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Current papers in developmental biology and gene function
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| ARCHIVE | Friday, June 28th, 2024 - Larval and Adult Neural Development, Structure, and Function |
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What's hot today
October 2024 September 2024 August 2024 July 2024 May 2024 April 2024 March 2024 February 2024 January 2024 December 2023 November 2023 October 2023 September 2023 August 2023 July 2023 June 2023 May 2023 April 2023 March 2023 February 2023 January 2023 December 2022 November 2022 October 2022 September 2022 August 2022 December 2021 December 2020 December 2019 December 2018 | Rathore, S., Stahl, A., Benoit, J. B., Buschbeck, E. K. (2023). Exploring the molecular makeup of support cells in insect camera eyes. BMC Genomics, 24(1):702 PubMed ID: 37993800
Summary: Animals typically have either compound eyes, or camera-type eyes, both of which have evolved repeatedly in the animal kingdom. Both eye types include two important kinds of cells: photoreceptor cells, which can be excited by light, and non-neuronal support cells (SupCs), which provide essential support to photoreceptors. At the molecular level deeply conserved genes that relate to the differentiation of photoreceptor cells have fueled a discussion on whether or not a shared evolutionary origin might be considered for this cell type. In contrast, only a handful of studies, primarily on the compound eyes of Drosophila melanogaster, have demonstrated molecular similarities in SupCs. D. melanogaster SupCs (Semper cells and primary pigment cells) are specialized eye glia that share several molecular similarities with certain vertebrate eye glia, including Muller glia. This led the authors to ask if there could be conserved molecular signatures of SupCs, even in functionally different eyes such as the image-forming larval camera eyes of the sunburst diving beetle Thermonectus marmoratus. To investigate this possibility, an in-depth comparative whole-tissue transcriptomics approach was used. Specifically, the larval principal camera eyes were dissected into SupC- and retina-containing regions, and the respective transcriptomes were generated. This analysis revealed several common features of SupCs including enrichment of genes that are important for glial function (e.g. gap junction proteins such as innexin 3), glycogen production (glycogenin), and energy metabolism (glutamine synthetase 1 and 2). To evaluate similarities, these transcriptomes with those of fly (Semper cells) and vertebrate (Muller glia) eye glia as well as respective retinas. T. marmoratus SupCs were found to have distinct genetic overlap with both fly and vertebrate eye glia. These results suggest that T. marmoratus SupCs are a form of glia, and like photoreceptors, may be deeply conserved. | Fuse, N., Hashiba, H., Ishibashi, K., Suzuki, T., Nguyen, Q. D., Fujii, K., Ikeda-Ohtsubo, W., Kitazawa, H., Tanimoto, H., Kurata, S. (2023). Neural control of redox response and microbiota-triggered inflammation in Drosophila gut. Frontiers in immunology. 14:1268611 PubMed ID: 37965334
Summary: The neural system plays a critical role in controlling gut immunity, and the gut microbiota contributes to this process. However, the roles and mechanisms of gut-brain-microbiota interactions remain unclear. To address this issue, Drosophila was employed as a model organism. Previous work has shown that NP3253 neurons, which are connected to the brain and gut, are essential for resistance to oral bacterial infections. This study aimed to investigate the role of NP3253 neurons, enteric neurons innervating the anterior midgut, in the regulation of gut immunity. RNA-seq analysis of the adult Drosophila gut was performed after genetically inactivating the NP3253 neurons. Flies were reared under oral bacterial infection and normal feeding conditions. In addition, samples were prepared under germ-free conditions to evaluate the role of the microbiota in gut gene expression. The genes regulated by NP3253 neurons were knocked down, and their susceptibility to oral bacterial infections was examined. Immune-related gene expression was found to be upregulated in NP3253 neuron-inactivated flies compared to the control. However, this upregulation was abolished in axenic flies, suggesting that the immune response was abnormally activated by the microbiota in NP3253 neuron-inactivated flies. In addition, redox-related gene expression was downregulated in NP3253 neuron-inactivated flies, and this downregulation was also observed in axenic flies. Certain redox-related genes were required for resistance to oral bacterial infections, suggesting that NP3253 neurons regulate the redox responses for gut immunity in a microbiota-independent manner. These results show that NP3253 neurons regulate the appropriate gene expression patterns in the gut and contribute to maintain homeostasis during oral infections. |
| Barth-Maron, A., D'Alessandro, I., Wilson, R. I. (2023). Interactions between specialized gain control mechanisms in olfactory processing. Curr Biol, 33(23):5109-5120.e5107 PubMed ID: 37967554
Summary: Gain control is a process that adjusts a system's sensitivity when input levels change. Neural systems contain multiple mechanisms of gain control, but it is not understood why so many mechanisms are needed or how they interact. This study investigated these questions in the Drosophila antennal lobe, where several types of inhibitory interneurons were identified with specialized gain control functions. Some interneurons are nonspiking, with compartmentalized calcium signals, and they specialize in intra-glomerular gain control. Conversely, other interneurons were found to be recruited by strong and widespread network input; they specialize in global presynaptic gain control. Using computational modeling and optogenetic perturbations, this study shows how these mechanisms can work together to improve stimulus discrimination while also minimizing temporal distortions in network activity. These results demonstrate how the robustness of neural network function can be increased by interactions among diverse and specialized mechanisms of gain control. | Gazso-Gerhat, G., Gombos, R., Toth, K., Kaltenecker, P., Szikora, S., Bíro, J., Csapo, E., Asztalos, Z., Mihaly, J. (2023). FRL and DAAM are required for lateral adhesion of interommatidial cells and patterning of the retinal floor. Development, 150(22) PubMed ID: 37997920
Summary: Optical insulation of the unit eyes (ommatidia) is an important prerequisite of precise sight with compound eyes. Separation of the ommatidia is ensured by pigment cells that organize into a hexagonal lattice in the Drosophila eye, forming thin walls between the facets. Cell adhesion, mediated by apically and latero-basally located junctional complexes, is crucial for stable attachment of these cells to each other and the basal lamina. Whereas former studies have focused on the formation and remodelling of the cellular connections at the apical region, this study reports a specific alteration of the lateral adhesion of the lattice cells, leaving the apical junctions largely unaffected. It was found that DAAM and FRL, two formin-type cytoskeleton regulatory proteins, play redundant roles in lateral adhesion of the interommatidial cells and patterning of the retinal floor. It was shown that formin-dependent cortical actin assembly is crucial for latero-basal sealing of the ommatidial lattice. It is expected that the investigation of these previously unreported eye phenotypes will pave the way toward a better understanding of the three-dimensional aspects of compound eye development. |
| Dweck, H. K. M., Carlson, J. R. (2023). Diverse mechanisms of taste coding in Drosophila. Sci Adv, 9(46):eadj7032 PubMed ID: 37976361
Summary: Taste systems encode chemical cues that drive vital behaviors. This study has elucidated noncanonical features of taste coding using an unconventional kind of electrophysiological analysis. Taste neurons of Drosophila are much more sensitive than previously thought. They have a low spontaneous firing frequency that depends on taste receptors. Taste neurons have a dual function as olfactory neurons: They are activated by most tested odorants, including N,N-diethyl-meta-toluamide (DEET), at a distance. DEET can also inhibit certain taste neurons, revealing that there are two modes of taste response: activation and inhibition. Electrophysiological OFF responses were characterized and it was found that the tastants that elicit them are related in structure. OFF responses link tastant identity to behavior: the magnitude of the OFF response elicited by a tastant correlated with the egg laying behavior it elicited. In summary, the sensitivity and coding capacity of the taste system are much greater than previously known. | Togashi, H., Davis, S. R., Sato, M. (2024). From soap bubbles to multicellular organisms: Unraveling the role of cell adhesion and physical constraints in tile pattern formation and tissue morphogenesis. Dev Biol, 506:1-6 PubMed ID: 37995916
Summary: Tile patterns, in which numerous cells are arranged in a regular pattern, are found in a variety of multicellular organisms and play important functional roles. Such regular arrangements of cells are regulated by various cell adhesion molecules. On the other hand, cell shape is also known to be regulated by physical constraints similar to those of soap bubbles. In particular, circumference minimization plays an important role, and cell adhesion negatively affects this process, thereby regulating tissue morphogenesis based on physical properties. This study focused on the Drosophila compound eye and the mouse auditory epithelium, and summarizes the mechanisms of tile pattern formation by cell adhesion molecules such as cadherins, Irre Cell Recognition Modules (IRMs), and nectins. Phenomena that cannot be explained by physical stability based on cortical tension alone have been reported in the tile pattern formation in the compound eye, suggesting that previously unexplored forces such as cellular concentric expansion force may play an important role. |
Thursday, June 27th - Cytoskeleton and Junctions |
| Ly, M., Schimmer, C., Hawkins, R., K, E. R., Fernandez-Gonzalez, R. (2024). Integrin-based adhesions promote cell-cell junction and cytoskeletal remodelling to drive embryonic wound healing. J Cell Sci, 137(5) PubMed ID: 37970744
Summary: Embryos repair wounds rapidly, with no inflammation or scarring. Embryonic wound healing is driven by the collective movement of the cells around the lesion. The cells adjacent to the wound polarize the cytoskeletal protein actin and the molecular motor non-muscle myosin II, which accumulate at the wound edge forming a supracellular cable around the wound. Adherens junction proteins, including E-cadherin, are internalized from the wound edge and localize to former tricellular junctions at the wound margin, in a process necessary for cytoskeletal polarity. The cells adjacent to wounds in the Drosophila embryonic epidermis polarized Talin, a core component of cell-extracellular matrix (ECM) adhesions, which preferentially accumulated at the wound edge. Integrin knockdown and inhibition of integrin binding delayed wound closure and reduced actin polarization and dynamics around the wound. Additionally, disrupting integrins caused a defect in E-cadherin reinforcement at tricellular junctions along the wound edge, suggesting crosstalk between integrin-based and cadherin-based adhesions. The results show that cell-ECM adhesion contributes to embryonic wound repair and reveal an interplay between cell-cell and cell-ECM adhesion in the collective cell movements that drive rapid wound healing. | Xu, Y., Wang, B., Bush, I., Saunders, H. A., Wildonger, J., Han, C. (2023). Light-induced trapping of endogenous proteins reveals spatiotemporal roles of microtubule and kinesin-1 in dendrite patterning of Drosophila sensory neurons. bioRxiv, PubMed ID: 37873262
Summary: Animal development involves numerous molecular events, whose spatiotemporal properties largely determine the biological outcomes. Conventional methods for studying gene function lack the necessary spatiotemporal resolution for precise dissection of developmental mechanisms. Optogenetic approaches are powerful alternatives, but most existing tools rely on exogenous designer proteins that produce narrow outputs and cannot be applied to diverse or endogenous proteins. To address this limitation, OptoTrap, a light-inducible protein trapping system was developed that allows manipulation of endogenous proteins tagged with GFP or split GFP. This system turns on fast and is reversible in minutes or hours. This study generated OptoTrap variants optimized for neurons and epithelial cells and demonstrate effective trapping of endogenous proteins of diverse sizes, subcellular locations, and functions. Furthermore, OptoTrap allowed instant disruption of microtubules and inhibits the kinesin-1 motor in specific dendritic branches of Drosophila sensory neurons. Using OptoTrap, direct evidence was obtained that microtubules support the growth of highly dynamic dendrites. Similarly, targeted manipulation of Kinesin heavy chain revealed differential spatiotemporal requirements of kinesin-1 in the patterning of low- and high-order dendritic branches, suggesting that different cargos are needed for the growth of these branches. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds great promise for studying developmental mechanisms in a wide range of cell types and developmental stages. |
| Christophers, B., Leahy, S. N., Soffar, D. B., von Saucken, V. E., Broadie, K., Baylies, M. K. (2023). Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission. bioRxiv, PubMed ID: 38045306
Summary: Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Previous work has shown Drosophila cofilin (DmCFL) knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. It was hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, an RNA sequencing analysis was conducted which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. DmCFL is enriched in the muscle postsynaptic compartment and DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, no significant changes were founs in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand our understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology. | Sato, Y., Yoshimura, K., Matsuda, K., Haraguchi, T., Marumo, A., Yamagishi, M., Sato, S., Ito, K., Yajima, J. (2023). Membrane-bound myosin IC drives the chiral rotation of the gliding actin filament around its longitudinal axis. Sci Rep, 13(1):19908 PubMed ID: 37963943
Summary: Myosin IC, a single-headed member of the myosin I family, specifically interacts with anionic phosphatidylinositol 4,5-bisphosphate (PI[4,5]P(2)) in the cell membrane via the pleckstrin homology domain located in the myosin IC tail. Myosin IC is widely expressed and physically links the cell membrane to the actin cytoskeleton; it plays various roles in membrane-associated physiological processes, including establishing cellular chirality, lipid transportation, and mechanosensing. This study evaluated the motility of full-length myosin IC of Drosophila melanogaster via the three-dimensional tracking of quantum dots bound to actin filaments that glided over a membrane-bound myosin IC-coated surface. The results revealed that myosin IC drove a left-handed rotational motion in the gliding actin filament around its longitudinal axis, indicating that myosin IC generated a torque perpendicular to the gliding direction of the actin filament. The quantification of the rotational motion of actin filaments on fluid membranes containing different PI(4,5)P(2) concentrations revealed that the rotational pitch was longer at lower PI(4,5)P(2) concentrations. These results suggest that the torque generated by membrane-bound myosin IC molecules can be modulated based on the phospholipid composition of the cell membrane. |
| Kroeger, B., Manning, S. A., Fonseka, Y., Oorschot, V., Crawford, S. A., Ramm, G., Harvey, K. F. (2023). Basal spot junctions of Drosophila epithelial tissues respond to morphogenetic forces and regulate Hippo signaling.. Dev Cell, PubMed ID: 38134928
Summary: Organ size is controlled by numerous factors including mechanical forces, which are mediated in part by the Hippo pathway. In growing Drosophila epithelial tissues, cytoskeletal tension influences Hippo signaling by modulating the localization of key pathway proteins to different apical domains. This study discovered a Hippo signaling hub at basal spot junctions, which form at the basal-most point of the lateral membranes and resemble adherens junctions in protein composition. Basal spot junctions recruit the central kinase Warts via Ajuba and E-cadherin, which prevent Warts activation by segregating it from upstream Hippo pathway proteins. Basal spot junctions are prominent when tissues undergo morphogenesis and are highly sensitive to fluctuations in cytoskeletal tension. They are distinct from focal adhesions, but the latter profoundly influences basal spot junction abundance by modulating the basal-medial actomyosin network and tension experienced by spot junctions. Thus, basal spot junctions couple morphogenetic forces to Hippo pathway activity and organ growth. | Ikawa, K., Hiro, S., Kondo, S., Ohsawa, S., Sugimura, K. (2023). Coronin-1 promotes directional cell rearrangement in Drosophila wing epithelium. Cell structure and function 48(2):251-257 PubMed ID: 38030242
Summary: Directional cell rearrangement is a critical process underlying correct tissue deformation during morphogenesis. Although the involvement of F-actin regulation in cell rearrangement has been established, the role and regulation of actin binding proteins (ABPs) in this process are not well understood. This study investigated the function of Coronin-1, a WD-repeat actin-binding protein, in controlling directional cell rearrangement in the Drosophila pupal wing. Transgenic flies expressing Coronin-1-EGFP were generated using CRISPR-Cas9. Coronin-1 was obserced to localize at the reconnecting junction during cell rearrangement, which is dependent on actin interacting protein 1 (AIP1) and cofilin, actin disassemblers and known regulators of wing cell rearrangement. Loss of Coronin-1 function reduces cell rearrangement directionality and hexagonal cell fraction. These results suggest that Coronin-1 promotes directional cell rearrangement via its interaction with AIP1 and cofilin, highlighting the role of ABPs in the complex process of morphogenesis. |
Wednesday, June 26th - Disease Models |
| Kang, J., Zhang, C., Wang, Y., Peng, J., Berger, B., Perrimon, N., Shen, J. (2024). Lipophorin receptors genetically modulate neurodegeneration caused by reduction of Psn expression in the aging Drosophila brain. Genetics, 226(1) PubMed ID: 37996068
Summary: Mutations in the Presenilin (PSEN) genes are the most common cause of early-onset familial Alzheimer's disease (FAD). Studies in cell culture, in vitro biochemical systems, and knockin mice showed that PSEN mutations are loss-of-function mutations, impairing γ-secretase activity. Mouse genetic analysis highlighted the importance of Presenilin (PS) in learning and memory, synaptic plasticity and neurotransmitter release, and neuronal survival, and Drosophila studies further demonstrated an evolutionarily conserved role of PS in neuronal survival during aging. However, molecular pathways that interact with PS in neuronal survival remain unclear. To identify genetic modifiers that modulate PS-dependent neuronal survival, this study developed a new Drosophila Psn model that exhibits age-dependent neurodegeneration and increases of apoptosis. Following a bioinformatic analysis, top ranked candidate genes were tested by selective knockdown (KD) of each gene in neurons using two independent RNAi lines in Psn KD models. Interestingly, 4 of the 9 genes enhancing neurodegeneration in Psn KD flies are involved in lipid transport and metabolism. Specifically, neuron-specific KD of lipophorin receptors, lpr1 and lpr2, dramatically worsens neurodegeneration in Psn KD flies, and overexpression of lpr1 or lpr2 does not alleviate Psn KD-induced neurodegeneration. Furthermore, lpr1 or lpr2 KD alone also leads to neurodegeneration, increased apoptosis, climbing defects, and shortened lifespan. Lastly, heterozygotic deletions of lpr1 and lpr2 or homozygotic deletions of lpr1 or lpr2 similarly lead to age-dependent neurodegeneration and further exacerbate neurodegeneration in Psn KD flies. These findings show that LpRs modulate Psn-dependent neuronal survival and are critically important for neuronal integrity in the aging brain. | Li, L., Wei, Z., Tang, Y., Jin, M., Yao, H., Li, X., Li, Q., Tan, J., Xiao, B. (2023). Icaritin greatly attenuates β-amyloid-induced toxicity in vivo. CNS neuroscience & therapeutics. PubMed ID: 37990437
Summary: The accumulation and deposition of β-amyloid (Aβ; see Drosophila Appl) has always been considered a major pathological feature of Alzheimer's disease (AD). The latest and mainstream amyloid cascade hypothesis indicates that all the main pathological changes in AD are attributed to the accumulation of soluble Aβ. However, the exploration of therapeutic drugs for Aβ toxicity has progressed slowly. This study aims to investigate the protective effects of Icaritin on the Aβ-induced Drosophila AD model and its possible mechanism. To identify the effects of Icaritin on AD, an excellent Drosophila AD model named Aβ(arc) (arctic mutant Aβ(42)) Drosophila. Climbing ability, flight ability, and longevity were used to evaluate the effects of Icaritin on AD phenotypes. Aβarc was determined by immunostaining and ELISA. To identify the effects of Icaritin on oxidative stress, the detection of ROS, hydrogen peroxide, MDA, SOD, catalase, GST, and Caspase-3 was performed. To identify the effects of Icaritin on energy metabolism, the detection of ATP and lactate was performed. Transcriptome analysis and qRT-PCR verifications were used to detect the genes directly involved in oxidative stress and energy metabolism. Mitochondrial structure and function were detected by an electron microscopy assay, a mitochondrial membrane potential assay, and a mitochondrial respiration assay. It was discovered that Icaritin almost completely rescues the climbing ability, flight ability, and longevity of Aβarc Drosophila. Aβarc was dramatically reduced by Icaritin treatment. It was also found that Icaritin significantly reduces oxidative stress and greatly improves impaired energy metabolism. Importantly, transcriptome analysis and qRT-PCR verifications showed that many key genes, directly involved in oxidative stress and energy metabolism, are restored by Icaritin. Next, it was found that Icaritin perfectly restores the integrity of mitochondrial structure and function damaged by Aβarc toxicity. This study suggested that Icaritin is a potential drug to deal with the toxicity of Aβarc, at least partially realized by restoring the mitochondria/oxidative stress/energy metabolism axis, and holds potential for translation to human AD. |
| Thorpe, H. J., Owings, K. G., Aziz, M. C., Haller, M., Coelho, E., Chow, C. Y. (2023). Drosophila models of PIGA-CDG mirror patient phenotypes. bioRxiv, PubMed ID: 37961693
Summary: Mutations in the phosphatidylinositol glycan biosynthesis class A (PIGA) gene cause a rare, X-linked recessive congenital disorder of glycosylation (CDG). PIGA-CDG is characterized by seizures, intellectual and developmental delay, and congenital malformations. The PIGA gene encodes an enzyme involved in the first step of GPI anchor biosynthesis. There are over 100 GPI anchored proteins that attach to the cell surface and are involved in cell signaling, immunity, and adhesion. Little is known about the pathophysiology of PIGA-CDG. This study describes the first Drosophila model of PIGA-CDG and demonstrate that loss of PIG-A function in Drosophila accurately models the human disease. As expected, complete loss of PIG-A function is larval lethal. Heterozygous null animals appear healthy, but when challenged, have a seizure phenotype similar to what is observed in patients. To identify the cell-type specific contributions to disease, neuron- and glia-specific knockdown of PIG-A were generated. Neuron-specific knockdown resulted in reduced lifespan and a number of neurological phenotypes, but no seizure phenotype. Glia-knockdown also reduced lifespan and, notably, resulted in a very strong seizure phenotype. RNAseq analyses demonstrated that there are fundamentally different molecular processes that are disrupted when PIG-A function is eliminated in different cell types. In particular, loss of PIG-A in neurons resulted in upregulation of glycolysis, but loss of PIG-A in glia resulted in upregulation of protein translation machinery. This study demonstrates that Drosophila is a good model of PIGA-CDG and provide new data resources for future study of PIGA-CDG and other GPI anchor disorders. | Khan, M. R., Yin, X., Kang, S. U., Mitra, J., Wang, H., Ryu, T., Brahmachari, S., Karuppagounder, S. S., Kimura, Y., Jhaldiyal, A., Kim, H. H., Gu, H., Chen, R., Redding-Ochoa, J., Troncoso, J., Na, C. H., Ha, T., Dawson, V. L., Dawson, T. M. (2023). Enhanced mTORC1 signaling and protein synthesis in pathologic α-synuclein cellular and animal models of Parkinson's disease. Science translational medicine, 15(724):eadd0499 PubMed ID: 38019930
Summary: Pathologic α-synuclein plays an important role in the pathogenesis of &alpha-synucleinopathies such as Parkinson's disease (PD). Disruption of proteostasis is thought to be central to pathologic α-synuclein toxicity; however, the molecular mechanism of this deregulation is poorly understood. Complementary proteomic approaches in cellular and animal models of PD were used to identify and characterize the pathologic α-synuclein interactome. This study reports that the highest biological processes that interacted with pathologic α-synuclein in mice included RNA processing and translation initiation. Regulation of catabolic processes that include autophagy were also identified. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein 2 (TSC2) and to trigger the activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which augmented mRNA translation and protein synthesis, leading to neurodegeneration. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescued the dopamine neuron loss, behavioral deficits, and aberrant biochemical signaling in the α-synuclein preformed fibril mouse model and Drosophila transgenic models of pathologic α-synuclein-induced degeneration. Pathologic α-synuclein furthermore led to a destabilization of the TSC1-TSC2 complex, which plays an important role in mTORC1 activity. Constitutive overexpression of TSC2 rescued motor deficits and neuropathology in α-synuclein flies. Biochemical examination of PD postmortem brain tissues also suggested deregulated mTORC1 signaling. These findings establish a connection between mRNA translation deregulation and mTORC1 pathway activation that is induced by pathologic α-synuclein in cellular and animal models of PD. |
| Na, D., Lim, D. H., Hong, J. S., Lee, H. M., Cho, D., Yu, M. S., Shaker, B., Ren, J., Lee, B., Song, J. G., Oh, Y., Lee, K., Oh, K. S., Lee, M. Y., Choi, M. S., Choi, H. S., Kim, Y. H., Bui, J. M., Lee, K., Kim, H. W., Lee, Y. S., Gsponer, J. (2023). A multi-layered network model identifies Akt1 as a common modulator of neurodegeneration. Molecular systems biology 19(12):e11801 PubMed ID: 37984409
Summary: The accumulation of misfolded and aggregated proteins is a hallmark of neurodegenerative proteinopathies. Although multiple genetic loci have been associated with specific neurodegenerative diseases (NDs), molecular mechanisms that may have a broader relevance for most or all proteinopathies remain poorly resolved. This study developed a multi-layered network expansion (MLnet) model to predict protein modifiers that are common to a group of diseases and, therefore, may have broader pathophysiological relevance for that group. When applied to the four NDs Alzheimer's disease (AD), Huntington's disease, and spinocerebellar ataxia types 1 and 3, multiple members of the insulin pathway, including PDK1, Akt1, InR, and sgg (GSK-3β), were predicted as common modifiers. These modifiers were validated with the help of four Drosophila ND models. Further evaluation of Akt1 in human cell-based ND models revealed that activation of Akt1 signaling by the small molecule SC79 increased cell viability in all models. Moreover, treatment of AD model mice with SC79 enhanced their long-term memory and ameliorated dysregulated anxiety levels, which are commonly affected in AD patients. These findings validate MLnet as a valuable tool to uncover molecular pathways and proteins involved in the pathophysiology of entire disease groups and identify potential therapeutic targets that have relevance across disease boundaries. MLnet can be used for any group of diseases and is available as a web tool. | Johnstone, J. N., Mirth, C. K., Johnson, T. K., Schittenhelm, R. B., Piper, M. D. W. (2023). GCN2 mediates access to stored amino acids for somatic maintenance during Drosophila ageing. bioRxiv, PubMed ID: 38014136
Summary: Many mechanistic theories of ageing argue that a progressive failure of somatic maintenance, the use of energy and resources to prevent and repair damage to the cell, underpins ageing. To sustain somatic maintenance an organism must acquire dozens of essential nutrients from the diet, including essential amino acids (EAAs), which are physiologically limiting for many animals. In Drosophila, adulthood deprivation of each individual EAA yields vastly different lifespan trajectories, and adulthood deprivation of one EAA, phenylalanine (Phe), has no associated lifespan cost; this is despite each EAA being strictly required for growth and reproduction. Moreover, survival under any EAA deprivation depends entirely on the conserved AA sensor GCN2, a component of the integrated stress response (ISR), suggesting that a novel ISR-mediated mechanism sustains lifelong somatic maintenance during EAA deprivation. Flies chronically deprived of dietary Phe continue to incorporate Phe into new proteins, and challenging flies to increase the somatic requirement for Phe shortens lifespan under Phe deprivation. Further, this study showed that autophagy is required for full lifespan under Phe deprivation, and that activation of the ISR can partially rescue the shortened lifespan of GCN2-nulls under Phe deprivation. A mechanism is proposed by which GCN2, via the ISR, activates autophagy during EAA deprivation, breaking down a larvally-acquired store of EAAs to support somatic maintenance. These data refine understanding of the strategies by which flies sustain lifelong somatic maintenance, which determines length of life in response to changes in the nutritional environment. |
Tuesday, June 25th - Enhancers and Transcriptional Regulation |
| Fischer, M. D., Graham, P., Pick, L. (2024). The ftz upstream element drives late ftz stripes but is not required for regulation of Ftz target genes. Dev Biol, 505:141-147 PubMed ID: 37977522
Summary: The regulation of gene expression in precise, rapidly changing spatial patterns is essential for embryonic development. Multiple enhancers have been identified for the evolving expression patterns of the cascade of Drosophila segmentation genes that establish the basic body plan of the fly. Classic reporter transgene experiments identified multiple cis-regulatory elements (CREs) that are sufficient to direct various aspects of the evolving expression pattern of the pair-rule gene fushi tarazu (ftz). These include enhancers that coordinately activate expression in all seven stripes and stripe-specific elements that activate expression in one or more ftz stripes. Of the two 7-stripe enhancers, analysis of reporter transgenes demonstrated that the upstream element (UPS) is autoregulatory, requiring direct binding of Ftz protein to direct striped expression. This study asked about the endogenous role of the UPS by precisely deleting this 7-stripe enhancer. In ftzΔUPS(7S) homozygotes, ftz stripes appear in the same order as wildtype, and all but stripe 4 are expressed at wildtype levels by the end of the cellular blastoderm stage. This suggests that the zebra element and UPS harbor information to direct stripe 4 expression, although previous deletion analyses failed to identify a stripe-specific CRE within these two 7-stripe enhancers. However, the UPS is necessary for late ftz stripe expression, with all 7 stripes decaying earlier than wildtype in ftzΔUPS(7S) homozygotes. Despite this premature loss of ftz expression, downstream target gene regulation proceeds as in wildtype, and segmentation is unperturbed in the overwhelming majority of animals. It is proposed that this late-acting enhancer provides a buffer against perturbations in gene expression but is not required for establishment of Ftz cell fates. Overall, these results demonstrate that multiple enhancers, each directing distinct aspects of an overall gene expression pattern, contribute to fine-tuning the complex patterns necessary for embryonic development. | Silver, B. D., Willett, C. G., Maher, K. A., Wang, D., Deal, R. B. (2023). Differences in transcription initiation directionality underlie distinctions between plants and animals in chromatin modification patterns at genes and cis-regulatory elements. bioRxiv, PubMed ID: 37961418
Summary: Transcriptional initiation is among the first regulated steps controlling eukaryotic gene expression. High-throughput profiling of fungal and animal genomes has revealed that RNA Polymerase II (Pol II) often initiates transcription in both directions at the promoter transcription start site (TSS), but generally only elongates productively into the gene body. Additionally, Pol II can initiate transcription in both directions at cis-regulatory elements (CREs) such as enhancers. These bidirectional Pol II initiation events can be observed directly with methods that capture nascent transcripts, and they are also revealed indirectly by the presence of transcription-associated histone modifications on both sides of the TSS or CRE. Previous studies have shown that nascent RNAs and transcription-associated histone modifications in the model plant Arabidopsis thaliana accumulate mainly in the gene body, suggesting that transcription does not initiate widely in the upstream direction from genes in this plant. Transcription-associated histone modifications and nascent transcripts at both TSSs and CREs were compared in Arabidopsis thaliana, Drosophila melanogaster, and Homo sapiens. The results provide evidence for mostly unidirectional Pol II initiation at both promoters and gene-proximal CREs of Arabidopsis thaliana, whereas bidirectional transcription initiation is observed widely at promoters in both Drosophila melanogaster and Homo sapiens, as well as CREs in Drosophila. Furthermore, the distribution of transcription-associated histone modifications around TSSs in the Oryza sativa (rice) and Glycine max (soybean) genomes suggests that unidirectional transcription initiation is the norm in these genomes as well. These results suggest that there are fundamental differences in transcriptional initiation directionality between flowering plant and metazoan genomes, which are manifested as distinct patterns of chromatin modifications around RNA polymerase initiation sites. |
| Taskiran, II, Spanier, K. I., Dickmanken, H., Kempynck, N., Pancikova, A., Eksi, E. C., Hulselmans, G., Ismail, J. N., Theunis, K., Vandepoel, R., Christiaens, V., Mauduit, D., Aerts, S. (2023). Cell-type-directed design of synthetic enhancers. Nature, PubMed ID: 38086419
Summary: Transcriptional enhancers act as docking stations for combinations of transcription factors and thereby regulate spatiotemporal activation of their target genes. It has been a long-standing goal in the field to decode the regulatory logic of an enhancer and to understand the details of how spatiotemporal gene expression is encoded in an enhancer sequence. This study shows that deep learning models(2-6), can be used to efficiently design synthetic, cell-type-specific enhancers, starting from random sequences, and that this optimization process allows detailed tracing of enhancer features at single-nucleotide resolution. The function of fully synthetic enhancers was evaluatednto specifically target Kenyon cells or glial cells in the fruit fly brain using transgenic animals. Enhancer design was further exploited to create 'dual-code' enhancers that target two cell types and minimal enhancers smaller than 50 base pairs that are fully functional. By examining the state space searches towards local optima, enhancer codes were characterized through the strength, combination and arrangement of transcription factor activator and transcription factor repressor motifs. Finally, the same strategies were applied to successfully design human enhancers, which adhere to enhancer rules similar to those of Drosophila enhancers. Enhancer design guided by deep learning leads to better understanding of how enhancers work and shows that their code can be exploited to manipulate cell states. | Cheng, Y., Chan, F., Kassis, J. A. (2023). The activity of engrailed imaginal disc enhancers is modulated epigenetically by chromatin and autoregulation. PLoS Genet, 19(11):e1010826 PubMed ID: 37967127
Summary: engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal discs; in contrast, small IDE-reporter transgenes are expressed mainly in the anterior compartment. En was found to bind to the IDEs, and suggesting that it may directly repress IDE function and modulate En expression levels. Two en IDEs, O and S were discrovered. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from an endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether these data suggest that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic memory set by the embryonic enhancers. This epigenetic regulation is similar to that of the Ultrabithorax IDEs and it is suggested that the activity of late-acting enhancers in other genes may be similarly regulated. |
| Wang, Y., Salazar, R., Simonetta, L., Sorrentino, V., Gatton, T. J., Wu, B., Vecsey, C. G., Carrillo, R. A. (2023). hkb is required for DIP-alpha expression and target recognition in the Drosophila neuromuscular circuit. bioRxiv, PubMed ID: 37905128
Summary: The human nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins (CSPs). In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth and cell survival. However, the upstream regulation and downstream signaling mechanisms of Dprs and DIPs are not clear. In the Drosophila larval neuromuscular system, DIP-α is expressed in the dorsal and ventral type-Is motor neurons (MNs). F1 dominant modifier genetic screen was conducted to identify regulators of Dprs and DIPs. The transcription factor, huckebein (hkb), was found to genetically interact with DIP-α and is important for target recognition specifically in the dorsal Is MN, but not the ventral Is MN. Loss of hkb led to complete removal of DIP-α expression. This specificity was confirmed to be through the dorsal Is MN specific transcription factor, even-skipped (eve), which acts downstream of hkb. Genetic interaction between hkb and eve revealed that they act in the same pathway to regulate dorsal Is MN connectivity. This study provides insight into the transcriptional regulation of DIP-α and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons. | Syed, S., Duan, Y., Lim, B. (2023). Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos. Elife, 12 PubMed ID: 37934571
Summary: It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. This study utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, a mutation in any one of these binding sites was found to cause a drastic reduction in transcriptional amplitude, resulting in a reduction in mRNA production of the target gene. Evidence is provided of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. It is proposed that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates. |
Monday, June 24th - Signaling |
| Tian, A., Wang, X. F., Xu, Y., Morejon, V., Huang, Y. C., Nwapuda, C., Deng, W. M. (2023). EGFR signaling controls directionality of epithelial multilayer formation upon loss of cell polarity. The EMBO journal, 42(24):e113856 PubMed ID: 37953688
Summary: Apical-basal polarity is maintained by distinct protein complexes that reside in membrane junctions, and polarity loss in monolayered epithelial cells can lead to formation of multilayers, cell extrusion, and/or malignant overgrowth. Yet, how polarity loss cooperates with intrinsic signals to control directional invasion toward neighboring epithelial cells remains elusive. Using the Drosophila ovarian follicular epithelium as a model, this study found that posterior follicle cells with loss of lethal giant larvae (lgl) or Discs large (Dlg) accumulate apically toward germline cells, whereas cells with loss of Bazooka (Baz) or atypical protein kinase C (aPKC) expand toward the basal side of wildtype neighbors. Further studies revealed that these distinct multilayering patterns in the follicular epithelium were determined by epidermal growth factor receptor (EGFR) signaling and its downstream target Pointed, a zinc-finger transcription factor. Additionally, Rho kinase was identified as a Pointed target that regulates formation of distinct multilayering patterns. These findings provide insight into how cell polarity genes and receptor tyrosine kinase signaling interact to govern epithelial cell organization and directional growth that contribute to epithelial tumor formation. | Shi, L., Ma, H., Wang, J., Ma, M., Zhao, H., Li, Z., Wang, J. H., Wu, S., Zhou, Z., Dong, M. Q., Li, Z. (2023). An EMC-Hpo-Yki axis maintains intestinal homeostasis under physiological and pathological conditions. Development, 150(24) PubMed ID: 38031990
Summary: alanced control of stem cell proliferation and differentiation underlines tissue homeostasis. Disruption of tissue homeostasis often results in many diseases. However, how endogenous factors influence the proliferation and differentiation of intestinal stem cells (ISCs) under physiological and pathological conditions remains poorly understood. This study found that the evolutionarily conserved endoplasmic reticulum membrane protein complex (EMC) negatively regulates ISC proliferation and intestinal homeostasis. Compromising EMC function in progenitors leads to excessive ISC proliferation and intestinal homeostasis disruption. Mechanistically, the EMC associates with and stabilizes Hippo (Hpo) protein, the key component of the Hpo signaling pathway. In the absence of EMC, Yorkie (Yki) is activated to promote ISC proliferation due to Hpo destruction. The EMC-Hpo-Yki axis also functions in enterocytes to maintain intestinal homeostasis. Importantly, the levels of the EMC are dramatically diminished in tunicamycin-treated animals, leading to Hpo destruction, thereby resulting in intestinal homeostasis disruption due to Yki activation. Thus, this study uncovers the molecular mechanism underlying the action of the EMC in intestinal homeostasis maintenance under physiological and pathological conditions and provides new insight into the pathogenesis of tunicamycin-induced tumorigenesis. |
| Jackson, J. A., Denk-Lobnig, M., Kitzinger, K. A., Martin, A. C. (2023). Change in RhoGAP and RhoGEF availability drives transitions in cortical patterning and excitability in Drosophila. bioRxiv, PubMed ID: 37986763
Summary: Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. While mechanisms have been established for individual cells' dynamic behaviors, mechanisms and specific molecules that result in developmental transitions in vivo are still poorly understood. This study took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. A RhoGEF (Pebble) and RhoGAP (RhoGAP15B) pair was identified whose relocalization from nucleus to cortex results in actomyosin waves in egg chambers. Furthermore, Overexpression of a different RhoGEF and RhoGAP pair resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly and RhoGAP recruitment by ~4 seconds. Overall, this study showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types. | Chen, J., Stork, T., Kang, Y., Nardone, K. A. M., Auer, F., Farrell, R. J., Jay, T. R., Heo, D., Sheehan, A., Paton, C., Nagel, K. I., Schoppik, D., Monk, K. R., Freeman, M. R. (2024). Astrocyte growth is driven by the Tre1/S1pr1 phospholipid-binding G protein-coupled receptor. Neuron, 112(1):93-112.e110 PubMed ID: 38096817
Summary: Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways regulating astrocyte morphogenesis remain poorly defined. This study shows the Drosophila lipid-binding G protein-coupled receptor (GPCR) Tre1 is required for astrocytes to establish their intricate morphology in vivo. The lipid phosphate phosphatases Wunen/Wunen2 also regulate astrocyte morphology and, via Tre1, mediate astrocyte-astrocyte competition for growth-promoting lipids. Loss of s1pr1, the functional analog of Tre1 in zebrafish, disrupts astrocyte process elaboration, and live imaging and pharmacology demonstrate that S1pr1 balances proper astrocyte process extension/retraction dynamics during growth. Loss of Tre1 in flies or S1pr1 in zebrafish results in defects in simple assays of motor behavior. Tre1 and S1pr1 are thus potent evolutionarily conserved regulators of the elaboration of astrocyte morphological complexity and, ultimately, astrocyte control of behavior. |
| Bakopoulos, D., Golenkina, S., Dark, C., Christie, E. L., Sanchez-Sanchez, B. J., Stramer, B. M., Cheng, L. Y. (2023). Convergent insulin and TGF-beta signalling drives cancer cachexia by promoting aberrant fat body ECM accumulation in a Drosophila tumour model. EMBO reports, 24(12):e57695 PubMed ID: 38014610
Summary: This study found that in the adipose tissue of wildtype animals, insulin and TGF-β signalling converge via a BMP antagonist short gastrulation (sog) to regulate ECM remodelling. In tumour bearing animals, Sog also modulates TGF-β signalling to regulate ECM accumulation in the fat body. TGF-β signalling causes ECM retention in the fat body and subsequently depletes muscles of fat body-derived ECM proteins. Activation of insulin signalling, inhibition of TGF-β signalling, or modulation of ECM levels via SPARC, Rab10 or Collagen IV in the fat body, is able to rescue tissue wasting in the presence of tumour. Together, our study highlights the importance of adipose ECM remodelling in the context of cancer cachexia. | Nakamura, M., Parkhurst, S. M. (2023). Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds. bioRxiv, PubMed ID: 38105960
Summary: To survive daily damage, the formation of actomyosin ring at the wound periphery is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. This study found that rapid recruitment of all three Drosophila calcium responding and phospholipid binding Annexin proteins (AnxB9, AnxB10, AnxB11) to distinct regions around the wound are regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, it was found that reduced extracellular calcium and depletion of intracellular calcium affect cell wound repair differently, despite these two conditions exhibiting similar GCaMP signals. Thus, the results suggest that, in addition to initiating repair events, both the quantity and sources of calcium influx are important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair. |
Friday, June 21st - Adult Neural Development, Structure, and Function |
| Munroe, J. A., Doe, C. Q. (2023). Imp is expressed in INPs and newborn neurons where it regulates neuropil targeting in the central complex. Neural Dev, 18(1):9 PubMed ID: 38031099
Summary: The generation of neuronal diversity remains incompletely understood. In Drosophila, the central brain is populated by neural stem cells derived from progenitors called neuroblasts (NBs). There are two types of NBs, type 1 and 2. T1NBs have a relatively simple lineage, whereas T2NBs expand and diversify the neural population with the generation of intermediate neural progenitors (INPs), contributing many neurons to the adult central complex, a brain region essential for navigation. However, it is not fully understood how neural diversity is created in T2NB and INP lineages. Imp, an RNA-binding protein, is expressed in T2NBs in a high-to-low temporal gradient, while the RNA-binding protein Syncrip forms an opposing gradient. It remains unknown if Imp expression is carried into INPs; whether it forms a gradient similar to NBs; and whether INP expression of Imp is required for generating neuronal identity or morphology. This study shows that Imp/Syp are both present in INPs, but not always in opposing gradients. Newborn INPs adopt their Imp/Syp levels from their parental T2NBs; that Imp and Syp are expressed in stage-specific high-to-low gradients in INPs. In addition, there is a late INP pulse of Imp. Neurons born from old INPs (E-PG and PF-R neurons) have altered morphology following both Imp knock-down and Imp overexpression. It is concluded that Imp functions in INPs and newborn neurons to determine proper neuronal morphology and central complex neuropil organization. | Rabah, Y., Frances, R., Minatchy, J., Guedon, L., Desnous, C., Placais, P. Y., Preat, T. (2023). Glycolysis-derived alanine from glia fuels neuronal mitochondria for memory in Drosophila. Nature metabolism, 5(11):2002-2019 PubMed ID: 37932430
Summary: Glucose is the primary source of energy for the brain; however, it remains controversial whether, upon neuronal activation, glucose is primarily used by neurons for ATP production or if it is partially oxidized in astrocytes, as proposed by the astrocyte-neuron lactate shuttle model for glutamatergic neurons. Thus, an in vivo picture of glucose metabolism during cognitive processes is missing. This study uncovered in Drosophila melanogaster a glia-to-neuron alanine transfer involving alanine aminotransferase that sustains memory formation. Following associative conditioning, glycolysis in glial cells produces alanine, which is back-converted into pyruvate in cholinergic neurons of the olfactory memory center to uphold their increased mitochondrial needs. Alanine, as a mediator of glia-neuron coupling, could be an alternative to lactate in cholinergic systems. In parallel, a dedicated glial glucose transporter imports glucose specifically for long-term memory, by directly transferring it to neurons for use by the pentose phosphate pathway. These results demonstrate in vivo the compartmentalization of glucose metabolism between neurons and glial cells during memory formation. |
| Tanaka, R., Zhou, B., Agrochao, M., Badwan, B. A., Au, B., Matos, N. C. B., Clark, D. A. (2023). Neural mechanisms to incorporate visual counterevidence in self-movement estimation. Curr Biol, 33(22):4960-4979. PubMed ID: 37918398
Summary: In selecting appropriate behaviors, animals should weigh sensory evidence both for and against specific beliefs about the world. For instance, animals measure optic flow to estimate and control their own rotation. However, existing models of flow detection can be spuriously triggered by visual motion created by objects moving in the world. This study shows that stationary patterns on the retina, which constitute evidence against observer rotation, suppress inappropriate stabilizing rotational behavior in the fruit fly Drosophila. In silico experiments show that artificial neural networks (ANNs) that are optimized to distinguish observer movement from external object motion similarly detect stationarity and incorporate negative evidence. Employing neural measurements and genetic manipulations, this study identified components of the circuitry for stationary pattern detection, which runs parallel to the fly's local motion and optic-flow detectors. The results show how the fly brain incorporates negative evidence to improve heading stability, exemplifying how a compact brain exploits geometrical constraints of the visual world. | Longden, K. D., Rogers, E. M., Nern, A., Dionne, H., Reiser, M. B. (2023). Different spectral sensitivities of ON- and OFF-motion pathways enhance the detection of approaching color objects in Drosophila. Nat Commun, 14(1):7693 PubMed ID: 38001097
Summary: Color and motion are used by many species to identify salient objects. They are processed largely independently, but color contributes to motion processing in humans, for example, enabling moving colored objects to be detected when their luminance matches the background. This study demonstrates an unexpected, additional contribution of color to motion vision in Drosophila. Behavioral ON-motion responses are more sensitive to UV than for OFF-motion, and cellular pathways were identified connecting UV-sensitive R7 photoreceptors to ON and OFF-motion-sensitive T4 and T5 cells, using neurogenetics and calcium imaging. Remarkably, this contribution of color circuitry to motion vision enhances the detection of approaching UV discs, but not green discs with the same chromatic contrast, and it was shown how this could generalize for systems with ON- and OFF-motion pathways. The results provide a computational and circuit basis for how color enhances motion vision to favor the detection of saliently colored objects. |
| Klose, M. K., Kim, J., Levitan, E. S. (2023). Activity-dependent capture of neuropeptide vesicles prepares clock neuron synapses for daily release. bioRxiv, PubMed ID: 38106047
Summary: Drosophila brain sLNv clock neurons release the neuropeptide PDF to control circadian rhythms. Strikingly, PDF content in sLNv terminals is rhythmic with a peak in the morning. Peak content drops because of activity-dependent release from dense-core vesicles (DCVs), but the mechanism for the daily increase in presynaptic PDF in the hours prior to release is unknown. Although transport from the soma was proposed to drive the daily increase in presynaptic PDF, live imaging in sLNv neurons shows that anterograde axonal DCV transport is constant throughout the day. Instead, capture of circulating DCVs, indicated by decreased retrograde axonal transport, rhythmically boosts presynaptic neuropeptide content. Genetic manipulations demonstrate that the late night increase in capture requires electrical activity but is independent of daily morphological changes. These results suggest that each day, during the hours of ongoing electrical activity, a toggle switches from inducing vesicle capture to triggering exocytosis, thereby maximizing daily rhythmic bursts of synaptic neuropeptide release by clock neurons. | Sanfilippo, P., Kim, A. J., Bhukel, A., Yoo, J., Mirshahidi, P. S., Pandey, V., Bevir, H., Yuen, A., Mirshahidi, P. S., Guo, P., Li, H. S., Wohlschlegel, J. A., Aso, Y., Zipursky, S. L. (2023). Mapping of multiple neurotransmitter receptor subtypes and distinct protein complexes to the connectome. bioRxiv, PubMed ID: 37873314
Summary: Neurons express different combinations of neurotransmitter receptor (NR) subunits and receive inputs from multiple neuron types expressing different neurotransmitters. Localizing NR subunits to specific synaptic inputs has been challenging. This study used epitope tagged endogenous NR subunits, expansion light-sheet microscopy, and EM connectomics to molecularly characterize synapses in Drosophila. In directionally selective motion sensitive neurons, different multiple NRs elaborated a highly stereotyped molecular topography with NR localized to specific domains receiving cell-type specific inputs. Developmental studies suggested that NRs or complexes of them with other membrane proteins determines patterns of synaptic inputs. In support of this model, a transmembrane protein associated selectively with a subset of spatially restricted synapses was identified, and through genetic analysis its requirement for synapse formation was demonstrate. It is proposed that mechanisms which regulate the precise spatial distribution of NRs provide a molecular cartography specifying the patterns of synaptic connections onto dendrites. |
Thursday, June 20th - Behavior |
| Puppato, S., Fiorenza, G., Carraretto, D., Gomulski, L. M., Gasperi, G., Caceres, C., Grassi, A., Mancini, M. V., De Cristofaro, A., Ioriatti, C., Guilhot, R., Malacrida, A. R. (2023). High promiscuity among females of the invasive pest species Drosophila suzukii. Mol Ecol, 32(22):6018-6026 PubMed ID: 37804145
Summary: Drosophila suzukii, the spotted-wing drosophila, is a highly invasive fruit fly that spread from Southern Asia across most regions of Asia and, in the last 15 years, has invaded Europe and the Americas. It is an economically important pest of small fruits such as berries and stone fruits. Drosophila suzukii speciated by adapting to cooler, mountainous, and forest environments. In temperate regions, it evolved seasonal polyphenism traits which enhanced its survival during stressful winter population bottlenecks. Consequently, in these temperate regions, the populations undergo seasonal reproductive dynamics. Despite its economic importance, no data are available on the behavioural reproductive strategies of this fly. The presence of polyandry, for example, has not been determined despite the important role it might play in the reproductive dynamics of populations. This study explored the presence of polyandry in an established population in Trentino, a region in northern Italy. In this area, D. suzukii overcomes the winter bottleneck and undergoes a seasonal reproductive fluctuation. A high remating frequency was observed in females during the late spring demographic explosion that led to the abundant summer population. The presence of a high degree of polyandry and shared paternity associated with the post-winter population increase raises the question of the possible evolutionary adaptive role of this reproductive behaviour in D. suzukii. | Mathejczyk, T. F., Babo E, J., Schonlein, E., Grinda, N. V., Greiner, A., Okroznik, N., Belusic, G., Wernet, M. F. (2023). Behavioral responses of free-flying Drosophila melanogaster to shiny, reflecting surfaces. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 209(6):929-941 PubMed ID: 37796303
Summary: Active locomotion plays an important role in the life of many animals, permitting them to explore the environment, find vital resources, and escape predators. Most insect species rely on a combination of visual cues such as celestial bodies, landmarks, or linearly polarized light to navigate or orient themselves in their surroundings. In nature, linearly polarized light can arise either from atmospheric scattering or from reflections off shiny non-metallic surfaces like water. Multiple reports have described different behavioral responses of various insects to such shiny surfaces. The goal of this study was to test whether free-flying Drosophila melanogaster, a molecular genetic model organism and behavioral generalist, also manifests specific behavioral responses when confronted with such polarized reflections. Fruit flies were placed in a custom-built arena with controlled environmental parameters (temperature, humidity, and light intensity). Flight detections and landings were quantified for three different stimuli: a diffusely reflecting matt plate, a small patch of shiny acetate film, and real water. Hydrated and dehydrated fly populations were compared, since the state of hydration may change the motivation of flies to seek or avoid water. This analysis reveals for the first time that flying fruit flies indeed use vision to avoid flying over shiny surfaces. |
| Berne, A., Zhang, T., Shomar, J., Ferrer, A. J., Valdes, A., Ohyama, T., Klein, M. (2023). Mechanical vibration patterns elicit behavioral transitions and habituation in crawling Drosophila larvae. Elife, 12 PubMed ID: 37855833
Summary: How animals respond to repeatedly applied stimuli, and how animals respond to mechanical stimuli in particular, are important questions in behavioral neuroscience. Adaptation to repeated mechanical agitation was studied using the Drosophila larva. Vertical vibration stimuli elicit a discrete set of responses in crawling larvae: continuation, pause, turn, and reversal. Through high-throughput larva tracking, this study characterize how the likelihood of each response depends on vibration intensity and on the timing of repeated vibration pulses. By examining transitions between behavioral states at the population and individual levels, how the animals habituate to the stimulus patterns was studied. Time constants associated with desensitization to prolonged vibration, with re-sensitization during removal of a stimulus, and additional layers of habituation were identified that operate in the overall response. Known memory-deficient mutants exhibit distinct behavior profiles and habituation time constants. An analogous simple electrical circuit suggests possible neural and molecular processes behind adaptive behavior. | Chowdhury, B., Abhilash, L., Ortega, A., Liu, S., Shafer, O. (2023). Homeostatic control of deep sleep and molecular correlates of sleep pressure in Drosophila. Elife, 12 PubMed ID: 37906092
Summary: Homeostatic control of sleep is typically addressed through mechanical stimulation-induced forced wakefulness and the measurement of subsequent increases in sleep. A major confound attends this approach: biological responses to deprivation may reflect a direct response to the mechanical insult rather than to the loss of sleep. Similar confounds accompany all forms of sleep deprivation and represent a major challenge to the field. This study describes a new paradigm for sleep deprivation in Drosophila that fully accounts for sleep-independent effects. The results reveal that deep sleep states are the primary target of homeostatic control and establish the presence of multi-cycle sleep rebound following deprivation. Furthermore, this study established that specific deprivation of deep sleep states results in state-specific homeostatic rebound. Finally, by accounting for the molecular effects of mechanical stimulation during deprivation experiments, it was shown that serotonin levels track sleep pressure in the fly's central brain. The results illustrate the critical need to control for sleep-independent effects of deprivation when examining the molecular correlates of sleep pressure and call for a critical reassessment of work that has not accounted for such non-specific effects. |
| Maruko, A., Iijima, K. M., Ando, K. (2023). Dissecting the daily feeding pattern: Peripheral CLOCK/CYCLE generate the feeding/fasting episodes and neuronal molecular clocks synchronize them. iScience, 26(11):108164 PubMed ID: 37915609
Summary: A 24-h rhythm of feeding behavior, or synchronized feeding/fasting episodes during the day, is crucial for survival. Internal clocks and light input regulate rhythmic behaviors, but how they generate feeding rhythms is not fully understood. This study aimed to dissect the molecular pathways that generate daily feeding patterns. By measuring the semidiurnal amount of food ingested by single flies, this study demonstrated that the generation of feeding rhythms under light:dark conditions requires quasimodo (qsm) but not molecular clocks. Under constant darkness, rhythmic feeding patterns consist of two components: CLOCK (CLK) in digestive/metabolic tissues generating feeding/fasting episodes, and the molecular clock in neurons synchronizing them to subjective daytime. Although CLK is a part of the molecular clock, the generation of feeding/fasting episodes by CLK in metabolic tissues was independent of molecular clock machinery. These results revealed novel functions of qsm and CLK in feeding rhythms in Drosophila. | Keles, M. F., Sapci, A., Brody, C., Palmer, I., Le, C., Tastan, O., Keles, S., Wu, M. N. (2023). Deep Phenotyping of Sleep in Drosophila. bioRxiv, PubMed ID: 37961473
Summary: Sleep is an evolutionarily conserved behavior, whose function is unknown. This paper presents a method for deep phenotyping of sleep in Drosophila, consisting of a high-resolution video imaging system, coupled with closed-loop laser perturbation to measure arousal threshold. To quantify sleep-associated microbehaviors, a deep-learning network was trained to annotate body parts in freely moving flies, and a semi-supervised computational pipeline was developed to classify behaviors. Quiescent flies exhibit a rich repertoire of microbehaviors, including proboscis pumping (PP) and haltere switches, which vary dynamically across the night. Using this system, the effects were characterized of optogenetically activating two putative sleep circuits. These data reveal that activating dFB neurons produces micromovements, inconsistent with sleep, while activating R5 neurons triggers PP followed by behavioral quiescence. These findings suggest that sleep in Drosophila is polyphasic with different stages and set the stage for a rigorous analysis of sleep and other behaviors in this species. |
Wednesday, June 19th - Evolution |
| Schrider, D. R. (2023). Allelic gene conversion softens selective sweeps. bioRxiv, PubMed ID: 38106127
Summary: The prominence of positive selection, in which beneficial mutations are favored by natural selection and rapidly increase in frequency, is a subject of intense debate. Positive selection can result in selective sweeps, in which the haplotype(s) bearing the adaptive allele "sweep" through the population, thereby removing much of the genetic diversity from the region surrounding the target of selection. Two models of selective sweeps have been proposed: classical sweeps, or "hard sweeps", in which a single copy of the adaptive allele sweeps to fixation, and "soft sweeps", in which multiple distinct copies of the adaptive allele leave descendants after the sweep. Soft sweeps can be the outcome of recurrent mutation to the adaptive allele, or the presence of standing genetic variation consisting of multiple copies of the adaptive allele prior to the onset of selection. Importantly, soft sweeps will be common when populations can rapidly adapt to novel selective pressures, either because of a high mutation rate or because adaptive alleles are already present. The prevalence of soft sweeps is especially controversial, and it has been noted that selection on standing variation or recurrent mutations may not always produce soft sweeps. This study shows that the inverse is true: selection on single-origin de novo mutations may often result in an outcome that is indistinguishable from a soft sweep. This is made possible by allelic gene conversion, which "softens" hard sweeps by copying the adaptive allele onto multiple genetic backgrounds, a process referred to as a "pseudo-soft" sweep. A simulation study was carried out, examining the impact of gene conversion on sweeps from a single de novo variant in models of human, Drosophila, and Arabidopsis populations. The fraction of simulations in which gene conversion had produced multiple haplotypes with the adaptive allele upon fixation was appreciable. Indeed, under realistic demographic histories and gene conversion rates, even if selection always acts on a single-origin mutation, sweeps involving multiple haplotypes are more likely than hard sweeps in large populations, especially when selection is not extremely strong. Thus, even when the mutation rate is low or there is no standing variation, hard sweeps are expected to be the exception rather than the rule in large populations. These results also imply that the presence of signatures of soft sweeps does not necessarily mean that adaptation has been especially rapid or is not mutation limited. | Stromberg, K. A., Spain, T., Tomlin, S. A., Powell, J., Amarillo, K. D., Schroeder, C. M. (2023). Evolutionary diversification reveals distinct somatic versus germline cytoskeletal functions of the Arp2 branched actin nucleator protein. Curr Biol, 33(24):5326-5339.e5327 PubMed ID: 37977138
Summary: Branched actin networks are critical in many cellular processes, including cell motility and division. Arp2, a protein within the seven-membered Arp2/3 complex, is responsible for generating branched actin. Given its essential roles, Arp2 evolves under stringent sequence conservation throughout eukaryotic evolution. Recurrent evolutionary diversification of Arp2 in Drosophila was unexpectedly discovered, yielding independently arising paralogs Arp2D in obscura species and Arp2D2 in montium species. Both paralogs are unusually testis-enriched in expression relative to Arp2. Whether their sequence divergence from canonical Arp2 led to functional specialization was investigated by replacing Arp2 in D. melanogaster with either Arp2D or Arp2D2. Despite their divergence, it was surprisingly found that both complement Arp2's essential function in somatic tissue, suggesting they have preserved the ability to polymerize branched actin even in a non-native species. However, it was found that Arp2D- and Arp2D2-expressing males display defects throughout sperm development, with Arp2D resulting in more pronounced deficiencies and subfertility, suggesting the Arp2 paralogs are cross-species incompatible in the testis. Focus was placed on Arp2D, and two highly diverged structural regions-the D-loop and C terminus were pinpointed, and it was found that they contribute to germline defects in D. melanogaster sperm development. However, while the Arp2D C terminus is suboptimal in the D. melanogaster testis, it is essential for Arp2D somatic function. Testis cytology of the paralogs' native species revealed striking differences in germline actin structures, indicating unique cytoskeletal requirements. These findings suggest canonical Arp2 function differs between somatic versus germline contexts, and Arp2 paralogs may have recurrently evolved for species-specialized actin branching in the testis. |
| Bitter, M. C., Berardi, S., Oken, H., Huynh, A., Schmidt, P., Petrov, D. A. (2023). Continuously fluctuating selection reveals extreme granularity and parallelism of adaptive tracking. bioRxiv, PubMed ID: 37904939
Summary: Temporally fluctuating environmental conditions are a ubiquitous feature of natural habitats. Yet, how finely natural populations adaptively track fluctuating selection pressures via shifts in standing genetic variation is unknown. This study generated high-frequency, genome-wide allele frequency data from a genetically diverse population of Drosophila melanogaster in extensively replicated field mesocosms from late June to mid-December, a period of ~12 generations. Adaptation throughout the fundamental ecological phases of population expansion, peak density, and collapse was underpinned by extremely rapid, parallel changes in genomic variation across replicates. Yet, the dominant direction of selection fluctuated repeatedly, even within each of these ecological phases. Comparing patterns of allele frequency change to an independent dataset procured from the same experimental system demonstrated that the targets of selection are predictable across years. In concert, the results reveal fitness-relevance of standing variation that is likely to be masked by inference approaches based on static population sampling, or insufficiently resolved time-series data. Such fine-scaled temporally fluctuating selection may be an important force maintaining functional genetic variation in natural populations and an important stochastic force affecting levels of standing genetic variation genome-wide. | Collet, J. M., Nidelet, S., Fellous, S. (2023). Genetic independence between traits separated by metamorphosis is widespread but varies with biological function. Proceedings Biological sciences, 290(2010):20231784 PubMed ID: 37935368
Summary: Why is metamorphosis so pervasive? Does it facilitate the independent (micro)evolution of quantitative traits in distinct life stages, similarly to how it enables some limbs and organs to develop at specific life stages? This hypothesis was tested by measuring the expression of 6400 genes in 41 Drosophila melanogaster inbred lines at larval and adult stages. Only 30% of the genes showed significant genetic correlations between larval and adult expression. By contrast, 46% of the traits showed some level of genetic independence between stages. Gene ontology terms enrichment revealed that across stages correlated traits were often involved in proteins synthesis, insecticide resistance and innate immunity, while a vast number of genes expression traits associated with energy metabolism were independent between life stages. These results were compared to a similar case: genetic constraints between males and females in gonochoric species (i.e. sexual antagonism). It was expected that selection for the separation between males and females to be higher than between juvenile and adult functions, as gonochorism is a more common strategy in the animal kingdom than metamorphosis. Surprisingly, it was found that inter-stage constraints were lower than inter-sexual genetic constraints. Overall, these results show that metamorphosis enables a large part of the transcriptome to evolve independently at different life stages. |
| Li, M., Chen, D. S., Junker, I. P., Szorenyi, F., Chen, G. H., Berger, A. J., Comeault, A. A., Matute, D. R., Ding, Y. (2023). Ancestral neural circuits potentiate the origin of a female sexual behavior. bioRxiv, PubMed ID: 38106147
Summary: Courtship interactions are remarkably diverse in form and complexity among species. How neural circuits evolve to encode new behaviors that are functionally integrated into these dynamic social interactions is unknown. This study reports a recently originated female sexual behavior in the island endemic Drosophila species D. santomea, where females signal receptivity to male courtship songs by spreading their wings, which in turn promotes prolonged songs in courting males. Copulation success depends on this female signal and correlates with males' ability to adjust his singing in such a social feedback loop. Functional comparison of sexual circuitry across species suggests that a pair of descending neurons, which integrates male song stimuli and female internal state to control a conserved female abdominal behavior, drives wing spreading in D. santomea. This co-option occurred through the refinement of a pre-existing, plastic circuit that can be optogenetically activated in an outgroup species. Combined, these results show that the ancestral potential of a socially-tuned key circuit node to engage the wing motor program facilitates the expression of a new female behavior in appropriate sensory and motivational contexts. More broadly, this work provides insights into the evolution of social behaviors, particularly female behaviors, and the underlying neural mechanisms. | Clark, J. M., Gibbs, A. G. (2023). Starvation selection reduces and delays larval ecdysone production and signaling. The Journal of experimental biology, 226(18) PubMed ID: 37671530
Summary: Previous studies have shown that selection for starvation resistance in Drosophila melanogaster results in delayed eclosion and increased adult fat stores. It is assumed that these traits are caused by the starvation selection pressure, but its mechanism is unknown. This study found that starvation-selected (SS) population stores more fat during larval development and has extended larval development and pupal development time. Developmental checkpoints in the third instar associated with ecdysteroid hormone pulses are increasingly delayed. The delay in the late larval period seen in the SS population is indicative of reduced and delayed ecdysone signaling. An enzyme immunoassay for ecdysteroids (with greatest affinity to the metabolically active 20-hydroxyecdysone and the α-ecdysone precursor) confirmed that the SS population had reduced and delayed hormone production compared with that of fed control (FC) flies. Feeding third instar larvae on food supplemented with α-ecdysone partially rescued the developmental delay and reduced subsequent adult starvation resistance. This work suggests that starvation selection causes reduced and delayed production of ecdysteroids in the larval stage and affects the developmental delay phenotype that contributes to subsequent adult fat storage and starvation resistance. |
Tuesday, July 18th - Chromatin |
| Khodursky, S., Zheng, E. B., Svetec, N., Durkin, S. M., Benjamin, S., Gadau, A., Wu, X., Zhao, L. (2023). The evolution and mutational robustness of chromatin accessibility in Drosophila. Genome Biol, 24(1):232 PubMed ID: 37845780
Summary: The evolution of genomic regulatory regions plays a critical role in shaping the diversity of life. While this process is primarily sequence-dependent, the enormous complexity of biological systems complicates the understanding of the factors underlying regulation and its evolution. This study applied deep neural networks as a tool to investigate the sequence determinants underlying chromatin accessibility in different species and tissues of Drosophila. Hybrid convolution-attention neural networks were trained to accurately predict ATAC-seq peaks using only local DNA sequences as input. These models generalize well across substantially evolutionarily diverged species of insects, implying that the sequence determinants of accessibility are highly conserved. Using this model to examine species-specific gains in accessibility, evidence was found suggesting that these regions may be ancestrally poised for evolution. Using in silico mutagenesis, it was shown that accessibility can be accurately predicted from short subsequences in each example. However, in silico knock-out of these sequences does not qualitatively impair classification, implying that accessibility is mutationally robust. Subsequently, it was shown that accessibility is predicted to be robust to large-scale random mutation even in the absence of selection. Conversely, simulations under strong selection demonstrate that accessibility can be extremely malleable despite its robustness. Finally, motifs predictive of accessibility were identified, recovering both novel and previously known motifs. These results demonstrate the conservation of the sequence determinants of accessibility and the general robustness of chromatin accessibility, as well as the power of deep neural networks to explore fundamental questions in regulatory genomics and evolution. | Anderson, J. T., Henikoff, S., Ahmad, K. (2023). Chromosome-specific maturation of the epigenome in the Drosophila male germline. Elife, 12 PubMed ID: 38032818
Summary: Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome, and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, active chromatin features in the testes were profiled from wildtype and meiotic arrest mutants, and this was integrated with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, widespread patterns associated with regulation of the sex chromosomes were detailed. The results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome. |
| Rogers, M. F., Marshall, O. J., Secombe, J. (2023). KDM5-mediated activation of genes required for mitochondrial biology is necessary for viability in Drosophila. Development, 150(21) PubMed ID: 37800333
Summary: Histone-modifying proteins play important roles in the precise regulation of the transcriptional programs that coordinate development. KDM5 family proteins interact with chromatin through demethylation of H3K4me3 as well as demethylase-independent mechanisms that remain less understood. To gain fundamental insights into the transcriptional activities of KDM5 proteins, this study examined the essential roles of the single Drosophila Kdm5 ortholog during development. KDM5 performs crucial functions in the larval neuroendocrine prothoracic gland, providing a model to study its role in regulating key gene expression programs. Integrating genome binding and transcriptomic data, this study identified that KDM5 regulates the expression of genes required for the function and maintenance of mitochondria, and it was found that loss of KDM5 causes morphological changes to mitochondria. This is key to the developmental functions of KDM5, as expression of the mitochondrial biogenesis transcription factor Ets97D, homolog of GABPα, is able to suppress the altered mitochondrial morphology as well as the lethality of Kdm5 null animals. Together, these data establish KDM5-mediated cellular functions that are important for normal development and could contribute to KDM5-linked disorders when dysregulated. | Melnikova, L., Molodina, V., Babosha, V., Kostyuchenko, M., Georgiev, P., Golovnin, A. (2023). The MADF-BESS Protein CP60 Is Recruited to Insulators via CP190 and Has Redundant Functions in Drosophila. Int J Mol Sci, 24(19) PubMed ID: 37834476
Summary: Drosophila CP190 and CP60 are transcription factors that are associated with centrosomes during mitosis. CP190 is an essential transcription factor and preferentially binds to housekeeping gene promoters and insulators through interactions with architectural proteins, including Su(Hw) and dCTCF. CP60 belongs to a family of transcription factors that contain the N-terminal MADF domain and the C-terminal BESS domain, which is characterized by the ability to homodimerize. This study shows that the conserved CP60 region adjacent to MADF is responsible for interacting with CP190. In contrast to the well-characterized MADF-BESS transcriptional activator Adf-1, CP60 is recruited to most chromatin sites through its interaction with CP190, and the MADF domain is likely involved in protein-protein interactions but not in DNA binding. The deletion of the Map60 gene showed that CP60 is not an essential protein, despite the strong and ubiquitous expression of CP60 at all stages of Drosophila development. Although CP60 is a stable component of the Su(Hw) insulator complex, the inactivation of CP60 does not affect the enhancer-blocking activity of the Su(Hw)-dependent gypsy insulator. Overall, these results indicate that CP60 has an important but redundant function in transcriptional regulation as a partner of the CP190 protein. |
| Dubruille, R., Herbette, M., Revel, M., Horard, B., Chang, C. H., Loppin, B. (2023). Histone removal in sperm protects paternal chromosomes from premature division at fertilization. Science, 382(6671):725-731 PubMed ID: 37943933
Summary: The global replacement of histones with protamines in sperm chromatin is widespread in animals, including insects, but its actual function remains enigmatic. In the Drosophila paternal effect mutant paternal loss (pal), sperm chromatin retains germline histones H3 and H4 genome wide without impairing sperm viability. However, after fertilization, pal sperm chromosomes are targeted by the egg chromosomal passenger complex and engage into a catastrophic premature division in synchrony with female meiosis II. pal encodes a rapidly evolving transition protein specifically required for the eviction of (H3-H4)(2) tetramers from spermatid DNA after the removal of H2A-H2B dimers. This study thus reveals an unsuspected role of histone eviction from insect sperm chromatin: safeguarding the integrity of the male pronucleus during female meiosis. | Lu, F., Park, B. J., Fujiwara, R., Wilusz, J. E., Gilmour, D. S., Lehmann, R., Lionnet, T. (2024). Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription. bioRxiv, PubMed ID: 37873455
Summary: Numerous components of the transcription machinery, including RNA polymerase II (Pol II), accumulate in regions of high local concentration known as clusters, which are thought to facilitate transcription. Using the histone locus of Drosophila nurse cells as a model, this study found that Pol II forms long-lived, transcriptionally poised clusters distinct from liquid droplets, which contain unbound and paused Pol II. Depletion of the Integrator complex endonuclease module, but not its phosphatase module or Pol II pausing factors disperses these Pol II clusters. Consequently, histone transcription fails to reach peak levels during S-phase and aberrantly continues throughout the cell cycle. It is proposed that Pol II clustering is a regulatory step occurring near promoters that limits rapid gene activation to defined times. Using the Drosophila histone locus as a model, this study has shown that clustered RNA polymerase II is poised for synchronous activation. |
Monday, June 16th - Genes, Proteins and Enzymes - Evolution, Structure, and Function |
| Summers, J. A., Yarbrough, M., Liu, M., McDonald, W. H., Hudson, B. G., Pastor-Pareja, J. C., Boudko, S. P. (2023). Collagen IV of basement membranes: IV. Adaptive mechanism of collagen IV scaffold assembly in Drosophila. J Biol Chem, 299(12):105394 PubMed ID: 37890775
Summary: Collagen IV is an essential structural protein in all metazoans. It provides a scaffold for the assembly of basement membranes, a specialized form of extracellular matrix, which anchors and signals cells and provides microscale tensile strength. Defective scaffolds cause basement membrane destabilization and tissue dysfunction. Scaffolds are composed of α-chains that coassemble into triple-helical protomers of distinct chain compositions, which in turn oligomerize into supramolecular scaffolds. Chloride ions mediate the oligomerization via NC1 trimeric domains, forming an NC1 hexamer at the protomer-protomer interface. The chloride concentration-'chloride pressure'-on the outside of cells is a primordial innovation that drives the assembly and dynamic stabilization of collagen IV scaffolds. However, a Cl-independent mechanism is operative in Ctenophora, Ecdysozoa, and Rotifera, which suggests evolutionary adaptations to environmental or tissue conditions. An understanding of these exceptions, such as the example of Drosophila, could shed light on the fundamentals of how NC1 trimers direct the oligomerization of protomers into scaffolds. This study investigated the NC1 assembly of Drosophila. The crystal structure of the NC1 hexamer was solved, the chain composition of protomers was determined, and Drosophila was found to adapt an evolutionarily unique mechanism of scaffold assembly that requires divalent cations. By studying the Drosophila case this study highlighted the mechanistic role of chloride pressure for maintaining functionality of the NC1 domain in humans. Moreover, it was discovered that the NC1 trimers encode information for homing protomers to distant tissue locations, providing clues for the development of protein replacement therapy for collagen IV genetic diseases. | Holst, J. D., Murphy, L. G., Gorman, M. J., Ragan, E. J. (2023). Comparison of insect and human cytochrome b561 proteins: Insights into candidate ferric reductases in insects. PLoS One, 18(12):e0291564 PubMed ID: 38039324
Summary: Cytochrome b561 (cytb561) proteins comprise a family of transmembrane oxidoreductases that transfer single electrons across a membrane. Most eukaryotic species, including insects, possess multiple cytb561 homologs. To learn more about this protein family in insects, a bioinformatics-based investigation of cytb561 family members was carried out from nine species representing eight insect orders. A phylogenetic analysis was performed to classify insect cytb561 ortholog groups. Then sequence analyses was conducted and protein models were analyzed to predict structural elements that may impact the biological functions and localization of these proteins, with a focus on possible ferric reductase activity. The study revealed three ortholog groups, designated CG1275, Nemy, and CG8399, and a fourth group of less-conserved genes. CG1275 and Nemy proteins are similar to a human ferric reductase, duodenal cytochrome b561 (Dcytb), and have many conserved amino acid residues that function in substrate binding in Dcytb. Notably, CG1275 and Nemy proteins contain a conserved histidine and other residues that play a role in ferric ion reduction by Dcytb. Nemy proteins were distinguished by a novel cysteine-rich cytoplasmic loop sequence. CG8399 orthologs are similar to a putative ferric reductase in humans, stromal cell-derived receptor 2. Like other members of the CYBDOM class of cytb561 proteins, these proteins contain reeler, DOMON, and cytb561 domains. Drosophila melanogaster CG8399 is the only insect cytb561 with known ferric reductase activity. Investigation of the DOMON domain in CG8399 proteins revealed a probable heme-binding site and a possible site for ferric reduction. The fourth group includes a subgroup of proteins with a conserved "KXXXXKXH" non-cytoplasmic loop motif that may be a substrate binding site and is present in a potential ferric reductase, human tumor suppressor cytochrome b561. This study provides a foundation for future investigations of the biological functions of cytb561 genes in insects. |
| Marshall, A. C., Cummins, J., Kobelke, S., Zhu, T., Widagdo, J., Anggono, V., Hyman, A., Fox, A. H., Bond, C. S., Lee, M. (2023). Different Low-complexity Regions of SFPQ Play Distinct Roles in the Formation of Biomolecular Condensates. J Mol Biol, 435(24):168364 PubMed ID: 37952770
Summary: Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism underlying the complex spatiotemporal organisation of molecules within the cell. Long disordered regions of low sequence complexity (LCRs) are a common feature of proteins that form liquid-like microscopic biomolecular condensates. In particular, RNA-binding proteins with prion-like regions have emerged as key drivers of liquid demixing to form condensates such as nucleoli, paraspeckles and stress granules. Splicing factor proline- and glutamine-rich (SFPQ) is an RNA- and DNA-binding protein essential for DNA repair and paraspeckle formation. SFPQ contains two LCRs of different length and composition. This study shows that the shorter C-terminal LCR of SFPQ is the main region responsible for the condensation of SFPQ in vitro and in the cell nucleus. In contrast, this study found that the longer N-terminal prion-like LCR of SFPQ attenuates condensation of the full-length protein, suggesting a more regulatory role in preventing aberrant condensate formation in the cell. The compositions of these respective LCRs are discussed with reference to current literature. Thesee data add nuance to the emerging understanding of biomolecular condensation, by providing the first example of a common multifunctional nucleic acid-binding protein with an extensive prion-like region that serves to regulate rather than drive condensate formation. | Vedelek, V., Vedelek, B., Lorincz, P., Juhasz, G., Sinka, R. (2023). A comparative analysis of fruit fly and human glutamate dehydrogenases in Drosophila melanogaster sperm development. Frontiers in cell and developmental biology, 11:1281487 PubMed ID: 38020911
Summary: Glutamate dehydrogenases are enzymes that take part in both amino acid and energy metabolism. Their role is clear in many biological processes, from neuronal function to cancer development. The putative testis-specific Drosophila glutamate dehydrogenase, Bb8, is required for male fertility and the development of mitochondrialGdh and the testis-specific Bb8. Both human GLUD1 and GLUD2 can rescue the bb8 (ms) mutant phenotype, with superior performance by GLUD2. The role was tested of three conserved amino acids observed in both Bb8 and GLUD2 in Gdh mutants, which showed their importance in the glutamate dehydrogenase function. The findings of this study indicate that Drosophila Bb8 and human GLUD2 could be novel examples of convergent molecular evolution. Furthermore, the importance of glutamate levels in mitochondrial homeostasis was tested during spermatogenesis by ectopic expression of the mitochondrial glutamate transporter Aralar1, which caused mitochondrial abnormalities in fly spermatids. The data presented in this study offer evidence supporting the significant involvement of glutamate metabolism in sperm development. |
| Bilska, B., Damulewicz, M., Abaquita, T. A. L., Pyza, E. (2023). Changes in heme oxygenase level during development affect the adult life of Drosophila melanogaster. Frontiers in cellular neuroscience, 17:1239101 PubMed ID: 37876913
Summary: Heme oxygenase (HO) has been shown to control various cellular processes in both mammals and Drosophila melanogaster. This study investigated how changes in HO levels in neurons and glial cells during development affect adult flies, by using the TARGET Drosophila system to manipulate the expression of the ho gene. The obtained data showed differences in adult survival, maximum lifespan, climbing, locomotor activity, and sleep, which depended on the level of HO (after ho up-regulation or downregulation), the timing of expression (chronic or at specific developmental stages), cell types (neurons or glia), sex (males or females), and age of flies. In addition to ho, the effects of changing the mRNA level of the Drosophila CNC factor gene (NRF2 homolog in mammals and master regulator of HO), were also examined to compare with those observed after changing ho expression. HO levels in neurons and glia must be maintained at an appropriate physiological level during development to ensure the well-being of adults. This study also found that the downregulation of ho in either neurons or glia in the brain is compensated by ho expressed in the retina. | Krishnan, H., Ahmed, S., Hubbard, S. R., Miller, W. T. (2024). Biochemical characterization of the Drosophila insulin receptor kinase and longevity-associated mutants. Faseb j, 38(1):e23355 PubMed ID: 38071609
Summary: Drosophila melanogaster (fruit fly) insulin receptor (D-IR) is highly homologous to the human counterpart. Like the human pathway, D-IR responds to numerous insulin-like peptides to activate cellular signals that regulate growth, development, and lipid metabolism in fruit flies. Allelic mutations in the D-IR kinase domain elevate life expectancy in fruit flies. This study developed a robust heterologous expression system to express and purify wild-type and longevity-associated mutant D-IR kinase domains to investigate enzyme kinetics and substrate specificities. D-IR exhibits remarkable similarities to the human insulin receptor kinase domain but diverges in substrate preferences. Longevity-associated mutations reduce D-IR catalytic activity. Deletion of the unique kinase insert domain portion or mutations proximal to activating tyrosines do not influence kinase activity, suggesting their potential role in substrate recruitment and downstream signaling. Through biochemical investigations, this study enhances comprehension of D-IR's role in Drosophila physiology, complementing genetic studies and expanding knowledge of the catalytic functions of this conserved signaling pathway. |
Friday, June 13th - Disease Models |
| Lai, Y., Reina-Gonzalez, P., Maor, G., Miller, G. W., Sarkar, S. (2023). Biotin rescues manganese-induced Parkinson's disease phenotypes and neurotoxicity. bioRxiv, PubMed ID: 38045419
Summary: Occupational exposure to manganese (Mn) induces manganism and has been widely linked as a contributing environmental factor to Parkinson's disease (PD), featuring dramatic signature overlaps between the two in motor symptoms and clinical hallmarks. However, the molecular mechanism underlying such link remains elusive, and for combating PD, effective mechanism-based therapies are lacking. This study developed an adult Drosophila model of Mn toxicity to recapitulate key parkinsonian features, spanning behavioral deficits, neuronal loss, and dysfunctions in lysosome and mitochondria. Global metabolomics was performed on flies at an early stage of toxicity, and metabolism of the B vitamin, biotin (vitamin B (7)) was identified as a master pathway underpinning Mn toxicity with systemic, body-brain increases in Mn-treated groups compared to the controls. Using Btnd (RNAi) mutant flies, it was shown that biotin depletion exacerbates Mn-induced neurotoxicity, parkinsonism, and mitochondrial dysfunction; while in Mn-exposed wild-type flies, biotin feeding dramatically ameliorates these pathophenotypes. It was further shown in human induced stem cells (iPSCs)- differentiated midbrain dopaminergic neurons that the supplemented biotin protects against Mn-induced neuronal loss, cytotoxicity, and mitochondrial dysregulation. Finally, human data profiling biotin-related proteins show for PD cases elevated circulating levels of biotin transporters but not of metabolic enzymes compared to healthy controls, suggesting humoral biotin transport as a key event involved in PD. Taken together, these findings identified compensatory biotin pathway as a convergent, systemic driver of Mn toxicity and parkinsonian pathology, providing new basis for devising effective countermeasures against manganism and PD. | Atilano, M. L., Hull, A., Romila, C. A., Adams, M. L., Wildfire, J., Ureña, E., Dyson, M., Ivan-Castillo-Quan, J., Partridge, L., Kinghorn, K. J. (2023). Autophagic dysfunction and gut microbiota dysbiosis cause chronic immune activation in a Drosophila model of Gaucher disease. PLoS Genet, 19(12):e1011063 PubMed ID: 38127816
Summary: Mutations in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD) and are the greatest known genetic risk factors for Parkinson's disease (PD). Communication between the gut and brain and immune dysregulation are increasingly being implicated in neurodegenerative disorders such as PD. This study shows that flies lacking the Gba1b gene, the main fly orthologue of GBA1, display widespread NF-kB signalling activation, including gut inflammation, and brain glial activation. Intestinal autophagic defects, gut dysfunction, and microbiome dysbiosis were also demonstrated. Remarkably, modulating the microbiome of Gba1b knockout flies, by raising them under germ-free conditions, partially ameliorates lifespan, locomotor and immune phenotypes. Moreover, modulation of the immune deficiency (IMD) pathway is detrimental to the survival of Gba1 deficient flies. It was also revealed that direct stimulation of autophagy by rapamycin treatment achieves similar benefits to germ-free conditions independent of gut bacterial load. Consistent with this, pharmacologically blocking autophagosomal-lysosomal fusion, mimicking the autophagy defects of Gba1 depleted cells, was shown to be sufficient to stimulate intestinal immune activation. Overall, these data elucidate a mechanism whereby an altered microbiome, coupled with defects in autophagy, drive chronic activation of NF-kB signaling in a Gba1 loss-of-function model. It also highlights that elimination of the microbiota or stimulation of autophagy to remove immune mediators, rather than prolonged immunosuppression, may represent effective therapeutic avenues for GBA1-associated disorders. |
| Chvilicek, M. M., Seguin, A., Lathen, D. R., Titos, I., Cummins-Beebe, P. N., Pabon, M. A., Miscevic, M., Nickel, E. A., Merrill, C. B., Rodan, A. R., Rothenfluh, A. (2023). Large genetic analysis of alcohol resistance and tolerance reveals an inverse correlation and suggests 'true' tolerance mutants. bioRxiv, PubMed ID: 37873285
Summary: Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce the same behavioral effects. Tolerance is historically not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and between labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. This study has analyzed a large amount of data to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes. Additionally, it was shown that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on this analysis, a method is providing for using a linear regression equation to assess the residuals of potential tolerance mutants. These residuals provide predictive insight into the likelihood of a mutant being a 'true' tolerance mutant, and a framework is offered for understanding the relationship between initial resistance and tolerance. | Liang, Y., Pan, C., Yin, T., Wang, L., Gao, X., Wang, E., Quang, H., Huang, D., Tan, L., Xiang, K., Wang, Y., Alexander, P. B., Li, Q. J., Yao, T. P., Zhang, Z., Wang, X. F. (2024). Branched-Chain Amino Acid Accumulation Fuels the Senescence-Associated Secretory Phenotype. Adv Sci (Weinh), 11(2):e2303489 PubMed ID: 37964763
Summary: The essential branched-chain amino acids (BCAAs) leucine, isoleucine, and valine play critical roles in protein synthesis and energy metabolism. Despite their widespread use as nutritional supplements, BCAAs' full effects on mammalian physiology remain uncertain due to the complexities of BCAA metabolic regulation. Here a novel mechanism linking intrinsic alterations in BCAA metabolism is identified to cellular senescence and the senescence-associated secretory phenotype (SASP), both of which contribute to organismal aging and inflammation-related diseases. Altered BCAA metabolism driving the SASP is mediated by robust activation of the BCAA transporters Solute Carrier Family 6 Members 14 and 15 as well as downregulation of the catabolic enzyme BCAA transaminase 1 during onset of cellular senescence, leading to highly elevated intracellular BCAA levels in senescent cells. This, in turn, activates the mammalian target of rapamycin complex 1 (mTORC1) to establish the full SASP program. Transgenic Drosophila models further indicate that orthologous BCAA regulators are involved in the induction of cellular senescence and age-related phenotypes in flies, suggesting evolutionary conservation of this metabolic pathway during aging. Finally, experimentally blocking BCAA accumulation attenuates the inflammatory response in a mouse senescence model, highlighting the therapeutic potential of modulating BCAA metabolism for the treatment of age-related and inflammatory diseases. |
| Yamaguchi, M., Huynh, M. A., Chiyonobu, T., Yoshida, H. (2023). Knockdown of Chronophage in the nervous system mimics features of neurodevelopmental disorders caused by BCL11A/B variants. Exp Cell Res, 433(2):113827 PubMed ID: 37926342
Summary: Neurodevelopmental disorders (NDD) are a group of disorders that include intellectual disability. Although several genes have been implicated in NDD, the molecular mechanisms underlying its pathogenesis remain unclear. Therefore, it is important to develop novel models to analyze the functions of NDD-causing genes in vivo. Recently, rare pathogenic variants of the B-cell lymphoma/leukemia11A/B (BCL11A/B) gene have been identified in several patients with NDD. Drosophila carries the Chronophage (Cph) gene, which has been predicted to be a homolog of BCL11A/B based on the conservation of the amino acid sequence. This study investigated whether nervous system-specific knockdown of Cph mimics NDD phenotypes in Drosophila. Nervous system-specific knockdown of Cph induced learning and locomotor defects in larvae and epilepsy-like behaviors in adults. The number of synaptic branches was also elevated in the larval neuromuscular junction without a corresponding increase in the number of boutons. Furthermore, the expression levels of putative target genes that are Drosophila homologs of the mammalian BCL11 target genes were decreased in Cph knockdown flies. These results suggest that Cph knockdown flies are a promising model for investigating the pathology of NDD-induced BCL11A/B dysfunction. | Sun, Y., Dai, H., Dai, X., Yin, J., Cui, Y., Liu, X., Gonzalez, G., Yuan, J., Tang, F., Wang, N., Perlegos, A. E., Bonini, N. M., Yang, X. W., Gu, W., Wang, Y. (2023). m(1)A in CAG repeat RNA binds to TDP-43 and induces neurodegeneration. Nature, 623(7987):580-587 PubMed ID: 37938769
Summary: Microsatellite repeat expansions within genes contribute to a number of neurological diseases such as ALS. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology. This study revealed that the adenosine in CAG repeat RNA can be methylated to N(1)-methyladenosine (m(1)A) by TRMT61A, and that m(1)A can be demethylated by ALKBH3. It was also observed that the m(1)A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m(1)A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m(1)A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, this study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m(1)A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion. |
Thursday, June 13th - RNAs and Transposons |
| Morillo, L., Paternina, T., Alasseur, Q., Genovesio, A., Schwartz, S., Le Hir, H. (2023). Comprhensive mapping of exon junction complex binding sites reveals universal EJC deposition in Drosophila. BMC Biol, 21(1):246 PubMed ID: 37936138
Summary: The exon junction complex (EJC) is involved in most steps of the mRNA life cycle, ranging from splicing to nonsense-mediated mRNA decay (NMD). It is assembled by the splicing machinery onto mRNA in a sequence-independent manner. A fundamental open question is whether the EJC is deposited onto all exon‒exon junctions or only on a subset of them. Several previous studies have made observations supportive of the latter, yet these have been limited by methodological constraints. This study sought to overcome these limitations via the integration of two different approaches for transcriptome-wide mapping of EJCs. The results revealed that nearly all, if not all, internal exons consistently harbor an EJC in Drosophila, demonstrating that EJC presence is an inherent consequence of the splicing reaction. Furthermore,this study underscores the limitations of eCLIP methods in fully elucidating the landscape of RBP (RNA-binding protein) binding sites. These findings highlight how highly specific (low false positive) methodologies can lead to erroneous interpretations due to partial sensitivity (high false negatives). This study contributes to understanding of EJC deposition and its association with pre-mRNA splicing. The universal presence of EJC on internal exons underscores its significance in ensuring proper mRNA processing. Additionally, these observations highlight the need to consider both specificity and sensitivity in RBP mapping methodologies. | Wang, M., Liang, A. M., Zhou, Z. Z., Pang, T. L., Fan, Y. J., Xu, Y. Z. (2023). Deletions of singular U1 snRNA gene significantly interfere with transcription and 3'-end mRNA formation. PLoS Genet, 19(11):e1011021 PubMed ID: 37917726
Summary: Small nuclear RNAs (snRNAs) are structural and functional cores of the spliceosome. In metazoan genomes, each snRNA has multiple copies/variants, up to hundreds in mammals. However, the expressions and functions of each copy/variant in one organism have not been systematically studied. Focusing on U1 snRNA genes, this study investigated all five copies in Drosophila melanogaster using two series of constructed strains. Analyses of transgenic flies that each have a U1 promoter-driven gfp revealed that U1:21D is the major and ubiquitously expressed copy, and the other four copies have specificities in developmental stages and tissues. Mutant strains that each have a precisely deleted copy of U1-gene exhibited various extents of defects in fly morphology or mobility, especially deletion of U1:82Eb. Interestingly, splicing was changed at limited levels in the deletion strains, while large amounts of differentially-expressed genes and alternative polyadenylation events were identified, showing preferences in the down-regulation of genes with 1-2 introns and selection of proximal sites for 3'-end polyadenylation. In vitro assays suggested that Drosophila U1 variants pulled down fewer SmD2 proteins compared to the canonical U1. This study demonstrates that all five U1-genes in Drosophila have physiological functions in development and play regulatory roles in transcription and 3'-end formation. |
| Ghosh, S., Chakraborti, S., Devi, D., Sahu, R., Mandal, S., Mandal, L. (2024). A conserved nutrient responsive axis mediates autophagic degradation of miRNA-mRNA hybrids in blood cell progenitors. Nucleic Acids Res, 52(1):385-403 PubMed ID: 37994707
Summary: In animals, microRNAs are amongst the primary non-coding RNAs involved in regulating the gene expression of a cell. Most mRNAs in a cell are targeted by one or many miRNAs. Although several mechanisms can be attributed to the degradation of miRNA and mRNA within a cell, but the involvement of autophagy in the clearance of miRNA and its target mRNA is not known. This study discovered a leucine-responsive axis in blood cell progenitors that can mediate an autophagy-directed degradation of miRNA-bound mRNA in Drosophila melanogaster and Homo sapiens. This previously unknown miRNA clearance axis is activated upon amino acid deprivation that can traffic miRNA-mRNA-loaded Argonaute for autophagic degradation in a p62-dependent manner. Thus, this research not only reports a novel axis that can address the turnover of a catalytically active miRISC but also elucidates a slicer-independent mechanism through which autophagy can selectively initiate the clearance of target mRNA. | Zhang, Y., Duan, Y. (2023). Genome-Wide Analysis on Driver and Passenger RNA Editing Sites Suggests an Underestimation of Adaptive Signals in Insects. Genes, 14(10) PubMed ID: 37895300
Summary: Adenosine-to-inosine (A-to-I) RNA editing leads to a similar effect to A-to-G mutations. RNA editing provides a temporo-spatial flexibility for organisms. Nonsynonymous (Nonsyn) RNA editing in insects is over-represented compared with synonymous (Syn) editing, suggesting adaptive signals of positive selection on Nonsyn editing during evolution. This study utilized the brain RNA editome of Drosophila melanogaster to systematically study the LD (r(2)) between editing sites and infer its impact on the adaptive signals of RNA editing. Pairs of editing sites (PESs) were identified from the transcriptome. For CDS PESs of two consecutive editing sites, their occurrence was significantly biased to type-3 PES (Syn-Nonsyn). The haplotype frequency of type-3 PES exhibited a significantly higher abundance of AG than GA, indicating that the rear Nonsyn site is the driver that promotes the editing of the front Syn site (passenger). The exclusion of passenger Syn sites dramatically amplifies the adaptive signal of Nonsyn RNA editing. This study for the first time quantitatively demonstrates that the linkage between RNA editing events comes from hitchhiking effects and leads to the underestimation of adaptive signals for Nonsyn editing. This work provides novel insights for studying the evolutionary significance of RNA editing events. |
| Luo, Y., He, P., Kanrar, N., Fejes Toth, K., Aravin, A. A. (2023). Maternally inherited siRNAs initiate piRNA cluster formation. Mol Cell, 83(21):3835-3851.e3837 PubMed ID: 37875112
Summary: PIWI-interacting RNAs (piRNAs) guide transposable element repression in animal germ lines. In Drosophila, piRNAs are produced from heterochromatic loci, called piRNA clusters, which act as information repositories about genome invaders. piRNA generation by dual-strand clusters depends on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, which is deposited on clusters guided by piRNAs, forming a positive feedback loop in which piRNAs promote their own biogenesis. However, how piRNA clusters are formed before cognate piRNAs are present remains unknown. This study reports spontaneous de novo piRNA cluster formation from repetitive transgenic sequences. Cluster formation occurs over several generations and requires continuous trans-generational maternal transmission of small RNAs. Maternally supplied small interfering RNAs (siRNAs) were found to trigger de novo cluster activation in progeny. In contrast, siRNAs are dispensable for cluster function after its establishment. These results reveal an unexpected interplay between the siRNA and piRNA pathways and suggest a mechanism for de novo piRNA cluster formation triggered by siRNAs. | Zhang, S., Wang, R., Zhu, X., Zhang, L., Liu, X., Sun, L. (2023). Characteristics and expression of lncRNA and transposable elements in Drosophila aneuploidy. iScience, 26(12):108494 PubMed ID: 38125016
Summary: Aneuploidy can globally affect the expression of the whole genome, which is detrimental to organisms. Dosage-sensitive regulators usually have multiple intermolecular interactions, and changes in their stoichiometry are responsible for the dysregulation of the regulatory network. Currently, studies on noncoding genes in aneuploidy are relatively rare. The characteristics and expression profiles of long noncoding RNAs (lncRNAs) and transposable elements (TEs) were studied in aneuploid Drosophila. It is found that lncRNAs and TEs are affected by genomic imbalance and appear to be more sensitive to an inverse dosage effect than mRNAs. Several dosage-sensitive lncRNAs and TEs were detected for their expression patterns during embryogenesis, and their biological functions in the ovary and testes were investigated using tissue-specific RNAi. This study advances understanding of the noncoding sequences in imbalanced genomes and provides a novel perspective for the study of aneuploidy-related human diseases such as cancer. |
Wednesday, June 12th - Junctions and Vesicles |
| Shaheen, A., Richter Gorey, C. L., Sghaier, A., Dason, J. S. (2023). Cholesterol is required for activity-dependent synaptic growth. J Cell Sci, 136(22) PubMed ID: 37902091
Summary: Changes in cholesterol content of neuronal membranes occur during development and brain aging. Little is known about whether synaptic activity regulates cholesterol levels in neuronal membranes and whether these changes affect neuronal development and function. This study generated transgenic flies that express the cholesterol-binding D4H domain of perfringolysin O toxin and found increased levels of cholesterol in presynaptic terminals of Drosophila larval neuromuscular junctions following increased synaptic activity. Reduced cholesterol impaired synaptic growth and largely prevented activity-dependent synaptic growth. Presynaptic knockdown of adenylyl cyclase phenocopied the impaired synaptic growth caused by reducing cholesterol. Furthermore, the effects of knocking down adenylyl cyclase and reducing cholesterol were not additive, suggesting that they function in the same pathway. Increasing cAMP levels using a dunce mutant with reduced phosphodiesterase activity failed to rescue this impaired synaptic growth, suggesting that cholesterol functions downstream of cAMP. A protein kinase A (PKA) sensor was used to show that reducing cholesterol levels reduced presynaptic PKA activity. Collectively, these results demonstrate that enhanced synaptic activity increased cholesterol levels in presynaptic terminals and that these changes likely activate the cAMP-PKA pathway during activity-dependent growth. | Guangming, G., Mei, C., Qinfeng, Y., Xiang, G., Chenchen, Z., Qingyuan, S., Wei, X., Junhua, G. (2023). Neurexin and neuroligins jointly regulate synaptic degeneration at the Drosophila neuromuscular junction based on TEM studies. Frontiers in cellular neuroscience, 17:1257347 PubMed ID: 38026694
Summary: The Drosophila larval neuromuscular junction (NMJ) is a well-known model system and is often used to study synapse development. This study shows synaptic degeneration at NMJ boutons, primarily based on transmission electron microscopy (TEM) studies. When degeneration starts, the subsynaptic reticulum (SSR) swells, retracts and folds inward, and the residual SSR then degenerates into a disordered, thin or linear membrane. The axon terminal begins to degenerate from the central region, and the T-bar detaches from the presynaptic membrane with clustered synaptic vesicles to accelerate large-scale degeneration. There are two degeneration modes for clear synaptic vesicles. In the first mode, synaptic vesicles without actin filaments degenerate on the membrane with ultrafine spots and collapse and disperse to form an irregular profile with dark ultrafine particles. In the second mode, clear synaptic vesicles with actin filaments degenerate into dense synaptic vesicles, form irregular dark clumps without a membrane, and collapse and disperse to form an irregular profile with dark ultrafine particles. Last, all residual membranes in NMJ boutons degenerate into a linear shape, and all the residual elements in axon terminals degenerate and eventually form a cluster of dark ultrafine particles. Swelling and retraction of the SSR occurs prior to degradation of the axon terminal, which degenerates faster and with more intensity than the SSR. NMJ bouton degeneration occurs under normal physiological conditions but is accelerated in Drosophila neurexin (dnrx) dnrx273, Drosophila neuroligin (dnlg) dnlg1 and dnlg4 mutants and dnrx83;dnlg3 and dnlg2;dnlg3 double mutants, which suggests that both neurexin and neuroligins play a vital role in preventing synaptic degeneration. |
| Chen, X., Perry, S., Wang, B., Wang, S., Hu, J., Loxterkamp, E., Dickman, D., Han, C. (2023). Tissue-specific knockout in Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles. bioRxiv, PubMed ID: 37808853
Summary: Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions in animal development. However, this approach has been successfully applied in only a small number of Drosophila tissues. The Drosophila motor nervous system is an excellent model system for studying the biology of neuromuscular junction (NMJ). To expand tissue-specific CRISPR to the Drosophila motor system, this study presents a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. The efficacy of this toolkit was validated by knocking out known genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. Using these tools, an essential role for SNARE pathways in NMJ maintenance was demonstrated. Furthermore, it was demonstrated that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating the retrograde Gbb signaling. Lastly, axon termini of motoneurons were found to rely on ESCRT-mediated intra-axonal membrane trafficking to lease extracellular vesicles at the NMJ. | Christophers, B., Leahy, S. N., Soffar, D. B., von Saucken, V. E., Broadie, K., Baylies, M. K. (2023). Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission. bioRxiv, PubMed ID: 38045306
Summary: Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Previous work has shown Drosophila cofilin (DmCFL) knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. It was hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, an RNA sequencing analysis was conducted which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. DmCFL was found to be enriched in the muscle postsynaptic compartment and that DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, no significant changes were found in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology |
| Xiao, C., M'Angale, P. G., Wang, S., Lemieux, A., Thomson, T. (2023). Identifying new players in structural synaptic plasticity through dArc1 interrogation. iScience, 26(11):108048 PubMed ID: 37876812
Summary: The formation, expansion, and pruning of synapses, known as structural synaptic plasticity, is needed for learning and memory, and perturbation of plasticity is associated with many neurological disorders and diseases. Previously, it was observed that the Drosophila homolog of Activity-regulated cytoskeleton-associated protein (dArc1), forms a capsid-like structure, associates with its own mRNA, and is transported across synapses. This transfer is needed for structural synaptic plasticity. To identify mRNAs that are modified by dArc1 in presynaptic neuron and postsynaptic muscle, the expression of dArc1 was disrupted, and genomic analysis was performed with deep sequencing. dArc1 was found to affects the expression of genes involved in metabolism, phagocytosis, and RNA-splicing. Through immunoprecipitation this study also identified potential mRNA cargos of dArc1 capsids. This study suggests that dArc1 acts as a master regulator of plasticity by affecting several distinct and highly conserved cellular processes. | Justs, K. A., Sempertegui, S., Riboul, D. V., Oliva, C. D., Durbin, R. J., Crill, S., Stawarski, M., Su, C., Renden, R. B., Fily, Y., Macleod, G. T. (2023). Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals. The Journal of physiology, 601(24):5705-5732 PubMed ID: 37942946
Summary: Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. In this study, through computer simulation and drawing on previously published empirical data, it was determined that motor neuron terminals in Drosophila larvae experience highly volatile power demands. It might not be surprising then, that the mitochondria in the motor neuron terminals of both Drosophila and mice were found to be heavily decorated with phosphagen kinases - a key element in an energy storage and buffering system well-characterized in fast-twitch muscle fibres. Knockdown of arginine kinase 1 (ArgK1) in Drosophila larval motor neurons led to several bioenergetic deficits, including mitochondrial matrix acidification and a faster decline in the cytosol ATP to ADP ratio during axon burst firing. |
Monday, June 10th - Gonads |
| Takashima, Y. A., Majane, A. C., Begun, D. J. (2023). Evolution of secondary cell number and position in the Drosophila accessory gland. PLoS One, 18(10):e0278811 PubMed ID: 37878630
Summary: In animals with internal fertilization, males transfer gametes and seminal fluid during copulation, both of which are required for successful reproduction. In Drosophila and other insects, seminal fluid is produced in the paired accessory gland (AG), the ejaculatory duct, and the ejaculatory bulb. The D. melanogaster AG has emerged as an important model system for this component of male reproductive biology. Seminal fluid proteins produced in the Drosophila AG are required for proper storage and use of sperm by the females, and are also critical for establishing and maintaining a suite of short- and long-term postcopulatory female physiological responses that promote reproductive success. The Drosophila AG is composed of two main cell types. The majority of AG cells, which are referred to as main cells, are responsible for production of many seminal fluid proteins. A minority of cells, about 4%, are referred to as secondary cells. These cells, which are restricted to the distal tip of the D. melanogaster AG, may play an especially important role in the maintenance of the long-term female post-mating response. Many studies of Drosophila AG evolution have suggested that the proteins produced in the gland evolve quickly, as does the transcriptome. This study investigated the evolution of secondary cell number and position in the AG in a collection of eight species spanning the entire history of the Drosophila genus. A heretofore underappreciated rapid evolutionary rate was documented for both number and position of these specialized AG cells, raising several questions about the developmental, functional, and evolutionary significance of this variation. | Anderson, J., Henikoff, S., Ahmad, K. (2023). Chromosome-specific maturation of the epigenome in the Drosophila male germline. bioRxiv, PubMed ID: 37873332
Summary: Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, active chromatin features were profiled in the testes from wildtype and meiotic arrest mutants, and this was integrated with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, widespread patterns associated with regulation of the sex chromosomes were detailed. The results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome. |
| Wenzel, M., Aquadro, C. F. (2023). Wolbachia infection at least partially rescues the fertility and ovary defects of several new Drosophila melanogaster bag of marbles protein-coding mutants. bioRxiv, PubMed ID: 37645949
Summary: The D. melanogaster protein coding gene bag of marbles (bam) plays a key role in early male and female reproduction by forming complexes with partner proteins to promote differentiation in gametogenesis. Like another germline gene, Sex lethal, bam genetically interacts with the endosymbiont Wolbachia, as Wolbachia rescues the reduced fertility of a bam hypomorphic mutant. This study explored the specificity of the bam-Wolbachia interaction by generating 22 new bam mutants, with ten mutants displaying fertility defects. Nine of these mutants trend towards rescue by the w Mel Wolbachia variant, with eight statistically significant at the fertility and/or cytological level. In some cases, fertility was increased a striking 20-fold. There is no specificity between the rescue and the known binding regions of bam, suggesting w Mel does not interact with one singular bam partner to rescue the reproductive phenotype. Whether Mel interacts with bam in a non-specific way, by increasing bam transcript levels or acting upstream in germline stem cells. A fertility assessment of a bam RNAi knockdown mutant reveals that w Mel rescue is specific to functionally mutant bam alleles and no obvious evidence was found of w Mel interaction with germline stem cells in bam mutants. | Francois, C. M., Pihl, T., Dunoyer de Segonzac, M., Herault, C., Hudry, B. (2023). Metabolic regulation of proteome stability via N-terminal acetylation controls male germline stem cell differentiation and reproduction. Nat Commun, 14(1):6737 PubMed ID: 37872135
Summary: The molecular mechanisms connecting cellular metabolism with differentiation remain poorly understood. This study found that metabolic signals contribute to stem cell differentiation and germline homeostasis during Drosophila melanogaster spermatogenesis. External citrate, originating outside the gonad, was found to fuel the production of Acetyl-coenzyme A by germline ATP-citrate lyase (dACLY). This pathway is essential during the final spermatogenic stages, where a high Acetyl-coenzyme A level promotes N-terminal acetyltransferase B (NatB)-dependent N-terminal protein acetylation. Using genetic and biochemical experiments, this study established that N-terminal acetylation shields key target proteins, essential for spermatid differentiation, from proteasomal degradation by the ubiquitin ligase dUBR1. This work uncovers crosstalk between metabolism and proteome stability that is mediated via protein post-translational modification. It is proposed that this system coordinates the metabolic state of the organism with gamete production. More broadly, modulation of proteome turnover by circulating metabolites may be a conserved regulatory mechanism to control cell functions. |
| Guntur, A. R., Smith, J. E., Brahmandam, A., DeBauche, P., Cronmiller, C., Lundell, M. J. (2023). ZFH-2 is required for Drosophila ovarian follicle development and is expressed at the band/interband boundaries of polytene chromosomes. Dev Biol, 504:1-11 PubMed ID: 37666353
Summary: The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. This study provides the first evidence that ZFH-2 has a role in oogenesis. ZFH-2 is expressed in the wild-type ovary, and a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. It was also shown that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. ZFH-2 was shown to be located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190. | Roach, T. V., Lenhart, K. F. (2023). Mating-induced ecdysone in the testis disrupts soma-germline contacts and stem cell cytokinesis. bioRxiv, PubMed ID: 37905121
Summary: Germline maintenance relies on adult stem cells to continually replenish lost gametes over a lifetime and respond to external cues altering the demands on the tissue. Mating worsens germline homeostasis over time, yet a negative impact on stem cell behavior has not been explored. Using extended live imaging of the Drosophila testis stem cell niche, this study found that short periods of mating in young males disrupts cytokinesis in germline stem cells (GSCs). This defect leads to failure of abscission, preventing release of differentiating cells from the niche. GSC abscission failure was found to be caused by increased ecdysone hormone signaling induced upon mating, which leads to disrupted somatic encystment of the germline. Abscission failure is rescued by isolating males from females but recurs with resumption of mating. Importantly, reiterative mating also leads to increased GSC loss, requiring increased restoration of stem cells via symmetric renewal and de-differentiation. Together, these results suggest a model whereby acute mating results in hormonal changes that negatively impact GSC cytokinesis but preserves the stem cell population. |
Friday, June 7th - Cytoskeleton and Junctions |
| Madan, V., Albacete-Albacete, L., Jin, L., Scaturro, P., Watson, J. L., Muschalik, N., Begum, F., Boulanger, J., Bauer, K., Kiebler, M. A., Derivery, E., Bullock, S. L. (2023). HEATR5B associates with dynein-dynactin and promotes motility of AP1-bound endosomal membranes. The EMBO journal, 42(23):e114473 PubMed ID: 37872872
Summary: The microtubule motor dynein mediates polarised trafficking of a wide variety of organelles, vesicles and macromolecules. These functions are dependent on the dynactin complex, which helps recruit cargoes to dynein's tail and activates motor movement. How the dynein-dynactin complex orchestrates trafficking of diverse cargoes is unclear. This study identified HEATR5B, an interactor of the adaptor protein-1 (AP1) clathrin adaptor complex, as a novel player in dynein-dynactin function. HEATR5B was recovered in a biochemical screen for proteins whose association with the dynein tail is augmented by dynactin. HEATR5B binds directly to the dynein tail and dynactin and stimulates motility of AP1-associated endosomal membranes in human cells. It was also demonstrated that the Drosophila HEATR5B homologue is an essential gene that selectively promotes dynein-based transport of AP1-bound membranes to the Golgi apparatus. As HEATR5B lacks the coiled-coil architecture typical of dynein adaptors, these data point to a non-canonical process orchestrating motor function on a specific cargo. It was additionally shown that HEATR5B promotes association of AP1 with endosomal membranes independently of dynein. Thus, HEATR5B co-ordinates multiple events in AP1-based trafficking. | McParland, E. D., Amber Butcher, T., Gurley, N. J., Johnson, R. I., Slep, K. C., Peifer, M. (2023). The Dilute domain of Canoe is not essential for Canoe's role in linking adherens junctions to the cytoskeleton but contributes to robustness of morphogenesis. bioRxiv, PubMed ID: 37905001
Summary: Robust linkage between cell-cell adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The multidomain protein Drosophila Canoe and its mammalian homolog Afadin are critical for this linkage, and in their absence many events of morphogenesis fail. To define underlying mechanisms, this study took Canoe apart, using Drosophila as a model. Canoe and Afadin share five folded protein domains, followed by a large intrinsically disordered region. The largest of these folded domains is the Dilute domain, which is found in Canoe/Afadin, their paralogs, and members of the MyosinV family. To define the roles of Canoe's Dilute domain this study combined biochemical, genetic and cell biological assays. Use of the AlphaFold tools revealed the predicted structure of the Canoe/Afadin Dilute domain, providing similarities and contrasts with that of MyosinV. The biochemical data suggest one potential shared function: the ability to dimerize. Drosophila mutants were generated with the Dilute domain cleanly deleted. Surprisingly, these mutants are viable and fertile, and CanoeΔDIL protein localizes to adherens junctions and is enriched at junctions under tension. However, when the dose of Canoe&DeltaDIL protein was reduced in a sensitized assay, it becomes clear it does not provide full wildtype function. Further, canoe&DeltalDIL mutants have defects in pupal eye development, another process that requires orchestrated cell rearrangements. Together, these data reveal the robustness in AJ-cytoskeletal connections during multiple embryonic and postembryonic events, and the power of natural selection to maintain protein structure even in robust systems. |
| Zhu, H., O'Shaughnessy, B. (2023). Actomyosin pulsing rescues embryonic tissue folding from disruption by myosin fluctuations. Research square, PubMed ID: 37886516
Summary: During early development, myosin II mechanically reshapes and folds embryo tissue. A much studied example is ventral furrow formation in Drosophila, marking the onset of gastrulation. Furrowing is driven by contraction of actomyosin networks on apical cell surfaces, but how the myosin patterning encodes tissue shape is unclear, and elastic models failed to reproduce essential features of experimental cell contraction profiles. The myosin patterning exhibits substantial cell-to-cell fluctuations with pulsatile time-dependence, a striking but unexplained feature of morphogenesis in many organisms. Using biophysical modeling this study found that viscous forces offer the principal resistance to actomyosin-driven apical constriction. In consequence, tissue shape is encoded in the direction-dependent curvature of the myosin patterning which orients an anterior-posterior furrow. Tissue contraction is highly sensitive to cell-to-cell myosin fluctuations, explaining furrowing failure in genetically perturbed embryos whose fluctuations are temporally persistent. In wild-type embryos this disastrous outcome is averted by pulsatile myosin time-dependence, which rescues furrowing by eliminating high frequencies in the fluctuation power spectrum. This low pass filter mechanism may underlie the usage of actomyosin pulsing in diverse morphogenetic processes across many organisms.
| LaFountain, J. R., Jr., Seaman, C. E., Cohan, C. S., Oldenbourg, R. (2023). Sliding of antiparallel microtubules drives bipolarization of monoastral spindles. PubMed ID: 37812128
Summary: Time-lapse imaging with liquid crystal polarized light (LC-PolScope) and fluorescent speckle microscopy (FSM) enabled this study of spindle microtubules in monoastral spindles that were produced in crane-fly spermatocytes through flattening-induced centrosome displacement. Monoastral spindles are found in several other contexts: after laser ablation of one of a cell's two centrosomes, in Drosophila "urchin" mutants, in Sciara males, and in RNAi variants of Drosophila S2 cells. In all cases, just one pole has a centrosome (the astral pole); the other lacks a centrosome (the anastral pole). Thus, the question: How is the anastral half-spindle, lacking a centrosome, constructed? It was learned that monoastral spindles are assembled in two phases: Phase I assembles the astral half-spindle composed of centrosomal microtubules, and Phase II assembles microtubules of the anastral half through extension of new microtubule polymerization outward from the spindle's equatorial mid-zone. That process uses plus ends of existing centrosomal microtubules as guiding templates to assemble anastral microtubules of opposite polarity. Anastral microtubules slide outward with their minus ends leading, thereby establishing proper bipolarity just like in normal biastral spindles that have two centrosomes. |
| Zhu, Z., Becam, I., Tovey, C. A., Elfarkouchi, A., Yen, E. C., Bernard, F., Guichet, A., Conduit, P. T. (2023). Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes. J Cell Biol, 222(10) PubMed ID: 37698931
Summary: Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. This study shows that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. These data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated. | Li, L., Zhang, N., Beati, S. A. H., De Las Heras Chanes, J., di Pietro, F., Bellaiche, Y., Muller, H. J., Grosshans, J. (2024). Kinesin-1 patterns Par-1 and Rho signaling at the cortex of syncytial embryos of Drosophila. J Cell Biol, 223(1) PubMed ID: 37955925
Summary: The cell cortex of syncytial Drosophila embryos is patterned into cap and intercap regions by centrosomes, specific sets of proteins that are restricted to their respective regions by unknown mechanisms. This study found that Kinesin-1 is required for the restriction of plus- and minus-ends of centrosomal and non-centrosomal microtubules to the cap region, marked by EB1 and Patronin/Shot, respectively. Kinesin-1 also directly or indirectly restricts proteins and Rho signaling to the intercap, including the RhoGEF Pebble, Dia, Myosin II, Capping protein-α, and the polarity protein Par-1. Furthermore, it was found that Par-1 is required for cap restriction of Patronin/Shot, and vice versa Patronin, for Par-1 enrichment at the intercap. In summary, these data support a model that Kinesin-1 would mediate the restriction of centrosomal and non-centrosomal microtubules to a region close to the centrosomes and exclude Rho signaling and Par-1. In addition, mutual antagonistic interactions would refine and maintain the boundary between cap and intercap and thus generate a distinct cortical pattern. |
Thursday, June 6th - Drosophila as a Model of Human Diseases |
| Filosevic Vujnovic, A., Saftic Martinovic, L., Medija, M., Andretic Waldowski, R. (2023). Distinct and Dynamic Changes in the Temporal Profiles of Neurotransmitters in Drosophila melanogaster Brain following Volatilized Cocaine or Methamphetamine Administrations. Pharmaceuticals (Basel, Switzerland), 16(10) PubMed ID: 37895961
Summary: Due to similarities in genetics, cellular response, and behavior, Drosophila is used as a model organism in addiction research. A well-described behavioral response examined in flies is the induced increase in locomotor activity after a single dose of volatilized cocaine (vCOC) and volatilized methamphetamine (vMETH), the sensitivity, and the escalation of the locomotor response after the repeated dose, the locomotor sensitization. However, knowledge about how vCOC and vMETH affect different neurotransmitter systems over time is scarce. This study used LC-MS/MS to systematically examine changes in the concentration of neurotransmitters, metabolites and non-metabolized COC and METH in the whole head homogenates of male flies one to seven hours after single and double vCOC or vMETH administrations. vMETH leads to complex changes in the levels of examined substances over time, while vCOC strongly and briefly increases concentrations of dopamine, tyramine and octopamine followed by a delayed degradation into N-acetyl dopamine and N-acetyl tyramine. The first exposure to psychostimulants leads to significant and dynamic changes in the concentrations relative to the second administration when they are more stable over several hours. Further investigations are needed to understand neurochemical and molecular changes post-psychostimulant administration. | Min, Y., Wang, X., Is, O., Patel, T. A., Gao, J., Reddy, J. S., Quicksall, Z. S., Nguyen, T., Lin, S., Tutor-New, F. Q., Chalk, J. L., Mitchell, A. O., Crook, J. E., Nelson, P. T., Van Eldik, L. J., Golde, T. E., Carrasquillo, M. M., Dickson, D. W., Zhang, K., Allen, M., Ertekin-Taner, N. (2023). Cross species systems biology discovers glial DDR2, STOM, and KANK2 as therapeutic targets in progressive supranuclear palsy. Nat Commun, 14(1):6801 PubMed ID: 37919278
Summary: Progressive supranuclear palsy (PSP) is a neurodegenerative parkinsonian disorder characterized by cell-type-specific tau lesions in neurons and glia. Prior work uncovered transcriptome changes in human PSP brains, although their cell-specificity is unknown. Further, systematic data integration and experimental validation platforms to prioritize brain transcriptional perturbations as therapeutic targets in PSP are currently lacking. This study, combined bulk tissue (n = 408) and single nucleus RNAseq (n = 34) data from PSP and control brains with transcriptome data from a mouse tauopathy and experimental validations in Drosophila tau models for systematic discovery of high-confidence expression changes in PSP with therapeutic potential. This study discovered, replicated, and annotated thousands of differentially expressed genes in PSP, many of which reside in glia-enriched co-expression modules and cells. DDR2, STOM, and KANK2 were prioritized as promising therapeutic targets in PSP with striking cross-species validations. These findings and data are shared via interactive application tool PSP RNAseq Atlas . These findings reveal robust glial transcriptome changes in PSP, provide a cross-species systems biology approach, and a tool for therapeutic target discoveries in PSP with potential application in other neurodegenerative diseases. |
| He, M. F., Liu, C. Q., Zhang, X. X., Lin, Y. M., Mao, Y. L., Qiao, J. D. (2023). Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy. J Vis Exp, (200) PubMed ID: 37902337
Summary: Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, a novel and efficient protocol are presed for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level. | Al-Ayari, E. A., Shehata, M. G., El-Hadidi, M., Shaalan, M. G. (2023). In silico SNP prediction of selected protein orthologues in insect models for Alzheimer's, Parkinson's, and Huntington's diseases. Sci Rep, 13(1):18986 PubMed ID: 37923901
Summary: Alzheimer's, Parkinson's, and Huntington's are the most common neurodegenerative diseases that are incurable and affect the elderly population. Discovery of effective treatments for these diseases is often difficult, expensive, and serendipitous. Previous comparative studies on different model organisms have revealed that most animals share similar cellular and molecular characteristics. The meta-SNP tool includes four different integrated tools (SIFT, PANTHER, SNAP, and PhD-SNP) was used to identify non synonymous single nucleotide polymorphism (nsSNPs). Prediction of nsSNPs was conducted on three representative proteins for Alzheimer's, Parkinson's, and Huntington's diseases; APPl in Drosophila melanogaster, LRRK1 in Aedes aegypti, and VCPl in Tribolium castaneum. With the possibility of using insect models to investigate neurodegenerative diseases. It is concluded from the protein comparative analysis between different insect models and nsSNP analyses that D. melanogaster is the best model for Alzheimer's representing five nsSNPs of the 21 suggested mutations in the APPl protein. Aedes aegypti is the best model for Parkinson's representing three nsSNPs in the LRRK1 protein. Tribolium castaneum is the best model for Huntington's disease representing 13 SNPs of 37 suggested mutations in the VCPl protein. This study aimed to improve human neural health by identifying the best insect to model Alzheimer's, Parkinson's, and Huntington's. |
| Nithianandam, V., Bukhari, H., Leventhal, M. J., Battaglia, R. A., Dong, X., Fraenkel, E., Feany, M. B. (2023). Integrative analysis reveals a conserved role for the amyloid precursor protein in proteostasis during aging. Nat Commun, 14(1):7034 PubMed ID: 37923712
Summary: Aβ peptides derived from the amyloid precursor protein (APP) have been strongly implicated in the pathogenesis of Alzheimer's disease. However, the normal function of APP and the importance of that role in neurodegenerative disease is less clear. The Drosophila ortholog of APP, Appl, was uncovered in an unbiased forward genetic screen for neurodegeneration mutants. Comprehensive single cell transcriptional and proteomic studies of Appl mutant flies were performed to investigate Appl function in the aging brain. An unexpected role was found for Appl in control of multiple cellular pathways, including translation, mitochondrial function, nucleic acid and lipid metabolism, cellular signaling and proteostasis. A role for Appl in regulating autophagy through TGFβ signaling was mechanistically defined, and the broader relevance of these findings was documented using mouse genetic, human iPSC and in vivo tauopathy models. The results demonstrate a conserved role for APP in controlling age-dependent proteostasis with plausible relevance to Alzheimer's disease. | Xu, J., Deng, Z., Shang, S., Wang, C., Han, H. (2023). FUNDC1 collaborates with PINK1 in regulating mitochondrial Fission and compensating for PINK1 deficiency. Biochem Biophys Res Commun, 687:149210 PubMed ID: 37931419
Summary: Parkinson's disease is presently thought to have its molecular roots in the alteration of PINK1-mediated mitophagy and mitochondrial dynamics. Finding new suppressors of the pathway is essential for developing cutting-edge treatment approaches. This study shows that FUNDC1 suppressed PINK1 mutant phenotypes in Drosophila. The restoration of PINK1-deficient phenotypes through FUNDC1 is not reliant on its LC3-binding motif Y (18)L (21) or autophagy-related pathway. Moreover, the absence of Drp1 affects the phenotypic restoration of PINK1 mediated by FUNDC1 in flies. In summary, these findings have unveiled a fresh mechanism through which FUNDC1 compensates for the loss of PINK1, operating independently of autophagy but exerting its influence via interaction with Drp1. |
Wednesday, June 5th - Homologs of Drosophila Proteins |
| Cowell, L. M., King, M., West, H., Broadsmith, M., Genever, P., Pownall, M. E., Isaacs, H. V. (2023). Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development. PLoS One, 18(10):e0286040 PubMed ID: 37856433
Summary: Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. It is proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor.This study investigated the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. Xenopus Cic was characterized and was shown to be widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with this hypothesis, it was shown that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. Two of these genes were investigated as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b, which encode a leucine zipper transcription factor and a ras family GTPase, respectively. Cic consensus binding sites were identified in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. It was shown that expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, it was shown that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. The data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding. | Torres, H. M., Hinojosa, L., VanCleave, A. M., Rodezno, T., Westendorf, J. J., Tao, J. (2023). Hdac1 and Hdac2 positively regulate Notch1 gain-of-function pathogenic signaling in committed osteoblasts of male mice. Birth defects research, PubMed ID: 37921375
Summary: Skeletal development requires precise extrinsic and intrinsic signals to regulate processes that form and maintain bone and cartilage. Notch1 is a highly conserved signaling receptor that regulates cell fate decisions by controlling the duration of transcriptional bursts. Epigenetic molecular events reversibly modify DNA and histone tails by influencing the spatial organization of chromatin and can fine-tune the outcome of a Notch1 transcriptional response. Histone deacetylase 1 and 2 (HDAC1 and HDAC2) are chromatin modifying enzymes that mediate osteoblast differentiation. While an HDAC1-Notch interaction has been studied in vitro and in Drosophila, its role in mammalian skeletal development and disorders is unclear. Osteosclerosis is a bone disorder with an abnormal increase in the number of osteoblasts and excessive bone formation. This study, tested whether Hdac1/2 contribute to the pathogenesis of osteosclerosis in a murine model of the disease owing to conditionally cre-activated expression of the Notch1 intracellular domain in immature osteoblasts. RESULTS: Importantly, selective homozygous deletions of Hdac1/2 in osteoblasts partially alleviate osteosclerotic phenotypes (Col2.3kb-Cre; TG(RosaN1ICD/+) ; Hdac1(flox/flox) ; Hdac2(flox/flox) ) with a 40% decrease in bone volume and a 22% decrease in trabecular thickness in 4 weeks old when compared to male mice with heterozygous deletions of Hdac1/2 (Col2.3 kb-Cre; TG(RosaN1ICD/+) ; Hdac1(flox/+) ; Hdac2(flox/+) ). Osteoblast-specific deletion of Hdac1/2 in male and female mice results in no overt bone phenotype in the absence of the Notch1 gain-of-function (GOF) allele. These results provide evidence that Hdac1/2 contribute to Notch1 pathogenic signaling in the mammalian skeleton. This study on epigenetic regulation of Notch1 GOF-induced osteosclerosis may facilitate further mechanistic studies of skeletal birth defects caused by Notch-related GOF mutations in human patients, such as Adams-Oliver disease, congenital heart disease, and lateral meningocele syndrome. |
| Sauty, S. M., Yankulov, K. (2023). Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-modal active/silent state at the sub-telomeres of S. cerevisiae.Epigenetics & chromatin, 16(1):40 PubMed ID: 37858268
Summary: Classical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. This study used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the different roles of PCNA and histone chaperones in the stability and the propagation of repressed states at the sub-telomeres of S. cerevisiae. Position dependent transient repression or bi-modal expression of reporter genes is shown at the VIIL sub-telomere. Mutations in the replicative clamp POL30 (PCNA) or the deletion of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the deletion of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. The physical interaction of CAF1 and RRM3 with PCNA was analyzed, and the implications of these findings for understanding of the stability and transmission of the epigenetic state of the genes id discussed. There are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. Yhe roles of CAF1, RRM3 and ASF1 in these modes of gene repression were analyzed. It is suggestd that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing. | Duan, D., Lyu, W., Chai, P., Ma, S., Wu, K., Wu, C., Xiong, Y., Sestan, N., Zhang, K., Koleske, A. J. (2023). Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation. Curr Biol, 33(21):4582-4598.e4510 PubMed ID: 37858340
Summary: Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both in vitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. This study reports that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. Abl2 was found to promote MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ688-790. It is proposed that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue. |
| Kushwaha, A., Thakur, M. K. (2024). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Molecular neurobiology. 61(1):487-497 PubMed ID: 37626270
Summary: Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ Suv39h1 (a mammalian ortholog of the Drosophila melanogaster SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It has been reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. In the current study, an attempt was made to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. It was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia. | Liu, Q., Bell, B. J., Kim, D. W., Lee, S. S., Keles, M. F., Liu, Q., Blum, I. D., Wang, A. A., Blank, E. J., Xiong, J., Bedont, J. L., Chang, A. J., Issa, H., Cohen, J. Y., Blackshaw, S., Wu, M. N. (2023). A clock-dependent brake for rhythmic arousal in the dorsomedial hypothalamus. Nat Commun, 14(1):6381 PubMed ID: 37821426
Summary: Circadian clocks generate rhythms of arousal, but the underlying molecular and cellular mechanisms remain unclear. In Drosophila, the clock output molecule WIDE AWAKE (WAKE) labels rhythmic neural networks and cyclically regulates sleep and arousal. This study shows, in a male mouse model, that mWAKE/ANKFN1 labels a subpopulation of dorsomedial hypothalamus (DMH) neurons involved in rhythmic arousal and acts in the DMH to reduce arousal at night. In vivo Ca(2+) imaging reveals elevated DMH(mWAKE) activity during wakefulness and rapid eye movement (REM) sleep, while patch-clamp recordings show that DMH(mWAKE) neurons fire more frequently at night. Chemogenetic manipulations demonstrate that DMH(mWAKE) neurons are necessary and sufficient for arousal. Single-cell profiling coupled with optogenetic activation experiments suggest that GABAergic DMH(mWAKE) neurons promote arousal. Surprisingly, the data suggest that mWAKE acts as a clock-dependent brake on arousal during the night, when mice are normally active. mWAKE levels peak at night under clock control, and loss of mWAKE leads to hyperarousal and greater DMH(mWAKE) neuronal excitability specifically at night. These results suggest that the clock does not solely promote arousal during an animal's active period, but instead uses opposing processes to produce appropriate levels of arousal in a time-dependent manner. |
Tuesday, June 4th - Enhancers and Transcriptional Regulation |
| Chaubal, A., Waldern, J. M., Taylor, C., Laederach, A., Marzluff, W. F., Duronio, R. J. (2023). Coordinated expression of replication-dependent histone genes from multiple loci promotes histone homeostasis in Drosophila. Mol Biol Cell, 34(12):ar118 PubMed ID: 37647143
Summary: Production of large amounts of histone proteins during S phase is critical for proper chromatin formation and genome integrity. This process is achieved in part by the presence of multiple copies of replication dependent (RD) histone genes that occur in one or more clusters in metazoan genomes. In addition, RD histone gene clusters are associated with a specialized nuclear body, the histone locus body (HLB), which facilitates efficient transcription and 3' end-processing of RD histone mRNA. How all five RD histone genes within these clusters are coordinately regulated such that neither too few nor too many histones are produced, a process referred to as histone homeostasis, is not fully understood. This study explored the mechanisms of coordinate regulation between multiple RD histone loci in Drosophila melanogaster and Drosophila virilis. Evidence is provided for functional competition between endogenous and ectopic transgenic histone arrays located at different chromosomal locations in D. melanogaster that helps maintain proper histone mRNA levels. Consistent with this model, in both species it was found that individual histone gene arrays can independently assemble an HLB that results in active histone transcription. These findings suggest a role for HLB assembly in coordinating RD histone gene expression to maintain histone homeostasis. | Masuda, L. H. P., Sabino, A. U., Reinitz, J., Ramos, A. F., Machado-Lima, A., Andrioli, L. P. (2024). Global repression by tailless during segmentation. Dev Biol, 505:11-23 PubMed ID: 37879494
Summary: The early expression of tll in two gap domains in the segmentation cascade of Drosophila is unusual even for most other insects. This study investigated tll regulation on pair-rule stripes. With ectopic misexpression of tll unexpected repression was detected of almost all pair-rule stripes of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz). Examining Tll embryonic ChIP-chip data with regions mapped as Cis-Regulatory Modules (CRMs) of pair-rule stripes this study verified Tll interactions to these regions. With the ChIP-chip data Tll interactions to the CRMs of gap domains and in the misexpression assay, Tll-mediated repression on Kruppel (Kr), kni (kni) and giant (gt) according to their differential sensitivity to Tll. These results with gap genes confirmed previous data from the literature and argue against indirect repression roles of Tll in the striped pattern. Moreover, the prediction of Tll binding sites in the CRMs of eve stripes and the mathematical modeling of their removal using an experimentally validated theoretical framework shows effects on eve stripes compatible with the absence of a repressor binding to the CRMs. In addition, modeling increased tll levels in the embryo results in the differential repression of eve stripes, agreeing well with the results of the misexpression assay. In genetic assays eve 5 was investigate; it is strongly repressed by the ectopic domain and representative of more central stripes not previously implied to be under direct regulation of tll. While this stripe is little affected in tll-, its posterior border is expanded in gt- but detected with even greater expansion in gt-;tll-. The paper ends with a discussion of tll with key roles in combinatorial repression mechanisms to contain the expression of medial patterns of the segmentation cascade in the extremities of the embryo. |
| Ramalingam, V., Yu, X., Slaughter, B. D., Unruh, J. R., Brennan, K. J., Onyshchenko, A., Lange, J. J., Natarajan, M., Buck, M., Zeitlinger, J. (2023). Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development. Nat Commun, 14(1):5862 PubMed ID: 37735176
Summary: While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor that is one of the more than 25 different splice isoforms from the lola locus, this study showed that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters. | Lovero, D., Porcelli, D., Giordano, L., Lo Giudice, C., Picardi, E., Pesole, G., Pignataro, E., Palazzo, A., Marsano, R. M. (2023). Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors. Biology, 12(8) PubMed ID: 37627011
Summary: Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. This work have analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. The conservation of a short sequence is highlighted in the genes encoding subunits of the V-ATPase complex, and their assembly factors were analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding knowledge on the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization. |
| Eggers, N., Gkountromichos, F., Krause, S., Campos-Sparr, A., Becker, P. B. (2023). Physical interaction between MSL2 and CLAMP assures direct cooperativity and prevents competition at composite binding sites. Nucleic Acids Res, 51(17):9039-9054 PubMed ID: 37602401
Summary: MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. This study explored the nature of the cooperative binding to these GA-rich, composite sequence elements in reconstituted naive embryonic chromatin. The cooperativity was found to requires physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. The cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in the reconstitution, such as roX2 lncRNA. | Haroush, N., Levo, M., Wieschaus, E. F., Gregor, T. (2023). Functional analysis of the Drosophila eve locus in response to non-canonical combinations of gap gene expression levels. Dev Cell, 58(23):2789-2801.e2785 PubMed ID: 37890488
Summary: Transcription factor combinations play a key role in shaping cellular identity. However, the precise relationship between specific combinations and downstream effects remains elusive. This relationship was examined within the context of the Drosophila eve locus, which is controlled by gap genes. Spatiotemporal levels of four gap genes was measured in heterozygous and homozygous gap mutant embryos, and they were correlated with the striped eve activity pattern. Although changes in gap gene expression extend beyond the manipulated gene, the spatial patterns of Eve expression closely mirror canonical activation levels in wild type. Interestingly, some combinations deviate from the wild-type repertoire but still drive eve activation. Although in homozygous mutants some Eve stripes exhibit partial penetrance, stripes consistently emerge at reproducible positions, even with varying gap gene levels. The findings suggest a robust molecular canalization of cell fates in gap mutants and provide insights into the regulatory constraints governing multi-enhancer gene loci. |
Monday, June 3rd - Larval and Adult Neural Structurel, Development and Function |
| Mitchell, J. W., Midillioglu, I., Schauer, E., Wang, B., Han, C., Wildonger, J. (2023). Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons. PLoS Genet, 19(11):e1011025 PubMed ID: 37943859
Summary: Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. This study investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. CRISPR-Cas9 genome engineering approaches were used to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, an over-active channel mutant was found to severely reduce dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. The data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on these data, it is proposed that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites. | Zhao, A., Nern, A., Koskela, S., Dreher, M., Erginkaya, M., Laughland, C. W., Ludwigh, H., Thomson, A., Hoeller, J., Parekh, R., Romani, S., Bock, D. D., Chiappe, E., Reiser, M. B. (2023). A comprehensive neuroanatomical survey of the Drosophila Lobula Plate Tangential Neurons with predictions for their optic flow sensitivity. bioRxiv, PubMed ID: 37904921
Summary: Flying insects exhibit remarkable navigational abilities controlled by their compact nervous systems. Optic flow, the pattern of changes in the visual scene induced by locomotion, is a crucial sensory cue for robust self-motion estimation, especially during rapid flight. The best-known optic-flow sensitive neurons are the large tangential cells of the dipteran lobula plate. Most of these studies have focused on the large, Horizontal and Vertical System neurons, yet the lobula plate houses a much larger set of 'optic-flow' sensitive neurons, many of which have been challenging to unambiguously identify or to reliably target for functional studies. This study reports the comprehensive reconstruction and identification of the Lobula Plate Tangential Neurons in an Electron Microscopy (EM) volume of a whole Drosophila brain. This catalog of 58 LPT neurons (per brain hemisphere) contains many neurons that are described here for the first time and provides a basis for systematic investigation of the circuitry linking self-motion to locomotion control. Leveraging computational anatomy methods, It was estimated the visual motion receptive fields of these neurons, and their tuning was compared to the visual consequence of body rotations and translational movements. These neurons were matched, in most cases on a one-for-one basis, to stochastically labeled cells in genetic driver lines, to the mirror-symmetric neurons in the same EM brain volume, and to neurons in an additional EM data set. Using cell matches across data sets, the integration of optic flow patterns by neurons downstream of the LPTs were analyzed, Most central brain neurons establish sharper selectivity for global optic flow patterns than their input neurons. Furthermore, self-motion information extracted from optic flow was found to be processed in distinct regions of the central brain, pointing to diverse foci for the generation of visual behaviors |
| Jovanoski, K. D., Duquenoy, L., Mitchell, J., Kapoor, I., Treiber, C. D., Croset, V., Dempsey, G., Parepalli, S., Cognigni, P., Otto, N., Felsenberg, J., Waddell, S. (2023). Dopaminergic systems create reward seeking despite adverse consequences. Nature, 623(7986):356-365 PubMed ID: 37880370
Summary: Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking. This study describes dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. It is proposed that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals. | Ganguly, I., Heckman, E. L., Litwin-Kumar, A., Clowney, E. J., Behnia, R. (2023). Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body. bioRxiv, PubMed ID: 37873086
Summary: The arthropod mushroom body is well-studied as an expansion layer that represents olfactory stimuli and links them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their tuning and function are poorly understood. This study used the FlyWire adult whole-brain connectome to identify inputs to visual Kenyon cells. The types of visual neurons identified are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual projection neurons presynaptic to Kenyon cells receive input from large swathes of visual space, while local visual interneurons, providing smaller fractions of input, receive more spatially restricted signals that may be tuned to specific features of the visual scene. Like olfactory Kenyon cells, visual Kenyon cells receive sparse inputs from different combinations of visual channels, including inputs from multiple optic lobe neuropils. The sets of inputs to individual visual Kenyon cells are consistent with random sampling of available inputs. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the expansion coding properties appear different, with a specific repertoire of visual inputs projecting onto a relatively small number of visual Kenyon cells. |
| Anthoney, N., Tainton-Heap, L., Luong, H., Notaras, E., Kewin, A. B., Zhao, Q., Perry, T., Batterham, P., Shaw, P. J., van Swinderen, B. (2023). Experimentally induced active and quiet sleep engage non-overlapping transcriptional programs in Drosophila. Elife, 12 PubMed ID: 37910019
Summary: Sleep in mammals can be broadly classified into two different physiological categories: rapid eye movement (REM) sleep and slow-wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. This study compared two commonly used approaches for studying sleep experimentally in Drosophila: optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, gaboxadol. These different sleep-induction methods have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions. | Yang, H. H., Brezovec, L. E., Capdevila, L. S., Vanderbeck, Q. X., Adachi, A., Mann, R. S., Wilson, R. I. (2023). Fine-grained descending control of steering in walking Drosophila. bioRxiv, PubMed ID: 37904997
Summary: . . Locomotion involves rhythmic limb movement patterns that originate in circuits outside the brain. Purposeful locomotion requires descending commands from the brain, but how these commands are structured is not understood. This study investigated this issue, focusing on the control of steering in walking Drosophila. First, different limb "gestures" are described associated with different steering maneuvers. Next, a set of descending neurons was identified whose activity predicts steering. Focusing on two descending cell types downstream from distinct brain networks, this study showed that they evoke specific limb gestures: one lengthens strides on the outside of a turn, while the other attenuates strides on the inside of a turn. Notably, a single descending neuron can have opposite effects during different locomotor rhythm phases, and networks were identified positioned to implement this phase-specific gating. Together, these results show how purposeful locomotion emerges from brain cells that drive specific, coordinated modulations of low-level patterns. |
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