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Friday, April 29th, 2022 - Larval and Adult Neural Function

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Jonaitis, J., MacLeod, J. and Pulver, S. R. (2022). Localization of muscarinic acetylcholine receptor-dependent rhythm-generating modules in the Drosophila larval locomotor network. J Neurophysiol 127(4): 1098-1116. PubMed ID: 35294308
Summary:
This study explored how muscarinic acetylcholine receptor (mAChR)-modulated rhythm-generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. Fictive motor patterns were measured in isolated CNS preparations, using a combination of Ca(2+) imaging and electrophysiology while manipulating mAChR signaling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca(2+) levels and abolished rhythmic activity. Overall, these results suggest that mAChR signaling plays a role in enabling rhythm generation at multiple sites in the larval CNS.
Farrugia, M., Vassallo, N. and Cauchi, R. J. (2022). Disruption of Survival Motor Neuron in Glia Impacts Survival but has no Effect on Neuromuscular Function in Drosophila. Neuroscience 491: 32-42. PubMed ID: 35314252
Summary:
Increasing evidence points to the involvement of cell types other than motor neurons in both ALS and SMA, the predominant motor neuron disease in adults and infants, respectively. The contribution of glia to ALS pathophysiology is well documented. This study asked whether the Smn protein, the causative factor for SMA, is required selectively in glia. Loss of Smn function in glia during development was shown to reduce survival to adulthood but did not affect motoric performance or neuromuscular junction (NMJ) morphology. In contrast, gain of ALS-linked TDP-43, FUS or C9orf72 function in glia induced significant defects in motor behaviour in addition to reduced survival. Furthermore, glia-specific gain of TDP-43 function caused both NMJ defects and muscle atrophy. Smn together with Gemins 2-8 and Unrip, form the Smn complex which is indispensable for the assembly of spliceosomal snRNPs. Glial-selective perturbation of Smn complex components or disruption of key snRNP biogenesis factors pICln and Tgs1, induce deleterious effects on adult fly viability. These findings suggest that the role of Smn in snRNP biogenesis as part of the Smn complex is required in glia for the survival of the organism, underscoring the importance of glial cells in SMA disease formation.
Suzuki, Y., Kurata, Y. and Sakai, T. (2022). Dorsal-lateral clock neurons modulate consolidation and maintenance of long-term memory in Drosophila. Genes Cells 27(4): 266-279. PubMed ID: 35094465
Summary:
A newly formed memory is initially unstable. However, if it is consolidated into the brain, the consolidated memory is stored as stable long-term memory (LTM). Despite the recent progress, the molecular and cellular mechanisms of LTM have not yet been fully elucidated. The fruitfly Drosophila melanogaster, for which various genetic tools are available, has been used to clarify the molecular mechanisms of LTM. Using the Drosophila courtship-conditioning assay as a memory paradigm, it was previously identified that the circadian clock gene period (per) plays a vital role in consolidating LTM, suggesting that per-expressing clock neurons are critically involved in LTM. However, it is still incompletely understood which clock neurons are essential for LTM. This study shows that dorsal-lateral clock neurons (LNds) play a crucial role in LTM. Using an LNd-specific split-GAL4 line, it wa confirmed that disruption of synaptic transmission in LNds impaired LTM maintenance. On the other hand, induction of per RNAi or the dominant-negative transgene of Per in LNds impaired LTM consolidation. These results reveal that transmitter release and Per function in LNds are involved in courtship memory processing.
Tanaka, R. and Clark, D. A. (2022). Identifying inputs to visual projection neurons in Drosophila lobula by analyzing connectomic data. eNeuro. PubMed ID: 35410869
Summary:
Electron microscopy-based connectomes provide important insights into how visual circuitry of fruit fly Drosophila computes various visual features, guiding and complementing behavioral and physiological studies. However, connectomic analyses of the lobula, a neuropil putatively dedicated to detecting object-like features, remains underdeveloped, largely because of incomplete data on the inputs to the brain region. This study attempted to map the columnar inputs into the Drosophila lobula neuropil by performing connectivity- and morphology-based clustering on a densely reconstructed connectome dataset. While the dataset mostly lacked visual neuropils other than lobula, which would normally help identify inputs to lobula, clustering analysis successfully extracted clusters of cells with homogeneous connectivity and morphology, likely representing genuine cell types. It was possible to draw a correspondence between the resulting clusters and previously identified cell types, revealing previously undocumented connectivity between lobula input and output neurons. While future, more complete connectomic reconstructions are necessary to verify the results presented in this study, they can serve as a useful basis for formulating hypotheses on mechanisms of visual feature detection in lobula.
Nakagawa, H., Maehara, S., Kume, K., Ohta, H. and Tomita, J. (2022). Biological functions of alpha2-adrenergic-like octopamine receptor in Drosophila melanogaster. Genes Brain Behav: e12807. PubMed ID: 35411674
Summary:
Octopamine regulates various physiological phenomena including memory, sleep, grooming and aggression in insects. In Drosophila, four types of octopamine receptors have been identified: Oamb, Oct/TyrR, OctβR and Octα2R. Among these receptors, Octalpha2R was recently discovered and pharmacologically characterized. However, the effects of the receptor on biological functions are still unknown. This study showed that Octα2R regulated several behaviors related to octopamine signaling. Octα2R hypomorphic mutant flies showed a significant decrease in locomotor activity. Octα2R expressed in the pars intercerebralis, which is a brain region projected by octopaminergic neurons, is involved in control of the locomotor activity. Besides, Octα2R hypomorphic mutants increased time and frequency of grooming and inhibited starvation-induced hyperactivity. These results indicated that Octα2R expressed in the central nervous system is responsible for the involvement in physiological functions.
Lin, C., Schneps, C. M., Chandrasekaran, S., Ganguly, A. and Crane, B. R. (2022). Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome. Structure. PubMed ID: 35397203
Summary:
Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM recognition. This study presents a generally applicable select western-blot-free tagged-protein interaction (SWFTI) assay that allowed the quantification of CRY binding to TIM in dark and light. The assay was used to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bound TIM constitutively, whereas those incapable of photoreduction bound TIM weakly. In response to the flavin redox state, two conserved histidine residues contributed to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. This approach provides an expeditious means to quantify the interactions of difficult-to-produce proteins.

Thursday, April 28th - Disease models

Jonaitis, J., MacLeod, J. and Pulver, S. R. (2022). Localization of muscarinic acetylcholine receptor-dependent rhythm-generating modules in the Drosophila larval locomotor network. J Neurophysiol 127(4): 1098-1116. PubMed ID: 35294308
Summary:
Mechanisms of rhythm generation have been extensively studied in motor systems that control locomotion over terrain in limbed animals; however, much less is known about rhythm generation in soft-bodied terrestrial animals. This study explored how muscarinic acetylcholine receptor (mAChR)-modulated rhythm-generating networks are distributed in the central nervous system (CNS) of soft-bodied Drosophila larvae. Fictive motor patterns were measured in isolated CNS preparations, using a combination of Ca(2+) imaging and electrophysiology while manipulating mAChR signaling pharmacologically. Bath application of the mAChR agonist oxotremorine potentiated bilaterally asymmetric activity in anterior thoracic regions and promoted bursting in posterior abdominal regions. Application of the mAChR antagonist scopolamine suppressed rhythm generation in these regions and blocked the effects of oxotremorine. Oxotremorine triggered fictive forward crawling in preparations without brain lobes. Oxotremorine also potentiated rhythmic activity in isolated posterior abdominal CNS segments as well as isolated anterior brain and thoracic regions, but it did not induce rhythmic activity in isolated anterior abdominal segments. Bath application of scopolamine to reduced preparations lowered baseline Ca(2+) levels and abolished rhythmic activity. Overall, these results suggest that mAChR signaling plays a role in enabling rhythm generation at multiple sites in the larval CNS. This work furthers understanding of motor control in soft-bodied locomotion and provides a foundation for study of rhythm-generating networks in an emerging genetically tractable locomotor system.
O'Neill, R. S. and Rusan, N. M. (2022). Traip controls mushroom body size by suppressing mitotic defects. Development 149(7). PubMed ID: 35297981
Summary:
Microcephaly is a failure to develop proper brain size and neuron number. Mutations in diverse genes are linked to microcephaly, including several with DNA damage repair (DDR) functions; however, it is not well understood how these DDR gene mutations limit brain size. One such gene is TRAIP, which has multiple functions in DDR. This study characterized the Drosophila TRAIP homolog nopo, hereafter traip, and found that traip mutants (traip-) have a brain-specific defect in the mushroom body (MB). traip- MBs were smaller and contained fewer neurons, but no neurodegeneration, consistent with human primary microcephaly. Reduced neuron numbers in traip- were explained by premature loss of MB neuroblasts (MB-NBs), in part via caspase-dependent cell death. Many traip- MB-NBs had prominent chromosome bridges in anaphase, along with polyploidy, aneuploidy or micronuclei. Traip localization during mitosis is sufficient for MB development, suggesting that Traip can repair chromosome bridges during mitosis if necessary. These results suggest that proper brain size is ensured by the recently described role for TRAIP in unloading stalled replication forks in mitosis, which suppresses DNA bridges and premature neural stem cell loss to promote proper neuron number.
Suzuki, Y., Kurata, Y. and Sakai, T. (2022). Dorsal-lateral clock neurons modulate consolidation and maintenance of long-term memory in Drosophila. Genes Cells 27(4): 266-279. PubMed ID: 35094465
Summary:
A newly formed memory is initially unstable. However, if it is consolidated into the brain, the consolidated memory is stored as stable long-term memory (LTM). Despite the recent progress, the molecular and cellular mechanisms of LTM have not yet been fully elucidated. The fruitfly Drosophila melanogaster, for which various genetic tools are available, has been used to clarify the molecular mechanisms of LTM. Using the Drosophila courtship-conditioning assay as a memory paradigm, it was previously identified that the circadian clock gene period (per) plays a vital role in consolidating LTM, suggesting that per-expressing clock neurons are critically involved in LTM. However, it is still incompletely understood which clock neurons are essential for LTM. This study shows that dorsal-lateral clock neurons (LNds) play a crucial role in LTM. Using an LNd-specific split-GAL4 line, it wa confirmed that disruption of synaptic transmission in LNds impaired LTM maintenance. On the other hand, induction of per RNAi or the dominant-negative transgene of Per in LNds impaired LTM consolidation. These results reveal that transmitter release and Per function in LNds are involved in courtship memory processing.
Tanaka, R. and Clark, D. A. (2022). Identifying inputs to visual projection neurons in Drosophila lobula by analyzing connectomic data. eNeuro. PubMed ID: 35410869
Summary:
Electron microscopy-based connectomes provide important insights into how visual circuitry of fruit fly Drosophila computes various visual features, guiding and complementing behavioral and physiological studies. However, connectomic analyses of the lobula, a neuropil putatively dedicated to detecting object-like features, remains underdeveloped, largely because of incomplete data on the inputs to the brain region. This study attempted to map the columnar inputs into the Drosophila lobula neuropil by performing connectivity- and morphology-based clustering on a densely reconstructed connectome dataset. While the dataset mostly lacked visual neuropils other than lobula, which would normally help identify inputs to lobula, clustering analysis successfully extracted clusters of cells with homogeneous connectivity and morphology, likely representing genuine cell types. It was possible to draw a correspondence between the resulting clusters and previously identified cell types, revealing previously undocumented connectivity between lobula input and output neurons. While future, more complete connectomic reconstructions are necessary to verify the results presented in this study, they can serve as a useful basis for formulating hypotheses on mechanisms of visual feature detection in lobula.
Nakagawa, H., Maehara, S., Kume, K., Ohta, H. and Tomita, J. (2022). Biological functions of alpha2-adrenergic-like octopamine receptor in Drosophila melanogaster. Genes Brain Behav: e12807. PubMed ID: 35411674
Summary:
Octopamine regulates various physiological phenomena including memory, sleep, grooming and aggression in insects. In Drosophila, four types of octopamine receptors have been identified: Oamb, Oct/TyrR, OctβR and Octα2R. Among these receptors, Octalpha2R was recently discovered and pharmacologically characterized. However, the effects of the receptor on biological functions are still unknown. This study showed that Octα2R regulated several behaviors related to octopamine signaling. Octα2R hypomorphic mutant flies showed a significant decrease in locomotor activity. Octα2R expressed in the pars intercerebralis, which is a brain region projected by octopaminergic neurons, is involved in control of the locomotor activity. Besides, Octα2R hypomorphic mutants increased time and frequency of grooming and inhibited starvation-induced hyperactivity. These results indicated that Octα2R expressed in the central nervous system is responsible for the involvement in physiological functions.
Lin, C., Schneps, C. M., Chandrasekaran, S., Ganguly, A. and Crane, B. R. (2022). Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome. Structure. PubMed ID: 35397203
Summary:
Cryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM recognition. This study presents a generally applicable select western-blot-free tagged-protein interaction (SWFTI) assay that allowed the quantification of CRY binding to TIM in dark and light. The assay was used to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bound TIM constitutively, whereas those incapable of photoreduction bound TIM weakly. In response to the flavin redox state, two conserved histidine residues contributed to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. This approach provides an expeditious means to quantify the interactions of difficult-to-produce proteins.

Thursday, April 28th - Disease model

Prifti, E., Tsakiri, E. N., Vourkou, E., Stamatakis, G., Samiotaki, M., Skoulakis, E. M. C. and Papanikolopoulou, K. (2022). Mical modulates Tau toxicity via cysteine oxidation in vivo. Acta Neuropathol Commun 10(1): 44. PubMed ID: 35379354
Summary:
Tau accumulation is clearly linked to pathogenesis in Alzheimer's disease and other Tauopathies. However, processes leading to Tau fibrillization and reasons for its pathogenicity remain largely elusive. Mical emerged as a novel interacting protein of human Tau expressed in Drosophila brains. Mical is characterized by the presence of a flavoprotein monooxygenase domain that generates redox potential with which it can oxidize target proteins. In the well-established Drosophila Tauopathy model, genetic interactions were used to show that Mical alters Tau interactions with microtubules and the Actin cytoskeleton and greatly affects Tau aggregation propensity and Tau-associated toxicity and dysfunction. Exploration of the mechanism was pursued using a Mical inhibitor, a mutation in Mical that selectively disrupts its monooxygenase domain, Tau transgenes mutated at cysteine residues targeted by Mical and mass spectrometry analysis to quantify cysteine oxidation. The collective evidence strongly indicates that Mical's redox activity mediates the effects on Tau via oxidation of Cys322. Importantly, this study also validate results from the fly model in human Tauopathy samples by showing that MICAL1 is up-regulated in patient brains and co-localizes with Tau in Pick bodies. This work provides mechanistic insights into the role of the Tau cysteine residues as redox-switches regulating the process of Tau self-assembly into inclusions in vivo, its function as a cytoskeletal protein and its effect on neuronal toxicity and dysfunction.
Kanel, P., Noll, G. A., Schroedter, K., Naffin, E., Kronenberg, J., Busswinkel, F., Twyman, R. M., Klambt, C. and Prufer, D. (2022). The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network. Aging (Albany NY) 14. PubMed ID: 35396341
Summary:
Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, this study expressed PEPBs in a heterologous context to determine their ectopic activity. Heterologous expression of the tobacco (Nicotiana tabacum) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, deregulated genes were identified encoding proteases that may contribute to proteome stability at equilibrium. The results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes that were observed.
Ring, J., Tadic, J., Ristic, S., Poglitsch, M., Bergmann, M., ..., Hansen, N., Sommer, C., Ninkovic, M., Seba, S., Rockenfeller, P., Vogtle, F. N., Dengjel, J., Meisinger, C., Keller, A., Sigrist, S. J., Eisenberg, T. and Madeo, F. (2022). The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity.EMBO Mol Med: e13952. PubMed ID: 35373908
Summary:
Amyloid beta 42 (Abeta42; see Drosophila Appl) is the principal trigger of neurodegeneration during Alzheimer's disease (AD). However, the etiology of its noxious cellular effects remains elusive. In a combinatory genetic and proteomic approach using a yeast model to study aspects of intracellular Abeta42 toxicity, this study identified the HSP40 family member Ydj1, the yeast orthologue of human DnaJA1, as a crucial factor in Abeta42-mediated cell death. It was demonstrate that Ydj1/DnaJA1 physically interacts with Abeta42 (in yeast and mouse), stabilizes Abeta42 oligomers, and mediates their translocation to mitochondria. Consequently, deletion of YDJ1 strongly reduces co-purification of Abeta42 with mitochondria and prevents Abeta42-induced mitochondria-dependent cell death. Consistently, purified DnaJ chaperone delays Abeta42 fibrillization in vitro, and heterologous expression of human DnaJA1 induces formation of Abeta42 oligomers and their deleterious translocation to mitochondria in vivo. Finally, downregulation of the Ydj1 fly homologue, Droj2, improves stress resistance, mitochondrial morphology, and memory performance in a Drosophila melanogaster AD model. These data reveal an unexpected and detrimental role for specific HSP40s in promoting hallmarks of Abeta42 toxicity.
Nath, S., Caron, N. S., May, L., Gluscencova, O. B., Kolesar, J., Brady, L., Kaufman, B. A., Boulianne, G. L., Rodriguez, A. R., Tarnopolsky, M. A. and Truant, R. (2022). Functional characterization of variants of unknown significance in a spinocerebellar ataxia patient using an unsupervised machine learning pipeline. Hum Genome Var 9(1): 10. PubMed ID: 35422034
Summary:
CAG-expanded ATXN7 has been previously defined in the pathogenesis of spinocerebellar ataxia type 7 (SCA7), a polyglutamine expansion autosomal dominant cerebellar ataxia. Pathology in SCA7 occurs as a result of a CAG triplet repeat expansion in excess of 37 in the first exon of ATXN7, which encodes ataxin-7 (see Drosophila Ataxin 7). SCA7 presents clinically with spinocerebellar ataxia and cone-rod dystrophy. This study presents a novel spinocerebellar ataxia variant occurring in a patient with mutations in both ATXN7 and TOP1MT, which encodes mitochondrial topoisomerase I (top1mt). Using machine-guided, unbiased microscopy image analysis, alterations were demonstrated in ataxin-7 subcellular localization, and through high-fidelity measurements of cellular respiration, bioenergetic defects in association with top1mt mutations. Ataxin-7 Q35P and top1mt R111W were identified as deleterious mutations, potentially contributing to disease states. These mutations were recapitulated through Drosophila genetic models. This work provides important insight into the cellular biology of ataxin-7 and top1mt and offers insight into the pathogenesis of spinocerebellar ataxia applicable to multiple subtypes of the illness. Moreover, this study demonstrates an effective pipeline for the characterization of previously unreported genetic variants at the level of cell biology.
Narayan, V. P., Wilson, A. J. and Chenoweth, S. F. (2022). Genetic and social contributions to sex differences in lifespan in Drosophila serrata. J Evol Biol 35(4): 657-663. PubMed ID: 35290690
Summary:
Sex differences in lifespan remain an intriguing puzzle in evolutionary biology. While explanations range from sex differences in selection to sex differences in the expression of recessive lifespan-altering mutations (via X-linkage), little consensus has been reached. One unresolved issue is the extent to which genetic influences on lifespan dimorphism are modulated by the environment. For example, studies have shown that sex differences in lifespan can either increase or decrease depending upon the social environment. This study took an experimental approach, manipulating multiple axes of the social environment across inbred long- and short-lived genotypes and their reciprocal F1s in the fly Drosophila serrata. The results reveal strong genetic effects and subtle yet significant genotype-by-environment interactions for male and female lifespan, specifically due to both population density and mating status. Further, the data do not support the idea that unconditional expression of deleterious X-linked recessive alleles in heterogametic males accounts for lower male lifespan.
Sun, Z. D., Hu, J. X., Wu, J. R., Zhou, B. and Huang, Y. P. (2022). Toxicities of amyloid-beta and tau protein are reciprocally enhanced in the Drosophila model. Neural Regen Res 17(10): 2286-2292. PubMed ID: 35259851
Summary:
Extracellular aggregation of amyloid-beta (Aβ) and intracellular tau tangles are two major pathogenic hallmarks and critical factors of Alzheimer's disease. A linear interaction between Aβ and tau protein has been characterized in several models. Aβ induces tau hyperphosphorylation through a complex mechanism; however, the master regulators involved in this linear process are still unclear. In a study with Drosophila melanogaster, was found to regulate tau hyperphosphorylation and toxicity by activating c-Jun N-terminal kinase. Importantly, Aβ toxicity was dependent on tau hyperphosphorylation, and flies with hypophosphorylated tau were insulated against Aβ-induced toxicity. Strikingly, tau accumulation reciprocally interfered with Aβ degradation and correlated with the reduction in mRNA expression of genes encoding Aβ-degrading enzymes, including dNep1, dNep3, dMmp2, dNep4, and dIDE. These results indicate that Aβ and tau protein work synergistically to further accelerate Alzheimer's disease progression and may be considered as a combined target for future development of Alzheimer's disease therapeutics.

Wednesday, April 27th - Signaling

Humbert, P. O., Pryjda, T. Z., Pranjic, B., Farrell, A., Fujikura, K., de Matos Simoes, R., Karim, R., Kozieradzki, I., Cronin, S. J. F., Neely, G. G., Meyer, T. F., Hagelkruys, A., Richardson, H. E. and Penninger, J. M. (2022). TSPAN6 is a suppressor of Ras-driven cancer. Oncogene 41(14): 2095-2105. PubMed ID: 35184157
Summary:
Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, this study identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. This study shows that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced Kras(G12D)-driven lung tumor initiation and malignant progression. Mechanistically, TSPAN6 binds to the EGFR (see Drosophila Egfr) and blocks EGFR-induced RAS activation. Moreover, inactivation of TSPAN6 induces an epithelial-to-mesenchymal transition and inhibits cell migration in vitro and in vivo. Finally, low TSPAN6 expression correlates with poor prognosis of patients with lung and pancreatic cancers with mesenchymal morphology. These results uncover TSPAN6 as a novel tumor suppressor receptor that controls epithelial cell identify and restrains RAS-driven epithelial cancer.
Stephenson, S. E. M., Costain, G., Blok, L. E. R., ..., Lockhart, P. J., Christodoulou, J. and Tan, T. Y. (2022). Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and a neurodevelopmental syndrome. Am J Hum Genet 109(4): 601-617. PubMed ID: 35395208
Summary:
Mammalian FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. Thirty-five individuals have been identified harboring de novo and inherited FBXW7 mutatyions associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. This study provides compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.
Long, M., Sanchez-Martinez, A., Longo, M., Suomi, F., Stenlund, H., Johansson, A. I., Ehsan, H., Salo, V. T., Montava-Garriga, L., Naddafi, S., Ikonen, E., Ganley, I. G., Whitworth, A. J. and McWilliams, T. G. (2022). DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron  depletion. Embo j: e109390. PubMed ID: 35411952
Summary:
Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. Temporal metabolomics was performed on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. These data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.
Kumari, A., Ghosh, A., Kolay, S. and Raghu, P. (2022). Septins tune lipid kinase activity and PI(4,5)P(2) turnover during G-protein-coupled PLC signalling in vivo. Life Sci Alliance 5(6). PubMed ID: 35277468
Summary:
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] hydrolysis by phospholipase C (PLC) is a conserved mechanism of signalling. Given the low abundance of PI(4,5)P(2), its hydrolysis needs to be coupled to resynthesis to ensure continued PLC activity; however, the mechanism by which depletion is coupled to resynthesis remains unknown. PI(4,5)P(2) synthesis is catalyzed by the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K). In Drosophila photoreceptors, photon absorption is transduced into PLC activity and during this process, PI(4,5)P(2) is resynthesized by a PIP5K. However, the mechanism by which PIP5K activity is coupled to PI(4,5)P(2) hydrolysis is unknown. This study, identified a unique isoform dPIP5K(L), that is both necessary and sufficient to mediate PI(4,5)P(2) synthesis during phototransduction. Depletion of PNUT, a non-redundant subunit of the septin family, enhances dPIP5K(L) activity in vitro and PI(4,5)P(2) resynthesis in vivo; co-depletion of dPIP5K(L) reverses the enhanced rate of PI(4,5)P(2) resynthesis in vivo. Thus, this work defines a septin-mediated mechanism through which PIP5K activity is coupled to PLC-mediated PI(4,5)P(2) hydrolysis.
de Vreede, G., Gerlach, S. U. and Bilder, D. (2022). Epithelial monitoring through ligand-receptor segregation ensures malignant cell elimination. Science 376(6590): 297-301. PubMed ID: 35420935
Summary:
Animals have evolved mechanisms, such as cell competition, to remove dangerous or nonfunctional cells from a tissue. Tumor necrosis factor signaling can eliminate clonal malignancies from Drosophila imaginal epithelia, but why this pathway is activated in tumor cells but not normal tissue is unknown. This study shows that the ligand that drives elimination, Eiger, is present in basolateral circulation but remains latent because it is spatially segregated from its apically localized receptor. Polarity defects associated with malignant transformation cause receptor mislocalization, allowing ligand binding and subsequent apoptotic signaling. This process occurs irrespective of the neighboring cells' genotype and is thus distinct from cell competition. Related phenomena at epithelial wound sites are required for efficient repair. This mechanism of polarized compartmentalization of ligand and receptor can generally monitor epithelial integrity to promote tissue homeostasis.
Deshpande, R., Lee, B., Qiao, Y. and Grewal, S. S. (2022). TOR signaling is required for host lipid metabolic remodelling and survival following enteric infection in Drosophila. Dis Model Mech. PubMed ID: 35363274
Summary:
When infected by enteric pathogenic bacteria, animals need to initiate local and whole-body defence strategies. While most attention has focused on the role innate immune anti-bacterial responses, less is known about how changes in host metabolism contribute to host defence. Using Drosophila as a model system, this study identified induction of intestinal target-of-rapamycin (TOR) kinase signaling as a key adaptive metabolic response to enteric infection. Enteric infection induces both local and systemic induction of TOR independently of the IMD innate immune pathway, and TOR functions together with IMD signaling to promote infection survival. These protective effects of TOR signaling are associated with re-modelling of host lipid metabolism. Thus, TOR is required to limit excessive infection-mediated wasting of host lipid stores by promoting an increase in the levels of gut- and fat body-expressed lipid synthesis genes. These data supports a model in which induction of TOR represents a host tolerance response to counteract infection-mediated lipid wasting in order to promote survival.

Tuesday, April 26th - Synapse and vesicles

Kamemura, K., Moriya, H., Ukita, Y., Okumura, M., Miura, M. and Chihara, T. (2022). Endoplasmic reticulum proteins Meigo and Gp93 govern dendrite targeting by regulating Toll-6 localization. Dev Biol 484: 30-39. PubMed ID: 35134382
Summary:
Neuronal target recognition is performed by numerous cell-surface transmembrane proteins. Correct folding of these proteins occurs in the endoplasmic reticulum (ER) lumen of the neuronal cells before being transported to the plasma membrane of axons or dendrites. Disturbance in this protein folding process in the ER leads to dysfunction of neuronal cell surface molecules, resulting in abnormal neuronal targeting. This study reports that the ER-resident protein Meigo in Drosophila, governs the dendrite targeting of olfactory projection neurons (PNs) along the mediolateral axis of the antennal lobe by regulating Toll-6 localization. Loss of Meigo causes Toll-6 mislocalization in the PNs and mediolateral dendrite targeting defects, which are suppressed by Toll-6 overexpression. Furthermore, this study found that the ER-chaperone protein, Gp93, also regulates the mediolateral targeting of PN dendrites by localization of the Toll-6 protein. Gp93 overexpression in the PN homozygous for the meigo mutation, partially rescued the dendrite targeting defect, while meigo knockdown decreased Gp93 expression levels in cultured cells. These results indicate that the ER-proteins Meigo and Gp93 regulate dendrite targeting by attenuating the amount and localization of cell surface receptors, including Toll-6, implying the unexpected but active involvement of ER proteins in neural wiring.
Nassari, S., Lacarriere-Keita, C., Levesque, D., Boisvert, F. M. and Jean, S. (2022). Rab21 in enterocytes participates in intestinal epithelium maintenance. Mol Biol Cell 33(4): ar32. PubMed ID: 35171715
Summary:
Membrane trafficking is defined as the vesicular transport of proteins into, out of, and throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways result in impaired function and are linked to diseases. However, how these trafficking pathways regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo system, this study investigated enterocyte-specific functions for the early endosomal machinery. Focus was placed on Rab21, which regulates specific steps in early endosomal trafficking. Depletion of Rab21 in enterocytes led to abnormalities in intestinal morphology, with deregulated cellular equilibrium associated with a gain in mitotic cells and increased cell death. Increases in apoptosis and Yorkie signaling were responsible for compensatory proliferation and tissue inflammation. Using an RNA interference screen, this study identified regulators of autophagy and membrane trafficking that phenocopied Rab21 knockdown. It was further shown that Rab21 knockdown-induced hyperplasia was rescued by inhibition of epidermal growth factor receptor signaling. Moreover, quantitative proteomics identified proteins affected by Rab21 depletion. Of these, changes were validated in apolipoprotein ApoLpp and the trehalose transporter Tret1-1, indicating roles for enterocyte Rab21 in lipid and carbohydrate homeostasis, respectively. These data shed light on an important role for early endosomal trafficking, and Rab21, in enterocyte-mediated intestinal epithelium maintenance.
Nair, A. G., Muttathukunnel, P. and Muller, M. (2021). Distinct molecular pathways govern presynaptic homeostatic plasticity. Cell Rep 37(11): 110105. PubMed ID: 34910905
Summary:
Presynaptic homeostatic plasticity (PHP) stabilizes synaptic transmission by counteracting impaired neurotransmitter receptor function through neurotransmitter release potentiation. PHP is thought to be triggered by impaired receptor function and to involve a stereotypic signaling pathway. However, this study demonstrates that different receptor perturbations that similarly reduce synaptic transmission result in different responses at the Drosophila neuromuscular junction. While receptor inhibition by the glutamate receptor (GluR) antagonist γ-D-glutamylglycine (γDGG) is not compensated by PHP, the GluR inhibitors Philanthotoxin-433 (PhTx) and Gyki-53655 (Gyki) induce compensatory PHP. Intriguingly, PHP triggered by PhTx and Gyki involve separable signaling pathways, including inhibition of distinct GluR subtypes, differential modulation of the active-zone scaffold Bruchpilot, and short-term plasticity. Moreover, while PHP upon Gyki treatment does not require genes promoting PhTx-induced PHP, it involves presynaptic protein kinase D. Thus, synapses not only respond differentially to similar activity impairments, but achieve homeostatic compensation via distinct mechanisms, highlighting the diversity of homeostatic signaling.
Song, C., Leahy, S. N., Rushton, E. M. and Broadie, K. (2022). RNA-binding FMRP and Staufen sequentially regulate the coracle scaffold to control synaptic glutamate receptor and bouton development. Development. PubMed ID: 35394012
Summary:
Both mRNA-binding Fragile X Mental Retardation Protein (FMRP) and mRNA-binding Staufen regulate synaptic bouton formation and glutamate receptor (GluR) levels at the Drosophila neuromuscular junction (NMJ) glutamatergic synapse. This study tested whether these RNA-binding proteins (RBPs) act jointly in a common mechanism. Both dfmr1 and staufen mutants, and trans-heterozygous double mutants, were shown to display increased synaptic bouton formation and GluRIIA accumulation. With cell-targeted RNAi, a downstream Staufen role within postsynaptic muscle. With immunoprecipitation, this study showed that FMRP binds staufen mRNA to stabilize postsynaptic transcripts. Staufen is known to target actin-binding, GluRIIA anchor Coracle, and this study confirmed that Staufen binds to coracle mRNA. FMRP and Staufen were shown to act sequentially to co-regulate postsynaptic Coracle expression, and show Coracle, in turn, controls GluRIIA levels and synaptic bouton development. Consistently, this study found dfmr1, staufen and coracle mutants elevate neurotransmission strength. FMRP, Staufen and Coracle all suppress pMad activation, providing a trans-synaptic signaling linkage between postsynaptic GluRIIA levels and presynaptic bouton development. This work supports an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulating structural and functional synapse development.
Barnes, C. L., Bonnery, D. and Cardona, A. (2022). Synaptic counts approximate synaptic contact area in Drosophila. PLoS One 17(4): e0266064. PubMed ID: 35377898
Summary:
The pattern of synaptic connections among neurons defines the circuit structure, which constrains the computations that a circuit can perform. The strength of synaptic connections is costly to measure yet important for accurate circuit modeling. Synaptic surface area has been shown to correlate with synaptic strength, yet in the emerging field of connectomics, most studies rely instead on the counts of synaptic contacts between two neurons. This study quantified the relationship between synaptic count and synaptic area as measured from volume electron microscopy of the larval Drosophila central nervous system. The total synaptic surface area, summed across all synaptic contacts from one presynaptic neuron to a postsynaptic one, can be accurately predicted solely from the number of synaptic contacts, for a variety of neurotransmitters. These findings support the use of synaptic counts for approximating synaptic strength when modeling neural circuits.
Gao, Y., Zhu, Y., Wang, H., Cheng, Y., Zhao, D., Sun, Q. and Chen, D. (2022). Lipid-mediated phase separation of AGO proteins on the ER controls nascent-peptide ubiquitination. Mol Cell 82(7): 1313-1328. PubMed ID: 35325613
Summary:
AGO/miRNA-mediated gene silencing and ubiquitin-mediated protein quality control represent two fundamental mechanisms that control proper gene expression. This study unexpectedly discovered that fly and human AGO proteins (see Drosophila Ago1), which are key components in the miRNA pathway, undergo lipid-mediated phase separation and condense into RNP granules on the endoplasmic reticulum (ER) membrane to control protein production. Phase separation on the ER is mediated by electrostatic interactions between a conserved lipid-binding motif within the AGOs and the lipid PI(4,5)P(2). The ER-localized AGO condensates recruit the E3 ubiquitin ligase Ltn1 to catalyze nascent-peptide ubiquitination and coordinate with the VCP-Ufd1-Npl4 complex to process unwanted protein products for proteasomal degradation. Collectively, this study provides insight into the understanding of post-transcription-translation coupling controlled by AGOs via lipid-mediated phase separation.

Monday April 25th - Adult and larval physiology and metabolism

Loya, A. K., Van Houten, S. K., Glasheen, B. M. and Swank, D. M. (2022). Shortening deactivation: quantifying a critical component of cyclical muscle contraction. Am J Physiol Cell Physiol 322(4): C653-c665. PubMed ID: 34965153
Summary:
A muscle undergoing cyclical contractions requires fast and efficient muscle activation and relaxation to generate high power with relatively low energetic cost. To enhance activation and increase force levels during shortening, some muscle types have evolved stretch activation (SA), a delayed increased in force following rapid muscle lengthening. SA's complementary phenomenon is shortening deactivation (SD), a delayed decrease in force following muscle shortening. SD increases muscle relaxation, which decreases resistance to subsequent muscle lengthening. Although it might be just as important to cyclical power output, SD has received less investigation than SA. To enable mechanistic investigations into SD and quantitatively compare it to SA, this study developed a protocol to elicit SA and SD from Drosophila and Lethocerus indirect flight muscles (IFM) and Drosophila jump muscle. When normalized to isometric tension, Drosophila IFM exhibited a 118% SD tension decrease, Lethocerus IFM dropped by 97%, and Drosophila jump muscle decreased by 37%. The same order was found for normalized SA tension: Drosophila IFM increased by 233%, Lethocerus IFM by 76%, and Drosophila jump muscle by only 11%. SD occurred slightly earlier than SA, relative to the respective length change, for both IFMs; but SD was exceedingly earlier than SA for jump muscle. These results suggest SA and SD evolved to enable highly efficient IFM cyclical power generation and may be caused by the same mechanism. However, jump muscle SA and SD mechanisms are likely different, and may have evolved for a role other than to increase the power output of cyclical contractions.
Salachan, P. V. and Sorensen, J. G. (2022). Molecular mechanisms underlying plasticity in a thermally varying environment. Mol Ecol. PubMed ID: 35397190
Summary:
Adaptation to environmental variability is a prerequisite for species' persistence in their natural environments. With climate change predicted to increase the frequency and severity of temperature fluctuations, ectothermic organisms may increasingly depend on acclimation capacity to accommodate thermal variability. To elucidate the molecular basis of fluctuating temperature induced phenotypic plasticity, this study investigated heat tolerance and the mechanisms induced by acclimation to thermal variability as compared to those seen at constant temperature. Genome-wide transcriptomic analysis was carried out on Drosophila melanogaster subjected to acclimation at constant (19 ± 0°C) and fluctuating (19 ± 8°CC) temperatures and contrasted the induction of molecular mechanisms in adult males, adult females, and larvae. Life stage and sex specific dynamics of the acclimation responses to fluctuating temperatures were found. Adult flies exposed to temperature fluctuations showed a constitutive improvement in heat tolerance while heat tolerance of larvae tracked thermal fluctuations. A constitutive down-regulation of gene expression was observed for several genes in the case of larvae exposed to fluctuations. These results for adult females showed that, for several genes, fluctuating temperature acclimation resulted in canalization of gene expression. Both transcriptional and post-transcriptional machinery were greatly affected by fluctuations in the case of adult males. Gene ontology analysis showed enrichment of heat stress response involving several major heat shock proteins in both larvae and adults exposed to fluctuating temperatures, even though fluctuations were in a benign range of temperatures. Finally, molecular mechanisms related to environmental sensing seem to be an important component of insect response to thermal variability.
Mishra, A., Tung, S., Sruti, V. R. S., Shreenidhi, P. M. and Dey, S. (2022). Desiccation Stress Acts as Cause as well as Cost of Dispersal in Drosophila melanogaster. Am Nat 199(4): E111-e123. PubMed ID: 35324379
Summary:
Environmental stress is one of the important causes of biological dispersal. At the same time, the process of dispersal itself can incur and/or increase susceptibility to stress for the dispersing individuals. Therefore, in principle, stress can serve as both a cause and a cost of dispersal. These potentially contrasting roles of a key environmental stress (desiccation) were studied using Drosophila melanogaster. By modulating water and rest availability, it was asked whether (a) dispersers are individuals that are more susceptible to desiccation stress, (b) dispersers pay a cost in terms of reduced resistance to desiccation stress, (c) dispersal evolution alters the desiccation cost of dispersal, and (d) females pay a reproductive cost of dispersal. Desiccation was was found to be a clear cause of dispersal in both sexes, as both male and female dispersal propensity increased with increasing duration of desiccation. However, the desiccation cost of dispersal was male biased, a trend unaffected by dispersal evolution. Instead, females paid a fecundity cost of dispersal. The complex relationship between desiccation and dispersal, which can lead to both positive and negative associations, were discussed. Furthermore, the sex differences highlighted here may translate into differences in movement patterns, thereby giving rise to sex-biased dispersal patterns.
Iyengar, A. S., Kulkarni, R. and Sheeba, V. (2022). Under warm ambient conditions, Drosophila melanogaster suppresses nighttime activity via the neuropeptide pigment dispersing factor. Genes Brain Behav 21(4): e12802. PubMed ID: 35285135
Summary:
Rhythmic locomotor behaviour of flies is controlled by an endogenous time-keeping mechanism, the circadian clock, and is influenced by environmental temperatures. Flies inherently prefer cool temperatures around 25°C, and under such conditions, time their locomotor activity to occur at dawn and dusk. Under relatively warmer conditions such as 30°C, flies shift their activity into the night, advancing their morning activity bout into the early morning, before lights-ON, and delaying their evening activity into early night. The molecular basis for such temperature-dependent behavioural modulation has been associated with core circadian clock genes, but the neuronal basis is not yet clear. Under relatively cool temperatures such as 25°C, the role of the circadian pacemaker ventrolateral neurons (LNvs), along with a major neuropeptide secreted by them, pigment dispersing factor (PDF), has been showed in regulating various aspects of locomotor activity rhythms. However, the role of the LNvs and PDF in warm temperature-mediated behavioural modulation has not been explored. This study shows that flies lacking proper PDF signalling or the LNvs altogether, cannot suppress their locomotor activity resulting in loss of sleep during the middle of the night, and thus describe a novel role for PDF signalling and the LNvs in behavioural modulation under warm ambient conditions. In a rapidly warming world, such behavioural plasticity may enable organisms to respond to harsh temperatures in the environment.
Garay, E., Schuth, N., Barbanente, A., Tejeda-Guzman, C., Vitone, D., Osorio, B., Clark, A. H., Nachtegaal, M., Haumann, M., Dau, H., Vela, A., Arnesano, F., Quintanar, L. and Missirlis, F. (2022). Tryptophan regulates Drosophila zinc stores. Proc Natl Acad Sci U S A 119(16): e2117807119. PubMed ID: 35412912
Summary:
Zinc deficiency is commonly attributed to inadequate absorption of the metal. Instead, this study shows that body zinc stores in Drosophila melanogaster depend on tryptophan consumption. Hence, a dietary amino acid regulates zinc status of the whole insect—a finding consistent with the widespread requirement of zinc as a protein cofactor. Specifically, the tryptophan metabolite kynurenine is released from insect fat bodies and induces the formation of zinc storage granules in Malpighian tubules, where 3-hydroxykynurenine and xanthurenic acid act as endogenous zinc chelators. Kynurenine functions as a peripheral zinc-regulating hormone and is converted into a 3-hydroxykynurenine–zinc–chloride complex, precipitating within the storage granules. Thus, zinc and the kynurenine pathway—well-known modulators of immunity, blood pressure, aging, and neurodegeneration—are physiologically connected.
Shaposhnikov, M. V., et al. (2022). Deletions of the cystathionine-beta-synthase (CBS) and cystathionine-γ-lyase (CSE) genes, involved in the control of hydrogen sulfide biosynthesis, significantly affect lifespan and fitness components of Drosophila melanogaster. Mech Ageing Dev 203: 111656. PubMed ID: 35247392
Summary:
The gasotransmitter hydrogen sulfide (H2S) is an important biological mediator, playing an essential role in many physiological and pathological processes. It is produced by transsulfuration - an evolutionarily highly conserved pathway for the metabolism of sulfur-containing amino acids methionine and cysteine. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in cysteine metabolism and H2S production. This study investigated the fitness components (longevity, stress resistance, viability of preimaginal stages, and reproductive function parameters) in D. melanogaster lines containing deletions of the CBS and CSE genes. Surprisingly, in most tests, CSE deletion improved, and CBS worsened the fitness. Lines with deletion of both CBS and CSE demonstrated better stress resistance and longevity than lines with single CBS deletion. At the same time, deletion of both CBS and CSE genes causes more serious disturbances of reproductive function parameters than single CBS deletion. Thus, a complex interaction of H2S-producing pathways and cellular stress response in determining the lifespan and fitness components of the whole organism was revealed.

Friday, April 22nd - Disease Models

Hilsabeck, T. A. U., Liu-Bryan, R., Guo, T., Wilson, K. A., Bose, N., Raftery, D., Beck, J. N., Lang, S., Jin, K., Nelson, C. S., Oron, T., Stoller, M., Promislow, D., Brem, R. B., Terkeltaub, R. and Kapahi, P. (2022). A fly GWAS for purine metabolites identifies human FAM214 homolog medusa, which acts in a conserved manner to enhance hyperuricemia-driven pathologies by modulating purine metabolism and the inflammatory response. Geroscience. PubMed ID: 35381951
Summary:
Elevated serum urate (hyperuricemia) promotes crystalline monosodium urate tissue deposits and gout, with associated inflammation and increased mortality. To identify modifiers of uric acid pathologies, a fly Genome-Wide Association Study (GWAS) was performed on purine metabolites using the Drosophila Genetic Reference Panel strains. The candidate genes were tested using the Drosophila melanogaster model of hyperuricemia and uric acid crystallization ("concretion formation") in the kidney-like Malpighian tubule. Medusa (mda) activity increased urate levels and inflammatory response programming. Conversely, whole-body mda knockdown decreased purine synthesis precursor phosphoribosyl pyrophosphate, uric acid, and guanosine levels; limited formation of aggregated uric acid concretions; and was sufficient to rescue lifespan reduction in the fly hyperuricemia and gout model. Levels of mda homolog FAM214A were elevated in inflammatory M1- and reduced in anti-inflammatory M2-differentiated mouse bone marrow macrophages, and influenced intracellular uric acid levels in human HepG2 transformed hepatocytes. In conclusion, mda/FAM214A (see Drosophila CG9005) acts in a conserved manner to regulate purine metabolism, promotes disease driven by hyperuricemia and associated tissue inflammation, and provides a potential novel target for uric acid-driven pathologies.
Thomas, E. O., Ramirez, P., Hyman, B. T., Ray, W. J. and Frost, B. (2021). Testing the neuroinflammatory role of tau-induced transposable elements in tauopathy. Alzheimers Dement 17 Suppl 2: e058664. Link to article: Thomas et al.
Summary:
Novel mechanisms through which pathogenic tau leads to neuronal toxicity remain largely unexplored as therapeutic targets for tauopathies. Activation of retrotransposons has been identified as a mechanism by which pathogenic tau contributes to neurotoxicity. This study has tested the hypothesis that transposable element-derived dsRNAs stimulate the immune response in tauopathy. Bioinformatic analysis of bidirectional transcription was completed on RNA-sequencing data from control and tau transgenic Drosophila. dsRNAs were quantified by J2 anti-dsRNA antibody via immunofluorescence and ELISA. Analysis of inflammatory markers in Drosophila was completed by RNA-sequencing and with a custom NanoString Gene Expression codeset. dsRNAs were found to be elevated in the brains of tau transgenic Drosophila, in the cortex of a mouse model of tauopathy, and in post-mortem brain tissue from human tauopathy. Retrotransposons were identified that are bidirectionally transcribed in tau transgenic Drosophila. RNA sequencing and NanoString analysis of tau transgenic flies reveals elevated levels of innate immune response gene transcripts. Loss of transposon transcriptional silencing results in similar dsRNA production and immune system activation found in tau transgenic Drosophila. The goal of this study was to determine if RNA intermediates from tau-induced transposable elements are a therapeutic target for tauopathies. Overall, the findings support a model where dsRNA production induced by loss of transcriptional transposon silencing contributes to inflammation in tauopathy.
Basu, I., Bar, S., Prasad, M. and Datta, R. (2022). Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR. Biochim Biophys Acta Mol Basis Dis 1868(7): 166399. PubMed ID: 35318126
Summary:
Mucopolysaccharidosis type VII (MPS VII) is a recessively inherited lysosomal storage disorder caused due to β-glucuronidase (β-GUS) enzyme deficiency. Prominent clinical symptoms include hydrops fetalis, musculoskeletal deformities, neurodegeneration and hepatosplenomegaly leading to premature death in most cases. Apart from these, MPS VII is also characterized as adipose storage deficiency disorder although the underlying mechanism of this lean phenotype in the patients or β-GUS-deficient mice still remains a mystery. This issue was addressed using a recently developed Drosophila model of MPS VII (the CG2135-/- fly), which also exhibited a significant loss of body fat. This study reports that the lean phenotype of the CG2135-/- larvae is due to fewer number of adipocytes, smaller lipid droplets and reduced adipogenesis. The data further revealed that there is an abnormal accumulation of autophagosomes in the CG2135-/- larvae due to autophagosome-lysosome fusion defect. Decreased lysosome-mediated turnover also led to attenuated mTOR activity in the CG2135-/- larvae. Interestingly, treatment of the CG2135-/- larvae with mTOR stimulators, 3BDO or glucose, led to the restoration of mTOR activity with simultaneous correction of the autophagy defect and adipose storage deficiency. These finding thus established a hitherto unknown mechanistic link between autophagy dysfunction, mTOR downregulation and reduced adiposity in MPS VII.
Prifti, E., Tsakiri, E. N., Vourkou, E., Stamatakis, G., Samiotaki, M., Skoulakis, E. M. C. and Papanikolopoulou, K. (2022). Systems genetic dissection of Alzheimer's disease brain gene expression networks. Mical modulates Tau toxicity via cysteine oxidation in vivo. Acta Neuropathol Commun 10(1): 44. PubMed ID: 35379354
Summary:
Tau accumulation is clearly linked to pathogenesis in Alzheimer's disease and other Tauopathies. However, processes leading to Tau fibrillization and reasons for its pathogenicity remain largely elusive. Mical emerged as a novel interacting protein of human Tau expressed in Drosophila brains. Mical is characterized by the presence of a flavoprotein monooxygenase domain that generates redox potential with which it can oxidize target proteins. In the well-established Drosophila Tauopathy model, genetic interactions were used to show that Mical alters Tau interactions with microtubules and the Actin cytoskeleton and greatly affects Tau aggregation propensity and Tau-associated toxicity and dysfunction. Exploration of the mechanism was pursued using a Mical inhibitor, a mutation in Mical that selectively disrupts its monooxygenase domain, Tau transgenes mutated at cysteine residues targeted by Mical and mass spectrometry analysis to quantify cysteine oxidation. The collective evidence strongly indicates that Mical's redox activity mediates the effects on Tau via oxidation of Cys322. Importantly, results from the fly model in human Tauopathy samples were validated by showing that MICAL1 is up-regulated in patient brains and co-localizes with Tau in Pick bodies. This work provides mechanistic insights into the role of the Tau cysteine residues as redox-switches regulating the process of Tau self-assembly into inclusions in vivo, its function as a cytoskeletal protein and its effect on neuronal toxicity and dysfunction.
Vasudevan, D., Katow, H., Huang, H. W., Tang, G. and Ryoo, H. D. (2021). A Protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oogenesis and wing development. Dis Model Mech. PubMed ID: 34919148
Summary:
Metazoans have evolved various quality control mechanisms to cope with cellular stress inflicted by external and physiological conditions. ATF4 is a major effector of the Integrated Stress Response (ISR), an evolutionarily conserved pathway that mediates adaptation to various cellular stressors. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and in adult tissue homeostasis thus remain poorly understood. This study reports that a protein-trap MiMIC insertion in the crc locus generates a crc-GFP fusion protein that allows visualization of crc activity in vivo. This allele also acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows crc-GFP induction in a Drosophila model for Retinitis Pigmentosa (RP). This crc allele renders flies more vulnerable to amino acid deprivation and age-dependent retinal degeneration. These mutants also show defects in wing veins and oocyte maturation. Together, these data reveal previously unknown roles for crc in development, cellular homeostasis and photoreceptor survival.
Johnson, S. L., Prifti, M. V., Sujkowski, A., Libohova, K., Blount, J. R., Hong, L., Tsou, W. L. and Todi, S. V. (2022). Drosophila as a Model of Unconventional Translation in Spinocerebellar Ataxia Type 3. Cells 11(7). PubMed ID: 35406787
Summary:
RNA toxicity contributes to diseases caused by anomalous nucleotide repeat expansions. Recent work demonstrated RNA-based toxicity from repeat-associated, non-AUG-initiated translation (RAN translation). RAN translation occurs around long nucleotide repeats that form hairpin loops, allowing for translation initiation in the absence of a start codon that results in potentially toxic, poly-amino acid repeat-containing proteins. Discovered in Spinocerebellar Ataxia Type (SCA) 8, RAN translation has been documented in several repeat-expansion diseases, including in the CAG repeat-dependent polyglutamine (polyQ) disorders. The ATXN3 gene, which causes SCA3, also known as Machado-Joseph Disease (MJD), contains a CAG repeat that is expanded in disease. ATXN3 mRNA possesses features linked to RAN translation. The potential contribution of RAN translation to SCA3/MJD was tested in Drosophila by using isogenic lines that contain homomeric or interrupted CAG repeats. No unconventional translation was observed in fly neurons or glia. However, these investigations indicate differential toxicity from ATXN3 protein-encoding mRNA that contains pure versus interrupted CAG repeats. Additional work suggests that this difference may be due in part to toxicity from homomeric CAG mRNA. It is concluded that Drosophila is not suitable to model RAN translation for SCA3/MJD, but offers clues into the potential pathogenesis stemming from CAG repeat-containing mRNA in this disorder.

Thursday, April 21 - Adult neural development and function

Frey, F., Sandakly, J., Ghannam, M., Doueiry, C., Hugosson, F., Berlandi, J., Ismail, J. N., Gayden, T., Hasselblatt, M., Jabado, N. and Shirinian, M. (2022). Drosophila Tet is required for maintaining glial homeostasis in developing and adult fly brains. eNeuro. PubMed ID: 35396259
Summary:
Ten eleven translocation (TET) proteins are crucial epigenetic regulators highly conserved in multicellular organisms. TETs' enzymatic function in demethylating 5-methyl cytosine in DNA is required for proper development and TETs are frequently mutated in cancer. Recently, Drosophila melanogaster Tet (dTet) was shown to be highly expressed in developing fly brains and discovered to play an important role in brain and muscle development as well as fly behavior. Furthermore, dTet was shown to have different substrate specificity compared to mammals. However, the exact role dTet plays in glial cells and how ectopic TET expression in glial cells contributes to tumorigenesis and glioma is still not clear. This study reports a novel role for dTet specifically in glial cell organization and number. Loss of dTet affects the organization of a specific glia population in the optic lobe, the "optic chiasm" glia. Additionally, irregularities were found in axon patterns in the ventral nerve cord (VNC) both, in the midline and longitudinal axons. These morphological glia and axonal defects were accompanied by locomotor defects in developing larvae escalating to immobility in adult flies. Furthermore, glia homeostasis was disturbed in dTet-deficient brains manifesting in gain of glial cell numbers and increased proliferation. Finally, a Drosophila model was established to understand the impact of human TET3 in glia and find that ectopic expression of hTET3 in dTet expressing cells causes glia expansion in larval brains and affects sleep/rest behavior and the circadian clock in adult flies.
Vulpe, A. and Menuz, K. (2021). Ir76b is a Co-receptor for Amine Responses in Drosophila Olfactory Neurons. Front Cell Neurosci 15: 759238. PubMed ID: 34867202
Summary:
Two large families of olfactory receptors, the Odorant Receptors (ORs) and Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odorant binding "tuning" OrX receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco. The situation for IRs is more complex. Different tuning IrX receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a co-receptor, but its function has not been systematically examined. Surprisingly, recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. This study demonstrates that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses. In contrast, amine responses persist in the absence of Ir76b or Ir25a in ac1 sensilla but are lost in a double mutant. Responses mediated by acid-sensing neurons do not require Ir76b, despite their expression of this co-receptor. This study also demonstrates that one population of coeloconic olfactory neurons exhibits Ir76b/Ir25a-dependent and Orco-dependent responses to distinct odorants. Together, the data establish the role of Ir76b as a bona fide co-receptor, which acts in partnership with Ir25a. Given that these co-receptors are among the most highly conserved olfactory receptors and are often co-expressed in chemosensory neurons, the data suggest Ir76b and Ir25a also work in tandem in other insects.
Zhang, Q., Zhang, B., Lim, N. K. H., Zhang, X., Meng, S., Nyengaard, J. R., Huang, F. and Wang, W. A. (2022). Hyccin/FAM126A deficiency reduces glial enrichment and axonal sheath, which are rescued by overexpression of a plasma membrane-targeting PI4KIIIα in Drosophila. Biochem Biophys Res Commun 589: 71-77. PubMed ID: 34894559
Summary:
Hyccin/FAM126A mutations are linked to hypomyelination and congenital cataract disease (HCC), but whether and how Hyccin/FAM126A deficiency causes hypomyelination remains undetermined. This study shows Hyccin/FAM126A expression was necessary for the expression of other components of the PI4KIIIalpha complex in Drosophila. Knockdown of Hyccin/FAM126A in glia reduced the enrichment of glial cells, disrupted axonal sheaths and visual ability in the visual system, and these defects could be fully rescued by overexpressing either human FAM126A or FAM126B, and partially rescued by overexpressing a plasma membrane-targeting recombinant mouse PI4KIIIα. Additionally, PI4KIIIα knockdown in glia phenocopied Hyccin/FAM126A knockdown, and this was partially rescued by overexpressing the recombinant PI4KIIIα, but not human FAM126A or FAM126B. This study establishes an animal model of HCC and indicates that Hyccin/FAM126A plays an essential role in glial enrichment and axonal sheath in a cell-autonomous manner in the visual system via controlling the expression and stabilization of the PI4KIIIα complex at the plasma membrane.
Jois, S., Chan, Y. B., Fernandez, M. P., Pujari, N., Janz, L. J., Parker, S. and Leung, A. K. (2022). Sexually dimorphic peripheral sensory neurons regulate copulation duration and persistence in male Drosophila. Sci Rep 12(1): 6177. PubMed ID: 35418584
Summary:
Peripheral sensory neurons are the gateway to the environment across species. In Drosophila, olfactory and gustatory senses are required to initiate courtship, as well as for the escalation of courtship patterns that lead to copulation. To be successful, copulation must last long enough to ensure the transfer of sperm and seminal fluid that ultimately leads to fertilization. The peripheral sensory information required to regulate copulation duration is unclear. This study employed genetic manipulations that allow driving gene expression in the male genitalia as a tool to uncover the role of these genitalia specific neurons in copulation. The fly genitalia contain sex-specific bristle hairs innervated by mechanosensory neurons. To date, the role of the sensory information collected by these peripheral neurons in male copulatory behavior is unknown. T these MSNs are cholinergic and co-express both fru and dsx. The sensory information received by the peripheral sensory neurons from the front legs (GRNs) and mechanosensory neurons (MSNs) at the male genitalia contribute to the regulation of copulation duration. Moreover, the results show that their function is required for copulation persistence, which ensures copulation is undisrupted in the presence of environmental stress before sperm transfer is complete.
Chen, S. C., Tang, X., Goda, T., Umezaki, Y., Riley, A. C., Sekiguchi, M., Yoshii, T. and Hamada, F. N. (2022). Dorsal clock networks drive temperature preference rhythms in Drosophila. Cell Rep 39(2): 110668. PubMed ID: 35417715
Summary:
Animals display a body temperature rhythm (BTR). Little is known about the mechanisms by which a rhythmic pattern of BTR is regulated and how body temperature is set at different times of the day. As small ectotherms, Drosophila exhibit a daily temperature preference rhythm (TPR), which generates BTR. This study demonstrates dorsal clock networks that play essential roles in TPR. Dorsal neurons 2 (DN2s) are the main clock for TPR. It was found that DN2s and posterior DN1s (DN1ps) contact and the extent of contacts increases during the day and that the silencing of DN2s or DN1ps leads to a lower temperature preference. The data suggest that temporal control of the microcircuit from DN2s to DN1ps contributes to TPR regulation. This study also identified anterior DN1s (DN1as) as another important clock for TPR. Thus, this study shows that the DN networks predominantly control TPR and determine both a rhythmic pattern and preferred temperatures.
Gkanias, E., McCurdy, L. Y., Nitabach, M. N. and Webb, B. (2022). An incentive circuit for memory dynamics in the mushroom body of Drosophila melanogaster. Elife 11. PubMed ID: 35363138
Summary:
Insects adapt their response to stimuli, such as odours, according to their pairing with positive or negative reinforcements, such as sugar or shock. Recent electrophysiological and imaging findings in Drosophila melanogaster allow detailed examination of the neural mechanisms supporting the acquisition, forgetting, and assimilation of memories. It is proposed that this data can be explained by the combination of a dopaminergic plasticity rule that supports a variety of synaptic strength change phenomena, and a circuit structure (derived from neuroanatomy) between dopaminergic and output neurons that creates different roles for specific neurons. Computational modelling shows that this circuit allows for rapid memory acquisition, transfer from short term to long term, and exploration/exploitation trade-off. The model can reproduce the observed changes in the activity of each of the identified neurons in conditioning paradigms and can be used for flexible behavioural control.

Wednesday, April 20th - Apoptosis and Autophagy

Takeuchi, K. I., Honda, D., Okumura, M., Miura, M. and Chihara, T. (2021). Systemic innate immune response induces death of olfactory receptor neurons in Drosophila. Genes Cells PubMed ID: 34921694
Summary:
Neural functions are known to decline during normal aging and neurodegenerative diseases. However, the mechanisms of functional impairment owing to the normal aging of the brain are poorly understood. It was previously reported that caspase-3-like protease, the protease responsible for inducing apoptosis, is activated in a subset of olfactory receptor neurons (ORNs), especially in Drosophila Or42b neurons, during normal aging. This study investigated the molecular mechanism underlying age-related caspase-3-like protease activation and cell death in Or42b neurons. Gene expression profiling of young and aged fly antenna showed that the expression of antimicrobial peptides was significantly upregulated, suggesting an activated innate immune response. Consistent with this observation, inhibition or activation of the innate immune pathway caused delayed or precocious cell death, respectively, in Or42b neurons. Accordingly, autonomous cell activation of the innate immune pathway in Or42b neurons is not likely required for their age-related death, whereas the systemic innate immune response induces caspase-3-like protease activation in Or42b neurons; this indicated that the death of these neurons is regulated non-cell autonomously. A possible link between the innate immune response and the death of olfactory neurons during normal aging is proposed.
Perez, E., Venkatanarayan, A. and Lundell, M. J. (2022). Hunchback prevents notch-induced apoptosis in the serotonergic lineage of Drosophila Melanogaster. Dev Biol. PubMed ID: 35381219
Summary:
The serotonergic lineage (NB7-3) in the Drosophila ventral nerve cord produces six cells during neurogenesis. Four of the cells differentiate into neurons: EW1, EW2, EW3 and GW. The other two cells undergo apoptosis. This simple lineage provides an opportunity to examine genes that are required to induce or repress apoptosis during cell specification. Previous studies have shown that Notch signaling induces apoptosis within the NB7-3 lineage. The three EW neurons are protected from Notch-induced apoptosis by asymmetric distribution of Numb protein, an inhibitor of Notch signaling. In a numb1 mutant EW2 and EW3 undergo apoptosis. The EW1 and GW neurons survive even in a numb1 mutant background suggesting that these cells are protected from Notch-induced apoptosis by some factor other than Numb. The EW1 and GW neurons are mitotic sister cells, and uniquely express the transcription factor Hunchback. Evidence is presented that Hunchback prevents apoptosis in NB7-3 lineage during normal CNS development and can rescue the two apoptotic cells in the lineage when it is ectopically expressed. hunchback overexpression produces ectopic cells that express markers similar to the EW2 neuron and changes the expression pattern of the EW3 neuron to a EW2 neuron. In addition this study shows that hunchback overexpression can override apoptosis that is genetically induced by the pro-apoptotic genes grim and hid.
Lee, S., Jo, M., Lee, H. E., Jeon, Y. M., Kim, S., Kwon, Y., Woo, J., Han, S., Mun, J. Y. and Kim, H. J. (2021). HEXA-018, a Novel Inducer of Autophagy, Rescues TDP-43 Toxicity in Neuronal Cells. Front Pharmacol 12: 747975. PubMed ID: 34925009
Summary:
The autophagy-lysosomal pathway is an essential cellular mechanism that degrades aggregated proteins and damaged cellular components to maintain cellular homeostasis. This study identified HEXA-018, a novel compound containing a catechol derivative structure, as a novel inducer of autophagy. HEXA-018 increased the LC3-I/II ratio, which indicates activation of autophagy. Consistent with this result, HEXA-018 effectively increased the numbers of autophagosomes and autolysosomes in neuronal cells. This study also found that the activation of autophagy by HEXA-018 is mediated by the AMPK-ULK1 pathway in an mTOR-independent manner. It was further shown that ubiquitin proteasome system impairment- or oxidative stress-induced neurotoxicity was significantly reduced by HEXA-018 treatment. Moreover, oxidative stress-induced mitochondrial dysfunction was strongly ameliorated by HEXA-018 treatment. In addition, the efficacy of HEXA-018 in models of TDP-43 proteinopathy was investigated. HEXA-018 treatment mitigated TDP-43 toxicity in cultured neuronal cell lines and Drosophila. These data indicate that HEXA-018 could be a new drug candidate for TDP-43-associated neurodegenerative diseases.
Lauzier, A., Bossanyi, M. F., Larcher, R., Nassari, S., Ugrankar, R., Henne, W. M. and Jean, S. (2022). Snazarus and its human ortholog SNX25 modulate autophagic flux. J Cell Sci 135(5). PubMed ID: 34821359
Summary:
Macroautophagy, the degradation and recycling of cytosolic components in the lysosome, is an important cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs impact autophagy. To improve understanding of their functions in vivo, all Drosophila SNXs were screened using inducible RNA interference in the fat body. Significantly, depletion of Snazarus (Snz) led to decreased autophagic flux. Interestingly, altered distribution of Vamp7-positive vesicles was observed with Snz depletion, and the roles of Snz were conserved in human cells. SNX25, the closest human ortholog to Snz, regulates both VAMP8 endocytosis and lipid metabolism. Through knockout-rescue experiments, it was demonstrated that these activities are dependent on specific SNX25 domains and that the autophagic defects seen upon SNX25 loss can be rescued by ethanolamine addition. The presence of differentially spliced forms of SNX14 and SNX25 was detected in cancer cells. This work identifies a conserved role for Snz/SNX25 as a regulator of autophagic flux and reveals differential isoform expression between paralogs.
Loreto, A., Angeletti, C., Gu, W., Osborne, A., Nieuwenhuis, B., Gilley, J., Merlini, E., Arthur-Farraj, P., Amici, A., Luo, Z., Hartley-Tassell, L., Ve, T., Desrochers, L. M., Wang, Q., Kobe, B., Orsomando, G. and Coleman, M. P. (2021). Neurotoxin-mediated potent activation of the axon degeneration regulator SARM1. Elife 10. PubMed ID: 34870595
Summary:
Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the NAD-consuming enzyme SARM1. This study reports a novel activator of SARM1, a metabolite of the pesticide and neurotoxin vacor. Removal of SARM1 completely rescues mouse neurons from vacor-induced neuron and axon death in vitro and in vivo. Crystal structure is presented of the Drosophila SARM1 regulatory domain complexed with this activator, the vacor metabolite VMN, which as the most potent activator yet known is likely to support drug development for human SARM1 and NMNAT2 disorders. This study indicates the mechanism of neurotoxicity and pesticide action by vacor, raises important questions about other pyridines in wider use today, provides important new tools for drug discovery, and demonstrates that removing SARM1 can robustly block programmed axon death induced by toxicity as well as genetic mutation.
Zheng, Q., Gao, N., Sun, Q., Li, X., Wang, Y. and Xiao, H. (2021). bfc, a novel serpent co-factor for the expression of croquemort, regulates efferocytosis in Drosophila melanogaster. PLoS Genet 17(12): e1009947. PubMed ID: 34860835
Summary:
Efferocytosis is the process by which phagocytes recognize, engulf, and digest (or clear) apoptotic cells during development. Impaired efferocytosis is associated with developmental defects and autoimmune diseases. In Drosophila melanogaster, recognition of apoptotic cells requires phagocyte surface receptors, including the scavenger receptor CD36-related protein, Croquemort (Crq, encoded by crq). In fact, Crq expression is upregulated in the presence of apoptotic cells, as well as in response to excessive apoptosis. This study identified a novel gene bfc (booster for croquemort), which plays a role in efferocytosis, specifically the regulation of the crq expression. Bfc protein interacts with the zinc finger domain of the GATA transcription factor Serpent (Srp), to enhance its direct binding to the crq promoter; thus, they function together in regulating crq expression and efferocytosis. Overall, this study shows that Bfc serves as a Srp co-factor to upregulate the transcription of the crq encoded receptor, and consequently boosts macrophage efferocytosis in response to excessive apoptosis. Therefore, this study clarifies how phagocytes integrate apoptotic cell signals to mediate efferocytosis.

Tuesday, April 19th - Stem Cells

Huang, J., Sheng, X., Zhuo, Z., Xiao, D., Wu, K., Wan, G. and Chen, H. (2022). ClC-c regulates the proliferation of intestinal stem cells via the EGFR signalling pathway in Drosophila. Cell Prolif 55(1): e13173. PubMed ID: 34952996
Summary:
Adult stem cells uphold a delicate balance between quiescent and active states, which is crucial for tissue homeostasis. Whereas many signalling pathways that regulate epithelial stem cells have been reported, many regulators remain unidentified. This study used flies to generate tissue-specific gene knockdown and gene knockout. qRT-PCR was used to assess the relative mRNA levels. Immunofluorescence was used to determine protein localization and expression patterns. Clonal analyses were used to observe the phenotype. RNA-seq was used to screen downstream mechanisms. It is reported that a member of the chloride channel family, ClC-c, which is specifically expressed in Drosophila intestinal stem/progenitor cells and regulates intestinal stem cell (ISC) proliferation under physiological conditions and upon tissue damage. Mechanistically, it was found that the ISC loss induced by the depletion of ClC-c in intestinal stem/progenitor cells is due to inhibition of the EGFR signalling pathway. These findings reveal an ISC-specific function of ClC-c in regulating stem cell maintenance and proliferation, thereby providing new insights into the functional links among the chloride channel family, ISC proliferation and tissue homeostasis.
Buddika, K., Huang, Y. T., Ariyapala, I. S., Butrum-Griffith, A., Norrell, S. A., O'Connor, A. M., Patel, V. K., Rector, S. A., Slovan, M., Sokolowski, M., Kato, Y., Nakamura, A. and Sokol, N. S. (2022). Coordinated repression of pro-differentiation genes via P-bodies and transcription maintains Drosophila intestinal stem cell identity. Curr Biol 32(2): 386-397.e386. PubMed ID: 34875230
Summary:
The role of processing bodies (P-bodies), key sites of post-transcriptional control, in adult stem cells remains poorly understood. This paper reports that adult Drosophila intestinal stem cells, but not surrounding differentiated cells such as absorptive enterocytes (ECs), harbor P-bodies that contain Drosophila orthologs of mammalian P-body components DDX6, EDC3, EDC4, and LSM14A/B. A targeted RNAi screen in intestinal progenitor cells identified 39 previously known and 64 novel P-body regulators, including Patr-1, a gene necessary for P-body assembly. Loss of Patr-1-dependent P-bodies leads to a loss of stem cells that is associated with inappropriate expression of EC-fate gene nubbin. Transcriptomic analysis of progenitor cells identifies a cadre of such weakly transcribed pro-differentiation transcripts that are elevated after P-body loss. Altogether, this study identifies a P-body-dependent repression activity that coordinates with known transcriptional repression programs to maintain a population of in vivo stem cells in a state primed for differentiation.
Gallaud, E., Richard-Parpaillon, L., Bataille, L., Pascal, A., Metivier, M., Archambault, V. and Giet, R. (2022). The spindle assembly checkpoint and the spatial activation of Polo kinase determine the duration of cell division and prevent tumor formation. PLoS Genet 18(4): e1010145. PubMed ID: 35377889
Summary:
The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Using asymmetrically dividing Drosophila larval neural stem cells (NSCs) as a model, it was shown that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, it was shown that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, these results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis.
Yuen, A. C., Hillion, K. H., Wang, R. and Amoyel, M. (2021). Germ cells commit somatic stem cells to differentiation following priming by PI3K/Tor activity in the Drosophila testis. PLoS Genet 17(12): e1009609. PubMed ID: 34898607
Summary:
How and when potential becomes restricted in differentiating stem cell daughters is poorly understood. While it is thought that signals from the niche are actively required to prevent differentiation, another model proposes that stem cells can reversibly transit between multiple states, some of which are primed, but not committed, to differentiate. In the Drosophila testis, somatic cyst stem cells (CySCs) generate cyst cells, which encapsulate the germline to support its development. CySCs were found to be maintained independently of niche self-renewal signals if activity of the PI3K/Tor pathway is inhibited. Conversely, PI3K/Tor is not sufficient alone to drive differentiation, suggesting that it acts to license cells for differentiation. Indeed, it was found that the germline is required for differentiation of CySCs in response to PI3K/Tor elevation, indicating that final commitment to differentiation involves several steps and intercellular communication. It is proposed that CySC daughter cells are plastic, that their fate depends on the availability of neighbouring germ cells, and that PI3K/Tor acts to induce a primed state for CySC daughters to enable coordinated differentiation with the germline.
Shin, M., Ferguson, M., Willms, R. J., Jones, L. O., Petkau, K. and Foley, E. (2022). Immune regulation of intestinal-stem-cell function in Drosophila. Stem Cell Reports 17(4): 741-755. PubMed ID: 35303435
Summary:
Intestinal progenitor cells integrate signals from their niche, and the gut lumen, to divide and differentiate at a rate that maintains an epithelial barrier to microbial invasion of the host interior. Despite the importance of evolutionarily conserved innate immune defenses to maintain stable host-microbe relationships, little is known about contributions of stem-cell immunity to gut homeostasis. Drosophila was used to determine the consequences of intestinal-stem-cell immune activity for epithelial homeostasis. Loss of stem-cell immunity greatly impacted growth and renewal in the adult gut. In particular, it was found that inhibition of stem-cell immunity impeded progenitor-cell growth and differentiation, leading to a gradual loss of stem-cell numbers with age and an impaired differentiation of mature enteroendocrine cells. These results highlight the importance of immune signaling in stem cells for epithelial function in the adult gut.
Spratford, C. M., Goins, L. M., Chi, F., Girard, J. R., Macias, S. N., Ho, V. W. and Banerjee, U. (2021). Intermediate progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants. Development 148(24). PubMed ID: 34918741
Summary:
Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated owing to lack of appropriate genetic tools. This study reports a Split GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs are a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. In addition, through their dynamic control of the Notch ligand Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. This study suggests that it would be useful to characterize such intermediate populations of cells in mammalian hematopoietic systems.

Monday, April 18th - Embryonic Development

Mendoza-Garcia, P., Basu, S., Sukumar, S. K., Arefin, B., Wolfstetter, G., Anthonydhason, V., Molander, L., Uçkun, E., Lindehell, H., Lebrero-Fernandez, C., Larsson, J., Larsson, E., Bemark, M. and Palmer, R. H. (2021). DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm. Development 148(23). PubMed ID: 34905617
Summary:
Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. This study used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). This study confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, this paper reports a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterizes the role of Kah in the regulation of embryonic midgut morphogenesis.
Skafida, E., Delidakis, C. and Monastirioti, M. (2021). Expression of Hey marks a subset of enteroendocrine cells in the Drosophila embryonic and larval midgut. Int J Dev Biol. PubMed ID: 34881798
Summary:
Hey is a conserved transcription factor of the bHLH-Orange family and it participates in the response to Notch signaling in certain tissues. Whereas three Hey paralogues exist in mammalian genomes, Drosophila possesses a single Hey gene. Fly Hey is expressed in the subset of newborn neurons that receive a Notch signal to differentiate them from their sibling cells after the asymmetric division of precursors called ganglion-mother-cells. A polyclonal anti-Hey antiserum and a GFP-tagged transgenic duplication of the Hey locus to examine its expression in tissues outside the nervous system were used in embryos and larvae. Robust Hey expression was detected in the embryonic midgut primordium at the time of birth of enteroendocrine cells, identified by expression of Prospero. About half of the Pros-positive cells were also Hey positive at mid-embryogenesis. By the end of embryogenesis, most enteroendocrine cells had downregulated Hey expression, although it was still detectable at low levels after hatching. Low levels of Hey were also detected in subsets of the epithelial enterocytes at different times. Embryo enteroendocrine Hey expression was found to be Notch dependent. In late third-instar larvae, when few new enteroendocrine cells are born, novel Hey expression was detected in one cell of each sibling pair. In conclusion, Hey is strongly expressed in one of each pair of newly-born enteroendocrine cells. This is consistent with a hypothesis that embryonic enteroendocrine cells are born by an asymmetric division of a precursor, where Notch/Hey probably distinguish between the subtypes of these cells upon their differentiation.
Torres-Zelada, E. F., George, S., Blum, H. R. and Weake, V. M. (2022). Chiffon triggers global histone H3 acetylation and expression of developmental genes in Drosophila embryos. J Cell Sci 135(2). PubMed ID: 34908116
Summary:
The histone acetyltransferase Gcn5 is critical for gene expression and development. In Drosophila, Gcn5 is part of four complexes (SAGA, ATAC, CHAT and ADA) that are essential for fly viability and have key roles in regulating gene expression. This study shows that although the SAGA, ADA and CHAT complexes play redundant roles in embryonic gene expression, the insect-specific CHAT complex uniquely regulates expression of a subset of developmental genes. A substantial decrease was observed in histone acetylation in chiffon mutant embryos that exceeds that observed in Ada2b, suggesting broader roles for Chiffon in regulating histone acetylation outside of the Gcn5 complexes. The chiffon gene encodes two independent polypeptides that nucleate formation of either the CHAT or Dbf4-dependent kinase (DDK) complexes. DDK includes the cell cycle kinase Cdc7, which is necessary for maternally driven DNA replication in the embryo. This study has identified a temporal switch between the expression of these chiffon gene products during a short window during the early nuclear cycles in embryos that correlates with the onset of zygotic genome activation, suggesting a potential role for CHAT in this process.
Chen, W. and He, B. (2022). Actomyosin activity-dependent apical targeting of Rab11 vesicles reinforces apical constriction. J Cell Biol 221(6). PubMed ID: 35404399
Summary:
During tissue morphogenesis, the changes in cell shape, resulting from cell-generated forces, often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. This study identified an actomyosin-dependent mechanism involving Rab11-mediated trafficking in regulating apical constriction in the Drosophila embryo. During Drosophila mesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures ("Rab11 vesicles") by promoting a directional bias in dynein-mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network, and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding.
Karkali, K., Tiwari, P., Singh, A., Tlili, S., Jorba, I., Navajas, D., Munoz, J. J., Saunders, T. E. and Martin-Blanco, E. (2022). Condensation of the Drosophila nerve cord is oscillatory and depends on coordinated mechanical interactions. Dev Cell 57(7): 867-882.e865. PubMed ID: 35413236
Summary:
During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. This study characterized the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. The process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, this study has defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.
Wen, F. L., Kwan, C. W., Wang, Y. C. and Shibata, T. (2021). Autonomous epithelial folding induced by an intracellular mechano-polarity feedback loop. PLoS Comput Biol 17(12): e1009614. PubMed ID: 34871312
Summary:
Epithelial tissues form folded structures during embryonic development and organogenesis. Whereas substantial efforts have been devoted to identifying mechanical and biochemical mechanisms that induce folding, whether and how their interplay synergistically shapes epithelial folds remains poorly understood. This paper proposes a mechano-biochemical model for dorsal fold formation in the early Drosophila embryo, an epithelial folding event induced by shifts of cell polarity. Based on experimentally observed apical domain homeostasis, this study coupled cell mechanics to polarity and found that mechanical changes following the initial polarity shifts alter cell geometry, which in turn influences the reaction-diffusion of polarity proteins, thus forming a feedback loop between cell mechanics and polarity. This model can induce spontaneous fold formation in silico, recapitulate polarity and shape changes observed in vivo, and confer robustness to tissue shape change against small fluctuations in mechanics and polarity. These findings reveal emergent properties of a developing epithelium under control of intracellular mechano-polarity coupling.

Friday, April 15th - Adult Physiology and metabolism

Voutyraki, C., Choromidis, A., Theodorou, V., Efraimoglou, C., Anagnostopoulos, G., Magadi, S. S., Grammenoudi, S., Zacharioudaki, E. and Delidakis, C. (2021). Repression of differentiation genes by Hes transcription factors fuels neural tumour growth in Drosophila. Int J Dev Biol. PubMed ID: 34881794
Summary:
Neural stem cells (NSC) divide asymmetrically to generate a cell that retains stem cell identity and another that is routed to differentiation. Prolonged mitotic activity of the NSCs gives rise to the plethora of neurons and glial cells that wire the brain and nerve cord. Genetic insults, such as excess of Notch signaling, perturb the normal NSC proliferation programs and trigger the formation of NSC hyperplasias, that can later progress to malignancies. Hes proteins are crucial mediators of Notch signaling and in the NSC context they act by repressing a cohort of early pro-differentiation transcription factors. Downregulation of these pro-differentiation factors makes NSC progeny cells susceptible to adopting an aberrant stem cell program. It has been recently shown that Hes overexpression in Drosophila leads to NSC hyperplasias that progress to malignant tumours after allografting to adult hosts. This study combined genetic analysis, tissue allografting and transcriptomic approaches to address the role of Hes genes in NSC malignant transformation. The E(spl) genes are important mediators in the progression of Notch hyperplasias to malignancy, since allografts lacking the E(spl) genes grow much slower. RNA profiling is presented of Hes-induced tumours at two different stages after allografting. The same cohort of differentiation-promoting transcription factors that are repressed in the primary hyperplasias continue to be downregulated after transplantation. This is accompanied by an upregulation of stress-response genes and metabolic reprogramming. It is concluded that the combination of dedifferentiation and cell physiology changes most likely drive tumour growth.
Zhang, P., Azad, P., Engelhart, D. C., Haddad, G. G. and Nigam, S. K. (2021). SLC22 Transporters in the Fly Renal System Regulate Response to Oxidative Stress In Vivo. Int J Mol Sci 22(24). PubMed ID: 34948211
Summary:
Several SLC22 transporters in the human kidney and other tissues are thought to regulate endogenous small antioxidant molecules such as uric acid, ergothioneine, carnitine, and carnitine derivatives. These transporters include those from the organic anion transporter (OAT), OCTN/OCTN-related, and organic cation transporter (OCT) subgroups. In mammals, it has been difficult to show a clear in vivo role for these transporters during oxidative stress. Ubiquitous knockdowns of related Drosophila SLC22s-including transporters homologous to those previously identified in mammals such as the "Fly-Like Putative Transporters" FLIPT1 (SLC22A15) and FLIPT2 (SLC22A16)-have shown modest protection against oxidative stress. However, these fly transporters tend to be broadly expressed, and it is unclear if there is an organ in which their expression is critical. Using two tissue-selective knockdown strategies, this study was able to demonstrate much greater and longer protection from oxidative stress compared to previous whole fly knockdowns as well as both parent and WT strains. Expression in the Malpighian tubule and likely other tissues as well (e.g., gut, fat body, nervous system) appear critical for managing oxidative stress. These four Drosophila SLC22 genes are similar to human SLC22 transporters (CG6126: SLC22A16, CG16727: SLC22A7, CG4630: SLC22A3, and CG6006: SLC22A1, etc.)-many of which are highly expressed in the kidney. Consistent with the Remote Sensing and Signaling Theory, this indicates an important in vivo role in the oxidative stress response for multiple SLC22 transporters within the fly renal system, perhaps through interaction with SLC22 counterparts in non-renal tissues. Many of the human relatives are well-known drug transporters. This work not only indicates the importance of SLC22 transporters in the fly renal system but also sets the stage for in vivo studies by examining their role in mammalian oxidative stress and organ crosstalk.
Voo, K., Ching, J. W. H., Lim, J. W. H., Chan, S. N., Ng, A. Y. E., Heng, J. Y. Y., Lim, S. S. and Pek, J. W. (2021). Maternal starvation primes progeny response to nutritional stress. PLoS Genet 17(11): e1009932. PubMed ID: 34843464
Summary:
Organisms adapt to environmental changes in order to survive. Mothers exposed to nutritional stresses can induce an adaptive response in their offspring. However, the molecular mechanisms behind such inheritable links are not clear. This study reports that in Drosophila, starvation of mothers primes the progeny against subsequent nutritional stress. RpL10Ab represses TOR pathway activity by genetically interacting with TOR pathway components TSC2 and Rheb. In addition, starved mothers produce offspring with lower levels of RpL10Ab in the germline, which results in higher TOR pathway activity, conferring greater resistance to starvation-induced oocyte loss. The RpL10Ab locus encodes RpL10Ab mRNA and a stable intronic sequence RNA (sisR-8), which collectively repress RpL10Ab pre-mRNA splicing in a negative feedback mechanism. During starvation, an increase in maternally deposited RpL10Ab and sisR-8 transcripts leads to the reduction of RpL10Ab expression in the offspring. This study suggests that the maternally deposited RpL10Ab and sisR-8 transcripts trigger a negative feedback loop that mediates intergenerational adaptation to nutritional stress as a starvation response.
Rand, D. M., Mossman, J. A., Spierer, A. N. and Santiago, J. A. (2021). Mitochondria as environments for the nuclear genome in Drosophila: mitonuclear GxGxE. J Hered. PubMed ID: 34964900
Summary:
Mitochondria evolved from a union of microbial cells belonging to distinct lineages that were likely anaerobic. The nutrients and oxygen that sustain eukaryotic metabolism today are processed in mitochondria through coordinated expression of 37 mitochondrial genes and over 1000 nuclear genes. This puts mitochondria at the nexus of gene-by-gene (GxG) and gene-by-environment (GxE) interactions that sustain life. This study used a Drosophila model of mitonuclear genetic interactions to explore the notion that mitochondria are environments for the nuclear genome, and vice versa. Factorial combinations of mtDNA and nuclear chromosomes were constructed to test for epistatic interactions (GxG), and expose these mitonuclear genotypes to altered dietary environments to examine GxE interactions. Development time and genome-wide RNAseq analyses was used to assess the relative contributions of mtDNA, nuclear chromosomes, and environmental effects on these traits (mitonuclear GxGxE). The nuclear transcriptional response to alternative mitochondrial "environments" (GxG) has significant overlap with the transcriptional response of mitonuclear genotypes to altered dietary environments. These analyses point to specific transcription factors (e.g., giant) that mediated these interactions, and identified coexpressed modules of genes that may account for the overlap in differentially expressed genes. Roughly 20% of the transcriptome includes GxG genes that are concordant with GxE genes, suggesting that mitonuclear interactions are part of an organism's environment.
Sultanova, Z., Ivimey-Cook, E. R., Chapman, T. and Maklakov, A. A. (2021). Fitness benefits of dietary restriction. Proc Biol Sci 288(1963): 20211787. PubMed ID: 34814748
Summary:
Dietary restriction (DR) improves survival across a wide range of taxa yet remains poorly understood. The key unresolved question is whether this evolutionarily conserved response to temporary lack of food is adaptive. Recent work suggests that early-life DR reduces survival and reproduction when nutrients subsequently become plentiful, thereby challenging adaptive explanations. A new hypothesis maintains that increased survival under DR results from reduced costs of overfeeding. This study tested the adaptive value of DR response in an outbred population of Drosophila melanogaster fruit flies. DR females did not suffer from reduced survival upon subsequent re-feeding and had increased reproduction and mating success compared to their continuously fully fed (FF) counterparts. The increase in post-DR reproductive performance was of sufficient magnitude that females experiencing early-life DR had the same total fecundity as continuously FF individuals. These results suggest that the DR response is adaptive and increases fitness when temporary food shortages cease.
Dhakal, S., Ren, Q., Liu, J., Akitake, B., Tekin, I., Montell, C. and Lee, Y. (2022). Drosophila TRPg is required in neuroendocrine cells for post-ingestive food selection. Elife 11. PubMed ID: 35416769
Summary:
The mechanism through which the brain senses the metabolic state, enabling an animal to regulate food consumption, and discriminate between nutritional and non-nutritional foods is a fundamental question. Flies choose the sweeter non-nutritive sugar, L-glucose, over the nutritive D-glucose if they are not starved. However, under starvation conditions, they switch their preference to D-glucose, and this occurs independent of peripheral taste neurons. This study found that eliminating the TRPgamma channel impairs the ability of starved flies to choose D-glucose. This food selection depends on trpgamma expression in neurosecretory cells in the brain that express Diuretic hormone 44 (DH44). Loss of trpgamma increases feeding, alters the physiology of the crop, which is the fly stomach equivalent, and decreases intracellular sugars and glycogen levels. Moreover, survival of starved trpgamma flies is reduced. Expression of trpgamma in DH44 neurons reverses these deficits. These results highlight roles for TRPgamma in coordinating feeding with the metabolic state through expression in DH44 neuroendocrine cells.

Thursday, April 14th - Signaling

Nagel, A. C., Muller, D., Zimmermann, M. and Preiss, A. (2021). The Membrane-Bound Notch Regulator Mnr Supports Notch Cleavage and Signaling Activity in Drosophila melanogaster. Biomolecules 11(11). PubMed ID: 34827670
Summary:
The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch signaling dynamics at the level of ligand-receptor interaction, endocytosis, and transcriptional regulation has been well studied, little is known about factors influencing Notch cleavage. EP555 was identified as a suppressor of the Notch antagonist Hairless (H). EP555 drives expression of CG32521 encoding membrane-bound proteins, which are accordingly renamed Membrane-bound Notch regulator (Mnr). Within the signal-receiving cell, upregulation of Mnr stimulates Notch receptor activation, whereas a knockdown reduces it, without apparent influence on ligand-receptor interaction. Evidence is provided that Mnr plays a role in &gamms;-secretase-mediated intramembrane cleavage of the Notch receptor. As revealed by a fly-eye-based reporter system, γ-secretase activity is stimulated by the overexpression of Mnr, and is inhibited by its knockdown. It is concluded that Mnr proteins support Notch signaling activity by fostering the cleavage of the Notch receptor. With Mnr, a membrane-bound factor has been identified that directly augments Notch intra-membrane processing, thereby acting as a positive regulator of Notch signaling activity.
Lubke, S., Braukmann, C., Rexer, K. H., Cigoja, L., Rout, P. and Onel, S. F. (2021). The Abl-interactor Abi suppresses the function of the BRAG2 GEF family member Schizo. Biol Open. PubMed ID: 34897417
Summary:
Guanine nucleotide exchange factors (GEF) of the BRAG subfamily activate small Arf GTPases, which are pivotal regulators of intracellular membrane traffic and actin dynamics. Consequently, BRAG proteins have been implicated to regulate the surface levels of adhesive and signaling receptors. However, not much is known about the mechanism leading to the regulation of these surface proteins. This study found that the Drosophila BRAG GEF Schizo interacts physically with the Abl-interactor (Abi). schizo mutants display severe defects in myoblast fusion during syncytial muscle formation and show increased amounts of the cell adhesion protein N-cadherin. The schizo myoblast fusion phenotype can be rescued by the expression of the Schizo GEF (Sec7) and membrane-binding (pleckstrin homology) domain. Furthermore, the expression of the Sec7-PH domain in a wild-type background decreases the amounts of N-cadherin and impairs myoblast fusion. These findings support the notion that the Sec7-PH domain serves as a constitutive-active form of Schizo. Using a yeast-two hybrid assay, it was shown that the SH3 domain of Abi interacts with the N-terminal region of Schizo. This region is also able to bind to the cytodomain of the cell adhesion molecule N-cadherin. To shed light on the function of Schizo and Abi in N-cadherin removal, epistasis experiments were employed in different developmental contexts of Drosophila. These studies point towards a new model for the regulation of Schizo. It is proposed that the binding of Abi to the N-terminal part of Schizo antagonizes Schizo function to inhibit N-cadherin removal.
Ochi, N., Nakamura, M., Nagata, R., Wakasa, N., Nakano, R. and Igaki, T. (2021). Cell competition is driven by Xrp1-mediated phosphorylation of eukaryotic initiation factor 2alpha. PLoS Genet 17(12): e1009958. PubMed ID: 34871307
Summary:
Cell competition is a context-dependent cell elimination via cell-cell interaction whereby unfit cells ('losers') are eliminated from the tissue when confronted with fitter cells ('winners'). Despite extensive studies, the mechanism that drives loser's death and its physiological triggers remained elusive. Through a genetic screen in Drosophila, this study found that endoplasmic reticulum (ER) stress causes cell competition. Mechanistically, ER stress upregulates the bZIP transcription factor Xrp1, which promotes phosphorylation of the eukaryotic translation initiation factor eIF2α via the kinase PERK, leading to cell elimination. Surprisingly, the genetic data show that different cell competition triggers such as ribosomal protein mutations or RNA helicase Hel25E mutations converge on upregulation of Xrp1, which leads to phosphorylation of eIF2α and thus causes reduction in global protein synthesis and apoptosis when confronted with wild-type cells. These findings not only uncover a core pathway of cell competition but also open the way to understanding the physiological triggers of cell competition.
Tripathi, S., Miyake, T., Kelebeev, J. and McDermott, J. C. (2022). TAZ exhibits phase separation properties and interacts with Smad7 and β-catenin to repress skeletal myogenesis. J Cell Sci 135(1). PubMed ID: 34859820
Summary:
Hippo signaling in Drosophila and mammals is prominent in regulating cell proliferation, death and differentiation. Hippo signaling effectors (YAP and TAZ; see Drosophila Yorkie) exhibit crosstalk with TGF-β-Smad and Wnt/β-catenin pathways. Previously, Smad7 and β-catenin were implicated in mammalian myogenesis. Therefore, this study assessed a potential role of TAZ on the Smad7-β-catenin complex in muscle cells. Functional interactions were documented between Smad7, TAZ and β-catenin in mouse myogenic cells. Ectopic TAZ expression resulted in repression of the muscle-specific creatine kinase muscle (Ckm) gene promoter and its corresponding protein level. Depletion of endogenous TAZ enhanced Ckm promoter activation. Ectopic TAZ, while potently active on a TEAD reporter (HIP-HOP), repressed myogenin (Myog) and Myod1 enhancer regions and myogenin protein level. Additionally, a Wnt/β-catenin readout (TOP flash) demonstrated TAZ-mediated inhibition of β-catenin activity. In myoblasts, TAZ was predominantly localized in nuclear speckles, while in differentiation conditions TAZ was hyperphosphorylated at Ser89, leading to enhanced cytoplasmic sequestration. Finally, live-cell imaging indicated that TAZ exhibits properties of liquid-liquid phase separation (LLPS). These observations indicate that TAZ, as an effector of Hippo signaling, suppresses the myogenic differentiation machinery.
Xu, M., Ding, L., Liang, J., Yang, X., Liu, Y., Wang, Y., Ding, M. and Huang, X. (2021). NAD kinase sustains lipogenesis and mitochondrial metabolism through fatty acid synthesis. Cell Rep 37(13): 110157. PubMed ID: 34965438
Summary:
Lipid storage in fat tissue is important for energy homeostasis and cellular functions. Through RNAi screening in Drosophila fat body, this study found that knockdown of a Drosophila NAD kinase (NADK), which phosphorylates NAD to synthesize NADP de novo, causes lipid storage defects. NADK sustains lipogenesis by maintaining the pool of NADPH. Promoting NADPH production rescues the lipid storage defect in the fat body of NADK RNAi animals. Furthermore, NADK and fatty acid synthase 1 (FASN1) regulate mitochondrial mass and function by altering the levels of acetyl-CoA and fatty acids. Reducing the level of acetyl-CoA or increasing the synthesis of cardiolipin (CL), a mitochondrion-specific phospholipid, partially rescues the mitochondrial defects of NADK RNAi. Therefore, NADK- and FASN1-mediated fatty acid synthesis coordinates lipid storage and mitochondrial function.
Zhou, J., Dabiri, Y., Gama-Brambila, R. A., Ghafoory, S., Altinbay, M., Mehrabi, A., Golriz, M., Blagojevic, B., Reuter, S., Han, K., Seidel, A., Dikic, I., Wolfl, S. and Cheng, X. (2022). pVHL-mediated SMAD3 degradation suppresses TGF-beta signaling. J Cell Biol 221(1). PubMed ID: 34860252
Summary:
The molecular mechanisms concerning the ubiquitin-related dynamic regulation of TGF-β signaling are not thoroughly understood. Using a combination of proteomics and an siRNA screen, this study identified pVHL as an E3 ligase for SMAD3 ubiquitination. pVHL directly interacts with conserved lysine and proline residues in the MH2 domain of SMAD3, triggering degradation. As a result, the level of pVHL expression negatively correlates with the expression and activity of SMAD3 in cells, Drosophila wing, and patient tissues. In Drosophila, loss of pVHL leads to the up-regulation of TGF-β targets visible in a downward wing blade phenotype, which is rescued by inhibition of SMAD activity. Drosophila pVHL expression exhibited ectopic veinlets and reduced wing growth in a similar manner as upon loss of TGF-β/SMAD signaling. Thus, this study demonstrates a conserved role of pVHL in the regulation of TGF-β/SMAD3 signaling in human cells and Drosophila wing development.

Wednesday, April 13th - Oogenesis

Tarikere, S., Ylla, G. and Extavour, C. G. (2021). Distinct gene expression dynamics in germ line and somatic tissue during ovariole morphogenesis in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 34849771
Summary:
The survival and evolution of a species is a function of the number of offspring it can produce. In insects the number of eggs that an ovary can produce is a major determinant of reproductive capacity. Insect ovaries are made up of tubular egg-producing subunits called ovarioles, whose number largely determines the number of eggs that can be potentially laid. Ovariole number is directly determined by the number of cellular structures called terminal filaments, which are stacks of cells that assemble in the larval ovary. Elucidating the developmental and regulatory mechanisms of terminal filament formation is thus key to understanding the regulation of insect reproduction through ovariole number regulation. This study systematically measured mRNA expression of all cells in the larval ovary at the beginning, middle and end of terminal filament formation. Somatic and germ line cells were separated during these stages and their tissue-specific gene expression was assessed during larval ovary development. Yhe number of differentially expressed somatic genes was highest during late stages of terminal filament formation and includes many signaling pathways that govern ovary development. It was also shown that germ line tissue, in contrast, shows greater differential expression during early stages of terminal filament formation, and highly expressed germ line genes at these stages largely control cell division and DNA repair. A tissue-specific and temporal transcriptomic dataset of gene expression in the developing larval ovary is provided as a resource to study insect reproduction.
Perillo, M., Swartz, S. Z. and Wessel, G. M. (2022). A conserved node in the regulation of Vasa between an induced and an inherited program of primordial germ cell specification. Dev Biol 482: 28-33. PubMed ID: 34863708
Summary:
Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. This study shows that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus. This post-translational activity occurs early in development, substantially prior to gastrulation. This process was tested in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation, it is concluded that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism.
Yildirim, K., Winkler, B., Pogodalla, N., Mackensen, S., Baldenius, M., Garcia, L., Naffin, E., Rodrigues, S. and Klambt, C. (2022). Redundant functions of the SLC5A transporters Rumpel, Bumpel, and Kumpel in ensheathing glial cells. Biol Open 11(1). PubMed ID: 34897385
Summary:
Neuronal processing is energy demanding and relies on sugar metabolism. To nurture the Drosophila nervous system, the blood-brain barrier forming glial cells take up trehalose from the hemolymph and then distribute the metabolic products further to all neurons. This function is provided by glucose and lactate transporters of the solute carrier (SLC) 5A family. This study identified three SLC5A genes that are specifically expressed in overlapping sets of CNS glial cells, rumpel, bumpel and kumpel. This study generated mutants in all genes and all mutants are viable and fertile, lacking discernible phenotypes. Loss of rumpel causes subtle locomotor phenotypes and flies display increased daytime sleep. In addition, in bumpel kumpel double mutants, and to an even greater extent in rumpel bumpel kumpel triple mutants, oogenesis is disrupted at the onset of the vitollegenic phase. This indicates a partially redundant function between these genes. Rescue experiments exploring this effect indicate that oogenesis can be affected by CNS glial cells. Moreover, expression of heterologous mammalian SLC5A transporters, with known transport properties, suggest that Bumpel and/or Kumpel transport glucose or lactate. Overall, these results imply a redundancy in SLC5A nutrient sensing functions in Drosophila glial cells, affecting ovarian development and behavior.
Valer, F. B., Spegiorim, G. C., Espreafico, E. M. and Ramos, R. G. P. (2022). The IRM cell adhesion molecules Hibris, Kin of irre and Roughest control egg morphology by modulating ovarian muscle contraction in Drosophila. J Insect Physiol 136: 104344. PubMed ID: 34896373
Summary:
The Irre Cell Recognition Module (IRM) is an evolutionarily conserved group of transmembrane glycoproteins required for cell-cell recognition and adhesion in metazoan development. In Drosophila melanogaster ovaries, four members of this group - Roughest (Rst), Kin of irre (Kirre), Hibris (Hbs) and Sticks and stones (Sns) - play important roles in germ cell encapsulation and muscle sheath organization during early pupal stages, as well as in the progression to late oogenesis in the adult. Females carrying some of the mutant rst alleles are viable but sterile, and previous work has identified defects in the organization of the peritoneal and epithelial muscle sheaths of these mutants that could underlie their sterile phenotype. In this study, besides further characterizing the sterility phenotype associated with rst mutants, the role of the IRM molecules Rst, Kirre and Hbs was investigated in maintaining the functionality of the ovarian muscle sheaths. Knocking down any of the three genes in these structures, either individually or in double heterozygous combinations, not only decreases contraction frequency but also irregularly increases contraction amplitude. Furthermore, these alterations can significantly impact the morphology of eggs laid by IRM-depleted females demonstrating a hitherto unknown role of IRM molecules in egg morphogenesis.
Russell, S. A., Laws, K. M. and Bashaw, G. J. (2021). Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis. Development 148(24). PubMed ID: 34910816
Summary:
The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. This study reports that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, these observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.
Pokrywka, N. J., Bush, S. and Nick, S. E. (2021). The R-SNARE Ykt6 is required for multiple events during oogenesis in Drosophila. Cells Dev 169: 203759. PubMed ID: 34856414
Summary:
Ykt6 has emerged as a key protein involved in a wide array of trafficking events, and has also been implicated in a number of human pathologies, including the progression of several cancers. It is a complex protein that simultaneously exhibits a high degree of structural and functional homology, and yet adopts differing roles in different cellular contexts. Because Ykt6 has been implicated in a variety of vesicle fusion events, this study characterized the role of Ykt6 in oogenesis by observing the phenotype of Ykt6 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins, organelles, and vesicular trafficking markers in mutant egg chambers. Ykt6 germline clones have morphological and actin defects affecting both the nurse cells and oocyte, consistent with a role in regulating membrane growth during mid-oogenesis. Additionally, these egg chambers exhibit defects in bicoid and oskar RNA localization, and in the trafficking of Gurken during mid-to-late oogenesis. Finally, it was shown that Ykt6 mutations result in defects in late endosomal pathways, including endo- and exocytosis. These findings suggest a role for Ykt6 in endosome maturation and in the movement of membranes to and from the cell surface.

Tuesday, April 11th - Adult Development

Svoysky, A. J., Bellah, J. L. and Johnston, L. A. (2021). Studies of Myc super-competition and clonal growth in Drosophila males and females. MicroPubl Biol 2021. PubMed ID: 34909609
Summary:
Cell competition is a cell selection process that arises in growing tissues as a result of interactions between cells of different fitness. This behavior is also observed in Myc super-competition, where healthy wild type cells in growing wing discs of Drosophila are outcompeted by nearby cells that express higher levels of the Myc oncogene. Most work on Myc super-competition has examined it in mixed populations of male and female larvae. However, as physiological and genetic differences between Drosophila males and females could affect the competitive behavior of cells, this study investigated whether sex differences affect the process. Both male and female wing disc cells were shown to be subject to Myc super-competition. Female disc cells appear to be more sensitive to competitive elimination than male cells, potentially due to differences in baseline cellular Myc levels between the sexes. This paper also reports sexual dimorphism of cell size and number between male and female growing wing discs that is independent of competition; wing discs and wing pouches from females are larger than males' due to larger cell size and cell number. It is suggested that separately examining male and female tissues in cell competition assays could enhance understanding of the effects of sex-specific pathways on cell and super-competition.
Noguchi, K., Yokozeki, K., Tanaka, Y., Suzuki, Y., Nakajima, K., Nishimura, T. and Goda, N. (2021). Sima, a Drosophila homolog of HIF-1alpha, in fat body tissue inhibits larval body growth by inducing Tribbles gene expression. Genes Cells. PubMed ID: 34918430
Summary:
Limited oxygen availability impairs normal body growth, although the underlying mechanisms are not fully understood. In Drosophila, hypoxic responses in the larval fat body (FB) disturb the secretion of insulin-like peptides from the brain, inhibiting body growth. However, the cell-autonomous effects of hypoxia on the insulin-signaling pathway in larval FB have been underexplored. This study aimed to examine the effects of overexpression of Sima, a Drosophila hypoxia-inducible factor-1 (HIF-1) α homolog and a key component of HIF-1 transcription factor essential for hypoxic adaptation, on the insulin-signaling pathway in larval FB. Forced expression of Sima in FB reduced the larval body growth with reduced Akt phosphorylation levels in FB cells and increased hemolymph sugar levels. Sima-mediated growth inhibition was reversed by overexpression of TOR or suppression of FOXO. After Sima overexpression, larvae showed higher expression levels of Tribbles, a negative regulator of Akt activity, and a simultaneous knockdown of Tribbles completely abolished the effects of Sima on larval body growth. Furthermore, a reporter analysis revealed Tribbles as a direct target gene of Sima. These results suggest that Sima in FB evokes Tribbles-mediated insulin resistance and consequently protects against aberrant insulin-dependent larval body growth under hypoxia.
Michailidi, M. R., Hadjivasiliou, Z., Aguilar-Hidalgo, D., Basagiannis, D., Seum, C., Dubois, M., Julicher, F. and Gonzalez-Gaitan, M. (2021). Morphogen gradient scaling by recycling of intracellular Dpp. Nature. PubMed ID: 34937053
Summary:
Morphogen gradients are fundamental to establish morphological patterns in developing tissues. During development, gradients scale to remain proportional to the size of growing organs. Scaling is a universal gear adjusting patterns to size in living organisms, yet its mechanisms remain unclear. In this study, focusing on the Dpp gradient in the Drosophila wing disc, a cell biological basis behind scaling has been unraveled. From small to large discs, scaling of the Dpp gradient is achieved by increasing the contribution of the internalized Dpp molecules to Dpp transport: to expand the gradient, endocytosed molecules are re-exocytosed to spread extracellularly. To regulate the contribution of endocytosed Dpp to the spreading extracellular pool during tissue growth, it is the Dpp binding rates that are progressively modulated by the extracellular factor Pentagone, driving scaling. Thus, for some morphogens, evolution may act on endocytic trafficking to regulate the range of the gradient and its scaling, which could allow adaptation of shape and pattern to different sizes of organs in different species.
Pechmann, M. and Prpic, N. M. (2022). The T-box gene optomotor-blind organizes proximodistal leg patterning in the beetle Tribolium castaneum by repressing dorsal Dpp pathway activity. Dev Biol 482: 124-134. PubMed ID: 34942194
Summary:
Leg axis formation in Drosophila is organized by Wingless (Wg) and Decapentaplegic (Dpp) that control a number of downstream factors to pattern the dorsoventral (DV) and proximodistal (PD) axis. The T-box genes are important downstream factors mainly involved in dorsoventral leg axis formation. The ventral side is specified by H15 and midline, whereas optomotor-blind (omb) and Dorsocross (Doc1) are factors to specify dorsal cell fates. This study shows that omb also organizes PD leg axis patterning in the beetle Tribolium castaneum. In the legs, Tc-omb is expressed along the dorsal side and represses ventral factors like wg and H15. Intriguingly, removing Tc-omb function leads to the activation of the Dpp pathway along the dorsal side of the legs, thus mimicking normal dpp expression in Drosophila. Dpp activity along the dorsal side leads to altered expression of proximal-distal patterning genes such as Distal-less (Dll) and dachshund (dac). These results indicate a cell-autonomous activation of Dll and repression of dac by dpp. These findings are compatible with the cross-regulatory "cascade model" of proximal-distal leg imaginal disc patterning of Drosophila.
Yamashita, S., Guirao, B. and Graner, F. (2021). From heterogeneous morphogenetic fields to homogeneous regions as a step towards understanding complex tissue dynamics. Development 148(23). PubMed ID: 34861038
Summary:
Within developing tissues, cell proliferation, cell motility and other cell behaviors vary spatially, and this variability gives a complexity to the morphogenesis. Recently, novel formalisms have been developed to quantify tissue deformation and underlying cellular processes. A major challenge for the study of morphogenesis now is to objectively define tissue sub-regions exhibiting different dynamics. This paper proposes a method to automatically divide a tissue into regions where the local deformation rate is homogeneous. This was achieved by several steps including image segmentation, clustering and region boundary smoothing. The use of the pipeline is demonstrated using a large dataset obtained during the metamorphosis of the Drosophila pupal notum. It was also adapted to determine regions in which the time evolution of the local deformation rate is homogeneous. Finally, its use generalized to find homogeneous regions for cellular processes such as cell division, cell rearrangement, or cell size and shape changes. Its use is also demonstrated on wing blade morphogenesis. This pipeline will contribute substantially to the analysis of complex tissue shaping, and the biochemical and biomechanical regulations driving tissue morphogenesis.
Laurichesse, Q., Moucaud, B., Laddada, L., Renaud, Y., Jagla, K. and Soler, C. (2021). Transcriptomic and Genetic Analyses Identify the Kruppel-Like Factor Dar1 as a New Regulator of Tube-Shaped Long Tendon Development. Front Cell Dev Biol 9: 747563. PubMed ID: 34977007
Summary:
To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During the leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. This study provides for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, the Kruppel-like factor Dar1 was identified as a critical actor of leg tendon development. Specifically expressed in the leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. These findings show that Dar1 acts downstream of Stripe and is required to set up the correct number of tendon progenitors.

Monday April 11th - Adult Development

Wierzbicki, F., Kofler, R. and Signor, S. (2021). Evolutionary dynamics of piRNA clusters in Drosophila. Mol Ecol. PubMed ID: 34878692
Summary:
Small RNAs produced from transposable element (TE)-rich sections of the genome, termed piRNA clusters, are a crucial component in the genomic defence against selfish DNA. In animals, it is thought the invasion of a TE is stopped when a copy of the TE inserts into a piRNA cluster, triggering the production of cognate small RNAs that silence the TE. Despite this importance for TE control, little is known about the evolutionary dynamics of piRNA clusters, mostly because these repeat-rich regions are difficult to assemble and compare. This study establish a framework for studying the evolution of piRNA clusters quantitatively. Previously introduced quality metrics and a newly developed software for multiple alignments of repeat annotations (Manna) allowed estimation of the level of polymorphism segregating in piRNA clusters and the divergence among homologous piRNA clusters. By studying 20 conserved piRNA clusters in multiple assemblies of four Drosophila species, it was shown that piRNA clusters are evolving rapidly. While 70%-80% of the clusters are conserved within species, the clusters share almost no similarity between species as closely related as D. melanogaster and D. simulans. Furthermore, abundant insertions and deletions are segregating within the Drosophila species. The evolution of clusters is shown to be mainly driven by large insertions of recently active TEs and smaller deletions mostly in older TEs. The effect of these forces is so rapid that homologous clusters often do not contain insertions from the same TE families.
Said, I., McGurk, M. P., Clark, A. G. and Barbash, D. A. (2022). Patterns of piRNA Regulation in Drosophila Revealed through Transposable Element Clade Inference. Mol Biol Evol 39(1). PubMed ID: 34921315
Summary:
Transposable elements (TEs) are self-replicating "genetic parasites" ubiquitous to eukaryotic genomes. In addition to conflict between TEs and their host genomes, TEs of the same family are in competition with each other. They compete for the same genomic niches while experiencing the same regime of copy-number selection. This suggests that competition among TEs may favor the emergence of new variants that can outcompete their ancestral forms. To investigate the sequence evolution of TEs, a method was developed to infer clades: collections of TEs that share SNP variants and represent distinct TE family lineages. This method was applied to a panel of 85 Drosophila melanogaster genomes and found that the genetic variation of several TE families shows significant population structure that arises from the population-specific expansions of single clades. Population genetic theory was used to classify these clades into younger versus older clades, and it was found that younger clades are associated with a greater abundance was found of sense and antisense piRNAs per copy than older ones. Further, the abundance of younger, but not older clades, is positively correlated with antisense piRNA production, suggesting a general pattern where hosts preferentially produce antisense piRNAs from recently active TE variants. Together these findings suggest a pattern whereby new TE variants arise by mutation and then increase in copy number, followed by the host producing antisense piRNAs that may be used to silence these emerging variants.
Han, S., Basting, P. J., Dias, G. B., Luhur, A., Zelhof, A. C. and Bergman, C. M. (2021). Transposable element profiles reveal cell line identity and loss of heterozygosity in Drosophila cell culture. Genetics 219(2). PubMed ID: 34849875
Summary:
Cell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabeling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. This study leveraged the classical observation that transposable elements (TEs) proliferate in cultured Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. Multiple cases were identified where TE profiles clarify the origin of Drosophila cell lines (Sg4, mbn2, and OSS_E) relative to published reports, and also provide evidence that insertions from only a subset of long-terminal repeat retrotransposon families are necessary to mark Drosophila cell line identity. A new bioinformatics approach was developed to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome sequencing data (called ngs_te_mapper2), which revealed loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. This work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.
Ilyin, A. A., Stolyarenko, A. D., Zenkin, N. and Klenov, M. S. (2021). Complex Genetic Interactions between Piwi and HP1a in the Repression of Transposable Elements and Tissue-Specific Genes in the Ovarian Germline. Int J Mol Sci 22(24). PubMed ID: 34948223
Summary:
Insertions of transposable elements (TEs) in eukaryotic genomes are usually associated with repressive chromatin, which spreads to neighbouring genomic sequences. In ovaries of Drosophila melanogaster, the Piwi-piRNA pathway plays a key role in the transcriptional silencing of TEs considered to be exerted mostly through the establishment of H3K9me3 histone marks recruiting Heterochromatin Protein 1a (HP1a). Using RNA-seq, this study investigated the expression of TEs and the adjacent genomic regions upon Piwi and HP1a germline knockdowns sharing a similar genetic background. The depletion of Piwi and HP1a led to the derepression of only partially overlapping TE sets. Several TEs were silenced predominantly by HP1a, whereas the upregulation of some other TEs was more pronounced upon Piwi knockdown and, surprisingly, was diminished upon a Piwi/HP1a double-knockdown. It was revealed that HP1a loss influenced the expression of thousands of protein-coding genes mostly not adjacent to TE insertions and, in particular, downregulated a putative transcriptional factor required for TE activation. Nevertheless, these results indicate that Piwi and HP1a cooperatively exert repressive effects on the transcription of euchromatic loci flanking the insertions of some Piwi-regulated TEs. It is suggested that this mechanism controls the silencing of a small set of TE-adjacent tissue-specific genes, preventing their inappropriate expression in ovaries.
Vedanayagam, J., Lin, C. J. and Lai, E. C. (2021). Rapid evolutionary dynamics of an expanding family of meiotic drive factors and their hpRNA suppressors. Nat Ecol Evol 5(12): 1613-1623. PubMed ID: 34862477
Summary:
Meiotic drivers are a class of selfish genetic elements whose existence is frequently hidden due to concomitant suppressor systems. Accordingly, little is known of their evolutionary breadth and molecular mechanisms. This study traced the evolution of the Dox meiotic drive system in Drosophila simulans, which affects male-female balance (sex ratio). Dox emerged via stepwise mobilization and acquisition of multiple D. melanogaster gene segments including from protamine, which mediates compaction of sperm chromatin. Moreover, novel Dox homologs and massive amplification was revealed of Dox superfamily genes on X chromosomes of its closest sisters D. mauritiana and D. sechellia. Emergence of Dox loci is tightly associated with 359-class satellite repeats that flank de novo genomic copies. In concert, coordinated diversification was found of autosomal hairpin RNA-class siRNA loci that target subsets of Dox superfamily genes. Overall, this study revealed fierce genetic arms races between meiotic drive factors and siRNA suppressors associated with recent speciation.
Karki, P., Carney, T. D., Maracci, C., Yatsenko, A. S., Shcherbata, H. R. and Rodnina, M. V. (2021). Tissue-specific regulation of translational readthrough tunes functions of the traffic jam transcription factor. Nucleic Acids Res. PubMed ID: 34897510
Summary:
Translational readthrough (TR) occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR has been identified in several eukaryotic organisms; however, its biological significance and mechanism remain unclear. This study quantified TR of several candidate genes in Drosophila melanogaster and characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj). Using CRISPR/Cas9-generated mutant flies, it was shown that the TR-generated Tj isoform is expressed in a subset of neural cells of the central nervous system and is excluded from the somatic cells of gonads. Control of TR in Tj is critical for preservation of neuronal integrity and maintenance of reproductive health. The tissue-specific distribution of a release factor splice variant, eRF1H, plays a critical role in modulating differential TR of leaky stop codon contexts. Fine-tuning of gene regulatory functions of transcription factors by TR provides a potential mechanism for cell-specific regulation of gene expression.

Friday, April 8th - Disease models

Tapia, A., Palomino-Schatzlein, M., Roca, M., Lahoz, A., Pineda-Lucena, A., Lopez Del Amo, V. and Galindo, M. I. (2021). Mild Muscle Mitochondrial Fusion Distress Extends Drosophila Lifespan through an Early and Systemic Metabolome Reorganization. Int J Mol Sci 22(22). PubMed ID: 34830014
Summary:
In a global aging population, it is important to understand the factors affecting systemic aging and lifespan. Mitohormesis, an adaptive response caused by different insults affecting the mitochondrial network, triggers a response from the nuclear genome inducing several pathways that promote longevity and metabolic health. Understanding the role of mitochondrial function during the aging process could help biomarker identification and the development of novel strategies for healthy aging. This study interfered the muscle expression of the Drosophila genes Marf and Opa1, two genes that encode for proteins promoting mitochondrial fusion, orthologues of human MFN2 and OPA1. Silencing of Marf and Opa1 in muscle increases lifespan, improves locomotor capacities in the long term, and maintains muscular integrity. A metabolomic analysis revealed that muscle down-regulation of Marf and Opa1 promotes a non-autonomous systemic metabolome reorganization, mainly affecting metabolites involved in the energetic homeostasis: carbohydrates, lipids and aminoacids. Interestingly, the differences are consistently more evident in younger flies, implying that there may exist an anticipative adaptation mediating the protective changes at the older age. This study demonstrates that mild mitochondrial muscle disturbance plays an important role in Drosophila fitness and reveals metabolic connections between tissues. This study opens new avenues to explore the link of mitochondrial dynamics and inter-organ communication, as well as their relationship with muscle-related pathologies, or in which muscle aging is a risk factor for their appearance. These results suggest that early intervention in muscle may prevent sarcopenia and promote healthy aging.
Srivastav, A. and Mohideen, S. S. (2021). Intestinal microbial dysbiosis induced tau accumulation establishes a gut-brain correlation for pathology Alzheimer's disease in Drosophila melanogaster. Alzheimers Dement 17 Suppl 2: e058708. PubMed ID: 34971141
Summary:
To analyze the concept of whether enteric dysbiosis contributes to the aggravation of tau protein, Drosophila melanogaster was used as a transgenic model organism. To study how the exposure of Pseudomonas sp., a gram-negative bacteria, influences tau accumulation and neurodegeneration thereby understanding the gut-brain correlation for the pathology of Alzheimer's Disease. The versatile Gal4-UAS gene expression system was used to express wild-type tau with the help of pan-eye specific GMR-Gal4 in the flies. All the flies were exposed to Pseudomonas sp. to initiate the gut microbial dysbiosis. The extent of neurodegeneration was quantified by automated analysis of the degenerated region in the eye, the number of aggregates, and immunofluorescence quantification in the medulla. To understand the response of glial cells upon bacterial infection in tauopathy-related dementia, tau was driven by glia-specific Repo-GAL4. Similarly, neuroinflammation which is found in tauopathies was studied using an eye-specific driver line GMR-GAL4, UAS- eiger/CyO. Upon exposure of Pseudomonas sp. on the wild type tau (tauwt) around the medulla, the degeneration was faster and, there was an increase in the levels of tau as compared to the control. Downregulation of Atg 1 and Atg 18 gene along with significant upregulation of Atg 12 is observed in tau expressing infected flies compared to tau expressing control flies. There was a significant degeneration as well as an increase in the number of aggregates in the medulla of the optic lobe. Similar degeneration was observed in flies expressing tauwt and egr along with an increased accumulation of tau in both retina and medulla. When the flies expressing tau are subjected to the pathogen, and aggravation in tau accumulation is observed along with neuroinflammation and changes in the homeostasis. Due to its role in maintaining of body's homeostasis, the gut-brain correlation can have both detrimental and beneficial effects on the brain and the survival of neurons.
Smith, C. A., Smith, H., Roberts, L., Coward, L., Gorman, G., Verma, A., Li, Q., Buford, T. W., Carter, C. S. and Jumbo-Lucioni, P. (2021). Probiotic Releasing Angiotensin (1-7) in a Drosophila Model of Alzheimer's Disease Produces Sex-Specific Effects on Cognitive Function. J Alzheimers Dis. PubMed ID: 34924372
Summary:
While extensive research on the brain has failed to identify effective therapies, using probiotics to target the gut microbiome has shown therapeutic potential in Alzheimer's disease (AD). Genetically modified probiotics (GMP) are a promising strategy to deliver key therapeutic peptides with high efficacy and tissue specificity. Angiotensin (Ang)-(1-7) levels inversely correlate to AD severity, but its administration is challenging. This lab has successfully established a GMP-based method of Ang-(1-7) delivery. Since Drosophila represents an excellent model to study the effect of probiotics on complex disorders in a high throughput manner, this study tested whether oral supplementation with Lactobacillus paracasei releasing Ang-(1-7) (LP-A) delays memory loss in a Drosophila AD model. Flies overexpressing the human amyloid-β protein precursor and its β-site cleaving enzyme in neurons were randomized to receive four 24-h doses of Lactobacillus paracasei alone (LP), LP-A or sucrose over 14 days. Memory was assessed via an aversive phototaxic suppression assay. Optimal dilution,1:2, was determined based on palatability. LP-A improved memory in trained AD males but worsened cognition in AD females. LP-supplementation experiments confirmed that Ang-(1-7) conferred additional cognitive benefits in males and was responsible for the deleterious cognitive effects in females. Sex-specific differences in the levels of angiotensin peptides and differential activation of the kynurenine pathway of tryptophan metabolism in response to supplementation may underlie this male-only therapeutic response. In summary, LP-A ameliorated the memory deficits of a Drosophila AD model, but effects were sex-specific. Dosage optimization may be required to address this differential response.
Singh, Y. P., Kumar, N., Priya, K., Chauhan, B. S., Shankar, G., Kumar, S., Singh, G. K., Srikrishna, S., Garg, P., Singh, G., Rai, G. and Modi, G. (2022). Exploration of Neuroprotective Properties of a Naturally Inspired Multifunctional Molecule (F24) against Oxidative Stress and Amyloid β Induced Neurotoxicity in Alzheimer's Disease Models. ACS Chem Neurosci 13(1): 27-42. PubMed ID: 34931800
Summary:
The pathological hallmarks of Alzheimer's disease (AD) are manifested as an increase in the level of oxidative stress and aggregation of the amyloid-β protein. In vitro, in vivo, and in silico experiments were designed and carried out with multifunctional cholinergic inhibitor, F24 (EJMC-7a) to explore its neuroprotective effects in AD models. The neuroprotection ability of F24 was tested in SH-SY5Y cells, a widely used neuronal cell line. The pretreatment and subsequent co-treatment of SH-SY5Y cells with different doses of F24 was effective in rescuing the cells from H2O2 induced neurotoxicity. F24 treated cells were found to be effective in the reduction of cellular reactive oxygen species, DNA damage, and Aβ(1-42) induced neurotoxicity, which validated its neuroprotective effectiveness. F24 exhibited efficacy in an in vivo Drosophila model by rescuing eye phenotypes from degeneration caused by Aβ toxicity. Further, computational studies were carried out to monitor the interaction between F24 and Aβ(1-42) aggregates. The computational studies corroborated in vitro and in vivo studies suggesting Aβ(1-42) aggregation modulation ability of F24. The brain entry ability of F24 was studied in the parallel artificial membrane permeability assay. Finally, F24 was tested at doses of 1 and 2.5 mg/kg in the Morris water maze AD model. The neuroprotective properties shown by F24 strongly suggest that multifunctional features of this molecule provide symptomatic relief and act as a disease-modifying agent in the treatment of AD. The results from these experiments strongly indicated that natural template-based F24 could serve as a lead molecule for further investigation to explore multifunctional therapeutic agents for AD management.
Tapia, A., Giachello, C. N., Palomino-Schatzlein, M., Baines, R. A. and Galindo, M. I. (2021). Generation and Characterization of the Drosophila melanogaster paralytic Gene Knock-Out as a Model for Dravet Syndrome. Life (Basel) 11(11). PubMed ID: 34833136
Summary:
Dravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. A knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, was made by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, an NMR metabolomic profiling of heads was performed that revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments.
Sheshadri, D., Onkar, A. and Ganesh, S. (2021). Alterations in brain glycogen levels influence life-history traits and reduce the lifespan in female Drosophila melanogaster. Biol Open 10(12). PubMed ID: 34817590
Summary:
Sexual dimorphism in lifespan, wherein females outlive males, is evident across all animal taxa. The longevity difference between sexes is controlled by multiple physiological processes with complex relationships to one another. In recent years, glycogen, the storage form of glucose, has been shown to cause rapid aging upon forced synthesis in healthy neurons. Glycogen in the form of corpora amylacea in the aging brain is also widely reported. While these studies did suggest a novel role for glycogen in aging, most of them have focused on pooled samples, and have not looked at sex-specific effects, if any. Given the widespread occurrence of sex-biased expression of genes and the underlying physiology, it is important to look at the sex-specific effects of metabolic processes. Using transgenic fly lines for the human glycogen synthase, this study investigated the sex-specific effects of glycogen on stress resistance, fitness, and survival. Drosophila melanogaster females with altered levels of glycogen in the brain were shown to display a shortened lifespan, increased resistance to starvation, and higher oxidative stress than male flies. The present study thus provides a novel insight into the sex-specific effect of glycogen in survival and aging and how differences in metabolic processes could contribute to sex-specific traits.

Thursday, April 7th - Adult neural development and function

Saitoe, M., Naganos, S., Miyashita, T., Matsuno, M. and Ueno, K. (2021). A non-canonical on-demand dopaminergic transmission underlying olfactory aversive learning. Neurosci Res. PubMed ID: 34973292
Summary:
Dopamine (DA) is involved in various brain functions including associative learning. However, it is unclear how a small number of DA neurons appropriately regulates various brain functions. DA neurons have a large number of release sites and release DA non-specifically to a large number of target neurons in the projection area in response to the activity of DA neurons. In contrast to this "broad transmission", recent studies in Drosophila ex vivo functional imaging studies have identified "on-demand transmission" that occurs independent on activity of DA neurons and releases DA specifically onto the target neurons that have produced carbon monoxide (CO) as a retrograde signal for DA release. Whereas broad transmission modulates the global function of the target area, on-demand transmission is suitable for modulating the function of specific circuits, neurons, or synapses. In Drosophila olfactory aversive conditioning, odor and shock information are associated in the brain region called mushroom body (MB) to form olfactory aversive memory. It has been suggested that DA neurons projecting to the MB mediate the transmission of shock information and reinforcement simultaneously. However, the circuit model based on on-demand transmission proposes that transmission of shock information and reinforcement are mediated by distinct neural mechanisms; while shock transmission is glutamatergic, DA neurons mediates reinforcement. On-demand transmission provides mechanical insights into how DA neurons regulate various brain functions.
Reinhard, N., Bertolini, E., Saito, A., Sekiguchi, M., Yoshii, T., Rieger, D. and Helfrich-Forster, C. (2021). The lateral posterior clock neurons of Drosophila melanogaster express three neuropeptides and have multiple connections within the circadian clock network and beyond. J Comp Neurol. PubMed ID: 34961936
Summary:
Drosophila's lateral posterior neurons (LPNs) belong to a small group of circadian clock neurons that is so far not characterized in detail. Thanks to a new highly specific split-Gal4 line, this study describes LPNs' morphology in fine detail, their synaptic connections, daily bimodal expression of neuropeptides, and a putative role of this cluster in controlling daily activity and sleep patterns is proposed. The three LPNs were found to be heterogeneous. Two of the neurons with similar morphology arborize in the superior medial and lateral protocerebrum and most likely promote sleep. One unique, possibly wakefulness-promoting, neuron with wider arborizations extends from the superior lateral protocerebrum toward the anterior optic tubercle. Both LPN types exhibit manifold connections with the other circadian clock neurons, especially with those that control the flies' morning and evening activity (M- and E-neurons, respectively). In addition, they form synaptic connections with neurons of the mushroom bodies, the fan-shaped body, and with many additional still unidentified neurons. Both LPN types rhythmically express three neuropeptides, Allostatin A, Allostatin C, and Diuretic Hormone 31 with maxima in the morning and the evening. The three LPN neuropeptides may, furthermore, signal to the insect hormonal center in the pars intercerebralis and contribute to rhythmic modulation of metabolism, feeding, and reproduction. These findings are discussed in the light of anatomical details gained by the recently published hemibrain of a single female fly on the electron microscopic level and of previous functional studies concerning the LPN.
Vallejos, M. J., Eadaim, A., Hahm, E. T. and Tsunoda, S. (2021). Age-related changes in Kv4/Shal and Kv1/Shaker expression in Drosophila and a role for reactive oxygen species. PLoS One 16(12): e0261087. PubMed ID: 34932577
Summary:
Age-related changes in ion channel expression are likely to affect neuronal signaling. This study examined how age affects Kv4/Shal and Kv1/Shaker K+ channel protein levels in Drosophila. Kv4/Shal protein levels decline sharply from 3 days to 10 days, then more gradually from 10 to 40 days after eclosion. In contrast, Kv1/Shaker protein exhibits a transient increase at 10 days that then stabilizes and eventually declines at 40 days. Data are presented that begin to show a relationship between reactive oxygen species (ROS), Kv4/Shal, and locomotor performance. Kv4/Shal levels are negatively affected by ROS, and that over-expression of Catalase or RNAi knock-down of the ROS-generating enzyme, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase (NOX), can attenuate the loss of Kv4/Shal protein. Finally, levels of Kv4.2 and Kv4.3 in the hippocampus, olfactory bulb, cerebellum, and motor cortex of mice aged 6 weeks and 1 year were compared. While there was no global decline in Kv4.2/4.3 that parallels what this study reports in Drosophila, it was found that Kv4.2/4.3 are differentially affected in various brain regions; this survey of changes may help inform mammalian studies that examine neuronal function with age.
Sengupta, S., Chan, Y. B., Palavicino-Maggio, C. B. and Kravitz, E. A. (2022). GABA transmission from mAL interneurons regulates aggression in Drosophila males. Proc Natl Acad Sci U S A 119(5). PubMed ID: 35082150
Summary:
Aggression is known to be regulated by pheromonal information in many species. But how central brain neurons processing this information modulate aggression is poorly understood. Using the fruit fly model of Drosophila melanogaster, this study systematically characterize the role of a group of sexually dimorphic GABAergic central brain neurons, popularly known as mAL, in aggression regulation. The mAL neurons are known to be activated by male and female pheromones. This report shows that mAL activation robustly increases aggression, whereas its inactivation decreases aggression and increases intermale courtship, a behavior considered reciprocal to aggression. GABA neurotransmission from mAL is crucial for this behavior regulation. Exploiting the genetic toolkit of the fruit fly model, a small group of approximately three to five GABA(+) central brain neurons were found with anatomical similarities to mAL. Activation of the mAL resembling group of neurons is necessary for increasing intermale aggression. Overall, these findings demonstrate how changes in activity of GABA(+) central brain neurons processing pheromonal information, such as mAL in Drosophila melanogaster, directly modulate the social behavior of aggression in male-male pairings.
Ren, Q. and Rao, Y. (2022). The exit of axons and glial membrane from the developing Drosophila retina requires integrins. Mol Brain 15(1): 2. PubMed ID: 34980203
Summary:
Coordinated development of neurons and glia is essential for the establishment of neuronal circuits during embryonic development. In the developing Drosophila visual system, photoreceptor (R cell) axons and wrapping glial (WG) membrane extend from the eye disc through the optic stalk into the optic lobe. Extensive studies have identified a number of genes that control the establishment of R-cell axonal projection pattern in the optic lobe. The molecular mechanisms directing the exit of R-cell axons and WG membrane from the eye disc, however, remain unknown. This study shows that integrins are required in R cells for the extension of R-cell axons and WG membrane from the eye disc into the optic stalk. Knockdown of integrins in R cells but not WG caused the stalling of both R-cell axons and WG membrane in the eye disc. Interfering with the function of Rhea (i.e. the Drosophila ortholog of vertebrate talin and a key player of integrin-mediated adhesion), caused an identical stalling phenotype. These results support a key role for integrins on R-cell axons in directing R-cell axons and WG membrane to exit the eye disc.
Rozenfeld, E., Tauber, M., Ben-Chaim, Y. and Parnas, M. (2021). GPCR voltage dependence controls neuronal plasticity and behavior. Nat Commun 12(1): 7252. PubMed ID: 34903750
Summary:
The G-protein coupled receptors (GPCRs) play a paramount role in diverse brain functions. Almost 20 years ago, GPCR activity was shown to be regulated by membrane potential in vitro, but whether the voltage dependence of GPCRs contributes to neuronal coding and behavioral output under physiological conditions in vivo has never been demonstrated. This study shows that muscarinic GPCR mediated neuronal potentiation in vivo is voltage dependent. This voltage dependent potentiation is abolished in mutant animals expressing a voltage independent receptor. Depolarization alone, without a muscarinic agonist, results in a nicotinic ionotropic receptor potentiation that is mediated by muscarinic receptor voltage dependency. Finally, muscarinic receptor voltage independence causes a strong behavioral effect of increased odor habituation. Together, this study identifies a physiological role for the voltage dependency of GPCRs by demonstrating crucial involvement of GPCR voltage dependence in neuronal plasticity and behavior. Thus, this study suggests that GPCR voltage dependency plays a role in many diverse neuronal functions including learning and memory.

Wednesday, April 6th - chromatin, DNA replication and chromosome dynamics

Schoelz, J. M., Feng, J. X. and Riddle, N. C. (2021). The Drosophila HP1 family is associated with active gene expression across chromatin contexts. Genetics 219(1). PubMed ID: 34849911
Summary:
Drosophila Heterochromatin Protein 1a (HP1a) is essential for heterochromatin formation and is involved in transcriptional silencing. However, certain loci require HP1a in order to be transcribed. One model posits that HP1a acts as a transcriptional silencer within euchromatin while acting as an activator within heterochromatin. However, HP1a has been observed as an activator of a set of euchromatic genes. Therefore, it is not clear whether, or how, chromatin context informs the function of HP1 proteins. To understand the role of HP1 proteins in transcription, the genome-wide binding profile of HP1a as well as two other Drosophila HP1 family members, HP1B and HP1C, were examined to determine whether coordinated binding of these proteins is associated with specific transcriptional outcomes. HP1 proteins were found to share many of their endogenous binding targets. These genes are marked by active histone modifications and are expressed at higher levels than nontarget genes in both heterochromatin and euchromatin. In addition, HP1 binding targets displayed increased RNA polymerase pausing compared with nontarget genes. Specifically, colocalization of HP1B and HP1C was associated with the highest levels of polymerase pausing and gene expression. Analysis of HP1 null mutants suggests these proteins coordinate activity at transcription start sites to regulate transcription. Depletion of HP1B or HP1C alters expression of protein-coding genes bound by HP1 family members. These data broaden understanding of the mechanism of transcriptional activation by HP1a and highlight the need to consider particular protein-protein interactions, rather than broader chromatin context, to predict impacts of HP1 at transcription start sites.
Kitzman, S. C., Duan, T., Pufall, M. A. and Geyer, P. K. (2021). Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants. G3 (Bethesda). PubMed ID: 34893833
Summary:
The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin binding protein Barrier-to-autointegration factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to deformation of NL structure and non-canonical activation of Checkpoint kinase 2 (Chk2). This study investigated contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf mis-regulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained mis-expressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global mis-expression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found up-regulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.
Parker, M. W., Kao, J. A., Huang, A., Berger, J. M. and Botchan, M. R. (2021). Molecular determinants of phase separation for Drosophila DNA replication licensing factors. Elife 10. PubMed ID: 34951585
Summary:
Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, it was shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. In this study, using D. melanogaster (Dm) Cdt1 as a model initiation factor, it was shown that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.
So, M., Stiban, J., Ciesielski, G. L., Hovde, S. L. and Kaguni, L. S. (2021). Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila Mitochondrial Replicative DNA Helicase. Front Genet 12: 790521. PubMed ID: 34950192
Summary:
Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). This report confirms the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, this study showed that the NTD also serves a role in membrane binding. It was demonstrated that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). It is suggested that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.
Lo Furno, E., Busseau, I., Aze, A., Lorenzi, C., Saghira, C., Danzi, M. C., Zuchner, S. and Maiorano, D. (2022). Translesion DNA synthesis-driven mutagenesis in very early embryogenesis of fast cleaving embryos. Nucleic Acids Res 50(2): 885-898. PubMed ID: 34939656
Summary:
In early embryogenesis of fast cleaving embryos, DNA synthesis is short and surveillance mechanisms preserving genome integrity are inefficient, implying the possible generation of mutations. This study analyzed mutagenesis in Xenopus laevis and Drosophila melanogaster early embryos. The occurrence of a high mutation rate in Xenopus is reported; it was shown to be dependent upon the translesion DNA synthesis (TLS) master regulator Rad18. Unexpectedly, a homology-directed repair contribution of Rad18 was observed in reducing the mutation load. Genetic invalidation of TLS in the pre-blastoderm Drosophila embryo resulted in reduction of both the hatching rate and single-nucleotide variations on pericentromeric heterochromatin in adult flies. Altogether, these findings indicate that during very early period Xenopus and Drosophila embryos TLS strongly contributes to the high mutation rate. This may constitute a previously unforeseen source of genetic diversity contributing to the polymorphisms of each individual with implications for genome evolution and species adaptation.
Larson, E. D., Komori, H., Gibson, T. J., Ostgaard, C. M., Hamm, D. C., Schnell, J. M., Lee, C. Y. and Harrison, M. M. (2021). Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila. Nat Commun 12(1): 7153. PubMed ID: 34887421
Summary:
During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. Zelda was shown to synergistically function with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is correlated with differences in chromatin accessibility and cofactor availability. It is proposed that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network driven by cell-type-specific enhancers.

Tuesday, April 5th - Evolution

Peng, J., Svetec, N. and Zhao, L. (2022). Intermolecular Interactions Drive Protein Adaptive and Coadaptive Evolution at Both Species and Population Levels. Mol Biol Evol 39(1). PubMed ID: 34878126
Summary:
Proteins are the building blocks for almost all the functions in cells. Understanding the molecular evolution of proteins and the forces that shape protein evolution is essential in understanding the basis of function and evolution. Previous studies have shown that adaptation frequently occurs at the protein surface, such as in genes involved in host-pathogen interactions. However, it remains unclear whether adaptive sites are distributed randomly or at regions associated with particular structural or functional characteristics across the genome, since many proteins lack structural or functional annotations. This study sought to tackle this question by combining large-scale bioinformatic prediction, structural analysis, phylogenetic inference, and population genomic analysis of Drosophila protein-coding genes. Protein sequence adaptation was found to be more relevant to function-related rather than structure-related properties. Interestingly, intermolecular interactions contribute significantly to protein adaptation. Intermolecular interactions, such as physical interactions, may play a role in the coadaptation of fast-adaptive proteins. It was found that strongly differentiated amino acids across geographic regions in protein-coding genes are mostly adaptive, which may contribute to the long-term adaptive evolution. This strongly indicates that a number of adaptive sites tend to be repeatedly mutated and selected throughout evolution in the past, present, and maybe future. These results highlight the important roles of intermolecular interactions and coadaptation in the adaptive evolution of proteins both at the species and population levels.
Singh, K., Kochar, E., Gahlot, P., Bhatt, K. and Prasad, N. G. (2021). Evolution of reproductive traits have no apparent life-history associated cost in populations of Drosophila melanogaster selected for cold shock resistance. BMC Ecol Evol 21(1): 219. PubMed ID: 34872492
Summary:
In insect species like Drosophila melanogaster, evolution of increased resistance or evolution of particular traits under specific environmental conditions can lead to energy trade-offs with other crucial life-history traits. Adaptation to cold stress can, in principle, involve modification of reproductive traits and physiological responses. This study has successfully selected replicate populations of Drosophila melanogaster for increased resistance to cold shock for over 33 generations. In these populations, the ability to recover from cold shock, mate, and lay fertile eggs 24 h post cold shock is under selection. To assess life-history cost, egg viability, mating frequency, longevity, lifetime fecundity, adult mortality, larva to adult development time, larvae to adults survival, and body weight were studied in the cold shock selected populations and their controls under two treatments (a) post cold shock and (b) without cold shock. Twenty-four hours post cold shock, the selected population had significantly higher egg viability and mating frequency compared to control populations indicating that they have higher cold shock resistance. Selected populations had significantly longer pre-adult development time compared to their control populations. Females from the selected populations had higher body weight compared to their control populations. However, no significant difference was found between the selected and control populations in longevity, lifetime fecundity, adult mortality, larvae to adults survival, and male body weight under the cold shock or no cold shock treatments. These findings suggest that cold shock selected populations have evolved higher mating frequency and egg viability. However, there is no apparent life-history associated cost with the evolution of egg viability and reproductive performances under the cold stress condition.
Rice, G. R., David, J. R., Gompel, N., Yassin, A. and Rebeiz, M. (2021). Resolving between novelty and homology in the rapidly evolving phallus of Drosophila. J Exp Zool B Mol Dev Evol. PubMed ID: 34958528
Summary:
The genitalia present some of the most rapidly evolving anatomical structures in the animal kingdom, possessing a variety of parts that can distinguish recently diverged species. In the Drosophila melanogaster group, the phallus is adorned with several processes, pointed outgrowths, that are similar in size and shape between species. However, the complex three-dimensional nature of the phallus can obscure the exact connection points of each process. Previous descriptions based upon adult morphology have primarily assigned phallic processes by their approximate positions in the phallus and have remained largely agnostic regarding their homology relationships. In the absence of clearly identified homology, it can be challenging to model when each structure first evolved. This study employed a comparative developmental analysis of these processes in eight members of the melanogaster species group to precisely identify the tissue from which each process forms. The results indicate that adult phallic processes arise from three pupal primordia in all species. It was found that in some cases the same primordia generate homologous structures whereas in other cases, different primordia produce phenotypically similar but remarkably non-homologous structures. This suggests that the same gene regulatory network may have been redeployed to different primordia to induce phenotypically similar traits. These results highlight how traits diversify and can be redeployed, even at short evolutionary scales.
Mo, W. Z., Li, Z. M., Deng, X. M., Chen, A. L., Ritchie, M. G., Yang, D. J., He, Z. B., Toda, M. J. and Wen, S. Y. (2021). Divergence and correlated evolution of male wing spot and courtship display between Drosophila nepalensis and D. trilutea. Insect Sci. PubMed ID: 34939317
Summary:
Male-specific wing spots are usually associated with wing displays in the courtship behavior of Drosophila and may play important roles in sexual selection. Two closely related species, D. nepalensis and D. trilutea, differ in wing spots and scissoring behavior. This study compared male morphological characters, pigmentation intensity of male wing spots, wing-scissoring behavior, courtship songs and reproductive isolation between two species. F1 fertile females and sterile males result from the cross between females of D. nepalensis and males of D. trilutea. The pigmentation of wing spots is significantly weaker in D. trilutea than in D. nepalensis and the F1 hybrid. Males scissor both wings in front of the female during courtship, with a posture spreading wings more widely, and at a faster frequency in D. nepalensis than in D. trilutea and the F1s. Males of D. trilutea vibrate wings to produce two types (A and B) of pulse songs, whereas D. nepalensis and the F1s sing only type B songs. The incidence of wing vibration and scissoring during courtship suggests that wing vibration is essential but scissoring is a facultative courtship element for successful mating in both species. The association between the darker wing spots with more elaborate scissoring might be the consequence of correlated evolution of these traits in D. nepalensis, however D. trilutea retains wing scissoring during courtship despite having weaker pigmentation of wing spots. The genetic architecture of two traits differs in the F1s, consistent with maternal or sex-linked effects for spots but non-additive effects for scissoring.
Ricchio, J., Uno, F. and Carvalho, A. B. (2021). New Genes in the Drosophila Y Chromosome: Lessons from D. willistoni. Genes (Basel) 12(11). PubMed ID: 34828421
Summary:
Y chromosomes play important roles in sex determination and male fertility. In several groups (e.g., mammals) there is strong evidence that they evolved through gene loss from a common X-Y ancestor, but in Drosophila the acquisition of new genes plays a major role. This conclusion came mostly from studies in two species. This study report the identification of the 22 Y-linked genes in D. willistoni. They all fit the previously observed pattern of autosomal or X-linked testis-specific genes that duplicated to the Y. The ratio of gene gains to gene losses is ~25 in D. willistoni, confirming the prominent role of gene gains in the evolution of Drosophila Y chromosomes. Four large segmental duplications (ranging from 62 kb to 303 kb) from autosomal regions to the Y, containing ~58 genes, were found. All but four of these duplicated genes became pseudogenes in the Y or disappeared. In the GK20609 gene the Y-linked copy remained functional, whereas its original autosomal copy degenerated, demonstrating how autosomal genes are transferred to the Y chromosome. Since the segmental duplication that carried GK20609 contained six other testis-specific genes, it seems that chance plays a significant role in the acquisition of new genes by the Drosophila Y chromosome.
Neal, S., McCulloch, K. J., Napoli, F. R., Daly, C. M., Coleman, J. H. and Koenig, K. M. (2022). Co-option of the limb patterning program in cephalopod eye development. BMC Biol 20(1): 1. PubMed ID: 34983491
Summary:
Across the Metazoa, similar genetic programs are found in the development of analogous, independently evolved, morphological features. The functional significance of this reuse and the underlying mechanisms of co-option remain unclear. Cephalopods have evolved a highly acute visual system with a cup-shaped retina and a novel refractive lens in the anterior, important for a number of sophisticated behaviors including predation, mating, and camouflage. Almost nothing is known about the molecular-genetics of lens development in the cephalopod. This study identified the co-option of the canonical bilaterian limb patterning program during cephalopod lens development, a functionally unrelated structure. This study shows radial expression of transcription factors SP6-9/sp1, Dlx/dll, Pbx/exdv, Meis/hthv, and a Prdl homolog in the squid Doryteuthis pealeii, similar to expression required in Drosophila limb development. This study assessed the role of Wnt signaling in the cephalopod lens, a positive regulator in the developing Drosophila limb, and found the regulatory relationship reversed, with ectopic Wnt signaling leading to lens loss. This regulatory divergence suggests that duplication of SP6-9 in cephalopods may mediate the co-option of the limb patterning program. Thus, it is suggested that this program could perform a more universal developmental function in radial patterning and highlights how canonical genetic programs are repurposed in novel structures.

Monday, April 4th - Behavior

Navas, C. A., Agudelo-Cantero, G. A. and Loeschcke, V. (2022). Thermal boldness: Volunteer exploration of extreme temperatures in fruit flies. J Insect Physiol 136: 104330. PubMed ID: 34848182
Summary:
A dominant perception is that small and motile ectothermic animals must use behavior to avoid exposure to critical or sub-critical temperatures impairing physiological performance. Concomitantly, volunteer exploration of extreme environments by some individuals may promote physiological adjustments and enhance ecological opportunity. This study introduces to the literature a Thermal Decision System (TDS) which is fully modular, thermally stable, versatile, and adaptable to study navigation through thermal landscapes in insects and other small motile animals. A specific setting of the TDS was used to investigate volunteer navigation through critical cold and hot temperatures in Drosophila melanogaster. It was demonstrated that a thermally bold behavior (volunteer crossings through a Critical Temperature Zone, CTZ) characterized a fraction of flies in a sample; such a fraction was higher in an outbred population relative to isofemale lines. As set, the TDS generated a thermal gradient within the cold and hot CTZs, and the exploration of this gradient by flies did not relate simply with a tendency to be thermally bold. Mild fasting affected thermal exploration and boldness in complex manners, but thermal boldness was evident in both fasted and fed flies. Also, thermal boldness was not associated with individual critical temperatures. Finally, some flies showed consistent thermal boldness, as flies that performed an extreme thermal cross were more likely to perform a second cross compared with untested flies. It is hypothesized that a simple 'avoidance principle' is not the only behavioral drive for D. melanogaster facing extreme temperatures over space, and that this pattern may characterize other small motile ectothermic animals with analogous natural history. The physiological correlates, genetic architecture, and interspecific variation of thermal boldness deserve further consideration.
Dollish, H. K., Kaladchibachi, S., Negelspach, D. C. and Fernandez, F. X. (2022). The Drosophila circadian phase response curve to light: Conservation across seasonally relevant photoperiods and anchorage to sunset. Physiol Behav 245: 113691. PubMed ID: 34958825
Summary:
Photic history, including the relative duration of day versus night in a 24-hour cycle, is known to influence subsequent circadian responses to light in mammals. Whether such modulation is present in Drosophila is currently unknown. To date, all photic phase-response curves (PRCs) generated from Drosophila have done so with animals housed under seasonally agnostic equatorial photoperiods with alternating 12-hour segments of light and darkness. However, the genus contains thousands of species, some of which populate high and low-latitude habitats (20-50° north or south of the Equator) where seasonal variations in the light-dark schedule are pronounced. This study addresses this disconnect by constructing the first high-resolution Drosophila seasonal atlas for light-induced circadian phase-resetting. Testing the light responses of over 4,000 Drosophila at 120 timepoints across 5 seasonally-relevant rectangular photoperiods (i.e., LD 8:16, 10:14, 12:12, 14:10, and 16:8; 24 hourly intervals surveyed in each), it was determined that many aspects of the fly circadian PRC waveform are conserved with increasing daylength. Surprisingly though, irrespective of LD schedule, the start of the PRCs always remained anchored to the timing of subjective sunset, creating a differential overlap of the advance zone with the morning hours after subjective sunrise that was maximized under summer photoperiods and minimized under winter photoperiods. These data suggest that there may be differences in flies versus mammals as to how the photoperiod modulates the waveform and amplitude of the circadian PRC to light. On the other hand, they support the possibility that the lights-off transition determines the phase-positioning of photic PRCs across seasons and across species. More work is necessary to test this claim and whether it might factor into the timing of seasonal light responses in humans.
Ravbar, P., Zhang, N. and Simpson, J. H. (2021). Behavioral evidence for nested central pattern generator control of Drosophila grooming. Elife 10. PubMed ID: 34936550
Summary:
Central pattern generators (CPGs) are neurons or neural circuits that produce periodic output without requiring patterned input. More complex behaviors can be assembled from simpler subroutines, and nested CPGs have been proposed to coordinate their repetitive elements, organizing control over different time scales. This study used behavioral experiments to establish that Drosophila grooming may be controlled by nested CPGs. On a short time scale (5-7 Hz, ~ 200 ms/movement), flies clean with periodic leg sweeps and rubs. More surprisingly, transitions between bouts of head sweeping and leg rubbing are also periodic on a longer time scale (0.3-0.6 Hz, ~2 s/bout). This study examined grooming at a range of temperatures to show that the frequencies of both oscillations increase-a hallmark of CPG control-and also that rhythms at the two time scales increase at the same rate, indicating that the nested CPGs may be linked. This relationship holds when sensory drive is held constant using optogenetic activation, but oscillations can decouple in spontaneously grooming flies, showing that alternative control modes are possible. Loss of sensory feedback does not disrupt periodicity but slow down the longer time scale alternation. Nested CPGs simplify the generation of complex but repetitive behaviors, and identifying them in Drosophila grooming presents an opportunity to map the neural circuits that constitute them.
Sinclair, C. S., Lisa, S. F. and Pischedda, A. (2021). Does sexual experience affect the strength of male mate choice for high-quality females in Drosophila melanogaster?. Ecol Evol 11(23): 16981-16992. PubMed ID: 34938486
Summary:
Although females are traditionally thought of as the choosy sex, there is increasing evidence in many species that males will preferentially court or mate with certain females over others when given a choice. In the fruit fly, Drosophila melanogaster, males discriminate between potential mating partners based on a number of female traits, including species, mating history, age, and condition. Interestingly, many of these male preferences are affected by the male's previous sexual experiences, such that males increase courtship toward types of females that they have previously mated with and decrease courtship toward types of females that have previously rejected them. D. melanogaster males also show courtship and mating preferences for larger females over smaller females, likely because larger females have higher fecundity. It is unknown, however, whether this preference shows behavioral plasticity based on the male's sexual history as is seen for other male preferences. This study manipulated the sexual experience of D. melanogaster males and tested whether this manipulation has any effect on the strength of male mate choice for large females. Sexually inexperienced males were found to have a robust courtship preference for large females that is unaffected by previous experience mating with, or being rejected by, females of differing sizes. Given that female body size is one of the most common targets of male mate choice across insect species, these experiments with D. melanogaster may provide insight into how these preferences develop and evolve.
Gordon, J. and Masek, P. (2021). Excessive energy expenditure due to acute physical restraint disrupts Drosophila motivational feeding response. Sci Rep 11(1): 24208. PubMed ID: 34921197
Summary:
To study the behavior of Drosophila, it is often necessary to restrain and mount individual flies. This requires removal from food, additional handling, anesthesia, and physical restraint. A strong positive correlation was found between the length of time flies are mounted and their subsequent reflexive feeding response, where one hour of mounting is the approximate motivational equivalent to ten hours of fasting. In an attempt to explain this correlation, anesthesia side-effects, handling, additional fasting, and desiccation were ruled out. Respirometric and metabolic techniques coupled with behavioral video scoring were used to assess energy expenditure in mounted and free flies. A specific behavior was isolated capable of exerting large amounts of energy in mounted flies, and it was identified as an attempt to escape from restraint. A model is presented where physical restraint leads to elevated activity and subsequent faster nutrient storage depletion among mounted flies. This ultimately further accelerates starvation and thus increases reflexive feeding response. In addition, it was shown that the consequences of the physical restraint profoundly alter aerobic activity, energy depletion, taste, and feeding behavior, and suggest that careful consideration is given to the time-sensitive nature of these highly significant effects when conducting behavioral, physiological or imaging experiments that require immobilization.
Meda, N., Menti, G. M., Megighian, A. and Zordan, M. A. (2021). A heuristic underlies the search for relief in Drosophila melanogaster. Ann N Y Acad Sci. PubMed ID: 34928521
Summary:
Humans rely on multiple types of sensory information to make decisions, and strategies that shorten decision-making time by taking into account fewer but essential elements of information are preferred to strategies that require complex analyses. Such shortcuts to decision making are known as heuristics. The identification of heuristic principles in species phylogenetically distant to humans would shed light on the evolutionary origin of speed-accuracy trade-offs and offer the possibility for investigating the brain representations of such trade-offs, urgency and uncertainty. By performing experiments on spatial learning in the invertebrate Drosophila melanogaster, this study showed that the fly's search strategies conform to a spatial heuristic-the nearest neighbor rule-to avoid bitter taste (a negative stimulation). That is, Drosophila visits a salient location closest to its current position to stop the negative stimulation; only if this strategy proves unsuccessful does the fly use other learned associations to avoid bitter taste. Characterizing a heuristic in D. melanogaster supports the view that invertebrates can, when making choices, operate on economic principles, as well as the conclusion that heuristic decision making dates to at least 600 million years ago.

Friday, April 1st - Immune response

Ozakman, Y., Raval, D. and Eleftherianos, I. (2021). Activin and BMP Signaling Activity Affects Different Aspects of Host Anti-Nematode Immunity in Drosophila melanogaster. Front Immunol 12: 795331. PubMed ID: 35003118
Summary:
Functional characterization of the interaction between TGF-β signaling activity and the mechanisms activated by the D. melanogaster immune response against parasitic nematode infection remains unexplored. This study investigated the participation of the TGF-β signaling branches, activin and bone morphogenetic protein (BMP), to host immune function against axenic or symbiotic Heterorhabditis bacteriophora nematodes (parasites lacking or containing their mutualistic bacteria, respectively). Using D. melanogaster larvae carrying mutations in the genes coding for the TGF-β extracellular ligands Daw and Dpp, this study analyzed the changes in survival ability, cellular immune response, and phenoloxidase (PO) activity during nematode infection. Infection with axenic H. bacteriophora decreases the mortality rate of dpp mutants, but not daw mutants. Following axenic or symbiotic H. bacteriophora infection, both daw and dpp mutants contain only plasmatocytes. Higher levels of Dual oxidase gene expression was detected in dpp mutants upon infection with axenic nematodes and Diptericin and Cecropin gene expression in daw mutants upon infection with symbiotic nematodes compared to controls. Finally, following symbiotic H. bacteriophora infection, daw mutants have higher PO activity relative to controls. Together, these findings reveal that while D. melanogaster Dpp/BMP signaling activity modulates the DUOX/ROS response to axenic H. bacteriophora infection, Daw/activin signaling activity modulates the antimicrobial peptide and melanization responses to axenic H. bacteriophora infection. Results from this study expand the current understanding of the molecular and mechanistic interplay between nematode parasites and the host immune system, and the involvement of TGF-β signaling branches in this process. Such findings will provide valuable insight on the evolution of the immune role of TGF-β signaling, which could lead to the development of novel strategies for the effective management of human parasitic nematodes.
Nunes, C., Koyama, T. and Sucena, E. (2021). Co-option of immune effectors by the hormonal signalling system triggering metamorphosis in Drosophila melanogaster. PLoS Genet 17(11): e1009916. PubMed ID: 34843450
Summary:
Insect metamorphosis is triggered by the production, secretion and degradation of 20-hydroxyecdysone (ecdysone). In addition to its role in developmental regulation, increasing evidence suggests that ecdysone is involved in innate immunity processes, such as phagocytosis and the induction of antimicrobial peptide (AMP) production. AMP regulation includes systemic responses as well as local responses at surface epithelia that contact with the external environment. At pupariation, Drosophila melanogaster increases dramatically the expression of three AMP genes, drosomycin (drs), drosomycin-like 2 (drsl2) and drosomycin-like 5 (drsl5). The systemic action of drs at pupariation is dependent on ecdysone signalling in the fat body and operates via the ecdysone downstream target, Broad. In parallel, ecdysone also regulates local responses, specifically through the activation of drsl2 expression in the gut. Finally, the relevance of this ecdysone dependent AMP expression for the control of bacterial load was confirmed by showing that flies lacking drs expression in the fat body have higher bacterial persistence over metamorphosis. In contrast, local responses may be redundant with the systemic effect of drs since reduction of ecdysone signalling or of drsl2 expression has no measurable negative effect on bacterial load control in the pupa. Together, these data emphasize the importance of the association between ecdysone signalling and immunity using in vivo studies and establish a new role for ecdysone at pupariation, which impacts developmental success by regulating the immune system in a stage-dependent manner. It is speculated that this co-option of immune effectors by the hormonal system may constitute an anticipatory mechanism to control bacterial numbers in the pupa, at the core of metamorphosis evolution.
Pennemann, F. L., Mussabekova, A., Urban, C., Stukalov, A., Andersen, L. L., Grass, V., Lavacca, T. M., Holze, C., Oubraham, L., Benamrouche, Y., Girardi, E., Boulos, R. E., Hartmann, R., Superti-Furga, G., Habjan, M., Imler, J. L., Meignin, C. and Pichlmair, A. (2021). Cross-species analysis of viral nucleic acid interacting proteins identifies TAOKs as innate immune regulators. Nat Commun 12(1): 7009. PubMed ID: 34853303
Summary:
The cell intrinsic antiviral response of multicellular organisms developed over millions of years and critically relies on the ability to sense and eliminate viral nucleic acids. This study used an affinity proteomics approach in evolutionary distant species (human, mouse and fly) to identify proteins that are conserved in their ability to associate with diverse viral nucleic acids. This approach shows a core of orthologous proteins targeting viral genetic material and species-specific interactions. Functional characterization of the influence of 181 candidates on replication of 6 distinct viruses in human cells and flies identifies 128 nucleic acid binding proteins with an impact on virus growth. The family of TAO kinases (TAOK1, -2 and -3) was identified as dsRNA-interacting antiviral proteins and show their requirement for type-I interferon induction. Depletion of TAO kinases in mammals or flies leads to an impaired response to virus infection characterized by a reduced induction of interferon stimulated genes in mammals and impaired expression of srg1 and diedel in flies. Overall, this study shows a larger set of proteins able to mediate the interaction between viral genetic material and host factors than anticipated so far, attesting to the ancestral roots of innate immunity and to the lineage-specific pressures exerted by viruses.
Cinege, G., Magyar, L. B., Kovacs, A. L., Lerner, Z., Juhasz, G., Lukacsovich, D., Winterer, J., Lukacsovich, T., Hegedus, Z., Kurucz, E., Hultmark, D., Foldy, C. and Ando, I. (2021). Broad Ultrastructural and Transcriptomic Changes Underlie the Multinucleated Giant Hemocyte Mediated Innate Immune Response against Parasitoids. J Innate Immun: 1-20. PubMed ID: 34864742
Summary:
Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously, this study showed that circulating MGHs have high motility and the interaction with the parasitoid rapidly triggers encapsulation. However, structural and molecular mechanisms behind these processes remained elusive. This study used detailed ultrastructural analysis and live cell imaging of MGHs to study encapsulation in Drosophila ananassae after parasitoid wasp infection. Dynamic structural changes were found, mainly driven by the formation of diverse vesicular systems and newly developed complex intracytoplasmic membrane structures, and abundant generation of giant cell exosomes in MGHs. In addition, RNA sequencing was used to study the transcriptomic profile of MGHs and activated plasmatocytes 72 h after infection, as well as the uninduced blood cells. This revealed that differentiation of MGHs was accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts related to vesicular function, cytoskeletal organization, and adhesion were enriched in MGHs. In addition, several orphan genes encoding for hemolysin-like proteins, pore-forming toxins of prokaryotic origin, were expressed at high level, which may be important for parasitoid elimination. These results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response.
Rodrigues, M. A., Merckelbach, A., Durmaz, E., Kerdaffrec, E. and Flatt, T. (2021). Transcriptomic evidence for a trade-off between germline proliferation and immunity in Drosophila. Evol Lett 5(6): 644-656. PubMed ID: 34917403
Summary:
Life-history theory posits that investment into reproduction might occur at the expense of investment into somatic maintenance, including immune function. If so, reduced or curtailed reproductive effort might be expected to increase immunity. In support of this notion, work in Caenorhabditis elegans has shown that worms lacking a germline exhibit improved immunity, but whether the antagonistic relation between germline proliferation and immunity also holds for other organisms is less well understood. This study reports that transgenic ablation of germ cells in late development or early adulthood in Drosophila melanogaster causes elevated baseline expression and increased induction of Toll and Imd immune genes upon bacterial infection, as compared to fertile flies with an intact germline. This study also identified immune genes whose expression after infection differs between fertile and germline-less flies in a manner that is conditional on their mating status. It is concluded that germline activity strongly impedes the expression and inducibility of immune genes and that this physiological trade-off might be evolutionarily conserved.
Silva, J. M. F., Nagata, T., Melo, F. L. and Elena, S. F. (2021). Heterogeneity in the Response of Different Subtypes of Drosophila melanogaster Midgut Cells to Viral Infections. Viruses 13(11). PubMed ID: 34835089
Summary:
Single-cell RNA sequencing (scRNA-seq) offers the possibility to monitor both host and pathogens transcriptomes at the cellular level. Public scRNA-seq datasets from Drosophila melanogaster midgut cells were used in this study to compare the differences in replication strategy and cellular responses between two fly picorna-like viruses, Thika virus (TV) and D. melanogaster Nora virus (DMelNV). TV exhibited lower levels of viral RNA accumulation but infected a higher number of cells compared to DMelNV. In both cases, viral RNA accumulation varied according to cell subtype. The cellular heat shock response to TV and DMelNV infection was cell-subtype- and virus-specific. Disruption of bottleneck genes at later stages of infection in the systemic response, as well as of translation-related genes in the cellular response to DMelNV in two cell subtypes, may affect the virus replication.

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