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| ARCHIVE | Thursday, May 16th - Drosophila as a model for human diseases |
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March 2024
February 2024 January 2024 December 2023 November 2023 October 2023 September 2023 August 2023 July 2023 June 2023 May 2023 April 2023 March 2023 February 2023 January 2023 December 2022 November 2022 October 2022 September 2022 August 2022 July 2022 June 2022 May 2022 April 2022 December 2021 December 2020 December 2019 December 2018 | Bennett, C. L., Dastidar, S., Arnold, F. J., McKinstry, S. U., Stockford, C., Freibaum, B. D., Sopher, B. L., Wu, M., Seidner, G., Joiner, W., Taylor, J. P., West, R. J. H., La Spada, A. R. (2023). Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity. Acta neuropathologica communications, 11(1):164 PubMed ID: 37845749
Summary: Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. This study considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, SETX fly lines were generated, and the effect of SETX in flies expressing either (G4C2)(58) repeats or glycine-arginine-50 [GR(50)] DPRs was evaluated. Dramatic suppression of disease phenotypes was observed in (G4C2)(58) and GR(50) Drosophila models, and a striking relocalization of GR(50) out of the nucleolus was detected in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. It is noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, the nucleolus in cells expressing GR-DPRs was directly assessed, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS. | Swinter, K., Salah, D., Rathnayake, R., Gunawardena, S. (2023). PolyQ-Expansion Causes Mitochondria Fragmentation Independent of Huntingtin and Is Distinct from Traumatic Brain Injury (TBI)/Mechanical Stress-Mediated Fragmentation Which Results from Cell Death. Cells, 12(19) PubMed ID: 37830620
Summary: Mitochondrial dysfunction has been reported in many Huntington's disease (HD) models; however, it is unclear how these defects occur. This study tested the hypothesis that excess pathogenic huntingtin (HTT) impairs mitochondrial homeostasis, using Drosophila genetics and pharmacological inhibitors in HD and polyQ-expansion disease models and in a mechanical stress-induced traumatic brain injury (TBI) model. Expression of pathogenic HTT caused fragmented mitochondria compared to normal HTT, but HTT did not co-localize with mitochondria under normal or pathogenic conditions. Expression of pathogenic polyQ (127Q) alone or in the context of Machado Joseph Disease (MJD) caused fragmented mitochondria. While mitochondrial fragmentation was not dependent on the cellular location of polyQ accumulations, the expression of a chaperone protein, excess of mitofusin (MFN), or depletion of dynamin-related protein 1 (DRP1) rescued fragmentation. Intriguingly, a higher concentration of nitric oxide (NO) was observed in polyQ-expressing larval brains and inhibiting NO production rescued polyQ-mediated fragmented mitochondria, postulating that DRP1 nitrosylation could contribute to excess fission. Furthermore, while excess PI3K, which suppresses polyQ-induced cell death, did not rescue polyQ-mediated fragmentation, it did rescue fragmentation caused by mechanical stress/TBI. Together, these observations suggest that pathogenic polyQ alone is sufficient to cause DRP1-dependent mitochondrial fragmentation upstream of cell death, uncovering distinct physiological mechanisms for mitochondrial dysfunction in polyQ disease and mechanical stress. |
| Roth, J. R., de Moraes, R. C. M., Xu, B. P., Crawley, S. R., Khan, M. A., Melkani, G. C. (2023). Rapamycin reduces neuronal mutant huntingtin aggregation and ameliorates locomotor performance in Drosophila. Frontiers in aging neuroscience, 15:1223911 PubMed ID: 37823007
Summary: Huntington's disease (HD) is a neurodegenerative disease characterized by movement and cognitive dysfunction. HD is caused by a CAG expansion in exon 1 of the HTT gene that leads to a polyglutamine (PQ) repeat in the huntingtin protein, which aggregates in the brain and periphery. Previously, Drosophila models were used to determine that Htt-PQ aggregation in the heart causes shortened lifespan and cardiac dysfunction that is ameliorated by promoting chaperonin function or reducing oxidative stress. The role of neuronal mutant huntingtin and how it affects peripheral function was further studied in this project. Normal (Htt-PQ25) or expanded mutant (Htt-PQ72) exon 1 of huntingtin was overexpressed in Drosophila neurons, and mutant huntingtin was found to cause age-dependent Htt-PQ aggregation in the brain and could cause a loss of synapsin. To determine if this neuronal dysfunction led to peripheral dysfunction,a negative geotaxis assay was performed to measure locomotor performance, and neuronal mutant huntingtin was found to cause an age-dependent decrease in locomotor performance. Next, it was found that rapamycin reduced Htt-PQ aggregation in the brain. These results demonstrate the role of neuronal Htt-PQ in dysfunction in models of HD, suggest that brain-periphery crosstalk could be important to the pathogenesis of HD, and show that rapamycin reduces mutant huntingtin aggregation in the brain. | Catterson, J. H., Minkley, L., Aspe, S., Judd-Mole, S., Moura, S., Dyson, M. C., Rajasingam, A., Woodling, N. S., Atilano, M. L., Ahmad, M., Durrant, C. S., Spires-Jones, T. L., Partridge, L. (2023). Protein retention in the endoplasmic reticulum rescues A β toxicity in Drosophila. Neurobiology of aging, 132:154-174 PubMed ID: 37837732
Summary: Amyloid β (A β; see Drosophila Appl) accumulation is a hallmark of Alzheimer's disease. In adult Drosophila brains, human A β overexpression harms climbing and lifespan. It's uncertain whether A β is intrinsically toxic or activates downstream neurodegeneration pathways. This study uncovers a novel protective role against A β toxicity: intra-endoplasmic reticulum (ER) protein accumulation with a focus on laminin and collagen subunits. Despite high A β, laminin B1 (LanB1) overexpression robustly counters toxicity, suggesting a potential A β resistance mechanism. Other laminin subunits and collagen IV also alleviate A β toxicity; combining them with LanB1 augments the effect. Imaging reveals ER retention of LanB1 without altering A β secretion. LanB1's rescue function operates independently of the IRE1α/XBP1 ER stress response. ER-targeted GFP overexpression also mitigates A β toxicity, highlighting broader ER protein retention advantages. Proof-of-principle tests in murine hippocampal slices using mouse Lamb1 demonstrate ER retention in transduced cells, indicating a conserved mechanism. Though ER protein retention generally harms, it could paradoxically counter neuronal A β toxicity, offering a new therapeutic avenue for Alzheimer's disease. |
| Han, J. E., Kang, K. H., Kim, H., Hong, Y. B., Choi, B. O., Koh, H. (2023). PINK1 and Parkin rescue motor defects and mitochondria dysfunction induced by a patient-derived HSPB3 mutant in Drosophila models. Biochem Biophys Res Commun, 682:71-76 PubMed ID: 37804589
Summary: Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with the α-crystalline domain that is critical to their chaperone activity. Within the sHSP family, three (HSPB1, HSPB3, and HSPB8) proteins are linked with inherited peripheral neuropathies, including distal hereditary motor neuropathy (dHMN) and Charco-Marie-Tooth disease (CMT). This study introduced the HSPB3 Y118H (HSPB3(Y118H)) mutant gene identified from the CMT2 family in Drosophila. With a missense mutation on its α-crystalline domain, this human HSPB3 mutant gene induced a loss of motor activity accompanied by reduced mitochondrial membrane potential in fly neuronal tissues. Moreover, mitophagy, a critical mechanism of mitochondrial quality control, is downregulated in fly motor neurons expressing HSPB3(Y118H). Surprisingly, ">PINK1 and Parkin, the core regulators of mitophagy, successfully rescued these motor and mitochondrial abnormalities in HSPB3 mutant flies. Results from the first animal model of HSPB3 mutations suggest that mitochondrial dysfunction plays a critical role in HSPB3-associated human pathology. | Nil, Z., Deshwar, A. R., Huang, Y., Barish, S., ..., Yamamoto, S., Costain, G., Bellen, H. J. (2023). Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies. American journal of human genetics, 110(11):1919-1937 PubMed ID: 37827158
Summary: Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L (homolog of Drosophila Grappa) is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. This study has identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. Thesee results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease. |
Wednesday, May 15th - Homologs of Drosophila Genes |
| Duan, D., Lyu, W., Chai, P., Ma, S., Wu, K., Wu, C., Xiong, Y., Sestan, N., Zhang, K., Koleske, A. J. (2023). Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation. Curr Biol, 33(21):4582-4598.e4510 PubMed ID: 37858340
Summary: Abl family kinases (see Drosophila Abl) are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both in vitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. This study reports that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ-790. It is proposed that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue. | Cowell, L. M., King, M., West, H., Broadsmith, M., Genever, P., Pownall, M. E., Isaacs, H. V. (2023). Regulation of gene expression downstream of a novel Fgf/Erk pathway during Xenopus development. PLoS One, 18(10):e0286040 PubMed ID: 37856433
Summary: Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf; see Drosophila Breathless) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. It has been proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor. This study investigated the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. Xenopus Cic was characterized and it was shown to be widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with the hypothesis, it was shown that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. Two of these genes were investigated as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b (see Drosophila Ras oncogene at 85D), which encode a leucine zipper transcription factor and a ras family GTPase, respectively. Cic consensus binding sites were identified in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. Expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, it was shown that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. These data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding. |
| Sauty, S. M., Yankulov, K. (2023). Analyses of POL30 (PCNA) reveal positional effects in transient repression or bi-modal active/silent state at the sub-telomeres of S. cerevisiae. Epigenetics & chromatin, 16(1):40 PubMed ID: 37858268
Summary: Classical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. This study used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the different roles of PCNA and histone chaperones in the stability and the propagation of repressed states are shown to at the sub-telomeres of S. cerevisiae. Position dependent transient repression or bi-modal expression of reporter genes were documented at the VIIL sub-telomere. This study also showed that mutations in the replicative clamp POL30 (PCNA) or the deletion of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the deletion of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. The physical interaction of CAF1 and RRM3 with PCNA was analyzed and the implications of these findings for understanding of the stability and transmission of the epigenetic state of the genes are discussed. There are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. This study characterised the roles of CAF1, RRM3 and ASF1 in these modes of gene repression. It is suggested that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing. | Liu, Q., Bell, B. J., Kim, D. W., Lee, S. S., Keles, M. F., Liu, Q., Blum, I. D., Wang, A. A., Blank, E. J., Xiong, J., Bedont, J. L., Chang, A. J., Issa, H., Cohen, J. Y., Blackshaw, S., Wu, M. N. (2023). A clock-dependent brake for rhythmic arousal in the dorsomedial hypothalamus. Nat Commun, 14(1):6381 PubMed ID: 37821426
Summary: Circadian clocks generate rhythms of arousal, but the underlying molecular and cellular mechanisms remain unclear. In Drosophila, the clock output molecule WIDE AWAKE (WAKE) labels rhythmic neural networks and cyclically regulates sleep and arousal. This study shows, in a male mouse model, that mWAKE/ANKFN1 labels a subpopulation of dorsomedial hypothalamus (DMH) neurons involved in rhythmic arousal and acts in the DMH to reduce arousal at night. In vivo Ca(2+) imaging reveals elevated DMH(mWAKE) activity during wakefulness and rapid eye movement (REM) sleep, while patch-clamp recordings show that DMH(mWAKE) neurons fire more frequently at night. Chemogenetic manipulations demonstrate that DMH(mWAKE) neurons are necessary and sufficient for arousal. Single-cell profiling coupled with optogenetic activation experiments suggest that GABAergic DMH(mWAKE) neurons promote arousal. Surprisingly, the data suggest that mWAKE acts as a clock-dependent brake on arousal during the night, when mice are normally active. mWAKE levels peak at night under clock control, and loss of mWAKE leads to hyperarousal and greater DMH(mWAKE) neuronal excitability specifically at night. These results suggest that the clock does not solely promote arousal during an animal's active period, but instead uses opposing processes to produce appropriate levels of arousal in a time-dependent manner. |
| Adiji, O. A., McConnell, B. S., Parker, M. W. (2024). The origin recognition complex requires chromatin tethering by a hypervariable intrinsically disordered region that is functionally conserved from sponge to man. Nucleic Acids Res, 52(8):4344-4360 PubMed ID: 38381902
Summary: The first step toward eukaryotic genome duplication is loading of the replicative helicase onto chromatin. This 'licensing' step initiates with the recruitment of the origin recognition complex (ORC) to chromatin, which is thought to occur via ORC's ATP-dependent DNA binding and encirclement activity. However, previous work has shown that ATP binding is dispensable for the chromatin recruitment of fly ORC, raising the question of how metazoan ORC binds chromosomes. This study shows that the intrinsically disordered region (IDR) of fly Orc1 is both necessary and sufficient for recruitment of ORC to chromosomes in vivo and demonstrate that this is regulated by IDR phosphorylation. Consistently, it was found that the IDR confers the ORC holocomplex with ATP-independent DNA binding activity in vitro. Using phylogenetic analysis, the surprising observation was made that metazoan Orc1 IDRs have diverged so markedly that they are unrecognizable as orthologs and yet these compositionally homologous sequences are functionally conserved. Altogether, these data suggest that chromatin is recalcitrant to ORC's ATP-dependent DNA binding activity, necessitating IDR-dependent chromatin tethering, which is proposed to poise ORC to opportunistically encircle nucleosome-free regions as they become available. | Kushwaha, A., Thakur, M. K. (2024). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Molecular neurobiology 61(1):487-497 PubMed ID: 37626270
Summary: Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ Suv39h1 (a mammalian ortholog of the Drosophila melanogaster SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It has been previously reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. In the current study an attempt was made to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. This attempt was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia. |
Tuesday, May 14th - Adult neural structure, development and function |
| Banach-Latapy, A., Rincheval, V., Briand, D., Guenal, I., Speder, P. (2023). Differential adhesion during development establishes individual neural stem cell niches and shapes adult behaviour in Drosophila. PLoS Biol, 21(11):e3002352 PubMed ID: 37943883
Summary: Neural stem cells (NSCs) reside in a defined cellular microenvironment, the niche, which supports the generation and integration of newborn neurons. The mechanisms building a sophisticated niche structure around NSCs and their functional relevance for neurogenesis are yet to be understood. In the Drosophila larval brain, the cortex glia (CG) encase individual NSC lineages in membranous chambers, organising the stem cell population and newborn neurons into a stereotypic structure. CG were found to wrap around lineage-related cells regardless of their identity, showing that lineage information builds CG architecture. It was then discovered that a mechanism of temporally controlled differential adhesion using conserved complexes supports the individual encasing of NSC lineages. An intralineage adhesion through homophilic Neuroglian interactions provides strong binding between cells of a same lineage, while a weaker interaction through Neurexin-IV and Wrapper exists between NSC lineages and CG. Loss of Neuroglian results in NSC lineages clumped together and in an altered CG network, while loss of Neurexin-IV/Wrapper generates larger yet defined CG chamber grouping several lineages together. Axonal projections of newborn neurons are also altered in these conditions. Further, the loss of these 2 adhesion complexes specifically during development is linked to locomotor hyperactivity in the resulting adults. Altogether, these findings identify a belt of adhesions building a neurogenic niche at the scale of individual stem cell and provide the proof of concept that niche properties during development shape adult behaviour. | Zhao, A., Nern, A., Koskela, S., Dreher, M., Erginkaya, M., Laughland, C. W., Ludwigh, H., Thomson, A., Hoeller, J., Parekh, R., Romani, S., Bock, D. D., Chiappe, E., Reiser, M. B. (2023). A comprehensive neuroanatomical survey of the Drosophila Lobula Plate Tangential Neurons with predictions for their optic flow sensitivity. bioRxiv, PubMed ID: 37904921
Summary: Flying insects exhibit remarkable navigational abilities controlled by their compact nervous systems. Optic flow, the pattern of changes in the visual scene induced by locomotion, is a crucial sensory cue for robust self-motion estimation, especially during rapid flight. The best-known optic-flow sensitive neurons are the large tangential cells of the dipteran lobula plate. This study reports the comprehensive reconstruction and identification of the Lobula Plate Tangential Neurons in an Electron Microscopy (EM) volume of a whole Drosophila brain. This catalog of 58 LPT neurons (per brain hemisphere) contains many neurons that are described here for the first time and provides a basis for systematic investigation of the circuitry linking self-motion to locomotion control. Leveraging computational anatomy methods, it is estimated the visual motion receptive fields of these neurons and compared their tuning to the visual consequence of body rotations and translational movements. These neurons were matched, in most cases on a one-for-one basis, to stochastically labeled cells in genetic driver lines, to the mirror-symmetric neurons in the same EM brain volume, and to neurons in an additional EM data set. Using cell matches across data sets, the integration of optic flow patterns by neurons downstream of the LPTs was analyzed and most central brain neurons were found to establish sharper selectivity for global optic flow patterns than their input neurons. Furthermore, it was found that self-motion information extracted from optic flow is processed in distinct regions of the central brain, pointing to diverse foci for the generation of visual behaviors. |
| Jovanoski, K. D., Duquenoy, L., Mitchell, J., Kapoor, I., Treiber, C. D., Croset, V., Dempsey, G., Parepalli, S., Cognigni, P., Otto, N., Felsenberg, J., Waddell, S. (2023). Dopaminergic systems create reward seeking despite adverse consequences. Nature, 623(7986):356-365 PubMed ID: 37880370
Summary: Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking. Although dysfunction of the dopaminergic valuation system of the brain is known to contribute towards unconstrained reward seeking, the underlying reasons for this behaviour are unclear. This study describes dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. It is proposed that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals. | Ganguly, I., Heckman, E. L., Litwin-Kumar, A., Clowney, E. J., Behnia, R. (2023). Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body. bioRxiv, PubMed ID: 37873086
Summary: The arthropod mushroom body is well-studied as an expansion layer that represents olfactory stimuli and links them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their tuning and function are poorly understood. This study used the FlyWire adult whole-brain connectome to identify inputs to visual Kenyon cells. The types of visual neurons identified are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual projection neurons presynaptic to Kenyon cells receive input from large swathes of visual space, while local visual interneurons, providing smaller fractions of input, receive more spatially restricted signals that may be tuned to specific features of the visual scene. Like olfactory Kenyon cells, visual Kenyon cells receive sparse inputs from different combinations of visual channels, including inputs from multiple optic lobe neuropils. The sets of inputs to individual visual Kenyon cells are consistent with random sampling of available inputs. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the expansion coding properties appear different, with a specific repertoire of visual inputs projecting onto a relatively small number of visual Kenyon cells. |
| Wang, C. M., Wu, C. Y., Lin, C. E., Hsu, M. C., Lin, J. C., Huang, C. C., Lien, T. Y., Lin, H. K., Chang, T. W., Chiang, H. C. (2023). Forgotten memory storage and retrieval in Drosophila. Nat Commun, 14(1):7153 PubMed ID: 37935667
Summary: Inaccessibility of stored memory in ensemble cells through the forgetting process causes animals to be unable to respond to natural recalling cues. While accumulating evidence has demonstrated that reactivating memory-stored cells can switch cells from an inaccessible state to an accessible form and lead to recall of previously learned information, the underlying cellular and molecular mechanisms remain elusive. The current study used Drosophila as a model to demonstrate that the memory of one-trial aversive olfactory conditioning, although inaccessible within a few hours after learning, is stored in KCαβ and retrievable after mild retraining. One-trial aversive conditioning triggers protein synthesis to form a long-lasting cellular memory trace, approximately 20 days, via creb in KCαβ, and a transient cellular memory trace, approximately one day, via orb in MBON-α3. PPL1-α3 negatively regulates forgotten one-trial conditioning memory retrieval. The current study demonstrated that KCαβ, PPL1-α3, and MBON-α3 collaboratively regulate the formation of forgotten one-cycle aversive conditioning memory formation and retrieval. | Yang, H. H., Brezovec, L. E., Capdevila, L. S., Vanderbeck, Q. X., Adachi, A., Mann, R. S., Wilson, R. I. (2023). Fine-grained descending control of steering in walking Drosophila. bioRxiv, PubMed ID: 37904997
Summary: Locomotion involves rhythmic limb movement patterns that originate in circuits outside the brain. Purposeful locomotion requires descending commands from the brain, but it is not understood how these commands are structured. Focusing on the control of steering in walking Drosophila. First, different limb "gestures" associated with different steering maneuvers are described. Next, this study identified a set of descending neurons whose activity predicts steering. Focusing on two descending cell types downstream from distinct brain networks, this study shows that they evoke specific limb gestures: one lengthens strides on the outside of a turn, while the other attenuates strides on the inside of a turn. Notably, a single descending neuron can have opposite effects during different locomotor rhythm phases, and networks positioned to implement this phase-specific gating were identified. Together, these results show how purposeful locomotion emerges from brain cells that drive specific, coordinated modulations of low-level patterns. |
Monday, May 13th - Transcriptional Regulation |
| Chaubal, A., Waldern, J. M., Taylor, C., Laederach, A., Marzluff, W. F., Duronio, R. J. (2023). Coordinated expression of replication-dependent histone genes from multiple loci promotes histone homeostasis in Drosophila. Mol Biol Cell, 34(12):ar118 PubMed ID: 37647143
Summary: Production of large amounts of histone proteins during S phase is critical for proper chromatin formation and genome integrity. This process is achieved in part by the presence of multiple copies of replication dependent (RD) histone genes that occur in one or more clusters in metazoan genomes. In addition, RD histone gene clusters are associated with a specialized nuclear body, the histone locus body (HLB), which facilitates efficient transcription and 3' end-processing of RD histone mRNA. How all five RD histone genes within these clusters are coordinately regulated such that neither too few nor too many histones are produced, a process referred to as histone homeostasis, is not fully understood. This study explored the mechanisms of coordinate regulation between multiple RD histone loci in Drosophila melanogaster and Drosophila virilis. Evidence ia provided for functional competition between endogenous and ectopic transgenic histone arrays located at different chromosomal locations in D. melanogaster that helps maintain proper histone mRNA levels. Consistent with this model, in both species it was found that individual histone gene arrays can independently assemble an HLB that results in active histone transcription. These findings suggest a role for HLB assembly in coordinating RD histone gene expression to maintain histone homeostasis. | Khanbabei, A., Segura, L., Petrossian, C., Lemus, A., Cano, I., Frazier, C., Halajyan, A., Ca, D., Loza-Coll, M. (2024). Experimental validation and characterization of putative targets of Escargot and STAT, two master regulators of the intestinal stem cells in Drosophila melanogaster. Dev Biol, 505:148-163 PubMed ID: 37952851
Summary: Many organs contain adult stem cells (ASCs) to replace cells due to damage, disease, or normal tissue turnover. ASCs can divide asymmetrically, giving rise to a new copy of themselves (self-renewal) and a sister that commits to a specific cell type (differentiation). Decades of research have led to the identification of pleiotropic genes whose loss or gain of function affect diverse aspects of normal ASC biology. Genome-wide screens of these so-called genetic "master regulator" (MR) genes, have pointed to hundreds of putative targets that could serve as their downstream effectors. This study experimentally validated and characterized the regulation of several putative targets of Escargot (Esg) and the Signal Transducer and Activator of Transcription (Stat92E, a.k.a. STAT), two known MRs in Drosophila intestinal stem cells (ISCs). The results indicate that regardless of bioinformatic predictions, most experimentally validated targets show a profile of gene expression that is consistent with co-regulation by both Esg and STAT, fitting a rather limited set of co-regulatory modalities. A bioinformatic analysis of proximal regulatory sequences in specific subsets of co-regulated targets identified additional transcription factors that might cooperate with Esg and STAT in modulating their transcription. Lastly, in vivo manipulations of validated targets rarely phenocopied the effects of manipulating Esg and STAT, suggesting the existence of complex genetic interactions among downstream targets of these two MR genes during ISC homeostasis. |
| Eggers, N., Gkountromichos, F., Krause, S., Campos-Sparr, A., Becker, P. B. (2023). Physical interaction between MSL2 and CLAMP assures direct cooperativity and prevents competition at composite binding sites. Nucleic Acids Res, 51(17):9039-9054 PubMed ID: 37602401
Summary: MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. This study explored the nature of the cooperative binding to these GA-rich, composite sequence elements in reconstituted naive embryonic chromatin. The cooperativity was found to require physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. The cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in the reconstitution, such as roX2 lncRNA. | Aguilera, J., Duan, J., Lee, S. M., Ray, M., Larschan, E. (2023). The CLAMP GA-binding transcription factor regulates heat stress-induced transcriptional repression by associating with 3D loop anchors. bioRxiv, PubMed ID: 37873306
Summary: In order to survive when exposed to heat stress (HS), organisms activate stress response genes and repress constitutive gene expression to prevent the accumulation of potentially toxic RNA and protein products. Although many studies have elucidated the mechanisms that drive HS-induced activation of stress response genes across species, little is known about repression mechanisms or how genes are targeted for activation versus repression context-specifically. The mechanisms of heat stress-regulated activation have been well-studied in Drosophila, in which the GA-binding transcription factor GAF is important for activating genes upon heat stress. This study shows that a functionally distinct GA-binding transcription factor (TF) protein, CLAMP (Chromatin-linked adaptor for MSL complex proteins), is essential for repressing constitutive genes upon heat stress but not activation of the canonical heat stress pathway. HS induces loss of CLAMP-associated 3D chromatin loop anchors associated with different combinations of GA-binding TFs prior to HS if a gene becomes repressed versus activated. Overall, this study demonstrated that CLAMP promotes repression of constitutive genes upon HS, and repression and activation are associated with the loss of CLAMP-associated 3D chromatin loops bound by different combinations of GA-binding TFs. |
| Ramalingam, V., Yu, X., Slaughter, B. D., Unruh, J. R., Brennan, K. J., Onyshchenko, A., Lange, J. J., Natarajan, M., Buck, M., Zeitlinger, J. (2023). Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development. Nat Commun, 14(1):5862 PubMed ID: 37735176
Summary: While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-, a Drosophila zinc finger transcription factor that us one of the more than 25 different splice isoforms from the lolaI locus, this study shows that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters. | Lovero, D., Porcelli, D., Giordano, L., Lo Giudice, C., Picardi, E., Pesole, G., Pignataro, E., Palazzo, A., Marsano, R. M. (2023). Structural and Comparative Analyses of Insects Suggest the Presence of an Ultra-Conserved Regulatory Element of the Genes Encoding Vacuolar-Type ATPase Subunits and Assembly Factors. Biology, 12(8) PubMed ID: 37627011
Summary: Gene and genome comparison represent an invaluable tool to identify evolutionarily conserved sequences with possible functional significance. This work analyzed orthologous genes encoding subunits and assembly factors of the V-ATPase complex, an important enzymatic complex of the vacuolar and lysosomal compartments of the eukaryotic cell with storage and recycling functions, respectively, as well as the main pump in the plasma membrane that energizes the epithelial transport in insects. This study involves 70 insect species belonging to eight insect orders. The conservation is highlighted of a short sequence in the genes encoding subunits of the V-ATPase complex and their assembly factors analyzed with respect to their exon-intron organization of those genes. This study offers the possibility to study ultra-conserved regulatory elements under an evolutionary perspective, with the aim of expanding knowledge of the regulation of complex gene networks at the basis of organellar biogenesis and cellular organization. |
Friday, May 10th - Adult physiology and metabolism |
| Vujnovic, A. F., Martinovic, L., S., Medija, M., Waldowski, R. A. (2023). Distinct and Dynamic Changes in the Temporal Profiles of Neurotransmitters in Drosophila melanogaster Brain following Volatilized Cocaine or Methamphetamine Administrations. Pharmaceuticals (Basel, Switzerland), 16(10) PubMed ID: 37895961
Summary: Due to similarities in genetics, cellular response, and behavior, Drosophila is used as a model organism in addiction research. A well-described behavioral response examined in flies is the induced increase in locomotor activity after a single dose of volatilized cocaine (vCOC) and volatilized methamphetamine (vMETH), the sensitivity, and the escalation of the locomotor response after the repeated dose, the locomotor sensitization. However, knowledge about how vCOC and vMETH affect different neurotransmitter systems over time is scarce. This study used LC-MS/MS to systematically examine changes in the concentration of neurotransmitters, metabolites and non-metabolized COC and METH in the whole head homogenates of male flies one to seven hours after single and double vCOC or vMETH administrations. vMETH leads to complex changes in the levels of examined substances over time, while vCOC strongly and briefly increases concentrations of dopamine, tyramine and octopamine followed by a delayed degradation into N-acetyl dopamine and N-acetyl tyramine. The first exposure to psychostimulants leads to significant and dynamic changes in the concentrations relative to the second administration when they are more stable over several hours. Further investigations are needed to understand neurochemical and molecular changes post-psychostimulant administration. | Alassaf, M., Rajan, A. (2023). Diet-induced glial insulin resistance impairs the clearance of neuronal debris in Drosophila brain. PLoS Biol, 21(11):e3002359 PubMed ID: 37934726
Summary: Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Using a Drosophila model this study shows that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, this study has established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris. |
| Brener, A., Lorber, D., Reuveny, A., Toledano, H., Porat-Kuperstein, L., Lebenthal, Y., Weizman, E., Olender, T., Volk, T. (2023). Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae. Cells, 12(19) PubMed ID: 37830547
Summary: The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi(1)) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. The findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating these findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases. | Torre, M., Bukhari, H., Nithianandam, V., Zanella, C. A., Mata, D. A., Feany, M. B. (2023). A Drosophila model relevant to chemotherapy-related cognitive impairment. Sci Rep, 13(1):19290 PubMed ID: 37935827
Summary: Chemotherapy-related cognitive impairment (CRCI) is a common adverse effect of treatment and is characterized by deficits involving multiple cognitive domains including memory. Despite the significant morbidity of CRCI and the expected increase in cancer survivors over the coming decades, the pathophysiology of CRCI remains incompletely understood, highlighting the need for new model systems to study CRCI. Given the powerful array of genetic approaches and facile high throughput screening ability in Drosophila, the goal of this study was to validate a Drosophila model relevant to CRCI. The chemotherapeutic agents cisplatin, cyclophosphamide, and doxorubicin were administered to adult Drosophila. Neurologic deficits were observed with all tested chemotherapies, with doxorubicin and in particular cisplatin also resulting in memory deficits. Histologic and immunohistochemical analysis was performed of cisplatin-treated Drosophila tissue, demonstrating neuropathologic evidence of increased neurodegeneration, DNA damage, and oxidative stress. Thus, the Drosophila model relevant to CRCI recapitulates clinical, radiologic, and histologic alterations reported in chemotherapy patients. This new Drosophila model can be used for mechanistic dissection of pathways contributing to CRCI (and chemotherapy-induced neurotoxicity more generally) and pharmacologic screens to identify disease-modifying therapies. |
| Brischigliaro, M., Cabrera-Orefice, A., Arnold, S., Viscomi, C., Zeviani, M., Fernandez-Vizarra, E. (2023). Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes. Elife, 12 PubMed ID: 37823874
Summary: Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. This study show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, it is concluded that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis. | Li, J., Dang, P., Li, Z., Zhao, T., Cheng, D., Pan, D., Yuan, Y., Song, W. (2023). Peroxisomal ERK mediates Akh/glucagon action and glycemic control. Cell Rep, 42(10):113200 PubMed ID: 37796662
Summary: he enhanced response of glucagon and its Drosophila homolog, Adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. This study found that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. Mammalian glucagon-induced hepatic ERK peroxisomal translocation was also observed in diabetic subjects. Therefore, these results conclude that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control. |
Thursday, May 9th - Tumors, Cancer and Growth |
| Khalili, D., Mohammed, M., Kunc, M., Sindlerova, M., Ankarklev, J., Theopold, U. (2023). Single-cell sequencing of tumor-associated macrophages in a Drosophila model. Frontiers in immunology, 14:1243797 PubMed ID: 37795097
Summary: Tumor-associated macrophages may act to either limit or promote tumor growth, yet the molecular basis for either path is poorly characterized. This study used a larval Drosophila model that expresses a dominant-active version of the Ras-oncogene (Ras(V12)) to study dysplastic growth during early tumor progression. Single-cell RNA-sequencing was performed of macrophage-like hemocytes to characterize these cells in tumor- compared to wild-type larvae. Hemocytes included manually extracted tumor-associated- and circulating cells. Five distinct hemocyte clusters were identified. In addition to Ras(V12) larvae, a tumor model was included where the activation of effector caspases was inhibited, mimicking an apoptosis-resistant setting. Circulating hemocytes from both tumor models differ qualitatively from control wild-type cells-they display an enrichment for genes involved in cell division, which was confirmed using proliferation assays. Split analysis of the tumor models further reveals that proliferation is strongest in the caspase-deficient setting. Similarly, depending on the tumor model, hemocytes that attach to tumors activate different sets of immune effectors-antimicrobial peptides dominate the response against the tumor alone, while caspase inhibition induces a shift toward members of proteolytic cascades. Finally, evidence is provided for transcript transfer between hemocytes and possibly other tissues. Taken together, these data support the usefulness of Drosophila to study the response against tumors at the organismic level. | Yu, K., Ramkumar, N., Wong, K. K. L., Tettweiler, G., Verheyen, E. M. (2023). The AMPK-like protein kinases Sik2 and Sik3 interact with Hipk and induce synergistic tumorigenesis in a Drosophila cancer model. Frontiers in cell and developmental biology, 11:1214539 PubMed ID: 37854071.
Summary: Homeodomain-interacting protein kinases (Hipks) regulate cell proliferation, apoptosis, and tissue development. Overexpression of Hipk in Drosophila causes tumorigenic phenotypes in larval imaginal discs. This study found that depletion of Salt-inducible kinases Sik2 or Sik3 can suppress Hipk-induced overgrowth. Furthermore, co-expression of constitutively active forms of Sik2 or Sik3 with Hipk caused significant tissue hyperplasia and tissue distortion, indicating that both Sik2 and Sik3 can synergize with Hipk to promote tumorous phenotypes, accompanied by elevated dMyc, Armadillo/β-catenin, and the Yorkie target gene expanded. Larvae expressing these hyperplastic growths also display an extended larval phase, characteristic of other Drosophila tumour models. Examination of total protein levels from fly tissues showed that Hipk proteins were reduced when Siks were depleted through RNAi, suggesting that Siks may regulate Hipk protein stability and/or activity. Conversely, expression of constitutively active Siks with Hipk leads to increased Hipk protein levels. Furthermore, Hipk can interact with Sik2 and Sik3 by co-immunoprecipitation. Co-expression of both proteins leads to a mobility shift of Hipk protein, suggesting it is post-translationally modified. In summary, this research demonstrates a novel function of Siks in synergizing with Hipk to promote tumour growth. |
| Bosch, P. S., Cho, B., Axelrod, J. D. (2023). Flamingo participates in multiple models of cell competition. bioRxiv, PubMed ID: 37790459
Summary: The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. This study shows that in Drosophila, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in pre-malignant cells as they grow among wildtype cells, in healthy cells to eliminate pre-malignant cells, and by supercompetitors to occupy excessive territory within wildtype tissues. "Would-be" winners that lack Fmi are unable to over-proliferate, and instead become losers. This study demonstrates that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well defined functions of Fmi. | Pranoto, I. K. A., Lee, J., Kwon, Y. V. (2023). The roles of the native cell differentiation program aberrantly recapitulated in Drosophila intestinal tumors. Cell Rep, 42(10):113245 PubMed ID: 37837622ID:
Summary: Many tumors recapitulate the developmental and differentiation program of their tissue of origin, a basis for tumor cell heterogeneity. Although stem-cell-like tumor cells are well studied, the roles of tumor cells undergoing differentiation remain to be elucidated. This study employ Drosophila genetics to demonstrate that the differentiation program of intestinal stem cells is crucial for enabling intestinal tumors to invade and induce non-tumor-autonomous phenotypes. The differentiation program that generates absorptive cells is aberrantly recapitulated in the intestinal tumors generated by activation of the Yap1 ortholog Yorkie. Inhibiting it allows stem-cell-like tumor cells to grow but suppresses invasiveness and reshapes various phenotypes associated with cachexia-like wasting by altering the expression of tumor-derived factors. This study provides insight into how a native differentiation program determines a tumor's capacity to induce advanced cancer phenotypes and suggests that manipulating the differentiation programs co-opted in tumors might alleviate complications of cancer, including cachexia. |
| Zheng, J., Guo, Y., Shi, C., Yang, S., Xu, W., Ma, X. (2023). Differential Ire1 determines loser cell fate in tumor-suppressive cell competition. Cell Rep, 42(11):113303 PubMed ID: 37924514
Summary: Tumor-suppressive cell competition (TSCC) is a conserved surveillance mechanism in which neighboring cells actively eliminate oncogenic cells. Despite overwhelming studies showing that the unfolded protein response (UPR) is dysregulated in various tumors, it remains debatable whether the UPR restrains or promotes tumorigenesis. Using Drosophila eye epithelium as a model, this study uncovered a surprising decisive role of the Ire1 branch of the UPR in regulating cell polarity gene scribble (scrib) loss-induced TSCC. Both mutation and hyperactivation of Ire1 accelerate elimination of scrib clones via inducing apoptosis and autophagy, respectively. Unexpectedly, relative Ire1 activity is also crucial for determining loser cell fate, as dysregulating Ire1 signaling in the surrounding healthy cells reversed the "loser" status of scrib clones by decreasing their apoptosis. Furthermore, it was shown that Ire1 is required for cell competition in mammalian cells. Together, these findings provide molecular insights into scrib-mediated TSCC and highlight Ire1 as a key determinant of loser cell fate. | Quintero, M., Bangi, E. (2023). Disruptions in cell fate decisions and transformed enteroendocrine cells drive intestinal tumorigenesis in Drosophila. Cell Rep, 42(11):113370 PubMed ID: 37924517
Summary: Most epithelial tissues are maintained by stem cells that produce the different cell lineages required for proper tissue function. Constant communication between different cell types ensures precise regulation of stem cell behavior and cell fate decisions. These cell-cell interactions are often disrupted during tumorigenesis, but mechanisms by which they are co-opted to support tumor growth in different genetic contexts are poorly understood. This study introduced PromoterSwitch, a genetic platform established to generate large, transformed clones derived from individual adult Drosophila intestinal stem/progenitor cells. Cancer-driving genetic alterations representing common colon tumor genome landscapes were shown to disrupt cell fate decisions within transformed tissue and result in the emergence of abnormal cell fates. It was also shown that transformed enteroendocrine cells, a differentiated, hormone-secreting cell lineage, support tumor growth by regulating intestinal stem cell proliferation through multiple genotype-dependent mechanisms, which represent potential vulnerabilities that could be exploited for therapy. |
Wednesday, May 8th - Drosophila as a model for human diseases |
| Min, Y., Wang, X., Is, O., Patel, T. A., Gao, J., Reddy, J. S., Quicksall, Z. S., Nguyen, T., Lin, S., Tutor-New, F. Q., Chalk, J. L., Mitchell, A. O., Crook, J. E., Nelson, P. T., Van Eldik, L. J., Golde, T. E., Carrasquillo, M. M., Dickson, D. W., Zhang, K., Allen, M., Ertekin-Taner, N. (2023). Cross species systems biology discovers glial DDR2, STOM, and KANK2 as therapeutic targets in progressive supranuclear palsy. Nat Commun, 14(1):6801 PubMed ID: 37919278
Summary: Progressive supranuclear palsy (PSP) is a neurodegenerative parkinsonian disorder characterized by cell-type-specific tau lesions in neurons and glia. Prior work uncovered transcriptome changes in human PSP brains, although their cell-specificity is unknown. Further, systematic data integration and experimental validation platforms to prioritize brain transcriptional perturbations as therapeutic targets in PSP are currently lacking. This study combined bulk tissue (n = 408) and single nucleus RNAseq (n = 34) data from PSP and control brains with transcriptome data from a mouse tauopathy and experimental validations in Drosophila tau models for systematic discovery of high-confidence expression changes in PSP with therapeutic potential. Thousands of differentially expressed genes were discovered, replicated, and annotated in PSP, many of which reside in glia-enriched co-expression modules and cells. DDR2, STOM, and KANK2 were were prioritized as promising therapeutic targets in PSP with striking cross-species validations. These findings and data are shared via an interactive application tool PSP RNAseq Atlas. The findings reveal robust glial transcriptome changes in PSP, provide a cross-species systems biology approach, and a tool for therapeutic target discoveries in PSP with potential application in other neurodegenerative diseases. | Yan, L., Zhou, J., Yuan, L., Ye, J., Zhao, X., Ren, G., Chen, H. (2023). Silibinin alleviates intestinal inflammation via inhibiting JNK signaling in Drosophila. Frontiers in pharmacology, 14:1246960 PubMed ID: 37781701
Summary: Inflammatory bowel diseases (IBDs) are characterized by chronic relapsing intestinal inflammation that causes digestive system dysfunction. For years, researchers have been working to find more effective and safer therapeutic strategies to treat these diseases. Silibinin (SIL), a flavonoid compound extracted from the seeds of milk thistle plants, possesses multiple biological activities and is traditionally applied to treat liver diseases. SIL is also widely used in the treatment of a variety of inflammatory diseases attributed to its excellent antioxidant and anti-inflammatory effects. However, the efficacy of SIL against IBDs and its mechanisms remain unclear. This study, using Drosophila melanogaster as a model organism, found that SIL can effectively relieve intestinal inflammation caused by dextran sulfate sodium (DSS). The results suggested that SIL supplementation can inhibit the overproliferation of intestinal stem cells (ISCs) induced by DSS, protect intestinal barrier function, acid-base balance, and intestinal excretion function, reduce intestinal reactive oxygen species (ROS) levels and inflammatory stress, and extend the lifespan of Drosophila. Furthermore, this study demonstrated that SIL ameliorates intestinal inflammation via modulating the c-Jun N-terminal kinase (JNK) signaling pathway in Drosophila. This research aims to provide new insight into the treatment of IBDs. |
| Aalto, A. L., Saadabadi, A., Lindholm, F., Kietz, C., Himmelroos, E., Marimuthu, P., Salo-Ahen, O. M. H., Eklund, P., Meinander, A. (2023). Stilbenoid compounds inhibit NF-κB-mediated inflammatory responses in the Drosophila intestine. Frontiers in immunology, 14:1253805 PubMed ID: 37809071
Summary: Stilbenoid compounds have been described to have anti-inflammatory properties in animal models in vivo, and have been shown to inhibit Ca2+-influx through the transient receptor potential ankyrin 1 (TrpA1). To study how stilbenoid compounds affect inflammatory signaling in vivo, the fruit fly, Drosophila melanogaster, was used as a model system. To induce intestinal inflammation in the fly, flies were fed with the intestinal irritant dextran sodium sulphate (DSS). DSS was found to induce severe changes in the bacteriome of the Drosophila intestine, and that this dysbiosis causes activation of the NF-κB transcription factor Relish. The DSS-model was used to study the anti-inflammatory properties of the stilbenoid compounds pinosylvin (PS) and pinosylvin monomethyl ether (PSMME). With the help of in vivo approaches, PS and PSMME were identified as transient receptor ankyrin 1 (TrpA1)-dependent antagonists of NF-κB-mediated intestinal immune responses in Drosophila were computationally predicted. The putative antagonist binding sites of these compounds at Drosophila TrpA1. Taken together, this study showed that the stilbenoids PS and PSMME have anti-inflammatory properties in vivo in the intestine and can be used to alleviate chemically induced intestinal inflammation in Drosophila. | Wang, C. W., Clemot, M., Hashimoto, T., Diaz, J. A., Goins, L. M., Goldstein, A. S., Nagaraj, R., Banerjee, U. (2023). A conserved mechanism for JNK-mediated loss of Notch function in advanced prostate cancer. Science signaling, 16(810):eabo5213 PubMed ID: 37934809
Summary: Dysregulated Notch signaling is a common feature of cancer; however, its effects on tumor initiation and progression are highly variable, with Notch having either oncogenic or tumor-suppressive functions in various cancers. To better understand the mechanisms that regulate Notch function in cancer, Notch signaling was studied in a Drosophila tumor model, prostate cancer-derived cell lines, and tissue samples from patients with advanced prostate cancer. It was demonstrated that increased activity of the Src-JNK pathway in tumors inactivated Notch signaling because of JNK pathway-mediated inhibition of the expression of the gene encoding the Notch S2 cleavage protease, Kuzbanian, which is critical for Notch activity. Consequently, inactive Notch accumulated in cells, where it was unable to transcribe genes encoding its target proteins, many of which have tumor-suppressive activities. These findings suggest that Src-JNK activity in tumors predicts Notch activity status and that suppressing Src-JNK signaling could restore Notch function in tumors, offering opportunities for diagnosis and targeted therapies for a subset of patients with advanced prostate cancer. |
| Diaw, S. H., Borsche, M., Streubel-Gallasch, L., Dulovic-Mahlow, M., Hermes, J., Lenz, I., Seibler, P., Klein, C., Bruggemann, N., Vos, M., Lohmann, K. (2023). Characterization of the pathogenic alpha-Synuclein Variant V15A in Parkinson´s disease. NPJ Parkinson's disease, 9(1):148 PubMed ID: 37903765
Summary: Despite being a major component of Lewy bodies and Lewy neurites, pathogenic variants in the gene encoding alpha-Synuclein (α-Syn) are rare. To date, only four missense variants in the SNCA gene, encoding α-Syn have unequivocally been shown to be disease-causing. This study describes a Parkinson´s disease patient with early cognitive decline carrying an as yet not fully characterized variant in SNCA (NM_001146055: c.44T > C, p.V15A). Different cellular models, including stably transfected neuroblastoma (SH-SY5Y) cell cultures, induced pluripotent stem cell (iPSC)-derived neuronal cultures, and a Drosophila model was generated to elucidate the impact of the p.V15A variant on α-Syn function and aggregation properties compared to other known pathogenic variants. This study demonstrated that p.V15A increased the aggregation potential of α-Syn and the levels of apoptotic markers, and impaired the mitochondrial network. Moreover, p.V15A affects the flying ability and survival of mutant flies. Thus, this study provides supporting evidence for the pathogenicity of the p.V15A variant, suggesting its inclusion in genetic testing approaches. | Zane, F., Bouzid, H., Sosa Marmol, S., Brazane, M., Besse, S., Molina, J. L., Cansell, C., Aprahamian, F., Durand, S., Ayache, J., Antoniewski, C., Todd, N., Carre, C., Rera, M. (2023). Smurfness-based two-phase model of ageing helps deconvolve the ageing transcriptional signature. Aging Cell, 22(11):e13946 PubMed ID: 37822253
Summary: Ageing is characterised at the molecular level by six transcriptional 'hallmarks of ageing', that are commonly described as progressively affected as time passes. By contrast, the 'Smurf' assay, which assesses food intake by the co-ingestion of a blue dye, which is not absorbed by the digestive tract, separates high-and-constant-mortality risk individuals from healthy, zero-mortality risk individuals, based on increased intestinal permeability. Performing whole body total RNA sequencing, it was found that Smurfness distinguishes transcriptional changes associated with chronological age from those associated with biological age. Transcriptional heterogeneity is shown to increase with chronological age in non-Smurf individuals preceding the other five hallmarks of ageing that are specifically associated with the Smurf state. Using this approach, targeted pro-longevity genetic interventions delaying entry in the Smurf state were devised. It is anticipated that increased attention to the evolutionary conserved Smurf phenotype will bring about significant advances in understanding of the mechanisms of ageing. |
Tuesday, May 7th - Embryonic Neural Development |
| Mitchell, J. W., Midillioglu, I., Schauer, E., Wang, B., Han, C., Wildonger, J. (2023). Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons. PLoS Genet, 19(11):e1011025 PubMed ID: 37943859
Summary: Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the localization of sensory ion channels to the dendritic membrane. However, it is not well understood when and how ion channels localize to growing sensory dendrites and whether their delivery is coordinated with growth of the dendritic arbor. This study investigated the localization of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in the peripheral sensory neurons of developing fruit flies. CRISPR-Cas9 genome engineering approaches were used to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Fluorescently tagged endogenous Ppk1 localizes to dendrites, as previously reported, and, unexpectedly, to axons and axon terminals. In dendrites, Ppk1 is present throughout actively growing dendrite branches and is stably integrated into the neuronal cell membrane during the expansive growth of the arbor. Although Ppk channels are dispensable for dendrite growth, it was found that an over-active channel mutant severely reduces dendrite growth, likely by acting at an internal membrane and not the dendritic membrane. These data reveal that the molecular motor dynein and recycling endosome GTPase Rab11 are needed for the proper trafficking of Ppk1 to dendrites. Based on these data, it is proposed that Ppk channel transport is coordinated with dendrite morphogenesis, which ensures proper ion channel density and distribution in sensory dendrites. | Hsiao, Y. L., Chen, H. W., Chen, K. H., Tan, B. C., Chen, C. H. and Pi, H. (2023). Actin-related protein 6 facilitates proneural protein-induced gene activation for rapid neural differentiation. Development 150(5). PubMed ID: 36897355
Summary: Neurogenesis is initiated by basic helix-loop-helix proneural proteins. This study showed that Actin-related protein 6 (Arp6), a core component of the H2A.Z exchange complex SWR1, interacts with proneural proteins and is crucial for efficient onset of proneural protein target gene expression. Arp6 mutants exhibit reduced transcription in sensory organ precursors (SOPs) downstream of the proneural protein patterning event. This leads to retarded differentiation and division of SOPs and smaller sensory organs. These phenotypes are also observed in proneural gene hypomorphic mutants. Hypomorphic ac sc mutant recapitulates Arp6 mutant phenotypes. Arp6 also interacted with Sc and Atonal (Ato) but failed to interact with the proneural protein heterodimeric partner Daughterless. Proneural protein expression is not reduced in Arp6 mutants. Enhanced proneural gene expression fails to rescue retarded differentiation in Arp6 mutants, suggesting that Arp6 acts downstream of or in parallel with proneural proteins. H2A.Z mutants display Arp6-like retardation in SOPs. Transcriptomic analyses demonstrate that loss of Arp6 and H2A.Z preferentially decreases expression of proneural protein-activated genes. H2A.Z enrichment in nucleosomes around the transcription start site before neurogenesis correlates highly with greater activation of proneural protein target genes by H2A.Z. It is proposed that upon proneural protein binding to E-box sites, H2A.Z incorporation around the transcription start site allows rapid and efficient activation of target genes, promoting rapid neural differentiation. |
| Singh, B. N., Tran, H., Kramer, J., Kirishenko, E., Changela, N., Wang, F., Feng, Y., Kumar, D., Tu, M., Lan, J., Bizet, M., Fuks, F. and Steward, R. (2023). Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates the axon guidance genes robo2 and slit in Drosophila. Res Sq. PubMed ID: 36824980
Summary: Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases enzymes catalyzing the transition of 5mC to 5hmC in DNA and have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila because Tet knock-out animals do not reach adulthood. The identification is described of Tet-target genes in the embryo and larval brain by determining Tet DNA-binding sites throughout the genome and by mapping the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC-modified sites can be found along the entire transcript and are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are frequently involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and are sensitized to reduced levels of slit. Both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. These results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs, primarily in developing nerve cells. | Sullivan, K. G., Bashaw, G. J. (2023). Commissureless acts as a substrate adapter in a conserved Nedd4 E3 ubiquitin ligase pathway to promote axon growth across the midline. bioRxiv. PubMed ID: 37905056
Summary: In both vertebrates and invertebrates, commissural neurons prevent premature responsiveness to the midline repellant Slit by downregulating surface levels of its receptor Roundabout1 (Robo1). In Drosophila, Commissureless (Comm) plays a critical role in this process; however, there is conflicting data on the underlying molecular mechanism. This study demonstrated that the conserved PY motifs in the cytoplasmic domain of Comm are required allow the ubiquitination and lysosomal degradation of Robo1. Disruption of these motifs prevents Comm from localizing to Lamp1 positive late endosomes and to promote axon growth across the midline in vivo. In addition, a role for Nedd4 in midline crossing was identified. Genetic analysis shows that nedd4 mutations result in midline crossing defects in the Drosophila embryonic nerve cord, which can be rescued by introduction of exogenous Nedd4. Biochemical evidence shows that Nedd4 incorporates into a three-member complex with Comm and Robo in a PY motif-dependent manner. Finally, genetic evidence is presented that Nedd4 acts with Comm in the embryonic nerve cord to downregulate Robo1 levels. Taken together, these findings demonstrate that Comm promotes midline crossing in the nerve cord by facilitating Robo ubiquitination by Nedd4, ultimately leading to its degradation. |
| Carranza, A., Howard, L. J., Brown, H. E., Ametepe, A. S. and Evans, T. A. (2023). Slit-independent guidance of longitudinal axons by Drosophila Robo3. bioRxiv. PubMed ID: 37214810
Summary: Drosophila Robo3 is a member of the evolutionarily conserved Roundabout (Robo) receptor family and one of three Drosophila Robo paralogs. During embryonic ventral nerve cord development, Robo3 does not participate in canonical Slit-dependent midline repulsion, but instead regulates the formation of longitudinal axon pathways at specific positions along the medial-lateral axis. Longitudinal axon guidance by Robo3 is hypothesized to be Slit dependent, but this has not been directly tested. In this study a series of Robo3 variants was created in which the N-terminal Ig1 domain is deleted or modified, in order to characterize the functional importance of Ig1 and Slit binding for Robo3's axon guidance activity. We show that Robo3 requires its Ig1 domain for interaction with Slit and for proper axonal localization in embryonic neurons, but deleting Ig1 from Robo3 only partially disrupts longitudinal pathway formation. Robo3 variants with modified Ig1 domains that cannot bind Slit retain proper localization and fully rescue longitudinal axon guidance. These results indicate that Robo3 guides longitudinal axons independently of Slit, and that sequences both within and outside of Ig1 contribute to this Slit-independent activity. | Karkali, K., Saunders, T. E., Panayotou, G. and Martín-Blanco, E. (2023). JNK signaling in pioneer neurons organizes ventral nerve cord architecture in Drosophila embryos. Nat Commun 14(1): 675. PubMed ID: 36750572
Summary: Morphogenesis of the Central Nervous System (CNS) is a complex process that obeys precise architectural rules. Yet, the mechanisms dictating these rules remain unknown. Analyzing morphogenesis of the Drosophila embryo Ventral Nerve Cord (VNC), this study observe that a tight control of JNK signaling is essential for attaining the final VNC architecture. JNK signaling in a specific subset of pioneer neurons autonomously regulates the expression of Fasciclin 2 (Fas 2) and Neurexin IV (Nrx IV) adhesion molecules, probably via the transcription factor zfh1. Interfering at any step in this cascade affects fasciculation along pioneer axons, leading to secondary cumulative scaffolding defects during the structural organization of the axonal network. The global disorder of architectural landmarks ultimately influences nervous system condensation. In summary, these data point to JNK signaling in a subset of pioneer neurons as a key element underpinning VNC architecture, revealing critical milestones on the mechanism of control of its structural organization. |
Monday, May 6th - Cell Cycle |
| Haseeb, M. A., Weng, K. A., Bickel, S. E. (2023). Chromatin-associated cohesin turns over extensively and forms new cohesive linkages during meiotic prophase. bioRxiv, PubMed ID: 37645916
Summary: In dividing cells, accurate chromosome segregation depends on sister chromatid cohesion, protein linkages that are established during DNA replication. Faithful chromosome segregation in oocytes requires that cohesion, first established in S phase, remain intact for days to decades, depending on the organism. Premature loss of meiotic cohesion in oocytes leads to the production of aneuploid gametes and contributes to the increased incidence of meiotic segregation errors as women age (maternal age effect). The prevailing model is that cohesive linkages do not turn over in mammalian oocytes. However, it has been reported that cohesion-related defects arise in Drosophila oocytes when individual cohesin subunits (see Verthandi) or cohesin regulators are knocked down after meiotic S phase. This study used two strategies to express a tagged cohesin subunit exclusively during mid-prophase in Drosophila oocytes and demonstrate that newly expressed cohesin is used to form de novo linkages after meiotic S phase. Moreover, nearly complete turnover of chromosome-associated cohesin occurs during meiotic prophase, with faster replacement on the arms than at the centromeres. Unlike S-phase cohesion establishment, the formation of new cohesive linkages during meiotic prophase does not require acetylation of conserved lysines within the Smc3 head. These findings indicate that maintenance of cohesion between S phase and chromosome segregation in Drosophila oocytes requires an active cohesion rejuvenation program that generates new cohesive linkages during meiotic prophase. | Koury, S. A. (2023). Female meiotic drive shapes the distribution of rare inversion polymorphisms in Drosophila melanogaster. Genetics. PubMed ID: 37616566
Summary: In all species, new chromosomal inversions are constantly being formed by spontaneous rearrangement and then stochastically eliminated from natural populations. In Drosophila, when new chromosomal inversions overlap with a pre-existing inversion in the population, their rate of elimination becomes a function of the relative size, position, and linkage phase of the gene rearrangements. These altered dynamics result from complex meiotic behavior wherein overlapping inversions generate asymmetric dyads that cause both meiotic drive/drag and segmental aneuploidy. In this context, patterns in rare inversion polymorphisms of a natural population can be modeled from the fundamental genetic processes of forming asymmetric dyads via crossing-over in meiosis I and preferential segregation from asymmetric dyads in meiosis II. A mathematical model was developed of crossover-dependent female meiotic drive and was parameterized with published experimental data from Drosophila melanogaster laboratory constructs. This mechanism is demonstrated to favor smaller, distal inversions and accelerate the elimination of larger, proximal inversions. Simulated sampling experiments indicate that the paracentric inversions directly observed in natural population surveys of Drosophila melanogaster are a biased subset that both maximizes meiotic drive and minimizes the frequency of lethal zygotes caused by this cytogenetic mechanism. Incorporating this form of selection into a population genetic model accurately predicts the shift in relative size, position, and linkage phase for rare inversions found in this species. The model and analysis presented in this study suggest that this weak form of female meiotic drive is an important process influencing the genomic distribution of rare inversion polymorphisms. |
| Warecki, B. and Tao, L. (2023). Centralspindlin-mediated transport of RhoGEF positions the cleavage plane for cytokinesis. Sci Signal 16(792): eadh0601. PubMed ID: 37402224
Summary: During cytokinesis, the cell membrane furrows inward along a cleavage plane. The positioning of the cleavage plane is critical to faithful cell division and is determined by the Rho guanine nucleotide exchange factor (RhoGEF)-mediated activation of the small guanosine triphosphatase RhoA and the conserved motor protein complex centralspindlin. This study explored whether and how centralspindlin mediates the positioning of RhoGEF. In dividing neuroblasts from Drosophila melanogaster, immediately before cleavage, first centralspindlin and then RhoGEF localized to the sites where cleavage subsequently initiated. Using in vitro assays with purified Drosophila proteins and stabilized microtubules, it was found that centralspindlin directly transported RhoGEF as cargo along single microtubules and sequestered it at microtubule plus-ends for prolonged periods of time. In addition, the binding of RhoGEF to centralspindlin appeared to stimulate centralspindlin motor activity. Thus, the motor activity and microtubule association of centralspindlin can translocate RhoGEF to areas where microtubule plus-ends are abundant, such as at overlapping astral microtubules, to locally activate RhoA and accurately position the cleavage plane during cell division. | Baker, C. C., Gallicchio, L., Matias, N. R., Porter, D. F., Parsanian, L., Taing, E., Tam, C., Fuller, M. T. (2023). Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase. Development, 150(22) PubMed ID: 37882771
Summary: During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. This study shows that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase. |
| Sperling, A. L., Fabian, D. K., Garrison, E. and Glover, D. M. (2023). A genetic basis for facultative parthenogenesis in Drosophila. Curr Biol. PubMed ID: 37516115
Summary: Facultative parthenogenesis enables sexually reproducing organisms to switch between sexual and asexual parthenogenetic reproduction. To gain insights into this phenomenon, the genomes of sexually reproducing and parthenogenetic strains of Drosophila mercatorum were sequenced, and differences were identified in the gene expression in their eggs. Then whether manipulating the expression of candidate gene homologs identified in Drosophila mercatorum could lead to facultative parthenogenesis in the non-parthenogenetic species Drosophila melanogaster was tested. This identified a polygenic system whereby increased expression of the mitotic protein kinase polo and decreased expression of a desaturase, Desat2, caused facultative parthenogenesis in the non-parthenogenetic species that was enhanced by increased expression of Myc. The genetically induced parthenogenetic Drosophila melanogaster eggs exhibit de novo centrosome formation, fusion of the meiotic products, and the onset of development to generate predominantly triploid offspring. Thus, this study demonstrated a genetic basis for sporadic facultative parthenogenesis in an animal. | Bakshi, A., Iturra, F. E., Alamban, A., Rosas-Salvans, M., Dumont, S., Aydogan, M. G. (2023). Cytoplasmic division cycles without the nucleus and mitotic CDK/cyclin complexes. Cell, 186(21):4694-4709. PubMed ID: 37832525
Summary: Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. This study demonstrates in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, evidence is presented that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. It is postulated that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes. |
Friday, May 3rd - RNAs and Transposons |
| Krzywinska, E., Ribeca, P., Ferretti, L., Hammond, A., Krzywinski, J. (2023). A novel factor modulating X chromosome dosage compensation in Anopheles. Curr Biol, 33(21):4697-4703. PubMed ID: 37774706
Summary: Dosage compensation (DC), a process countering chromosomal imbalance in individuals with heteromorphic sex chromosomes, has been molecularly characterized only in mammals, Caenorhabditis elegans, and fruit flies. In Drosophila melanogaster males, it is achieved by an approximately 2-fold hypertranscription of the monosomic X chromosome mediated by the MSL complex. The complex is not assembled on female X chromosomes because production of its key protein MSL-2 is prevented due to intron retention and inhibition of translation by Sex-lethal, a female-specific protein operating at the top of the sex determination pathway. It remains unclear how DC is mechanistically regulated in other insects. In the malaria mosquito Anopheles gambiae, an approximately 2-fold hypertranscription of the male X also occurs by a yet-unknown molecular mechanism distinct from that in D. melanogaster. This study shows that a male-specifically spliced gene called 007, which arose by a tandem duplication in the Anopheles ancestral lineage, is involved in the control of DC in males. Homozygous 007 knockouts lead to a global downregulation of the male X, phenotypically manifested by a slower development compared to wild-type mosquitoes or mutant females-however, without loss of viability or fertility. In females, a 007 intron retention promoted by the sex determination protein Femaleless, known to prevent hypertranscription from both X chromosomes, introduces a premature termination codon apparently rendering the female transcripts non-productive. In addition to providing a unique perspective on DC evolution, the 007, with its conserved properties, may represent an important addition to a genetic toolbox for malaria vector control. | Yushkova, E. (2024). Interaction effect of mutations in the genes (piwi and aub) of the Argonaute family and hobo transposons on the integral survival parameters of Drosophila melanogaster. Biogerontology, 25(1):131-146 PubMed ID: 37864608
Summary: The Argonaute family genes (piwi and aub) involved in the production of small RNAs are responsible for the regulation of many cellular processes, including the suppression of genome instability, modulation of gene activity, and transposable elements. Dysfunction of these genes and the associated activation of transposable elements adversely affect reproductive development and quality of life. The role of transposons in contrast to retrotransposons and their interaction with genes of the Argonaute family in aging processes have not been studied. This study considers a scenario in which the piwi and aub genes in the presence of functional hobo transposons can modify the effects from the level of DNA damage to lifespan. The simultaneous presence of mutation (piwi or aub) and hobo (regardless of size) in the genome has practically no effect or (less often) leads to a decrease in the level of DNA damage in ovarian cells. A high level of sterility and low ovarian reserve were noted mainly with a combination of mutations and full-sized hobo elements. The combination of these two genetic factors negatively affects the fertility of young females and embryonic survival. Isolated cases of restoration of reproductive functions with age were noted but only in females that had low fertility in the early period of life. The presence of hobo transposons contributed to an increase in the lifespan of both mutant and non-mutant females. Dysfunction of the piwi and aub genes (without hobo) can reduce the lifespan of both sexes. Together, each mutation and hobo transposons act antagonistically/additively (in females) and synergistically/antagonistically (in males) to change the lifespan. In parameters of locus-specific instability, hobo activation was more pronounced in piwi gene dysfunction. The results obtained complement data on the study of new functions of Argonaute family genes and their interactions with transposable elements in the aging process. |
| Kour, S., Fortuna, T., Anderson, E. N., Mawrie, D., Bilstein, J., Sivasubramanian, R., Ward, C., Roy, R., Rajasundaram, D., Sterneckert, J., Pandey, U. B. (2023). Drosha-dependent microRNAs modulate FUS-mediated neurodegeneration in vivo. Nucleic Acids Res, 51(20):11258-11276 PubMed ID: 37791873
Summary: Mutations in the Fused in Sarcoma (FUS) gene cause the familial and progressive form of amyotrophic lateral sclerosis (ALS). FUS is a nuclear RNA-binding protein involved in RNA processing and the biogenesis of a specific set of microRNAs. This study reports that Drosha and two previously uncharacterized Drosha-dependent miRNAs are strong modulators of FUS expression and prevent the cytoplasmic segregation of insoluble mutant FUS in vivo. Depletion of Drosha mitigates FUS-mediated degeneration, survival and motor defects in Drosophila. Mutant FUS strongly interacts with Drosha and causes its cytoplasmic mis-localization into the insoluble FUS inclusions. Reduction in Drosha levels increases the solubility of mutant FUS. Interestingly, two Drosha dependent microRNAs, miR-378i and miR-6832-5p, which differentially regulate the expression, solubility and cytoplasmic aggregation of mutant FUS in iPSC neurons and mammalian cells. More importantly, different modes of action are reported of these miRNAs against mutant FUS. Whereas miR-378i may regulate mutant FUS inclusions by preventing G3BP-mediated stress granule formation, miR-6832-5p may affect FUS expression via other proteins or pathways. Overall, this research reveals a possible association between ALS-linked FUS mutations and the Drosha-dependent miRNA regulatory circuit, as well as a useful perspective on potential ALS treatment via microRNAs. | Liu, M., Xie, X. J., Li, X., Ren, X., Sun, J., Lin, Z., Hemba-Waduge, R. U., Ji, J. Y. (2023). Transcriptional coupling of telomeric retrotransposons with the cell cycle. bioRxiv, PubMed ID: 37808851
Summary: Instead of employing telomerases to safeguard chromosome ends, dipteran species maintain their telomeres by transposition of telomeric-specific retrotransposons (TRs): in Drosophila , these are HeT-A, TART, and TAHRE. Previous studies have shown how these TRs create tandem repeats at chromosome ends, but the exact mechanism controlling TR transcription has remained unclear. This study reports the identification of multiple subunits of the transcription cofactor Mediator complex and transcriptional factors Scalloped (Sd, the TEAD homolog in flies) and E2F1-Dp as novel regulators of TR transcription and telomere length in Drosophila. Depletion of multiple Mediator subunits, Dp, or Sd increased TR expression and telomere length, while over-expressing E2F1-Dp or knocking down the E2F1 regulator Rbf1 (Retinoblastoma-family protein 1) stimulated TR transcription, with Mediator and Sd affecting TR expression through E2F1-Dp. The CUT&RUN analysis revealed direct binding of CDK8, Dp, and Sd to telomeric repeats. These findings highlight the essential role of the Mediator complex in maintaining telomere homeostasis by regulating TR transcription through E2F1-Dp and Sd, revealing the intricate coupling of TR transcription with the host cell-cycle machinery, thereby ensuring chromosome end protection and genomic stability during cell division. |
| Matzkin, L. M., Bono, J. M., Pigage, H. K., Allan, C. W., Diaz, F., McCoy, J. R., Green, C. C., Callan, J. B., Delahunt, S. P. (2023). Females translate male mRNA transferred during mating. bioRxiv, PubMed ID: 37790342
Summary: Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. This study developed an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. Strong evidence is found for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction. | Coronado-Zamora, M., Gonzalez, J. (2023). Transposons contribute to the functional diversification of the head, gut, and ovary transcriptomes across Drosophila natural strains. Genome research, 33(9):1541-1553 PubMed ID: 37793782
Summary: Transcriptomes are dynamic, with cells, tissues, and body parts expressing particular sets of transcripts. Transposable elements (TEs) are a known source of transcriptome diversity; however, studies often focus on a particular type of chimeric transcript, analyze single body parts or cell types, or are based on incomplete TE annotations from a single reference genome. This work has implemented a method based on de novo transcriptome assembly that minimizes the potential sources of errors while identifying a comprehensive set of gene-TE chimeras. This method was applied to the head, gut, and ovary dissected from five Drosophila melanogaster natural strains, with individual reference genomes available. ~19% of body part-specific transcripts were found to be gene-TE chimeras. Overall, chimeric transcripts contribute a mean of 43% to the total gene expression, and they provide protein domains for DNA binding, catalytic activity, and DNA polymerase activity. This comprehensive data set is a rich resource for follow-up analysis. Moreover, because TEs are present in virtually all species sequenced to date, their role in spatially restricted transcript expression is likely not exclusive to the species analyzed in this work. |
Thursday, May 2nd - Enzyme and protein expression, evolution, structure, and function |
| Yoon, H. J., Price, B. E., Parks, R. K., Ahn, S. J., Choi, M. Y. (2023). Diuretic hormone 31 activates two G protein-coupled receptors with differential second messengers for diuresis in Drosophila suzukii. Insect biochemistry and molecular biology, 162:104025 PubMed ID: 37813200
Summary: Diuretic hormones (DHs) bind to G protein-coupled receptors (GPCRs), regulating water and ion balance to maintain homeostasis in animals. Two distinct DHs are known in insects: calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. This study identified and characterized DH31 and two DH31 GPCR variants, DH31-Ra and DH31-Rb, from spotted-wing drosophila, Drosophila suzukii, a globally prevalent vinegar fly causing severe damage to small fruits. Both GPCRs are active, but DH31-Ra is the dominant receptor based on gene expression analyses and DH31 peptide binding affinities. A notable difference between the two variants lies in 1) the GPCR structures of their C-termini and 2) the utilization of second messengers, and the amino acid sequences of the two variants are identical. DH31-Ra contains 12 additional amino acids, providing different intracellular C-terminal configurations. DH31-Ra utilizes both cAMP and Ca(2+) as second messengers, whereas DH31-Rb utilizes only cAMP; this is the first time reported for an insect CT-like DH31 peptide. DH31 stimulated fluid secretion in D. suzukii adults, and secretion increased in a dose-dependent manner. However, when the fly was injected with a mixture of DH31 and CAPA, an anti-diuretic hormone, fluid secretion was suppressed. The structures of the DH31 receptors and the differential signaling pathways, including second messengers, involved in fly diuresis, are discussed. These findings provide fundamental insights into the characterization of D. suzukii DH31 and DH31-Rs, and facilitate the identification of potential biological targets for D. suzukii management. | Yarikipati, P., Jonusaite, S., Pleinis, J. M., Dominicci Cotto, C., Sanchez-Hernandez, D., Morrison, D. E., Goyal, S., Schellinger, J., Penalva, C., Curtiss, J., Rodan, A. R., Jenny, A. (2023). Unanticipated domain requirements for Drosophila Wnk kinase in vivo. PLoS Genet, 19(10):e1010975 PubMed ID: 37819975
Summary: WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate canonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. This study shows that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. The only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. Our data thus provide novel insights into unexpected in vivo roles of specific WNK domains. |
| Sayeesh, P. M., Iguchi, M., Suemoto, Y., Inoue, J., Inomata, K., Ikeya, T., Ito, Y. (2023). Interactions of the N- and C-Terminal SH3 Domains of Drosophila Drk with the Proline-Rich Peptides from Sos and Dos. Int J Mol Sci, 24(18) PubMed ID: 37762438
Summary: Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. A previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, NMR titration experiments were conducted using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. The results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. We have also performed docking simulations based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, the various modes in the interactions of Drk with Sos/Dos are discussed. | Gupta, K., Chakrabarti, S., Janardan, V., Gogia, N., Banerjee, S., Srinivas, S., Mahishi, D., Visweswariah, S. S. (2023). Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response. PLoS Genet, 19(9):e1010962 PubMed ID: 37733787
Summary: Evolutionarily conserved genes often play critical roles in organismal physiology. This study describes multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. It was observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism. |
| Keller, S. H., Deng, H., Lim, B. (2023). Regulation of the dynamic RNA Pol II elongation rate in Drosophila embryos. Cell Rep, 42(10):113225 PubMed ID: 37837623
Summary: An increasing number of studies have shown the key role that RNA polymerase II (RNA Pol II) elongation plays in gene regulation. This study systematically examine how various enhancers, promoters, and gene body composition influence the RNA Pol II elongation rate through a single-cell-resolution live imaging assay. By using reporter constructs containing 5' MS2 and 3' PP7 repeating stem loops, the rate of RNA Pol II elongation in live Drosophila embryos was quantified. Promoters and exonic gene lengths have no effect on elongation rate, while enhancers and the presence of long introns may significantly change how quickly RNA Pol II moves across a gene. Furthermore, it was observed in multiple constructs that the RNA Pol II elongation rate accelerates after the transcriptional onset of nuclear cycle 14 in Drosophila embryos. This study provides a single-cell view of various mechanisms that affect the dynamic RNA Pol II elongation rate, ultimately affecting the rate of mRNA production. | Viola, C. M., Frittmann, O., Jenkins, H. T., Shafi, T., De Meyts, P., Brzozowski, A. M. (2023). Structural conservation of insulin/IGF signalling axis at the insulin receptors level in Drosophila and humans. Nat Commun, 14(1):6271 PubMed ID: 37805602
Summary: The insulin-related hormones regulate key life processes in Metazoa, from metabolism to growth, lifespan and aging, through an evolutionarily conserved insulin signalling axis (IIS). In humans the IIS axis is controlled by insulin, two insulin-like growth factors, two isoforms of the insulin receptor (hIR-A and -B), and its homologous IGF-1R. In Drosophila, this signalling engages seven insulin-like hormones (DILP1-7) and a single receptor (dmIR). This report describes the cryoEM structure of the dmIR ectodomain:DILP5 complex, revealing high structural homology between dmIR and hIR. The excess of DILP5 yields dmIR complex in an asymmetric 'T' conformation, similar to that observed in some complexes of human IRs. However, dmIR binds three DILP5 molecules in a distinct arrangement, showing also dmIR-specific features. This work adds structural support to evolutionary conservation of the IIS axis at the IR level, and also underpins a better understanding of an important model organism. |
Tuesday, April 29th - Disease Models |
| Willot, Q., du Toit, A., de Wet, S., Huisamen, E. J., Loos, B., Terblanche, J. S. (2023). Exploring the connection between autophagy and heat-stress tolerance in Drosophila melanogaster. Proceedings Biological sciences, 290(2006):20231305 PubMed ID: 37700658
Summary: Mechanisms aimed at recovering from heat-induced damages are closely associated with the ability of ectotherms to survive exposure to stressful temperatures. Autophagy, a ubiquitous stress-responsive catabolic process, has recently gained renewed attention as one of these mechanisms. By increasing the turnover of cellular structures as well as the clearance of long-lived protein and protein aggregates, the induction of autophagy has been linked to increased tolerance to a range of abiotic stressors in diverse ectothermic organisms. However, whether a link between autophagy and heat-tolerance exists in insect models remains unclear despite broad ecophysiological implications thereof. This study explored the putative association between autophagy and heat-tolerance using Drosophila melanogaster as a model. It was hypothesized that (i) heat-stress would cause an increase of autophagy in flies' tissues, and (ii) rapamycin exposure would trigger a detectable autophagic response in adults and increase their heat-tolerance. In line with this hypothesis, flies exposed to heat-stress present signs of protein aggregation and appear to trigger an autophagy-related homoeostatic response as a result. It was further shown that rapamycin feeding causes the systemic effect associated with target of rapamycin (TOR) inhibition, induces autophagy locally in the fly gut, and increases the heat-stress tolerance of individuals. These results argue in favour of a substantial contribution of autophagy to the heat-stress tolerance mechanisms of insects. | Yoo, J., Dombrovski, M., Mirshahidi, P., Nern, A., LoCascio, S. A., Zipursky, S. L., Kurmangaliyev, Y. Z. (2023). Brain wiring determinants uncovered by integrating connectomes and transcriptomes. Curr Biol, 33(18):3998-4005. PubMed ID: 37647901
Summary: Advances in brain connectomics have demonstrated the extraordinary complexity of neural circuits. Developing neurons encounter the axons and dendrites of many different neuron types and form synapses with only a subset of them. During circuit assembly, neurons express cell-type-specific repertoires comprising many cell adhesion molecules (CAMs) that can mediate interactions between developing neurites. Many CAM families have been shown to contribute to brain wiring in different ways. It has been challenging, however, to identify receptor-ligand pairs directly matching neurons with their synaptic targets. This study integrated the synapse-level connectome of the neural circuit with the developmental expression patterns and binding specificities of CAMs on pre- and postsynaptic neurons in the Drosophila visual system. To overcome the complexity of neural circuits, focus was placed on pairs of genetically related neurons that make differential wiring choices. In the motion detection circuit, closely related subtypes of T4/T5 neurons choose between alternative synaptic targets in adjacent layers of neuropil. This choice correlates with the matching expression in synaptic partners of different receptor-ligand pairs of the Beat and Side families of CAMs. Genetic analysis demonstrated that presynaptic Side-II and postsynaptic Beat-VI restrict synaptic partners to the same layer. Removal of this receptor-ligand pair disrupts layers and leads to inappropriate targeting of presynaptic sites and postsynaptic dendrites. It is proposed that different Side/Beat receptor-ligand pairs collaborate with other recognition molecules to determine wiring specificities in the fly brain. Combining transcriptomes, connectomes, and protein interactome maps allow unbiased identification of determinants of brain wiring. |
| Yamada, T., Yoshinari, Y., Tobo, M., Habara, O., Nishimura, T. (2023). Nacalpha protects the larval fat body from cell death by maintaining cellular proteostasis in Drosophila. Nat Commun, 14(1):5328 PubMed ID: 37658058
Summary: Protein homeostasis (proteostasis) is crucial for the maintenance of cellular homeostasis. Impairment of proteostasis activates proteotoxic and unfolded protein response pathways to resolve cellular stress or induce apoptosis in damaged cells. However, the responses of individual tissues to proteotoxic stress and evoking cell death program have not been extensively explored in vivo. This study shows that a reduction in Nascent polypeptide-associated complex protein alpha subunit (Nacα) specifically and progressively induces cell death in Drosophila fat body cells. Nacα mutants disrupt both ER integrity and the proteasomal degradation system, resulting in caspase activation through JNK and p53. Although forced activation of the JNK and p53 pathways was insufficient to induce cell death in the fat body, the reduction of Nacα sensitized fat body cells to intrinsic and environmental stresses. Reducing overall protein synthesis by mTor inhibition or Minute mutants alleviated the cell death phenotype in Nacα mutant fat body cells. This work revealed that Nacα is crucial for protecting the fat body from cell death by maintaining cellular proteostasis, thus demonstrating the coexistence of a unique vulnerability and cell death resistance in the fat body. | Cachoux, V. M. L., Balakireva, M., Gracia, M., Bosveld, F., Lopez-Gay, J. M., Maugarny, A., Gaugue, I., di Pietro, F., Rigaud, S. U., Noiret, L., Guirao, B., Bellaiche, Y. (2023). Epithelial apoptotic pattern emerges from global and local regulation by cell apical area. Curr Biol, 33(22):4807-4826.e4806 PubMed ID: 37827152
Summary: Geometry is a fundamental attribute of biological systems, and it underlies cell and tissue dynamics. Cell geometry controls cell-cycle progression and mitosis and thus modulates tissue development and homeostasis. In sharp contrast and despite the extensive characterization of the genetic mechanisms of caspase activation, little is known about whether and how cell geometry controls apoptosis commitment in developing tissues. This study combined multiscale time-lapse microscopy of developing Drosophila epithelium, quantitative characterization of cell behaviors, and genetic and mechanical perturbations to determine how apoptosis is controlled during epithelial tissue development. Early in cell lives and well before extrusion, apoptosis commitment is linked to two distinct geometric features: a small apical area compared with other cells within the tissue and a small relative apical area with respect to the immediate neighboring cells. These global and local geometric characteristics are sufficient to recapitulate the tissue-scale apoptotic pattern. Furthermore, this study established that the coupling between these two geometric features and apoptotic cells is dependent on the Hippo/YAP and Notch pathways. Overall, by exploring the links between cell geometry and apoptosis commitment, this work provides important insights into the spatial regulation of cell death in tissues and improves understanding of the mechanisms that control cell number and tissue size. |
| Pai, Y. L., Lin, Y. J., Peng, W. H., Huang, L. T., Chou, H. Y., Wang, C. H., Chien, C. T., Chen, G. C. (2023). The deubiquitinase Leon/USP5 interacts with Atg1/ULK1 and antagonizes autophagy. Cell Death Dis, 14(8):540 PubMed ID: 37607937
Summary: Accumulating evidence has shown that the quality of proteins must be tightly monitored and controlled to maintain cellular proteostasis. Misfolded proteins and protein aggregates are targeted for degradation through the ubiquitin proteasome (UPS) and autophagy-lysosome systems. The ubiquitination and deubiquitinating enzymes (DUBs) have been reported to play pivotal roles in the regulation of the UPS system. However, the function of DUBs in the regulation of autophagy remain to be elucidated. This study found that knockdown of Leon/USP5 caused a marked increase in the formation of autophagosomes and autophagic flux under well-fed conditions. Genetic analysis revealed that overexpression of Leon suppressed Atg1-induced cell death in Drosophila. Immunoblotting assays further showed a strong interaction between Leon/USP5 and the autophagy initiating kinase Atg1/ULK1. Depletion of Leon/USP5 led to increased levels of Atg1/ULK1. These findings indicate that Leon/USP5 is an autophagic DUB that interacts with Atg1/ULK1, negatively regulating the autophagic process. | Li, C., Zhu, X., Sun, X., Guo, X., Li, W., Chen, P., Shidlovskii, Y. V., Zhou, Q., Xue, L. (2023). Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis. Cell division. 18(1):16 PubMed ID: 37794497
Summary: The c-Jun N-terminal kinase (JNK) pathway is an evolutionarily conserved regulator of cell death, which is essential for coordinating tissue homeostasis. This study has characterized the Drosophila Ste20-like kinase Slik as a novel modulator of JNK pathway-mediated apoptotic cell death. First, ectopic JNK signaling-triggered cell death is enhanced by slik depletion whereas suppressed by Slik overexpression. Second, loss of slik activates JNK signaling, which results in enhanced apoptosis and impaired tissue homeostasis. In addition, genetic epistasis analysis suggests that Slik acts upstream of or in parallel to Hep to regulate JNK-mediated apoptotic cell death. Moreover, Slik is necessary and sufficient for preventing physiologic JNK signaling-mediated cell death in development. Furthermore, introduction of STK10, the human ortholog of Slik, into Drosophila restores slik depletion-induced cell death and compromised tissue homeostasis. Lastly, knockdown of STK10 in human cancer cells also leads to JNK activation, which is cancelled by expression of Slik. This study has uncovered an evolutionarily conserved role of Slik/STK10 in blocking JNK signaling, which is required for cell death inhibition and tissue homeostasis maintenance in development. |
Monday, April 29th - Disease Models |
| Bennett, C. L., Dastidar, S., Arnold, F. J., McKinstry, S. U., Stockford, C., Freibaum, B. D., Sopher, B. L., Wu, M., Seidner, G., Joiner, W., Taylor, J. P., West, R. J. H., La Spada, A. R. (2023). Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity. Acta neuropathologica communications, 11(1):164 PubMed ID: 37845749
Summary: Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. This study considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, SETX fly lines were generated and the effect of SETX was evaluated in flies expressing either (G4C2)(58) repeats or glycine-arginine-50 [GR(50)] DPRs. Dramatic suppression was observed of disease phenotypes in (G4C2)(58) and GR(50) Drosophila models, and a striking relocalization of GR(50) out of the nucleolus was detected in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. It is noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, the nucleolus in cells expressing GR-DPRs was directly assessed, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS. | Swinter, K., Salah, D., Rathnayake, R., Gunawardena, S. (2023). PolyQ-Expansion Causes Mitochondria Fragmentation Independent of Huntingtin and Is Distinct from Traumatic Brain Injury (TBI)/Mechanical Stress-Mediated Fragmentation Which Results from Cell Death. Cells, 12(19) PubMed ID: 37830620
Summary: Mitochondrial dysfunction has been reported in many Huntington's disease (HD) models; however, it is unclear how these defects occur. This study tested the hypothesis that excess pathogenic huntingtin (HTT) impairs mitochondrial homeostasis, using Drosophila genetics and pharmacological inhibitors in HD and polyQ-expansion disease models and in a mechanical stress-induced traumatic brain injury (TBI) model. Expression of pathogenic HTT caused fragmented mitochondria compared to normal HTT, but HTT did not co-localize with mitochondria under normal or pathogenic conditions. Expression of pathogenic polyQ (127Q) alone or in the context of Machado Joseph Disease (MJD) caused fragmented mitochondria. While mitochondrial fragmentation was not dependent on the cellular location of polyQ accumulations, the expression of a chaperone protein, excess of mitofusin (MFN), or depletion of dynamin-related protein 1 (DRP1) rescued fragmentation. Intriguingly, a higher concentration of nitric oxide (NO) was observed in polyQ-expressing larval brains and inhibiting NO production rescued polyQ-mediated fragmented mitochondria, postulating that DRP1 nitrosylation could contribute to excess fission. Furthermore, while excess PI3K, which suppresses polyQ-induced cell death, did not rescue polyQ-mediated fragmentation, it did rescue fragmentation caused by mechanical stress/TBI. Together, these observations suggest that pathogenic polyQ alone is sufficient to cause DRP1-dependent mitochondrial fragmentation upstream of cell death, uncovering distinct physiological mechanisms for mitochondrial dysfunction in polyQ disease and mechanical stress. |
| Catterson, J. H., Minkley, L., Aspe, S., Judd-Mole, S., Moura, S., Dyson, M. C., Rajasingam, A., Woodling, N. S., Atilano, M. L., Ahmad, M., Durrant, C. S., Spires-Jones, T. L., Partridge, L. (2023). Protein retention in the endoplasmic reticulum rescues A β toxicity in Drosophila. Neurobiology of aging, 132:154-174 PubMed ID: 37837732
Summary: Amyloid β (A β) accumulation is a hallmark of Alzheimer's disease. In adult Drosophila brains, human A β overexpression harms climbing and lifespan. It's uncertain whether A β is intrinsically toxic or activates downstream neurodegeneration pathways. This study uncovers a novel protective role against A β toxicity: intra-endoplasmic reticulum (ER) protein accumulation with a focus on laminin and collagen subunits. Despite high A β, laminin B1 (LanB1) overexpression robustly counters toxicity, suggesting a potential A β resistance mechanism. Other laminin subunits and collagen IV also alleviate A β toxicity; combining them with LanB1 augments the effect. Imaging reveals ER retention of LanB1 without altering A β secretion. LanB1's rescue function operates independently of the IRE1 α/XBP1 ER stress response. ER-targeted GFP overexpression also mitigates A β toxicity, highlighting broader ER protein retention advantages. Proof-of-principle tests in murine hippocampal slices using mouse Lamb1 demonstrate ER retention in transduced cells, indicating a conserved mechanism. Though ER protein retention generally harms, it could paradoxically counter neuronal A β toxicity, offering a new therapeutic avenue for Alzheimer's disease. | Roth, J. R., de Moraes, R. C. M., Xu, B. P., Crawley, S. R., Khan, M. A., Melkani, G. C. (2023). Rapamycin reduces neuronal mutant huntingtin aggregation and ameliorates locomotor performance in Drosophila. Frontiers in aging neuroscience, 15:1223911 PubMed ID: 37823007
Summary: Huntington's disease (HD) is a neurodegenerative disease characterized by movement and cognitive dysfunction. HD is caused by a CAG expansion in exon 1 of the HTT gene that leads to a polyglutamine (PQ) repeat in the huntingtin protein, which aggregates in the brain and periphery. Drosophila models have been used to determine that Htt-PQ aggregation in the heart causes shortened lifespan and cardiac dysfunction that is ameliorated by promoting chaperonin function or reducing oxidative stress. The role of neuronal mutant huntingtin and how it affects peripheral function was further studied. Normal (Htt-PQ25) or expanded mutant (Htt-PQ72) exon 1 of huntingtin was overexpressed in Drosophila neurons and mutant huntingtin was found to cause age-dependent Htt-PQ aggregation in the brain and could cause a loss of synapsin. To determine if this neuronal dysfunction led to peripheral dysfunction, a negative geotaxis assay was performed to measure locomotor performance and it was found that neuronal mutant huntingtin caused an age-dependent decrease in locomotor performance. Next, it was found that rapamycin reduced Htt-PQ aggregation in the brain. These results demonstrate the role of neuronal Htt-PQ in dysfunction in models of HD, suggest that brain-periphery crosstalk could be important to the pathogenesis of HD, and show that rapamycin reduces mutant huntingtin aggregation in the brain. |
| Han, J. E., Kang, K. H., Kim, H., Hong, Y. B., Choi, B. O., Koh, H. (2023). PINK1 and Parkin rescue motor defects and mitochondria dysfunction induced by a patient-derived HSPB3 mutant in Drosophila models. Biochem Biophys Res Commun, 682:71-76 PubMed ID: 37804589
Summary: Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with the α-crystalline domain that is critical to their chaperone activity. Within the sHSP family, three (HSPB1, HSPB3, and HSPB8) proteins are linked with inherited peripheral neuropathies, including distal hereditary motor neuropathy (dHMN) and Charco-Marie-Tooth disease (CMT). This study introduced the HSPB3 Y118H (HSPB3(Y118H)) mutant gene identified from the CMT2 family in Drosophila. With a missense mutation on its α-crystalline domain, this human HSPB3 mutant gene induced a loss of motor activity accompanied by reduced mitochondrial membrane potential in fly neuronal tissues. Moreover, mitophagy, a critical mechanism of mitochondrial quality control, is downregulated in fly motor neurons expressing HSPB3(Y118H). Surprisingly, PINK1 and Parkin, the core regulators of mitophagy, successfully rescued these motor and mitochondrial abnormalities in HSPB3 mutant flies. Results from the first animal model of HSPB3 mutations suggest that mitochondrial dysfunction plays a critical role in HSPB3-associated human pathology. | Nil, Z., Deshwar, A. R., Huang, Y., ..., Mao, X., Wegner, D. J., Sisco, K., Shinawi, M., Wangler, M. F., Weksberg, R., Yamamoto, S., Costain, G., Bellen, H. J. (2023). Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies. American journal of human genetics, 110(11):1919-1937 PubMed ID: 37827158
Summary: Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. This study has identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. These results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease. |
Friday, April 26th - Adult neural structure, development and function |
| Sharma, Y., Jacobs, J. S., Sivan-Loukianova, E., Lee, E., Kernan, M. J., Eberl, D. F. (2023). The retrograde IFT dynein is required for normal function of diverse mechanosensory cilia in Drosophila. Frontiers in molecular neuroscience, 16:1263411 PubMed ID: 37808471
Summary: Cilia biogenesis relies on intraflagellar transport (IFT), a conserved transport mechanism which functions bi-directionally to bring protein complexes to the growing ciliary tip and recycle signaling and transport proteins between the cilium and cell body. In Drosophila, anterograde IFT is critical for assembly of sensory cilia in the neurons of both chordotonal (ch) organs, which have relatively long ciliary axonemes, and external sensory (es) organs, which have short axonemal segments with microtubules in distal sensory segments forming non-axonemal bundles. Previous work has isolated the beethoven (btv) mutant in a mutagenesis screen for auditory mutants. Although many btv mutant flies are deaf, some retain a small residual auditory function as determined both by behavior and by auditory electrophysiology. This study molecularly characterized the btv gene and demonstrated that it encodes the IFT-associated dynein-2 heavy chain Dync2h1. Morphological changes in Johnston's organ are described as flies age to 30 days, and it was found that morphological and electrophysiological phenotypes in this ch organ of btv mutants become more severe with age. NompB protein, encoding the conserved IFT88 protein, an IFT complex B component, fails to be cleared from chordotonal cilia in btv mutants, instead accumulating in the distorted cilia. In macrochaete bristles, a class of es organ, btv mutants show a 50% reduction in mechanoreceptor potentials. Thus, the btv-encoded Dync2h1 functions as the retrograde IFT motor in the assembly of long ciliary axonemes in ch organs and is also important for normal function of the short ciliary axonemes in es organs. | Mannino, M. C., Cassidy, M. B., Florez, S., Rusan, Z., Chakraborty, S., Schoborg, T. (2023). Mutations in abnormal spindle disrupt temporal transcription factor expression and trigger immune responses in the Drosophila brain. Genetics, 225(4) PubMed ID: 37831641
Summary: The coordination of cellular behaviors during neurodevelopment is critical for determining the form, function, and size of the central nervous system (CNS). Mutations in the vertebrate Abnormal Spindle-Like, Microcephaly Associated (ASPM) gene and its Drosophila melanogaster ortholog abnormal spindle (asp) lead to microcephaly (MCPH), a reduction in overall brain size whose etiology remains poorly defined. This study provides the neurodevelopmental transcriptional landscape for a Drosophila model for autosomal recessive primary microcephaly-5 (MCPH5) and extend findings into the functional realm to identify the key cellular mechanisms responsible for Asp-dependent brain growth and development. Multiple transcriptomic signatures, including new patterns of coexpressed genes were identified in the developing CNS. Defects in optic lobe neurogenesis were detected in larval brains through downregulation of temporal transcription factors (tTFs) and Notch signaling targets, which correlated with a significant reduction in brain size and total cell numbers during the neurogenic window of development. This study also found inflammation as a hallmark of asp mutant brains, detectable throughout every stage of CNS development, which also contributes to the brain size phenotype. Finally, apoptosis was shown not to be a primary driver of the asp mutant brain phenotypes, further highlighting an intrinsic Asp-dependent neurogenesis promotion mechanism that is independent of cell death. Collectively, these results suggest that the etiology of the asp mutant brain phenotype is complex and that a comprehensive view of the cellular basis of the disorder requires an understanding of how multiple pathway inputs collectively determine tissue size and architecture. |
| Trombley, S., Powell, J., Guttipatti, P., Matamoros, A., Lin, X., O'Harrow, T., Steinschaden, T., Miles, L., Wang, Q., Wang, S., Qiu, J., Li, Q., Li, F., Song, Y. (2023). Glia instruct axon regeneration via a ternary modulation of neuronal calcium channels in Drosophila. Nat Commun, 14(1):6490 PubMed ID: 37838791
Summary: A neuron's regenerative capacity is governed by its intrinsic and extrinsic environment. Both peripheral and central neurons exhibit cell-type-dependent axon regeneration, but the underlying mechanism is unclear. Glia provide a milieu essential for regeneration. However, the routes of glia-neuron signaling remain underexplored. This study shows that regeneration specificity is determined by the axotomy-induced Ca(2+) transients only in the fly regenerative neurons, which is mediated by L-type calcium channels, constituting the core intrinsic machinery. Peripheral glia regulate axon regeneration via a three-layered and balanced modulation. Glia-derived tumor necrosis factor acts through its neuronal receptor to maintain calcium channel expression after injury. Glia sustain calcium channel opening by enhancing membrane hyperpolarization via the inwardly-rectifying potassium channel (Irk1). Glia also release adenosine which signals through neuronal adenosine receptor (AdoR) to activate HCN channels (Ih) and dampen Ca(2+) transients. Together, this study identified a multifaceted glia-neuron coupling which can be hijacked to promote neural repair. | Sun, Y., Li, M., Geng, J., Meng, S., Tu, R., Zhuang, Y., Sun, M., Rui, M., Ou, M., Xing, G., Johnson, T. K., Xie, W. (2023). Neuroligin 2 governs synaptic morphology and function through RACK1-cofilin signaling in Drosophila. Communications biology, 6(1):1056 PubMed ID: 37853189
Summary: Neuroligins are transmembrane cell adhesion proteins well-known for their genetic links to autism spectrum disorders. Neuroligins can function by regulating the actin cytoskeleton, however the factors and mechanisms involved are still largely unknown. Here, using the Drosophila neuromuscular junction as a model, it was revealed that F-Actin assembly at the Drosophila NMJ is controlled through Cofilin signaling mediated by an interaction between DNlg2 and RACK1, factors not previously known to work together. The deletion of DNlg2 displays disrupted RACK1-Cofilin signaling pathway with diminished actin cytoskeleton proteo-stasis at the terminal of the NMJ, aberrant NMJ structure, reduced synaptic transmission, and abnormal locomotion at the third-instar larval stage. Overexpression of wildtype and activated Cofilin in muscles are sufficient to rescue the morphological and physiological defects in dnlg2 mutants, while inactivated Cofilin is not. Since the DNlg2 paralog DNlg1 is known to regulate F-actin assembly mainly via a specific interaction with WAVE complex, the present work suggests that the orchestration of F-actin by Neuroligins is a diverse and complex process critical for neural connectivity. |
| Sun, M., Ma, M., Deng, B., Li, N., Peng, Q., Pan, Y. (2023). A neural pathway underlying hunger modulation of sexual receptivity in Drosophila females. Cell Rep, 42(10):113243 PubMed ID: 37819758
Summary: Accepting or rejecting a mate is one of the most crucial decisions a female will make, especially when faced with food shortage. Previous studies have identified the core neural circuity from sensing male courtship or mating status to decision-making for sexual receptivity in Drosophila females, but how hunger and satiety states modulate female receptivity is poorly understood. This study identified the neural circuit and its neuromodulation underlying the hunger modulation of female receptivity. Adipokinetic hormone receptor (AkhR)-expressing neurons inhibit sexual receptivity in a starvation-dependent manner. AkhR neurons are octopaminergic and act on a subset of Octβ1R-expressing LH421 neurons. Knocking down Octβ1R expression in LH421 neurons eliminates starvation-induced suppression of female receptivity. It was further found that LH421 neurons inhibit the sex-promoting pC1 neurons via GABA-resistant to dieldrin (Rdl) signaling. pC1 neurons also integrate courtship stimulation and mating status and thus serve as a common integrator of multiple internal and external cues for decision-making. | Nakano, H., Sakai, T. (2023). Impact of Drosophila LIM homeodomain protein Apterous on the morphology of the adult mushroom body. Biochem Biophys Res Commun, 682:77-84 PubMed ID: 37804590
Summary: A LIM homeodomain transcription factor Apterous (Ap) regulates embryonic and larval neurodevelopment in Drosophila. Although Ap is still expressed in the adult brain, it remains elusive whether Ap is involved in neurodevelopmental events in the adult brain because flies homozygous for ap mutations are usually lethal before they reach the adult stage. In this study, using adult escapers of ap knockout (KO) homozygotes, whether the complete lack of ap expression affects the morphology of the mushroom body (MB) neurons and Pigment-dispersing factor (Pdf)-positive clock neurons in the adult brain was examined. Although ap KO escapers showed severe structural defects of MB neurons, no clear morphological defects were found in Pdf-positive clock neurons. These results suggest that Ap in the adult brain is essential for the neurodevelopment of specific ap-positive neurons, but it is not necessarily involved in the development of all ap-positive neurons. |
Thursday, April 25th - Synapse and Vesicles |
| Yamada, D., Davidson, A. M., Hige, T. (2024). Cyclic nucleotide-induced bidirectional long-term synaptic plasticity in Drosophila mushroom body. bioRxiv, PubMed ID: 37808762
Summary: Activation of the cAMP pathway is one of the common mechanisms underlying long-term potentiation (LTP). In the Drosophila mushroom body, simultaneous activation of odor-coding Kenyon cells (KCs) and reinforcement-coding dopaminergic neurons activates adenylyl cyclase in KC presynaptic terminals, which is believed to trigger synaptic plasticity underlying olfactory associative learning. However, learning induces long-term depression (LTD) at these synapses, contradicting the universal role of cAMP as a facilitator of transmission. A system was developed to electrophysiologically monitor both short-term and long-term synaptic plasticity at KC output synapses and demonstrate that they are indeed an exception where activation of the cAMP/protein kinase A pathway induces LTD. Contrary to the prevailing model, cAMP imaging finds no evidence for synergistic action of dopamine and KC activity on cAMP synthesis. Furthermore, it was found that forskolin-induced cAMP increase alone is insufficient for plasticity induction; it additionally requires simultaneous KC activation to replicate the presynaptic LTD induced by pairing with dopamine. On the other hand, activation of the cGMP pathway paired with KC activation induces slowly developing LTP, proving antagonistic actions of the two second-messenger pathways predicted by behavioral study. Finally, KC subtype-specific interrogation of synapses reveals that different KC subtypes exhibit distinct plasticity duration even among synapses on the same postsynaptic neuron. Thus, this work not only revises the role of cAMP in synaptic plasticity by uncovering the unexpected convergence point of the cAMP pathway and neuronal activity, but also establishes the methods to address physiological mechanisms of synaptic plasticity in this important model. | Wells, A., Mendes, C. C., Castellanos, F., Mountain, P., Wright, T., Wainwright, S. M., Stefana, M. I., Harris, A. L., Goberdhan, D. C. I., Wilson, C. (2023). A Rab6 to Rab11 transition is required for dense-core granule and exosome biogenesis in Drosophila secondary cells. PLoS Genet, 19(10):e1010979 PubMed ID: 37844085
Summary: Secretory cells in glands and the nervous system frequently package and store proteins destined for regulated secretion in dense-core granules (DCGs), which disperse when released from the cell surface. Despite the relevance of this dynamic process to diseases such as diabetes and human neurodegenerative disorders, mechanistic understanding is relatively limited, because of the lack of good cell models to follow the nanoscale events involved. This study employed the prostate-like secondary cells (SCs) of the Drosophila male accessory gland to dissect the cell biology and genetics of DCG biogenesis. These cells contain unusually enlarged DCGs, which are assembled in compartments that also form secreted nanovesicles called exosomes. Known conserved regulators of DCG biogenesis, including the small G-protein Arf1 and the coatomer complex AP-1, play key roles in making SC DCGs. Using real-time imaging, this study found that the aggregation events driving DCG biogenesis are accompanied by a change in the membrane-associated small Rab GTPases which are major regulators of membrane and protein trafficking in the secretory and endosomal systems. Indeed, a transition from trans-Golgi Rab6 to recycling endosomal protein Rab11, which requires conserved DCG regulators like AP-1, is essential for DCG and exosome biogenesis. These data allow development of a model for DCG biogenesis that brings together several previously disparate observations concerning this process and highlights the importance of communication between the secretory and endosomal systems in controlling regulated secretion. |
| Biton, T., Scher, N., Carmon, S., Elbaz-Alon, Y., Schejter, E. D., Shilo, B. Z., Avinoam, O. (2023). Fusion pore dynamics of large secretory vesicles define a distinct mechanism of exocytosis. J Cell Biol, 222(11) PubMed ID: 37707500
Summary: Exocrine cells utilize large secretory vesicles (LSVs) up to 10 μm in diameter. LSVs fuse with the apical surface, often recruiting actomyosin to extrude their content through dynamic fusion pores. The molecular mechanism regulating pore dynamics remains largely uncharacterized. This study observed that the fusion pores of LSVs in the Drosophila larval salivary glands expand, stabilize, and constrict. Arp2/3 is essential for pore expansion and stabilization, while myosin II is essential for pore constriction. Several Bin-Amphiphysin-Rvs (BAR) homology domain proteins were identified that regulate fusion pore expansion and stabilization. The I-BAR protein Missing-in-Metastasis (MIM) localizes to the fusion site and is essential for pore expansion and stabilization. The MIM I-BAR domain is essential but not sufficient for localization and function. It is concluded that MIM acts in concert with actin, myosin II, and additional BAR-domain proteins to control fusion pore dynamics, mediating a distinct mode of exocytosis, which facilitates actomyosin-dependent content release that maintains apical membrane homeostasis during secretion. | Wang, R., Fortier, T. M., Chai, F., Miao, G., Shen, J. L., Restrepo, L. J., DiGiacomo, J. J., Velentzas, P. D., Baehrecke, E. H. (2023). PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development. Cell, 186(19):4172-4188.e4118 PubMed ID: 37633267
Summary: Selective clearance of organelles, including endoplasmic reticulum (ER) and mitochondria, by autophagy plays an important role in cell health. This study describes a developmentally programmed selective ER clearance by autophagy. Parkinson's disease-associated PINK1, as well as Atl, Rtnl1, and Trp1 receptors, regulate ER clearance by autophagy. The E3 ubiquitin ligase Parkin functions downstream of PINK1 and is required for mitochondrial clearance while having the opposite function in ER clearance. By contrast, Keap1 and the E3 ubiquitin ligase Cullin3 function downstream of PINK1 to regulate ER clearance by influencing Rtnl1 and Atl. PINK1 regulates a change in Keap1 localization and Keap1-dependent ubiquitylation of the ER-phagy receptor Rtnl1 to facilitate ER clearance. Thus, PINK1 regulates the selective clearance of ER and mitochondria by influencing the balance of Keap1- and Parkin-dependent ubiquitylation of substrates that determine which organelle is removed by autophagy. |
| Ham, S. J., Yoo, H., Woo, D., Lee, D. H., Park, K. S., Chung, J. (2023). PINK1 and Parkin regulate IP(3)R-mediated ER calcium release. Nat Commun, 14(1):5202 PubMed ID: 37626046
Summary: Although defects in intracellular calcium homeostasis are known to play a role in the pathogenesis of Parkinson's disease (PD), the underlying molecular mechanisms remain unclear. This study shows that loss of PTEN-induced kinase 1 (PINK1) and Parkin leads to dysregulation of inositol 1,4,5-trisphosphate receptor (IP(3)R) activity, robustly increasing ER calcium release. In addition, CDGSH iron sulfur domain 1 (CISD1, also known as mitoNEET) functions downstream of Parkin to directly control IP(3)R. Both genetic and pharmacologic suppression of CISD1 and its Drosophila homolog CISD (also known as Dosmit) restore the increased ER calcium release in PINK1 and Parkin null mammalian cells and flies, respectively, demonstrating the evolutionarily conserved regulatory mechanism of intracellular calcium homeostasis by the PINK1-Parkin pathway. More importantly, suppression of CISD in PINK1 and Parkin null flies rescues PD-related phenotypes including defective locomotor activity and dopaminergic neuronal degeneration. Based on these data, it is proposed that the regulation of ER calcium release by PINK1 and Parkin through CISD1 and IP(3)R is a feasible target for treating PD pathogenesis. | Mrestani, A., Dannhauser, S., Pauli, M., Kollmannsberger, P., Hubsch, M., Morris, L., Langenhan, T., Heckmann, M., Paul, M. M. (2023). Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging. Life science alliance, 6(12) PubMed ID: 37696575
Summary: Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca(2+)-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. CRISPR/Cas9-assisted genome engineering of the rim locus was used to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rimV5-Znf but not in rimHA-Znf. In addition, rimHA-Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, ∼10 RIM(HA-Znf) subclusters were identified with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation. |
Wednesday, April 24th - Adult Physiology |
| Heidarian, Y., Tourigny, J. P., Fasteen, T. D., Mahmoudzadeh, N. H., Hurlburt, A. J., Nemkov, T., Reisz, J. A., D'Alessandro, A., Tennessen, J. M. (2023). Metabolomic analysis of Drosophila melanogaster larvae lacking pyruvate kinase. G3 (Bethesda), 14(1) PubMed ID: 37792629
Summary: Pyruvate kinase (Pyk) is a rate-limiting enzyme that catalyzes the final metabolic reaction in glycolysis. The importance of this enzyme, however, extends far beyond ATP production, as Pyk is also known to regulate tissue growth, cell proliferation, and development. Studies of this enzyme in Drosophila melanogaster are complicated by the fact that the fly genome encodes 6 Pyk paralogs whose functions remain poorly defined. To address this issue, sequence distance and phylogenetic approaches were used to demonstrate that the gene Pyk encodes the enzyme most similar to the mammalian Pyk orthologs, while the other 5 Drosophila Pyk paralogs have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of 2 different Pyk mutant strains revealed that larvae lacking Pyk exhibit a severe block in glycolysis, with a buildup of glycolytic intermediates upstream of pyruvate. However, this analysis also unexpectedly reveals that pyruvate levels are unchanged in Pyk mutants, indicating that larval metabolism maintains pyruvate pool size despite severe metabolic limitations. Consistent with these metabolomic findings, a complementary RNA-seq analysis revealed that genes involved in lipid metabolism and protease activity are elevated in Pyk mutants, again indicating that loss of this glycolytic enzyme induces compensatory changes in other aspects of metabolism. Overall, this study provides both insight into how Drosophila larval metabolism adapts to disruption of glycolytic metabolism as well as immediate clinical relevance, considering that Pyk deficiency is the most common congenital enzymatic defect in humans. | Sriskanthadevan-Pirahas, S., Tinwala, A. Q., Turingan, M. J., Khan, S., Grewal, S. S. (2023). Mitochondrial metabolism in Drosophila macrophage-like cells regulates body growth via modulation of cytokine and insulin signaling. Biol Open, 12(11) PubMed ID: 37850733
Summary: Macrophages play critical roles in regulating and maintaining tissue and whole-body metabolism in normal and disease states. While the cell-cell signaling pathways that underlie these functions are becoming clear, less is known about how alterations in macrophage metabolism influence their roles as regulators of systemic physiology. This was investigated by examining Drosophila macrophage-like cells called hemocytes. Knockdown of TFAM, a mitochondrial genome transcription factor, to reduce mitochondrial OxPhos activity specifically in larval hemocytes. This reduction in hemocyte OxPhos leads to a decrease in larval growth and body size. These effects are associated with a suppression of systemic insulin, the main endocrine stimulator of body growth. It was also found that TFAM knockdown leads to decreased hemocyte JNK signaling and decreased expression of the TNF alpha homolog, Eiger in hemocytes. Furthermore, genetic knockdown of hemocyte JNK signaling or Eiger expression mimics the effects of TFAM knockdown and leads to a non-autonomous suppression of body size without altering hemocyte numbers. These data suggest that modulation of hemocyte mitochondrial metabolism can determine their non-autonomous effects on organismal growth by altering cytokine and systemic insulin signaling. Given that nutrient availability can control mitochondrial metabolism, these findings may explain how macrophages function as nutrient-responsive regulators of tissue and whole-body physiology and homeostasis. |
| Abhilash, L., Shafer, O. T. (2023). Parametric effects of light acting via multiple photoreceptors contribute to circadian entrainment in Drosophila melanogaster. Proceedings Biological sciences, 290(2006):20230149 PubMed ID: 37700655
Summary: Circadian rhythms in physiology and behaviour have near 24 h periodicities that must adjust to the exact 24 h geophysical cycles on earth to ensure adaptive daily timing. Such adjustment is called entrainment. One major mode of entrainment is via the continuous modulation of circadian period by the prolonged presence of light. Although Drosophila melanogaster is a prominent insect model of chronobiology, there is little evidence for such continuous effects of light in the species. This study demonstrates that prolonged light exposure at specific times of the day shapes the daily timing of activity in flies. It was also established that continuous UV- and blue-blocked light lengthens the circadian period of Drosophila, and evidence is provided that this is produced by the combined action of multiple photoreceptors which, includes the cell-autonomous photoreceptor Cryptochrome. Finally, ramped light cycles were introducted as an entrainment paradigm that produces light entrainment that lacks the large light-driven startle responses typically displayed by flies and requires multiple days for entrainment to shifted cycles. These features are reminiscent of entrainment in mammalian models systems and make possible new experimental approaches to understanding the mechanisms underlying entrainment in the fly. | Dorogova, N. V., Fedorova, S. A., Bolobolova, E. U., Baricheva, E. M. (2023). The misregulation of mitochondria-associated genes caused by GAGA-factor lack promotes autophagic germ cell death in Drosophila testes. Genetica, 151(6):349-355 PubMed ID: 37819589 :
Summary: The Drosophila GAGA-factor encoded by the Trithorax-like (Trl) gene is DNA-binding protein with unusually wide range of applications in diverse cell contexts. In Drosophila spermatogenesis, reduced GAGA expression caused by Trl mutations induces mass autophagy leading to germ cell death. This work investigated the contribution of mitochondrial abnormalities to autophagic germ cell death in Trl gene mutants. Using a cytological approach, in combination with an analysis of high-throughput RNA sequencing (RNA-seq) data, it was demonstrated that the GAGA deficiency led to considerable defects in mitochondrial ultrastructure, by causing misregulation of GAGA target genes encoding essential components of mitochondrial molecular machinery. Mitochondrial anomalies induced excessive production of reactive oxygen species and their release into the cytoplasm, thereby provoking oxidative stress. Changes in transcription levels of some GAGA-independent genes in the Trl mutants indicated that testis cells experience ATP deficiency and metabolic aberrations, that may trigger extensive autophagy progressing to cell death. |
| Wang, L., Lin, J., Yang, K., Wang, W., Lv, Y., Zeng, X., Zhao, Y., Yu, J., Pan, L. (2023). Perilipin 1 Deficiency Prompts Lipolysis in Lipid Droplets and Aggravates the Pathogenesis of Persistent Immune Activation in Drosophila. Journal of innate immunity, 15(1):697-708 PubMed ID: 37742619
Summary: Lipid droplets (LDs) are highly dynamic intracellular organelles, which are involved in numerous of biological processes. However, the dynamic morphogenesis and functions of intracellular LDs during persistent innate immune responses remain obscure. This study induce long-term systemic immune activation in Drosophila through genetic manipulation. Then, the dynamic pattern of LDs is traced in the Drosophila fat body. Deficiency of Plin1, a key regulator of LDs' reconfiguration, blocks LDs minimization at the initial stage of immune hyperactivation but enhances LDs breakdown at the later stage of sustained immune activation via recruiting the lipase Brummer (Bmm, homologous to human ATGL). The high wasting in LDs shortens the lifespan of flies with high-energy-cost immune hyperactivation. Therefore, these results suggest a critical function of LDs during long-term immune activation and provide a potential treatment for the resolution of persistent inflammation. | Nunes, R. D., Drummond-Barbosa, D. (2023). A high-sugar diet, but not obesity, reduces female fertility in Drosophila melanogaster. Development, 150(20) PubMed ID: 37795747
Summary: Obesity is linked to reduced fertility in various species, from Drosophila to humans. Considering that obesity is often induced by changes in diet or eating behavior, it remains unclear whether obesity, diet, or both reduce fertility. This study shows that Drosophila females on a high-sugar diet become rapidly obese and less fertile as a result of increased death of early germline cysts and vitellogenic egg chambers (or follicles). They also have high glycogen, glucose and trehalose levels and develop insulin resistance in their fat bodies (but not ovaries). By contrast, females with adipocyte-specific knockdown of the anti-obesity genes brummer or adipose are obese but have normal fertility. Remarkably, females on a high-sugar diet supplemented with a separate source of water have mostly normal fertility and glucose levels, despite persistent obesity, high glycogen and trehalose levels, and fat body insulin resistance. These findings demonstrate that a high-sugar diet affects specific processes in oogenesis independently of insulin resistance, that high glucose levels correlate with reduced fertility on a high-sugar diet, and that obesity alone does not impair fertility. |
Tuesday, April 16th - Disease Models |
| Dhankhar, J., Shrivastava, A., Agrawal, N. (2023). Amendment of Altered Immune Response by Curcumin in Drosophila Model of Huntington's Disease. Journal of Huntington's disease, 12(4):335-354 PubMed ID: 37781812
Summary: Though primarily classified as a brain disorder, surplus studies direct Huntington's disease (HD) to be a multi-system disorder affecting various tissues and organs, thus affecting overall physiology of host. Recently, it has been reported that neuronal expression of mutant huntingtin induces immune dysregulation in Drosophila and may pose chronic threat to challenged individuals. Therefore, the polyphenolic compound curcumin was tested to circumvent the impact of immune dysregulation in Drosophila model of HD. The present study examined the molecular basis underlying immune derangements and immunomodulatory potential of curcumin in HD. UAS-GAL4 system was used to imitate the HD symptoms in Drosophila, and the desired female progenies (elav > Httex1pQ25; control and elav > Httex1pQ93; diseased) were cultured on food mixed without and with 10 μM concentration of curcumin since early development. Effect of curcumin supplementation was investigated by monitoring the hemocytes' count and their functional abilities in diseased condition. Reactive oxygen species (ROS) level in cells was assessed by DHE staining and mitochondrial dysfunction was assessed by CMXros red dye. In addition, transcript levels of pro-inflammatory cytokines and anti-microbial peptides were monitored by qRT-PCR. Curcumin supplementation was found to effectively reduced higher crystal cell count and phenoloxidase activity in diseased flies. Interestingly, curcumin significantly managed altered plasmatocytes count, improved their phagocytic activity by upregulating the expression of key phagocytic receptors in HD condition. Moreover, substantial alleviation of ROS levels and mitochondria dysfunction was observed in plasmatocytes of diseased flies upon curcumin supplementation. Furthermore, curcumin administration effectively attenuated transcriptional expression of pro-inflammatory cytokines and AMPs in diseased flies. These results indicate that curcumin efficiently attenuates immune derangements in HD flies and may prove beneficial in alleviating complexities associated with HD. | Godfrey, R. K., Alsop, E., Bjork, R. T., Chauhan, B. S., Ruvalcaba, H. C., Antone, J., Gittings, L. M., Michael, A. F., Williams, C., Hala'ufia, G., Blythe, A. D., Hall, M., Sattler, R., Van Keuren-Jensen, K., Zarnescu, D. C. (2023). Modelling TDP-43 proteinopathy in Drosophila uncovers shared and neuron-specific targets across ALS and FTD relevant circuits. Acta neuropathologica communications, 11(1):168 PubMed ID: 37864255
Summary: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comprise a spectrum of neurodegenerative diseases linked to TDP-43 (see Drosophila TDPH) proteinopathy, which at the cellular level, is characterized by loss of nuclear TDP-43 and accumulation of cytoplasmic TDP-43 inclusions that ultimately cause RNA processing defects including dysregulation of splicing, mRNA transport and translation. Complementing previous work in motor neurons, this study reports a novel model of TDP-43 proteinopathy based on overexpression of TDP-43 in a subset of Drosophila Kenyon cells of the mushroom body (MB), a circuit with structural characteristics reminiscent of vertebrate cortical networks. This model recapitulates several aspects of dementia-relevant pathological features including age-dependent neuronal loss, nuclear depletion and cytoplasmic accumulation of TDP-43, and behavioral deficits in working memory and sleep that occur prior to axonal degeneration. RNA immunoprecipitations identify several candidate mRNA targets of TDP-43 in MBs, some of which are unique to the MB circuit and others that are shared with motor neurons. Among the latter is the glypican Dally-like-protein (Dlp), which exhibits significant TDP-43 associated reduction in expression during aging. Using genetic interactions iy was shown that overexpression of Dlp in MBs mitigates TDP-43 dependent working memory deficits, conistent with Dlp acting as a mediator of TDP-43 toxicity. Substantiating these findings in the fly model, it was found that the expression of GPC6 mRNA, a human ortholog of dlp, is specifically altered in neurons exhibiting the molecular signature of TDP-43 pathology in FTD patient brains. These findings suggest that circuit-specific Drosophila models provide a platform for uncovering shared or disease-specific molecular mechanisms and vulnerabilities across the spectrum of TDP-43 proteinopathies. |
| Olmos, V., Thompson, E. N., Gogia, N., Luttik, K., Veeranki, V., Ni, L., Sim, S., Chen, K., Krause, D. S., Lim, J. (2024). Dysregulation of alternative splicing in spinocerebellar ataxia type 1. Hum Mol Genet, 33(2):138-149 PubMed ID: 37802886
Summary: Spinocerebellar ataxia type 1 is caused by an expansion of the polyglutamine tract in ATAXIN-1. Ataxin-1 is broadly expressed throughout the brain and is involved in regulating gene expression. However, it is not yet known if mutant ataxin-1 can impact the regulation of alternative splicing events. W RNA sequencing was performed in mouse models of spinocerebellar ataxia type 1 and mutant ataxin-1 expression abnormally leads to diverse splicing events in the mouse cerebellum of spinocerebellar ataxia type 1. The diverse splicing events occurred in a predominantly cell autonomous manner. A majority of the transcripts with misregulated alternative splicing events were previously unknown, thus allowing identification of overall new biological pathways that are distinctive to those affected by differential gene expression in spinocerebellar ataxia type 1. Evidence is provided that the splicing factor Rbfox1 mediates the effect of mutant ataxin-1 on misregulated alternative splicing and that genetic manipulation of Rbfox1 expression modifies neurodegenerative phenotypes in a Drosophila model of spinocerebellar ataxia type 1 in vivo. Together, this study provides novel molecular mechanistic insight into the pathogenesis of spinocerebellar ataxia type 1 and identifies potential therapeutic strategies for spinocerebellar ataxia type 1. | Eberwein, A. E., Kulkarni, S. S., Rushton, E., Broadie, K. (2023). Glycosphingolipids are linked to elevated neurotransmission and neurodegeneration in a Drosophila model of Niemann Pick type C. Disease models & mechanisms, 16(10) PubMed ID: 37815467
Summary: The lipid storage disease Niemann Pick type C (NPC) causes neurodegeneration owing primarily to loss of NPC1. This study employed a Drosophila model to test links between glycosphingolipids, neurotransmission and neurodegeneration. Npc1a nulls had elevated neurotransmission at the glutamatergic neuromuscular junction (NMJ), which was phenocopied in brainiac (brn) mutants, impairing mannosyl glucosylceramide (MacCer) glycosylation. Npc1a; brn double mutants had the same elevated synaptic transmission, suggesting that Npc1a and brn function within the same pathway. Glucosylceramide (GlcCer) synthase inhibition with miglustat prevented elevated neurotransmission in Npc1a and brn mutants, further suggesting epistasis. Synaptic MacCer did not accumulate in the NPC model, but GlcCer levels were increased, suggesting that GlcCer is responsible for the elevated synaptic transmission. Null Npc1a mutants had heightened neurodegeneration, but no significant motor neuron or glial cell death, indicating that dying cells are interneurons and that elevated neurotransmission precedes neurodegeneration. Glycosphingolipid synthesis mutants also had greatly heightened neurodegeneration, with similar neurodegeneration in Npc1a; brn double mutants, again suggesting that Npc1a and brn function in the same pathway. These findings indicate causal links between glycosphingolipid-dependent neurotransmission and neurodegeneration in this NPC disease model. |
| Blount, J. R., Patel, N. C., Libohova, K., Harris, A. L., Tsou, W. L., Sujkowski, A., Todi, S. V. (2023). Lysine 117 on ataxin-3 modulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3. Journal of the neurological sciences, 454:120828 PubMed ID: 37865002
Summary: Ataxin-3 (Atxn3) is a deubiquitinase with a polyglutamine (polyQ) repeat tract whose abnormal expansion causes the neurodegenerative disease, Spinocerebellar Ataxia Type 3 (SCA3; also known as Machado-Joseph Disease). The ubiquitin chain cleavage properties of Atxn3 are enhanced when the enzyme is itself ubiquitinated at lysine (K) at position 117: in vitro, K117-ubiqutinated Atxn3 cleaves poly-ubiquitin markedly more rapidly compared to its unmodified counterpart. How polyQ expansion causes SCA3 remains unclear. To gather insights into the biology of disease of SCA3, the following question was posited: is K117 important for toxicity caused by pathogenic Atxn3? To answer this question, transgenic Drosophila lines were generated that express full-length, human, pathogenic Atxn3 with 80 polyQ with an intact or mutated K117. It was found that mutating K117 mildly enhances the toxicity and aggregation of pathogenic Atxn3. An additional transgenic line that expresses Atxn3 without any K residues confirms increased aggregation of pathogenic Atxn3 whose ubiquitination is perturbed. These findings suggest that Atxn3 ubiquitination is a regulatory step of SCA3, in part by modulating its aggregation. | Sodders, M. J., Shen, M., Olsen, A. L. (2023). Measuring Constipation in a Drosophila Model of Parkinson's Disease. J Vis Exp, (199) PubMed ID: 37811970
Summary: Non-motor symptoms in Parkinson's disease (PD) are common, difficult to treat, and significantly impair quality of life. One prevalent non-motor symptom is constipation, which can precede the diagnosis of PD by years or even decades. Constipation has been underexplored in animal models of PD and lacks specific therapies. This assay utilizes a Drosophila model of PD in which human alpha-synuclein is expressed under a pan-neuronal driver. Flies expressing alpha-synuclein develop the hallmark features of PD: the loss of dopaminergic neurons, motor impairment, and alpha-synuclein inclusions. This protocol outlines a method for studying constipation in these flies. Flies are placed on fly food with a blue color additive overnight and then transferred to standard food the following day. They are subsequently moved to new vials with standard fly food every hour for 8 h. Before each transfer, the percentage of blue-colored fecal spots compared to the total fecal spots on the vial wall is calculated. Control flies that lack alpha-synuclein expel all the blue dye hours before flies expressing alpha-synuclein. Additionally, the passage of blue-colored food from the gut can be monitored with simple photography. The simplicity of this assay enables its use in forward genetic or chemical screens to identify modifiers of constipation in Drosophila. |
Monday, April 15th - Evolution |
| Shpak, M., Ghanavi, H. R., Lange, J. D., Pool, J. E., Stensmyr, M. C. (2023). Genomes from historical Drosophila melanogaster specimens illuminate adaptive and demographic changes across more than 200 years of evolution. PLoS Biol, 21(10):e3002333 PubMed ID: 37824452
Summary: The ability to perform genomic sequencing on long-dead organisms is opening new frontiers in evolutionary research. These opportunities are especially notable in the case of museum collections, from which countless documented specimens may now be suitable for genomic analysis-if data of sufficient quality can be obtained. This study reports 25 newly sequenced genomes from museum specimens of the model organism Drosophila melanogaster, including the oldest extant specimens of this species. By comparing historical samples ranging from the early 1800s to 1933 against modern-day genomes, this study documents evolution across thousands of generations, including time periods that encompass the species' initial occupation of northern Europe and an era of rapidly increasing human activity. It was also found that the Lund, Sweden population underwent local genetic differentiation during the early 1800s to 1933 interval (potentially due to drift in a small population) but then became more similar to other European populations thereafter (potentially due to increased migration). Within each century-scale time period, temporal sampling allows documentation of compelling candidates for recent natural selection. In some cases, insights were gained regarding previously implicated selection candidates, such as ChKov1, for which the inferred timing of selection favors the hypothesis of antiviral resistance over insecticide resistance. Other candidates are novel, such as the circadian-related gene Ahcy, which yields a selection signal that rivals that of the DDT resistance gene Cyp6g1. These insights deepen understanding of recent evolution in a model system, and highlight the potential of future museomic studies. | Corpuz, R. L., Bellinger, M. R., Veillet, A., Magnacca, K. N., Price, D. K. (2023). The Transmission Patterns of the Endosymbiont Wolbachia within the Hawaiian Drosophilidae Adaptive Radiation. Genes, 14(8) PubMed ID: 37628597
Summary: The evolution of endosymbionts and their hosts can lead to highly dynamic interactions with varying fitness effects for both the endosymbiont and host species. Wolbachia, a ubiquitous endosymbiont of arthropods and nematodes, can have both beneficial and detrimental effects on host fitness. This study documented the occurrence and patterns of transmission of Wolbachia within the Hawaiian Drosophilidae and examined the potential contributions of Wolbachia to the rapid diversification of their hosts. Screens for Wolbachia infections across a minimum of 140 species of Hawaiian Drosophila and Scaptomyza revealed species-level infections of 20.0%, and across all 399 samples, a general infection rate of 10.3%. Among the 44 Wolbachia strains identified using a modified Wolbachia multi-locus strain typing scheme, 30 (68.18%) belonged to supergroup B, five (11.36%) belonged to supergroup A, and nine (20.45%) had alleles with conflicting supergroup assignments. Co-phylogenetic reconciliation analysis indicated that Wolbachia strain diversity within their endemic Hawaiian Drosophilidae hosts can be explained by vertical (e.g., co-speciation) and horizontal (e.g., host switch) modes of transmission. Results from stochastic character trait mapping suggest that horizontal transmission is associated with the preferred oviposition substrate of the host, but not the host's plant family or island of occurrence. For Hawaiian Drosophilid species of conservation concern, with 13 species listed as endangered and one listed as threatened, knowledge of Wolbachia strain types, infection status, and potential for superinfection could assist with conservation breeding programs designed to bolster population sizes, especially when wild populations are supplemented with laboratory-reared, translocated individuals. Future research aimed at improving the understanding of the mechanisms of Wolbachia transmission in nature, their impact on the host, and their role in host species formation may shed light on the influence of Wolbachia as an evolutionary driver, especially in Hawaiian ecosystems. |
| Depetris-Chauvin, A., Galagovsky, D., Keesey, I. W., Hansson, B. S., Sachse, S., Knaden, M. (2023). Evolution at multiple processing levels underlies odor-guided behavior in the genus Drosophila. Curr Biol, 33(22):4771-4785 PubMed ID: 37804828
Summary: Olfaction is a fundamental sense guiding animals to their food. How the olfactory system evolves and influences behavior is still poorly understood. This study selected five drosophilid species, including Drosophila melanogaster, inhabiting different ecological niches to compare their olfactory systems at multiple levels. First ecologically relevant natural food odorants from every species were identified and species-specific odorant preferences were identified. To compare odor coding in sensory neurons, the antennal lobe (AL) structure was analyzed, glomerular atlases were generated, and GCaMP transgenic lines were developed for all species. Although subsets of glomeruli showed distinct tuning profiles, odorants inducing species-specific preferences were coded generally similarly. Species distantly related or occupying different habitats showed more evident differences in odor coding, and further analysis revealed that changes in olfactory receptor (OR) sequences partially explain these differences. The results demonstrate that genetic distance in phylogeny and ecological niche occupancy are key determinants in the evolution of ORs, AL structures, odor coding, and behavior. Interestingly, changes in odor coding among species could not be explained by evolutionary changes at a single olfactory processing level but rather are a complex phenomenon based on changes at multiple levels. | Erkosar, B., Dupuis, C., Cavigliasso, F., Savary, L., Kremmer, L., Gallart-Ayala, H., Ivanisevic, J., Kawecki, T. J. (2023). Evolutionary adaptation to juvenile malnutrition impacts adult metabolism and impairs adult fitness in Drosophila. Elife, 12 PubMed ID: 37847744
Summary: Juvenile undernutrition has lasting effects on adult metabolism of the affected individuals, but it is unclear how adult physiology is shaped over evolutionary time by natural selection driven by juvenile undernutrition. RNAseq, targeted metabolomics, and genomics were combined to study the consequences of evolution under juvenile undernutrition for metabolism of reproductively active adult females of Drosophila melanogaster. Compared to Control populations maintained on standard diet, Selected populations maintained for over 230 generations on a nutrient-poor larval diet evolved major changes in adult gene expression and metabolite abundance, in particular affecting amino acid and purine metabolism. The evolved differences in adult gene expression and metabolite abundance between Selected and Control populations were positively correlated with the corresponding differences previously reported for Selected versus Control larvae. This implies that genetic variants affect both stages similarly. Even when well fed, the metabolic profile of Selected flies resembled that of flies subject to starvation. Finally, selected flies had lower reproductive output than controls even when both were raised under the conditions under which the selected populations evolved. These results imply that evolutionary adaptation to juvenile undernutrition has large pleiotropic consequences for adult metabolism, and that they are costly rather than adaptive for adult fitness. Thus, juvenile and adult metabolism do not appear to evolve independently from each other even in a holometabolous species where the two life stages are separated by a complete metamorphosis. |
| Mahdjoub, H., Khelifa, R., Roy, J., Sbilordo, S. H., Zeender, V., Perdigon Ferreira, J., Gourgoulianni, N., Lupold, S. (2023). Interplay between male quality and male-female compatibility across episodes of sexual selection. Sci Adv, 9(39):eadf5559 PubMed ID: 37774022
Summary: The processes underlying mate choice profoundly influence the dynamics of sexual selection and the evolution of male sexual traits. Consistent preference for certain phenotypes may erode genetic variation in populations through directional selection, whereas divergent preferences (e.g., genetically compatible mates) provide one mechanism to maintain such variation. However, the relative contributions of these processes across episodes of selection remain unknown. Using Drosophila melanogaster, this study followed the fate of male genotypes, previously scored for their overall reproductive value and their compatibility with different female genotypes, across pre- and postmating episodes of selection. When pairs of competitor males differed in their intrinsic quality and their compatibility with the female, both factors influenced outcomes from mating success to paternity but to a varying degree between stages. These results add further dimensions to understanding of how the interactions between genotypes and forms of selection shape reproductive outcomes and ultimately reproductive trait evolution. | Dey, M., Brown, E., Charlu, S., Keene, A., Dahanukar, A. (2023). Evolution of fatty acid taste in drosophilids. Cell Rep, 42(10):113297 PubMed ID: 37864792
Summary: Comparative studies of related but ecologically distinct species can reveal how the nervous system evolves to drive behaviors that are particularly suited to certain environments. Drosophila melanogaster is a generalist that feeds and oviposits on most overripe fruits. A sibling species, D. sechellia, is an obligate specialist of Morinda citrifolia (noni) fruit, which is rich in fatty acids (FAs). To understand evolution of noni taste preference, this study characterized behavioral and cellular responses to noni-associated FAs in three related drosophilids. Mixtures of sugar and noni FAs evoke strong aversion in the generalist species but not in D. sechellia. Surveys of taste sensory responses reveal noni FA- and species-specific differences in at least two mechanisms-bitter neuron activation and sweet neuron inhibition-that correlate with shifts in noni preference. Chemoreceptor mutant analysis in D. melanogaster predicts that multiple genetic changes account for evolution of gustatory preference in D. sechellia. |
Monday, April 8th - Signaling |
| Zhao, A., Varady, S., O'Kelley-Bangsberg, M., Deng, V., Platenkamp, A., Wijngaard, P., Bern, M., Gormley, W., Kushkowski, E., Thompson, K., Tibbetts, L., Conner, A. T., Noeckel, D., Teran, A., Ritz, A., Applewhite, D. A. (2023). From network analysis to experimental validation: identification of regulators of non-muscle myosin II contractility using the folded-gastrulation signaling pathway. BMC molecular and cell biology, 24(1):32 PubMed ID: 37821823
Summary: The morphogenetic process of apical constriction, which relies on non-muscle myosin II (NMII; Zipper) generated constriction of apical domains of epithelial cells, is key to the development of complex cellular patterns. Apical constriction occurs in almost all multicellular organisms, but one of the most well-characterized systems is the Folded-gastrulation (Fog)-induced apical constriction that occurs in Drosophila. The binding of Fog to its cognizant receptors Mist/Smog results in a signaling cascade that leads to the activation of NMII-generated contractility. Despite knowledge of key molecular players involved in Fog signaling, this study sought to explore whether other proteins have an undiscovered role in its regulation. A computational method was developed to predict unidentified candidate NMII regulators using a network of pairwise protein-protein interactions called an interactome. First a Drosophila interactome of over 500,000 protein-protein interactions was constructed from several databases that curate high-throughput experiments. Next, several graph-based algorithms were implemented that predicted 14 proteins potentially involved in Fog signaling. To test these candidates, RNAi depletion was used in combination with a cellular contractility assay in Drosophila S2R + cells, which respond to Fog by contracting in a stereotypical manner. Of the candidates dcreened using this assay, two proteins, the serine/threonine phosphatase Flapwing and the putative guanylate kinase CG11811 (oya) were demonstrated to inhibit cellular contractility when depleted, suggestive of their roles as novel regulators of the Fog pathway. | Keyan, K. S., Salim, S., Gowda, S., Abdelrahman, D., Amir, S. S., Islam, Z., Vargas, C., Bengoechea-Alonso, M. T., Alwa, A., Dahal, S., Kolatkar, P. R., Da'as, S., Torrisani, J., Ericsson, J., Mohammad, F., Khan, O. M. (2023). Control of TGFbeta signalling by ubiquitination independent function of E3 ubiquitin ligase TRIP12. Cell Death Dis, 14(10):692 PubMed ID: 37863914
Summary: The TGFβ pathway is a master regulator of cell proliferation, differentiation, and death. Deregulation of TGFβ signalling is well established in several human diseases including autoimmune disorders and cancer. Thus, understanding molecular pathways governing TGFβ signalling may help better understand the underlying causes of some of those conditions. This study shows that a HECT domain E3 ubiquitin ligase TRIP12 controls TGFβ signalling in multiple models. Interestingly, TRIP12 control of TGFβ signalling is completely independent of its E3 ubiquitin ligase activity. Instead, TRIP12 recruits SMURF2 to SMAD4, which is most likely responsible for inhibitory monoubiquitination of SMAD4, since SMAD4 monoubiquitination and its interaction with SMURF2 were dramatically downregulated in TRIP12(-/-) cells. Additionally, genetic inhibition of TRIP12 in human and murine cells leads to robust activation of TGFβ signalling which was rescued by re-introducing wildtype TRIP12 or a catalytically inactive C1959A mutant. Importantly, TRIP12 control of TGFβ signalling is evolutionary conserved. Indeed, genetic inhibition of Drosophila TRIP12 orthologue, ctrip, in gut leads to a reduced number of intestinal stem cells which was compensated by the increase in differentiated enteroendocrine cells. These effects were completely normalised in Drosophila strain where ctrip was co-inhibited together with Drosophila SMAD4 orthologue, Medea. Similarly, in murine 3D intestinal organoids, CRISPR/Cas9 mediated genetic targeting of Trip12 enhances TGFβ mediated proliferation arrest and cell death. Finally, CRISPR/Cas9 mediated genetic targeting of TRIP12 in MDA-MB-231 breast cancer cells enhances the TGFβ induced migratory capacity of these cells which was rescued to the wildtype level by re-introducing wildtype TRIP12. This work establishes TRIP12 as an evolutionary conserved modulator of TGFβ signalling in health and disease. |
| Krejxova, G., Morgantini, C., Zemanova, H., Lauschke, V. M., Kovarova, J., Kubasek, J., Nedbalova, P., Kamps-Hughes, N., Moos, M., Aouadi, M., Dolezal, T., Bajgar, A. (2023). Macrophage-derived insulin antagonist ImpL2 induces lipoprotein mobilization upon bacterial infection. The EMBO journal, 42(23):e114086 PubMed ID: 37807855
Summary: The immune response is an energy-demanding process that must be coordinated with systemic metabolic changes redirecting nutrients from stores to the immune system. Although this interplay is fundamental for the function of the immune system, the underlying mechanisms remain elusive. The data of this study show that the pro-inflammatory polarization of Drosophila macrophages is coupled to the production of the insulin antagonist ImpL2 through the activity of the transcription factor HIF1α. ImpL2 production, reflecting nutritional demands of activated macrophages, subsequently impairs insulin signaling in the fat body, thereby triggering FOXO-driven mobilization of lipoproteins. This metabolic adaptation is fundamental for the function of the immune system and an individual's resistance to infection. This study demonstrated that analogically to Drosophila, mammalian immune-activated macrophages produce ImpL2 homolog IGFBP7 in a HIF1α-dependent manner and that enhanced IGFBP7 production by these cells induces mobilization of lipoproteins from hepatocytes. Hence, the production of ImpL2/IGFBP7 by macrophages represents an evolutionarily conserved mechanism by which macrophages alleviate insulin signaling in the central metabolic organ to secure nutrients necessary for their function upon bacterial infection. | Chiang, M. H., Lin, Y. C., Chen, S. F., Lee, P. S., Fu, T. F., Wu, T., Wu, C. L. (2023). Independent insulin signaling modulators govern hot avoidance under different feeding states. PLoS Biol, 21(10):e3002332 PubMed ID: 37847673
Summary: Thermosensation is critical for the survival of animals. However, mechanisms through which nutritional status modulates thermosensation remain unclear. This study shows that hungry Drosophila exhibit a strong hot avoidance behavior (HAB) compared to food-sated flies. Hot stimulus increases the activity of α'β' mushroom body neurons (MBns), with weak activity in the sated state and strong activity in the hungry state. Furthermore, it was shown that α'β' MBn receives the same level of hot input from the mALT projection neurons via cholinergic transmission in sated and hungry states. Differences in α'β' MBn activity between food-sated and hungry flies following heat stimuli are regulated by distinct Drosophila insulin-like peptides (Dilps). Dilp2 is secreted by insulin-producing cells (IPCs) and regulates HAB during satiety, whereas Dilp6 is secreted by the fat body and regulates HAB during the hungry state. Dilp2 induces PI3K/AKT signaling, whereas Dilp6 induces Ras/ERK signaling in α'β' MBn to regulate HAB in different feeding conditions. Finally, it was shown that the 2 α'β'-related MB output neurons (MBONs), MBON-α'3 and MBON-β'1, are necessary for the output of integrated hot avoidance information from α'β' MBn. These results demonstrate the presence of dual insulin modulation pathways in α'β' MBn, which are important for suitable behavioral responses in Drosophila during thermoregulation under different feeding states. |
| Nemoto, K., Masuko, K., Fuse, N., Kurata, S. (2023). . Dilp8 and its candidate receptor, Drl, are involved in the transdetermination of the Drosophila imaginal disc. Genes to cells 28(12):857-867 PubMed ID: 37817293
Summary: Drosophila imaginal disc cells can change their identity under stress conditions through transdetermination (TD). Research on TD can help elucidate the in vivo process of cell fate conversion. Previous work showed that the overexpression of winged eye (wge) induces eye-to-wing TD in the eye disc and that an insulin-like peptide, Dilp8, is then highly expressed in the disc. Although Dilp8 is known to mediate systemic developmental delay via the Lgr3 receptor, its role in TD remains unknown. This study showed that Dilp8 is expressed in specific cells that do not express eye or wing fate markers during Wge-mediated TD and that the loss of Dilp8 impairs the process of eye-to-wing transition. Thus, Dilp8 plays a pivotal role in the cell fate conversion under wge overexpression. Furthermore, this study found that instead of Lgr3, another candidate receptor, Drl, is involved in Wge-mediated TD and acts locally in the eye disc cells. A model is proposed in which Dilp8-Drl signaling organizes cell fate conversion in the imaginal disc during TD. | Trammell, C. E., Rowe, E. H., Char, A. B., Jones, B. J., Fawcett, S., Ahlers, L. R. H., Goodman, A. G. (2023). Insulin-mediated endothelin signaling is antiviral during West Nile virus infection. J Virol, 97(10):e0111223 PubMed ID: 37796127
Summary: Arboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. This study identified a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling (In insulin resistance the high levels of blood insulin results in increased production and activity of endothelin-1, which promotes vasoconstriction and elevates blood pressure). Additionally, it was demonstrated that results obtained from D. melanogaster can be successfully translated into the more relevant human system. These results add to the growing field of insulin-mediated antiviral immunity and identify potential biomarkers or intervention targets to better address West Nile virus infection and severe disease. |
Thursday, April 4th - Behavior |
| Mishra, S., Sharma, N., Lone, S. R. (2023). Understanding the impact of sociosexual interactions on sleep using Drosophila melanogaster as a model organism. Frontiers in physiology, 14:1220140 PubMed ID: 37670770
Summary: Sleep is conserved across species, and it is believed that a fixed amount of sleep is needed for normal neurobiological functions. Sleep rebound follows sleep deprivation; however, continuous sleep deprivation for longer durations is believed to be detrimental to the animal's wellbeing. Under some physiologically demanding situations, such as migration in birds, the birth of new offspring in cetaceans, and sexual interactions in pectoral sandpipers, animals are known to forgo sleep. The mechanisms by which animals forgo sleep without having any obvious negative impact on the proper functioning of their neurobiological processes are yet unknown. Therefore, a simple assay is needed to study how animals forgo sleep. The assay should be ecologically relevant so it can offer insights into the physiology of the organisms. Equally important is that the organism should be genetically amenable, which helps in understanding the cellular and molecular processes that govern such behaviors. This paper presents a simple method of sociosexual interaction to understand the process by which animals forgo sleep. In the case of Drosophila melanogaster, when males and females are in proximity, they are highly active and lose a significant amount of sleep. In addition, there is no sleep rebound afterward, and instead, males engaged in sexual interactions continue to show normal sleep. Thus, sexual drive in the fruit flies is a robust assay to understand the underlying mechanism by which animals forgo sleep. | Strunov, A., Schoenherr, C., Kapun, M. (2023). Wolbachia has subtle effects on thermal preference in highly inbred Drosophila melanogaster which vary with life stage and environmental conditions. Sci Rep, 13(1):13792 PubMed ID: 37612420
Summary: Temperature fluctuations are challenging for ectotherms which are not able to regulate body temperature by physiological means and thus have to adjust their thermal environment via behavior. However, little is yet known about whether microbial symbionts influence thermal preference (T(p)) in ectotherms by modulating their physiology. Several recent studies have demonstrated substantial effects of Wolbachia infections on host T(p) in different Drosophila species. These data indicate that the direction and strength of thermal preference variation is strongly dependent on host and symbiont genotypes and highly variable among studies. By employing highly controlled experiments, this study investigated the impact of several environmental factors including humidity, food quality, light exposure, and experimental setup that may influence T(p) measurements in adult Drosophila melanogaster flies. Additionally, the effects were assessed of Wolbachia infection on T(p) of Drosophila at different developmental stages, which has not been done before. Only subtle effects were found of Wolbachia on host T(p) which are strongly affected by experimental variation in adult, but not during juvenile life stages. An in-depth analyses show that environmental variation has a substantial influence on T(p) which demonstrates the necessity of careful experimental design and cautious interpretations of T(p) measurements together with a thorough description of the methods and equipment used to conduct behavioral studies. |
| Roemschied, F. A., Pacheco, D. A., Aragon, M. J., Ireland, E. C., Li, X., Thieringer, K., Pang, R., Murthy, M. (2023). Flexible circuit mechanisms for context-dependent song sequencing. Nature, 622(7984):794-801 PubMed ID: 37821705
Summary: Sequenced behaviours, including locomotion, reaching and vocalization, are patterned differently in different contexts, enabling animals to adjust to their environments. How contextual information shapes neural activity to flexibly alter the patterning of actions is not fully understood. Previous work has indicated that this could be achieved via parallel motor circuits, with differing sensitivities to context. This study demonstrated that a single pathway operates in two regimes dependent on recent sensory history. The Drosophila song production system was leveraged to investigate the role of several neuron types in song patterning near versus far from the female fly. Male flies sing 'simple' trains of only one mode far from the female fly but complex song sequences comprising alternations between modes when near her. Ventral nerve cord (VNC) circuits are shaped by mutual inhibition and rebound excitability between nodes driving the two song modes. Brief sensory input to a direct brain-to-VNC excitatory pathway drives simple song far from the female, whereas prolonged input enables complex song production via simultaneous recruitment of functional disinhibition of VNC circuitry. Thus, female proximity unlocks motor circuit dynamics in the correct context. A compact circuit model was constructed to demonstrate that the identified mechanisms suffice to replicate natural song dynamics. These results highlight how canonical circuit motifs can be combined to enable circuit flexibility required for dynamic communication. | Salem, W., Cellini, B., Jaworski, E., Mongeau, J. M. (2023). Flies adaptively control flight to compensate for added inertia. Proceedings Biological sciences, 290(2008):20231115 PubMed ID: 37817597
Summary: Animal locomotion is highly adaptive, displaying a large degree of flexibility, yet how this flexibility arises from the integration of mechanics and neural control remains elusive. For instance, animals require flexible strategies to maintain performance as changes in mass or inertia impact stability. Compensatory strategies to mechanical loading are especially critical for animals that rely on flight for survival. To shed light on the capacity and flexibility of flight neuromechanics to mechanical loading, the performance of fruit flies (Drosophila) was pushed near its limit, and a control theoretic framework was implemented. Flies with added inertia were placed inside a virtual reality arena which permitted free rotation about the vertical (yaw) axis. Adding inertia increased the fly's response time yet had little influence on overall gaze stabilization performance. Flies maintained stability following the addition of inertia by adaptively modulating both visuomotor gain and damping. By contrast, mathematical modelling predicted a significant decrease in gaze stabilization performance. Adding inertia altered saccades, however, flies compensated for the added inertia by increasing saccade torque. Taken together, in response to added inertia flies increase reaction time but maintain flight performance through adaptive neural control. Overall, adding inertia decreases closed-loop flight robustness. This work highlights the flexibility and capacity of motor control in flight. |
| Baker, C. A., Guan, X. J., Choi, M., Murthy, M. (2024). The role of fruitless in specifying courtship behaviors across divergent Drosophila species. Sci Adv, 10(11):eadk1273 PubMed ID: 38478605
Summary: Sex-specific behaviors are critical for reproduction and species survival. The sex-specifically spliced transcription factor fruitless (fru) helps establish male courtship behaviors in invertebrates. Forcing male-specific fru (fruM) splicing in Drosophila melanogaster females produces male-typical behaviors while disrupting female-specific behaviors. However, whether the joint role of fru in specifying male and inhibiting female behaviors is conserved across species is unknown. CRISPR-Cas9 was used to force FruM expression in female Drosophila virilis, a species in which males and females produce sex-specific songs. In contrast to D. melanogaster, in which one fruM allele is sufficient to generate male behaviors in females, two alleles are needed in D. virilis females. D. virilis females expressing FruM maintain the ability to sing female-typical song as well as lay eggs, whereas D. melanogaster FruM females cannot lay eggs. These results reveal potential differences in fru function between divergent species and underscore the importance of studying diverse behaviors and species for understanding the genetic basis of sex differences. | Amin, H., Nolte, S. S., Swain, B., von Philipsborn, A. C. (2023). GABAergic signaling shapes multiple aspects of Drosophila courtship motor behavior. iScience, 26(11):108069 PubMed ID: 37860694
Summary: Inhibitory neurons are essential for orchestrating and structuring behavior. This study used one of the best studied behaviors in Drosophila, male courtship, to analyze how inhibitory, GABAergic neurons shape the different steps of this multifaceted motor sequence. RNAi-mediated knockdown of the GABA-producing enzyme GAD1 and the ionotropic receptor Rdl in sex specific, fruitless expressing neurons in the ventral nerve cord causes uncoordinated and futile copulation attempts, defects in wing extension choice and severe alterations of courtship song. Altered song of GABA depleted males fails to stimulate female receptivity, but rescue of song patterning alone is not sufficient to rescue male mating success. Knockdown of GAD1 and Rdl in male brain circuits abolishes courtship conditioning. The around 220 neurons coexpressing GAD1 and Fruitless in the Drosophila male nervous system were characterized, and inhibitory circuit motifs underlying key features of courtship behavior were proposed based on the observed phenotypes. |
Monday, April 1st - Larval and Adult Neural Development, Structure and Function |
| Tello, J. A., Jiang, L., Zohar, Y., Restifo, L. L. (2023). Drosophila CASK regulates brain size and neuronal morphogenesis, providing a genetic model of postnatal microcephaly suitable for drug discovery. Neural Dev, 18(1):6 PubMed ID: 37805506
Summary: CASK-related neurodevelopmental disorders are untreatable. Affected children show variable severity, with microcephaly, intellectual disability (ID), and short stature as common features. X-linked human CASK shows dosage sensitivity with haploinsufficiency in females. CASK protein has multiple domains, binding partners, and proposed functions at synapses and in the nucleus. Human and Drosophila CASK show high amino-acid-sequence similarity in all functional domains. Flies homozygous for a hypomorphic CASK mutation (∆18) have motor and cognitive deficits. A Drosophila genetic model of CASK-related disorders could have great scientific and translational value. It ws demonstrated that Δ18 homozygous flies have small brains, small heads, and short bodies. When neurons from developing CASK-mutant CNS were cultured in vitro, they grew small neurite arbors with a distinctive, quantifiable 'bushy' morphology that was significantly rescued by transgenic CASK(+). As in humans, the bushy phenotype showed dosage-sensitive severity. To overcome the limitations of manual tissue trituration for neuronal culture, the design and operation of a microfluidic system was devised for standardized, automated dissociation of CNS tissue into individual viable neurons. Neurons from CASK-mutant CNS dissociated in the microfluidic system recapitulate the bushy morphology. Moreover, for any given genotype, device-dissociated neurons grew larger arbors than did manually dissociated neurons. This automated dissociation method is also effective for rodent CNS. These biological and engineering advances set the stage for drug discovery using the Drosophila model of CASK-related disorders. The bushy phenotype provides a cell-based assay for compound screening. Nearly a dozen genes encoding CASK-binding proteins or transcriptional targets also have brain-development mutant phenotypes, including ID. Hence, drugs that improve CASK phenotypes might also benefit children with disorders due to mutant CASK partners. | Maksymchuk, N., Sakurai, A., Cox, D. N., Cymbalyuk, G. S. (2023). Cold-Temperature Coding with Bursting and Spiking Based on TRP Channel Dynamics in Drosophila Larva Sensory Neurons. Int J Mol Sci, 24(19) PubMed ID: 37834085
Summary: Temperature sensation involves thermosensitive TRP (thermoTRP) and non-TRP channels. Drosophila larval Class III (CIII) neurons serve as the primary cold nociceptors and express a suite of thermoTRP channels i mplicated in noxious cold sensation. How CIII neurons code temperature remains unclear.This study combined computational and electrophysiological methods to address this question. In electrophysiological experiments, two basic cold-evoked patterns of CIII neurons were identified: bursting and spiking. In response to a fast temperature drop to noxious cold, CIII neurons distinctly mark different phases of the stimulus. Bursts frequently occurred along with the fast temperature drop, forming a peak in the spiking rate and likely coding the high rate of the temperature change. Single spikes dominated at a steady temperature and exhibited frequency adaptation following the peak. When temperature decreased slowly to the same value, mainly spiking activity was observed, with bursts occurring sporadically throughout the stimulation. The spike and the burst frequencies positively correlated with the rate of the temperature drop. Using a computational model, the distinction is explained in the occurrence of the two CIII cold-evoked patterns bursting and spiking, using the dynamics of a thermoTRP current. A two-parameter activity map (Temperature, constant TRP current conductance) marks parameters that support silent, spiking, and bursting regimes. Projecting on the map the instantaneous TRP conductance, governed by activation and inactivation processes, reflects temperature coding responses as a path across silent, spiking, or bursting domains on the map. The map sheds light on how various parameter sets for TRP kinetics represent various types of cold-evoked responses. Together, these results indicate that bursting detects the high rate of temperature change, whereas tonic spiking could reflect both the rate of change and magnitude of steady cold temperature. |
| Lago-Baldaia, I., Cooper, M., Seroka, A., Trivedi, C., Powell, G. T., Wilson, S. W., Ackerman, S. D., Fernandes, V. M. (2023). A Drosophila glial cell atlas reveals a mismatch between transcriptional and morphological diversity. PLoS Biol, 21(10):e3002328 PubMed ID: 37862379
Summary: glia display diverse morphologies, both across and within glial classes. This study explored the relationship between glial morphology and transcriptional signature using the Drosophila CNS, where glia are categorised into 5 main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes). Single-cell RNA sequencing data of Drosophila glia were analysedin 2 well-characterised tissues from distinct developmental stages containing distinct circuit types: the embryonic ventral nerve cord (VNC) (motor) and the adult optic lobes (sensory). This analysis identified a new morphologically and transcriptionally distinct surface glial population in the VNC. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While extensive within-class transcriptomic diversity was detected for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, this study generated a single-cell transcriptomic atlas of glia in Drosophila, and this extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level. | Nakamizo-Dojo, M., Ishii, K., Yoshino, J., Tsuji, M., Emoto, K. (2023). Descending GABAergic pathway links brain sugar-sensing to peripheral nociceptive gating in Drosophila. Nat Commun, 14(1):6515 PubMed ID: 37845214
Summary: Although painful stimuli elicit defensive responses including escape behavior for survival, starved animals often prioritize feeding over escape even in a noxious environment. This behavioral priority is typically mediated by suppression of noxious inputs through descending control in the brain, yet underlying molecular and cellular mechanisms are incompletely understood. This study identified a cluster of GABAergic neurons in Drosophila larval brain, designated as SEZ-localized Descending GABAergic neurons (SDGs), that project descending axons onto the axon terminals of the peripheral nociceptive neurons and prevent presynaptic activity through GABA(B) receptors. Remarkably, glucose feeding to starved larvae causes sustained activation of SDGs through glucose-sensing neurons and subsequent insulin signaling in SDGs, which attenuates nociception and thereby suppresses escape behavior in response to multiple noxious stimuli. These findings illustrate a neural mechanism by which sugar sensing neurons in the brain engages descending GABAergic neurons in nociceptive gating to achieve hierarchical interaction between feeding and escape behavior. |
| Chua, N. J., Makarova, A. A., Gunn, P., Villani, S., Cohen, B., Thasin, M., Wu, J., Shefter, D., Pang, S., Xu, C. S., Hess, H. F., Polilov, A. A., Chklovskii, D. B. (2023). A complete reconstruction of the early visual system of an adult insect. Curr Biol, 33(21):4611-4623.e4614 PubMed ID: 37774707
Summary: For most model organisms in neuroscience, research into visual processing in the brain is difficult because of a lack of high-resolution maps that capture complex neuronal circuitry. The microinsect Megaphragma viggianii, because of its small size and non-trivial behavior, provides a unique opportunity for tractable whole-organism connectomics. This study imaged the Drosophila whole head using serial electron microscopy. Its compound eye was reconstructed, and the optical properties of the ommatidia as well as the connectome of the first visual neuropil-the lamina was analyze. Compared with the fruit fly and the honeybee, Megaphragma visual system is highly simplified: it has 29 ommatidia per eye and 6 lamina neuron types. Features are reported that are both stereotypical among most ommatidia and specialized to some. By identifying the "barebones" circuits critical for flying insects, these results will facilitate constructing computational models of visual processing in insects. | Poe, A. R., Zhu, L., Tang, S. H., Valencia, E., Kayser, M. S. (2023). Energetic Demands Regulate Sleep-Wake Rhythm Circuit Development. bioRxiv, PubMed ID: 37786713
Summary: Sleep and feeding patterns lack a clear daily rhythm during early life. As diurnal animals mature, feeding is consolidated to the day and sleep to the night. Circadian sleep patterns begin with formation of a circuit connecting the central clock to arousal output neurons; emergence of circadian sleep also enables long-term memory (LTM). However, the cues that trigger the development of this clock-arousal circuit are unknown. This study identify a role for nutritional status in driving sleep-wake rhythm development in Drosophila larvae. In the 2(nd) instar (L2) period, sleep and feeding are spread across the day; these behaviors become organized into daily patterns by L3. Forcing mature (L3) animals to adopt immature (L2) feeding strategies disrupts sleep-wake rhythms and the ability to exhibit LTM. In addition, the development of the clock (DN1a)-arousal (Dh44) circuit itself is influenced by the larval nutritional environment. Finally, it was demonstrated that larval arousal Dh44 neurons act through glucose metabolic genes to drive onset of daily sleep-wake rhythms. Together, these data suggest that changes to energetic demands in developing organisms triggers the formation of sleep-circadian circuits and behaviors. |
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