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Brantley, S. E., Stouthamer, C. M., Kr, P., Fischer, M. L., Hill, J., Schlenke, T. A., Mortimer, N. T. (2024). Host JAK-STAT activity is a target of parasitoid wasp virulence strategies. PLoS pathogens, 20(7):e1012349 PubMed ID: 38950076
Summary:
Innate immune responses that allow hosts to survive infection depend on the action of multiple conserved signaling pathways. Pathogens and parasites in turn have evolved virulence factors to target these immune signaling pathways in an attempt to overcome host immunity. Consequently, the interactions between host immune molecules and pathogen virulence factors play an important role in determining the outcome of an infection. The immune responses of Drosophila melanogaster provide a valuable model to understand immune signaling and host-pathogen interactions. Flies are commonly infected by parasitoid wasps and mount a coordinated cellular immune response following infection. This response is characterized by the production of specialized blood cells called lamellocytes that form a tight capsule around wasp eggs in the host hemocoel. The conserved JAK-STAT signaling pathway has been implicated in lamellocyte proliferation and is required for successful encapsulation of wasp eggs. Activity of Stat92E, the D. melanogaster STAT ortholog, is induced in immune tissues following parasitoid infection. Virulent wasp species are able to suppress Stat92E activity during infection, suggesting they target JAK-STAT pathway activation as a virulence strategy. Furthermore, two wasp species (Leptopilina guineaensis and Ganaspis xanthopoda) suppress phenotypes associated with a gain-of-function mutation in hopscotch, the D. melanogaster JAK ortholog, indicating that they inhibit the activity of the core signaling components of the JAK-STAT pathway. These data suggest that parasitoid wasp virulence factors block JAK-STAT signaling to overcome fly immune defenses.
| Chasse, A. Y., Bandyadka, S., Wertheimer, M. C., Serizier, S. B., McCall, K. (2024). Professional phagocytes are recruited for the clearance of obsolete nonprofessional phagocytes in the Drosophila ovary. Frontiers in immunology 15:1389674 PubMed ID: 38994369
Summary:
Cell death is an important process in the body, as it occurs throughout every tissue during development, disease, and tissue regeneration. Phagocytes are responsible for clearing away dying cells and are typically characterized as either professional or nonprofessional phagocytes. Professional phagocytes, such as macrophages, are found in nearly every part of the body while nonprofessional phagocytes, such as epithelial cells, are found in every tissue type. However, there are organs that are considered "immune-privileged" as they have little to no immune surveillance and rely on nonprofessional phagocytes to engulf dying cells. These organs are surrounded by barriers to protect the tissue from viruses, bacteria, and perhaps even immune cells. The Drosophila ovary is considered immune-privileged, however the presence of hemocytes, the macrophages of Drosophila, around the ovary suggests they may have a potential function. This study analyze hemocyte localization and potential functions in response to starvation-induced cell death in the ovary. Hemocytes were found to accumulate in the oviduct in the vicinity of mature eggs and follicle cell debris. Genetic ablation of hemocytes revealed that the presence of hemocytes affects oogenesis and that they phagocytose ovarian cell debris and in their absence fecundity decreases. Unpaired3, an IL-6 like cytokine, was found to be required for the recruitment of hemocytes to the oviduct to clear away obsolete follicle cells. These findings demonstrate a role for hemocytes in the ovary, providing a more thorough understanding of phagocyte communication and cell clearance in a previously thought immune-privileged organ.
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Yao, X., He, Y., Zhu, C., Yang, S., Wu, J., Ma, F., Jin, P. (2024). miR-190 restores the innate immune homeostasis of Drosophila by directly inhibiting Tab2 in Imd pathway. Microbes and infection:105399 PubMed ID: 39084397
Summary:
The Drosophila Imd pathways are well-known mechanisms involved in innate immunity responsible for Gram-negative (G-) bacterial infection. The intensity and durability of immunity need to be finely regulated to keep sufficient immune activation meanwhile avoid excessive immune response. This study firstly demonstrated that miR-190 can downregulate the expression levels of antimicrobial peptides (AMPs) in the Imd immune pathway after Escherichia coli infection using the miR-190 overexpression flies and the miR-190KO/+ flies. Secondly, miR-190 overexpression significantly reduces while miR-190 KO increases Drosophila survival rates upon lethal Enterobacter cloacae infection. Thirdly, this study further demonstrated that miR-190 negatively regulates innate immune responses by directly targeting both RA/RB and RC isoforms of Tab2. In addition, the dynamic expression pattern of AMPs (Dpt, AttA, CecA1), miR-190 and Tab2 in the wild-type flies reveals that miR-190 play an important role in Drosophila immune homeostasis restoration at the late stage of E. coli infection. Collectively, this study reveals that miR-190 can downregulate the expression of AMPs by targeting Tab2 and promote immune homeostasis restoration in Drosophila Imd pathway. This study provides new insights into the regulatory mechanism of animal innate immune homeostasis.
| Chauhan, M., Martinak, P. E., Hollenberg, B. M., Goodman, A. G. (2024). Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus. bioRxiv, PubMed ID: 38948804
Summary:
The Toll pathway plays a pivotal role in innate immune responses against pathogens. The evolutionary conserved pathogen recognition receptors (PRRs), including Toll like receptors (TLRs), play a crucial role in recognition of pathogen associated molecular patterns (PAMPs). The Drosophila genome encodes nine Toll receptors that are orthologous to mammalian TLRs. While mammalian TLRs directly recognize PAMPs, most Drosophila Tolls recognize the proteolytically cleaved ligand Spatzle to activate downstream signaling cascades. This study demonstrated that Toll-9 is crucial for antiviral immunity against Drosophila C virus (DCV), a natural pathogen of Drosophila A transposable element insertion in the Toll-9 gene renders the flies more susceptible to DCV. The stable expression of Toll-9 in S2 cells confers resistance against DCV infection by upregulation of the RNAi pathway. Toll-9 promotes the dephosphorylation of AKT, resulting in the induction of antiviral RNAi genes to inhibit DCV replication. Toll-9 localizes to the endosome where it binds dsRNA, suggesting its role to detect viral dsRNA. Toll-9 also induces apoptosis during DCV infection, contributing to its antiviral role. Together, this work identifies the role of Toll-9 in antiviral immunity against DCV infection through its ability to bind dsRNA and induce AKT-mediated RNAi antiviral immunity.
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Mallick, S., Kenney, E., Eleftherianos, I. (2024). The Activin Branch Ligand Daw Regulates the Drosophila melanogaster Immune Response and Lipid Metabolism against the Heterorhabditis bacteriophora Serine Carboxypeptidase. Int J Mol Sci, 25(14) PubMed ID: 39063211
Summary:
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Despite impressive advances in the broad field of innate immunity, understanding of the molecules and signaling pathways that control the host immune response to nematode infection remains incomplete. Recent work has shown that Transforming Growth Factor-β (TGF-β) signaling in the fruit fly Drosophila melanogaster is activated by nematode infection and certain TGF-β superfamily members regulate the D. melanogaster anti-nematode immune response. This study investigated the effect of an entomopathogenic nematode infection factor on host TGF-β pathway regulation and immune function. Heterorhabditis bacteriophora serine carboxypeptidase activates the Activin branch in D. melanogaster adults and the immune deficiency pathway in Activin-deficient flies. The carboxypeptidase affects hemocyte numbers and survival in flies deficient for Activin (Dawdle) signaling, and causes increased intestinal steatosis in Activin-deficient flies. Thus, insights into the D. melanogaster signaling pathways and metabolic processes interacting with H. bacteriophora pathogenicity factors will be applicable to entomopathogenic nematode infection of important agricultural insect pests and vectors of disease.
| Wang, X., Wei, D., Pan, Y., Liu, J., Xiao, X., Xia, Q., Wang, F. (2024). A cryptic homotypic interaction motif of insect STING is required for its antiviral signaling. Dev Comp Immunol, 159:105224 PubMed ID: 38969190
Summary:
Stimulator of interferon genes (STING) mediates innate immune response upon binding to cyclic GMP-AMP (cGAMP). It recruits tank-binding kinase 1 (TBK1) and transcription factor interferon regulatory factor 3 (IRF3) through its C-terminal tail and facilitates TBK1-dependent phosphorylation of IRF3 via forming STING polymers in mammalian cells. However, the mechanism behind STING-mediated activation of NF-kappaB transcription factor, Relish, in insect cells is unknown. This study revealed that insect STING formed oligomers and the cryptic RIP homotypic interaction motif (cRHIM) was required for its oligomerization and its anti-viral functions. Cells expressing cRHIM-deficient mutants exhibited lower levels of anti-viral molecules, higher viral load after viral infection and weak activation of Relish. Moreover, under cGAMP stimulation, insect STING interacted with IMD, and deletion of the cRHIM motif on either protein prevented this interaction. Finally, this study demonstrated that cGAMP enhanced the amyloid-like property of insect STING aggregates by ThT staining. In summary, this research showed that insect STING employed a homotypic motif to form intermolecular interactions that are essential for its antiviral signaling.
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Monday, June 9th - Enhancers and Transcriptional regulation |
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Hodkinson, L. J., Rieder, L. E. (2024). Cis element length variability does not confer differential transcription factor occupancy at the D. melanogaster histone locus. bioRxiv, PubMed ID: 38979213
Summary:
Histone genes require precise regulation to maintain histone homeostasis and ensure nucleosome stoichiometry. Animal histone genes often have unique clustered genomic organization. However, there is variability of histone gene number and organization as well as differential regulation of the histone genes across species. The Drosophila melanogaster histone locus has unique organizational characteristics as it exists as a series of ~100 highly regular, tandemly repeated arrays of the 5 replication-dependent histone genes at a single locus. Yet D. melanogaster are viable with only 12 transgenic histone gene arrays. It was hypothesized that the histone genes across the locus are differentially regulated. The GA-repeat within the H3/H4 promoter was discovered to be the only variable sequence across the histone gene arrays. The H3/H4 promoter GA-repeat is targeted by CLAMP to promote histone gene expression. This study also showed two additional GA-binding transcription factors, GAGA Factor and Pipsqueak, target the GA-repeat. When CLAMP and GAF targeting was examined, neither CLAMP nor GAF show bias for any GA-repeat lengths. Furthermore, it was found that the distribution of GA-repeats targeted by both CLAMP and GAF do not change throughout early development. Together the results suggest that the transcription factors targeting the H3/H4 GA-repeat do not impact differential regulation of the histone genes, but indicate that future studies should interrogate additional cis elements or factors that impact histone gene regulation.
| Fogarty, E. A., Buchert, E. M., Ma, Y., Nicely, A. B., Buttitta, L. A. (2024). Transcriptional repression and enhancer decommissioning silence cell cycle genes in postmitotic tissues. bioRxiv, PubMed ID: 38766255
Summary:
The mechanisms that maintain a non-cycling status in postmitotic tissues are not well understood. Many cell cycle genes have promoters and enhancers that remain accessible even when cells are terminally differentiated and in a non-cycling state, suggesting their repression must be maintained long term. In contrast, enhancer decommissioning has been observed for rate-limiting cell cycle genes in the Drosophila wing, a tissue where the cells die soon after eclosion, but it has been unclear if this also occurs in other contexts of terminal differentiation. This study shows that enhancer decommissioning also occurs at specific, rate-limiting cell cycle genes in the long-lived tissues of the Drosophila eye and brain, and it is proposed that this loss of chromatin accessibility may help maintain a robust postmitotic state.The decommissioned enhancers were examined at specific rate-limiting cell cycle genes; they were shown to encode dynamic temporal and spatial expression patterns that include shared, as well as tissue-specific elements, resulting in broad gene expression with developmentally controlled temporal regulation. This analysis was extended to cell cycle gene expression and chromatin accessibility in the mammalian retina using a published dataset, and the principles of cell cycle gene regulation identified in terminally differentiating Drosophila tissues were found to be conserved in the differentiating mammalian retina. A robust, non-cycling status is proposed to be maintained in long-lived postmitotic tissues through a combination of stable repression at most cell cycle gens, alongside enhancer decommissioning at specific rate-limiting cell cycle genes.
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Versluis, P., Graham, T. G. W., Eng, V., Ebenezer, J., Darzacq, X., Zipfel, W. R., Lis, J. T. (2024). Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation. Mol Cell, 84(15):2856-2869 PubMed ID: 39121843
Summary:
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. To investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins were constructed using CRISPR-Cas9, allowing tracking a population of molecules at the induced .htm">Hsp70 loci in Drosophila melanogaster polytene chromosomes. Early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
| Sloutskin, A., Itzhak, D., Vogler, G., Pozeilov, H., Ideses, D., Alter, H., Adato, O., Shachar, H., Doniger, T., Shohat-Ophir, G., Frasch, M., Bodmer, R., Duttke, S. H., Juven-Gershon, T. (2024). From promoter motif to cardiac function: a single DPE motif affects transcription regulation and organ function in vivo. Development, 151(14) PubMed ID: 38958007
Summary:
Transcription initiates at the core promoter, which contains distinct core promoter elements. This study highlights the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. The importance is demonstrate in vivo of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach using both CRISPR and nascent transcriptomics, the effects are shown of mutating a single core promoter element within the natural context. Specifically, the downstream core promoter element (DPE) was targeted of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7 bp substitution mutation results in massive perturbation of the Tinman regulatory network that orchestrates dorsal musculature, which is manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features, and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation.
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Hodkinson, L. J., Gross, J., Schmidt, C. A., Diaz-Saldana, P. P., Aoki, T., Rieder, L. E. (2024). Sequence reliance of the Drosophila context-dependent transcription factor CLAMP. Genetics, 227(3) PubMed ID: 38775472
Summary:
Despite binding similar cis elements in multiple locations, a single transcription factor (TF) often performs context-dependent functions at different loci. How factors integrate cis sequence and genomic context is still poorly understood and has implications for off-target effects in genetic engineering. The Drosophila context-dependent TF chromatin-linked adaptor for male-specific lethal proteins (CLAMP) targets similar GA-rich cis elements on the X-chromosome and at the histone gene locus but recruits very different, locus-specific factors. This study discovered that CLAMP leverages information from both cis element and local sequence to perform context-specific functions. These observations imply the importance of other cues, including protein-protein interactions and the presence of additional cofactors.
| DeHaro-Arbona, F. J., Roussos, C., Baloul, S., Townson, J., Gomez Lamarca, M. J., Bray, S. (2024). Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind. Elife, 12 PubMed ID: 38727722
Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, this study revealed that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.
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Friday, June 6th - Synapse and Vesicles |
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Chang, Y. C., Peng, Y. J., Lee, J. Y., Chag, K. T. (2024). Peripheral glia and neurons jointly regulate activity-induced synaptic remodeling at the Drosophila neuromuscular junction. bioRxiv, PubMed ID: 39005352
Summary:
In the nervous system, reliable communication depends on the ability of neurons to adaptively remodel their synaptic structure and function in response to changes in neuronal activity. While neurons are the main drivers of synaptic plasticity, glial cells are increasingly recognized for their roles as active modulators. However, the underlying molecular mechanisms remain unclear. In this study, using Drosophila neuromuscular junction as a model system for a tripartite synapse, this study showed that peripheral glial cells collaborate with neurons at the NMJ to regulate activity-induced synaptic remodeling, in part through a protein called shriveled (Shv). Shv is an activator of integrin signaling previously shown to be released by neurons during intense stimulation at the fly NMJ to regulate activity-induced synaptic remodeling. This study demonstrate that Shv is also present in peripheral glia, and glial Shv is both necessary and sufficient for synaptic remodeling. However, unlike neuronal Shv, glial Shv does not activate integrin signaling at the NMJ. Instead, it regulates synaptic plasticity in two ways: 1) maintaining the extracellular balance of neuronal Shv proteins to regulate integrin signaling, and 2) controlling ambient extracellular glutamate concentration to regulate postsynaptic glutamate receptor abundance. Loss of glial cells showed the same phenotype as loss of Shv in glia. Together, these results reveal that neurons and glial cells homeostatically regulate extracellular Shv protein levels to control activity-induced synaptic remodeling. Additionally, peripheral glia maintains postsynaptic glutamate receptor abundance and contribute to activity-induced synaptic remodeling by regulating ambient glutamate concentration at the fly NMJ.
| Kaneko, T., Li, R., He, Q., Yang, L., Ye, B. (2024). Transsynaptic BMP Signaling Regulates Fine-Scale
Topography between Adjacent Sensory Neurons. eNeuro, 11(8) PubMed ID: 39137988
Summary:
Sensory axons projecting to the central nervous system are organized into topographic maps that represent the locations of sensory stimuli. In some sensory systems, even adjacent sensory axons are arranged topographically, forming "fine-scale" topographic maps. Although several broad molecular gradients are known to instruct coarse topography, little is known about the molecular signaling that regulates fine-scale topography at the level of two adjacent axons. This study provides evidence that transsynaptic bone morphogenetic protein (BMP) signaling mediates local interneuronal communication to regulate fine-scale topography in the nociceptive system of Drosophila larvae. The topographic separation of the axon terminals of adjacent nociceptors requires their common postsynaptic target, the A08n neurons. This phenotype is recapitulated by knockdown of the BMP ligand, Decapentaplegic (Dpp), in these neurons. In addition, removing the Type 2 BMP receptors or their effector (Mad transcription factor) in single nociceptors impairs the fine-scale topography, suggesting the contribution of BMP signaling originated from A08n. This signaling is likely mediated by phospho-Mad in the presynaptic terminals of nociceptors to ensure local interneuronal communication. Finally, reducing Dpp levels in A08n reduces the nociceptor-A08n synaptic contacts. These data support that transsynaptic BMP signaling establishes the fine-scale topography by facilitating the formation of topographically correct synapses. Local BMP signaling for synapse formation may be a developmental strategy that independently regulates neighboring axon terminals for fine-scale topography.
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Boerner, J., Robbins, K., Murphey, R. (2024). Laser Cell Ablation in Intact Drosophila Larvae Reveals Synaptic Competition. J Vis Exp, (209) PubMed ID: 39141541
Summary:
The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3(rd) instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.
| Welch, L. G., Muschalik, N., Munro, S. (2024). The FAM114A proteins are adaptors for the recycling of Golgi enzymes. J Cell Sci, 137(17) PubMed ID: 39129673
Summary:
Golgi-resident enzymes remain in place while their substrates flow through from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the Golgi to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and one COPI adaptor protein, GOLPH3 (see Drosophila sauron), acts to recruit enzymes into vesicles in part of the stack. This study used proximity biotinylation to identify further components of intra-Golgi vesicles and found FAM114A2, a cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1, showed that they bind to Golgi-resident membrane proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not cause substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Drosophila mutants lacking FAM114A have defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins bind Golgi enzymes and are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.
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Xu, C., Li, Z., Lyu, C., Hu, Y., McLaughlin, C. N., Wong, K. K. L., Xie, Q., Luginbuhl, D. J., Li, H., Udeshi, N. D., Svinkina, T., Mani, D. R., Han, S., Li, T., Li, Y., Guajardo, R., Ting, A. Y., Carr, S. A., Li, J., Luo, L. (2024). Molecular and cellular mechanisms of teneurin signaling in synaptic partner matching. Cell, 187(18):5081-5101 PubMed ID: 38996528
Summary:
In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. HThis study used in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced understanding of both cellular and molecular mechanisms of synaptic partner matching.
| Mou, W., Cui, Y. (2024). Enhancing neuronal reticulophagy: a strategy for combating aging and APP toxicity. Autophagy, 20(12):2819-2820 PubMed ID: 38963012
Summary:
Reticulophagy, which directs the endoplasmic reticulum (ER) to the phagophore for sequestration within an autophagosome and subsequent lysosomal degradation via specific receptors, is essential for ER quality control and is implicated in various diseases. This study utilizes Drosophila to establish an in vivo model for reticulophagy. Starvation-induced reticulophagy is detected across multiple tissues in Drosophila. Whole-body upregulation or downregulation of the expression of reticulophagy receptors, atl and Rtnl1, negatively affects fly health. Notably, moderate upregulation of reticulophagy in neuronal tissues by overexpressing these receptors reduces age-related degeneration. In a Drosophila Alzheimer model expressing human APP (amyloid beta precursor protein), reticulophagy is compromised. Correcting reticulophagy by enhancing atl and Rtnl1 expression in the neurons promotes APP degradation, significantly reducing neurodegenerative symptoms. However, overexpression of mutated atl and Rtnl1 , which disrupts the interaction of the corresponding proteins with Atg8, does not alleviate these symptoms, emphasizing the importance of receptor functionality. These findings support modulating reticulophagy as a therapeutic strategy for aging and neurodegenerative diseases associated with ER protein accumulation.
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Wednesday June 4th - Disease Models |
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Yuan, Y., Yu, L., Zhuang, X., Wen, D., He, J., Hong, J., Xie, J., Ling, S., Du, X., Chen, W., Wang, X. (2025). Drosophila models used to simulate human ATP1A1 gene mutations that cause Charcot-Marie-Tooth type 2 disease and refractory seizures. Neural Regen Res, 20(1):265-276 PubMed ID: 38767491
Summary:
Certain amino acids changes in the human Na+/K+-ATPase pump, ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1), cause Charcot-Marie-Tooth disease type 2 (CMT2) disease and refractory seizures. To develop in vivo models to study the role of Na+/K+-ATPase in these diseases, the Drosophila gene homolog, Atpα, was modified to mimic the human ATP1A1 gene mutations that cause CMT2. Mutations located within the helical linker region of human ATP1A1 (I592T, A597T, P600T, and D601F) were simultaneously introduced into endogenous Drosophila Atpα by CRISPR/Cas9-mediated genome editing, generating the AtpαTTTF model. In addition, the same strategy was used to generate the corresponding single point mutations in flies (AtpαI571T, AtpαA576T, AtpαP579T, and AtpαD580F). Moreover, a deletion mutation (Atpαmut) that causes premature termination of translation was generated as a positive control. Of these alleles, two were found that could be maintained as homozygotes (AtpαI571T and AtpαP579T). Three alleles (AtpαA576T, AtpαP579 and AtpαD580F) can form heterozygotes with the Atpαmut allele. The Atpα allele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila. Flies heterozygous for AtpαTTTF mutations have motor performance defects, a reduced lifespan, seizures, and an abnormal neuronal morphology. These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.
| Catterson, J. H., Mouofo, E. N., Lopez De Toledo Soler, I., Lean, G., Dlamini, S., Liddell, P., Voong, G., Katsinelos, T., Wang, Y. C., Schoovaerts, N., Verstreken, P., Spires-Jones, T. L., Durrant, C. S. (2024). Drosophila appear resistant to trans-synaptic tau propagation. Brain communications, 6(4):fcae256 PubMed ID: 39130515
Summary:
Alzheimer's disease is the most common cause of dementia in the elderly, prompting extensive efforts to pinpoint novel therapeutic targets for effective intervention. Among the hallmark features of Alzheimer's disease is the development of neurofibrillary tangles comprised of hyperphosphorylated tau protein, whose progressive spread throughout the brain is associated with neuronal death. Trans-synaptic propagation of tau has been observed in mouse models, and indirect evidence for tau spread via synapses has been observed in human Alzheimer's disease. Halting tau propagation is a promising therapeutic target for Alzheimer's disease; thus, a scalable model system to screen for modifiers of tau spread would be very useful for the field. To this end, this study sought to emulate the trans-synaptic spread of human tau in Drosophila melanogaster. Employing the trans-Tango circuit mapping technique, this study investigated whether tau spreads between synaptically connected neurons. Immunohistochemistry and confocal imaging were used to look for tau propagation. Examination of hundreds of flies expressing four different human tau constructs in two distinct neuronal populations reveals a robust resistance in Drosophila to the trans-synaptic spread of human tau. This resistance persisted in lines with concurrent expression of amyloid-β, in lines with global human tau knock-in to provide a template for human tau in downstream neurons, and with manipulations of temperature. These negative data are important for the field as this study established that Drosophila expressing human tau in subsets of neurons are unlikely to be useful to perform screens to find mechanisms to reduce the trans-synaptic spread of tau. The inherent resistance observed in Drosophila may serve as a valuable clue, offering insights into strategies for impeding tau spread in future studies.
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Lewis, S. A., Forstrom, J., Tavani, J., Schafer, R., Tiede, Z., Padilla-Lopez, S. R., Kruer, M. C. (2024). eIF2alpha phosphorylation evokes dystonia-like movements with D2-receptor and cholinergic origin and abnormal neuronal connectivity. bioRxiv, PubMed ID: 38798458
Summary:
Dystonia is the 3(rd) most common movement disorder. Dystonia is acquired through either injury or genetic mutations, with poorly understood molecular and cellular mechanisms. Eukaryotic initiation factor alpha (eIF2α) controls cell state including neuronal plasticity via protein translation control and expression of ATF4. Dysregulated eIF2α phosphorylation (eIF2α-P) occurs in dystonia patients and models including DYT1 (Drosophila Torsin), but the consequences are unknown. This study increased/decreased eIF2α-P and tested motor control and neuronal properties in a Drosophila model. Bidirectionally altering eIF2α-P produced dystonia-like abnormal posturing and dyskinetic movements in flies. These movements were also observed with expression of the DYT1 risk allele. Cholinergic and D2-receptor neuroanatomical origins were identified of these dyskinetic movements caused by genetic manipulations to dystonia molecular candidates eIF2α-P, ATF4, or DYT1, with evidence for decreased cholinergic release. In vivo, increased and decreased eIF2α-P increase synaptic connectivity at the NMJ with increased terminal size and bouton synaptic release sites. Long-term treatment of elevated eIF2α-P with ISRIB restored adult longevity, but not performance in a motor assay. Disrupted eIF2α-P signaling may alter neuronal connectivity, change synaptic release, and drive motor circuit changes in dystonia.
| Hendricks, E. L., Linskey, N., Smith, I. R., Liebl, F. L. W. (2024). Kismet/CHD7/CHD8 and Amyloid Precursor Protein-like Regulate Synaptic Levels of Rab11 at the Drosophila Neuromuscular Junction. Int J Mol Sci, 25(15) PubMed ID: 39125997
Summary:
The transmembrane protein β-amyloid precursor protein (APP) is central to the pathophysiology of Alzheimer's disease (AD). The β-amyloid hypothesis posits that aberrant processing of APP forms neurotoxic β-amyloid aggregates, which lead to the cognitive impairments observed in AD. Although numerous additional factors contribute to AD, there is a need to better understand the synaptic function of APP. This study found that Drosophila APP-like (APPL) has both shared and non-shared roles at the synapse with Kismet (Kis), a chromatin helicase binding domain (CHD) protein. Kis is the homolog of CHD7 and CHD8, both of which are implicated in neurodevelopmental disorders including CHARGE Syndrome and autism spectrum disorders, respectively. Loss of function mutations in kis> and animals expressing human APP and BACE in their central nervous system show reductions in the glutamate receptor subunit, GluRIIC, the GTPase Rab11, and the bone morphogenetic protein (BMP), pMad, at the Drosophila larval neuromuscular junction (NMJ). Similarly, processes like endocytosis, larval locomotion, and neurotransmission are deficient in these animals. These pharmacological and epistasis experiments indicate that there is a functional relationship between Kis and APPL, but Kis does not regulate appl expression at the larval NMJ. Instead, Kis likely influences the synaptic localization of APPL, possibly by promoting rab11 transcription. These data identify a potential mechanistic connection between chromatin remodeling proteins and aberrant synaptic function in AD.
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Nair, S., Jiang, Y., Marchal, I. S., Chernobelsky, E., Huang, H. W., Suh, S., Pan, R., Kong, X. P., Ryoo, H. D., Sigurdsson, E. M. (2024). Anti-tau single domain antibodies clear pathological tau and attenuate its toxicity and related functional defects. Cell Death Dis, 15(7):543 PubMed ID: 39079958
Summary:
Tauopathies are a group of neurodegenerative diseases characterized by the presence of tau inclusions. This study has developed over fifty anti-tau single-domain antibodies (sdAbs) derived from phage display libraries of a llama immunized with recombinant and pathological tau immunogens. This study examined the therapeutic potential of four of these sdAbs in a Drosophila tauopathy model following their transgenic expression either in all neurons or neuronal subtypes. Three of these sdAbs showed therapeutic potential in various assays, effectively clearing pathological tau and attenuating or preventing tau-induced phenotypes that typically manifest as defects in neuronal axonal transport, neurodegeneration, functional impairments, and shortened lifespan. Of these three, one sdAb was superior in every assay, which may at least in part be attributed to its tau-binding epitope. These findings support its development as a gene therapy for tauopathies.
| Singh, M. K., Ryu, T. H., Nguyen, M. N., Yu, K. (2024). Inhibition of high-fat diet-induced miRNA ameliorates tau toxicity in Drosophila. Biochem Biophys Res Commun, 733:150446 PubMed ID: 39067249
Summary:
Alzheimer's disease (AD), caused by amyloid β (Aβ) plaques and Tau tangles, is a neurodegenerative disease characterized by progressive memory impairment and cognitive dysfunction. High-fat diet (HFD), which induces type 2 diabetes, exacerbates Aβ plaque deposition in the brain. To investigate the function of HFD in Tau-mediated AD, an HFD was fed to the Drosophila Tau model and found that HFD aggravates Tau-induced neurological phenotypes. Since microRNAs (miRNAs) are biomarkers for diabetes and AD, the expression levels of common miRNAs of HFD and AD was evaluated in HFD-fed Tau model fly brains. Among the common miRNAs, the expression levels of Let-7 and miR-34 were increased. The inhibition of these miRNAs alleviates Tau-mediated AD phenotypes. This research provides valuable insights into how HFD accelerates tau toxicity. Additionally, this work highlights the therapeutic potential of targeting Let-7 and miR-34 to develop innovative treatment approaches for AD.
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Tuesday June 3rd - Adult physiology, metabolism, and homeostasis |
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Xie, N., Bickley, B. A., Gross, A. D. (2024). GABA-gated chloride channel mutation (Rdl) induces cholinergic physiological compensation resulting in cross resistance in Drosophila melanogaster. Pesticide biochemistry and physiology, 203:105972 PubMed ID: 39084765
Summary:
The Drosophila melanogaster MD-RR strain contains an Rdl mutation (A301S) resulting in resistance to several insecticide classes viz. phenyl pyrazoles (e.g., fipronil), cyclodienes (e.g., dieldrin), and chlorinated aliphatic hydrocarbons (e.g., lindane). Fitness costs are commonly observed with resistant insect populations as side effects of the genetic change conferring the resistant phenotype. Because of fitness costs, reversion from the resistant to susceptible genotype and phenotype is common. However, the Rdl genotype in D. melanogaster appears to allow the flies to maintain the resistant genotype/phenotype without selective pressure and with minimal fitness costs. Evidence is provided that compensation for the Rdl mutation influences the cholinergic system, where an increase in acetylcholineesterase gene expression and enzyme activity results in neurophysiological changes and cross resistance to a carbamate insecticide (propoxur oral resistance ratio (RR) of 63) and an organophosphate insecticide (dichlorvos oral RR of 7). Such cross resistance was not previously reported with the initial collection and testing of this strain. In addition to acetylcholinesterase, the Rdl mutation influences the expression of the muscarinic acetylcholine receptor subtype-B, resulting in resistance to non-selective muscarinic compounds (pilocarpine and atropine). Collectively, these results indicate that the Rdl mutation (A301S) at GABA-gated ionophore complex influences the physiology of the cholinergic system, leading to resistance to established insecticide classes. Additionally, this mutation may impact the effectiveness of insecticides targeting novel sites, like muscarinic receptors.
| Zhao, B., Luo, J., Wang, H., Li, Y., Li, D., Bi, X. (2024). In vivo RNAi screening identifies multiple deubiquitinases required for the maintenance of intestinal homeostasis in Drosophila. Insect biochemistry and molecular biology, 172:104162 PubMed ID: 39067716
Summary:
Deubiquitinases (DUBs) are essential for the maintenance of protein homeostasis and assembly of proteins into functional complexes. Despite growing interest in DUBs biological functions, the roles of DUBs in regulating intestinal stem cells (ISCs) and gut homeostasis remain largely unknown. This study performed an in vivo RNAi screen through induced knock-down of DUBs expression in adult midgut ISCs and enteroblasts (EBs) to identify DUB regulators of intestinal homeostasis in Drosophila. 43 DUBs were identified and 8 DUBs were identified that are required for ISCs homeostasis. Knocking-down of usp1, CG7857, usp5, rpn8, usp10 and csn5 decreases the number of ISCs/EBs, while knocking-down of CG4968 and usp8 increases the number of ISCs/EBs. Moreover, knock-down of usp1, CG4968, CG7857, or rpn8 in ISCs/EBs disrupts the intestinal barrier integrity and shortens the lifespan, indicating the requirement of these DUBs for the maintenance of gut homeostasis. Furthermore, evidences are provided that USP1 mediates ISC lineage differentiation via modulating the Notch signaling activity. This study identifies, for the first time, the deubiquitinases required for the maintenance of intestinal homeostasis in Drosophila, and provide new insights into the functional links between the DUBs and intestinal homeostasis.
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Lenhart, A., Ahsan, A., McHaty, M., Bergland, A. O. (2024). Improvement of starvation resistance via periodic fasting is genetically variable in Drosophila melanogaster. Physiological entomology, 49(3):270-278 PubMed ID: 39130127
Summary:
Organisms subjected to periodic nutrient limitation early in life exhibit improvements in aspects of survival, including resistance to some environmental stressors. Recent findings indicate that forms of periodic fasting such as intermittent fasting and time restricted feeding can improve starvation resistance. However, it remains unclear to what extent this survival improvement persists across different genetic backgrounds. This study examined fasting-induced starvation resistance across a broad survey of wild-derived lineages and documented genetic variation within this trait. A standard dietary intervention was adopted and improvement was shown to starvation resistance within a common laboratory lineage, replicating previous results. Next, fasting-induced starvation resistance was adopted across isofemale lines collected across latitudes and in different seasons, and among inbred lines derived from flies collected on different continents. Genetic variation was discovered of fasting-induced starvation resistance, and it was shown that fasting improved starvation resistance as often as it worsened starvation resistance. Fasted flies generally showed reduced fat concentration, and their starvation survival varied with sex, season of collection, and geographic origin. While specific lineages common to the laboratory can show a specific fasting-induced phenotype, this study showed that this result is not consistent across genetic backgrounds, reinforcing the idea that phenotypes observed in historic laboratory strains may not be conserved across a species.
| Darby, A. M., Okoro, D. O., Aredas, S., Frank, A. M., Pearson, W. H., Dionne, M. S., Lazzaro, B. P. (2024). High sugar diets can increase susceptibility to bacterial infection in Drosophila melanogaster. PLoS pathogens, 20(8):e1012447 PubMed ID: 39133760
Summary:
Overnutrition with dietary sugar can worsen infection outcomes in diverse organisms including insects and humans, through generally unknown mechanisms. In the present study, adult Drosophila melanogaster fed high-sugar diets became more susceptible to infection by the Gram-negative bacteria Providencia rettgeri and Serratia marcescens. P. rettgeri and S. marcescens were found to proliferate more rapidly in D. melanogaster fed a high-sugar diet, resulting in increased probability of host death. D. melanogaster become hyperglycemic on the high-sugar diet, and evidence was shown that the extra carbon availability may promote S. marcescens growth within the host. However, no evidence was found that increased carbon availability directly supports greater P. rettgeri growth. D. melanogaster on both diets fully induce transcription of antimicrobial peptide (AMP) genes in response to infection, but D. melanogaster provided with high-sugar diets show reduced production of AMP protein. Thus, overnutrition with dietary sugar may impair host immunity at the level of AMP translation. These results demonstrate that dietary sugar can shape infection dynamics by impacting both host and pathogen, depending on the nutritional requirements of the pathogen and by altering the physiological capacity of the host to sustain an immune response.
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Bretscher, H., O'Connor, M. B. (2024). Glycogen homeostasis and mtDNA expression require motor neuron to muscle TGFβ Activin Signaling in Drosophila. bioRxiv, PubMed ID: 39131342
Summary:
>Maintaining metabolic homeostasis requires coordinated nutrient utilization between intracellular organelles and across multiple organ systems. Many organs rely heavily on mitochondria to generate (ATP) from glucose, or stored glycogen. Proteins required for ATP generation are encoded in both nuclear and mitochondrial DNA (mtDNA). Motoneuron to muscle signaling by the TGFβ/Activin family member Actβ positively regulates glycogen levels during Drosophila development. Remarkably, this study found that levels of stored glycogen are unaffected by altering cytoplasmic glucose catabolism. Instead, Actβ loss reduces levels of mtDNA and nuclearly encoded genes required for mtDNA replication, transcription and translation. Direct RNAi mediated knockdown of these same nuclearly encoded mtDNA expression factors also results in decreased glycogen stores. Lastly, expressing an activated form of the type I receptor Baboon in muscle was shown to restore both glycogen and mtDNA levels in actβ mutants, thereby confirming a direct link between Actβ signaling, glycogen homeostasis and mtDNA expression.
| Tanaka, T., Yano, T., Usuki, S., Seo, Y., Mizuta, K., Okaguchi, M., Yamaguchi, M., Hanyu-Nakamura, K., Toyama-Sorimachi, N., Bruckner, K., Nakamura, A. (2024). Endocytosed dsRNAs induce lysosomal membrane permeabilization that allows cytosolic dsRNA translocation for Drosophila RNAi responses. Nat Commun, 15(1):6993 PubMed ID: 39143098
Summary:
RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. This study showed that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl(-)/H(+) antiporter DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.
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Thursday, May 29th - Stem Cells |
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Wen, P., Lei, H., Deng, H., Deng, S., Rodriguez Tirado, C., Wang, M., Mu, P., Zheng, Y., Pan, D. (2024). Hyd/UBR5 defines a tumor suppressor pathway that links Polycomb repressive complex to regulated protein degradation in tissue growth control and tumorigenesis. Genes Dev, 38(13-14):675-691 PubMed ID: 39137945
Summary:
Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. This study shows that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. This study further identified the epigenetic regulator as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm. Consistent with these molecular studies, this study showed that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. Evidence is also provided that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.
| Nagai, H., Adachi, Y., Nakasugi, T., Takigawa, E., Ui, J., Makino, T., Miura, M., Nakajima, Y. I. (2024). Highly regenerative species-specific genes improve age-associated features in the adult Drosophila midgut. BMC Biol, 22(1):157 PubMed ID: 39090637
Summary:
The remarkable regenerative abilities observed in planarians and cnidarians are closely linked to the active proliferation of adult stem cells and the precise differentiation of their progeny, both of which typically deteriorate during aging in low regenerative animals. While regeneration-specific genes conserved in highly regenerative organisms may confer regenerative abilities and long-term maintenance of tissue homeostasis, it remains unclear whether introducing these regenerative genes into low regenerative animals can improve their regeneration and aging processes. This study ectopically express highly regenerative species-specific JmjC domain-encoding genes (HRJDs) in Drosophila, a widely used low regenerative model organism. Two HRJD orthologs HRJDa and HRJDb were examined from two planarian species, Dugesia japonica and Schmidtea mediterranea, where both HRJDs contain only the JmjC domain and the EF hand motif. Surprisingly, HRJD expression impedes tissue regeneration in the developing wing disc but extends organismal lifespan when expressed in the intestinal stem cell lineages of the adult midgut under non-regenerative conditions. Notably, HRJDs enhance the proliferative activity of intestinal stem cells while maintaining their differentiation fidelity, ameliorating age-related decline in gut barrier functions. These findings together suggest that the introduction of highly regenerative species-specific genes can improve stem cell functions and promote a healthy lifespan when expressed in aging animals.
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Perales, I. E., Jones, S. D., Duan, T., Geyer, P. K. (2024). Maintenance of germline stem cell homeostasis despite severe nuclear distortion. Dev Biol, 515:139-150 PubMed ID: 39038593
Summary:
Stem cell loss in aging and disease is associated with nuclear deformation. Yet, how nuclear shape influences stem cell homeostasis is poorly understood. This study investigated this connection using Drosophila germline stem cells, as survival of these stem cells is compromised by dysfunction of the nuclear lamina, the extensive protein network that lines the inner nuclear membrane and gives shape to the nucleus. To induce nuclear distortion in germline stem cells, the GAL4-UAS system was used to increase expression of the permanently farnesylated nuclear lamina protein, Kugelkern, a rate limiting factor for nuclear growth. Elevated Kugelkern levels cause severe nuclear distortion in germline stem cells, including extensive thickening and lobulation of the nuclear envelope and nuclear lamina, as well as alteration of internal nuclear compartments. Despite these changes, germline stem cell number, proliferation, and female fertility are preserved, even as females age. Collectively, these data demonstrate that disruption of nuclear architecture does not cause a failure of germline stem cell survival or homeostasis, revealing that nuclear deformation does not invariably promote stem cell loss.
| Lin, K. Y., Gujar, M. R., Lin, J., Ding, W. Y., Huang, J., Gao, Y., Tan, Y. S., Teng, X., Christine, L. S. L., Kanchanawong, P., Toyama, Y., Wang, H. (2024). Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling. bioRxiv, PubMed ID: 38903085
Summary:
The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. This study revealed the fine F-actin structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. F-actin polymerization promotes the nuclear translocation of Mrtf, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G-protein-coupled receptor (GPCR) Smog, G-protein αq subunit, Rho1 GTPase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand Fog to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, this study establishes that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, a NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.
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Sun, H., Shami Shah, A., Chiu, D. C., Bonfini, A., Buchon, N., Baskin, J. M. (2024). Wnt/β-catenin signaling within multiple cell types dependent upon kramer regulates Drosophila intestinal stem cell proliferation. iScience, 27(6):110113 PubMed ID: 38952681
Summary:
The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/β-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, this study examined the cellular determinants of ISC proliferation, harnessing kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. Wnt signaling within Prospero-positive cells supports ISC proliferation, and kramer regulates Wnt signaling in this context by antagonizing kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes kramer as a physiological regulator of Wnt/β-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/β-catenin signaling.
| Warder, B. N., Nelson, K. A., Sui, J., Anllo, L., DiNardo, S. (2024). An actomyosin network organizes niche morphology and responds to feedback from recruited stem cells. Curr Biol, PubMed ID: 39137785
Summary:
Stem cells often rely on signals from a niche, which in many tissues adopts a precise morphology. What remains elusive is how niches are formed and how morphology impacts function. To address this, the Drosophila gonadal niche, which affords genetic tractability and live-imaging, was leverage. Previous work showed mechanisms dictating niche cell migration to their appropriate position within the gonad and the resultant consequences on niche function. This study showed that once positioned, niche cells robustly polarize filamentous actin (F-actin) and non-muscle myosin II (MyoII) toward neighboring germ cells. Actomyosin tension along the niche periphery generates a highly reproducible smoothened contour. Without contractility, niches are misshapen and exhibit defects in their ability to regulate germline stem cell behavior. Germ cells were shown to aid in polarizing MyoII within niche cells, and extrinsic input is required for niche morphogenesis and function. This work reveals a feedback mechanism where stem cells shape the niche that guides their behavior.
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Monday, May 26th - Adult Neural Structure, Development and Function |
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Sekxiguchi, M., Reinhard, N., Fukuda, A., Katoh, S., Rieger, D., Helfrich-Forster, C., Yoshii, T. (2024). A Detailed Re-Examination of the Period Gene Rescue Experiments Shows That Four to Six Cryptochrome-Positive Posterior Dorsal Clock Neurons (DN(1p)) of Drosophila melanogaster Can Control Morning and Evening Activity. J Biol Rhythms:7487304241263130 PubMed ID: 39082442
Summary:
Animal circadian clocks play a crucial role in regulating behavioral adaptations to daily environmental changes. The fruit fly Drosophila melanogaster exhibits 2 prominent peaks of activity in the morning and evening, known as morning (M) and evening (E) peaks. These peaks are controlled by 2 distinct circadian oscillators located in separate groups of clock neurons in the brain. To investigate the clock neurons responsible for the M and E peaks, a cell-specific gene expression system, the GAL4-UAS system, has been commonly employed. This study re-examined the two-oscillator model for the M and E peaks of Drosophila by utilizing more than 50 Gal4 lines in conjunction with the UAS-period16 line, which enables the restoration of the clock function in specific cells in the period (per) null mutant background. Previous studies have indicated that the group of small ventrolateral neurons (s-LNv) is responsible for controlling the M peak, while the other group, consisting of the 5th ventrolateral neuron (5th LNv) and the three cryptochrome (CRY)-positive dorsolateral neurons (LNd), is responsible for the E peak. Furthermore, the group of posterior dorsal neurons 1 (DN1p) is thought to also contain M and E oscillators. This studyfound that Gal4 lines directed at the same clock neuron groups can lead to different results, underscoring the fact that activity patterns are influenced by many factors. Nevertheless, it was possible to confirm previous findings that the entire network of circadian clock neurons controls M and E peaks, with the lateral neurons playing a dominant role. In addition, it was demonstrated that 4 to 6 CRY-positive DN1p cells are sufficient to generate M and E peaks in light-dark cycles and complex free-running rhythms in constant darkness. Ultimately, this detailed screening could serve as a catalog to choose the best Gal4 lines that can be used to rescue per in specific clock neurons.
| Jonaitis, J., Hibbard, K. L., McCafferty Layte, K., Hiramoto, A., Cardona, A., Truman, J. W., Nose, A., Zwart, M. F., Pulver, S. R. (2024). STEERING FROM THE REAR: COORDINATION OF CENTRAL PATTERN GENERATORS UNDERLYING NAVIGATION BY ASCENDING INTERNEURONS. bioRxiv, PubMed ID: 38948859
Summary:
Understanding how animals coordinate movements to achieve goals is a fundamental pursuit in neuroscience. This study explores how neurons that reside in posterior lower-order regions of a locomotor system project to anterior higher-order regions to influence steering and navigation. The anatomy and functional role was tested of a population of ascending interneurons in the ventral nerve cord of Drosophila larvae. Through electron microscopy reconstructions and light microscopy, The cholinergic 19f cells were determined to receive input primarily from premotor interneurons and synapse upon a diverse array of postsynaptic targets within the anterior segments including other 19f cells. Calcium imaging of 19f activity in isolated central nervous system (CNS) preparations in relation to motor neurons revealed that 19f neurons are recruited into most larval motor programmes. 19f activity lags behind motor neuron activity and as a population, the cells encode spatio-temporal patterns of locomotor activity in the larval CNS. Optogenetic manipulations of 19f cell activity in isolated CNS preparations revealed that they coordinate the activity of central pattern generators underlying exploratory headsweeps and forward locomotion in a context and location specific manner. In behaving animals, activating 19f cells suppressed exploratory headsweeps and slowed forward locomotion, while inhibition of 19f activity potentiated headsweeps, slowing forward movement. Inhibiting activity in 19f cells ultimately affected the ability of larvae to remain in the vicinity of an odor source during an olfactory navigation task. Overall, these findings provide insights into how ascending interneurons monitor motor activity and shape interactions amongst rhythm generators underlying complex navigational tasks.
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Stentiford, R., Knight, J. C., Nowotny, T., Philippides, A., Graham, P. (2024). Estimating orientation in natural scenes: A spiking neural network model of the insect central complex.. PLoS Comput Biol, 20(8):e1011913 PubMed ID: 39146374
Summary:
The central complex of insects contains cells, organised as a ring attractor, that encode head direction. The 'bump' of activity in the ring can be updated by idiothetic cues and external sensory information. Plasticity at the synapses between these cells and the ring neurons, that are responsible for bringing sensory information into the central complex, has been proposed to form a mapping between visual cues and the heading estimate which allows for more accurate tracking of the current heading, than if only idiothetic information were used. In Drosophila, ring neurons have well characterised non-linear receptive fields. This work produces synthetic versions of these visual receptive fields using a combination of excitatory inputs and mutual inhibition between ring neurons. These receptive fields were used to bring visual information into a spiking neural network model of the insect central complex based on the recently published Drosophila connectome. Previous modelling work has focused on how this circuit functions as a ring attractor using the same type of simple visual cues commonly used experimentally. While this study initially tested the model on these simple stimuli, it the went on to apply the model to complex natural scenes containing multiple conflicting cues. This simple visual filtering provided by the ring neurons is sufficient to form a mapping between heading and visual features and maintain the heading estimate in the absence of angular velocity input. The network is successful at tracking heading even when presented with videos of natural scenes containing conflicting information from environmental changes and translation of the camera.
| Odierna, G. L., Kerwin, S. K., Shin, G. J., Millard, S. S. (2024). Drosophila larval motor patterning relies on regulated alternative splicing of Dscam2. Frontiers in molecular neuroscience, 17:1415207 PubMed ID: 39092203
Summary:
Recent studies capitalizing on the newly complete nanometer-resolution Drosophila larval connectome have made significant advances in identifying the structural basis of motor patterning. However, the molecular mechanisms utilized by neurons to wire these circuits remain poorly understood. This study explored how cell-specific expression of two Dscam2 isoforms, which mediate isoform-specific homophilic binding, contributes to motor patterning and output of Drosophila larvae. Ablating Dscam2 isoform diversity resulted in impaired locomotion. Electrophysiological assessment at the neuromuscular junction during fictive locomotion indicated that this behavioral defect was largely caused by weaker bouts of motor neuron activity. Morphological analyses of single motor neurons using MultiColour FlpOut revealed severe errors in dendrite arborization and assessment of cholinergic and GABAergic projections to the motor domain revealed altered morphology of interneuron processes. Loss of Dscam2 did not affect locomotor output, motor neuron activation or dendrite targeting. These findings thus suggest that locomotor circuit phenotypes arise specifically from inappropriate Dscam2 interactions between premotor interneurons and motor neurons when they express the same isoform. Indeed, this study reports that first-order premotor interneurons express Dscam2A. Since motor neurons express Dscam2B, these results provide evidence that Dscam2 isoform expression alternates between synaptic partners in the nerve cord. This study demonstrates the importance of cell-specific alternative splicing in establishing the circuitry that underlies neuromotor patterning without inducing unwanted intercellular interactions.
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Ichinose, T., Kondo, S., Kanno, M., Shichino, Y., Mito, M., Iwasaki, S., Tanimoto, H. (2024)s. Translational regulation enhances distinction of cell types in the nervous system. Elife, 12 PubMed ID: 39010741
Summary:
Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, this study revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5' leaders in glia. Using transgenic reporter strains, evidence is provided that the small upstream open-reading frames in the 5' leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.
| Gautham, A. K., Miner, L. E., Franco, M. N., Thornquist, S. C., Crickmore, M. A. (2024). Dopamine biases decisions by limiting temporal integration. Nature, 632(8026):850-857 PubMed ID: 39085606
Summary:
Motivations bias human responses to stimuli, producing behavioural outcomes that match needs and goals. This study describes a mechanism behind this phenomenon: adjusting the time over which stimulus-derived information is permitted to accumulate towards a decision. As a Drosophila copulation progresses, the male becomes less likely to continue mating through challenges. This study shows that a set of copulation decision neurons (CDNs) flexibly integrates information about competing drives to mediate this decision. Early in mating, dopamine signalling restricts CDN integration time by potentiating Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation in response to stimulatory inputs, imposing a high threshold for changing behaviours. Later into mating, the timescale over which the CDNs integrate termination-promoting information expands, increasing the likelihood of switching behaviours. Scalable windows of temporal integration at dedicated circuit nodes are suggested as a key but underappreciated variable in state-based decision-making.
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Thursday, May 22nd - Adult Physiology |
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Szabla, N., Maria Labecka, A., Antot, A., Sobczyk, L., Angilletta, M. J., Czarnoleski, M. (2024). Evolution and development of Drosophila melanogaster under different thermal conditions affected cell sizes and sensitivity to paralyzing hypoxia. J Insect Physiol, 157:104671 PubMed ID: 38972633
Summary:
Environmental gradients cause evolutionary and developmental changes in the cellular composition of organisms, but the physiological consequences of these effects are not well understood. Experimental populations of Drosophila melanogaster were studied that had evolved in one of three selective regimes: constant 16°C, constant 25°C, or intergenerational shifts between 16°C and 25°C. Genotypes from each population were reared at three developmental temperatures (16°C, 20.5°C, and 25°C). As adults, thorax length and cell sizes in the Malpighian tubules and wing epithelia of flies from each combination of evolutionary and developmental temperatures were measured. Flies from these treatments were exposed to a short period of nearly complete oxygen deprivation to measure hypoxia tolerance. For genotypes from any selective regime, development at a higher temperature resulted in smaller flies with smaller cells, regardless of the tissue. At every developmental temperature, genotypes from the warm selective regime had smaller bodies and smaller wing cells but had larger tubule cells than did genotypes from the cold selective regime. Genotypes from the fluctuating selective regime were similar in size to those from the cold selective regime, but their cells of either tissue were the smallest among the three regimes. Evolutionary and developmental treatments interactively affected a fly's sensitivity to short-term paralyzing hypoxia. Genotypes from the cold selective regime were less sensitive to hypoxia after developing at a higher temperature. Genotypes from the other selective regimes were more sensitive to hypoxia after developing at a higher temperature. These results show that thermal conditions can trigger evolutionary and developmental shifts in cell size, coupled with changes in body size and hypoxia tolerance. These patterns suggest links between the cellular composition of the body, levels of hypoxia within cells, and the energetic cost of tissue maintenance.
| Lee, M., Park, S. H., Joo, K. M., Kwon, J. Y., Lee, K. H., Kang, K. (2024). Drosophila HCN mediates gustatory homeostasis by preserving sensillar transepithelial potential in sweet environments. Elife, 13 PubMed ID: 39073076
Summary:
Establishing transepithelial ion disparities is crucial for sensory functions in animals. In insect sensory organs called sensilla, a transepithelial potential, known as the sensillum potential (SP), arises through active ion transport across accessory cells, sensitizing receptor neurons such as mechanoreceptors and chemoreceptors. Because multiple receptor neurons are often co-housed in a sensillum and share SP, niche-prevalent overstimulation of single sensory neurons can compromise neighboring receptors by depleting SP. However, how such potential depletion is prevented to maintain sensory homeostasis remains unknown. This study found that the Ih-encoded hyperpolarization-activated cyclic nucleotide-gated (HCN) channel bolsters the activity of bitter-sensing gustatory receptor neurons (bGRNs), albeit acting in sweet-sensing GRNs (sGRNs). For this task, HCN maintains SP despite prolonged sGRN stimulation induced by the diet mimicking their sweet feeding niche, such as overripe fruit. Evidence is precented that Ih-dependent demarcation of sGRN excitability is implemented to throttle SP consumption, which may have facilitated adaptation to a sweetness-dominated environment. Thus, HCN expressed in sGRNs serves as a key component of a simple yet versatile peripheral coding that regulates bitterness for optimal food intake in two contrasting ways: sweet-resilient preservation of bitter aversion and the previously reported sweet-dependent suppression of bitter taste.
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Liu, M., Hemba-Waduge, R. U., Li, X., Huang, X., Liu, T. H., Han, X., Wang, Y., Ji, J. Y. (2024)s. Wnt/Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression.. JProc Natl Acad Sci U S A, 121(28):e2322066121 PubMed ID: 38968125
Summary:
The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. This study reports that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid β-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila. Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, a set of Wnt target genes responsible for intracellular lipid homeostasis was identified. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid β-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid β-oxidation.
| Pool, K. R., Gajanayakage, R. H., Connolly, C., Blache, D. (2024). Ancestral lineages of dietary exposure to an endocrine disrupting chemical drive distinct forms of transgenerational subfertility in an insect model. Sci Rep, 14(1):18153 PubMed ID: 39103404
Summary:
Across the globe, many species of insects are facing population decline. This is largely driven by anthropogenic changes to the environment, including the widespread exposure of invertebrates to endocrine disrupting chemicals (EDCs), which impair fertility. To test whether generations of Drosophila melanogaster born from parents exposed to a common dietary EDC, equol, could recover reproductive function, this study identified the reproductive capacity of the two subsequent generations. Using a novel suite of flow cytometry assays to assess sperm functionality in real time, sperm function was compromised across three generations, even after non-exposed in individuals contribute to the breeding population. Though the sex ratio alters in response to EDC exposure, favouring the survival of female offspring, most lineages with ancestral EDC exposure exhibit persistent subfertility in both the male and female. Male offspring with ancestral EDC exposure present with reduced fertility and dysfunctional spermatozoa, whereby spermatozoa are metabolically stressed, lack DNA integrity and present with permanent epigenetic alterations. Across generations, male and female offspring demonstrate distinct patterns of reproductive characteristics, depending upon the specific lineage of EDC exposure. These results illustrate how dietary EDCs present in agricultural plants could promote transgenerational subfertility and contribute to declining insect populations.
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Scott, J. G., Dressel, A. E., Mertz, R. W., Hesler, S., Loeb, G. (2024). Monitoring of the nAChRsα6 G275A spinetoram resistance allele in Drosophila melanogaster populations from New York vineyards. Pest Manag Sci, PubMed ID: 38989631
Summary:
Spinosyns are a group of naturally occurring and semi-synthetic insecticides with widespread utility in agriculture, including organic production systems. One example is spinetoram (Delegate), which is the only registered insecticide in New York State (for control of Drosophila melanogaster in vineyards) to which vinegar flies have not yet evolved high levels of resistance. However, low levels of resistance have been found in vineyard populations of D. melanogaster, and a highly resistant strain was obtained after only five selections (in the laboratory). This study identified the nAChR α6 mutation (G275A) responsible for the resistance and developed a rapid, high-throughput assay for resistance.Surveys of collections made in 2023 show low levels of the resistance allele in four populations. A correlation was observed between vineyard use of spinetoram and frequency of the resistance allele, but not between county-wide use of spinosyns and frequency of the resistance allele. One of the sites monitored was previously surveyed in 2019 and the frequency of the resistance allele detected in 2023 had increased. Implications of these findings to resistance management of D. melanogaster are discussed.
| Ma, P., Zhang, Y., Yin, Y., Wang, S., Chen, S., Liang, X., Li, Z., Deng, H. (2024). Gut microbiota metabolite tyramine ameliorates high-fat diet-induced insulin resistance via increased Ca(2+) signaling. The EMBO journal, 43(16):3466-3493 PubMed ID: 38965418
Summary:
The gut microbiota and their metabolites are closely linked to obesity-related diseases, such as type 2 diabetes, but their causal relationship and underlying mechanisms remain largely elusive. This study found that dysbiosis-induced tyramine (TA) suppresses high-fat diet (HFD)-mediated insulin resistance in both Drosophila and mice. In Drosophila, HFD increases cytosolic Ca(2+) signaling in enterocytes, which, in turn, suppresses intestinal lipid levels. 16 S rRNA sequencing and metabolomics revealed that HFD leads to increased prevalence of tyrosine decarboxylase (Tdc)-expressing bacteria and resulting tyramine production. Tyramine acts on the tyramine receptor, TyrR1, to promote cytosolic Ca(2+) signaling and activation of the CRTC-CREB complex to transcriptionally suppress dietary lipid digestion and lipogenesis in enterocytes, while promoting mitochondrial biogenesis. Furthermore, the tyramine-induced cytosolic Ca(2+) signaling is sufficient to suppress HFD-induced obesity and insulin resistance in Drosophila. In mice, tyramine intake also improves glucose tolerance and insulin sensitivity under HFD. These results indicate that dysbiosis-induced tyramine suppresses insulin resistance in both flies and mice under HFD, suggesting a potential therapeutic strategy for related metabolic disorders, such as diabetes.
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Wednesday, May 21st - Gonads |
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Gallicchio, L., Matias, N. R., Morales-Polanco, F., Nava, I., Stern, S., Zeng, Y., Fuller, M. T. (2024). A developmental mechanism to regulate alternative polyadenylation in an adult stem cell lineage. Genes Dev, 38(13-14):655-674 PubMed ID: 39111825
Summary:
Alternative cleavage and polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3' UTRs from the same genetic locus, potentially impacting mRNA translation, localization, and stability. Developmentally regulated APA can thus make major contributions to cell type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, ~500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. This study shows that upregulation of PCF11 and Cbc, the two components of cleavage factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knockdown of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. These findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell type-specific APA at selected genes.
| Duan, X., Wang, H., Cao, Z., Su, N., Wang, Y., Zheng, Y. (2024). Deficiency of ValRS-m Causes Male Infertility in Drosophila melanogaster. Int J Mol Sci, 25(13) PubMed ID: 39000597
Summary:
Drosophila spermatogenesis involves the renewal of germline stem cells, meiosis of spermatocytes, and morphological transformation of spermatids into mature sperm. Previous work demonstrated that Ocnus (ocn) plays an essential role in spermatogenesis. The ValRS-m (Valyl-tRNA synthetase, mitochondrial) gene was down-regulated in ocn RNAi testes. ValRS-m-knockdown was found to induce complete sterility in male flies. The depletion of ValRS-m blocked mitochondrial behavior and ATP synthesis, thus inhibiting the transition from spermatogonia to spermatocytes, and eventually, inducing the accumulation of spermatogonia during spermatogenesis. To understand the intrinsic reason for this, transcriptome-sequencing analysis was conducted for control and ValRS-m-knockdown testes. The differentially expressed genes (DEGs) between these two groups were selected with a fold change of ≥2 or ≤1/2. Compared with the control group, 4725 genes were down-regulated (dDEGs) and 2985 genes were up-regulated (uDEGs) in the ValRS-m RNAi group. The dDEsGs were mainly concentrated in the glycolytic pathway and pyruvate metabolic pathway, and the uDEGs were primarily related to ribosomal biogenesis. A total of 28 DEGs associated with mitochondria and 6 meiosis-related genes were verified to be suppressed when ValRS-m was deficient. Overall, these results suggest that ValRS-m plays a wide and vital role in mitochondrial behavior and spermatogonia differentiation in Drosophila.
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Milano, S. N., Bayer, L. V., Ko, J. J., Casella, C. E., Bratu, D. P. (2024). The role of ER exit sites in maintaining P-body organization and transmitting ER stress response during Drosophila melanogaster oogenesis. bioRxiv, PubMed ID: 39005311
Summary:
Processing bodies (P-bodies) are cytoplasmic membrane-less organelles which host multiple mRNA processing events. While the fundamental principles of P-body organization are beginning to be elucidated in vitro, a nuanced understanding of how their assembly is regulated in vivo remains elusive. This study investigated the potential link between ER exit sites and P-bodies in Drosophila melanogaster egg chambers. Employing a combination of live and super-resolution imaging, this study found that P-bodies associated with ER exit sites are larger and less mobile than cytoplasmic P-bodies, indicating that they constitute a distinct class of P-bodies which are more mature than their cytoplasmic counterparts. Moreover, altering the composition of ER exit sites has differential effects on core P-body proteins (Me31B, Cup, and Trailer Hitch) suggesting a potential role for ER exit sites in P-body organization. In the absence of ER exit sites, P-body integrity is compromised and the stability and translational repression efficiency of the maternal mRNA, oskar, are reduced. Finally, ER stress was shown to be communicated to P-bodies via ER exit sites, highlighting the pivotal role of ER exit sites as a bridge between membrane-bound and membrane-less organelles in ER stress response. Together, these data unveils the significance of ER exit sites not only in governing P-body organization, but also in facilitating inter-organellar communication during stress, potentially bearing implications for a variety of disease pathologies.
| Kong, R., Zhao, H., Li, J., Ma, Y., Li, N., Shi, L., Li, Z. (2024). A regulatory loop of JAK/STAT signalling and its downstream targets represses cell fate conversion and maintains male germline stem cell niche homeostasis. Cell proliferation:e13648 PubMed ID: 38987866
Summary:
A specialised microenvironment, termed niche, provides extrinsic signals for the maintenance of residential stem cells. However, how residential stem cells maintain niche homeostasis and whether stromal niche cells could convert their fate into stem cells to replenish lost stem cells upon systemic stem cell loss remain largely unknown. Through systemic identification of JAK/STAT downstream targets in adult Drosophila testis, this study showed that Escargot (Esg), a member of the Snail family of transcriptional factors, is a putative JAK/STAT downstream target. esg is intrinsically required in cyst stem cells (CySCs) but not in germline stem cells (GSCs). esg depletion in CySCs results in CySC loss due to differentiation and non-cell autonomous GSC loss. Interestingly, hub cells are gradually lost by delaminating from the hub and converting into CySCs in esg-defective testes. Mechanistically, esg directly represses the expression of socs36E, the well-known downstream target and negative regulator of JAK/STAT signalling. Finally, further depletion of socs36E completely rescues the defects observed in esg-defective testes. Collectively, JAK/STAT target Esg suppresses SOCS36E to maintain CySC fate and repress niche cell conversion. Thus, this work uncovers a regulatory loop between JAK/STAT signalling and its downstream targets in controlling testicular niche homeostasis under physiological conditions.
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Chatterjee, P., Mukherjee, S., Majumder, P. (2024). Shaping Drosophila eggs: unveiling the roles of Arpc1 and cpb in morphogenesis. Functional & integrative genomics, 24(4):120 PubMed ID: 38960936
Summary:
The Drosophila egg chamber (EC) starts as a spherical tissue at the beginning. With maturation, the outer follicle cells of EC collectively migrate in a direction perpendicular to the anterior-posterior axis, to shape EC from spherical to ellipsoidal. Filamentous actin (F-actin) plays a significant role in shaping individual migratory cells to the overall EC shape, like in every cell migration. The primary focus of this study waS to unveil the function of different Actin Binding Proteins (ABPs) in regulating mature Drosophila egg shape. 66 ABPs were screened, and the genetic screening data revealed that individual knockdown of Arp2/3 complex genes and the capping protein β (cpb) gene have severely altered the egg phenotype. Arpc1 and cpb RNAi mediated knockdown resulted in the formation of spherical eggs which are devoid of dorsal appendages. Studies also showed the role of Arpc1 and cpb on the number of laid eggs and follicle cell morphology. Furthermore, the depletion of Arpc1 and cpb resulted in a change in F-actin quantity. Together, the data indicate that Arpc1 and cpb regulate Drosophila egg shape, F-actin management, egg-laying characteristics and dorsal appendages formation.
| Wetzker, C., Froschauer, C., Massino, C., Reinhardt, K. (2024). Drosophila melanogaster sperm turn more oxidative in the female. The Journal of experimental biology, 227(15) PubMed ID: 39023115
Summary:
Males and females of many species store sperm for extended periods. During storage, sperm are predicted to undergo cellular and functional changes, especially towards glycolytic energy metabolism because oxygen radicals derived from oxidative phosphorylation can affect sperm motility and fertilisation ability. However, not all species can use both major energy metabolism pathways. This study examined the fruit fly Drosophila melanogaster and asked whether sperm metabolism can be fuelled by both glycolysis and oxidative phosphorylation, and to what extent metabolism changes during storage. Inhibiting glycolysis in vitro led to a more oxidative state of sperm; inhibiting oxidative phosphorylation increased the glycolytic component, assessed by multi-photon autofluorescence lifetime imaging (FLIM) of NAD(P)H. Sperm in male and female sperm storage organs were examined using FLIM of NAD(P)H and FAD. In intact storage organs, it was found that, unexpectedly, (i) sperm were more oxidative in females than in males, and (ii) oxidative phosphorylation increased with storage duration in females. The observation that the relative contribution of the two major energy metabolic pathways in D. melanogaster sperm differs in males and females and over storage time has important evolutionary implications.
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Friday, May 16th - Embryonic Development |
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Gillard, G., Roper, K. (2024). beta-H-Spectrin is a key component of an apical-medial hub of proteins during cell wedging in tube morphogenesis. J Cell Sci, 137(15) PubMed ID: 38988298
Summary:
Coordinated cell shape changes are a major driver of tissue morphogenesis, with apical constriction of epithelial cells leading to tissue bending. Previous work identified that interplay between the apical-medial actomyosin, which drives apical constriction, and the underlying longitudinal microtubule array has a key role during tube budding of salivary glands in the Drosophila embryo. At this microtubule-actomyosin interface, a hub of proteins accumulates, and previous work showed that this hub includes the microtubule-actin crosslinker Shot and the microtubule minus-end-binding protein Patronin. This study identified two actin-crosslinkers, β-heavy (H)-Spectrin (also known as Karst) and Filamin (also known as Cheerio), and the multi-PDZ-domain protein Big bang as components of the protein hub. Tissue-specific degradation of β-H-Spectrin leads to reduction of apical-medial F-actin, Shot, Patronin and Big bang, as well as concomitant defects in apical constriction, but that residual Patronin is still sufficient to assist microtubule reorganisation. Unlike Patronin and Shot, neither β-H-Spectrin nor Big bang was found to be require microtubules for their localisation. β-H-Spectrin is instead recruited via binding to apical-medial phosphoinositides, and overexpression of the C-terminal pleckstrin homology domain-containing region of β-H-Spectrin (β-H-33) displaces endogenous β-H-Spectrin and leads to strong morphogenetic defects. This protein hub therefore requires the synergy and coincidence of membrane- and microtubule-associated components for its assembly and function in sustaining apical constriction during tubulogenesis.
| Briney, C. A., Henriksen, J. C., Lin, C., Jones, L. A., Benner, L., Rains, A. B., Gutierrez, R., Gafken, P. R., Rissland, O. S. (2024). Muskelin acts as a substrate receptor of the highly regulated Drosophila CTLH E3 ligase during the maternal-to-zygotic transition. bioRxiv, PubMed ID: 39005399
Summary:
The maternal-to-zygotic transition (MZT) is a conserved developmental process where the maternally-derived protein and mRNA cache is replaced with newly made zygotic gene products. Previous work showed that in Drosophila the deposited RNA-binding proteins ME31B, Cup, and Trailer Hitch (TRAL) are ubiquitylated by the CTLH E3 ligase and cleared. However, the organization and regulation of the CTLH complex remain poorly understood in flies. In particular, Drosophila lacks an identifiable substrate adaptor, and the mechanisms restricting degradation of ME31B and its cofactors to the MZT are unknown. This study shows that the developmental specificity of the CTLH complex is mediated by multipronged regulation, including transcriptional control by the transcription factor OVO and autoinhibition of the E3 ligase. One major regulatory target is the subunit Muskelin, which acts as a substrate adaptor for the Drosophila CTLH complex. Although conserved, Muskelin has structural roles in other species, suggesting a surprising functional plasticity. Finally, this study found that Muskelin has few targets beyond the three known RNA binding proteins, showing exquisite target specificity. Thus, multiple levels of integrated regulation restrict the activity of the embryonic CTLH complex to early embryogenesis, seemingly with the goal of regulating three important RNA binding proteins.
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Vanderleest, T. E., Xie, Y., Budhathoki, R., Linvill, K., Hobson, C., Heddleston, J., Loerke, D., Blankenship, J. T. (2024). Lattice light sheet microscopy reveals 4D force propagation dynamics and leading-edge behaviors in an embryonic epithelium in Drosophila. Curr Biol, 34(14):3165-3177. PubMed ID: 38959881
Summary:
How pulsed contractile dynamics drive the remodeling of cell and tissue topologies in epithelial sheets has been a key question in development and disease. Due to constraints in imaging and analysis technologies, studies that have described the in vivo mechanisms underlying changes in cell and neighbor relationships have largely been confined to analyses of planar apical regions. Thus, how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface in three dimensions, including especially the apical-basal axis, is unclear. Lattice light sheet microscopy (LLSM)-based analysis during axis elongation (germband extension) was performed to determine how far and fast forces propagate across different apical-basal layers, as well as where topological changes initiate from in a columnar epithelium. These datasets are highly time- and depth-resolved and reveal that topology-changing forces are spatially entangled, with contractile force generation occurring across the observed apical-basal axis in a pulsed fashion, while the conservation of cell volumes constrains instantaneous cell deformations. Leading layer behaviors occur opportunistically in response to favorable phasic conditions, with lagging layers "zippering" to catch up as new contractile pulses propel further changes in cell topologies. These results argue against specific zones of topological initiation and demonstrate the importance of systematic 4D-based analysis in understanding how forces and deformations in cell dimensions propagate in a three-dimensional environment.
| Wang, X., Cupo, C. M., Ostvar, S., Countryman, A. D., Kasza, K. E. (2024). E-cadherin tunes tissue mechanical behavior before and during morphogenetic tissue flows. bioRxiv, PubMed ID: 38766260
Summary:
Adhesion between epithelial cells enables the remarkable mechanical behavior of epithelial tissues during morphogenesis. However, it remains unclear how cell-cell adhesion influences mechanics in static as well as in dynamically flowing epithelial tissues. This study systematically modulate E-cadherin-mediated adhesion in the Drosophila embryo, and the effects on the mechanical behavior of the germband epithelium was performed before and during dramatic tissue remodeling and flow associated with body axis elongation. Before axis elongation, increasing E-cadherin levels was found to produce tissue comprising more elongated cells and predicted to be more fluid-like, providing reduced resistance to tissue flow. During axis elongation, the dominant effect of E-cadherin was found to be tuning the speed at which cells proceed through rearrangement events, revealing potential roles for E-cadherin in generating friction between cells. Before and during axis elongation, E-cadherin levels influence patterns of actomyosin-dependent forces, supporting the notion that E-cadherin tunes tissue mechanics in part through effects on actomyosin. Taken together, these findings reveal dual-and sometimes opposing-roles for E-cadherin-mediated adhesion in controlling tissue structure and dynamics in vivo that result in unexpected relationships between adhesion and flow.
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Sun, J., Durmaz, A. D., Babu, A., Macabenta, F., Stathopoulos, A. (2024). Two sequential gene expression programs bridged by cell division support long-distance collective cell migration. Development, 151(10) PubMed ID: 38646822
Summary:
The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. This study focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, the cell cycle effector transcription factor E2F1 was found to be a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. This polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.
| Herault, C., Pihl, T., Hudry, B. (2024). Cellular sex throughout the organism underlies somatic sexual differentiation. Nat Commun, 15(1):6925 PubMed ID: 39138201
Summary:
Sex chromosomes underlie the development of male or female sex organs across species. While systemic signals derived from sex organs prominently contribute to sex-linked differences, it is unclear whether the intrinsic presence of sex chromosomes in somatic tissues has a specific function. This study used genetic tools to show that cellular sex is crucial for sexual differentiation throughout the body in Drosophila melanogaster. Every somatic cell converts the intrinsic presence of sex chromosomes into the active production of a sex determinant, a female specific serine- and arginine-rich (SR) splicing factor. This discovery dismisses the mosaic model which posits that only a subset of cells has the potential to sexually differentiate. Using cell-specific sex reversals, this study shows that this prevalence of cellular sex drives sex differences in organ size and body weight and is essential for fecundity. These findings demonstrate that cellular sex drives differentiation programs at an organismal scale and highlight the importance of cellular sex pathways in sex trait evolution.
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Thursday, May 15th - Evolution |
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Krittika, S., Yadav, P. (2024). Correlated changes in stress resistance and biochemical parameters in response to long-term protein restriction in Drosophila melanogaster. R Soc Open Sci, 11(6):231741 PubMed ID: 39100164
Summary:
Studies in fruit flies, Drosophila melanogaster, have observed considerable variation in the effect of dietary protein restriction (PR) on various fitness traits. In addition, not only are there inconsistent results relating lifespan to stress resistance. PR implementation was studied across generations (long term) hypothesizing that it will be beneficial for fitness traits, stress resistance and storage reserves due to nutritional plasticity transferred by parents to offspring in earlier Drosophila studies. By imposing two concentrations of PR diets (50% and 70% of control protein) from the pre-adult and adult (age 1 day) stages of the flies, the stage-specific and long-term effect of the imposed PR were assessed. All long-term PR flies showed increased resistance against the tested stressors (starvation, desiccation, H(2)O(2)-induced oxidative stress). In addition, this study also found long-term PR-induced increased stress resistance across generations. The PR flies also possessed higher protein and triglyceride (TG) content, reduced glucose and unaffected glycogen levels. The effect of returning the PR flies to control (AL) food for a single generation was assayed, their biochemical parameters were assessed to witness the transient PR effect. It was seen that TG content upon reversal was similar to AL flies except for PRI70 males; however, the glucose levels of PR males increased, while they were consistently lower in females. Taken altogether, this study suggests that long-term PR implementation contributes to increased stress resistance and was found to influence storage reserves in D. melanogaster.
| Hubert, D. L., Arnold, K. R., Greenspan, Z. G., Pupo, A., Robinson, R. D., Chavarin, V. V., Barter, T. B., Djukovic, D., Raftery, D., Vue, Z., Hinton, A., McReynolds, M. R., Harrison, B. R., Phillips, M. A. (2024). Selection for early reproduction leads to accelerated aging and extensive metabolic remodeling in Drosophila melanogaster populations.. bioRxiv
PubMed ID: 39005259
Summary:
Experimental evolution studies that feature selection on life-history characters are a proven approach for studying the evolution of aging and variation in rates of senescence. Recently, the incorporation of genomic and transcriptomic approaches into this framework has led to the identification of hundreds of genes associated with different aging patterns. However, understanding of the specific molecular mechanisms underlying these aging patterns remains limited. This study incorporated extensive metabolomic profiling into this framework to generate mechanistic insights into aging patterns in Drosophila melanogaster. Specifically, this study characterized metabolomic change over time associated with accelerated aging in populations of D. melanogaster under selection for early reproduction compared to their controls. Using this data this study i) evaluated the evolutionary repeatability across the metabolome; ii) evaluated the value of the metabolome as a predictor of "biological age" in this system; and iii) identified specific metabolic pathways associated with accelerated aging. Generally, the findings suggest that the metabolome is a reliable predictor of age and senescence in populations that share a recent evolutionary history. Metabolomic analysis revealed that generations of selection for early reproduction resulted in highly repeatable alterations to the metabolome. Specifically, changes in carbohydrate, amino acid, and TCA cycle-related metabolite abundances over time point to metabolic remodeling that favors rapid early reproduction with long-term consequences for carbohydrate and protein utilization.
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Yamamoto, A., Huang, W., Carbone, M. A., Anholt, R. R. H., Mackay, T. F. C. (2024). The genetic basis of incipient sexual isolation in Drosophila melanogaster. Proceedings Biological sciences, 291(2027):20240672 PubMed ID: 39045689
Summary:
Speciation is a fundamental evolutionary process but the genetic changes accompanying speciation are difficult to determine since true species do not produce viable and fertile offspring. Partially reproductively isolated incipient species are useful for assessing genetic changes that occur prior to speciation. Drosophila melanogaster from Zimbabwe, Africa are partially sexually isolated from other D. melanogaster populations whose males have poor mating success with Zimbabwe females. The North American D. melanogaster Genetic Reference Panel (DGRP) was used to show that there is significant genetic variation in mating success of DGRP males with Zimbabwe females, to map genetic variants and genes associated with variation in mating success and to determine whether mating success to Zimbabwe females is associated with other quantitative traits previously measured in the DGRP. Incipient sexual isolation is highly polygenic and associated with the common African inversion In(3R)K and the amount of the sex pheromone 5,9-heptacosadiene in DGRP females. the effect of eight candidate genes using RNA interference to provide testable hypotheses for future studies investigating the molecular genetic basis of incipient sexual isolation in D. melanogaster.
Speciation is a fundamental evolutionary process but the genetic changes accompanying speciation are difficult to determine since true species do not produce viable and fertile offspring. Partially reproductively isolated incipient species are useful for assessing genetic changes that occur prior to speciation. Drosophila melanogaster from Zimbabwe, Africa are partially sexually isolated from other D. melanogaster populations whose males have poor mating success with Zimbabwe females. The North American D. melanogaster Genetic Reference Panel (DGRP) was used to show that there is significant genetic variation in mating success of DGRP males with Zimbabwe females, to map genetic variants and genes associated with variation in mating success and to determine whether mating success to Zimbabwe females is associated with other quantitative traits previously measured in the DGRP. Incipient sexual isolation is highly polygenic and associated with the common African inversion In(3R)K and the amount of the sex pheromone 5,9-heptacosadiene in DGRP females. the effect of eight candidate genes using RNA interference to provide testable hypotheses for future studies investigating the molecular genetic basis of incipient sexual isolation in D. melanogaster.
Speciation is a fundamental evolutionary process but the genetic changes accompanying speciation are difficult to determine since true species do not produce viable and fertile offspring. Partially reproductively isolated incipient species are useful for assessing genetic changes that occur prior to speciation. Drosophila melanogaster from Zimbabwe, Africa are partially sexually isolated from other D. melanogaster populations whose males have poor mating success with Zimbabwe females. The North American D. melanogaster Genetic Reference Panel (DGRP) was used to show that there is significant genetic variation in mating success of DGRP males with Zimbabwe females, to map genetic variants and genes associated with variation in mating success and to determine whether mating success to Zimbabwe females is associated with other quantitative traits previously measured in the DGRP. Incipient sexual isolation is highly polygenic and associated with the common African inversion In(3R)K and the amount of the sex pheromone 5,9-heptacosadiene in DGRP females. The effect of eight candidate genes was functionally validated using RNA interference to provide testable hypotheses for future studies investigating the molecular genetic basis of incipient sexual isolation in D. melanogaster.
| Ma, L., Zheng, C., Liu, J., Song, F., Tian, L., Cai, W., Li, H., Duan, Y. (2024). Learning from the Codon Table: Convergent Recoding Provides Novel Understanding on the Evolution of A-to-I RNA Editing. J Mol Evol, 92(4):488-504 PubMed ID: 39012510
Summary:
Adenosine-to-inosine (A-to-I) RNA editing recodes the genetic information. Apart from diversifying the proteome, another tempting advantage of RNA recoding is to correct deleterious DNA mutation and restore ancestral allele. Solid evidences for beneficial restorative editing are very rare in animals. By searching for "convergent recoding" under a phylogenetic context, this term was proposed for judging the potential restorative functions of particular editing site. For the well-known mammalian Gln>Arg (Q>R) recoding site, its ancestral state in vertebrate genomes was the pre-editing Gln, and all 470 available mammalian genomes strictly avoid other three equivalent ways to achieve Arg in protein. The absence of convergent recoding from His>Arg, or synonymous mutations on Gln codons, could be attributed to the strong maintenance on editing motif and structure, but the absence of direct A-to-G mutation were found to be extremely unexpected. With similar ideas, cases of convergent recoding in Drosophila genus, reducing the possibility of their restorative function. In summary, this study defined an interesting scenario of convergent recoding, the occurrence of which could be used as preliminary judgements for whether a recoding site has a sole restorative role. This work provides novel insights to the natural selection and evolution of RNA editing.
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Bitter, M. C., Berardi, S., Oken, H., Huynh, A., Lappo, E., Schmidt, P., Petrov, D. A. (2024). Continuously fluctuating selection reveals fine granularity of adaptation. Nature, PubMed ID: 39143223
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. Genome-wide allele frequency data was generated every 1-2 generations from a genetically diverse population of Drosophila melanogaster in extensively replicated field mesocosms from late June to mid-December (a period of approximately 12 total 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 change in allele frequency 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 a 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 contributing to the maintenance of functional genetic variation in natural populations and an important stochastic force impacting genome-wide patterns of diversity at linked neutral sites, akin to genetic draft.
| Asgari, D., Stewart, A. J., Meisel, R. P. (2024). The role of uncertainty and negative feedback loops in the evolution of induced immune defenses. G3 (Bethesda), PubMed ID: 39106431
Summary:
Organisms use constitutive or induced defenses against pathogens and other external threats. Constitutive defenses are constantly on, whereas induced defenses are activated when needed. Each of these strategies has costs and benefits, which can affect the type of defense that evolves in response to pathogens. In addition, induced defenses are usually regulated by multiple negative feedback mechanisms that prevent overactivation of the immune response. However, it is unclear how negative feedback affects the costs, benefits, and evolution of induced responses. To address this gap, a mechanistic model was developed of the well-characterized Drosophila melanogaster immune signaling network that includes three separate mechanisms of negative feedback as a representative of the widespread phenomenon of muti-level regulation of induced responses. Under stochastic fly-bacteria encounters, an induced defense is favored when bacterial encounters are rare or uncertain, but in ways that depend on the bacterial proliferation rate. This model also predicts that the specific negative regulators that optimize the induced response depend on the bacterial proliferation rate, linking negative feedback mechanisms to the factors that favor induction.
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Tuesday, May 12th - Signaling |
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Armirola-Ricaurte, C., Morant, L., Adant, I., ..., Reilly, M. M., Rasic, V. M., Jordanova, A. (2024). Biallelic variants in COX18 cause a mitochondrial disorder primarily manifesting as peripheral neuropathy. medRxiv : the preprint server for health sciences, PubMed ID: 39006432
Summary:
Defects in mitochondrial dynamics are a common cause of Charcot-Marie-Tooth disease (CMT), while primary deficiencies in the mitochondrial respiratory chain (MRC) are rare and atypical for this etiology. This study aims to report COX18 as a novel CMT-causing gene. This gene encodes an assembly factor of mitochondrial Complex IV (CIV) that translocates the C-terminal tail of MTCO2 across the mitochondrial inner membrane. Exome sequencing was performed in four affected individuals. The patients and available family members underwent thorough neurological and electrophysiological assessment. The impact of one of the identified variants on splicing, protein levels, and mitochondrial bioenergetics was investigated in patient-derived lymphoblasts. The functionality of the mutant protein was assessed using a Proteinase K protection assay and immunoblotting. Neuronal relevance of COX18 was assessed in a Drosophila melanogaster knockdown model. Exome sequencing coupled with homozygosity mapping revealed a homozygoussplice variant c.435-6A>G in COX18 in two siblings with early-onset progressive axonal sensory-motor peripheral neuropathy. By querying external databases,two additional families were identified with rare deleterious biallelic variants in COX18. All affected individuals presented with axonal CMT and some patients also exhibited central nervous system symptoms, such as dystonia and spasticity. Functional characterization of the c.435-6A>pG variant demonstrated that it leads to the expression of an alternative transcript that lacks exon 2, resulting in a stable but defective COX18 isoform. The mutant protein impairs CIV assembly and activity, leading to a reduction in mitochondrial membrane potential. Downregulation of the COX18 homolog in Drosophila melanogaster displayed signs of neurodegeneration, including locomotor deficit and progressive axonal degeneration of sensory neurons. THIS study presents genetic and functional evidence that supports COX18 as a newly identified gene candidate for autosomal recessive axonal CMT with or without central nervous system involvement. These findings emphasize the significance of peripheral neuropathy within the spectrum of primary mitochondrial disorders and the role of mitochondrial CIV in the development of CMT. Thia research has important implications for the diagnostic workup of CMT patients.
| Adebowale, A., Oyaluna, Z., Falobi, A. A., Abolaji, A. O., Olaiya, C. O., Ojo, O. O. (2024). Magainin-AM2 inhibits sucrose-induced hyperglycaemia, oxidative stress, and cognitive dysfunction in Drosophila melanogaster. Free radical biology & medicine, 222:414-423 PubMed ID: 38964592
Summary:
Hyperglycaemia-induced oxidative stress plays significant roles in the development of type 2 diabetes and its complications. This study investigates effects of magainin-AM2, an amphibian host-defense peptide on high-sucrose diet induced redox imbalance and cognitive impairment in Drosophila melanogaster. Effects of various concentrations of sucrose, magainin-AM2 (a peptide) or a combination of both agents on mortality, eclosion rate, generation of reactive oxygen and nitrogen species, activities of antioxidant enzymes, thiol system, and markers of cognitive functions in control and treated flies were examined. Results showed that the exposure of flies to high sucrose diet increased mortality rate and levels of glucose (1.8-1.9-fold), hydrogen peroxide (1.4-1.5-fold) and nitrite/nitrate (1.2-fold). Decreased levels of total thiol non-protein thiols , catalase activities and glutathione-s-transferase activities (31-43) were also observed. Magainin-AM2 (0-10 μM/kg diet) did not affect fly mortality rate, levels of hydrogen peroxide and nitrite/nitrate, and activities of catalase and glutathione-s-transferase. However, the peptide produced a dose-dependent increase in total thiol 1.2-1.6-fold)and increases non-protein thiol levels at 10 μM/kg diet (2.0-fold). Magainin-AM2 inhibited sucrose-induced elevation of glucose, hydrogen peroxide and nitrite/nitrate. The peptide prevented sucrose-induced reduction in total and non-protein thiols (1.9-2.0-fold) levels and activities of catalase (2.3-3.1-fold) and glutathione-s-transferase (1.8-2.8-fold-0.05). Magainin-AM2 inhibited sucrose-induced reduction in acetylcholinesterase activities (3.6-4.0-fold), eclosion rate and negative geotaxis (1.3-14-fold). These results indicate that beneficial actions of magainin-AM2 may also involve the prevention of hyperglycaemia-induced oxidative damage and encourage its further development as an anti-diabetic agent.
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Schultheis, N., Connell, A., Kapral, A., Becker, R. J., Mueller, R., Shah, S., O'Donnell, M., Roseman, M., Swanson, L., DeGuara, S., Wang, W., Yin, F., Saini, T., Weiss, R. J., Selleck, S. B. (2024). Altering heparan sulfate suppresses cell abnormalities and neuron loss in Drosophila presenilin model of Alzheimer Disease. iScience, 27(7):110256 PubMed ID: 39109174
Summary:
This study examined the function of heparan-sulfate-modified proteoglycans (HSPGs) in pathways affecting Alzheimer disease (AD)-related cell pathology in human cell lines and mouse astrocytes. Mechanisms of HSPG influences on presenilin-dependent cell loss were evaluated in Drosophila using knockdown of the presenilin homolog, Psn, together with partial loss-of-function of sulfateless (sfl), a gene specifically affecting HS sulfation. HSPG modulation of autophagy, mitochondrial function, and lipid metabolism were shown to be conserved in human cell lines, Drosophila, and mouse astrocytes. RNA interference (RNAi) of Ndst1 reduced intracellular lipid levels in wild-type mouse astrocytes or those expressing humanized variants of APOE, APOE3, and APOE4. Neuron-directed knockdown of Psn in Drosophila produced apoptosis and cell loss in the brain, phenotypes suppressed by reductions in sfl expression. Abnormalities in mitochondria, liposomes, and autophagosome-derived structures in animals with Psn knockdown were also rescued by reduction of sfl. These findings support the direct involvement of HSPGs in AD pathogenesis.
| Li, M., Macro, J., Huggins, B. J., Meadows, K., Mishra, D., Martin, D., Kannan, K., Rogina, B. (2024). Extended lifespan in female Drosophila melanogaster through late-life calorie restriction. GeroScience, 46(5):4017-4035 PubMed ID: 38954128
Summary:
Calorie restriction has many beneficial effects on healthspan and lifespan in a variety of species. However, how late in life application of caloric restriction can extend fly life is not clear. This study showed that late-life calorie restriction increases lifespan in female Drosophila melanogaster aged on a high-calorie diet. This shift results in rapid decrease in mortality rate and extends fly lifespan. In contrast, shifting female flies from a low- to a high-calorie diet leads to a rapid increase in mortality and shorter lifespan. These changes are mediated by immediate metabolic and physiological adaptations. One of such adaptation is rapid adjustment in egg production, with flies directing excess energy towards egg production when shifted to a high diet, or away from reproduction in females shifted to low-caloric diet. However, lifelong female fecundity reveals no associated fitness cost due to CR when flies are shifted to a high-calorie diet. In view of high conservation of the beneficial effects of CR on physiology and lifespan in a wide variety of organisms, including humans, these findings could provide valuable insight into CR applications that could provide health benefits later in life.
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Lobato, A. G., Ortiz-Vega, N., Canic, T., ...., Zuchner, S., Syed, S., Zhai, R. G. (2024). Loss of Fic causes progressive neurodegeneration in a Drosophila model of hereditary spastic paraplegia.. Biochimica et biophysica acta Molecular basis of disease, 1870(7):167348 PubMed ID: 38986817
Summary:
Hereditary Spastic Paraplegia (HSP) is a group of rare inherited disorders characterized by progressive weakness and spasticity of the legs. Recent newly discovered biallelic variants in the gene FICD were found in patients with a highly similar phenotype to early onset HSP. FICD encodes filamentation induced by cAMP domain protein. FICD is involved in the AMPylation and deAMPylation protein modifications of the endoplasmic reticulum (ER) chaperone BIP, a major constituent of the ER that regulates the unfolded protein response. Although several biochemical properties of FICD have been characterized, the neurological function of FICD and the pathological mechanism underlying HSP are unknown. This study established a Drosophila model to gain mechanistic understanding of the function of FICD in HSP pathogenesis, and specifically the role of BIP in neuromuscular physiology. These studies on Drosophila Fic null mutants uncovered that loss of Fic resulted in locomotor impairment and reduced levels of BIP in the motor neuron circuitry, as well as increased reactive oxygen species (ROS) in the ventral nerve cord of Fic null mutants. Finally, feeding Drosophila Fic null mutants with chemical chaperones PBA or TUDCA, or treatment of patient fibroblasts with PBA, reduced the ROS accumulation. The neuronal phenotypes of Fic null mutants recapitulate several clinical features of HSP patients and further reveal cellular patho-mechanisms. By modeling FICD in Drosophila, this study has provided potential targets for intervention for HSP, and advance fundamental biology that is important for understanding related rare and common neuromuscular diseases.
| Chen, J., Nouzova, M., Noriega, F. G., Tatar, M. (2024). Gut-to-brain regulation of Drosophila aging through neuropeptide F, insulin and juvenile hormone. Proc Natl Acad Sci. PubMed ID: 39413128
Summary:
Dietary restriction slows aging in many animals, while in some cases the sensory signals from diet alone are sufficient to retard or accelerate lifespan. The digestive tract is a candidate location to sense nutrients, where neuropeptides secreted by enteroendocrine cells (EEC) produce systemic signals in response to food. This study measured how Drosophila neuropeptide F (NPF) is secreted into adult circulation by enteroendocrine cells and found that specific enteroendocrine cells differentially respond to dietary sugar and yeast. Lifespan is increased when gut NPF is genetically depleted, and this manipulation is sufficient to blunt the longevity benefit conferred by dietary restriction. Depletion of NPF receptors at insulin producing neurons of the brain also increases lifespan, consistent with observations where loss of gut NPF decreases neuronal insulin secretion. The longevity conferred by repressing gut NPF and brain NPF receptors is reversed by treating adults with a juvenile hormone (JH) analog. JH is produced by the adult corpora allata, and inhibition of the insulin receptor at this tissue decreases JH titer and extends lifespan, while this longevity is restored to wild type by treating adults with a JH analog. Overall, enteroendocrine cells of the gut modulate Drosophila aging through interorgan communication mediated by a gut-brain-corpora allata axis, and insulin produced in the brain impacts lifespan through its control of JH titer. These data suggest that a consideration should be made of how human incretins and their analogs, which are used to treat obesity and diabetes, may impact aging.
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Monday, May 12th - Signaling |
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Tobita, H., Kiuchi, T. (2024). Knockout of cryptochrome 1 disrupts circadian rhythm and photoperiodic diapause induction in the silkworm, Bombyx mori. Insect biochemistry and molecular biology, 172:104153. PubMed ID: 38964485
Summary:
Most insects enter diapause, a state of physiological dormancy crucial for enduring harsh seasons, with photoperiod serving as the primary cue for its induction, ensuring proper seasonal timing of the process. The involvement of clock gene cryptochrome 1 (cry1), which functions as a photoreceptor implicated in photoentrainment remains unclear. In bivoltine strains of the silkworm, Bombyx mori, embryonic diapause is maternally controlled and affected by environmental conditions experienced by mother moths during embryonic and larval stages. This study focused on the involvement of cry1 gene in B. mori photoperiodism. Drosophila-type cry (cry1) and mammalian-type cry (cry2) genes are present in the B. mori genome, akin to other lepidopterans. Temporal expression analysis revealed higher cry1 gene expression during the photophase and lower expression during the scotophase, with knockouts of core clock genes (per, tim, Clk and cyc) disrupting this temporal expression pattern. This study established a cry1 knockout strain in p50T, a bivoltine strain exhibiting clear photoperiodism during both embryonic and larval stages. The cry1 knockout strain exhibited arrhythmic eclosion, implicating B. mori cry1 in the circadian clock feedback loop governing behavior rhythms. Females of the cry1 knockout strain failed to control photoperiodic diapause induction during both embryonic and larval stages, mirroring the diapause phenotype of the wild-type individuals reared under constant darkness, indicating that B. mori CRY1 contributes to photoperiodic time measurement as a photoreceptor. Furthermore, photoperiodic diapause induction during the larval stage was abolished in a cry1/tim double-knockout strain, suggesting that photic information received by CRY1 is relayed to the circadian clock. Overall, this study represents the first evidence of cry1 involvement in insect photoperiodism, specifically in diapause induction.
| Jordan, T., Baker, F., Harman, J., Walton, B., Ajamu-Johnson, A., Alashqar, R., Struhl, G., Langridge, P. D. (2024). An in vivo screen for proteolytic switch domains that can mediate Notch activation by force. bioRxiv, PubMed ID: 39026694
Summary:
Notch proteins are single pass transmembrane receptors that are activated by proteolytic cleavage, allowing their cytosolic domains to function as transcription factors in the nucleus. Upon binding, Delta/Serrate/LAG-2 (DSL) ligands activate Notch by exerting a "pulling" force across the intercellular ligand/receptor bridge. This pulling force is generated by Epsin-mediated endocytosis of ligand into the signal-sending cells, and results in cleavage of the force-sensing Negative Regulatory Region (NRR) of the receptor by an ADAM10 protease [Kuzbanian (Kuz) in Drosophila ]. This study used chimeric Notch and DSL proteins to screen for other domains that can substitute for the NRR in the developing Drosophila wing. While many of the tested domains are either refractory to cleavage or constitutively cleaved, several were identified that mediate Notch activation in response to ligand. These NRR analogues derive from widely divergent source proteins and have strikingly different predicted structures. Yet, almost all depend on force exerted by Epsin-mediated ligand endocytosis and cleavage catalyzed by Kuz. This study positted that the sequence space of protein domains that can serve as force-sensing proteolytic switches in Notch activation is unexpectedly large, a conclusion that has implications for the mechanism of target recognition by Kuz/ADAM10 proteases and is consistent with a more general role for force dependent AD8u,AM10 proteolysis in other cell contact-dependent signaling mechanisms. The results also validate the screen for increasing the repertoire of proteolytic switches available for synthetic Notch (synNotch) therapies and tissue engineering.
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Yang, S., Tian, M., Dai, Y., Wang, R., Yamada, S., Feng, S., Wang, Y., Chhangani, D., Ou, T., Li, W., Guo, X., McAdow, J., Rincon-Limas, D. E., Yin, X., Tai, W., Cheng, G., Johnson, A. (2024). Infection and chronic disease activate a systemic brain-muscle signaling axis. Science immunology, 9(97):eadm7908 PubMed ID: 38996009
Summary:
Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show nonneural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood. Multiple models were developed to investigate the impact of CNS stressors on motor function, and Escherichia coli infections and SARS-CoV-2 protein expression were found to cause reactive oxygen species (ROS) to accumulate in the brain. ROS induced expression of the cytokine Unpaired 3 (Upd3) in Drosophila and its ortholog, IL-6, in mice. CNS-derived Upd3/IL-6 activated the JAK-STAT pathway in skeletal muscle, which caused muscle mitochondrial dysfunction and impaired motor function. Similar phenotypes after expressing toxic amyloid-β (Aβ42) in the CNS. Infection and chronic disease therefore activate a systemic brain-muscle signaling axis in which CNS-derived cytokines bypass the connectome and directly regulate muscle physiology, highlighting IL-6 as a therapeutic target to treat disease-associated muscle dysfunction.
| Zhang, P., Pronovost, S. M., Marchetti, M., Zhang, C., Kang, X., Kandelouei, T., Li, C., Edgar, B. A. (2024). Inter-cell type interactions that control JNK signaling in the Drosophila intestine. Nat Commun, 15(1):5493 PubMed ID: 38944657
Summary:
JNK signaling is a critical regulator of inflammation and regeneration, but how it is controlled in specific tissue contexts remains unclear. This study showed that, in the Drosophila intestine, the TNF-type ligand, Eiger (Egr), is expressed exclusively by intestinal stem cells (ISCs) and enteroblasts (EBs), where it is induced by stress and during aging. Egr preferentially activates JNK signaling in a paracrine fashion in differentiated enterocytes (ECs) via its receptor, Grindelwald (Grnd). N-glycosylation genes (Alg3, Alg9) restrain this activation, and stress -induced downregulation of Alg3 and Alg3 correlates with JNK activation, suggesting a regulatory switch. JNK activity in ECs induces expression of the intermembrane protease Rhomboid (Rho), driving secretion of EGFR ligands Keren (Krn) and Spitz (Spi), which in turn activate EGFR signaling in progenitor cells (ISCs and EBs) to stimulate their growth and division, as well as to produce more Egr. This study uncovers an N-glycosylation-controlled, paracrine JNK-EGFR-JNK feedforward loop that sustains ISC proliferation during stress-induced gut regeneration.
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Vidaurre, V., Song, A., Li, T., Ku, W. L., Zhao, K., Qian, J., Chen, X. (2024). The Drosophila histone methyltransferase SET1 coordinates multiple signaling pathways in regulating male germline stem cell maintenance and differentiation. Development, 151(15) PubMed ID: 39007366
Summary:
Many tissue-specific adult stem cell lineages maintain a balance between proliferation and differentiation. This paper is a study how the H3K4me3 methyltransferase Set1 regulates early-stage male germ cells in Drosophila. Early-stage germline-specific knockdown of Set1 results in temporally progressive defects, arising as germ cell loss and developing into overpopulated early-stage germ cells. These germline defects also impact the niche architecture and cyst stem cell lineage non-cell-autonomously. Additionally, wild-type Set1, but not the catalytically inactive Set1, rescues the Set1 knockdown phenotypes, highlighting the functional importance of the methyltransferase activity of Set1. Further, RNA-sequencing experiments reveal key signaling pathway components, such as the JAK-STAT pathway gene Stat92E and the BMP pathway gene Mad, which are upregulated upon Set1 knockdown. Genetic interaction assays support the functional relationships between Set1 and JAK-STAT or BMP pathways, as both Stat92E and Mad mutations suppress the Set1 knockdown phenotypes. These findings enhance understanding of the balance between proliferation and differentiation in an adult stem cell lineage. The phenotype of germ cell loss followed by over-proliferation when inhibiting a histone methyltransferase also raises concerns about using their inhibitors in cancer therapy.
| Soares, C. C., Rizzo, A., Maresma, M. F., Meier, P. (2024). Autocrine glutamate signaling drives cell competition in Drosophila. Dev Cell, PubMed ID: 39047739
Summary:
Cell competition is an evolutionarily conserved quality control process that eliminates suboptimal or potentially dangerous cells. Although differential metabolic states act as direct drivers of competition, how these are measured across tissues is not understood. This study demonstrates that vesicular glutamate transporter (VGlut) and autocrine glutamate signaling are required for cell competition and Myc-driven super-competition in the Drosophila epithelia. This study found that the loss of glutamate-stimulated VGlut>NMDAR>CaMKII>CrebB signaling triggers loser status and cell death under competitive settings via the autocrine induction of TNF. This in turn drives TNFR>JNK activation, triggering loser cell elimination and PDKLDH-dependent metabolic reprogramming. Inhibiting caspases or preventing loser cells from transferring lactate to their neighbors nullifies cell competition. Further, in a Drosophila model for premalignancy, Myc-overexpressing clones co-opt this signaling circuit to acquire super-competitor status. Targeting glutamate signaling converts Myc "super-competitor" clones into "losers," highlighting new therapeutic opportunities to restrict the evolution of fitter clones.
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Friday, May 9th - Genes, RNAs, enzymes, and protein expression, evolution, structure, and function |
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Sonn, J. Y., Kim, W., Iwanaszko, M., Aoi, Y., Li, Y., Parkitny, L., Brissette, J. L., Weiner, L., Al-Ramahi, I., Botas, J., Shilatifard, A., Zoghbi, H. Y. (2024). MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription. bioRxiv, PubMed ID: 39005382
Summary:
Loss-of-function mutations in methyl-CpG binding protein 2 (MECP2) cause Rett syndrome, a postnatal neurodevelopmental disorder that occurs in ~1/10,000 live female births. MeCP2 binds to methylated cytosines across genomic DNA and recruits various partners to regulate gene expression. MeCP2 has been shown to repress transcription in vitro and interacts with co-repressors such as the Sin3A and NCoR complexes. Based on these observations, MeCP2 has been largely considered as a repressor of transcription. However, a mouse model of RTT displays many down-regulated genes, and those same genes are up-regulated in a MECP2 duplication mouse model. Furthermore, TCF20, which has been associated with transcriptional activation, have recently been identified as a protein interactor of MeCP2. These data broaden the potential functions of MeCP2 as a regulator of gene expression. Yet, the molecular mechanisms underlying MeCP2-dependent gene regulation remain largely unknown. Using a human MECP2 gain-of-function Drosophila model, this study screened for genetic modifiers of MECP2 -induced phenotypes. This approach identified several subunits of the Drosophila super elongation complex, a P-TEFb containing RNA polymerase II (RNA pol II) elongation factor required for the release of promoter-proximally paused RNA pol II, as genetic interactors of MECP2. This study discovered that MeCP2 physically interacts with the SEC in human cells and in the mouse brain. Furthermore, MeCP2 directly binds AFF4, the scaffold of the SEC, via the transcriptional repression domain. Finally, loss of MeCP2 in the mouse cortex caused reduced binding of AFF4 specifically on a subset of genes involved in the regulation of synaptic function, which also displayed the strongest decrease in RNA pol II binding in the genebody. Taken together, this study reveals a previously unrecognized mechanism through which MeCP2 regulates transcription, providing a new dimension to its regulatory role in gene expression.
| Baumgartner, L., Ipsaro, J. J., Hohmann, U., Handler, D., Schleiffer, A., Duchek, P., Brennecke, J. (2024). Evolutionary adaptation of an HP1-protein chromodomain integrates chromatin and DNA sequence signals. Elife, 13 PubMed ID: 38995818
Summary:
Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, it was shown that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C(2)H(2) zinc finger protein named Kipferl. This study elucidates the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, a single amino acid change was identified within Rhino's chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino's chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.
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Lin, M. H., Jensen, M. K., Elrod, N. D., Chu, H. F., Haseley, M., Beam, A. C., Huang, K. L., Chiang, W., Russell, W. K., Williams, K., Proschel, C., Wagner, E. J., Tong, L. (2024). Cytoplasmic binding partners of the Integrator endonuclease INTS11 and its paralog CPSF73 are required for their nuclear function. Mol Cell, 84(15):2900-2917. PubMed ID: 39032490
Summary:
INTS11 and CPSF73 are metal-dependent endonucleases for Integrator and pre-mRNA 3'-end processing, respectively. This study shows that the INTS11 binding partner BRAT1/CG7044, a factor important for neuronal fitness, stabilizes INTS11 in the cytoplasm and is required for Integrator function in the nucleus. Loss of BRAT1 in neural organoids leads to transcriptomic disruption and precocious expression of neurogenesis-driving transcription factors. The structures of the human INTS9-INTS11-BRAT1 and Drosophila dIntS11-CG7044 complexes reveal that the conserved C terminus of BRAT1/CG7044 is captured in the active site of INTS11, with a cysteine residue directly coordinating the metal ions. Inspired by these observations, this study found that UBE3D is a binding partner for CPSF73, and UBE3D likely also uses a conserved cysteine residue to directly coordinate the active site metal ions. These studies have revealed binding partners for INTS11 and CPSF73 that behave like cytoplasmic chaperones with a conserved impact on the nuclear functions of these enzymes.
| Baek, S. E., Kwon, Y., Yoon, J. W., Kim, H. S., Yang, J. Y., Lee, D. S., Yeom, E. (2024). The overexpression of DSP1 in neurons induces neuronal dysfunction and neurodegeneration phenotypes in Drosophila. Mol Brain, 17(1):43 PubMed ID: 39003465
Summary:
Dorsal switch protein 1(DSP1), a mammalian homolog of HMGB1, is firstly identified as a dorsal co-repressor in 1994. DSP1 contains HMG-box domain and functions as a transcriptional regulator in Drosophila melanogaster. It plays a crucial role in embryonic development, particularly in dorsal-ventral patterning during early embryogenesis, through the regulation of gene expression. Moreover, DSP1 is implicated in various cellular processes, including cell fate determination and tissue differentiation, which are essential for embryonic development. While the function of DSP1 in embryonic development has been relatively well-studied, its role in the adult Drosophila brain remains less understood. This study investigated the role of DSP1 in the brain by using neuronal-specific DSP1 overexpression flies. Climbing ability and life span are decreased in DSP1-overexpressed flies. Furthermore, these flies demonstrated neuromuscular junction (NMJ) defect, reduced eye size and a decrease in tyrosine hydroxylase (TH)-positive neurons, indicating neuronal toxicity induced by DSP1 overexpression. Thw data suggest that DSP1 overexpression leads to neuronal dysfunction and toxicity, positioning DSP1 as a potential therapeutic target for neurodegenerative diseases.
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Pedersen, C. N., Yang, F., Ita, S., Xu, Y., Akunuri, R., Trampari, S., Neumann, C. M. T., Desdorf, L. M., Schiott, B., Salvino, J. M., Mortensen, O. V., Nissen, P., Shahsavar, A. (2024). Cryo-EM structure of the dopamine transporter with a novel atypical non-competitive inhibitor bound to the orthosteric site. J Neurochem, PubMed ID: 39010681
Summary:
The regulation of dopamine (DA) removal from the synaptic cleft is a crucial process in neurotransmission and is facilitated by the sodium- and chloride-coupled dopamine transporter DAT. Psychostimulant drugs, cocaine, and amphetamine, both block the uptake of DA, while amphetamine also triggers the release of DA. As a result, they prolong or even amplify neurotransmitter signaling. Atypical inhibitors of DAT lack cocaine-like rewarding effects and offer a promising strategy for the treatment of drug use disorders. This study presents the 3.2 Å resolution cryo-electron microscopy structure of the Drosophila melanogaster dopamine transporter (dDAT) in complex with the atypical non-competitive inhibitor AC-4-248. The inhibitor partially binds at the central binding site, extending into the extracellular vestibule, and locks the transporter in an outward open conformation. These findings propose mechanisms for the non-competitive inhibition of DAT and attenuation of cocaine potency by AC-4-248 and provide a basis for the rational design of more efficacious atypical inhibitors.
| Dolezal, D. M., Joiner, M. A., Eberl, D. F. (2024). Two distinct functions of Lim1 in the Drosophila antenna. microPublication biology, 2024 PubMed ID: 38957438
Summary:
The Lim1 transcription factor is required in Drosophila for patterning the eye-antennal disk. At the adult stage, Lim1 is strongly expressed in Johnston's Organ (JO) neurons, the antennal auditory organ. Using RNAi-mediated knockdown of Lim1 using a strong neuronal driver, this study found a significant reduction in electrophysiological responses to auditory stimuli, recorded from the antennal nerve. This reduction can be accounted for by Lim1 knockdown in the auditory subset of JO neurons, with no effect of knockdown in JO neuron subsets associated with wind or gravity detection. Conversely, Lim1 knockdown in JO sense organ precursors had no effect on hearing. Mosaic animals with antennal clones of the Lim1 E9 null mutation showed morphological defects in the antenna, and significant auditory electrophysiological defects. These results are consistent with two distinct functions for Lim1 in the antenna, including an early patterning function in the eye-antennal disk, and a later neural differentiation function in the JO neurons.
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Tuesday, May 6th - Adult neural development, Strucure, and functions |
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Lancaster, C. L., Yalamanchili, P. S., Goldy, J. N., Leung, S. W., Corbett, A. H., Moberg, K. H. (2024). The RNA-binding protein Nab2 regulates levels of the RhoGEF Trio to govern axon and dendrite morphology. Mol Biol Cell, 35(8):ar109 PubMed ID: 38985523
Summary:
The Drosophila RNA-binding protein (RBP) Nab2 acts in neurons to regulate neurodevelopment and is orthologous to the human intellectual disability-linked RBP, ZC3H14. Nab2 governs axon projection in mushroom body neurons and limits dendritic arborization of class IV sensory neurons in part by regulating splicing events in ~150 mRNAs. Analysis of the Sex-lethal (Sxl) mRNA revealed that Nab2 promotes an exon-skipping event and regulates m(6)A methylation on Sxl pre-mRNA by the Mettl3 methyltransferase. Mettl3 heterozygosity broadly rescues Nab2(null) phenotypes implying that Nab2 acts through similar mechanisms on other RNAs, including unidentified targets involved in neurodevelopment. This study shows that Nab2 and Mettl3 regulate the removal of a 5'UTR (untranslated region) intron in the trio pre-mRNA. Trio utilizes two GEF domains to balance Rac and RhoGTPase activity. Intriguingly, an isoform of Trio containing only the RhoGEF domain, GEF2, is depleted in Nab2(null) nervous tissue. Expression of Trio-GEF2 rescues projection defects in Nab2(null) axons and dendrites, while the GEF1 Rac1-regulatory domain exacerbates these defects, suggesting Nab2-mediated regulation Trio-GEF activities. Collectively, these data indicate that Nab2-regulated processing of trio is critical for balancing Trio-GEF1 and -GEF2 activity and show that Nab2, Mettl3, and Trio function in a common pathway that shapes axon and dendrite morphology.
| Comyn, T., Preat, T., Pavlowsky, A., Placais, P. Y. (2024). PKCδ is an activator of neuronal mitochondrial metabolism that mediates the spacing effect on memory consolidation. bioRxiv, PubMed ID: 38948698
Summary:
Relevance-based selectivity and high energy cost are two distinct features of long-term memory (LTM) formation that warrant its default inhibition. Spaced repetition of learning is a highly conserved cognitive mechanism that can lift this inhibition. This study questioned how the spacing effect integrates experience selection and energy efficiency at the cellular and molecular levels. Drosophila that spaced training triggers LTM formation were investigated by extending over several hours an increased mitochondrial metabolic activity in neurons of the associative memory center, the mushroom body. This effect is mediated by PKCδ, a member of the so-called 'novel PKC' family of enzymes, which uncovers the critical function of PKCδ in neurons as a regulator of mitochondrial metabolism for LTM. Additionally, PKCδ activation and translocation to mitochondria result from LTM-specific dopamine signaling on MB neurons. By bridging experience-dependent neuronal circuit activity with metabolic modulation of memory-encoding neurons, PKCδ signaling binds the cognitive and metabolic constraints underlying LTM formation into a unified gating mechanism.
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Halty-deLeon, L., Pal Mahadevan, V., Wiesel, E., Hansson, B. S., Wicher, D. (2024). Response Plasticity of Drosophila Olfactory Sensory Neurons. Int J Mol Sci, 25(13) PubMed ID: 39000230
Summary:
In insect olfaction, sensitization refers to the amplification of a weak olfactory signal when the stimulus is repeated within a specific time window. In the vinegar fly, Drosophila melanogaster, this occurs already at the periphery, at the level of olfactory sensory neurons (OSNs) located in the antenna. This study investigated whether sensitization is a widespread property in a set of seven types of OSNs, as well as the mechanisms involved. First,the differences in spontaneous activity, response velocity and response dynamics, were characterized and compared among the selected OSN types. These express different receptors with distinct tuning properties and behavioral relevance. Second, it was shown that sensitization is not a general property. Among the selected OSN types, it occurs in those responding to more general food odors, while OSNs involved in very specific detection of highly specific ecological cues like pheromones and warning signals show no sensitization. Moreover, mitochondria were shown to play an active role in sensitization by contributing to the increase in intracellular Ca(2+) upon weak receptor activation. Thus, by using a combination of single sensillum recordings (SSRs), calcium imaging and pharmacology, this study widens the understanding of how the olfactory signal is processed at the periphery.
| Lee, J. Y., Gala, D. S., Kiourlappou, M., Olivares-Abril, J., Joha, J., Titlow, J. S., Teodoro, R. O., Davis, I. (2024). Murine glial protrusion transcripts predict localized Drosophila glial mRNAs involved in plasticity. J Cell Biol, 223(10) PubMed ID: 39037431
Summary:
The polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia are both known to contain long cytoplasmic processes, while localized transcripts have only been studied extensively in neurons, not glia, especially in intact nervous systems. This study predicts 1,740 localized Drosophila glial transcripts by extrapolating from this meta-analysis of seven existing studies characterizing the localized transcriptomes and translatomes of synaptically associated mammalian glia. The localization of mRNAs in mammalian glial projections strongly predicts the localization of their high-confidence Drosophila homologs in larval motor neuron-associated glial projections and are highly statistically enriched for genes associated with neurological diseases. Some of these localized glial transcripts are specifically required in glia for structural plasticity at the nearby neuromuscular junction synapses. It is concluded that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important in disease.
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Asefa, W. R., Woo, J. N., Kim, S. Y., Choi, H., Sung, H., Choi, M. S., Choi, M., Yoon, S. E., Kim, Y. J., Suh, B. C., Kang, K., Kwon, J. Y. (2024). Molecular and cellular basis of sodium sensing in Drosophila labellum. iScience, 27(7):110248 PubMed ID: 39015148
Summary:
Appropriate ingestion of salt is essential for physiological processes such as ionic homeostasis and neuronal activity. Generally, low concentrations of salt elicit attraction, while high concentrations elicit aversive responses. This study observed that sugar neurons in the L sensilla of the Drosophila labellum cf. responses to NaCl, while sugar neurons in the S-c sensilla do not respond to NaCl, suggesting that gustatory receptor neurons involved in NaCl sensing may employ diverse molecular mechanisms. Through an RNAi screen of the entire Ir and ppk gene families and molecular genetic approaches, this study identified IR76b, IR25a, and IR56b as necessary components for NaCl sensing in the Drosophila labellum. Co-expression of these three IRs in heterologous systems such as S2 cells or Xenopus oocytes resulted in a current in response to sodium stimulation, suggesting formation of a sodium-sensing complex. These results should provide insights for research on the diverse combinations constituting salt receptor complexes.
| Imoto, K., Ishikawa, Y., Aso, Y., Funke, J., Tanaka, R., Kamikouchi, A. (2024). Neural-circuit basis of song preference learning in fruit flies. iScience, 27(7):110266 PubMed ID: 39040064
Summary:
As observed in human language learning and song learning in birds, the fruit fly Drosophila melanogaster changes its auditory behaviors according to prior sound experiences. This phenomenon, known as song preference learning in flies, requires GABAergic input to pC1 neurons in the brain, with these neurons playing a key role in mating behavior. The neural circuit basis of this GABAergic input, however, is not known. This study found that GABAergic neurons expressing the sex-determination gene doublesex are necessary for song preference learning. In the brain, only four doublesex-expressing GABAergic neurons exist per hemibrain, identified as pCd-2 neurons. pCd-2 neurons directly, and in many cases mutually, connect with pC1 neurons, suggesting the existence of reciprocal circuits between them. Moreover, GABAergic and dopaminergic inputs to doublesex-expressing GABAergic neurons are necessary for song preference learning. Together, this study provides a neural circuit model that underlies experience-dependent auditory plasticity at a single-cell resolution.
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Monday, May 5th - Chromatin |
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Lv, P., Zhao, Z., Hirano, Y., Du, J. (2024). The CoREST complex regulates multiple histone modifications temporal-specifically in clock neurons. Open biology, 14(7):230355 PubMed ID: 38981515
Summary:
Epigenetic regulation is important for circadian rhythm. In previous studies, multiple histone modifications were found at the Period (Per) locus. However, most of these studies were not conducted in clock neurons. A screen revealed that a CoREST mutation resulted in defects in circadian rhythm by affecting Per transcription. Based on previous studies, it was hypothesized that CoREST regulates circadian rhythm by regulating multiple histone modifiers at the Per locus. Genetic and physical interaction experiments supported these regulatory relationships. Moreover, through tissue-specific chromatin immunoprecipitation assays in clock neurons, the CoREST mutation was found to lead to time-dependent changes in corresponding histone modifications at the Per locus. Finally, a model is proposed indicating the role of the CoREST complex in the regulation of circadian rhythm. This study revealed the dynamic changes of histone modifications at the Per locus specifically in clock neurons. Importantly, it provides insights into the role of epigenetic factors in the regulation of dynamic gene expression changes in circadian rhythm.
| Li, Y., Jiang, Z., Xu, Y., Yan, J., Wu, Q., Huang, S., Wang, L., Xie, Y., Wu, X., Wang, Y., Li, Y., Fan, X., Li, F., Yuan, W. (2024). Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila. Int J Mol Sci, 25(11) PubMed ID: 38892188
Summary:
Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, CRISPR/Cas9 technology was used to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.
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Crain, A. T., Butler, M. B., Hill, C. A., Huynh, M., McGinty, R. K., Duronio, R. J. (2024). Drosophila melanogaster Set8 and L(3)mbt function in gene expression independently of histone H4 lysine 20 methylation. Genes Dev, 38(9-10):455-472 PubMed ID: 38866557
Summary:
Monomethylation of lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me binding proteins. This model was interrogated in a developing animal by comparing in parallel the transcriptomes of Set8 (null) , H4 (K20R/A) , and l(3)mbt mutant Drosophila melanogaster. The gene expression profiles of H4 (K20A) and H4 (K20R) larvae are markedly different than Set8 (null) larvae despite similar reductions in H4K20me1. Set8 (null) mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo, but these phenotypes are not recapitulated by mutation of H4 (K20) , indicating that the developmental defects of Set8 (null) animals are largely due to H4K20me1-independent effects on gene expression. Furthermore, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. It is concluded that gene expression changes in Set8 (null) and H4 (K20) mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin and therefore that H4K20me1 does not play a large role in gene expression.
| Wernig-Zorc, S., Kugler, F., Schmutterer, L., Rao, P., Hausmann, C., Holzinger, S., Langst, G., Schwartz, U. (2024. nucMACC: An MNase-seq pipeline to identify structurally altered nucleosomes in the genome. Sci Adv, 10(27):eadm9740 PubMed ID: 38959309
Summary:
Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. This study presents the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. The regulatory nucleosome landscape was characterized in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. Unstable nucleosomes were shown to present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.
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Doronin, S. A., Ilyin, A. A., Kononkova, A. D., Solovyev, M. A., Olenkina, O. M., Nenasheva, V. V., Mikhaleva, E. A., Lavrov, S. A., Ivannikova, A. Y., Simonov, R. A., Fedotova, A. A., Khrameeva, E. E., Ulianov, S. V., Razin, S. V., Shevelyov, Y. Y. (2024). Nucleoporin Elys attaches peripheral chromatin to the nuclear pores in interphase nuclei. Communications biology, 7(1):783 PubMed ID: 38951619
Summary:
Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Using DamID-seq, Elys binding sites were identified in Drosophila late embryos, and they eeveloped into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. These findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin (Doronin, 2024).
| Chavan, A., Isenhart, R., Nguyen, S. C., Kotb, N. M., Harke, J., Sintsova, A., Ulukaya, G., Uliana, F., Ashiono, C., Kutay, U., Pegoraro, G., Rangan, P., Joyce, E. F., Jagannathan, M. (2024). A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate.Genes Dev, 38(9-10):415-435 PubMed ID: 38866555
Summary:
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The association of genomic loci to the nuclear periphery is proposed to facilitate cell type-specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. This study used an Oligopaint-based HiDRO screen targeting ~1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. The heterochromatin-associated protein Stonewall (Stwl) as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for Stwl's known role in GSC maintenance and ovary homeostasis. Thus, this study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.
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