizing enzyme. PCB 28-induced mortality in flies was accompanied by locomotor impairment, a common phenotype of neurodegenerative disorders. Along this line, it was shown that PCB 28-initiated caspase activation in differentiated fly neurons. This suggested the loss of neurons through apoptosis. These findings in flies are congruent with observation in human exposed to high PCB levels. In plasma samples of PCB exposed humans, levels of the neurofilament light chain increase after LC-PCB exposure, indicating neuronal damage. In summary these findings demonstrate parallels between Drosophila and the human systems with respect to CYP mediated metabolism and PCB mediated neurotoxicity. |
| Jacomin, A. C., Gohel, R., Hussain, Z., Varga, A., Maruzs, T., Eddison, M., Sica, M., Jain, A., Moffat, K. G., Johansen, T., Jenny, A., Juhasz, G. and Nezis, I. P. (2021). Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila. Life Sci Alliance 4(2). PubMed ID: 33318080
Summary: Hunger drives food-seeking behaviour and controls adaptation of organisms to nutrient availability and energy stores. Lipids constitute an essential source of energy in the cell that can be mobilised during fasting by autophagy. Selective degradation of proteins by autophagy is made possible essentially by the presence of LIR and KFERQ-like motifs. Using in silico screening of Drosophila proteins that contain KFERQ-like motifs, the adaptor protein Arouser, which functions to regulate fat storage and mobilisation and is essential during periods of food deprivation, was identified and characterized. Hypomorphic arouser mutants are not satiated, are more sensitive to food deprivation, and are more aggressive, suggesting an essential role for Arouser in the coordination of metabolism and food-related behaviour. This analysis shows that Arouser functions in the fat body through nutrient-related signalling pathways and is degraded by endosomal microautophagy. Arouser degradation occurs during feeding conditions, whereas its stabilisation during non-feeding periods is essential for resistance to starvation and survival. In summary, these data describe a novel role for endosomal microautophagy in energy homeostasis, by the degradation of the signalling regulatory protein Arouser. | Klepsatel, P., Girish, T. N. and Galikova, M. (2020). Acclimation temperature affects thermal reaction norms for energy reserves in Drosophila. Sci Rep 10(1): 21681. PubMed ID: 33303846
Summary: Organisms have evolved various physiological mechanisms to cope with unfavourable environmental conditions. The ability to tolerate non-optimal thermal conditions can be substantially improved by acclimation. This study examined how an early-life acclimation to different temperatures (19 °C, 25 °C and 29 °C) influences thermal reaction norms for energy stores in Drosophila adults. The results show that acclimation temperature has a significant effect on the amount of stored fat and glycogen (and their relative changes) and the optimal temperature for their accumulation. Individuals acclimated to 19 °C had, on average, more energy reserves than flies that were initially maintained at 25 °C or 29 °C. In addition, acclimation caused a shift in optimal temperature for energy stores towards acclimation temperature. Significant population differences in this response were detected. The effect of acclimation on the optimal temperature for energy stores was more pronounced in flies from the temperate climate zone (Slovakia) than in individuals from the tropical zone (India). Overall, it was found that the acclimation effect was stronger after acclimation to low (19 °C) than to high (29 °C) temperature. The observed sensitivity of thermal reaction norms for energy reserves to acclimation temperature can have important consequences for surviving periods of food scarcity, especially at suboptimal temperatures. |
| Gnainsky, Y., Zfanya, N., Elgart, M., Omri, E., Brandis, A., Mehlman, T., Itkin, M., Malitsky, S., Adamski, J. and Soen, Y. (2021). Systemic Regulation of Host Energy and Oogenesis by Microbiome-Derived Mitochondrial Coenzymes. Cell Rep 34(1): 108583. PubMed ID: 33406416
Summary: Gut microbiota have been shown to promote oogenesis and fecundity, but the mechanistic basis of remote influence on oogenesis remained unknown. This study reports a systemic mechanism of influence mediated by bacterial-derived supply of mitochondrial coenzymes. Removal of microbiota decreased mitochondrial activity and ATP levels in the whole-body and ovary, resulting in repressed oogenesis. Similar repression was caused by RNA-based knockdown of mitochondrial function in ovarian follicle cells. Reduced mitochondrial function in germ-free (GF) females was reversed by bacterial recolonization or supplementation of riboflavin, a precursor of FAD and FMN. Metabolomics analysis of GF females revealed a decrease in oxidative phosphorylation and FAD levels and an increase in metabolites that are degraded by FAD-dependent enzymes (e.g., amino and fatty acids). Riboflavin supplementation opposed this effect, elevating mitochondrial function, ATP, and oogenesis. These findings uncover a bacterial-mitochondrial axis of influence, linking gut bacteria with systemic regulation of host energy and reproduction. | Lee, H. J., Lee, S. H., Lee, J. H., Kim, Y., Seong, K. M., Jin, Y. W. and Min, K. J. (2020). Role of Commensal Microbes in the gamma-Ray Irradiation-Induced Physiological Changes in Drosophila melanogaster. Microorganisms 9(1). PubMed ID: 33374132
Summary: Ionizing radiation induces biological/physiological changes and affects commensal microbes, but few studies have examined the relationship between the physiological changes induced by irradiation and commensal microbes. This study investigated the role of commensal microbes in the γ-ray irradiation-induced physiological changes in Drosophila melanogaster. The bacterial load was increased in 5 Gy irradiated flies, but irradiation decreased the number of operational taxonomic units. The mean lifespan of conventional flies showed no significant change by irradiation, whereas that of axenic flies was negatively correlated with the radiation dose. γ-Ray irradiation did not change the average number of eggs in both conventional and axenic flies. Locomotion of conventional flies was decreased after 5 Gy radiation exposure, whereas no significant change in locomotion activity was detected in axenic flies after irradiation. γ-Ray irradiation increased the generation of reactive oxygen species in both conventional and axenic flies, but the increase was higher in axenic flies. Similarly, the amounts of mitochondria were increased in irradiated axenic flies but not in conventional flies. These results suggest that axenic flies are more sensitive in their mitochondrial responses to radiation than conventional flies, and increased sensitivity leads to a reduced lifespan and other physiological changes in axenic flies. |
| Fan, X., Zeng, Y., Fan, Z., Cui, L., Song, W., Wu, Q., Gao, Y., Yang, D., Mao, X., Zeng, B., Zhang, M., Ni, Q., Li, Y., Wang, T., Li, D. and Yang, M. (2020). Dihydromyricetin promotes longevity and activates the transcription factors FOXO and AOP in Drosophila. Aging (Albany NY) 12. PubMed ID: 33291074
Summary: Drugs or compounds have been shown to promote longevity in various approaches. This study used Drosophila to explore novel natural compounds can be applied to anti-aging. A flavonoid named Dihydromyricetin can increase stress that tolerance and lipid levels, slow down gut dysfunction and extend Drosophila lifespan. Dihydromyricetin can also lessen pERK and pAKT signaling, consequently activating FOXO and AOP to modulate longevity. These results suggested that DHM could be used as an effective compound for anti-aging intervention, which could likely be applied to both mammals and humans. | Kashio, S. and Miura, M. (2020). Kynurenine Metabolism in the Fat Body Non-autonomously Regulates Imaginal Disc Repair in Drosophila. iScience 23(12): 101738. PubMed ID: 33376969
Summary: issue interactions are critical for maintaining homeostasis; however, little is known about how remote tissue regulates regeneration. Previous work established a genetic dual system that induces cell ablation in Drosophila larval imaginal discs and simultaneously manipulates genes in non-damaged tissues. Using humoral metabolome analysis and a genetic damage system, this study found that the Tryptophan (Trp)-Kynurenine (Kyn) pathway in the fat body is required for disc repair. Genetic manipulation of Trp-Kyn metabolism in the fat body impaired disc regeneration without affecting wing development. In particular, the fat body-derived humoral kynurenic acid (KynA) was required for disc repair. The impairment of S-adenosylmethionine (SAM) synthesis from methionine (Met) in the fat body hampers the maintenance of KynA levels in hemolymph at the early stage of disc repair, suggesting a connection between Met-SAM and Trp-Kyn metabolisms. These data indicate KynA from the fat body acts as a permissive metabolite for tissue repair and regeneration. |
Friday, February 12th - Adult neural function |
| Hsu, C. T., Choi, J. T. Y. and Sehgal, A. (2020). Manipulations of the olfactory circuit highlight the role of sensory stimulation in regulating sleep amount. Sleep. PubMed ID: 33313876
Summary: While wake duration is a major sleep driver, an important question is if wake quality also contributes to controlling sleep. In particular, this study sought to determine whether changes in sensory stimulation affect sleep in Drosophila. As Drosophila rely heavily on their sense of smell, this study focused on manipulating olfactory input and the olfactory sensory pathway. Sensory deprivation was first performed by removing antennae or applying glue to antennae. Sleep was then measured in response to neural activation, via TRPA1, or inhibition, via KIR2.1, of subpopulations of neurons in the olfactory pathway. Genetically restricting manipulations to adult animals prevented developmental effects. This study found that olfactory deprivation reduces sleep, largely independently of mushroom bodies that integrate olfactory signals for memory consolidation and have previously been implicated in sleep. However, specific neurons in the lateral horn, the other third order target of olfactory input, affect sleep. Also, activation of inhibitory second order projection neurons increases sleep. No single neuronal population in the olfactory processing pathway was found to bidirectionally regulate sleep, and reduced sleep in response to olfactory deprivation may be masked by temperature changes. These findings demonstrate that Drosophila sleep is sensitive to sensory stimulation, and identify novel sleep-regulating neurons in the olfactory circuit. Scaling of signals across the circuit may explain the lack of bidirectional effects when neuronal activity is manipulated. It is proposed that olfactory inputs act through specific circuit components to modulate sleep in flies. | Hsu, J. M., Kang, Y., Corty, M. M., Mathieson, D., Peters, O. M. and Freeman, M. R. (2020). Injury-Induced Inhibition of Bystander Neurons Requires dSarm and Signaling from Glia. Neuron. PubMed ID: 33296670
Summary: Nervous system injury and disease have broad effects on the functional connectivity of the nervous system, but how injury signals are spread across neural circuits remains unclear. This study explored how axotomy changes the physiology of severed axons and adjacent uninjured "bystander" neurons in a simple in vivo nerve preparation. Within hours after injury, suppression of axon transport was observed in all axons, whether injured or not, and decreased mechano- and chemosensory signal transduction was observed in uninjured bystander neurons. Unexpectedly, it was found the axon death molecule dSarm, but not its NAD(+) hydrolase activity, was required cell autonomously for these early changes in neuronal cell biology in bystander neurons, as were the voltage-gated calcium channel Cacophony (Cac) and the mitogen-activated protein kinase (MAPK) signaling cascade. Bystander neurons functionally recovered at later time points, while severed axons degenerated via α/Armadillo/Toll-interleukin receptor homology domain (dSarm)/Axundead signaling, and independently of Cac/MAPK. Interestingly, suppression of bystander neuron function required Draper/MEGF10 signaling in glia, indicating glial cells spread injury signals and actively suppress bystander neuron function. This work identifies a new role for dSarm and glia in suppression of bystander neuron function after injury and defines two genetically and temporally separable phases of dSarm signaling in the injured nervous system. |
| Kessissoglou, I. A., Langui, D., Hasan, A., Maral, M., Dutta, S. B., Hiesinger, P. R. and Hassan, B. A. (2020). The Drosophila amyloid precursor protein homologue mediates neuronal survival and neuroglial interactions. PLoS Biol 18(12): e3000703. PubMed ID: 33290404
Summary: The amyloid precursor protein (APP) is a structurally and functionally conserved transmembrane protein whose physiological role in adult brain function and health is still unclear. Because mutations in APP cause familial Alzheimer's disease (fAD), most research focuses on this aspect of APP biology. This study investigated the physiological function of APP in the adult brain using the fruit fly Drosophila melanogaster, which harbors a single APP homologue called APP Like (APPL). Previous studies have provided evidence for the implication of APPL in neuronal wiring and axonal growth through the Wnt signaling pathway during development. However, like APP, APPL continues to be expressed in all neurons of the adult brain where its functions and their molecular and cellular underpinnings are unknown. This study reports that APPL loss of function (LOF) results in the dysregulation of endolysosomal function in neurons, with a notable enlargement of early endosomal compartments followed by neuronal cell death and the accumulation of dead neurons in the brain during a critical period at a young age. These defects can be rescued by reduction in the levels of the early endosomal regulator Rab5, indicating a causal role of endosomal function for cell death. Finally, this study shows that the secreted extracellular domain of APPL interacts with glia and regulates the size of their endosomes, the expression of the Draper engulfment receptor, and the clearance of neuronal debris in an axotomy model. It is proposes that APP proteins represent a novel family of neuroglial signaling factors required for adult brain homeostasis. | Wang, Y., Lobb-Rabe, M., Ashley, J., Anand, V. and Carrillo, R. A. (2021). Structural and functional synaptic plasticity induced by convergent synapse loss in the Drosophila neuromuscular circuit. J Neurosci. PubMed ID: 33402422
Summary: Throughout the nervous system, the convergence of two or more presynaptic inputs on a target cell is commonly observed. The question asked in this study is to what extent converging inputs influence each other's structural and functional synaptic plasticity. In complex circuits, isolating individual inputs is difficult because postsynaptic cells can receive thousands of inputs. An ideal model to address this question is the Drosophila larval neuromuscular junction (NMJ) where each postsynaptic muscle cell receives inputs from two glutamatergic types of motor neurons (MNs), known as 1b and 1s MNs. Notably, each muscle is unique and receives input from a different combination of 1b and 1s MNs; multiple muscles were surveyed for this reason. A cell-specific promoter was identified that allows ablation of 1s MNs post-innervation; structural and functional responses of convergent 1b NMJs were measured using microscopy and electrophysiology. For all muscles examined in both sexes, ablation of 1s MNs resulted in NMJ expansion and increased spontaneous neurotransmitter release at corresponding 1b NMJs. This demonstrates that 1b NMJs can compensate for the loss of convergent 1s MNs. However, only a subset of 1b NMJs showed compensatory evoked neurotransmission, suggesting target-specific plasticity. Silencing 1s MNs led to similar plasticity at 1b NMJs, suggesting that evoked neurotransmission from 1s MNs contributes to 1b synaptic plasticity. Finally, 1s innervation in male larvae was genetically blocked and robust 1b synaptic plasticity was eliminated, raising the possibility that 1s NMJ formation is required to set up a reference for subsequent synaptic perturbations. |
| Hou, X., Outhwaite, I. R., Pedi, L. and Long, S. B. (2020). Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation. Elife 9. PubMed ID: 33252040
Summary: The calcium release-activated calcium channel Orai regulates Ca(2+) entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca(2+) release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), low (6.7 Å) resolution X-ray structural information was obtained on Drosophila melanogaster Orai in an open conformation. This paper presents a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-electron microscopy. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel's gate. Structured M1-M2 turrets, not evident previously, form the channel's extracellular entrance. | Zhao, K., Hong, H., Zhao, L., Huang, S., Gao, Y., Metwally, E., Jiang, Y., Sigrist, S. J. and Zhang, Y. Q. (2020). Postsynaptic cAMP signalling regulates the antagonistic balance of Drosophila glutamate receptor subtypes. Development 147(24). PubMed ID: 33234716
Summary: The balance among different subtypes of glutamate receptors (GluRs) is crucial for synaptic function and plasticity at excitatory synapses. This study shows that the two subtypes of GluRs (A and B) expressed at Drosophila neuromuscular junction synapses mutually antagonize each other in terms of their relative synaptic levels and affect subsynaptic localization of each other. Upon temperature shift-induced neuromuscular junction plasticity, GluR subtype A increased but subtype B decreased with a timecourse of hours. Inhibition of the activity of GluR subtype A led to imbalance of GluR subtypes towards more GluRIIA. To gain a better understanding of the signalling pathways underlying the balance of GluR subtypes, an RNA interference screen of candidate genes was performed and postsynaptic-specific knockdown of dunce, which encodes cAMP phosphodiesterase, was found to increase levels of GluR subtype A but decreased subtype B. Furthermore, bidirectional alterations of postsynaptic cAMP signalling resulted in the same antagonistic regulation of the two GluR subtypes. These findings thus identify a direct role of postsynaptic cAMP signalling in control of the plasticity-related balance of GluRs. |
Thursday February 11th - Signaling |
| Khor, S. and Cai, D. (2020). Control of lifespan and survival by Drosophila NF-κB signaling through neuroendocrine cells and neuroblasts. Aging (Albany NY) 12(24): 24604-24622. PubMed ID: 33232282
Summary: This paper reports a comparative analysis of the effects of immune activation in the fly nervous system using genetic activation models to target Drosophila NF-κB within Toll versus Imd pathways. Genetic gain-of-function models for either pathway pan-neuronally, as well as in discrete subsets of neural cells including neuroendocrine insulin-producing cells (IPCs) or neuroblasts, reduce fly lifespan, however, these phenotypes in IPCs and neuroblasts are stronger with Toll activation than Imd activation. Of note, while aging is influenced more by Toll/NF-κB activation in IPCs during adulthood, neuroblasts influence aging more substantially during development. The study then focused on Toll/NF-κB inhibition, revealing that IPCs or neuroblasts are important for the effects of lifespan and healthspan extension but in a life stage-dependent manner while some of these effects display sexual dimorphism. Importantly, co-inhibition of Toll/NF-κB pathway in IPCs and neuroblasts increased fly lifespan greater than either cell population, suggesting that independent mechanisms might exist. Toll/NF-κB inhibition in IPCs was also sufficient to enhance survival under various fatal stresses, supporting the additional benefits to fly healthspan. In conclusion, IPCs and neuroblasts are important for Drosophila NF-κB for controlling lifespan. | Kim, M. J. and O'Connor, M. B. (2021). Drosophila Activin signaling promotes muscle growth through InR/TORC1-dependent and -independent processes. Development 148(1). PubMed ID: 33234715
Summary: The Myostatin/Activin branch of the TGF-β superfamily acts as a negative regulator of vertebrate skeletal muscle size, in part, through downregulation of insulin/insulin-like growth factor 1 (IGF-1) signaling. Surprisingly, recent studies in Drosophila indicate that motoneuron-derived Activin signaling acts as a positive regulator of muscle size. This study demonstrates that Drosophila Activin signaling promotes the growth of muscle cells along all three axes: width, thickness and length. Activin signaling positively regulates the insulin receptor (InR)/TORC1 pathway and the level of Myosin heavy chain (Mhc), an essential sarcomeric protein, via increased Pdk1 and Akt1 expression. Enhancing InR/TORC1 signaling in the muscle of Activin pathway mutants restores Mhc levels close to those of the wild type, but only increases muscle width. In contrast, hyperactivation of the Activin pathway in muscles increases overall larval body and muscle fiber length, even when Mhc levels are lowered by suppression of TORC1. Together, these results indicate that the Drosophila Activin pathway regulates larval muscle geometry and body size via promoting InR/TORC1-dependent Mhc production and the differential assembly of sarcomeric components into either pre-existing or new sarcomeric units depending on the balance of InR/TORC1 and Activin signals |
| Karman, Z., Rethi-Nagy, Z., Abraham, E., Fabri-Ordogh, L., Csonka, A., Vilmos, P., Debski, J., Dadlez, M., Glover, D. M. and Lipinszki, Z. (2020). Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase. Open Biol 10(12): 200343. PubMed ID: 33352067
Summary: Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. This study identified novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel and demonstrated that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. It is proposed that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, the data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance understanding of the substrate-recognition mechanisms and function of PP4. | Jagga, B., Edwards, M., Pagin, M., Wagstaff, K. M., Aragao, D., Roman, N., Nanson, J. D., Raidal, S. R., Dominado, N., Stewart, M., Jans, D. A., Hime, G. R., Nicolis, S. K., Basler, C. F. and Forwood, J. K. (2021). Structural basis for nuclear import selectivity of pioneer transcription factor SOX2. Nat Commun 12(1): 28. PubMed ID: 33397924
Summary: SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, this study found that these two distantly positioned NLSs of SOX2 (Drosophila homolog: SoxN) contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPalpha3 (Drosophila homolog: karyopherin alpha3). This study identified key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, this study identified a structural basis for the preference of SOX2 binding to IMPalpha3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface. |
| Kaya-Copur, A., Marchiano, F., Hein, M. Y., Alpern, D., Russeil, J., Luis, N. M., Mann, M., Deplancke, B., Habermann, B. H. and Schnorrer, F. (2021). The Hippo pathway controls myofibril assembly and muscle fiber growth by regulating sarcomeric gene expression. Elife 10. PubMed ID: 33404503
Summary: Skeletal muscles are composed of gigantic cells called muscle fibers, packed with force-producing myofibrils. During development the size of individual muscle fibers must dramatically enlarge to match with skeletal growth. How muscle growth is coordinated with growth of the contractile apparatus is not understood. This study used the large Drosophila flight muscles to mechanistically decipher how muscle fiber growth is controlled. Regulated activity of core members of the Hippo pathway were found to be required to support flight muscle growth. Interestingly, Dlg5 and Slmap were identified as regulators of the STRIPAK phosphatase, which negatively regulates Hippo to enable post-mitotic muscle growth. Mechanistically, the Hippo pathway was shown to control timing and levels of sarcomeric gene expression during development and thus regulates the key components that physically mediate muscle growth. Since Dlg5, STRIPAK and the Hippo pathway are conserved a similar mechanism may contribute to muscle or cardiomyocyte growth in humans. | Destalminil-Letourneau, M., Morin-Poulard, I., Tian, Y., Vanzo, N. and Crozatier, M. (2021). The vascular niche controls Drosophila hematopoiesis via fibroblast growth factor signaling. Elife 10. PubMed ID: 33395389
Summary: In adult mammals, hematopoiesis, the production of blood cells from hematopoietic stem and progenitor cells (HSPCs), is tightly regulated by extrinsic signals from the microenvironment called 'niche'. Bone marrow HSPCs are heterogeneous and controlled by both endosteal and vascular niches. The Drosophila hematopoietic lymph gland is located along the cardiac tube which corresponds to the vascular system. In the lymph gland, the niche called Posterior Signaling Center controls only a subset of the heterogeneous hematopoietic progenitor population indicating that additional signals are necessary. This study reports that the vascular system acts as a second niche to control lymph gland homeostasis. The FGF ligand Branchless produced by vascular cells activates the FGF pathway in hematopoietic progenitors. By regulating intracellular calcium levels, FGF signaling maintains progenitor pools and prevents blood cell differentiation. This study reveals that two niches contribute to the control of Drosophila blood cell homeostasis through their differential regulation of progenitors. |
Wednesday, February 10th - Chromatin |
| Kalashnikova, D. A., Maksimov, D. A., Romanov, S. E., Laktionov, P. P. and Koryakov, D. E. (2020). SetDB1 and Su(var)3-9 play non-overlapping roles in somatic cell chromosomes of Drosophila melanogaster. J Cell Sci. PubMed ID: 33288549
Summary: We explored functional roles of two H3K9-specific histone methyltransferases SetDB1 and Su(var)3-9. Using DamID approach, the binding profile for SetDB1 in Drosophila salivary gland chromosomes was generated, and it was matched to the profile of Su(var)3-9. Unlike Su(var)3-9, SetDB1 turned out to be an euchromatic protein that is absent from repeated DNA compartment, and is largely restricted to TSSes and 5'UTRs of ubiquitously expressed genes. Significant SetDB1 association is also observed at insulator protein CP190 binding sites. SetDB1 and H3K9me2/3-enriched sites tend to display poor overlap. At the same time, SetDB1 has clear connection with the distribution of H3K27me3 mark. SetDB1 binds outside the domains possessing this modification, and about half of the borders of H3K27me3 domains are decorated by SetDB1 together with actively transcribed genes. On the basis of poor correlation between the distribution of SetDB1 and H3K9 methylation marks, it is speculated that in somatic cells, SetDB1 may contribute to the methylation of a broader set of chromosomal proteins than just H3K9. In addition, SetDB1 can be expected to play a role in the establishment of chromatin functional domains. | Khan, C., Muliyil, S., Ayyub, C. and Rao, B. J. (2020). spn-A/rad51 mutant exhibits enhanced genomic damage, cell death and low temperature sensitivity in somatic tissues. Chromosoma. PubMed ID: 33222024
Summary: Homologous recombination (HR) is one of the key pathways to repair double-strand breaks (DSBs). Rad51 serves an important function of catalysing strand exchange between two homologous sequences in the HR pathway. In higher organisms, rad51 function is indispensable with its absence leading to early embryonic lethality, thus precluding any mechanistic probing of the system. In contrast, the absence of Drosophila rad51 (spn-A/rad51) has been associated with defects in the germline, without any reported detrimental consequences to Drosophila somatic tissues. A systematic analysis was performed of developmental defects in somatic tissues of spn-A mutant flies by using genetic complementation between multiple spn-A alleles. This study uncovers a requirement for spn-A in somatic tissue maintenance during both larval and pupal stages. Also, this study shows that spn-A mutant exhibits patterning defects in abdominal cuticle in the stripes and bristles, while there appear to be only subtle defects in the adult wing and eye. Interestingly, spn-A mutant shows a discernible phenotype of low temperature sensitivity, suggesting a role of spn-A in temperature sensitive cellular processes. In summary, this study describes the important role played by spn-A/rad51 in Drosophila somatic tissues. |
| Hatkevich, T., Miller, D. E., Turcotte, C. A., Miller, M. C. and Sekelsky, J. (2021). A pathway for error-free non-homologous end joining of resected meiotic double-strand breaks. Nucleic Acids Res. PubMed ID: 33406239
Summary: Programmed DNA double-strand breaks (DSBs) made during meiosis are repaired by recombination with the homologous chromosome to generate, at selected sites, reciprocal crossovers that are critical for the proper separation of homologs in the first meiotic division. Backup repair processes can compensate when the normal meiotic recombination processes are non-functional. This paper describes a novel backup repair mechanism that occurs when the homologous chromosome is not available in Drosophila melanogaster meiosis. In the presence of a previously described mutation (Mcm5A7) that disrupts chromosome pairing, DSB repair is initiated by homologous recombination but is completed by non-homologous end joining (NHEJ). Remarkably, this process yields precise repair products. These results provide support for a recombination intermediate recently proposed in mouse meiosis, in which an oligonucleotide bound to the Spo11 protein that catalyzes DSB formation remains bound after resection. It is proposed that this oligonucleotide functions as a primer for fill-in synthesis to allow scarless repair by NHEJ. It is argued that this is a conserved repair mechanism that is likely to be invoked to overcome occasional challenges in normal meiosis. | Levitsky, V. G., Zykova, T. Y., Moshkin, Y. M. and Zhimulev, I. F. (2020). Nucleosome Positioning around Transcription Start Site Correlates with Gene Expression Only for Active Chromatin State in Drosophila Interphase Chromosomes. Int J Mol Sci 21(23). PubMed ID: 33291385
Summary: This study analyzed the whole-genome experimental maps of nucleosomes in Drosophila melanogaster and classified genes by the expression level in S2 cells (RPKM value, reads per kilobase million) as well as the number of tissues in which a gene was expressed (breadth of expression, BoE). Chromatin in 5'-regions of genes were classified into four states according to the hidden Markov model (4HMM). Only the Aquamarine chromatin state was considered as Active, while the remaining three states were defined as Non-Active. Surprisingly, about 20/40% of genes with 5'-regions mapped to Active/Non-Active chromatin possessed the minimal/at least modest RPKM and BoE. Regardless of RPKM/BoE the genes of Active chromatin possessed the regular nucleosome arrangement in 5'-regions, while genes of Non-Active chromatin did not show respective specificity. Only for genes of Active chromatin the RPKM/BoE positively correlates with the number of nucleosome sites upstream/around TSS and negatively with that downstream TSS. It is proposed that for genes of Active chromatin, regardless of RPKM value and BoE the nucleosome arrangement in 5'-regions potentiates transcription, while for genes of Non-Active chromatin, the transcription machinery does not require the substantial support from nucleosome arrangement to influence gene expression. |
| Rickels, R., Wang, L., Iwanaszko, M., Ozark, P. A., Morgan, M. A., Piunti, A., Khalatyan, N., Soliman, S. H. A., Rendleman, E. J., Savas, J. N., Smith, E. R. and Shilatifard, A. (2020). A small UTX stabilization domain of Trr is conserved within mammalian MLL3-4/COMPASS and is sufficient to rescue loss of viability in null animals. Genes Dev 34(21-22): 1493-1502. PubMed ID: 33033055
Summary: Catalytic-inactivating mutations within the Drosophila enhancer H3K4 mono-methyltransferase Trr and its mammalian homologs, MLL3/4, cause only minor changes in gene expression compared with whole-gene deletions for these COMPASS members. To identify essential histone methyltransferase-independent functions of Trr, this study screened to identify a minimal Trr domain sufficient to rescue Trr-null lethality and demonstrate that this domain binds and stabilizes Utx in vivo. Using the homologous MLL3/MLL4 human sequences, a short ~80-amino-acid UTX stabilization domain (USD) was mapped that promotes UTX stability in the absence of the rest of MLL3/4. Nuclear UTX stability is enhanced when the USD is fused with the MLL4 HMG-box. Thus, COMPASS-dependent UTX stabilization is an essential noncatalytic function of Trr/MLL3/MLL4, suggesting that stabilizing UTX could be a therapeutic strategy for cancers with MLL3/4 loss-of-function mutations. | Cherezov, R. O., Vorontsova, J. E. and Simonova, O. B. (2020). TBP-Related Factor 2 as a Trigger for Robertsonian Translocations and Speciation. Int J Mol Sci 21(22). PubMed ID: 33238614
Summary: Robertsonian (centric-fusion) translocation is the form of chromosomal translocation in which two long arms of acrocentric chromosomes are fused to form one metacentric. These translocations reduce the number of chromosomes while preserving existing genes and are considered to contribute to speciation. This study asked whether hypomorphic mutations in genes that disrupt the formation of pericentromeric regions could lead to centric fusion. TBP-related factor 2 (Trf2) encodes an alternative general transcription factor. A decrease of TRF2 expression disrupts the structure of the pericentromeric regions and prevents their association into chromocenter. This study revealed several centric fusions in two lines of Drosophila melanogaster with weak Trf2 alleles in genetic experiments. An RNAi-mediated knock-down of Trf2 was performed in Drosophila and S2 cells; Trf2 was shown to upregulate expression of D1-one of the major genes responsible for chromocenter formation and nuclear integrity in Drosophila. These data indicate that Trf2 may be involved in transcription program responsible for structuring of pericentromeric regions and may contribute to new karyotypes formation in particular by promoting centric fusion. |
Tuesday, February 9th - Apoptosis and Autophagy |
| Xu, T., Nicolson, S., Sandow, J. J., Dayan, S., Jiang, X., Manning, J. A., Webb, A. I., Kumar, S. and Denton, D. (2020). Cp1/cathepsin L is required for autolysosomal clearance in Drosophila. Autophagy: 1-16. PubMed ID: 33112206
Summary: Macroautophagy/autophagy is a highly conserved lysosomal degradative pathway important for maintaining cellular homeostasis. Much of the current knowledge of autophagy is focused on the initiation steps in this process. Recently, an understanding of later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling, is becoming evident. Autophagy can function in both cell survival and cell death, however, the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remain to be established. Using proteomic analysis of Drosophila larval midguts during degradation, this study identified a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. Cp1/cathepsin L-deficient larval midgut cells accumulate aberrant autophagic vesicles due to a block in autophagic flux, yet later stages of midgut degradation are not compromised. The accumulation of large aberrant autolysosomes in the absence of Cp1 appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material, rather than a defect in autophagosome-lysosome fusion. Finally, this study found that other cathepsins may also contribute to proper autolysosomal degradation in Drosophila larval midgut cells. These findings provide evidence that cathepsins play an essential role in the autolysosome to maintain basal autophagy flux by balancing autophagosome production and turnover. | Bjedov, I., Cocheme, H. M., Foley, A., Wieser, D., Woodling, N. S., Castillo-Quan, J. I., Norvaisas, P., Lujan, C., Regan, J. C., Toivonen, J. M., Murphy, M. P., Thornton, J., Kinghorn, K. J., Neufeld, T. P., Cabreiro, F. and Partridge, L. (2020). Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila. PLoS Genet 16(11): e1009083. PubMed ID: 33253201
Summary: Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. This paper reports that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. It was first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. Next autophagy was directly induced by over-expressing the major autophagy kinase Atg1; a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention. |
| Deehan, M., Lin, W., Blum, B., Emili, A. and Frydman, H. (2021). Intracellular Density of Wolbachia Is Mediated by Host Autophagy and the Bacterial Cytoplasmic Incompatibility Gene cifB in a Cell Type-Dependent Manner in Drosophila melanogaster. mBio 12(1). PubMed ID: 33436431
Summary: Autophagy is an intracellular degradation pathway involved in innate immunity. Pathogenic bacteria have evolved several mechanisms to escape degradation or exploit autophagy to acquire host nutrients. In the case of endosymbionts, which often have commensal or mutualistic interactions with the host, autophagy is not well characterized. This study utilized tissue-specific autophagy mutants to determine if Wolbachia, a vertically transmitted obligate endosymbiont of Drosophila melanogaster, is regulated by autophagy in somatic and germ line cell types. This analysis revealed core autophagy proteins Atg1 and Atg8 and a selective autophagy-specific protein Ref(2)p negatively regulate Wolbachia in the hub, a male gonad somatic cell type. Furthermore, it was determined that the Wolbachia effector protein, CifB, modulates autophagy-Wolbachia interactions, identifying a new host-related pathway which these bacterial proteins interact with. In the female germ line, the cell type necessary for inheritance of Wolbachia through vertical transmission, it was discovered that bulk autophagy mediated by Atg1 and Atg8 positively regulates Wolbachia density, whereas Ref(2)p had no effect. Global metabolomics of fly ovaries deficient in germ line autophagy revealed reduced lipid and carbon metabolism, implicating metabolites from these pathways as positive regulators of Wolbachia. This work provides further understanding of how autophagy affects bacteria in a cell type-dependent manner. | Chou, H. Y., Lee, Y. T., Lin, Y. J., Wen, J. K., Peng, W. H., Hsieh, P. L., Lin, S. Y., Hung, C. C. and Chen, G. C. (2020). PTPN9-mediated dephosphorylation of VTI1B promotes ATG16L1 precursor fusion and autophagosome formation. Autophagy: 1-16. PubMed ID: 33112705
Summary: Macroautophagy/autophagy is an evolutionarily conserved intracellular pathway for the degradation of cytoplasmic materials. Under stress conditions, autophagy is upregulated and double-membrane autophagosomes are formed by the expansion of phagophores. The ATG16L1 (Drosophila homolog: ATG16) precursor fusion contributes to development of phagophore structures and is critical for the biogenesis of autophagosomes. This study discovered a novel role of the protein tyrosine phosphatase PTPN9 in the regulation of homotypic ATG16L1 vesicle fusion and early autophagosome formation. Depletion of PTPN9 and its Drosophila homolog Ptpmeg2 impaired autophagosome formation and autophagic flux. PTPN9 colocalized with ATG16L1 and was essential for homotypic fusion of ATG16L1(+) vesicles during starvation-induced autophagy. This study further identified the Q-SNARE VTI1B as a substrate target of PTPN9 phosphatase. Like PTPN9, the VTI1B nonphosphorylatable mutant but not the phosphomimetic mutant enhanced SNARE complex assembly and autophagic flux. These findings highlight the important role of PTPN9 in the regulation of ATG16L1(+) autophagosome precursor fusion and autophagosome biogenesis through modulation of VTI1B phosphorylation status. |
| Jipa, A., Vedelek, V., Merenyi, Z., Urmosi, A., Takats, S., Kovacs, A. L., Horvath, G. V., Sinka, R. and Juhasz, G. (2020). Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles. Autophagy: 1-11. PubMed ID: 33249988
Summary: Yeast Atg8 and its homologs are involved in autophagosome biogenesis in all eukaryotes. These are the most widely used markers for autophagy thanks to the association of their lipidated forms with autophagic membranes. The Atg8 protein family expanded in animals and plants, with most Drosophila species having two Atg8 homologs. This study uses clear-cut genetic analysis in Drosophila melanogaster to show that lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. In contrast, expression of Atg8b is restricted to the male germline and its loss causes male sterility without affecting autophagy. High expression of non-lipidated Atg8b in the male germline is required for fertility. Consistent with these non-canonical functions of Atg8 proteins, loss of Atg genes required for Atg8 lipidation lead to autophagy defects but do not cause lethality or male sterility. | Murakawa, T., Kiger, A. A., Sakamaki, Y., Fukuda, M. and Fujita, N. (2020). An autophagy-dependent tubular lysosomal network synchronizes degradative activity required for muscle remodeling. J Cell Sci 133(21). PubMed ID: 33077556
Summary: Lysosomes are compartments for the degradation of both endocytic and autophagic cargoes. The shape of lysosomes changes with cellular degradative demands; however, there is limited knowledge about the mechanisms or significance that underlies distinct lysosomal morphologies. This study found an extensive tubular autolysosomal network in Drosophila abdominal muscle remodeling during metamorphosis. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein Syntaxin17 and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, this study revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling. |
Monday February 8th - Adult physiology |
| Idda, T., Bonas, C., Hoffmann, J., Bertram, J., Quinete, N., Schettgen, T., Fietkau, K., Esser, A., Stope, M. B., Leijs, M. M., Baron, J. M., Kraus, T., Voigt, A. and Ziegler, P. (2020). Metabolic activation and toxicological evaluation of polychlorinated biphenyls in Drosophila melanogaster. Sci Rep 10(1): 21587. PubMed ID: 33299007
Summary: Degradation of polychlorinated biphenyls (PCBs) is initiated by cytochrome P450 (CYP) enzymes and includes PCB oxidation to OH-metabolites, which often display a higher toxicity than their parental compounds. In search of an animal model reflecting PCB metabolism and toxicity, Drosophila melanogaster, a well-known model system for genetics and human disease, was tested. Feeding Drosophila with lower chlorinated (LC) PCB congeners 28, 52 or 101 resulted in the detection of a human-like pattern of respective OH-metabolites in fly lysates. Feeding flies high PCB 28 concentrations caused lethality. Thus selected CYPs were silenced via RNA interference, and the effect on PCB 28-derived metabolite formation was analyzed by assaying 3-OH-2',4,4'-trichlorobiphenyl (3-OHCB 28) and 3'-OH-4',4,6'-trichlorobiphenyl (3'-OHCB 28) in fly lysates. Several Drosophila CYPs (dCYPs) were identified whose knockdown reduced PCB 28-derived OH-metabolites and suppressed PCB 28 induced lethality including dCYP1A2. Following in vitro analysis using a liver-like CYP-cocktail, containing human orthologues of dCYP1A2, human CYP1A2 was confirmed as a PCB 28 metabolizing enzyme. PCB 28-induced mortality in flies was accompanied by locomotor impairment, a common phenotype of neurodegenerative disorders. Along this line, PCB 28-initiated caspase activation was shown in differentiated fly neurons. This suggested the loss of neurons through apoptosis. These findings in flies are congruent with observation in human exposed to high PCB levels. In plasma samples of PCB exposed humans, levels of the neurofilament light chain increase after LC-PCB exposure, indicating neuronal damage. In summary these findings demonstrate parallels between Drosophila and the human systems with respect to CYP mediated metabolism and PCB mediated neurotoxicity. | Hofbauer, H. F., Heier, C., Sen Saji, A. K. and Kuhnlein, R. P. (2020). Lipidome remodeling in aging normal and genetically obese Drosophila males. Insect Biochem Mol Biol: 103498. PubMed ID: 33221388
Summary: Lipid homeostasis is essential for insects to maintain phospholipid (PL)-based membrane integrity and to provide on-demand energy supply throughout life. Triacylglycerol (TAG) is the major lipid class used for energy production and is stored in lipid droplets, the universal cellular fat storage organelles. Accumulation and mobilization of TAG are strictly regulated since excessive accumulation of TAG leads to obesity and has been correlated with adverse effects on health- and lifespan across phyla. Little is known, however, about when during adult life and why excessive storage lipid accumulation restricts lifespan. This study used genetically obese Drosophila mutant males, which were all shown to be short-lived compared to control males and applied single fly mass spectrometry-based lipidomics to profile TAG, diacylglycerol and major membrane lipid signatures throughout adult fly life from eclosion to death. This comparative approach revealed distinct phases of lipidome remodeling throughout aging. Quantitative and qualitative compositional changes of TAG and PL species, which are characterized by the length and saturation of their constituent fatty acids, were pronounced during young adult life. In contrast, lipid signatures of adult and senescent flies were remarkably stable. Genetically obese flies displayed both quantitative and qualitative changes in TAG species composition, while PL signatures were almost unaltered compared to normal flies at all ages. Collectively, this suggests a tight control of membrane composition throughout lifetime largely uncoupled from storage lipid metabolism. Finally, the first evidence is presented for a characteristic lipid signature of moribund flies, likely generated by a rapid and selective storage lipid depletion close to death. Of note, the analytical power to monitor lipid species profiles combined with high sensitivity of this single fly lipidomics approach is universally applicable to address developmental or behavioral lipid signature modulations of importance for insect life. |
| Gnainsky, Y., Zfanya, N., Elgart, M., Omri, E., Brandis, A., Mehlman, T., Itkin, M., Malitsky, S., Adamski, J. and Soen, Y. (2021). Systemic Regulation of Host Energy and Oogenesis by Microbiome-Derived Mitochondrial Coenzymes. Cell Rep 34(1): 108583. PubMed ID: 33406416
Summary: Gut microbiota have been shown to promote oogenesis and fecundity, but the mechanistic basis of remote influence on oogenesis remained unknown. This study reports a systemic mechanism of influence mediated by bacterial-derived supply of mitochondrial coenzymes. Removal of microbiota decreased mitochondrial activity and ATP levels in the whole-body and ovary, resulting in repressed oogenesis. Similar repression was caused by RNA-based knockdown of mitochondrial function in ovarian follicle cells. Reduced mitochondrial function in germ-free (GF) females was reversed by bacterial recolonization or supplementation of riboflavin, a precursor of FAD and FMN. Metabolomics analysis of GF females revealed a decrease in oxidative phosphorylation and FAD levels and an increase in metabolites that are degraded by FAD-dependent enzymes (e.g., amino and fatty acids). Riboflavin supplementation opposed this effect, elevating mitochondrial function, ATP, and oogenesis. These findings uncover a bacterial-mitochondrial axis of influence, linking gut bacteria with systemic regulation of host energy and reproduction. | Jacomin, A. C., Gohel, R., Hussain, Z., Varga, A., Maruzs, T., Eddison, M., Sica, M., Jain, A., Moffat, K. G., Johansen, T., Jenny, A., Juhasz, G. and Nezis, I. P. (2021). Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila. Life Sci Alliance 4(2). PubMed ID: 33318080
Summary: Hunger drives food-seeking behaviour and controls adaptation of organisms to nutrient availability and energy stores. Lipids constitute an essential source of energy in the cell that can be mobilised during fasting by autophagy. Selective degradation of proteins by autophagy is made possible essentially by the presence of LIR and KFERQ-like motifs. Using in silico screening of Drosophila proteins that contain KFERQ-like motifs, this study identified and characterized the adaptor protein Arouser, which functions to regulate fat storage and mobilisation and is essential during periods of food deprivation. Hypomorphic arouser mutants are not satiated, are more sensitive to food deprivation, and are more aggressive, suggesting an essential role for Arouser in the coordination of metabolism and food-related behaviour. This analysis shows that Arouser functions in the fat body through nutrient-related signalling pathways and is degraded by endosomal microautophagy. Arouser degradation occurs during feeding conditions, whereas its stabilisation during non-feeding periods is essential for resistance to starvation and survival. In summary, these data describe a novel role for endosomal microautophagy in energy homeostasis, by the degradation of the signalling regulatory protein Arouser. |
| Fan, X., Zeng, Y., Fan, Z., Cui, L., Song, W., Wu, Q., Gao, Y., Yang, D., Mao, X., Zeng, B., Zhang, M., Ni, Q., Li, Y., Wang, T., Li, D. and Yang, M. (2020). Dihydromyricetin promotes longevity and activates the transcription factors FOXO and AOP in Drosophila. Aging (Albany NY) 12. PubMed ID: 33291074
Summary: Drugs or compounds have been shown to promote longevity in various approaches. This study used Drosophila to explore novel natural compounds can be applied to anti-aging. A flavonoid named Dihydromyricetin can increase stress tolerance and lipid levels, slow down gut dysfunction and extend Drosophila lifespan. Dihydromyricetin can also lessen pERK and pAKT signaling, consequently activating FOXO and AOP to modulate longevity. These results suggested that DHM could be used as an effective compound for anti-aging intervention, which could likely be applied to both mammals and humans. | Dhillon, A., Chowdhury, T., Morbey, Y. E. and Moehring, A. J. (2020). Reproductive consequences of an extra long-term sperm storage organ. BMC Evol Biol 20(1): 159. PubMed ID: 33256600
Summary: Sperm storage plays a key role in the reproductive success of many sexually-reproducing organisms, and the capacity of long-term sperm storage varies across species. To date there are no empirical tests of the reproductive consequences of additional long-term sperm storage. While Dipterans ancestrally have three long-term sperm organs, known as the spermathecae, Drosophila contain only two. This study identified a candidate gene, spermathreecae (sp3), in which a disruption cause the development of three functional spermathecae rather than the usual two in Drosophila. This disruption was used to test the reproductive consequences of having an additional long-term sperm storage organ. Compared to females with two spermathecae, females with three spermathecae store a greater total number of sperm and can produce offspring a greater length of time. However, they did not produce a greater total number of offspring. Thus, additional long-term sperm storage in insects may increase female fitness through extending the range of conditions where she produces offspring, or through increasing the quality of offspring via enhanced local sperm competition at fertilization. |
Friday, February 5th - Adult neural development and function |
| Ganguly, A., Qi, C., Bajaj, J. and Lee, D. (2020). Serotonin receptor 5-HT7 in Drosophila mushroom body neurons mediates larval appetitive olfactory learning. Sci Rep 10(1): 21267. PubMed ID: 33277559
Summary: Serotonin (5-HT) and dopamine are critical neuromodulators known to regulate a range of behaviors in invertebrates and mammals, such as learning and memory. Effects of both serotonin and dopamine are mediated largely through their downstream G-protein coupled receptors through cAMP-PKA signaling. While the role of dopamine in olfactory learning in Drosophila is well described, the function of serotonin and its downstream receptors on Drosophila olfactory learning remain largely unexplored. This study showed that the output of serotonergic neurons, possibly through points of synaptic contacts on the mushroom body (MB), is essential for training during olfactory associative learning in Drosophila larvae. Additionally, it was demonstrated that the regulation of olfactory associative learning by serotonin is mediated by its downstream receptor (d5-HT7) in a cAMP-dependent manner. d5-HT7 expression specifically in the MB, an anatomical structure essential for olfactory learning in Drosophila, is critical for olfactory associative learning. Importantly this work shows that spatio-temporal restriction of d5-HT7 expression to the MB is sufficient to rescue olfactory learning deficits in a d5-HT7 null larvae. In summary, these results establish a critical, and previously unknown, role of d5-HT7 in olfactory learning. | Golovin, R. M., Vest, J. and Broadie, K. (2021). Neuron-Specific FMRP Roles in Experience-Dependent Remodeling of Olfactory Brain Innervation During an Early-life Critical Period. J Neurosci. PubMed ID: 33402421
Summary: Critical periods are developmental windows during which neural circuits effectively adapt to the new sensory environment. Animal models of Fragile X syndrome (FXS) exhibit profound impairments of sensory experience-driven critical periods. However, it is not known whether the causative Fragile X Mental Retardation Protein (FMRP) acts uniformly across neurons, or instead manifests neuron-specific functions. This study used antennal lobe (AL) olfactory circuit to investigate neuron-specific FMRP roles in the odorant experience-dependent remodeling of the olfactory sensory neuron (OSN) innervation. Targeted OSN class-specific FMRP RNAi was found to impair innervation remodeling within AL synaptic glomeruli, whereas global dfmr1 null mutants display relatively normal odorant-driven refinement. Both OSN cell autonomous and cell non-autonomous FMRP functions were found to mediate odorant experience-dependent remodeling, with AL circuit FMRP imbalance causing defects in overall glomerulus innervation refinement. OSN class-specific FMRP levels were found to bidirectionally regulate critical period remodeling, with odorant experience selectively controlling OSN synaptic terminals in AL glomeruli. OSN class-specific FMRP loss impairs critical period remodeling by disrupting responses to lateral modulation from other odorant-responsive OSNs mediating overall AL gain control. Silencing glutamatergic AL interneurons reduces OSN remodeling, while conversely, interfering with the OSN class-specific GABA(A) signaling enhances remodeling. These findings reveal control of OSN synaptic remodeling by FMRP with neuron-specific circuit functions, and indicate how neural circuitry can compensate for global FMRP loss to reinstate normal critical period brain circuit remodeling. |
| Feng, K., Sen, R., Minegishi, R., Dubbert, M., Bockemuhl, T., Buschges, A. and Dickson, B. J. (2020). Distributed control of motor circuits for backward walking in Drosophila. Nat Commun 11(1): 6166. PubMed ID: 33268800
Summary: How do descending inputs from the brain control leg motor circuits to change how an animal walks? Conceptually, descending neurons are thought to function either as command-type neurons, in which a single type of descending neuron exerts a high-level control to elicit a coordinated change in motor output, or through a population coding mechanism, whereby a group of neurons, each with local effects, act in combination to elicit a global motor response. The Drosophila Moonwalker Descending Neurons (MDNs), which alter leg motor circuit dynamics so that the fly walks backwards, exemplify the command-type mechanism. This study identified several dozen MDN target neurons within the leg motor circuits showed that two of them mediate distinct and highly-specific changes in leg muscle activity during backward walking: LBL40 neurons provide the hindleg power stroke during stance phase; LUL130 neurons lift the legs at the end of stance to initiate swing. Through these two effector neurons, MDN directly controls both the stance and swing phases of the backward stepping cycle. These findings suggest that command-type descending neurons can also operate through the distributed control of local motor circuits. | Fernandez-Chiappe, F., Frenkel, L., Colque, C. C., Ricciuti, A., Hahm, B., Cerredo, K., Muraro, N. I. and Ceriani, M. F. (2020). High frequency neuronal bursting is essential for circadian and sleep behaviors in Drosophila. J Neurosci. PubMed ID: 33262246
Summary: Circadian rhythms have been extensively studied in Drosophila, however, still little is known about how the electrical properties of clock neurons are specified. A behavioral genetic screen was performed in this study through the downregulation of candidate ion channels in the lateral ventral neurons (LNvs); it was shown that the hyperpolarization-activated cation current I(h) is important for the behaviors that the LNvs influence: temporal organization of locomotor activity, analyzed in males, and sleep, analyzed in females. Using whole-cell patch clamp electrophysiology this study demonstrated that small LNvs are bursting neurons, and that I(h) is necessary to achieve the high frequency bursting firing pattern characteristic of both types of LNvs in females. Since firing in bursts has been associated to neuropeptide release, it is hypothesized that I(h) would be important for LNvs communication. Indeed, this study demonstrates that I(h) is fundamental for the recruitment of PDF filled dense core vesicles to the terminals at the dorsal protocerebrum and for their timed release, and hence for the temporal coordination of circadian behaviors. |
| Ferreira Castro, A., Baltruschat, L., Sturner, T., Bahrami, A., Jedlicka, P., Tavosanis, G. and Cuntz, H. (2020). Achieving functional neuronal dendrite structure through sequential stochastic growth and retraction. Elife 9. PubMed ID: 33241995
Summary: Class I ventral posterior dendritic arborisation (c1vpda) proprioceptive sensory neurons respond to contractions in the Drosophila larval body wall during crawling. Their dendritic branches run along the direction of contraction, possibly a functional requirement to maximise membrane curvature during crawling contractions. Although the molecular machinery of dendritic patterning in c1vpda has been extensively studied, the process leading to the precise elaboration of their comb-like shapes remains elusive. To link dendrite shape with its proprioceptive role, a long-term, non-invasive, in vivo time-lapse imaging was perfomred of c1vpda embryonic and larval morphogenesis to reveal a sequence of differentiation stages. Computer models and dendritic branch dynamics tracking were used to propose that distinct sequential phases of stochastic growth and retraction achieve efficient dendritic trees both in terms of wire and function. This study shows how dendrite growth balances structure-function requirements, shedding new light on general principles of self-organisation in functionally specialised dendrites. | Gunawardhana, K. L., Rivas, G. B. S., Caster, C. and Hardin, P. E. (2021). Crosstalk between vrille transcripts, proteins, and regulatory elements controlling circadian rhythms and development in Drosophila. iScience 24(1): 101893. PubMed ID: 33364582
Summary: The vrille (vri) gene encodes a transcriptional repressor required for Drosophila development as well as circadian behavior in adults. Alternate first exons produce vri transcripts predicted to produce a short VRI isoform during development and long VRI in adults. A vri mutant (vri &Delta679) lacking long VRI transcripts is viable, confirming that short VRI is sufficient for developmental functions, yet behavioral rhythms in vri &Delta679 flies persist, showing that short VRI is sufficient for clock output. E-box regulatory elements that drive rhythmic long VRI transcript expression are required for developmental expression of short VRI transcripts. Surprisingly, long VRI transcripts primarily produce short VRI in adults, apparently due to a poor Kozak sequence context, demonstrating that short VRI drives circadian behavior. Thus, E-box-driven long VRI transcripts primarily control circadian rhythms via short VRI, whereas the same E-boxes drive short VRI transcripts that control developmental functions using short VRI. |
Thursday, February 4th |
| Buchberger, E., Bilen, A., Ayaz, S., Salamanca, D., Matas de Las Heras, C., Niksic, A., Almudi, I., Torres-Oliva, M., Casares, F. and Posnien, N. (2021). Variation in pleiotropic hub gene expression is associated with interspecific differences in head shape and eye size in Drosophila. Mol Biol Evol. PubMed ID: 33386848
Summary: Revealing the mechanisms underlying the breath-taking morphological diversity observed in nature is a major challenge in Biology. It has been established that recurrent mutations in hotspot genes cause the repeated evolution of morphological traits, such as body pigmentation or the gain and loss of structures. To date, however, it remains elusive whether hotspot genes contribute to natural variation in the size and shape of organs. Since natural variation in head morphology is pervasive in Drosophila, the molecular and developmental basis of differences in compound eye size and head shape was studied in two closely related Drosophila species. Differences were shown in the progression of retinal differentiation between species, and comparative transcriptomics and chromatin accessibility data were applied to identify the GATA transcription factor Pannier (Pnr) as central factor associated with these differences. Although the genetic manipulation of Pnr affected multiple aspects of dorsal head development, the effect of natural variation is restricted to a subset of the phenotypic space. Data is presented suggesting that this developmental constraint is caused by the co-evolution of expression of pnr and its co-factor u-shaped (ush). It is proposed that natural variation in expression or function of highly connected developmental regulators with pleiotropic functions is a major driver for morphological evolution, and implications on gene regulatory network evolution are discussed. In comparison to previous findings, the data strongly suggests that evolutionary hotspots are not the only contributors to the repeated evolution of eye size and head shape in Drosophila. | Conner, W. R., Delaney, E. K., Bronski, M. J., Ginsberg, P. S., Wheeler, T. B., Richardson, K. M., Peckenpaugh, B., Kim, K. J., Watada, M., Hoffmann, A. A., Eisen, M. B., Kopp, A., Cooper, B. S. and Turelli, M. (2020). A phylogeny for the Drosophila montium species group: a model clade for comparative analyses. Mol Phylogenet Evol: 107061. PubMed ID: 33387647
Summary: The Drosophila montium species group is a clade of 94 named species closely related to the model species D. melanogaster. The montium species group is distributed over a broad geographic range throughout Asia, Africa, and Australasia. Species of this group possess a wide range of morphologies, mating behaviors, and endosymbiont associations, making this clade useful for comparative analyses. This study used genomic data from 42 available species to estimate the phylogeny and relative divergence times within the montium species group, and its relative divergence time from D. melanogaster. To assess the robustness of the phylogenetic inferences, three non-overlapping sets of 20 single-copy coding sequences were used, and all 60 genes were analyzed with both Bayesian and maximum likelihood methods. This analyses support monophyly of the group. Apart from the uncertain placement of a single species, D. baimaii, this analyses also support the monophyly of all seven subgroups proposed within the montium group. Phylograms and relative chronograms provide a highly resolved species tree, with discordance restricted to estimates of relatively short branches deep in the tree. In contrast, age estimates for the montium crown group, relative to its divergence from D. melanogaster, depend critically on prior assumptions concerning variation in rates of molecular evolution across branches, and hence have not been reliably determined. Methodological issues are discussed that limit phylogenetic resolution - even when complete genome sequences are available - as well as the utility of the current phylogeny for understanding the evolutionary and biogeographic history of this clade. |
| Wigby, S., Brown, N. C., Allen, S. E., Misra, S., Sitnik, J. L., Sepil, I., Clark, A. G. and Wolfner, M. F. (2020). The Drosophila seminal proteome and its role in postcopulatory sexual selection. Philos Trans R Soc Lond B Biol Sci 375(1813): 20200072. PubMed ID: 33070726
Summary: Postcopulatory sexual selection (PCSS), comprised of sperm competition and cryptic female choice, has emerged as a widespread evolutionary force among polyandrous animals. There is abundant evidence that PCSS can shape the evolution of sperm. However, sperm are not the whole story: they are accompanied by seminal fluid substances that play many roles, including influencing PCSS. Foremost among seminal fluid models is Drosophila melanogaster, which displays ubiquitous polyandry, and exhibits intraspecific variation in a number of seminal fluid proteins (Sfps) that appear to modulate paternity share. This study first consolidated current information on the identities of D. melanogaster Sfps. Comparing between D. melanogaster and human seminal proteomes, evidence is found of similarities between many protein classes and individual proteins, including some D. melanogaster Sfp genes linked to PCSS, suggesting evolutionary conservation of broad-scale functions. Experimental evidence for the functions of D. melanogaster Sfps in PCSS and sexual conflict is reviewed. Gaps are identified in current knowledge and areas for future research, including an enhanced identification of PCSS-related Sfps, their interactions with rival sperm and with females, the role of qualitative changes in Sfps and mechanisms of ejaculate tailoring. This article is part of the theme issue 'Fifty years of sperm competition'. | Bogaerts-Marquez, M., Guirao-Rico, S., Gautier, M. and Gonzalez, J. (2020). Temperature, rainfall and wind variables underlie environmental adaptation in natural populations of Drosophila melanogaster. Mol Ecol. PubMed ID: 33350518
Summary: While several studies in a diverse set of species have shed light on the genes underlying adaptation, knowledge on the selective pressures that explain the observed patterns lags behind. Drosophila melanogaster is a valuable organism to study environmental adaptation because this species originated in Southern Africa and has recently expanded worldwide, and also because it has a functionally well-annotated genome. This work aimed to decipher which environmental variables are relevant for adaptation of D. melanogaster natural populations in Europe and North America. 36 whole-genome pool-seq samples of D. melanogaster natural populations were ezamined, collected in 20 European and 11 North American locations. The BayPass software was used to identify SNPs and transposable elements showing signature of adaptive differentiation across populations, as well as significant associations with 59 environmental variables related to temperature, rainfall, evaporation, solar radiation, wind, daylight hours, and soil type. Besides temperature and rainfall, wind related variables are also relevant for D. melanogaster environmental adaptation. Interestingly, 23% to 51% of the genes that showed significant associations with environmental variables were not found overly differentiated across populations. Besides SNPs, ten reference transposable element insertions associated with environmental variables were identified. These results showed that genome-environment association analysis can identify adaptive genetic variants that are undetected by population differentiation analysis while also allowing the identification of candidate environmental drivers of adaptation. |
| Bruce, H. S. and Patel, N. H. (2020). Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments. Nat Ecol Evol 4(12): 1703-1712. PubMed ID: 33262517
Summary: The origin of insect wings has long been debated. Central to this debate is whether wings are a novel structure on the body wall resulting from gene co-option, or evolved from an exite (outgrowth; for example, a gill) on the leg of an ancestral crustacean. This study reports the phenotypes for the knockout of five leg patterning genes in the crustacean Parhyale hawaiensis and compares these with their previously published phenotypes in Drosophila and other insects. This leads to an alignment of insect and crustacean legs that suggests that two leg segments that were present in the common ancestor of insects and crustaceans were incorporated into the insect body wall, moving the proximal exite of the leg dorsally, up onto the back, to later form insect wings. These results suggest that insect wings are not novel structures, but instead evolved from existing, ancestral structures. | Ferreira, E. A., Lambert, S., Verrier, T., Marion-Poll, F. and Yassin, A. (2020). Soft Selective Sweep on Chemosensory Genes Correlates with Ancestral Preference for Toxic Noni in a Specialist Drosophila Population. Genes (Basel) 12(1). PubMed ID: 33383708
Summary: Understanding how organisms adapt to environmental changes is a major question in evolution and ecology. In particular, the role of ancestral variation in rapid adaptation remains unclear because its trace on genetic variation, known as soft selective sweep, is often hardly recognizable from genome-wide selection scans. This study investigate the evolution of chemosensory genes in Drosophila yakuba mayottensis, a specialist subspecies on toxic noni (Morinda citrifolia) fruits on the island of Mayotte. Population genomics analyses and behavioral assays were combined to evaluate the level of divergence in chemosensory genes and perception of noni chemicals between specialist and generalist subspecies of D. yakuba. A signal of soft selective sweep was identified on a handful of genes, with the most diverging ones involving a cluster of gustatory receptors expressed in bitter-sensing neurons. These results highlight the potential role of ancestral genetic variation in promoting host plant specialization in herbivorous insects and identify a number of candidate genes underlying behavioral adaptation. |
Wednesday, February 3rd - RNA and transposons |
| Erkelenz, S., Stankovic, D., Mundorf, J., Bresser, T., Claudius, A. K., Boehm, V., Gehring, N. H. and Uhlirova, M. (2021). Ecd promotes U5 snRNP maturation and Prp8 stability. Nucleic Acids Res. PubMed ID: 33444449
Summary: Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs), the biogenesis of which remains only partially understood. This study defines the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches, this study demonstrates the Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd. The predominant task of Ecd is to deliver Prp8 to the emerging U5 snRNPs in the cytoplasm. Ecd deficiency, on the other hand, leads to reduced Prp8 protein levels and compromised U5 snRNP biogenesis, causing loss of splicing fidelity and transcriptome integrity. Based on these findings, it is propose that Ecd chaperones Prp8 to the forming U5 snRNP allowing completion of the cytoplasmic part of the U5 snRNP biogenesis pathway necessary to meet the cellular demand for functional spliceosomes. | Xia, X., Fu, X., Du, J., Wu, B., Zhao, X., Zhu, J. and Zhao, Z. (2020). Regulation of circadian rhythm and sleep by miR-375-timeless interaction in Drosophila. Faseb J. PubMed ID: 33078445
Summary: MicroRNAs are important coordinators of circadian regulation that mediate the fine-tuning of gene expression. The global functional miRNA-mRNA interaction network involved in the circadian system remains poorly understood. This study used CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs)-CLIP (Cross-Linking and Immuno-Precipitation) to explore the regulatory functions of miRNAs in the circadian system by comparing the miRNA-mRNA interactions between Drosophila wild-type strain W(1118) and a mutant of the key circadian transcriptional regulator Clock (Clk(jrk)). This experimental approach unambiguously identified tens of thousands of miRNA-mRNA interactions in both the head and body. The miRNA-mRNA interactome showed dramatic changes in the Clk(jrk) flies. Particularly, among ~300 miRNA-mRNA circadian relevant interactions, multiple interactions involving core clock genes pdp1, tim, and vri displayed distinct changes as a result of the Clk mutation. Based on the CLEAR-CLIP analysis, this study found a novel regulation of the circadian rhythm and sleep by the miR-375-timeless interaction. The results indicated that Clk disruption abolished normal rhythmic expression of miR-375 and the functional regulation occurred in the l-LNv neurons, where miR-375 modulated the circadian rhythm and sleep via targeting timeless. This work provides the first global view of miRNA regulation in the circadian rhythm. |
| Bhagavatula, S. and Knust, E. (2020). A putative stem-loop structure in Drosophila crumbs is required for mRNA localisation in epithelia and germline cells. J Cell Sci. PubMed ID: 33310910
Summary: Crumbs (Crb) is an evolutionarily conserved transmembrane protein localised in the apical membrane of epithelial cells. Loss or mis-localisation of Crb is often associated with disruption of apico-basal cell polarity. crb mRNA is also apically enriched in epithelial cells, and, as shown in this study, accumulates in the oocyte of developing egg chambers. The Localization Element (LE) of crb mRNA was narrowed down to 47 nucleotides forming a putative stem-loop structure, suggesting to be recognised by Egalitarian (Egl). Mutations in conserved nucleotides abrogate apical transport. crb mRNA enrichment in the oocyte is affected in egl mutant egg chambers. A CRISPR based genomic deletion of the crb locus that includes the LE disrupts asymmetric crb mRNA localisation in epithelia and prevents its accumulation in the oocyte during early stages of oogenesis, but does not affect Crb protein localisation in embryonic and follicular epithelia. However, flies lacking the LE show ectopic Crb protein expression in the nurse cells. These data suggest an additional role of the Drosophila 3'-UTR in regulating translation in a tissue specific manner. | Ellison, C. E., Kagda, M. S. and Cao, W. (2020). Telomeric TART elements target the piRNA machinery in Drosophila. PLoS Biol 18(12): e3000689. PubMed ID: 33347429
Summary: Coevolution between transposable elements (TEs) and their hosts can be antagonistic, where TEs evolve to avoid silencing and the host responds by reestablishing TE suppression, or mutualistic, where TEs are co-opted to benefit their host. The TART-A TE functions as an important component of Drosophila telomeres but has also reportedly inserted into the Drosophila melanogaster nuclear export factor gene nxf2. Rather than inserting into nxf2, TART-A has actually captured a portion of nxf2 sequence. TART-A produces abundant Piwi-interacting small RNAs (piRNAs), some of which are antisense to the nxf2 transcript, and the TART-like region of nxf2 was found to be evolving rapidly. Furthermore, in D. melanogaster, TART-A is present at higher copy numbers, and nxf2 shows reduced expression, compared to the closely related species Drosophila simulans. It is proposed that capturing nxf2 sequence allowed TART-A to target the nxf2 gene for piRNA-mediated repression and that these 2 elements are engaged in antagonistic coevolution despite the fact that TART-A is serving a critical role for its host genome. |
| Song, W., Ressl, S. and Tracey, W. D. (2020). Loss of Pseudouridine Synthases in the RluA Family Causes Hypersensitive Nociception in Drosophila. G3 (Bethesda). PubMed ID: 33028630
Summary: Nociceptive neurons of Drosophila melanogaster larvae are characterized by highly branched dendritic processes whose proper morphogenesis relies on a large number of RNA-binding proteins. Post-transcriptional regulation of RNA in these dendrites has been found to play an important role in their function. This study investigatde the neuronal functions of two putative RNA modification genes, RluA-1 and RluA-2, which are predicted to encode pseudouridine synthases. RluA-1 is specifically expressed in larval sensory neurons while RluA-2 expression is ubiquitous. Nociceptor-specific RNAi knockdown of RluA-1 caused hypersensitive nociception phenotypes, which were recapitulated with genetic null alleles. These were rescued with genomic duplication and nociceptor-specific expression of UAS-RluA-1-cDNA As with RluA-1, RluA-2 loss of function mutants also displayed hyperalgesia. Interestingly, nociceptor neuron dendrites showed a hyperbranched morphology in the RluA-1 mutants. The latter may be a cause or a consequence of heightened sensitivity in mutant nociception behaviors. | Bansal, P., Madlung, J., Schaaf, K., Macek, B. and Bono, F. (2020). An Interaction Network of RNA-Binding Proteins Involved in Drosophila Oogenesis. Mol Cell Proteomics 19(9): 1485-1502. PubMed ID: 33451731
Summary: During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, protein-protein associations were examined of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells led to identification of 26 previously unknown associations, besides recovering several well characterized interactions. This study identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented in this study provides the foundation for future functional and structural studies of mRNA localization across metazoans. |
Tuesday, February 2nd - Disease Models |
| Han, S. Y., Pandey, A., Moore, T., Galeone, A., Duraine, L., Cowan, T. M. and Jafar-Nejad, H. (2020). A conserved role for AMP-activated protein kinase in NGLY1 deficiency. PLoS Genet 16(12): e1009258. PubMed ID: 33315951
Summary: Mutations in human N-glycanase 1 (NGLY1) cause the first known congenital disorder of deglycosylation (CDDG). Patients with this rare disease, which is also known as NGLY1 deficiency, exhibit global developmental delay and other phenotypes including neuropathy, movement disorder, and constipation. NGLY1 is known to regulate proteasomal and mitophagy gene expression through activation of a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1). Loss of NGLY1 has also been shown to impair energy metabolism, but the molecular basis for this phenotype and its in vivo consequences are not well understood. Using a combination of genetic studies, imaging, and biochemical assays, this study reports that loss of NGLY1 in the visceral muscle of the Drosophila larval intestine results in a severe reduction in the level of AMP-activated protein kinase α (AMPKα), leading to energy metabolism defects, impaired gut peristalsis, failure to empty the gut, and animal lethality. Ngly1-/- mouse embryonic fibroblasts and NGLY1 deficiency patient fibroblasts also show reduced AMPKα levels. Moreover, pharmacological activation of AMPK signaling significantly suppressed the energy metabolism defects in these cells. Importantly, the reduced AMPKα level and impaired energy metabolism observed in NGLY1 deficiency models are not caused by the loss of NFE2L1 activity. Taken together, these observations identify reduced AMPK signaling as a conserved mediator of energy metabolism defects in NGLY1 deficiency and suggest AMPK signaling as a therapeutic target in this disease. | Dahl-Halvarsson, M., Olive, M., Pokrzywa, M., Norum, M., Ejeskar, K. and Tajsharghi, H. (2020). Impaired muscle morphology in a Drosophila model of myosin storage myopathy was supressed by overexpression of an E3 ubiquitin ligase. Dis Model Mech 13(12). PubMed ID: 33234710
Summary: Myosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/β-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases of myosin storage myopathy. This study developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of the R1845W mutation. A higher expression level of the mutated allele was shown to be concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was suppressed by expression of Thin (herein referred to as Abba), an E3 ubiquitin ligase. This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities, including extensive loss of thick filaments with selective A-band loss, and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Furthermore, the impaired muscle morphology associated with the mutant allele was suppressed by expression of Abba. These findings suggest that modification of the ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients. |
| Cunningham, K. M., Maulding, K., Ruan, K., Senturk, M., Grima, J. C., Sung, H., Zuo, Z., Song, H., Gao, J., Dubey, S., Rothstein, J. D., Zhang, K., Bellen, H. J. and Lloyd, T. E. (2020). TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS. Elife 9. PubMed ID: 33300868
Summary: Disrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which disrupted NCT causes neurodegeneration remain unclear. A Drosophila screen identified ref(2)P/p62, a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. These data suggest that the C9orf72-HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD. | Cowan, C. M., Sealey, M. A. and Mudher, A. (2020). Suppression of tau-induced phenotypes by vitamin E demonstrates the dissociation of oxidative stress and phosphorylation in mechanisms of tau toxicity. J Neurochem. PubMed ID: 33251603
Summary: Various lines of evidence implicate oxidative stress in the pathogenic mechanism(s) underpinning tauopathies. Consequently, antioxidant therapies have been considered in clinical practice for the treatment of tauopathies such as Alzheimer's disease (AD), but with mixed results. Previous studies have reported increased protein oxidation upon expression of both human 0N3R (hTau(0N3R)) and 0N4R (hTau(0N4R)) tau (see Drosophila Tau) in vivo. Building on these studies, this study demonstrates here the suppression of hTau(0N3R) associated phenotypes in Drosophila melanogaster after treatment with vitamin C or vitamin E. Curiously the rescue of phenotype was seen without alteration in total tau level or alteration in phosphorylation at a number of disease-associated sites. Moreover, treatment with paraquat, a pro-oxidant drug, did not exacerbate the hTau(0N3R) phenotypes. This result following paraquat treatment is reminiscent of previous findings with hTau(0N4R) which also causes greater oxidative stress when compared to hTau(0N3R) but has a milder phenotype. Collectively these data imply that the role of oxidative stress in tau-mediated toxicity is not straight forward and there may be isoform-specific effects as well as contribution of other factors. This may explain the ambiguous effects of anti-oxidant treatments on clinical outcome in dementia patients. |
| Benmimoun, B., Papastefanaki, F., Perichon, B., Segklia, K., Roby, N., Miriagou, V., Schmitt, C., Dramsi, S., Matsas, R. and Speder, P. (2020). An original infection model identifies host lipoprotein import as a route for blood-brain barrier crossing. Nat Commun 11(1): 6106. PubMed ID: 33257684
Summary: Pathogens able to cross the blood-brain barrier (BBB) induce long-term neurological sequelae and death. Understanding how neurotropic pathogens bypass this strong physiological barrier is a prerequisite to devise therapeutic strategies. This paper proposes an innovative model of infection in the developing Drosophila brain, combining whole brain explants with in vivo systemic infection. Several mammalian pathogens are able to cross the Drosophila BBB, including Group B Streptococcus (GBS). Amongst GBS surface components, lipoproteins, and in particular the B leucine-rich Blr, are important for BBB crossing and virulence in Drosophila. Further, (V)LDL receptor LpR2, expressed in the BBB, was identified as a host receptor for Blr, allowing GBS translocation through endocytosis. Finally, this study show that Blr is required for BBB crossing and pathogenicity in a murine model of infection. These results demonstrate the potential of Drosophila for studying BBB crossing by pathogens and identify a new mechanism by which pathogens exploit the machinery of host barriers to generate brain infection. | Brunet, M. A., Jacques, J. F., Nassari, S., Tyzack, G. E., McGoldrick, P., Zinman, L., Jean, S., Robertson, J., Patani, R. and Roucou, X. (2020). The FUS gene is dual-coding with both proteins contributing to FUS-mediated toxicity. EMBO Rep 22(1): e50640. PubMed ID: 33226175
Summary: Novel functional coding sequences (altORFs) are camouflaged within annotated ones (CDS) in a different reading frame. This study shows that an altORF is nested in the FUS CDS, encoding a conserved 170 amino acid protein, altFUS. AltFUS is endogenously expressed in human tissues, notably in the motor cortex and motor neurons. Over-expression of wild-type FUS and/or amyotrophic lateral sclerosis-linked FUS mutants is known to trigger toxic mechanisms in different models. These include inhibition of autophagy, loss of mitochondrial potential and accumulation of cytoplasmic aggregates. altFUS, not FUS, is responsible for the inhibition of autophagy, and pivotal in mitochondrial potential loss and accumulation of cytoplasmic aggregates. Suppression of altFUS expression in a Drosophila model of FUS-related toxicity protects against neurodegeneration. Some mutations found in ALS patients are overlooked because of their synonymous effect on the FUS protein. Yet, this study shows they exert a deleterious effect causing missense mutations in the overlapping altFUS protein. These findings demonstrate that FUS is a bicistronic gene and suggests that both proteins, FUS and altFUS, cooperate in toxic mechanisms. |
Monday February 1st - Adult neural function |
| Chouhan, N. S., Griffith, L. C., Haynes, P. and Sehgal, A. (2020). Availability of food determines the need for sleep in memory consolidation. Nature. PubMed ID: 33268891
Summary: Sleep remains a major mystery of biology, with little understood about its basic function. One of the most commonly proposed functions of sleep is the consolidation of memory. However, as conditions such as starvation require the organism to be awake and active, the ability to switch to a memory consolidation mechanism that is not contingent on sleep may confer an evolutionary advantage. This study identified an adaptive circuit-based mechanism that enables Drosophila to form sleep-dependent and sleep-independent memory. Flies fed after appetitive conditioning needed increased sleep for memory consolidation, but flies starved after training did not require sleep to form memories. Memory in fed flies is mediated by the anterior-posterior α'/β' neurons of the mushroom body, while memory under starvation is mediated by medial α'/β' neurons. Sleep-dependent and sleep-independent memory rely on distinct dopaminergic neurons and corresponding mushroom body output neurons. However, sleep and memory are coupled such that mushroom body neurons required for sleep-dependent memory also promote sleep. Flies lacking Neuropeptide F display sleep-dependent memory even when starved, suggesting that circuit selection is determined by hunger. This plasticity in memory circuits enables flies to retain essential information in changing environments. | Buhl, E., Kottler, B., Hodge, J. J. L. and Hirth, F. (2021). Thermoresponsive motor behavior is mediated by ring neuron circuits in the central complex of Drosophila. JSci Rep 11(1): 155. PubMed ID: 33420240
Summary: Insects are ectothermal animals that are constrained in their survival and reproduction by external temperature fluctuations which require either active avoidance of or movement towards a given heat source. In Drosophila, different thermoreceptors and neurons have been identified that mediate temperature sensation to maintain the animal's thermal preference. However, less is known how thermosensory information is integrated to gate thermoresponsive motor behavior. This study used transsynaptic tracing together with calcium imaging, electrophysiology and thermogenetic manipulations in freely moving Drosophila exposed to elevated temperature and identify different functions of ellipsoid body ring neurons, R1-R4, in thermoresponsive motor behavior. The results show that warming of the external surroundings elicits calcium influx specifically in R2-R4 but not in R1, which evokes threshold-dependent neural activity in the outer layer ring neurons. In contrast to R2, R3 and R4d neurons, thermogenetic inactivation of R4m and R1 neurons expressing the temperature-sensitive mutant allele of dynamin, shibire(TS), results in impaired thermoresponsive motor behavior at elevated 31 °C. trans-Tango mediated transsynaptic tracing together with physiological and behavioral analyses indicate that integrated sensory information of warming is registered by neural activity of R4m as input layer of the ellipsoid body ring neuropil and relayed on to R1 output neurons that gate an adaptive motor response. Together these findings imply that segregated activities of central complex ring neurons mediate sensory-motor transformation of external temperature changes and gate thermoresponsive motor behavior in Drosophila. |
| Chiu, H., Hoopfer, E. D., Coughlan, M. L. and Anderson, D. J. (2020). A circuit logic for sexually shared and dimorphic aggressive behaviors in Drosophila. Cell. PubMed ID: 33382967
Summary: Aggression involves both sexually monomorphic and dimorphic actions. How the brain implements these two types of actions is poorly understood. This study has identified three cell types that regulate aggression in Drosophila: one type is sexually shared, and the other two are sex specific. Shared common aggression-promoting (CAP) neurons mediate aggressive approach in both sexes, whereas functionally downstream dimorphic but homologous cell types, called male-specific aggression-promoting (MAP) neurons in males and fpC1 in females, control dimorphic attack. These symmetric circuits underlie the divergence of male and female aggressive behaviors, from their monomorphic appetitive/motivational to their dimorphic consummatory phases. The strength of the monomorphic → dimorphic functional connection is increased by social isolation in both sexes, suggesting that it may be a locus for isolation-dependent enhancement of aggression. Together, these findings reveal a circuit logic for the neural control of behaviors that include both sexually monomorphic and dimorphic actions, which may generalize to other organisms. | Alizzi, R. A., Xu, D., Tenenbaum, C. M., Wang, W. and Gavis, E. R. (2020). The ELAV/Hu protein Found in neurons regulates cytoskeletal and ECM adhesion inputs for space-filling dendrite growth. PLoS Genet 16(12): e1009235. PubMed ID: 33370772
Summary: Dendritic arbor morphology influences how neurons receive and integrate extracellular signals. This study shows that he ELAV/Hu family RNA-binding protein Found in neurons (Fne) is required for space-filling dendrite growth to generate highly branched arbors of Drosophila larval class IV dendritic arborization neurons. Dendrites of fne mutant neurons are shorter and more dynamic than in wild-type, leading to decreased arbor coverage. These defects result from both a decrease in stable microtubules and loss of dendrite-substrate interactions within the arbor. Identification of transcripts encoding cytoskeletal regulators and cell-cell and cell-ECM interacting proteins as Fne targets using TRIBE further supports these results. Analysis of one target, encoding the cell adhesion protein Basigin, indicates that the cytoskeletal defects contributing to branch instability in fne mutant neurons are due in part to decreased Basigin expression. The ability of Fne to coordinately regulate the cytoskeleton and dendrite-substrate interactions in neurons may shed light on the behavior of cancer cells ectopically expressing ELAV/Hu proteins. |
| Currier, T. A., Matheson, A. M. and Nagel, K. I. (2020). Encoding and control of orientation to airflow by a set of Drosophila fan-shaped body neurons. Elife 9. PubMed ID: 33377868
Summary: The insect central complex (CX) is thought to underlie goal-oriented navigation but its functional organization is not fully understood. This study recorded from genetically-identified CX cell types in Drosophila and presented directional visual, olfactory, and airflow cues known to elicit orienting behavior. A group of neurons targeting the ventral fan-shaped body (ventral P-FNs) was found to be robustly tuned for airflow direction. Ventral P-FNs did not generate a 'map' of airflow direction. Instead, cells in each hemisphere were tuned to 45° ipsilateral, forming a pair of orthogonal bases. Imaging experiments suggest that ventral P-FNs inherit their airflow tuning from neurons that provide input from the lateral accessory lobe (LAL) to the noduli (NO). Silencing ventral P-FNs prevented flies from selecting appropriate corrective turns following changes in airflow direction. These results identify a group of CX neurons that robustly encode airflow direction and are required for proper orientation to this stimulus. | Deutsch, D., Pacheco, D., Encarnacion-Rivera, L., Pereira, T., Fathy, R., Clemens, J., Girardin, C., Calhoun, A., Ireland, E., Burke, A., Dorkenwald, S., McKellar, C., Macrina, T., Lu, R., Lee, K., Kemnitz, N., Ih, D., Castro, M., Halageri, A., Jordan, C., Silversmith, W., Wu, J., Seung, H. S. and Murthy, M. (2020). The neural basis for a persistent internal state in Drosophila females. Elife 9. PubMed ID: 33225998
Summary: Sustained changes in mood or action require persistent changes in neural activity, but it has been difficult to identify the neural circuit mechanisms that underlie persistent activity and contribute to long-lasting changes in behavior. This study shows that a subset of Doublesex+ pC1 neurons in the Drosophila female brain, called pC1d/e, can drive minutes-long changes in female behavior in the presence of males. Using automated reconstruction of a volume electron microscopic (EM) image of the female brain, all inputs and outputs to both pC1d and pC1e were mapped. This reveals strong recurrent connectivity between, in particular, pC1d/e neurons and a specific subset of Fruitless+ neurons called aIPg. This study additionally found that pC1d/e activation drives long-lasting persistent neural activity in brain areas and cells overlapping with the pC1d/e neural network, including both Doublesex+ and Fruitless+ neurons. This work thus links minutes-long persistent changes in behavior with persistent neural activity and recurrent circuit architecture in the female brain. |
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