What's hot today:
Current papers in developmental biology and gene function


Saturday, October 29th, 2022 - Embryonic Development

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Vanderleest, T. E., Xie, Y., Smits, C., Blankenship, J. T. and Loerke, D. (2022). Interface extension is a continuum property suggesting a linkage between AP contractile and DV lengthening processes. Mol Biol Cell: mbcE21070352. PubMed ID: 36129772
In the early Drosophila embryo, the elongation of the anterior-posterior body axis is driven by cell intercalation in the germband epithelium. Neighboring cells intercalate through the contraction of AP interfaces (between anterior-posterior neighbors) into higher-order vertices, which then resolve through the extension of new DV interfaces (between dorsal-ventral neighbors). Although interface contraction has been extensively studied, less is known about how new interfaces are established. This study shows that DV interface elongation behaviors initiate at the same time as AP contractions, and that DV interfaces which are newly created from resolution of higher-order vertices do not appear to possess a unique 'identity'; instead, all horizontal interfaces undergo lengthening, elongating through ratchet-like sliding behaviors analogous to those found in AP interfaces. Cortical F-actin networks are essential for high area oscillation amplitudes required for effective ratcheting. These results suggest that, contrary to canonical models, the elongation of new DV interfaces is not produced by a mechanistically separate process. Instead, medial Myosin populations drive oscillating radial forces in the cells to generate transient force asymmetries at all tricellular vertices, which - combined with planar polarized stabilization - produce directional ratcheted sliding to generate both AP interface contraction and DV interface elongation.
Bourne, C. M., Lai, D. C. and Schottenfeld-Roames, J. (2022). Regulators of the secretory pathway have distinct inputs into single-celled branching morphogenesis and seamless tube formation in the Drosophila trachea. Dev Biol 490: 100-109. PubMed ID: 35870495
Biological tubes serve as conduits through which gas, nutrients and other important fluids are delivered to tissues. Most biological tubes consist of multiple cells connected by epithelial junctions. Unlike these multicellular tubes, seamless tubes are unicellular and lack junctions. Seamless tubes are present in various organ systems, including the vertebrate vasculature, C.elegans excretory system, and Drosophila tracheal system. The Drosophila tracheal system is a network of air-filled tubes that delivers oxygen to all tissues. Specialized cells within the tracheal system, called terminal cells, branch extensively and form seamless tubes. Terminal tracheal tubes are polarized; the lumenal membrane has apical identity whereas the outer membrane exhibits basal characteristics. Although various aspects of membrane trafficking have been implicated in terminal cell morphogenesis, the precise secretory pathway requirements for basal and apical membrane growth have yet to be elucidated. This study, demonstrated that anterograde trafficking, retrograde trafficking and Golgi-to-plasma membrane vesicle fusion are each required for the complex branched architecture of the terminal cell, but their inputs during seamless lumen formation are more varied. The COPII subunit, Sec31, and ER exit site protein, Sec16, are critical for subcellular tube architecture, whereas the SNARE proteins Syntaxin 5, Syntaxin 1 and Syntaxin 18 are more generally required for seamless tube growth and maintenance. These data suggest that distinct components of the secretory pathway have differential contributions to basal and apical membrane growth and maintenance during terminal cell morphogenesis.
Ojha, S. and Tapadia, M. G. (2022). Nonapoptotic role of caspase-3 in regulating Rho1GTPase-mediated morphogenesis of epithelial tubes of Drosophila renal system. Dev Dyn 251(5): 777-794. PubMed ID: 34773432
Cells trigger caspase-mediated apoptosis to eliminate themselves from the system when tissue needs to be sculptured, or they detect any abnormality within them, thus preventing irreparable damage to the host. However, nonapoptotic activities of caspases are also involved in many cellular functions. Interestingly, Drosophila Malpighian tubules (MTs) express apoptotic proteins, without succumbing to cell death. Yhis stuy showd apoptosis-independent role of executioner caspase-3, Drice, in MT morphogenesis. Drice is required for precise cytoskeleton organization and convergent extension, failing which morphology, size, cell number, and arrangement get affected. Furthermore, characteristic stellate cell shape transformation in MTs is also governed by Drice. Genetic interaction study shows that Drice mediates its action by regulating Rho1GTPase functionally, and localization of polarity protein Disc large. Subsequently, downregulation of Rho1GTPase in Drice mutants significantly rescues the cystic MTs phenotype. The study shows a mechanism by which Drice governs tubulogenesis via Rho1GTPase-mediated coordinated organization of actin cytoskeleton and membrane stabilization. Collectively these findings suggest a nonapoptotic function of caspase-3 in fine-tuning of cellular rearrangement during tubule development, and these results will add to the growing understanding of diverse roles of caspases during its evolution in metazoans.
Stern, T., Shvartsman, S. Y. and Wieschaus, E. F. (2022). Deconstructing gastrulation at single-cell resolution. Curr Biol 32(8): 1861-1868. PubMed ID: 35290798
Gastrulation movements in all animal embryos start with regulated deformations of patterned epithelial sheets, which are driven by cell divisions, cell shape changes, and cell intercalations. Each of these behaviors has been associated with distinct aspects of gastrulation and has been a subject of intense research using genetic, cell biological, and more recently, biophysical approaches Most of these studies, however, focus either on cellular processes driving gastrulation or on large-scale tissue deformations. Recent advances in microscopy and image processing create a unique opportunity for integrating these complementary viewpoints. This study has taken a step toward bridging these complementary strategies and deconstruct the early stages of gastrulation in the entire Drosophila embryo. This approach relies on an integrated computational framework for cell segmentation and tracking and on efficient algorithms for event detection. The detected events are then mapped back onto the blastoderm shell, providing an intuitive visual means to examine complex cellular activity patterns within the context of their initial anatomic domains was identified. By analyzing these maps, The loss of nearly half of surface cells to invaginations is compensated primarily by transient mitotic rounding. In addition, by analyzing mapped cell intercalation events, direct quantitative relations were derived between intercalation frequency and the rate of axis elongation. This work is setting the stage for systems-level dissection of a pivotal step in animal development.
Toth, K., Foldi, I. and Mihaly, J. (2022). A Comparative Study of the Role of Formins in Drosophila Embryonic Dorsal Closure. Cells 11(9). PubMed ID: 35563844
Dorsal closure is a late embryogenesis process required to seal the epidermal hole on the dorsal side of the Drosophila embryo. This process involves the coordination of several forces generated in the epidermal cell layer and in the amnioserosa cells, covering the hole. Ultimately, these forces arise due to cytoskeletal rearrangements that induce changes in cell shape and result in tissue movement. While a number of cytoskeleton regulatory proteins have already been linked to dorsal closure, this study has expanded this list by demonstrating that four of the six Drosophila formin type actin assembly factors are needed to bring about the proper fusion of the epithelia. An analysis of the morphological and dynamic properties of dorsal closure in formin mutants revealed a differential contribution for each formin, although this study found evidence for functional redundancies as well. Therefore, it is proposed that the four formins promote the formation of several, and only partly identical, actin structures each with a specific role in the mechanics of dorsal closure.
Warecki, B., Titen, S. W. A., Alam, M. S., Vega, G., Lemseffer, N., Hug, K., Minden, J. S. and Sullivan, W. (2022). Wolbachia action in the sperm produces developmentally deferred chromosome segregation defects during the Drosophila mid-blastula transition. Elife 11. PubMed ID: 36149408
Wolbachia, a vertically transmitted endosymbiont infecting many insects, spreads rapidly through uninfected populations by a mechanism known as cytoplasmic incompatibility (CI). In CI, a paternally delivered modification of the sperm leads to chromatin defects and lethality during and after the first mitosis of embryonic development in multiple species. However, whether CI-induced defects in later stage embryos are a consequence of the first division errors or caused by independent defects remains unresolved. To address this question, this study focused on ~1/3 of embryos from CI crosses in Drosophila simulans that develop apparently normally through the first and subsequent pre-blastoderm divisions before exhibiting mitotic errors during the mid-blastula transition and gastrulation. Single embryo PCR was developed and whole genome sequencing to find a large percentage of these developed CI-derived embryos bypass the first division defect. Using fluorescence in situ hybridization, this study found increased chromosome segregation errors in gastrulating CI-derived embryos that had avoided the first division defect. Thus, Wolbachia action in the sperm induces developmentally deferred defects that are not a consequence of the first division errors. Like the immediate defect, the delayed defect is rescued through crosses to infected females. These studies inform current models on the molecular and cellular basis of CI.

Friday, October 28th - Disease Models

Ogunsuyi, O. B., Aro, O. P., Oboh, G. and Olagoke, O. C. (2022). Curcumin improves the ability of donepezil to ameliorate memory impairment in Drosophila melanogaster: involvement of cholinergic and cnc/Nrf2-redox systems. Drug Chem Toxicol: 1-9. PubMed ID: 36069210
One of the well-established models for examining neurodegeneration and neurotoxicity is the Drosophila melanogaster model of aluminum-induced toxicity. Anti-cholinesterase drugs have been combined with other neuroprotective agents to improve Alzheimer's disease management, but there is not much information on the combination of anti-cholinesterases with dietary polyphenols to combat memory impairment. This study assessed how curcumin influences some of the critical therapeutic effects of donepezil (a cholinesterase inhibitor) in AlCl(3)-treated Drosophila melanogaster. Harwich strain flies were exposed to 40 mM AlCl(3) - alone or in combination with curcumin (1 mg/g) and/or donepezil (12.5 &mi;g/g and 25 &mi;g/g) - for seven days. The flies' behavioral evaluations (memory index and locomotor performance) were analyzed. Thereafter, the flies were processed into homogenates for the quantification of acetylcholinesterase (AChE), catalase, total thiol, and rate of lipid peroxidation, as well as the mRNA levels of acetylcholinesterase (ACE1) and cnc/NRF2. Results showed that AlCl(3)-treated flies presented impaired memory and increased activities of acetylcholinesterase and lipid peroxidation, while there were decrease in total thiol levels and catalase activity when compared to the control. Also, the expression of ACE1 was significantly increased while that of cnc/NRF2 was significantly decreased. However, combinations of curcumin and donepezil, especially at lower dose of donepezil, significantly improved the memory index and biochemical parameters compared to donepezil alone. Thus, curcumin plus donepezil offers unique therapeutic effects during memory impairment in the D. melanogaster model of neurotoxicity.
O'Hanlon, M. E., Tweedy, C., Scialo, F., Bass, R., Sanz, A. and Smulders-Srinivasan, T. K. (2022). Mitochondrial electron transport chain defects modify Parkinson's disease phenotypes in a Drosophila model. Neurobiol Dis 171: 105803. PubMed ID: 35764292
Mitochondrial defects have been implicated in Parkinson's disease (PD) More evidence of mitochondrial involvement arose when many of the genes whose mutations caused inherited PD were discovered to be subcellularly localized to mitochondria or have mitochondrial functions. The aim of this study was to better understand mitochondrial dysfunction in PD by evaluating mitochondrial respiratory complex mutations in a Drosophila model of PD. This study conducted a targeted heterozygous enhancer/suppressor screen using Drosophila mutations within mitochondrial electron transport chain (ETC) genes against a null PD mutation in parkin. The interactions were assessed by climbing assays at 2-5 days as an indicator of motor function. A strong enhancer mutation in COX5A was examined further for L-dopa rescue, oxygen consumption, mitochondrial content, and reactive oxygen species. A later timepoint of 16-20 days was also investigated for both COX5A and a suppressor mutation in cyclope. Mutations in individual genes for subunits within the mitochondrial respiratory complexes were found to have interactions with parkin, while others do not, irrespective of complex. One intriguing mutation in a complex IV subunit (cyclope) shows a suppressor rescue effect at early time points, improving the gross motor defects caused by the PD mutation, providing a strong candidate for drug discovery. Most mutations, however, show varying degrees of enhancement or slight suppression of the PD phenotypes. Thus, individual mitochondrial mutations within different oxidative phosphorylation complexes have different interactions with PD with regard to degree and direction. Upon further investigation of the strongest enhancer (COX5A), the mechanism by which these interactions occur initially does not appear to be based on defects in ATP production, but rather may be related to increased levels of reactive oxygen species. This work highlights some key subunits potentially involved in mechanisms underlying PD pathogenesis, implicating ETC complexes other than complex I in PD.
Shaw, N. M., Rios-Monterrosa, J. L., Fedorchak, G. R., Ketterer, M. R., Coombs, G. S., Lammerding, J. and Wallrath, L. L. (2022). Effects of mutant lamins on nucleo-cytoskeletal coupling in Drosophila models of LMNA muscular dystrophy. Front Cell Dev Biol 10: 934586. PubMed ID: 36120560
The nuclei of multinucleated skeletal muscles experience substantial external force during development and muscle contraction. Protection from such forces is partly provided by lamins (see Drosophila Lamin), intermediate filaments that form a scaffold lining the inner nuclear membrane. Lamins play a myriad of roles, including maintenance of nuclear shape and stability, mediation of nuclear mechanoresponses, and nucleo-cytoskeletal coupling. This study investigated how disease-causing mutant lamins alter myonuclear properties in response to mechanical force. This was accomplished via a novel application of a micropipette harpooning assay applied to larval body wall muscles of Drosophila models of lamin-associated muscular dystrophy. The assay enables the measurement of both nuclear deformability and intracellular force transmission between the cytoskeleton and nuclear interior in intact muscle fibers. These studies revealed that specific mutant lamins increase nuclear deformability while other mutant lamins cause nucleo-cytoskeletal coupling defects, which were associated with loss of microtubular nuclear caging. Microtubule caging of the nucleus was found to depended on Msp300, a KASH domain protein that is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Taken together, these findings identified residues in lamins required for connecting the nucleus to the cytoskeleton and suggest that not all muscle disease-causing mutant lamins produce similar defects in subcellular mechanics.
Singh, A. and Agrawal, N. (2022). Progressive transcriptional changes in metabolic genes and altered fatbody homeostasis in Drosophila model of Huntington's disease. Metab Brain Dis. PubMed ID: 36121619
Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder marked by progressive neuronal atrophy, particularly in striatum and cerebral cortex. Although predominant manifestations of the disease include loss in the triad of motor, cognitive and behavioral capabilities, metabolic dysfunction in patients and HD models are being increasingly recognized. Patients display progressive body weight loss, which aggravates the disease and leads to cachexia in the terminal stages. Using the Drosophila model of HD, it has been reported earlier that diseased flies exhibit an atypical pattern of lipid gain and loss with progression along with exhibiting extensive mitochondrial dysfunction, impaired calcium homeostasis and heightened apoptosis in the fatbody. This study first monitored the structural changes that abdominal fatbody undergoes with disease progression. Further, the transcriptional changes of key metabolic genes in whole fly were checked as well as genes regulating mitochondrial function, apoptosis, autophagy and calcium homeostasis in the abdominal fatbody. Extensive alterations were found in whole-body and fatbody-specific transcriptional profile of the diseased flies, which was in consort with their stage-specific physiological state. Additionally, lysosome-mediated autophagy was assessed in the fatbody of diseased flies in order to ascertain the mechanisms contributing to fatbody atrophy at the terminal stage. Interestingly, elevated autophagy was found in fatbody of flies throughout disease progression. This study provides new insights into the effect on peripheral metabolism due to degeneration of neurons in the neurodegenerative disease, thereby discerns novel mechanisms leading to cachexia in diseased flies and advocates for the need of managing metabolic dysfunctions in HD.
Rai, P. and Roy, J. K. (2022). Rab11 regulates mitophagy signaling pathway of Parkin and Pink1 in the Drosophila model of Parkinson's disease. Biochem Biophys Res Commun 626: 175-186. PubMed ID: 35994827
Parkinson's disease (PD) is a common neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. The pathophysiology of this disease is the formation of the Lewy body, mostly consisting of alpha-synuclein and dysfunctional mitochondria. There are two common PD-associated genes, Pink1 (encoding a mitochondrial ser/thr kinase) and Parkin (encoding cytosolic E3-ubiquitin ligase), involved in the mitochondrial quality control pathway. They assist in removing damaged mitochondria via selective autophagy (mitophagy) which if unchecked, results in the formation of protein aggregates in the cytoplasm. The role of Rab11, a small Ras-like GTPase associated with recycling endosomes, in PD is still unclear. The present study used the PD model of Drosophila melanogaster and found that Rab11 has a crucial role in the regulation of mitochondrial quality control and endo-lysosomal pathways in association with Parkin and Pink1 and Rab11 acting downstream of Parkin. Additionally, overexpression of Rab11 in parkin mutant rescued the mitochondrial impairment, suggesting the therapeutic potential of Rab11 in PD pathogenesis.
Sanchez Marco, S. B., Buhl, E., Firth, R., Zhu, B., Gainsborough, M., Beleza-Meireles, A., Moore, S., Caswell, R., Stals, K., Ellard, S., Kennedy, C., Hodge, J. J. L. and Majumdar, A. (2022). Hereditary spastic paraparesis presenting as cerebral palsy due to ADD3 variant with mechanistic insight provided by a Drosophila γ-adducin model. Clin Genet. PubMed ID: 36046955
Cerebral palsy (CP) causes neurological disability in early childhood. Hypoxic-ischaemic injury plays a major role in its aetiology. Mutations in ADD3 (encoding γ-adducin) gene have been described in a monogenic form of spastic quadriplegic cerebral palsy (OMIM 601568). A 16-year-old male with spastic diplegia was studied. An animal model was generated using Drosophila to study the effects of γ-adducin loss and gain of function. WES revealed a biallelic variant in the ADD3 gene, NM_016824.5(ADD3): c.1100G > A, p.(Gly367Asp). Mutations in this gene have been described as an ultra-rare autosomal recessive, which is a known form of inherited cerebral palsy. Molecular modelling suggests that this mutation leads to a loss of structural integrity of γ-adducin and is therefore expected to result in a decreased level of functional protein. Pan-neuronal over-expression or knock-down of the Drosophila ortholog of ADD3 called hts caused a reduction of life span and impaired locomotion thereby phenocopying aspects of the human disease. The animal experiments present a starting point to understand the biological processes underpinning the clinical phenotype and pathogenic mechanisms, to gain insights into potential future methods for treating or preventing ADD3 related spastic quadriplegic cerebral palsy.

Thursday, October 27th - Vesicles and Synapse

Park, H. G., Kim, Y. D., Cho, E., Lu, T. Y., Yao, C. K., Lee, J. and Lee, S. (2022). Vav independently regulates synaptic growth and plasticity through distinct actin-based processes. J Cell Biol 221(10). PubMed ID: 35976098
Modulation of presynaptic actin dynamics is fundamental to synaptic growth and functional plasticity; yet the underlying molecular and cellular mechanisms remain largely unknown. At Drosophila NMJs, the presynaptic Rac1-SCAR pathway mediates BMP-induced receptor macropinocytosis to inhibit BMP growth signaling. This study shows that the Rho-type GEF Vav acts upstream of Rac1 to inhibit synaptic growth through macropinocytosis. Evidence is presented that Vav-Rac1-SCAR signaling has additional roles in tetanus-induced synaptic plasticity. Presynaptic inactivation of Vav signaling pathway components, but not regulators of macropinocytosis, impairs post-tetanic potentiation (PTP) and enhances synaptic depression depending on external Ca2+ concentration. Interfering with the Vav-Rac1-SCAR pathway also impairs mobilization of reserve pool (RP) vesicles required for tetanus-induced synaptic plasticity. Finally, treatment with an F-actin-stabilizing drug completely restores RP mobilization and plasticity defects in Vav mutants. It is proposed that actin-regulatory Vav-Rac1-SCAR signaling independently regulates structural and functional presynaptic plasticity by driving macropinocytosis and RP mobilization, respectively.
Park, S. Y., Muschalik, N., Chadwick, J. and Munro, S. (2022). In vivo characterization of Drosophila golgins reveals redundancy and plasticity of vesicle capture at the Golgi apparatus. Curr Biol. PubMed ID: 36103876
The Golgi is the central sorting station in the secretory pathway. One set of proteins proposed to direct vesicle arrival at the Golgi are the golgins, long coiled-coil proteins localized to specific parts of the Golgi stack. In mammalian cells, three of the golgins, TMF, golgin-84, and GMAP-210, can capture intra-Golgi transport vesicles when placed in an ectopic location. However, the individual golgins are not required for cell viability, and mouse knockout mutants only have defects in specific tissues. To further illuminate this system, this study examined the Drosophila orthologs of these three intra-Golgi golgins. The study shows that ectopic forms can capture intra-Golgi transport vesicles, but strikingly, the cargo present in the vesicles captured by each golgin varies between tissues. Loss-of-function mutants show that the golgins are individually dispensable, although the loss of TMF recapitulates the male fertility defects observed in mice. However, the deletion of multiple golgins results in defects in glycosylation and loss of viability. Examining the vesicles captured by a particular golgin when another golgin is missing reveals that the vesicle content in one tissue changes to resemble that of a different tissue. This reveals a plasticity in Golgi organization between tissues, providing an explanation for why the Golgi is sufficiently robust to tolerate the loss of many of the individual components of its membrane traffic machinery.
Orr, B. O., Fetter, R. D. and Davis, G. W. (2022). Activation and expansion of presynaptic signaling foci drives presynaptic homeostatic plasticity. Neuron. PubMed ID: 36087584
Presynaptic homeostatic plasticity (PHP) adaptively regulates synaptic transmission in health and disease. Despite identification of numerous genes that are essential for PHP, a dynamic framework to explain how PHP is initiated, potentiated, and limited to achieve precise control of vesicle fusion is lacking. Utilizing both mice and Drosophila, this study demonstrates that PHP progresses through the assembly and physical expansion of presynaptic signaling foci where activated integrins biochemically converge with trans-synaptic Semaphorin2b/PlexinB signaling. Each component of the identified signaling complexes, including alpha/beta-integrin, Semaphorin2b, PlexinB, talin, and focal adhesion kinase (FAK), and their biochemical interactions, are essential for PHP. Complex integrity requires the Sema2b ligand and complex expansion includes a ∼2.5-fold expansion of active-zone associated puncta composed of the actin-binding protein talin. Finally, complex pre-expansion is sufficient to accelerate the rate and extent of PHP. A working model is proposed incorporating signal convergence with dynamic molecular assemblies that instruct PHP.
Linnemannstons, K., Karuna, M. P., Witte, L., Choezom, D., Honemann-Capito, M., Lagurin, A. S., Schmidt, C. V., Shrikhande, S., Steinmetz, L. K., Wiebke, M., Lenz, C. and Gross, J. C. (2022). Microscopic and biochemical monitoring of endosomal trafficking and extracellular vesicle secretion in an endogenous in vivo model. J Extracell Vesicles 11(9): e12263. PubMed ID: 36103151
Extracellular vesicle (EV) secretion enables cell-cell communication in multicellular organisms. During development, EV secretion and the specific loading of signalling factors in EVs contributes to organ development and tissue differentiation. This study present an in vivo model to study EV secretion using the fat body and the haemolymph of the fruit fly, Drosophila melanogaster. The system makes use of tissue-specific EV labelling and is amenable to genetic modification by RNAi. This allows the unique combination of microscopic visualisation of EVs in different organs and quantitative biochemical purification to study how EVs are generated within the cells and which factors regulate their secretion in vivo. Characterisation of the system revealed that secretion of EVs from the fat body is mainly regulated by Rab11 and Rab35, highlighting the importance of recycling Rab GTPase family members for EV secretion. It was furthermore discovered a so far unknown function of Rab14 along with the kinesin Klp98A in EV biogenesis and secretion.
Song, J., Mizrak, A., Lee, C. W., Cicconet, M., Lai, Z. W., Tang, W. C., Lu, C. H., Mohr, S. E., Farese, R. V., Jr. and Walther, T. C. (2022). Identification of two pathways mediating protein targeting from ER to lipid droplets. Nat Cell Biol 24(9): 1364-1377. PubMed ID: 36050470
Pathways localizing proteins to their sites of action are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, how proteins, such as metabolic enzymes, target from the endoplasmic reticulum (ER) to cellular lipid droplets (LDs) is poorly understood. This study identified two distinct pathways for ER-to-LD protein targeting: early targeting at LD formation sites during formation, and late targeting to mature LDs after their formation. Using systematic, unbiased approaches in Drosophila cells, this study identified specific membrane-fusion machinery, including regulators, a tether and SNARE proteins, that are required for the late targeting pathway. Components of this fusion machinery localize to LD-ER interfaces and organize at ER exit sites. Multiple cargoes were identified for early and late ER-to-LD targeting pathways. These findings provide a model for how proteins target to LDs from the ER either during LD formation or by protein-catalysed formation of membrane bridges.
Vuilleumier, R., Miao, M., Medina-Giro, S., Ell, C. M., Flibotte, S., Lian, T., Kauwe, G., Collins, A., Ly, S., Pyrowolakis, G., Haghighi, A. P. and Allan, D. W. (2022). Dichotomous cis-regulatory motifs mediate the maturation of the neuromuscular junction by retrograde BMP signaling. Nucleic Acids Res 50(17): 9748-9764. PubMed ID: 36029115
Retrograde bone morphogenetic protein (BMP) signaling at the Drosophila neuromuscular junction (NMJ) has served as a paradigm to study TGF-β-dependent synaptic function and maturation. Yet, how retrograde BMP signaling transcriptionally regulates these functions remains unresolved. This study uncovered a gene network, enriched for neurotransmission-related genes, that is controlled by retrograde BMP signaling in motor neurons through two Smad-binding cis-regulatory motifs, the BMP-activating (BMP-AE) and silencer (BMP-SE) elements. Unpredictably, both motifs mediate direct gene activation, with no involvement of the BMP derepression pathway regulators Schnurri and Brinker. Genome editing of candidate BMP-SE and BMP-AE within the locus of the active zone gene bruchpilot, and a novel Ly6 gene witty, demonstrated the role of these motifs in upregulating genes required for the maturation of pre- and post-synaptic NMJ compartments. These findings uncover how Smad-dependent transcriptional mechanisms specific to motor neurons directly orchestrate a gene network required for synaptic maturation by retrograde BMP signaling.

Wednesday, October 26th - Adult Physiology and Metabolism

Prakash, A., Monteith, K. M. and Vale, P. F. (2022). Mechanisms of damage prevention, signalling and repair impact disease tolerance. Proc Biol Sci 289(1981): 20220837. PubMed ID: 35975433
The insect gut is frequently exposed to pathogenic threats and must not only clear these potential infections, but also tolerate relatively high microbe loads. In contrast to the mechanisms that eliminate pathogens, less is currently known about the mechanisms of disease tolerance. This study investigated how well-described mechanisms that prevent, signal, control or repair damage during infection contribute to the phenotype of disease tolerance. Enteric infections were established with the bacterial pathogen Pseudomonas entomophila in transgenic lines of Drosophila melanogaster fruit flies affecting dcy (a major component of the peritrophic matrix), upd3 (a cytokine-like molecule), irc (a negative regulator of reactive oxygen species) and egfr (epithelial growth factor receptor). Flies lacking dcy experienced the highest mortality, while loss of function of either irc or upd3 reduced tolerance in both sexes. The disruption of egfr resulted in a severe loss in tolerance in male flies but had no substantial effect on the ability of female flies to tolerate P. entomophila infection, despite carrying greater microbe loads than males. Together, these findings provide evidence for the role of damage limitation mechanisms in disease tolerance and highlight how sexual dimorphism in these mechanisms could generate sex differences in infection outcomes.
Sangsuwan, T., Mannervik, M. and Haghdoost, S. (2022). Transgenerational effects of gamma radiation dose and dose rate on Drosophila flies irradiated at an early embryonal stage. Mutat Res Genet Toxicol Environ Mutagen 881: 503523. PubMed ID: 36031335
Ionizing radiation (IR) kills cells mainly through induction of DNA damages and the surviving cells may suffer from mutations. Transgenerational effects of IR are well documented, but the exact mechanisms underlying them are less well understood; they include induction of mutations in germ cells and epigenetic inheritance. In the present project, short- and long-term effects of low-dose-rate (LDR; 50 and 97 mGy/h) and high-dose-rate (HDR; 23.4, 47.1 and 495 Gy/h) IR in Drosophila embryos were investigated. Also, the survival of larvae, pupae and adults developed from embryos irradiated at an early stage (30 min after egg laying) was studied. The larval crawling and pupation height assays were applied to investigate radiation effects on larval locomotion and pupation behavior, respectively. In parallel, the offspring from 3 Gy irradiated early-stage embryos were followed up to 12 generations and abnormal phenotypes were studied. Acute exposure of embryos at different stages of development showed that the early stage embryo is the most sensitive. The effects on larval locomotion showed no significant differences between the dose rates but a significant decrease of locomotion activity above 7 Gy was observed. The results indicate that embryos exposed to the low dose rates have shorter eclosion times. At the same cumulative dose (1 up to 7 Gy), HDR is more embryotoxic than LDR. A radiation-induced depigmentation was also found on males (A5 segment of the dorsal abdomen, A5pig(-)) that can be transmitted up to 12 generations. The phenomenon does not follow the classical Mendelian laws of segregation.
Perlegos, A. E., Shields, E. J., Shen, H., Liu, K. F. and Bonini, N. M. (2022). Mettl3-dependent m(6)A modification attenuates the brain stress response in Drosophila. Nat Commun 13(1): 5387. PubMed ID: 36104353
N(6)-methyladenosine (m(6)A), the most prevalent internal modification on eukaryotic mRNA, plays an essential role in various stress responses. The brain is uniquely vulnerable to cellular stress, thus defining how m(6)A sculpts the brain's susceptibility may provide insight to brain aging and disease-related stress. This study investigate the impact of m(6)A mRNA methylation in the adult Drosophila brain with stress. m(6)A is enriched in the adult brain and increases with heat stress. Through m(6)A-immunoprecipitation sequencing, this study showed 5'UTR Mettl3-dependent m(6)A is enriched in transcripts of neuronal processes and signaling pathways that increase upon stress. Mettl3 knockdown results in increased levels of m(6)A targets and confers resilience to stress. Loss of Mettl3 results in decreased levels of nuclear m(6)A reader Ythdc1, and knockdown of Ythdc1 also leads to stress resilience. Overall, these data suggest that m(6)A modification in Drosophila dampens the brain's biological response to stress.
Li, Y., Pan, L., Li, P., Yu, G., Li, Z., Dang, S., Jin, F. and Nan, Y. (2022). Microbiota aggravates the pathogenesis of Drosophila acutely exposed to vehicle exhaust. Heliyon 8(9): e10382. PubMed ID: 36060467
Vehicle exhaust (VE) is the primary cause of urban air pollution, which adversely affects the respiratory system, exacerbates lung diseases, and results in high mortality rates. However, the underlying mechanism of the pathogenesis is largely unclear. This study developed a Drosophila model to systematically investigate the effects of VE on their health and physiology. VE was found to significantly impaired life span and locomotion in Drosophila. Interestingly, there was an increase in bacterial load in the guts upon VE exposure, suggesting VE is able to induce dysbiosis in the guts. Microbiota depletion can ameliorate the impairment of life span and locomotion. VE causes permeability of intestinal epithelial cells and increases proliferation of intestinal cells, suggesting VE disrupts intestinal homeostasis. This study elucidated the underlying mechanism by which VE triggers Imd and DUOX gene expression. Taken together, this Drosophila model provides insight into the pathogenesis of Drosophila exposure to VE, enabling a better understanding of the specific role of microbiota.
Okuda, K., Silva Costa Franco, M. M., Yasunaga, A., Gazzinelli, R., Rabinovitch, M., Cherry, S. and Silverman, N. (2022). Leishmania amazonensis sabotages host cell SUMOylation for intracellular survival. iScience 25(9): 104909. PubMed ID: 36060064
Leishmania parasites use elaborate virulence mechanisms to invade and thrive in macrophages. These virulence mechanisms inhibit host cell defense responses and generate a specialized replicative niche, the parasitophorous vacuole. A genome-wide RNAi screen was performed in Drosophila macrophage-like cells to identify the host factors necessary for Leishmania amazonensis infection. This screen identified 52 conserved genes required specifically for parasite entry, including several components of the SUMOylation machinery (See Sumo). Further studies in mammalian macrophages found that L. amazonensis infection inhibited SUMOylation within infected macrophages and this inhibition enhanced parasitophorous vacuole growth and parasite proliferation through modulation of multiple genes especially ATP6V0D2, which in turn affects CD36 expression and cholesterol levels. Together, these data suggest that parasites actively sabotage host SUMOylation and alter host transcription to improve their intracellular niche and enhance their replication.
Mhatre, S. D., Iyer, J., Petereit, J., Dolling-Boreham, R. M., Tyryshkina, A., Paul, A. M., Gilbert, R., Jensen, M., Woolsey, R. J., Anand, S., Sowa, M. B., Quilici, D. R., Costes, S. V., Girirajan, S. and Bhattacharya, S. (2022). Artificial gravity partially protects space-induced neurological deficits in Drosophila melanogaster. Cell Rep 40(10): 111279. PubMed ID: 36070701
Spaceflight poses risks to the central nervous system (CNS), and understanding neurological responses is important for future missions. This study reports CNS changes in Drosophila aboard the International Space Station in response to spaceflight microgravity (SFμg) and artificially simulated Earth gravity (SF1g) via inflight centrifugation as a countermeasure. While inflight behavioral analyses of SFμg exhibit increased activity, postflight analysis displays significant climbing defects, highlighting the sensitivity of behavior to altered gravity. Multi-omics analysis shows alterations in metabolic, oxidative stress and synaptic transmission pathways in both SFμg and SF1g; however, neurological changes immediately postflight, including neuronal loss, glial cell count alterations, oxidative damage, and apoptosis, are seen only in SFμg. Additionally, progressive neuronal loss and a glial phenotype in SF1g and SF&mug brains, with pronounced phenotypes in SFμg, are seen upon acclimation to Earth conditions. Overall, these results indicate that artificial gravity partially protects the CNS from the adverse effects of spaceflight.

Tuesday, October 25 Larval and adult neural development and function

Poppinga, H., Çoban, B., Meltzer, H., Mayseless, O., Widmann, A., Schuldiner, O. and Fiala, A. (2022). Pruning deficits of the developing Drosophila mushroom body result in mild impairment in associative odour learning and cause hyperactivity. Open Biol 12(9): 220096. PubMed ID: 36128716
The principles of how brain circuits establish themselves during development are largely conserved across animal species. Connections made during embryonic development that are appropriate for an early life stage are frequently remodelled later in ontogeny via pruning and subsequent regrowth to generate adult-specific connectivity. The mushroom body of the fruit fly Drosophila melanogaster is a well-established model circuit for examining the cellular mechanisms underlying neurite remodelling. This central brain circuit integrates sensory information with learned and innate valences to adaptively instruct behavioural decisions. Thereby, the mushroom body organizes adaptive behaviour, such as associative learning. However, little is known about the specific aspects of behaviour that require mushroom body remodelling. This study used genetic interventions to prevent the intrinsic neurons of the larval mushroom body (γ-type Kenyon cells) from remodelling. It was asked to what degree remodelling deficits resulted in impaired behaviour. Deficits were found to cause hyperactivity and mild impairment in differential aversive olfactory learning, but not appetitive learning. Maintenance of circadian rhythm and sleep were not affected. It is concluded that neurite pruning and regrowth of γ-type Kenyon cells is not required for the establishment of circuits that mediate associative odour learning per se, but it does improve distinct learning tasks.
Rojo-Cortes, F., Fuenzalida-Uribe, N., Tapia-Valladares, V., Roa, C. B., Hidalgo, S., Gonzalez-Ramirez, M. C., Oliva, C., Campusano, J. M. and Marzolo, M. P. (2022). Lipophorin receptors regulate mushroom body development and complex behaviors in Drosophila. BMC Biol 20(1): 198. PubMed ID: 36071487
Drosophila melanogaster lipophorin receptors (LpRs), LpR1 and LpR2, are members of the LDLR family known to mediate lipid uptake in a range of organisms from Drosophila to humans. The vertebrate orthologs of LpRs, ApoER2 and VLDL-R, function as receptors of a glycoprotein involved in development of the central nervous system, Reelin, which is not present in flies. ApoER2 and VLDL-R are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain, as well as with neurodevelopmental and neurodegenerative disorders linked to those regions. It is currently unknown whether LpRs play similar roles in the Drosophila brain. This study reports that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the mushroom bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurite arborization. This effect depends on LpRs and Dab, the Drosophila ortholog of the Reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin, suggesting that Drosophila LpRs interact with human Reelin to induce downstream cellular events. These findings demonstrate that LpRs contribute to MB development and function, supporting the existence of a LpR-dependent signaling in Drosophila, and advance understanding of the molecular factors functioning in neural systems to generate complex behaviors in this model. These results further emphasize the importance of Drosophila as a model to investigate the alterations in specific genes contributing to neural disorders.
Ray, A. and Li, X. (2022). A Notch-dependent transcriptional mechanism controls expression of temporal patterning factors in Drosophila medulla. Elife 11. PubMed ID: 36040415
Temporal patterning is an important mechanism for generating a great diversity of neuron subtypes from a seemingly homogenous progenitor pool in both vertebrates and invertebrates. Drosophila neuroblasts are temporally patterned by sequentially expressed Temporal Transcription Factors (TTFs). These TTFs are proposed to form a transcriptional cascade based on mutant phenotypes, although direct transcriptional regulation between TTFs has not been verified in most cases. Furthermore, it is not known how the temporal transitions are coupled with the generation of the appropriate number of neurons at each stage. This study used neuroblasts of the Drosophila optic lobe medulla to address these questions and show that the expression of TTFs Sloppy-paired 1/2 (Slp1/2) is directly regulated at the transcriptional level by two other TTFs and the cell-cycle dependent Notch signaling through two cis-regulatory elements. It was also shown that supplying constitutively active Notch can rescue the delayed transition into the Slp stage in cell cycle arrested neuroblasts. These findings reveal a novel Notch-pathway dependent mechanism through which the cell cycle progression regulates the timing of a temporal transition within a TTF transcriptional cascade.
Prasad, A. R., Lago-Baldaia, I., Bostock, M. P., Housseini, Z. and Fernandes, V. M. (2022). Differentiation signals from glia are fine-tuned to set neuronal numbers during development.. Elife 11. PubMed ID: 36094172
Neural circuit formation and function require that diverse neurons are specified in appropriate numbers. Known strategies for controlling neuronal numbers involve regulating either cell proliferation or survival. This study used the Drosophila visual system to probe how neuronal numbers are set. Photoreceptors from the eye-disc induce their target field, the lamina, such that for every unit eye there is a corresponding lamina unit (column). Although each column initially contains ~6 post-mitotic lamina precursors, only 5 differentiate into neurons, called L1-L5; the 'extra' precursor, which is invariantly positioned above the L5 neuron in each column, undergoes apoptosis. This study showed that a glial population called the outer chiasm giant glia (xg(O)), which resides below the lamina, secretes multiple ligands to induce L5 differentiation in response to epidermal growth factor (EGF) from photoreceptors. By forcing neuronal differentiation in the lamina, it was uncovered that though fated to die, the 'extra' precursor is specified as an L5. Therefore, two precursors are specified as L5s but only one differentiates during normal development. It was found that the row of precursors nearest to xg(O) differentiate into L5s and, in turn, antagonise differentiation signalling to prevent the 'extra' precursors from differentiating, resulting in their death. Thus, an intricate interplay of glial signals and feedback from differentiating neurons defines an invariant and stereotyped pattern of neuronal differentiation and programmed cell death to ensure that lamina columns each contain exactly one L5 neuron.
Odell, S. R., Clark, D., Zito, N., Jain, R., Gong, H., Warnock, K., Carrion-Lopez, R., Maixner, C., Prieto-Godino, L. and Mathew, D. (2022). Internal state affects local neuron function in an early sensory processing center to shape olfactory behavior in Drosophila larvae. Sci Rep 12(1): 15767. PubMed ID: 36131078
Crawling insects, when starved, tend to have fewer head wavings and travel in straighter tracks in search of food. This study used the Drosophila melanogaster larva to investigate whether this flexibility in the insect's navigation strategy arises during early olfactory processing and, if so, how. A critical role is demonstrated for Keystone-LN, an inhibitory local neuron in the antennal lobe, in implementing head-sweep behavior. Keystone-LN responds to odor stimuli, and its inhibitory output is required for a larva to successfully navigate attractive and aversive odor gradients. Insulin signaling in Keystone-LN likely mediates the starvation-dependent changes in head-sweep magnitude, shaping the larva's odor-guided movement. These findings demonstrate how flexibility in an insect's navigation strategy can arise from context-dependent modulation of inhibitory neurons in an early sensory processing center. They raise new questions about modulating a circuit's inhibitory output to implement changes in a goal-directed movement.
Naganos, S., Ueno, K., Horiuchi, J. and Saitoe, M. (2022). Dopamine activity in projection neurons regulates short-lasting olfactory approach memory in Drosophila. Eur J Neurosci 56(5): 4558-4571. PubMed ID: 35815601
Survival in many animals requires the ability to associate certain cues with danger and others with safety. In a Drosophila melanogaster aversive olfactory conditioning paradigm, flies are exposed to two odours, one presented coincidentally with electrical shocks, and a second presented 45 s after shock cessation. When flies are later given a choice between these two odours, they avoid the shock-paired odour and prefer the unpaired odour. While many studies have examined how flies learn to avoid the shock-paired odour through formation of odour-fear associations, this study demonstrates that conditioning also causes flies to actively approach the second odour. In contrast to fear memories, which are longer lasting and requires activity of D1-like dopamine receptors only in the mushroom bodies, approach memory is short-lasting and requires activity of D1-like dopamine receptors in projection neurons originating from the antennal lobes, primary olfactory centers. Further, while recall of fear memories requires activity of the mushroom bodies, recall of approach memories does not. These data suggest that olfactory approach memory is formed using different mechanisms in different brain locations compared to aversive and appetitive olfactory memories.

Monday, October 24th - Chromatin

Huang, W., Zhu, J. Y., Fu, Y., van de Leemput, J. and Han, Z. (2022). Lpt, trr, and Hcf regulate histone mono- and dimethylation that are essential for Drosophila heart development. Dev Biol 490: 53-65. PubMed ID: 35853502
Mammalian KMT2C, KMT2D, and HCFC1 are expressed during heart development and have been associated with congenital heart disease, but their roles in heart development remain elusive. This study found that the Drosophila Lpt and trr genes encode the N-terminal and C-terminal homologs, respectively, of mammalian KMT2C or KMT2D. Lpt and trr mutant embryos showed reduced cardiac progenitor cells. Silencing of Lpt, trr, or both simultaneously in the heart led to similar abnormal cardiac morphology, tissue fibrosis, and cardiac functional defects. Like KMT2D, Lpt and trr were found to modulate histone H3K4 mono- and dimethylation, but not trimethylation. Investigation of downstream genes regulated by mouse KMT2D in the heart showed that their fly homologs are similarly regulated by Lpt or trr in the fly heart, suggesting that Lpt and trr regulate an evolutionarily conserved transcriptional network for heart development. Moreover, this study showed that cardiac silencing of Hcf, the fly homolog of mammalian HCFC1, leads to heart defects similar to those observed in Lpt and trr silencing, as well as reduced H3K4 monomethylation. These findings suggest that Lpt and trr function together to execute the conserved function of mammalian KMT2C and KMT2D in histone H3 lysine K4 mono- and dimethylation required for heart development. Possibly aided by Hcf, which plays a related role in H3K4 methylation during fly heart development.
Ohhara, Y., Kato, Y., Kamiyama, T. and Yamakawa-Kobayashi, K. (2022). Su(var)2-10- and Su(var)205-dependent upregulation of the heterochromatic gene neverland is required for developmental transition in Drosophila. Genetics. PubMed ID: 36149288
Animals develop from juveniles to sexually mature adults through the action of steroid hormones. In insect metamorphosis, a surge of the steroid hormone ecdysone prompts the transition from the larval to the adult stage. Ecdysone is synthesized by a series of biosynthetic enzymes that are specifically expressed in an endocrine organ, the prothoracic gland. At the late larval stage, the expression levels of ecdysone biosynthetic enzymes are upregulated through the action of numerous transcription factors, thus initiating metamorphosis. In contrast, the mechanism by which chromatin regulators support the expression of ecdysone biosynthetic genes is largely unknown. This study demonstrates that Su(var)2-10 and Su(var)205, suppressor of variegation [Su(var)] genes encoding a chromatin regulator Su(var)2-10 and non-histone heterochromatic protein 1a (HP1a), respectively, regulate the transcription of one of the heterochromatic ecdysone biosynthetic genes, neverland, in Drosophila melanogaster. Knockdown of Su(var)2-10 and Su(var)205 in the prothoracic gland caused a decrease in neverland expression, resulting in a defect in larval-to-prepupal transition. Furthermore, overexpression of neverland and administration of 7-dehydrocholesterol, a biosynthetic precursor of ecdysone produced by Neverland, rescued developmental defects in Su(var)2-10 and Su(var)205 knockdown animals. These results indicate that Su(var)2-10- and Su(var)205-mediated proper expression of neverland is required for the initiation of metamorphosis. Given that Su(var)2-10-positive puncta are juxtaposed with the pericentromeric heterochromatic region, it is proposed that Su(var)2-10- and Su(var)205-dependent regulation of inherent heterochromatin structure at the neverland gene locus is essential for its transcriptional activation.
Hannaford, M. R., Liu, R., Billington, N., Swider, Z. T., Galletta, B. J., Fagerstrom, C. J., Combs, C., Sellers, J. R. and Rusan, N. M. (2022). Pericentrin interacts with Kinesin-1 to drive centriole motility. J Cell Biol 221(9). PubMed ID: 35929834
Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosomal microtubules (MTs) with motor proteins anchored at the cortex or the nuclear surface. However, it remains unknown how centrioles migrate in cellular contexts in which they do not nucleate MTs. This study demonstrates that during interphase, inactive centrioles move directly along the interphase MT network as Kinesin-1 cargo. Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP. Finally, these studies of neural stem cell asymmetric divisions in the Drosophila brain show that the PLP-Kinesin-1 interaction is essential for the timely separation of centrioles, the asymmetry of centrosome activity, and the age-dependent centrosome inheritance.
Hendy, O., Serebreni, L., Bergauer, K., Muerdter, F., Huber, L., Nemcko, F. and Stark, A. (2022). Developmental and housekeeping transcriptional programs in Drosophila require distinct chromatin remodelers. Mol Cell. PubMed ID: 36113480
Gene transcription is a highly regulated process in all animals. In Drosophila, two major transcriptional programs, housekeeping and developmental, have promoters with distinct regulatory compatibilities and nucleosome organization. However, it remains unclear how the differences in chromatin structure relate to the distinct regulatory properties and which chromatin remodelers are required for these programs. Using rapid degradation of core remodeler subunits in Drosophila melanogaster S2 cells, this study demonstrates that developmental gene transcription requires SWI/SNF-type complexes, primarily to maintain distal enhancer accessibility. In contrast, wild-type-level housekeeping gene transcription requires the Iswi and Ino80 remodelers to maintain nucleosome positioning and phasing at promoters. These differential remodeler dependencies relate to different DNA-sequence-intrinsic nucleosome affinities, which favor a default ON state for housekeeping but a default OFF state for developmental gene transcription. Overall, these results demonstrate how different transcription-regulatory strategies are implemented by DNA sequence, chromatin structure, and remodeler activity.
Kang, H., Cabrera, J. R., Zee, B. M., Kang, H. A., Jobe, J. M., Hegarty, M. B., Barry, A. E., Glotov, A., Schwartz, Y. B. and Kuroda, M. I. (2022). Variant Polycomb complexes in Drosophila consistent with ancient functional diversity. Sci Adv 8(36): eadd0103. PubMed ID: 36070387
Polycomb group (PcG) mutants were first identified in Drosophila on the basis of their failure to maintain proper Hox gene repression during development. The proteins encoded by the corresponding fly genes mainly assemble into one of two discrete Polycomb repressive complexes: PRC1 or PRC2. However, biochemical analyses in mammals have revealed alternative forms of PRC2 and multiple distinct types of noncanonical or variant PRC1. Through a series of proteomic analyses, this study identified analogous PRC2 and variant PRC1 complexes in Drosophila, as well as a broader repertoire of interactions implicated in early development. These data provide strong support for the ancient diversity of PcG complexes and a framework for future analysis in a longstanding and versatile genetic system.
Gotz, M., Messina, O., Espinola, S., Fiche, J. B. and Nollmann, M. (2022). Multiple parameters shape the 3D chromatin structure of single nuclei at the doc locus in Drosophila. Nat Commun 13(1): 5375. PubMed ID: 36104317
The spatial organization of chromatin at the scale of topologically associating domains (TADs) and below displays large cell-to-cell variations. Up until now, how this heterogeneity in chromatin conformation is shaped by chromatin condensation, TAD insulation, and transcription has remained mostly elusive. This study used Hi-M, a multiplexed DNA-FISH imaging technique providing developmental timing and transcriptional status, to show that the emergence of TADs at the ensemble level partially segregates the conformational space explored by single nuclei during the early development of Drosophila embryos. Surprisingly, a substantial fraction of nuclei display strong insulation even before TADs emerge. Moreover, active transcription within a TAD leads to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affects insulation to the neighboring TAD. Overall, these results indicate that multiple parameters contribute to shaping the chromatin architecture of single nuclei at the TAD scale.

Friday, October 21, Disease Models

Milosavljevic, J., Lempicki, C., Lang, K., Heinkele, H., Kampf, L., Leroy, C., Chen, M., Gerstner, L., Spitz, D., Wang, M., Knob, A., Kayser, S., Helmstädter, M., Walz, G., Pollak, M. and Hermle, T. (2022). Nephrotic Syndrome Gene TBC1D8B is Required for Endosomal Maturation and Nephrin Endocytosis in Drosophila. J Am Soc Nephrol. PubMed ID: 36137753
Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear. A stable deletion was generated in TBC1D8B using microhomology-mediated end joining genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulated endocytic regulators in transgenic mice provided a comprehensive functional analysis of TBC1D8B. A null allele of Drosophila TBC1D8B exhibited nephrocyte-restricted nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from TBC1D8B bearing the edited deletion predominantly localized to mature early endosomes and late endosomes and was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of TBC1D8B Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, four novel TBC1D8B variants were discovered within a cohort of 363 FSGS patients, and functional impact was validated of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. It is concluded that variants in TBC1D8B are not infrequent among FSGS patients. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking.
Fevga, C., Tesson, C., Mascaro, A. C., ..., Mandemakers, W., Brice, A. and Bonifati, V. (2022). PTPA variants and impaired PP2A activity in early-onset parkinsonism with intellectual disability. Brain. PubMed ID: 36073231
The protein phosphatase 2A complex (PP2A), the major Ser/Thr phosphatase in the brain, is involved in a number of signaling pathways and functions, including the regulation of crucial proteins for neurodegeneration, such as alpha-synuclein, tau, and LRRK2. This study reports the identification of variants in the PTPA/PPP2R4 gene, encoding a major PP2A activator, in two families with early-onset parkinsonism and intellectual disability. Functional studies were performed on the disease-associated variants in cultured cells and knock-down of ptpa in Drosophila melanogaster. A homozygous PTPA variant, c.893T > G (p.Met298Arg), was identified in patients from a South African family with early-onset parkinsonism and intellectual disability. Screening of a large series of additional families yielded a second homozygous variant, c.512C > A (p.Ala171Asp), was identified in a Libyan family with a similar phenotype. Both variants co-segregate with disease in the respective families. The affected subjects display juvenile-onset parkinsonism and intellectual disability. The motor symptoms were responsive to treatment with levodopa and deep brain stimulation of the subthalamic nucleus. In overexpression studies, both the PTPA p.Ala171Asp and p.Met298Arg variants were associated with decreased PTPA RNA stability and decreased PTPA protein levels; the p.Ala171Asp variant additionally displayed decreased PTPA protein stability. Crucially, expression of both variants was associated with decreased PP2A complex levels and impaired PP2A phosphatase activation. PTPA ortholog knock-down in Drosophila neurons induced a significant impairment of locomotion in the climbing test. This defect was age-dependent and fully reversed by L-DOPA treatment. It is conclude that bi-allelic missense PTPA variants associated with impaired activation of the PP2A phosphatase cause autosomal recessive early-onset parkinsonism with intellectual disability. Thee findings might also provide new insights for understanding the role of the PP2A complex in the pathogenesis of more common forms of neurodegeneration.
Huang, Y., Lemire, G., Briere, L. C., Liu, F., Wessels, M. W., Wang, X., Osmond, M., Kanca, O., Lu, S., High, F. A., Walker, M. A., Rodan, L. H., Kernohan, K. D., Sweetser, D. A., Boycott, K. M. and Bellen, H. J. (2022). The recurrent de novo c.2011C>T missense variant in MTSS2 causes syndromic intellectual disability. Am J Hum Genet. PubMed ID: 36067766
MTSS2, also known as MTSS1L, binds to plasma membranes and modulates their bending. MTSS2 is highly expressed in the central nervous system (CNS) and appears to be involved in activity-dependent synaptic plasticity. Variants in MTSS2 have not yet been associated with a human phenotype in OMIM. This study reports five individuals with the same heterozygous de novo variant in MTSS2 (GenBank: NM_138383.2: c.2011C>T [p.Arg671Trp]) identified by exome sequencing. The individuals present with global developmental delay, mild intellectual disability, ophthalmological anomalies, microcephaly or relative microcephaly, and shared mild facial dysmorphisms. Immunoblots of fibroblasts from two affected individuals revealed that the variant does not significantly alter MTSS2 levels. The variant was modeled in Drosophila; the fly ortholog missing-in-metastasis (mim) was widely expressed in most neurons and a subset of glia of the CNS. Loss of mim led to a reduction in lifespan, impaired locomotor behavior, and reduced synaptic transmission in adult flies. Expression of the human MTSS2 reference cDNA rescued the mim loss-of-function (LoF) phenotypes, whereas the c.2011C>T variant had decreased rescue ability compared to the reference, suggesting it is a partial LoF allele. However, elevated expression of the variant, but not the reference MTSS2 cDNA, led to similar defects as observed by mim LoF, suggesting that the variant is toxic and may act as a dominant-negative allele when expressed in flies. In summary, these findings support that mim is important for appropriate neural function, and that the MTSS2 c.2011C>T variant causes a syndromic form of intellectual disability.
Mattison, K. A., Tossing, G., Mulroe, F., Simmons, C., Butler, K. M., Schreiber, A., Alsadah, A., Neilson, D. E., Naess, K., Wedell, A., Wredenberg, A., Sorlin, A., McCann, E., Burghel, G. J., Menendez, B., Hoganson, G. E., Botto, L. D., Filloux, F. M., Aledo-Serrano, Á., Gil-Nagel, A., Tatton-Brown, K., Verbeek, N. E., van Hirtum-Das, M., Breckpot, J., Hammer, T. B., Møller, R. S., Whitney, A., Douglas, A. G. L., Kharbanda, M., Brunetti-Pierri, N., Morleo, M., Nigro, V., May, H. J., Tao, J. X., Argili, E., Sherr, E. H., Dobyns, W. B., Consortium, G. E. R., Baines, R. A., Warwicker, J., Parker, J. A., Banka, S., Campeau, P. M. and Escayg, A. (2022). ATP6V0C variants impair vacuolar V-ATPase causing a neurodevelopmental disorder often associated with epilepsy. Brain. PubMed ID: 36074901
The vacuolar H+-ATPase (V-ATPase) is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. This study describes heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the V-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modeling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased V-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behavior, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction, and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder, and provides insight into disease mechanisms.
Mulroe, F., Lin, W. H., Mackenzie-Gray Scott, C., Aourz, N., Fan, Y. N., Coutts, G., Parrish, R. R., Smolders, I., Trevelyan, A., Wykes, R., Allan, S., Freeman, S. and Baines, R. A. (2022). Targeting firing rate neuronal homeostasis can prevent seizures. Dis Model Mech. PubMed ID: 36073607
Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. This study used a combination of Drosophila and mouse and, in the latter, both a pentylenetetrazole (PTZ) induced seizure model, and an electrically induced seizure model for refractory seizures to evaluate anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode-of-action includes increased expression of the firing rate homeostatic regulator PUMILIO (PUM). Knock-down of PUM expression, in Drosophila, blocks anticonvulsive effects of 4-TBB, whilst analysis of validated PUM targets in mouse brain show significant reductions following exposure to this compound. A structure-activity study identifies the active parts of the molecule and, further, shows the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-, and electrically-induced, seizures. This study provides a proof-of-principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development.
Guleria, V. S., Parit, R., Quadri, N., Das, R. and Upadhyai, P. (2022). The intraflagellar transport protein IFT52 associated with short-rib thoracic dysplasia is essential for ciliary function in osteogenic differentiation in vitro and for sensory perception in Drosophila. Exp Cell Res 418(2): 113273. PubMed ID: 35839863
Primary cilia are non-motile sensory cell-organelle that are essential for organismal development, differentiation, and postnatal homeostasis. Their biogenesis and function are mediated by the intraflagellar transport (IFT) system. Pathogenic variants in IFT52, a central component of the IFT-B complex is associated with short-rib thoracic dysplasia with or without polydactyly 16 (SRTD16), with major skeletal manifestations, in addition to other features, this study sought to examine the role of IFT52 in osteoblast differentiation. Using lentiviral shRNA interference Ift52 was depleted in C3H10T1/2 mouse mesenchymal stem cells. This led to the disruption of the IFT-B anterograde trafficking machinery that impaired primary ciliogenesis and blocked osteogenic differentiation. In Ift52 silenced cells, Hedgehog (Hh) pathway upregulation during osteogenesis was attenuated and despite Smoothened Agonist (SAG) based Hh activation, osteogenic differentiation was incompletely restored. Further IFT52 activity was investigated in Drosophila, wherein the only ciliated somatic cells are the bipolar sensory neurons of the peripheral nervous system. Knockdown of IFT52 in Drosophila neuronal tissues reduced lifespan with the loss of embryonic chordotonal cilia, and produced severe locomotion, auditory and proprioceptive defects in larva and adults. Together these findings improve knowledge of the role of IFT52 in various physiological contexts and its associated human disorder.

Thursday, October 20th - Synapse and Vesicles

Liu, C. H., Chen, M. Y., Cheng, J., Chuang, T. N., Liu, H. P. and Lin, W. Y. (2022). Imidacloprid Impairs Glutamatergic Synaptic Plasticity and Desensitizes Mechanosensitive, Nociceptive, and Photogenic Response of Drosophila melanogaster by Mediating Oxidative Stress, Which Could Be Rescued by Osthole. Int J Mol Sci 23(17). PubMed ID: 36077576
Imidacloprid (IMD) is a widely used neonicotinoid-targeting insect nicotine acetylcholine receptors (nAChRs). However, off-target effects raise environmental concerns, including the IMD's impairment of the memory of honeybees and rodents. Although the down-regulation of inotropic glutamate receptor (iGluR) was proposed as the cause, whether IMD directly manipulates the activation or inhibition of iGluR is unknown. Using electrophysiological recording on fruit fly neuromuscular junction (NMJ), this study found that IMD of 0.125 and 12.5 mg/L did not activate glutamate receptors nor inhibit the glutamate-triggered depolarization of the glutamatergic synapse. However, chronic IMD treatment attenuated short-term facilitation (STF) of NMJ by more than 20%. Moreover, by behavioral assays, it was found that IMD desensitized the fruit flies' response to mechanosensitive, nociceptive, and photogenic stimuli. Finally, the treatment of the antioxidant osthole rescued the chronic IMD-induced phenotypes. It was clarified that IMD is neither agonist nor antagonist of glutamate receptors, but chronic treatment with environmental-relevant concentrations impairs glutamatergic plasticity of the NMJ of fruit flies and interferes with the sensory response by mediating oxidative stress.
Chen, S., Venkatesan, A., Lin, Y. Q., Xie, J., Neely, G., Banerjee, S. and Bhat, M. A. (2022). Drosophila Homolog of the Human Carpenter Syndrome Linked Gene, MEGF8, Is Required for Synapse Development and Function. J Neurosci 42(37): 7016-7030. PubMed ID: 35944997
Drosophila multiple epidermal growth factor-like domains 8 (dMegf8) is a homolog of human MEGF8. MEGF8 encodes a multidomain transmembrane protein which is highly conserved across species. In humans, MEGF8 mutations cause a rare genetic disorder called Carpenter syndrome, which is frequently associated with abnormal left-right patterning, cardiac defects, and learning disabilities. MEGF8 is also associated with psychiatric disorders. The presence of intellectual disabilities in Carpenter syndrome patients and association of MEGF8 with psychiatric disorders indicate that mutations in MEGF8 cause underlying defects in synaptic structure and functions. This study investigated the role of Drosophila dMegf8 in the larval neuromuscular junctions (NMJ). dMegf8 localizes to NMJ synapses and is required for proper synaptic growth. dMegf8 mutant larvae and adults show severe motor coordination deficits. At the NMJ, dMegf8 mutants show altered localization of presynaptic and postsynaptic proteins, defects in synaptic ultrastructure, and neurotransmission. Interestingly, dMegf8 mutants have reduced levels of the Type II BMP receptor Wishful thinking (Wit). dMegf8 displays genetic interactions with neurexin-1 (dnrx) and wit, and in association with Dnrx and Wit plays an essential role in synapse organization. These studies provide insights into human MEGF8 functions and potentially into mechanisms that may underlie intellectual disabilities observed in Carpenter syndrome as well as MEGF8-related synaptic structural and/or functional deficits in psychiatric disorders.
Kim, Y. J. (2022). Activity-induced synaptic structural modifications by Akt. Biochem Biophys Res Commun 621: 94-100. PubMed ID: 35820284
The activity-dependent regulation of synaptic structures plays a key role in synaptic development and plasticity; however, the signaling mechanisms involved remain largely unknown. The serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K), plays a pivotal role in a wide range of physiological functions. This study focused on the importance of Akt in rapid synaptic structural changes after stimulation at the Drosophila neuromuscular junction, a well-studied model synapse. Compared with wild-type larvae, akt mutants showed significantly reduced muscle size and an increased number of boutons per area, suggesting that Akt is required for proper pre- and postsynaptic growth. In addition, the level of cysteine string protein (CSP) was significantly increased, and its distribution was different in akt mutants. After high K(+) single stimulation, the CSP level of akt mutant NMJs increased dramatically compared with that of wild-type NMJs. Interestingly, ghost boutons without postsynaptic specialization were found in akt mutant NMJs, and the number of these boutons was significantly increased by patterned stimulation. In contrast, the postsynaptic change in the subsynaptic reticulum (SSR) in the akt mutant occurred independent of stimulation. These results suggest that Akt functions in both pre- and postsynaptic growth and differentiation, and in particular, presynaptic action occurs in an activity-dependent manner.
Krzystek, T. J., White, J. A., Rathnayake, R., Thurston, L., Hoffmar-Glennon, H., Li, Y. and Gunawardena, S. (2022). HTT (huntingtin) and RAB7 co-migrate retrogradely on a signaling LAMP1-containing late endosome during axonal injury. Autophagy: 1-22. PubMed ID: 36048753
HTT (huntingtin) is a 350-kDa protein of unknown function. While HTT moves bidirectionally within axons and HTT loss/reduction causes axonal transport defects, the identity of cargo-containing vesicles that HTT helps move remain elusive. Previous work found an axonal retrogradely moving HTT-Rab7 vesicle complex; however, its biological relevance is unclear. Using Drosophila genetics, in vivo microscopy, membrane isolation and pharmacological inhibition, this study identified that adaptors Hip1 and Rilpl aid the retrograde motility of LAMP1-containing HTT-Rab7 late endosomes, not autophagosomes. Reduction of Syx17 and chloroquine- or bafilomycin A1-mediated pharmacological inhibition, but not reduction of Atg5, disrupted the in vivo motility of these vesicles. Further, because HTT-Rab7 vesicles colocalized with long-distance signaling components (BMP signaling: tkv-wit, injury: wnd) and move in a retrograde direction after Drosophila nerve crush, it is proposed that these vesicles likely traffic damage signals following axonal injury. Together, these findings support a previously unknown role for HTT in the retrograde movement of a Rab7-LAMP1-containing signaling late endosome.
Schmidt, S. G., Malle, M. G., Nielsen, A. K., Bohr, S. S., Pugh, C. F., Nielsen, J. C., Poulsen, I. H., Rand, K. D., Hatzakis, N. S. and Loland, C. J. (2022). The dopamine transporter antiports potassium to increase the uptake of dopamine. Nat Commun 13(1): 2446. PubMed ID: 35508541
The dopamine transporter facilitates dopamine reuptake from the extracellular space to terminate neurotransmission. The transporter belongs to the neurotransmitter:sodium symporter family, which includes transporters for serotonin, norepinephrine, and GABA that utilize the Na(+) gradient to drive the uptake of substrate. Decades ago, it was shown that the serotonin transporter also antiports K(+), but investigations of K(+)-coupled transport in other neurotransmitter:sodium symporters have been inconclusive. This study shows that ligand binding to the Drosophila- and human dopamine transporters are inhibited by K(+), and the conformational dynamics of the Drosophila dopamine transporter in K(+) are divergent from the apo- and Na(+)-states. Furthermore, it was found that K(+) increases dopamine uptake by the Drosophila dopamine transporter in liposomes, and visualize Na(+) and K(+) fluxes in single proteoliposomes using fluorescent ion indicators. These results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K(+) on other transporters in this pharmacologically important family.
Tan, W., Zhang, Q., Quinones-Frías, M. C., Hsu, A. Y., Zhang, Y., Rodal, A., Hong, P., Luo, H. R. and Xu, B. (2022). Enzyme-Responsive Peptide Thioesters for Targeting Golgi Apparatus. J Am Chem Soc 144(15): 6709-6713. PubMed ID: 35404599
The Golgi apparatus (GA) is the hub of intracellular trafficking, but selectively targeting GA remains a challenge. This study shows an unconventional types of peptide thioesters, consisting of an aminoethyl thioester and acting as substrates of thioesterases, for instantly targeting the GA of cells. The peptide thioesters, above or below their critical micelle concentrations, enter cells mainly via caveolin-mediated endocytosis or macropinocytosis, respectively. After being hydrolyzed by GA-associated thioesterases, the resulting thiopeptides form dimers and accumulate in the GA. After saturating the GA, the thiopeptides are enriched in the endoplasmic reticulum (ER). Their buildup in ER and GA disrupts protein trafficking, thus leading to cell death via multiple pathways. The peptide thioesters target the GA of a wide variety of cells, including human, murine, and Drosophila cells. Changing d-diphenylalanine to l-diphenylalanine in the peptide maintains the GA-targeting ability. In addition, targeting GA redirects protein (e.g., NRAS) distribution. This work illustrates a thioesterase-responsive and redox-active molecular platform for targeting the GA and controlling cell fates.

Wednesday October 19th - Behavior

Gonzalez-Suarez, A. D., Zavatone-Veth, J. A., Chen, J., Matulis, C. A., Badwan, B. A. and Clark, D. A. (2022). Excitatory and inhibitory neural dynamics jointly tune motion detection. Curr Biol 32(17): 3659-3675. PubMed ID: 35868321
Neurons integrate excitatory and inhibitory signals to produce their outputs, but the role of input timing in this integration remains poorly understood. Motion detection is a paradigmatic example of this integration, since theories of motion detection rely on different delays in visual signals. These delays allow circuits to compare scenes at different times to calculate the direction and speed of motion. Different motion detection circuits have different velocity sensitivity, but it remains untested how the response dynamics of individual cell types drive this tuning. This study sped up or slowed down specific neuron types in Drosophila's motion detection circuit by manipulating ion channel expression. Altering the dynamics of individual neuron types upstream of motion detectors increased their sensitivity to fast or slow visual motion, exposing distinct roles for excitatory and inhibitory dynamics in tuning directional signals, including a role for the amacrine cell CT1. A circuit model constrained by functional data and anatomy qualitatively reproduced the observed tuning changes. Overall, these results reveal how excitatory and inhibitory dynamics together tune a canonical circuit computation.
Martinez-Cervantes, J., Shah, P., Phan, A. and Cervantes-Sandoval, I. (2022). High order unimodal olfactory sensory preconditioning in Drosophila. Elife 11. PubMed ID: 36129180
Learning and memory storage is a complex process that has proven challenging to tackle. It is likely that, in nature, the instructive value of reinforcing experiences is acquired rather than innate. The association between seemingly neutral stimuli increases the gamut of possibilities to create meaningful associations and the predictive power of moment-by-moment experiences. This study reports physiological and behavioral evidence of olfactory unimodal sensory preconditioning in fruit flies. The presentation of a pair of odors (S1 and S2) before one of them (S1) is associated with electric shocks elicits a conditional response not only to the trained odor (S1) but to the odor previously paired with it (S2). This occurs even if the S2 odor was never presented in contiguity with the aversive stimulus. In addition, this study showa that inhibition of the small G protein Rac1, a known forgetting regulator, facilitates the association between S1/S2 odors. These results indicate that flies can infer value to olfactory stimuli based on the previous associative structure between odors, and that inhibition of Rac1 lengthens the time window of the olfactory 'sensory buffer', allowing the establishment of associations between odors presented in sequence.
Gong, J., Chen, J., Gu, P., Shang, Y., Ruppell, K. T., Yang, Y., Wang, F., Wen, Q. and Xiang, Y. (2022). Shear stress activates nociceptors to drive Drosophila mechanical nociception. Neuron. PubMed ID: 36087585
Mechanical nociception is essential for animal survival. However, the forces involved in nociceptor activation and the underlying mechanotransduction mechanisms remain elusive. This study addressed these problems by investigating nocifensive behavior in Drosophila larvae. Strong poking was shown to stimulate nociceptors with a mixture of forces including shear stress and stretch. Unexpectedly, nociceptors are selectively activated by shear stress, but not stretch. Both the shear stress responses of nociceptors and nocifensive behavior require transient receptor potential A1 (TrpA1), which is specifically expressed in nociceptors. It was further demonstrated that expression of mammalian or Drosophila TrpA1 in heterologous cells confers responses to shear stress but not stretch. Finally, shear stress activates TrpA1 in a membrane-delimited manner, through modulation of membrane fluidity. Together, this study reveals TrpA1 as an evolutionarily conserved mechanosensitive channel specifically activated by shear stress and suggests a critical role of shear stress in activating nociceptors to drive mechanical nociception.
Hobin, M., Dorfman, K., Adel, M., Rivera-Rodriguez, E. J., Kuklin, E. A., Ma, D. and Griffith, L. C. (2022). The Drosophila microRNA bantam regulates excitability in adult mushroom body output neurons to promote early night sleep. iScience 25(9): 104874. PubMed ID: 36034229
Sleep circuitry evolved to have both dedicated and context-dependent modulatory elements. Identifying modulatory subcircuits and understanding their molecular machinery is a major challenge for the sleep field. Previously, Twenty-five sleep-regulating microRNAs were identified in Drosophila melanogaster, including the developmentally important microRNA bantam. This study showed that bantam acts in the adult to promote early nighttime sleep through a population of glutamatergic neurons that is intimately involved in applying contextual information to behaviors, the γ5β'2a/β'2mp/β'2mp_bilateral Mushroom Body Output Neurons (MBONs). Calcium imaging revealed that bantam inhibits the activity of these cells during the early night, but not the day. Blocking synaptic transmission in these MBONs rescued the effect of bantam knockdown. This suggests bantam promotes early night sleep via inhibition of the γ5β'2a/β'2mp/β'2mp_bilateral MBONs. RNAseq identifies Kelch and CCHamide-2 receptor as possible mediators, establishing a new role for bantam as an active regulator of sleep and neural activity in the adult fly.
Inami, S. and Sakai, T. (2022). Circadian photoreceptors are required for light-dependent maintenance of long-term memory in Drosophila. Neurosci Res. PubMed ID: 36096270
In the fruit fly Drosophila melanogaster, environmental light is required for maintaining long-term memory (LTM). Furthermore, the Pigment dispersing factor (Pdf), which is a circadian neuropeptide, and the neuronal activity of Pdf neurons are essential for light-dependent maintenance of courtship LTM. Since Pdf neurons can sense light directly via circadian photoreceptors [Rhodopsin 7 (Rh7) and Cryptochrome (Cry)], it is possible that Rh7 and Cry in Pdf neurons are involved in the maintenance of LTM. In this study, using a courtship conditioning assay, it was demonstrated that circadian photoreceptors in Pdf neurons are required for maintaining courtship LTM.
Han, C., Peng, Q., Sun, M., Jiang, X., Su, X., Chen, J., Ma, M., Zhu, H., Ji, X. and Pan, Y. (2022). The doublesex gene regulates dimorphic sexual and aggressive behaviors in Drosophila. Proc Natl Acad Sci U S A 119(37): e2201513119. PubMed ID: 36067320
Most animal species display dimorphic sexual behaviors and male-biased aggressiveness. This study shows that the doublesex (dsx) gene, which expresses male-specific Dsx(M) and female-specific Dsx(F) transcription factors, functions in the nervous system to control both male and female sexual and aggressive behaviors. This study found that Dsx is not only required in central brain neurons for male and female sexual behaviors, but also functions in approximately eight pairs of male-specific neurons to promote male aggressiveness and approximately two pairs of female-specific neurons to inhibit female aggressiveness. Dsx(F) knockdown females fight more frequently, even with males. These findings reveal crucial roles of dsx, which is broadly conserved from worms to humans, in a small number of neurons in both sexes to establish dimorphic sexual and aggressive behaviors.

Tuesday October 18th - Adult Neural Development and function

Kandasamy, R., Costea, P. I., Stam, L. and Nesterov, A. (2022). TRPV channel nanchung and TRPA channel water witch form insecticide-activated complexes. Insect Biochem Mol Biol 149: 103835. PubMed ID: 36087889
It has been shown that insect vanilloid-type transient receptor potential (TRPV) channels Nanchung (Nan) and Inactive (Iav) form complexes, which can be over-stimulated and eventually silenced by commercial insecticides, afidopyropen, pymetrozine and pyrifluquinazon. Silencing of the TRPV channels by the insecticides perturbs function of the mechano-sensory organs, chordotonal organs, disrupting sound perception, gravitaxis, and feeding. In addition to TRPV channels, chordotonal organs express an ankyrin-type transient receptor potential (TRPA) channel, Water witch (Wtrw). Genetic data implicate Wtrw in sound and humidity sensing, although the signaling pathway, which links Wtrw to these functions has not been clearly defined. This study shows that, in heterologous system, Nan and Wtrw form calcium channels, which can be activated by afidopyropen, pymetrozine and an endogenous agonist, nicotinamide. Analogous to Nan-Iav heteromers, Nan forms the main binding interface for afidopyropen, whereas co-expression of Wtrw dramatically increases its binding affinity. Pymetrozine competes with afidopyropen for binding to Nan-Wtrw complexes, suggesting that these compounds have overlapping binding sites. Analysis of Drosophila single-nucleus transcriptomic atlas revealed co-expression of nan and wtrw in audio- and mechanosensory neurons. The observation that Nan can form insecticide-sensitive heteromers with more than one type of TRP channels, raises a possibility that Nan may partner with some other TRP channel(s). In addition, it was show that Wtrw can be activated by plant-derived reactive electrophiles, allyl isothiocyanate and cinnamaldehyde, defining new molecular target for these repellents.
Lankinen, P., Kastally, C. and Hoikkala, A. (2022). Plasticity in Photoperiodism: Drosophila montana Females Have a Life-Long Ability to Switch From Reproduction to Diapause. J Biol Rhythms 37(5): 516-527. PubMed ID: 35924307
Photoperiodic reproductive diapause is an essential part of female life cycle in several insect species living on high latitudes, where overwintering in reproductive stage involves high risks for survival and progeny production. The sensitive period (SP), during which photoperiodic cues can trigger the switch from direct development to diapause, can last from a few hours or days after emergence to the entire life span of females. Moreover, in some species, sexually mature females can enter post-reproductive diapause as a response to decreasing day length and/or temperature. The duration of SP for diapause induction and the females' ability to enter post-reproductive diapause at short day lengths in Drosophila montana strains was assessed from different latitudes in Europe, North America, and Japan. This study shows that the females of this species have a life-long SP and that they retain an ability to switch between reproduction and diapause as a response to back-and-forth changes in day length for at least 3 months. D. montana strains from different latitudes showed high variation in females' ability to enter post-reproductive diapause; females of the southern strains generally requiring longer time and/or lower temperature to enter this stage than those of the northern strains. Moreover, the proportion of females that switched to post-reproductive diapause in 3 weeks in short day conditions at 16 °C showed positive correlation with the critical day length (CDL) for diapause induction and the latitudinal and continental origin of the strains. Life-long SP increases females' flexibility to respond to short-term changes in environmental conditions and enables reproducing females to switch to post-reproductive diapause when the days get shorter and colder toward the autumn. This ability can play a major role in species phenology and should be taken into account in theoretical and empirical studies on insect adaptation to seasonal variation.
Hewitt, V. L., Miller-Fleming, L., Twyning, M. J., Andreazza, S., Mattedi, F., Prudent, J., Polleux, F., Vagnoni, A. and Whitworth, A. J. (2022). Decreasing pdzd8-mediated mito-ER contacts improves organismal fitness and mitigates Aβ(42) toxicity. Life Sci Alliance 5(11). PubMed ID: 35831024
Mitochondria<>/a-ER contact sites (MERCs) orchestrate many important cellular functions including regulating mitochondrial quality control through mitophagy and mediating mitochondrial calcium uptake. This study identified and functionally characterize the Drosophila ortholog of the recently identified mammalian MERC protein, Pdzd8. Reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion, and reduces lifespan. Although depletion of pdzd8 prolongs the survival of flies fed with mitochondrial toxins, it is also sufficient to rescue locomotor defects of a fly model of Alzheimer's disease expressing Amyloid β(42) (Aβ(42)). Together, these results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.
Linh, D. M., Anh, H. M., Hanh Dan, V. T., Masamitsu, Y. and Thao, D. T. P. (2022). Crucial roles of UCH-L1 on insulin-producing cells and carbohydrate metabolism in Drosophila melanogaster model. Exp Cell Res 419(2): 113321. PubMed ID: 35985499
Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a highly expressed protein in β cells and has been implicated in β cells viability and function, however, the role of UCH-L1 in β cells remains unclear. This study examined the functions of UCH-L1 in β cells by utilizing the Drosophila melanogaster model. The results showed that specific knockdown of dUCH (D.melanogaster homolog of UCH-L1) in Drosophila Insulin-producing cells (D.melanogaster homolog of β cells) induced mitochondria fusion, IPCs death/degeneration, interfered with DILP2 secretion, and triggered the rise of glycogen storage and body weight. Strikingly, the impairment in IPCs cellular activities can be rescued by vitamin C- a strong antioxidant compound, which suggested the relationship between knockdown dUCH and oxidative stress in IPCs; and the potential of this model in screening compounds for β cells function moderation. Since carbohydrate metabolism is an important function of beta cells, this study continued to examine the ability to regulate carbohydrate metabolism of knockdown dUCH flies. The results showed that knockdown dUCH caused the decline of IPCs number under a high-sucrose diet, which finally led to metabolic and physiological disturbances, including total lipid rise, glycogen storage reduction, circulating carbohydrate increase, and weight loss. These symptoms could be early indications of metabolic disorders, particularly β cell dysfunction-related diseases. Taken together, these results indicate that dUCH is essential in the viability and functions of IPCs through the regulation of carbohydrate metabolism in the Drosophila model.
Henry, L. P. and Ayroles, J. F. (2022). Drosophila melanogaster microbiome is shaped by strict filtering and neutrality along a latitudinal cline. Mol Ecol. PubMed ID: 36094780
Microbiomes affect many aspects of host biology, but the eco-evolutionary forces that shape their diversity in natural populations remain poorly understood. Geographical gradients, such as latitudinal clines, generate predictable patterns in biodiversity at macroecological scales, but whether these macroscale processes apply to host-microbiome interactions is an open question. To address this question, this study sampled the microbiomes of 13 natural populations of Drosophila melanogaster along a latitudinal cline in the eastern United States. The microbiomes were surprisingly consistent across the cline, as latitude did not predict either alpha or beta diversity. Only a narrow taxonomic range of bacteria were present in all microbiomes, indicating that strict taxonomic filtering by the host and neutral ecological dynamics are the primary factors shaping the fly microbiome. These findings reveal the complexity of eco-evolutionary interactions shaping microbial variation in D. melanogaster and highlight the need for additional sampling of the microbiomes in natural populations along environmental gradients.
Ghosh, A. C., Hu, Y., Tattikota, S. G., Liu, Y., Comjean, A. and Perrimon, N. (2022). Modeling exercise using optogenetically contractible Drosophila larvae. BMC Genomics 23(1): 623. PubMed ID: 36042416
The pathophysiological effects of a number of metabolic and age-related disorders can be prevented to some extent by exercise and increased physical activity. However, the molecular mechanisms that contribute to the beneficial effects of muscle activity remain poorly explored. Availability of a fast, inexpensive, and genetically tractable model system for muscle activity and exercise will allow the rapid identification and characterization of molecular mechanisms that mediate the beneficial effects of exercise. This study reports the development and characterization of an optogenetically-inducible muscle contraction (OMC) model in Drosophila larvae that was used to study acute exercise-like physiological responses. To characterize muscle-specific transcriptional responses to acute exercise, bulk mRNA-sequencing was performed, revealing striking similarities between acute exercise-induced genes in flies and those previously identified in humans. This larval muscle contraction model opens a path for rapid identification and characterization of exercise-induced factors.

Friday, October 14th - Adult Physiology and Metabolism

Lepeta, K., Roubinet, C., Bauer, M., Vigano, M. A., Aguilar, G., Kanca, O., Ochoa-Espinosa, A., Bieli, D., Cabernard, C., Caussinus, E. and Affolter, M. (2022). Engineered kinases as a tool for phosphorylation of selected targets in vivo. J Cell Biol 221(10). PubMed ID: 36102907
Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, this study has created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. The detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. It is presumed that after careful optimization, the novel approach described in this study can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.
Jozwick, L. M. and Bidwai, A. P. (2022). Protein kinase CK2 phosphorylates a conserved motif in the Notch effector E(spl)-Mgamma.. Mol Cell Biochem. PubMed ID: 36087252
Across metazoan animals, the effects of Notch signaling are mediated via the Enhancer of Split (E(spl)/HES) basic Helix-Loop-Helix-Orange (bHLH-O) repressors. Although these repressors are generally conserved, their sequence diversity is, in large part, restricted to the C-terminal domain (CtD), which separates the Orange (O) domain from the penultimate WRPW tetrapeptide motif that binds the obligate co-repressor Groucho. While the kinases CK2 and MAPK target the CtD and regulate Drosophila E(spl)-M8 and mammalian HES6, the generality of this regulation to other E(spl)/HES repressors has remained unknown. To determine the broader impact of phosphorylation on this large family of repressors,bioinformatics, evolutionary, and biochemical analyses were conducted. These studies identify E(spl)-Mγ as a new target of native CK2 purified from Drosophila embryos, reveal that phosphorylation is specific to CK2 and independent of the regulatory CK2-β\β subunit, and identify that the site of phosphorylation is juxtaposed to the WRPW motif, a feature unique to and conserved in the Mγ homologues over 50 × 10(6) years of Drosophila evolution. Thus, a preponderance of E(spl) homologues (four out of seven total) in Drosophila are targets for CK2, and the distinct positioning of the CK2 and MAPK sites raises the prospect that phosphorylation underlies functional diversity of bHLH-O proteins.
Xu, D. C., Wang, L., Yamada, K. M. and Baena-Lopez, L. A. (2022). Non-apoptotic activation of Drosophila caspase-2/9 modulates JNK signaling, the tumor microenvironment, and growth of wound-like tumors. Cell Rep 39(3): 110718. PubMed ID: 35443185
Resistance to apoptosis due to caspase deregulation is considered one of the main hallmarks of cancer. However, the discovery of novel non-apoptotic caspase functions has revealed unknown intricacies about the interplay between these enzymes and tumor progression. To investigate this biological problem, this study capitalized on a Drosophila tumor model with human relevance based on the simultaneous overactivation of the EGFR and the JAK/STAT signaling pathways. The data indicate that widespread non-apoptotic activation of initiator caspases limits JNK signaling and facilitates cell fate commitment in these tumors, thus preventing the overgrowth and exacerbation of malignant features of transformed cells. Intriguingly, caspase activity also reduces the presence of macrophage-like cells with tumor-promoting properties in the tumor microenvironment. These findings assign tumor-suppressing activities to caspases independent of apoptosis, while providing molecular details to better understand the contribution of these enzymes to tumor progression.
Koehler, S., Huber, T. B. and Denholm, B. (2022). A protective role for Drosophila Filamin in nephrocytes via Yorkie mediated hypertrophy. Life Sci Alliance 5(12). PubMed ID: 35922155
Podocytes are specialized epithelial cells of the kidney glomerulus and are an essential part of the filtration barrier. Because of their position, they are exposed to constant biomechanical forces such as shear stress and hydrostatic pressure. These forces increase during disease, resulting in podocyte injury. It is likely podocytes have adaptative responses to help buffer against deleterious mechanical force and thus reduce injury. However, these responses remain largely unknown. In this study, using the Drosophila model, this study show the mechanosensor Cheerio (dFilamin) provides a key protective role in nephrocytes. Expression of an activated mechanosensitive variant of Cheerio rescued filtration function and induced compensatory and hypertrophic growth in nephrocytes depleted of the nephrocyte diaphragm proteins Sns or Duf. Delineating the protective pathway downstream of Cheerio it wa found that repression of the Hippo pathway induces nephrocyte hypertrophy, whereas Hippo activation reversed the Cheerio-mediated hypertrophy. Furthermore, it was found that Yorkie was activated upon expression of active Cheerio. Taken together, these data suggest that Cheerio acts via the Hippo pathway to induce hypertrophic growth, as a protective response in abnormal nephrocytes.
Lu, J., Wang, Y., Wang, X., Wang, D., Pflugfelder, G. O. and Shen, J. (2022). The Tbx6 Transcription Factor Dorsocross Mediates Dpp Signaling to Regulate Drosophila Thorax Closure. Int J Mol Sci 23(9). PubMed ID: 35562934
Movement and fusion of separate cell populations are critical for several developmental processes, such as neural tube closure in vertebrates or embryonic dorsal closure and pupal thorax closure in Drosophila. Fusion failure results in an opening or groove on the body surface. Drosophila pupal thorax closure is an established model to investigate the mechanism of tissue closure. This study reports the identification of T-box transcription factor genes Dorsocross (Doc) as Decapentaplegic (Dpp) targets in the leading edge cells of the notum in the late third instar larval and early pupal stages. Reduction of Doc in the notum region results in a thorax closure defect, similar to that in dpp loss-of-function flies. Nine genes are identified as potential downstream targets of Doc in regulating thorax closure by molecular and genetic screens. These results reveal a novel function of Doc in Drosophila development. The candidate target genes provide new clues for unravelling the mechanism of collective cell movement.
Goncalves Antunes, M., Sanial, M., Contremoulins, V., Carvalho, S., Plessis, A. and Becam, I. (2022). High hedgehog signaling is transduced by a multikinase-dependent switch controlling the apico-basal distribution of the GPCR smoothened. Elife 11. PubMed ID: 36083801
The oncogenic G-protein-coupled receptor (GPCR) Smoothened (SMO) is a key transducer of the Hedgehog (HH) morphogen, which plays an essential role in the patterning of epithelial structures. This study examined how HH controls SMO subcellular localization and activity in a polarized epithelium using the Drosophila wing imaginal disc as a model. Evidence is provided that HH promotes the stabilization of SMO by switching its fate after endocytosis toward recycling. This effect involves the sequential and additive action of protein kinase A, casein kinase I, and the Fused (FU) kinase. Moreover, in the presence of very high levels of HH, the second effect of FU leads to the local enrichment of SMO in the most basal domain of the cell membrane. Together, these results link the morphogenetic effects of HH to the apico-basal distribution of SMO and provide a novel mechanism for the regulation of a GPCR.

Thursday October 13th - Signaling

Wei, K. H., Mai, D., Chatla, K. and Bachtrog, D. (2022). Dynamics and Impacts of Transposable Element Proliferation in the Drosophila nasuta Species Group Radiation. Mol Biol Evol 39(5). PubMed ID: 35485457
Transposable element (TE) mobilization is a constant threat to genome integrity. Eukaryotic organisms have evolved robust defensive mechanisms to suppress their activity, yet TEs can escape suppression and proliferate, creating strong selective pressure for host defense to adapt. This genomic conflict fuels a never-ending arms race that drives the rapid evolution of TEs and recurrent positive selection of genes involved in host defense; the latter has been shown to contribute to postzygotic hybrid incompatibility. However, how TE proliferation impacts genome and regulatory divergence remains poorly understood. This paper reports the highly complete and contiguous genome assemblies of seven closely related Drosophila species that belong to the nasuta species group-a poorly studied group of flies that radiated in the last 2 My. A high-quality de novo TE library was constructed and germline RNA-seq data was gathered, allowing a comprehensive annotation, and TE insertion patterns were compared between the species, and the evolutionary forces controlling their spread were inferred. A strong negative association was found between TE insertion frequency and expression of genes nearby; this likely reflects survivor bias from reduced fitness impact of TEs inserting near lowly expressed, nonessential genes, with limited TE-induced epigenetic silencing. Phylogenetic analyses of insertions of 147 TE families reveal that 53% of them show recent amplification in at least one species. The most highly amplified TE is a nonautonomous DNA element (Drosophila INterspersed Element; DINE) which has gone through multiple bouts of expansions with thousands of full-length copies littered throughout each genome. Across all TEs, this study found that TEs expansions are significantly associated with high expression in the expanded species consistent with suppression escape. Thus, whereas horizontal transfer followed by the invasion of a naive genome has been highlighted to explain the long-term survival of TEs, this analysis suggests that evasion of host suppression of resident TEs is a major strategy to persist over evolutionary times. Altogether, these results shed light on the heterogenous and context-dependent nature in which TEs affect gene regulation and the dynamics of rampant TE proliferation amidst a recently radiated species group.
He, T., Fan, Y., Wang, Y., Liu, M. and Zhu, A. J. (2022). Dissection of the microRNA Network Regulating Hedgehog Signaling in Drosophila. Front Cell Dev Biol 10: 866491. PubMed ID: 35573695
The evolutionarily conserved Hedgehog (Hh) signaling plays a critical role in embryogenesis and adult tissue homeostasis. Aberrant Hh signaling often leads to various forms of developmental anomalies and cancer. Since altered microRNA (miRNA) expression is associated with developmental defects and tumorigenesis, it is not surprising that several miRNAs have been found to regulate Hh signaling. However, these miRNAs are mainly identified through small-scale in vivo screening or in vitro assays. As miRNAs preferentially reduce target gene expression via the 3' untranslated region, this study analyzed the effect of reduced expression of core components of the Hh signaling cascade on downstream signaling activity, and generated a transgenic Drosophila toolbox of in vivo miRNA sensors for core components of Hh signaling, including hh, patched (ptc), smoothened (smo), costal 2 (cos2), fused (fu), Suppressor of fused (Su(fu)), and cubitus interruptus (ci). With these tools in hand, a genome-wide in vivo miRNA overexpression screen was performed in the developing Drosophila wing imaginal disc. Of the twelve miRNAs identified, seven were not previously reported in the in vivo Hh regulatory network. Moreover, these miRNAs may act as general regulators of Hh signaling, as their overexpression disrupts Hh signaling-mediated cyst stem cell maintenance during spermatogenesis. To identify direct targets of these newly discovered miRNAs, the miRNA sensor toolbox was used to show that >miR-10 and miR-958 directly target fu and smo, respectively, while the other five miRNAs act through yet-to-be-identified targets other than the seven core components of Hh signaling described above. Importantly, through loss-of-function analysis, this study found that endogenous miR-10 and miR-958 target fu and smo, respectively, whereas deletion of the other five miRNAs leads to altered expression of Hh signaling components, suggesting that these seven newly discovered miRNAs regulate Hh signaling in vivo. Given the powerful effects of these miRNAs on Hh signaling, it is believed that identifying their bona fide targets of the other five miRNAs will help reveal important new players in the Hh regulatory network.
Pegoraro, M., Fishman, B., Zonato, V., Zouganelis, G., Francis, A., Kyriacou, C. P. and Tauber, E. (2022). Photoperiod-Dependent Expression of MicroRNA in Drosophila. Int J Mol Sci 23(9). PubMed ID: 35563325
Like many other insects in temperate regions, Drosophila melanogaster exploits the photoperiod shortening that occurs during the autumn as an important cue to trigger a seasonal response. Flies survive the winter by entering a state of reproductive arrest (diapause), which drives the relocation of resources from reproduction to survival. This study profiled the expression of microRNA (miRNA) in long and short photoperiods and identified seven differentially expressed miRNAs (dme-mir-2b, dme-mir-11, dme-mir-34, dme-mir-274, dme-mir-184, dme-mir-184*, and dme-mir-285). Misexpression of dme-mir-2b, dme-mir-184, and dme-mir-274 in pigment-dispersing, factor-expressing neurons largely disrupted the normal photoperiodic response, suggesting that these miRNAs play functional roles in photoperiodic timing. This study also analyzed the targets of photoperiodic miRNA by both computational predication and by Argonaute-1-mediated immunoprecipitation of long- and short-day RNA samples. Together with global transcriptome profiling, these results expand existing data on other Drosophila species, identifying genes and pathways that are differentially regulated in different photoperiods and reproductive status. These data suggest that post-transcriptional regulation by miRNA is an important facet of photoperiodic timing.
Francis, D., Burguete, A. S. and Ghabrial, A. (2022). Regulation of Archease by the mTOR-vATPase axis. Development. PubMed ID: 36111596
Larval terminal cells of the Drosophila tracheal system generate extensive branched tubes, requiring a huge increase in apical membrane. Terminal cells were compromised for apical membrane expansion - mTOR-vATPase axis and apical polarity mutants - were invaded by the neighboring stalk cell. The invading cell grows and branches, replacing the original single intercellular junction between stalk and terminal cell with multiple intercellular junctions. This study characterize disjointed, a mutation in the same phenotypic class. disjointed encodes Drosophila Archease, required for RNA ligase (RtcB) function critical for tRNA maturation and ER stress-regulated nonconventional splicing of Xbp1 mRNA. The steady-state subcellular localization of Archease is principally nuclear, and dependent upon TOR-vATPase activity. In tracheal cells mutant for Rheb or vATPase, Archease localization shifted dramatically from nucleus to cytoplasm. Further, this study found that blocking tRNA maturation by knockdown of tRNAse z also induced compensatory branching. Taken together, these data suggest that the TOR-vATPase axis promotes apical membrane growth in part through nuclear localization of Archease, where Archease is required for tRNA maturation.
Kingston, E. R., Blodgett, L. W. and Bartel, D. P. (2022). Endogenous transcripts direct microRNA degradation in Drosophila, and this targeted degradation is required for proper embryonic development. Mol Cell. PubMed ID: 36150386
MicroRNAs (miRNAs) typically direct degradation of their mRNA targets. However, some targets have unusual miRNA-binding sites that direct degradation of cognate miRNAs. Although this target-directed miRNA degradation (TDMD) is thought to shape the levels of numerous miRNAs, relatively few sites that endogenously direct degradation have been identified. This study identified six sites, five in mRNAs and one in a noncoding RNA named Marge, which serve this purpose in Drosophila cells or embryos. These six sites direct miRNA degradation without collateral target degradation, helping explain the effectiveness of this miRNA-degradation pathway. Mutations that disrupt this pathway are lethal, with many flies dying as embryos. Concomitant derepression of miR-3 and its paralog miR-309 appears responsible for some of this lethality, whereas the loss of Marge-directed degradation of miR-310 miRNAs causes defects in embryonic cuticle development. Thus, TDMD is implicated in the viability of an animal and is required for its proper development.

Tuesday, October 11th - Larval and Adult Physiology and Metabolism

Morimoto, J., Wenzel, M., Derous, D., Henry, Y. and Colinet, H. (2022). The transcriptomic signature of responses to larval crowding in Drosophila melanogaster. Insect Sci. PubMed ID: 36115064
Intraspecific competition at the larval stage is an important ecological factor affecting life-history, adaptation and evolutionary trajectory in holometabolous insects. However, the molecular pathways underpinning these ecological processes are poorly characterised. Drosophila melanogaster were reared at three egg densities (5, 60 and 300 eggs/mL), and the transcriptomes were sequenced of pooled third-instar larvae. Emergence time, egg-to-adult viability, adult mass and adult sex-ratio were also measured at each density. Medium crowding had minor detrimental effects on adult phenotypes compared to low density and yielded 24 differentially expressed genes (DEGs) including several chitinase enzymes. In contrast, high crowding had substantial detrimental effects on adult phenotypes and yielded 2107 DEGs. Among these, upregulated gene sets were enriched in sugar, steroid and amino acid metabolism as well as DNA replication pathways, whereas downregulated gene sets were enriched in ABC transporters, Taurine, Toll/Imd signalling and P450 xenobiotics metabolism pathways. Overall, these findings show that larval crowding has a large consistent effect on several molecular pathways (i.e., core responses) with few pathways displaying density-specific regulation (i.e., idiosyncratic responses). This provides important insights into how holometabolous insects respond to intraspecific competition during development.
Chen, J. S., Tsaur, S. C., Ting, C. T. and Fang, S. (2022). Dietary Utilization Drives the Differentiation of Gut Bacterial Communities between Specialist and Generalist Drosophilid Flies. Microbiol Spectr 10(4): e0141822. PubMed ID: 35863034
Gut bacteria play vital roles in the dietary detoxification, digestion, and nutrient supplementation of hosts during dietary specialization. The roles of gut bacteria in the host can be unveiled by comparing communities of specialist and generalist bacterial species. However, these species usually have a long evolutionary history, making it difficult to determine whether bacterial community differentiation is due to host dietary adaptation or phylogenetic divergence. In this regard, this study investigated the bacterial communities from two Araceae-feeding Colocasiomyia species and further performed a meta-analysis by incorporating the published data from Drosophila bacterial community studies. The compositional and functional differentiation of bacterial communities was uncovered by comparing three (Araceae-feeding, mycophagous, and cactophilic) specialists with generalist flies. The compositional differentiation showed that Bacteroidetes and Firmicutes inhabited specialists, while more Proteobacteria lived in generalists. The functional prediction based on the bacterial community compositions suggested that amino acid metabolism and energy metabolism are overrepresented pathways in specialists and generalists, respectively. The differences were mainly associated with the higher utilization of structural complex carbohydrates, protein utilization, vitamin B(12) acquisition, and demand for detoxification in specialists than in generalists. The complementary roles of bacteria reveal a connection between gut bacterial communities and fly dietary specialization.
Strilbytska, O., Strutynska, T., Semaniuk, U., Burdyliyk, N., Bubalo, V. and Lushchak, O. (2022). Dietary Sucrose Determines Stress Resistance, Oxidative Damages, and Antioxidant Defense System in Drosophila. Scientifica (Cairo) 2022: 7262342. PubMed ID: 35547569
Varied nutritional interventions affect lifespan and metabolic health. Abundant experimental evidence indicates that the carbohydrate restriction in the diet induces changes to support long-lived phenotypes. Reactive oxygen species (ROS) are among the main mechanisms that mediate the effect of nutrient consumption on the aging process. This study tested the influence of sucrose concentration in the diet on stress resistance, antioxidant defense systems, and oxidative stress markers in D. melanogaster. High sucrose concentration in the fly medium was found to lead to enhanced resistance to starvation, oxidative, heat, and cold stresses. However, flies that were raised on low sucrose food displayed increased levels of low-molecular-mass thiols, lipid peroxides in females, and higher activity of antioxidant enzymes, indicating that the consumption of a low carbohydrate diet could induce oxidative stress in the fruit fly. The consumption of sucrose-enriched diet increased protein carbonyl level, which may indicate about the activation of glycation processes. The results highlight a strong dependence of oxidative metabolism in D. melanogaster from dietary carbohydrates.
Mitra, A., Vo, L., Soukar, I., Chaubal, A., Greenberg, M. L. and Pile, L. A. (2022). Isoforms of the transcriptional cofactor SIN3 differentially regulate genes necessary for energy metabolism and cell survival. jBiochim Biophys Acta Mol Cell Res 1869(10): 119322. PubMed ID: 35820484
The SIN3 scaffolding protein is a conserved transcriptional regulator known to fine-tune gene expression. In Drosophila, there are two major isoforms of SIN3, SIN3 220 and SIN3 187, which each assemble into multi-subunit histone modifying complexes. The isoforms have distinct developmental expression patterns and non-redundant functions. The SIN3 187 isoform uniquely regulates a subset of pathways including post-embryonic development, phosphate metabolism and apoptosis. Target genes in the phosphate metabolism pathway include nuclear-encoded mitochondrial genes coding for proteins responsible for oxidative phosphorylation. This study investigated the physiological effects of SIN3 isoforms on energy metabolism and cell survival. Ectopic expression of SIN3 187 represses expression of several nuclear-encoded mitochondrial genes affecting production of ATP and generation of reactive oxygen species (ROS). Forced expression of SIN3 187 also activates several pro-apoptotic and represses a few anti-apoptotic genes. In the SIN3 187 expressing cells, these gene expression patterns are accompanied with an increased sensitivity to paraquat-mediated oxidative stress. These findings indicate that SIN3 187 influences the regulation of mitochondrial function, apoptosis and oxidative stress response.
Tian, Q., Wang, P., Xie, C., Pang, P., Zhang, Y., Gao, Y., Cao, Z., Wu, Y., Li, W., Zhu, M. X., Li, D. and Yao, J. (2022). Identification of an arthropod molecular target for plant-derived natural repellents. Proc Natl Acad Sci U S A 119(18): e2118152119. PubMed ID: 35452331
Arthropods maintain ecosystem balance while also contributing to the spread of disease. Plant-derived natural repellents represent an ecological method of pest control, but their direct molecular targets in arthropods remain to be further elucidated. Occupying a critical phylogenetic niche in arthropod evolution, scorpions retain an ancestral genetic profile. In this study, using a behavior-guided screening of the Mesobuthus martensii genome, a scorpion transient receptor potential (sTRP1) channel was identified that senses Cymbopogon-derived natural repellents, while remaining insensitive to the synthetic chemical pesticide DEET. Scrutinizing orthologs of sTRP1 in Drosophila melanogaster, dTRPγ ion channel was further demonstrated as a chemosensory receptor of natural repellents to mediate avoidance behavior. This study sheds light on arthropod molecular targets of natural repellents, exemplifying the arthropod–plant adaptation. It should also help the rational design of insect control strategy and in conserving biodiversity.
Chen, Y., Xu, W., Chen, Y., Han, A., Song, J., Zhou, X. and Song, W. (2022). Renal NF-κB activation impairs uric acid homeostasis to promote tumor-associated mortality independent of wasting. Immunity 55(9): 1594-1608. PubMed ID: 36029766
Tumor-induced host wasting and mortality are general phenomena across species. Many groups have previously demonstrated endocrinal impacts of malignant tumors on host wasting in rodents and Drosophila. Whether and how environmental factors and host immune response contribute to tumor-associated host wasting and survival, however, are largely unknown. This study reports that flies bearing malignant yki3SA-gut tumors exhibited the exponential increase of commensal bacteria, which were mostly acquired from the environment, and systemic IMD-NF-kappaB activation due to suppression of a gut antibacterial amidase PGRP-SC2. Either gut microbial elimination or specific IMD-NF-kappaB blockade in the renal-like Malpighian tubules potently improved mortality of yki3SA-tumor-bearing flies in a manner independent of host wasting. It was further indicate that renal IMD-NF-kappaB activation caused uric acid (UA) overload to reduce survival of tumor-bearing flies. Therefore, these results uncover a fundamental mechanism whereby gut commensal dysbiosis, renal immune activation, and UA imbalance potentiate tumor-associated host death.

Monday, October 10 - Evolution

da Silva Ribeiro, T., Galvan, J. A. and Pool, J. E. (2022). Maximum SNP FST outperforms full-window statistics for detecting soft sweeps in local adaptation. Genome Biol Evol. PubMed ID: 36152314
Local adaptation can lead to elevated genetic differentiation at the targeted genetic variant and nearby sites. Selective sweeps come in different forms, and depending on the initial and final frequencies of a favored variant, very different patterns of genetic variation may be produced. If local selection favors an existing variant that had already recombined onto multiple genetic backgrounds, then the width of elevated genetic differentiation (high FST) may be too narrow to detect using a typical windowed genome scan, even if the targeted variant becomes highly differentiated. Therefore a simulation approach was used to investigate the power of SNP-level FST (specifically, the maximum SNP FST value within a window, or FST_MaxSNP) to detect diverse scenarios of local adaptation, and compared it against whole-window FST and the Comparative Haplotype Identity statistic. It was found that FST_MaxSNP had superior power to detect complete or mostly complete soft sweeps, but lesser power than full-window statistics to detect partial hard sweeps. Nonetheless, the power of FST_MaxSNP depended highly on sample size, and confident outliers depend on robust precautions and quality control. To investigate the relative enrichment of FST_MaxSNP outliers from real data, the two FST statistics were applied to a panel of Drosophila melanogaster populations. FST_MaxSNP had a genome-wide enrichment of outliers compared to demographic expectations, and though it yielded a lesser enrichment than window FST, it detected mostly unique outlier genes and functional categories. These results suggest that FST_MaxSNP is highly complementary to typical window-based approaches for detecting local adaptation, and merits inclusion in future genome scans and methodologies.
Navarro-Dominguez, B., Chang, C. H., Brand, C. L., Muirhead, C. A., Presgraves, D. C. and Larracuente, A. M. (2022). Epistatic selection on a selfish Segregation Distorter supergene - drive, recombination, and genetic load. Elife 11. PubMed ID: 35486424
Meiotic drive supergenes are complexes of alleles at linked loci that together subvert Mendelian segregation resulting in preferential transmission. In males, the most common mechanism of drive involves the disruption of sperm bearing one of a pair of alternative alleles. While at least two loci are important for male drive-the driver and the target-linked modifiers can enhance drive, creating selection pressure to suppress recombination. This work investigates the evolution and genomic consequences of an autosomal, multilocus, male meiotic drive system, Segregation Distorter (SD) in the fruit fly, Drosophila melanogaster. In African populations, the predominant SD chromosome variant, SD-Mal, is characterized by two overlapping, paracentric inversions on chromosome arm 2R and nearly perfect (~100%) transmission. The SD-Mal system in was studied in detail, exploring its components, chromosomal structure, and evolutionary history. The findings reveal a recent chromosome-scale selective sweep mediated by strong epistatic selection for haplotypes carrying Sd, the main driving allele, and one or more factors within the double inversion. While most SD-Mal chromosomes are homozygous lethal, SD-Mal haplotypes can recombine with other, complementing haplotypes via crossing over, and with wildtype chromosomes via gene conversion. SD-Mal chromosomes have nevertheless accumulated lethal mutations, excess non-synonymous mutations, and excess transposable element insertions. Therefore, SD-Mal haplotypes evolve as a small, semi-isolated subpopulation with a history of strong selection. These results may explain the evolutionary turnover of SD haplotypes in different populations around the world and have implications for supergene evolution broadly.
Auer, T. O., Alvarez-Ocana, R., Cruchet, S., Benton, R. and Arguello, J. R. (2022). Copy number changes in co-expressed odorant receptor genes enable selection for sensory differences in drosophilid species. Nat Ecol Evol 6(9): 1343-1353. PubMed ID: 35864227
Despite numerous examples of chemoreceptor gene family expansions and contractions, how these relate to modifications in the sensory neuron populations in which they are expressed remains unclear. Drosophila melanogaster's odorant receptor (Or) family is ideal for addressing this question because most Ors are expressed in distinct olfactory sensory neuron (OSN) types. Between-species changes in Or copy number may therefore indicate increases or reductions in the number of OSN populations. This study investigated the Or67a subfamily, which exhibits copy number variation in D. melanogaster and its closest relatives: D. simulans, D. sechellia and D. mauritiana. These species' common ancestor had three Or67a paralogues that had already diverged adaptively. Following speciation, two Or67a paralogues were lost independently in D. melanogaster and D. sechellia, with ongoing positive selection shaping the intact genes. Unexpectedly, the functionally diverged Or67a paralogues in D. simulans are co-expressed in a single neuron population, which projects to a glomerulus homologous to that innervated by Or67a neurons in D. melanogaster. Thus, while sensory pathway neuroanatomy is conserved, independent selection on co-expressed receptors has contributed to species-specific peripheral coding. This work reveals a type of adaptive change largely overlooked for olfactory evolution, raising the possibility that similar processes influence other cases of insect Or co-expression.
Corbel, Q., Serra, M., Garcia-Roa, R. and Carazo, P. (2022). Male Adaptive Plasticity Can Explain the Evolution of Sexual Perception Costs. Am Nat 200(3): E110-e123. PubMed ID: 35977789
Sensory perception of environmental cues has been shown to trigger plastic responses that can induce important fitness costs, including the dramatic modulation of aging across distant taxa. For example, male Drosophila melanogaster suffer a marked decrease in fitness, characterized by faster reproductive and actuarial aging, if they perceive female cues but fail to mate shortly after (aging via sexual perception). While this has been a breakthrough for in understanding of the mechanisms of aging, it raises the question of why such plastic responses evolved. This study used D. melanogaster to ask whether sexual perception costs may be a by-product of adaptive plastic responses to female cues. It was found that (A) short-term perception (1 day) of female cues before mating opportunities increases male relative lifetime reproductive success in a competitive environment, (B) medium-term perception (3-7 days) is neutral, and (C) long-term perception (15 days) leads to reproductive costs. Mathematical simulations under a wide range of sociosexual and demographic scenarios showed that such plastic male responses can be adaptive whenever mating rates fluctuate within the range experienced by D. melanogaster and other insects in the wild, suggesting that this may be a widespread strategy in nature. Finally, this study showed that, because the short-term benefits of plastic responses will be acquired mostly by high-quality males while long-term costs will be paid mostly by low-quality males, sexual perception can significantly magnify sexual selection (15%-27% average increase in the opportunity for selection).
Singh, A. and Agrawal, A. F. (2022). Sex-Specific Variance in Fitness and the Efficacy of Selection. Am Nat 199(5): 587-602. PubMed ID: 35472021
Variance in fitness is thought to be greater in males than in females in many species. If this is so, there are two potentially contradictory consequences on the efficacy of selection (N(e)s): greater variance in fitness may allow stronger selection (i.e., increased s), but it will also cause stronger genetic drift (i.e., reduced N(e)). A simple model was developed to ask how the stronger condition dependency of fitness in males than in females affects selection and fitness variance in each sex to examine the net effect on the efficacy of selection. The phenotypic variance in fitness was measured for each sex in Drosophila melanogaster in different environmental and mating contexts. Variance in fitness was only approximately one and a half to two times higher in males than in females; juvenile mortality likely dampens the difference in variation between the sexes. Combining these results with previous studies of sex-specific selection on mutations, it is inferred that the increased drift due to males counterbalances the stronger selection on males in this species, leaving N(e)s similar to what would be expected if both sexes were "female-like" with respect to selection and variance in fitness. Reasons why this could differ in other species are discussed.
Deppisch, P., Helfrich-Forster, C. and Senthilan, P. R. (2022). The Gain and Loss of Cryptochrome/Photolyase Family Members during Evolution. Genes (Basel) 13(9). PubMed ID: 36140781
The cryptochrome/photolyase (CRY/PL) family represents an ancient group of proteins fulfilling two fundamental functions. While photolyases repair UV-induced DNA damages, cryptochromes mainly influence the circadian clock. This study took advantage of the large number of already sequenced and annotated genes available in databases and systematically searched for the protein sequences of CRY/PL family members in all taxonomic groups primarily focusing on metazoans and limiting the number of species per taxonomic order to five. Using BLASTP searches and subsequent phylogenetic tree and motif analyses, five distinct photolyases (CPDI, CPDII, CPDIII, 6-4 photolyase, and the plant photolyase PPL) and six cryptochrome subfamilies (DASH-CRY, mammalian-type MCRY, Drosophila-type DCRY, cnidarian-specific ACRY, plant-specific PCRY, and the putative magnetoreceptor CRY4) were identified. Manually assigning the CRY/PL subfamilies to the species studied, it was noted that over evolutionary history, an initial increase of various CRY/PL subfamilies was followed by a decrease and specialization. Thus, in more primitive organisms (e.g., bacteria, archaea, simple eukaryotes, and in basal metazoans), relatively few CRY/PL members are found. As species become more evolved (e.g., cnidarians, mollusks, echinoderms, etc.), the CRY/PL repertoire also increases, whereas it appears to decrease again in more recent organisms (humans, fruit flies, etc.). Moreover, this study indicates that all cryptochromes, although largely active in the circadian clock, arose independently from different photolyases, explaining their different modes of action.

Friday, October 7th - Disease Models

Bhanja, A., Nayak, N., Mukherjee, S., Sutar, P. P. and Mishra, M. (2022). Treating the Onset of Diabetes Using Probiotics Along with Prebiotic from Pachyrhizus erosus in High-Fat Diet Fed Drosophila melanogaster. Probiotics Antimicrob Proteins 14(5): 884-903. PubMed ID: 35710863
The increasing mortality due to hypertension and hypercholesterolemia is directly linked with type-2 diabetes. This shows the lethality of the disease. Reports suggest that the prebiotics along with probiotics help in lowering the effects of type-2 diabetes. Prebiotic like inulin is best known for its anti-diabetic effect. The current study utilizes jicama extract as prebiotic source of inulin along with the bacterial strains with probiotic properties (Lactiplantibacillus plantarum and Enterococcus faecium) for treating type-2 diabetes in high-fat diet-induced Drosophila melanogaster model. The high-fat diet-induced Drosophila showed deposition of lipid droplets and formation of micronuclei in the gut. The larva and adult treated with probiotics and synbiotic (probiotic + prebiotic- inulin) comparatively reduced the lipid deposition and micronuclei number in the gut. The increased amount of triglyceride in the whole body of the fatty larva and adult indicated the onset of diabetes. The overexpression of insulin-like genes (Dilp 2) and (Dilp 5) confirmed the insulin resistance, whereas the expression was reduced in the larva and adult supplemented with probiotics and synbiotic. The reactive oxygen species level was reduced with the supplementation of probiotics. The weight, larva size, crawling speed and climbing were also altered in high-fat diet-induced Drosophila melanogaster. The study confirmed the effects of probiotics and synbiotic in successfully lowering diabetes in Drosophila. The study also proved the anti-diabetic potential of the probiotics. Further, it was also confirmed that the probiotics work better in the presence of prebiotic.
Bartoszewski, S., Dawidziuk, M., Kasica, N., Durak, R., Jurek, M., Podwysocka, A., Guilbride, D. L., Podlasz, P., Winata, C. L. and Gawlinski, P. (2022). A Zebrafish/Drosophila Dual System Model for Investigating Human Microcephaly. Cells 11(17). PubMed ID: 36078134
Microcephaly presents in neurodevelopmental disorders with multiple aetiologies, including bi-allelic mutation in TUBGCP2, a component of the biologically fundamental and conserved microtubule-nucleation complex, γ-TuRC. This study presents a simple vertebrate/invertebrate dual system to investigate fundamental TUBGCP2-related processes driving human microcephaly and associated developmental traits. Antisense morpholino knockdown (KD) of the Danio rerio homolog, tubgcp2, recapitulates human TUBGCP2-associated microcephaly. Co-injection of wild type mRNA pre-empts microcephaly in 55% of KD zebrafish larvae, confirming causality. Body shortening observed in morphants is also rescued. Drosophila melanogaster double knockouts (KO) for TUBGCP2 homologs Grip84/cg7716 also develop microcephalic brains with general microsomia. Exacerbated Grip84/cg7716-linked developmental aberration versus single mutations strongly suggests interactive or coinciding gene functions. It is inferred that tubgcp2 and Grip84/cg7716 affect brain size similarly to TUBGCP2 and recapitulate both microcephaly and microcephaly-associated developmental impact, validating the zebrafish/fly research model for human microcephaly. Given the conserved cross-phyla homolog function, the data also strongly support mitotic and/or proliferative disruption linked to aberrant microtubule nucleation in progenitor brain cells as key mechanistic defects for human microcephaly.
Dubey, S. K., Maulding, K., Sung, H. and Lloyd, T. E. (2022). Nucleoporins are degraded via upregulation of ESCRT-III/Vps4 complex in Drosophila models of C9-ALS/FTD. Cell Rep 40(12): 111379. PubMed ID: 36130523
Disruption of the nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) have been implicated in the pathogenesis of neurodegenerative diseases. A GGGGCC hexanucleotide repeat expansion (HRE) in an intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, but the mechanism by which the HRE disrupts NCT is incompletely understood. This study found that expression of GGGGCC repeats in Drosophila neurons induces proteasome-mediated degradation of select nucleoporins of the NPC. This process requires the Vps4 ATPase and the endosomal-sorting complex required for transport complex-III (ESCRT-III), as knockdown of ESCRT-III/Vps4 genes rescues nucleoporin levels, normalizes NCT, and suppresses GGGGCC-mediated neurodegeneration. GGGGCC expression upregulates nuclear ESCRT-III/Vps4 expression, and expansion microscopy demonstrates that the nucleoporins are translocated into the cytoplasm before undergoing proteasome-mediated degradation. These findings demonstrate a mechanism for nucleoporin degradation and NPC dysfunction in neurodegenerative disease.
Dias, K. R., Carlston, C. M.,..., Harvey, R. J. and Roscioli, T. (2022). De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental disorder with cardiac malformations. Genet Med 24(9): 1952-1966. PubMed ID: 35916866
ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. This study delineated a novel neurocognitive disorder caused by variants in the ZMYND8 gene. An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the Drosophila ZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. This study presents genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.
Caglayan, A. O., Tuysuz, B., Gul, E., Alkaya, D. U., Yalcinkaya, C., Gleeson, J. G., Bilguvar, K. and Gunel, M. (2022). Biallelic BICD2 variant is a novel candidate for Cohen-like syndrome. J Hum Genet 67(9): 553-556. PubMed ID: 35338243
Heterozygous mutations in Bicaudal D2 Drosophila homolog 2 (BICD2) gene (see Drosophila BicD), encodes a vesicle transport protein involved in dynein-mediated movement along microtubules, are responsible for an exceedingly rare autosomal dominant spinal muscular atrophy type 2A which starts in the childhood and predominantly effects lower extremities. Recently, a more severe form, type 2B, has also been described. This study presents a patient born to a consanguineous union and who suffered from intellectual disability, speech delay, epilepsy, happy facial expression, truncal obesity with tappering fingers, and joint hypermobility. Whole-exome sequencing analysis revealed a rare, homozygous missense mutation (c.731T>C; p.Leu244Pro) in BICD2 gene. This finding presents the first report in the literature for homozygous BICD2 mutations and its association with a Cohen-Like syndrome. Patients presenting with Cohen-Like phenotypes should be further interrogated for mutations in BICD2.
Damschroder, D., Zapata-Perez, R., Richardson, K., Vaz, F. M., Houtkooper, R. H. and Wessells, R. (2022). Stimulating the sir2-pgc-1alpha axis rescues exercise capacity and mitochondrial respiration in Drosophila tafazzin mutants. Dis Model Mech. PubMed ID: 36107830
Cardiolipin (CL) is a phospholipid required for proper mitochondrial function. Tafazzin remodels CL to create highly unsaturated fatty acid chains. However, when tafazzin is mutated, CL remodeling is impeded, leading to mitochondrial dysfunction and the disease Barth syndrome. Patients with Barth syndrome often have severe exercise intolerance, which negatively impacts their overall quality of life. Boosting NAD+ levels can improve symptoms of other mitochondrial diseases, but its effect in the context of Barth syndrome has not been examined. This study demonstrates for the first time that nicotinamide riboside (NR) can rescue exercise tolerance and mitochondrial respiration in a Drosophila tafazzin mutant and that the beneficial effects are dependent on sir2 and pgc-1α. Overexpressing pgc-1α increased the total abundance of cardiolipin in mutants. In addition, muscles and neurons were identified as key targets for future therapies because sir2 or pgc-1α overexpression in either of these tissues is sufficient to restore the exercise capacity of Drosophila tafazzin mutants.

Thursday, October 6th - Chromatin DNA and Chromosomes

de Lima, L. G. and Ruiz-Ruano, F. J. (2022). In-Depth Satellitome Analyses of 37 Drosophila Species Illuminate Repetitive DNA Evolution in the Drosophila Genus. Genome Biol Evol 14(5). PubMed ID: 35511582
Satellite DNAs (SatDNA) are ubiquitously present in eukaryotic genomes and have been recently associated with several biological roles. Understanding the evolution and significance of SatDNA requires an extensive comparison across multiple phylogenetic depths. This study combined the RepeatExplorer pipeline and cytogenetic approaches to conduct a comprehensive identification and analysis of the satellitome in 37 species from the genus Drosophila. 188 SatDNA-like families were identified, 112 of them being characterized for the first time. Repeat analysis within a phylogenetic framework has revealed the deeply divergent nature of SatDNA sequences in the Drosophila genus. The SatDNA content varied from 0.54% of the D. arizonae genome to 38.8% of the D. albomicans genome, with the SatDNA content often following a phylogenetic signal. Monomer size and guanine-cytosine-content also showed extreme variation ranging 2-570 bp and 9.1-71.4%, respectively. SatDNA families are shared among closely related species, consistent with the SatDNA library hypothesis. However, the emergence of species-specific SatDNA families was uncovered through amplification of unique or low abundant sequences in a lineage. Finally, genome sizes of the Sophophora subgenus were found to be positively correlated with transposable element content, whereas genome size in the Drosophila subgenus is positively correlated with SatDNA. This finding indicates genome size could be driven by different categories of repetitive elements in each subgenus. Altogether, this study conducted the most comprehensive satellitome analysis in Drosophila from a phylogenetic perspective and generated the largest catalog of SatDNA sequences to date, enabling future discoveries in SatDNA evolution and Drosophila genome architecture.
Bredesen-Aa, B. A. and Rehmsmeier, M. (2022). Gnocis: An integrated system for interactive and reproducible analysis and modelling of cis-regulatory elements in Python 3. PLoS One 17(9): e0274338. PubMed ID: 36084008
Gene expression is regulated through cis-regulatory elements (CREs), among which are promoters, enhancers, Polycomb/Trithorax Response Elements (PREs), silencers and insulators. Computational prediction of CREs can be achieved using a variety of statistical and machine learning methods combined with different feature space formulations. Although Python packages for DNA sequence feature sets and for machine learning are available, no existing package facilitates the combination of DNA sequence feature sets with machine learning methods for the genome-wide prediction of candidate CREs. This paper presents Gnocis, a Python package that streamlines the analysis and the modelling of CRE sequences by providing extensible Application Programming Interfaces (APIs) and implementing the glue required for combining feature sets and models for genome-wide prediction. Gnocis implements a variety of base feature sets, including motif pair occurrence frequencies and the k-spectrum mismatch kernel. It integrates with Scikit-learn and TensorFlow for state-of-the-art machine learning. Gnocis additionally implements a broad suite of tools for the handling and preparation of sequence, region and curve data, which can be useful for general DNA bioinformatics in Python. Deep-MOCCA, a neural network architecture inspired by SVM-MOCCA that achieves moderate to high generalization without prior motif knowledge, is presented. To demonstrate the use of Gnocis, multiple machine learning methods were applied to the modelling of D. melanogaster PREs, including a Convolutional Neural Network (CNN), making this the first study to model PREs with CNNs. The models are readily adapted to new CRE modelling problems and to other organisms. In order to produce a high-performance, compiled package for Python 3, Gnocis was implemented in Cython. Gnocis can be installed using the PyPI package manager by running 'pip install gnocis'.
Patchigolla, V. S. P. and Mellone, B. G. (2022). Enrichment of Non-B-Form DNA at D. melanogaster Centromeres. Genome Biol Evol 14(5). PubMed ID: 35441684
Centromeres are essential chromosomal regions that mediate the accurate inheritance of genetic information during eukaryotic cell division. Despite their conserved function, centromeres do not contain conserved DNA sequences and are instead epigenetically marked by the presence of the centromere-specific histone H3 variant centromeric protein A. The functional contribution of centromeric DNA sequences to centromere identity remains elusive. Previous work found that dyad symmetries with a propensity to adopt noncanonical secondary DNA structures are enriched at the centromeres of several species. These findings lead to the proposal that noncanonical DNA structures may contribute to centromere specification. This study analyzed the predicted secondary structures of the recently identified centromere DNA sequences of Drosophila melanogaster. Although dyad symmetries are enriched only on the Y centromere, this study found that other types of noncanonical DNA structures, including melted DNA and G-quadruplexes, are common features of all D. melanogaster centromeres. This work is consistent with previous models suggesting that noncanonical DNA secondary structures may be conserved features of centromeres with possible implications for centromere specification.
Bernues, J., Izquierdo-Boulstridge, A., Reina, O., ..., Climent-Canto, P. and Azorin, F. (2022). Lysine 27 dimethylation of Drosophila linker histone dH1 contributes to heterochromatin organization independently of H3K9 methylation. Nucleic Acids Res 50(16): 9212-9225. PubMed ID: 36039761
Post-translational modifications (PTMs) of core histones are important epigenetic determinants that correlate with functional chromatin states. Thus study addresses the function of PTMs in Drosophila that encodes a single somatic linker histone, dH1. It has been reported that dH1 is dimethylated at K27 (dH1K27me2). This study shows that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase, it is also detected at intercalary heterochromatin. ChIPseq experiments show that >98% of dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. Moreover, expression of a mutated dH1K27A form, which impairs dH1K27me2, alters heterochromatin organization, upregulates expression of heterochromatic transposable elements and results in the accumulation of RNA:DNA hybrids (R-loops) in heterochromatin, without affecting H3K9 methylation and HP1a binding. The pattern of dH1K27me2 is H3K9 methylation independent, as it is equally detected in flies carrying a H3K9R mutation, and is not affected by depletion of Su(var)3-9, HP1a or Su(var)4-20. Altogether these results suggest that dH1K27me2 contributes to heterochromatin organization independently of H3K9 methylation.
Cheng, Q. and Xie, H. (2022). Genome-wide analysis of bivalent histone modifications during Drosophila embryogenesis. Genesis: e23502. PubMed ID: 36125264
In eukaryotes, histone modifications are key epigenetic regulators that are associated with distinct chromatin features. Bivalent histone modifications describe a situation where a subset of promoters have with both activating (H3K4me3) and repressive (H3K27me3) markers in pluripotent cells (e.g., ESCs). However, it remains to be understood whether bivalent histone modifications are stable throughout developmental stages. In this study, by systematically analyzing ChIP-seq data of H3K4me3 and H3K27me3, the first panoramic view is provided of bivalent histone modifications in Drosophila from embryonic 0-4 to 20-24 hr. In this study, it was found that bivalent histone modifications occur at other locations in the genome in addition to the promoter region. Additionally, the different genomic regions occupied by bivalent histone modifications exhibit spatiotemporal specificity at each stage. Furthermore, gene ontology and motif analysis reflected continuous and gradual changes of target genes during different developmental process. In summary, it is suggested that bivalent histone modifications have potential regulatory functions throughout Drosophila embryonic stage.
Amankwaa, B., Schoborg, T. and Labrador, M. (2022). Drosophila insulator proteins exhibit in vivo liquid-liquid phase separation properties. Life Sci Alliance 5(12). PubMed ID: 35853678
Mounting evidence implicates liquid-liquid phase separation (LLPS), the condensation of biomolecules into liquid-like droplets in the formation and dissolution of membraneless intracellular organelles (MLOs). Cells use MLOs or condensates for various biological processes, including emergency signaling and spatiotemporal control over steady-state biochemical reactions and heterochromatin formation. Insulator proteins are architectural elements involved in establishing independent domains of transcriptional activity within eukaryotic genomes. In Drosophila, insulator proteins form nuclear foci known as insulator bodies in response to osmotic stress. However, the mechanism through which insulator proteins assemble into bodies is yet to be investigated. This study identified signatures of LLPS by insulator bodies, including high disorder tendency in insulator proteins, scaffold-client-dependent assembly, extensive fusion behavior, sphericity, and sensitivity to 1,6-hexanediol. The cohesin subunit Rad21 is shown to be a component of insulator bodies, adding to the known insulator protein constituents and γH2Av. These data suggest a concerted role of cohesin and insulator proteins in insulator body formation and under physiological conditions. A mechanism is proposed whereby these architectural proteins modulate 3D genome organization through LLPS.

Wednesday, October 5th - Disease Models

Zarate, R. V., Hidalgo, S., Navarro, N., Molina-Mateo, D., Arancibia, D., Rojo-Cortes, F., Oliva, C., Andres, M. E., Zamorano, P. and Campusano, J. M. (2022). An Early Disturbance in Serotonergic Neurotransmission Contributes to the Onset of Parkinsonian Phenotypes in Drosophila melanogaster. Cells 11(9). PubMed ID: 35563850
Parkinson's disease (PD) is a neurodegenerative disease characterized by motor symptoms and dopaminergic cell loss. A pre-symptomatic phase characterized by non-motor symptoms precedes the onset of motor alterations. Two recent PET studies in human carriers of mutations associated with familial PD demonstrate an early serotonergic commitment-alteration in . PubMed ID: SERT binding-before any dopaminergic or motor dysfunction, that is, at putative PD pre-symptomatic stages. These findings support the hypothesis that early alterations in the serotonergic system could contribute to the progression of PD, an idea difficult to be tested in humans. Some components of the serotonergic system were investigated during the pre-symptomatic phase in a well-characterized Drosophila PD model, Pink1B9 mutant flies. Lower brain serotonin content was detected in Pink1B9 flies, accompanied by reduced activity of SERT before the onset of motor dysfunctions. The consequences of a brief early manipulation of the serotonergic system in the development of motor symptoms was explored later in aged animals. Feeding young Pink1B9 flies with fluoxetine, a SERT blocker, prevents the loss of dopaminergic neurons and ameliorates motor impairment observed in aged mutant flies. Surprisingly, the same pharmacological manipulation in young control flies results in aged animals exhibiting a PD-like phenotype. These findings support that an early dysfunction in the serotonergic system precedes and contributes to the onset of the Parkinsonian phenotype in Drosophila.
Thackray, A. M., Lam, B., McNulty, E. E., Nalls, A. V., Mathiason, C. K., Magadi, S. S., Jackson, W. S., Andreoletti, O., Marrero-Winkens, C., Schatzl, H. and Bujdoso, R. (2022). Clearance of variant Creutzfeldt-Jakob disease prions in vivo by the Hsp70 disaggregase system. Brain. PubMed ID: 35446941
The metazoan Hsp70 disaggregase protects neurons from proteotoxicity that arises from the accumulation of misfolded protein aggregates. Hsp70 and its co-chaperones disassemble and extract polypeptides from protein aggregates for refolding or degradation. The effectiveness of the chaperone system decreases with age and leads to accumulation rather than removal of neurotoxic protein aggregates. Therapeutic enhancement of the Hsp70 protein disassembly machinery is proposed to counter late-onset protein misfolding neurodegenerative disease that may arise. In the context of prion disease, it is not known whether stimulation of protein aggregate disassembly paradoxically leads to enhanced formation of seeding competent species of disease-specific proteins and acceleration of neurodegenerative disease. This study tested the hypothesis that modulation of Hsp70 disaggregase activity perturbs mammalian prion-induced neurotoxicity and prion seeding activity. To do so PrP transgenic Drosophila were used that authentically replicate mammalian prions. RNASeq identified that Hsp70, DnaJ-1 and Hsp110 gene expression was down-regulated in prion-exposed PrP Drosophila. RNAi knockdown of Hsp110 or DnaJ-1 gene expression in variant CJD prion-exposed human PrP Drosophila enhanced neurotoxicity, whereas over-expression mitigated toxicity. Strikingly, prion seeding activity in variant CJD prion-exposed human PrP Drosophila was ablated or reduced by Hsp110 or DnaJ-1 over-expression, respectively. Similar effects were seen in scrapie prion-exposed ovine PrP Drosophila with modified Hsp110 or DnaJ-1 gene expression. These unique observations show that the metazoan Hsp70 disaggregase facilitates the clearance of mammalian prions and that its enhanced activity is a potential therapeutic strategy for human prion disease.
Ader, F., Russi, M., Tixier-Cardoso, L., Jullian, E., Martin, E., Richard, P., Villard, E. and Monnier, V. (2022). Drosophila CRISPR/Cas9 mutants as tools to analyse cardiac filamin function and pathogenicity of human FLNC variants. Biol Open 11(9). PubMed ID: 36066120
Filamins are large proteins with actin binding properties. Mutations in FLNC, one of the three filamin genes in humans, have recently been implicated in dominant cardiomyopathies, but the underlying mechanisms are not well understood. This study aimed to use Drosophila melanogaster as a new in vivo model to study these diseases. First, it was shown that adult-specific cardiac RNAi-induced depletion of Drosophila Filamin (dFil) induced cardiac dilatation, impaired systolic function and sarcomeric alterations, highlighting its requirement for cardiac function and maintenance of sarcomere integrity in the adult stage. Next, using CRISPR/Cas9 gene editing, three missense variants, previously identified in patients with hypertrophic cardiomyopathy were introduced into the cheerio gene. Flies carrying these variants did not exhibit cardiac defects or increased propensity to form filamin aggregates, arguing against their pathogenicity. Finally, this study showed that deletions of the C-term part of dFil carrying the last four Ig-like domains are dispensable for cardiac function. Collectively, these results highlight the relevance of this model to explore the cardiac function of filamins and increase understanding of physio-pathological mechanisms involved in FLNC-related cardiomyopathies.
Altassan, R., Qudair, A., Alokaili, R., Alhasan, K., Faqeih, E. A., Alhashem, A., Alowain, M., Alsayed, M., Rahbeeni, Z., Albadi, L., Alkuraya, F. S., Anderson, E. N., Rajan, D. and Pandey, U. B. (2022). Further delineation of GEMIN4 related neurodevelopmental disorder with microcephaly, cataract, and renal abnormalities syndrome. Am J Med Genet A 188(10): 2932-2940. PubMed ID: 35861185
Pathogenic variants in GEMIN4 have recently been linked to an inherited autosomal recessive neurodevelopmental disorder characterized with microcephaly, cataracts, and renal abnormalities (NEDMCR syndrome). This report provides a retrospective review of 16 patients from 11 unrelated Saudi consanguineous families with GEMIN4 mutations. The cohort comprises 11 new and unpublished clinical details from five previously described patients. Only two missense, homozygous, pathogenic variants were found in all affected patients, suggesting a founder effect. All patients shared global developmental delay with variable ophthalmological, renal, and skeletal manifestations. In addition, endogenous Drosophila GEMIN4 was knocked down in neurons to further investigate the mechanism of the functional defects in affected patients. The fly model findings demonstrated developmental defects and motor dysfunction suggesting that loss of GEMIN4 function is detrimental in vivo; likely similar to human patients. To date, this study presents the largest cohort of patients affected with GEMIN4 mutations. Considering that identifying GEMIN4 defects in patients presenting with neurodevelopmental delay and congenital cataract will help in early diagnosis, appropriate management and prevention plans that can be made for affected families.
Aiello, G., Sabino, C., Pernici, D., Audano, M., Antonica, F., Gianesello, M., Ballabio, C., Quattrone, A., Mitro, N., Romanel, A., Soldano, A. and Tiberi, L. (2022). Transient rapamycin treatment during developmental stage extends lifespan in Mus musculus and Drosophila melanogaster. EMBO Rep 23(9): e55299. PubMed ID: 35796299
Lifespan is determined by complex and tangled mechanisms that are largely unknown. The early postnatal stage has been proposed to play a role in lifespan, but its contribution is still controversial. This show that a short rapamycin treatment during early life can prolong lifespan in Mus musculus and Drosophila melanogaster. Notably, the same treatment at later time points has no effect on lifespan, suggesting that a specific time window is involved in lifespan regulation. It was also found that sulfotransferases are upregulated during early rapamycin treatment both in newborn mice and in Drosophila larvae, and transient dST1 overexpression in Drosophila larvae extends lifespan. These findings unveil a novel link between early-life treatments and long-term effects on lifespan.
Aharoni, S., Proskorovski-Ohayon, R., Krishnan, R. K., Yogev, Y., Wormser, O., Hadar, N., Bakhrat, A., Alshafee, I., Gombosh, M., Agam, N., Gradstein, L., Shorer, Z., Zarivach, R., Eskin-Schwartz, M., Abdu, U. and Birk, O. S. (2022). PSMC1 variant causes a novel neurological syndrome. Clin Genet 102(4): 324-332. PubMed ID: 35861243.
Proteasome 26S, the eukaryotic proteasome, serves as the machinery for cellular protein degradation. It is composed of the 20S core particle and one or two 19S regulatory particles, composed of a base and a lid. This study now delineates an autosomal recessive syndrome of failure to thrive, severe developmental delay and intellectual disability, spastic tetraplegia with central hypotonia, chorea, hearing loss, micropenis and undescended testes, as well as mild elevation of liver enzymes. None of the affected individuals achieved verbal communication or ambulation. Ventriculomegaly was evident on MRI. Homozygosity mapping combined with exome sequencing revealed a disease-associated p.I328T PSMC1 variant. Protein modeling demonstrated that the PSMC1 variant is located at the highly conserved putative ATP binding and hydrolysis domain, and is suggested to interrupt a hydrophobic core within the protein. Fruit flies in which the Drosophila ortholog Rpt2 was silenced specifically in the eye exhibited an apparent phenotype that was highly rescued by the human wild-type PSMC1, yet only partly by the mutant PSMC1, proving the functional effect of the p.I328T disease-causing variant.

Tuesday, October 5th - Immune Response

Gordon, K. E., Wolfner, M. F. and Lazzaro, B. P. (2022). A single mating is sufficient to induce persistent reduction of immune defense in mated female Drosophila melanogaster. J Insect Physiol 140: 104414. PubMed ID: 35728669
In many species, female reproductive investment comes at a cost to immunity and resistance to infection. Mated Drosophila melanogaster females are more susceptible to bacterial infection than unmated females. Transfer of the male seminal fluid protein Sex Peptide reduces female post-mating immune defense. Sex Peptide is known to cause both short- and long-term changes to female physiology and behavior. While previous studies showed that females were less resistant to bacterial infection as soon as 2.5 h and as long as 26.5 h after mating, it is unknown whether this is a binary switch from mated to unmated state or whether females can recover to unmated levels of immunity. It is additionally unknown whether repeated mating causes progressive reduction in defense capacity. The immune defense of mated females when infected at 2, 4, 7, or 10 days after mating was compared to that of unmated females and no recovery of immune capacity was seen regardless of the length of time between mating and infection. Because D. melanogaster females can mate multiply, whether a second mating, and therefore a second transfer of seminal fluids, caused deeper reduction in immune performance was additionally tested. Females mated either once or twice before infection were found to survive at equal proportions, both with significantly lower probability than unmated females. It is concluded that a single mating event is sufficient to persistently suppress the female immune system. Interestingly, it was observed that induced levels of expression of genes encoding antimicrobial peptides (AMPs) decreased with age in both experiments, partially obscuring the effects of mating. Collectively, the data indicate that being reproductively active versus reproductively inactive are alternative binary states with respect to female D. melanogaster immunity. The establishment of a suppressed immune status in reproductively active females can inform understanding of the regulation of immune defense and the mechanisms of physiological trade-offs.
Chen, K., Wang, X., Wei, X., Chen, J., Wei, Y., Jiang, H., Lu, Z. and Feng, C. (2022). Nitric Oxide-Induced Calcineurin A Mediates Antimicrobial Peptide Production Through the IMD Pathway. Front Immunol 13: 905419. PubMed ID: 35663981
Nitric oxide (NO) at a high concentration is an effector to kill pathogens during insect immune responses, it also functions as a second messenger at a low concentration to regulate antimicrobial peptide (AMP) production in insects. Drosophila calcineurin subunit CanA1 is a ubiquitous serine/threonine protein phosphatase involved in NO-induced AMP production. However, it is unclear how NO regulates AMP expression. In this study, a lepidopteran pest Ostrinia furnacalis and Drosophila S2 cells were used to investigate how NO signaling affects the AMP production. Bacterial infections upregulated the transcription of nitric oxide synthase 1/2 (NOS1/2), CanA and AMP genes and increased NO concentration in larval hemolymph. Inhibition of NOS or CanA activity reduced the survival of bacteria-infected O. furnacalis. NO donor increased NO level in plasma and upregulated the production of CanA and certain AMPs. In S2 cells, killed Escherichia coli induced NOS transcription and boosted NO production, whereas knockdown of NOS blocked the NO level increase caused by E. coli. As in O. furnacalis larvae, supplementation of the NO donor increased NO level in the culture medium and AMP expression in S2 cells. Suppression of the key pathway genes showed that the IMD (but not Toll) pathway was involved in the upregulation of CecropinA1, Defensin, Diptericin, and Drosomycin by killed E. coli. Knockdown of NOS also reduced the expression of CanA1 and AMPs induced by E. coli, indicative of a role of NO in the AMP expression. Furthermore, CanA1 RNA interference and inhibition of its phosphatase activity significantly reduced NO-induced AMP expression, and knockdown of IMD suppressed NO-induced AMP expression. Together, these results suggest that NO-induced AMP production is mediated by CanA1 via the IMD pathway.
Waring, A. L., Hill, J., Allen, B. M., Bretz, N. M., Le, N., Kr, P., Fuss, D. and Mortimer, N. T. (2022). Meta-Analysis of Immune Induced Gene Expression Changes in Diverse Drosophila melanogaster Innate Immune Responses. Insects 13(5). PubMed ID: 35621824
Organisms are commonly infected by a diverse array of pathogens and mount functionally distinct responses to each of these varied immune challenges. Host immune responses are characterized by the induction of gene expression, however, the extent to which expression changes are shared among responses to distinct pathogens is largely unknown. To examine this, meta-analysis was performed of gene expression data collected from Drosophila melanogaster following infection with a wide array of pathogens. Sixty-two genes were identified that are significantly induced by infection. While many of these infection-induced genes encode known immune response factors, 21 genes were identified that have not been previously associated with host immunity. Examination of the upstream flanking sequences of the infection-induced genes lead to the identification of two conserved enhancer sites. These sites correspond to conserved binding sites for GATA and nuclear factor κB (NFκB) family transcription factors and are associated with higher levels of transcript induction. Thirty-one genes were further identified with predicted functions in metabolism and organismal development that are significantly downregulated following infection by diverse pathogens. This study identifies conserved gene expression changes in Drosophila melanogaster following infection with varied pathogens, and transcription factor families that may regulate this immune induction.
Zhou, H., Wu, S., Liu, L., Li, R., Jin, P. and Li, S. (2022). Drosophila Relish Activating lncRNA-CR33942 Transcription Facilitates Antimicrobial Peptide Expression in Imd Innate Immune Response. Front Immunol 13: 905899. PubMed ID: 35720331
Long noncoding RNAs (lncRNAs) are an emerging class of regulators that play crucial roles in regulating the strength and duration of innate immunity. However, little is known about the regulation of Drosophila innate immunity-related lncRNAs. In this study, it was first revealed that overexpression of lncRNA-CR33942 could strengthen the expression of the Imd pathway antimicrobial peptide (AMP) genes Diptericin (Dpt) and Attacin-A (AttA) after infection, and vice versa. Secondly, RNA-seq analysis of lncRNA-CR33942-overexpressing flies post Gram-negative bacteria infection confirmed that lncRNA-CR33942 positively regulated the Drosophila immune deficiency (Imd) pathway. Mechanistically, it was found that lncRNA-CR33942 interacts and enhances the binding of NF-κB transcription factor Relish to Dpt and AttA promoters, thereby facilitating Dpt and AttA expression. Relish could also directly promote lncRNA-CR33942 transcription by binding to its promoter. Finally, rescue experiments and dynamic expression profiling post-infection demonstrated the vital role of the Relish/lncRNA-CR33942/AMP regulatory axis in enhancing Imd pathway and maintaining immune homeostasis. This study elucidates novel mechanistic insights into the role of lncRNA-CR33942 in activating Drosophila Imd pathway and the complex regulatory interaction during the innate immune response of animals.
Yu, J., Xiao, K., Chen, X., Deng, L., Zhang, L., Li, Y., Gao, A., Gao, J., Wu, C., Yang, X., Zhou, Q., Yang, J., Bao, C., Jiao, J., Cheng, S., Guo, Z., Xu, W., Cao, X., Guo, Z., Dai, J., Hu, J., Fu, Z. and Cao, G. (2022). Neuron-derived neuropeptide Y fine-tunes the splenic immune responses. Neuron 110(8): 1327-1339. PubMed ID: 35139365
The nervous and immune systems are closely entwined to maintain the immune balance in health and disease. This study showed that LPS can activate suprarenal and celiac ganglia (SrG-CG) neurons and upregulate NPY expression in rats. Single-cell sequencing analysis revealed that knockdown of the NPY gene in SrG-CG altered the proliferation and activation of splenic lymphocytes. In a neuron and splenocyte coculture system and in vivo experiments, neuronal NPY in SrG-CG attenuated the splenic immune response. Notably, it was demonstrated that neuronal NPF in Drosophila exerted a conservative immunomodulatory effect. Moreover, numerous SNPs in NPY and its receptors were significantly associated with human autoimmune diseases, which was further supported by the autoimmune disease patients and mouse model experiments. Together, this study demonstrated that NPY is an ancient language for nervous-immune system crosstalk and might be utilized to alleviate inflammatory storms during infection and to modulate immune balance in autoimmune diseases.
Gallo, M., Vento, J. M., Joncour, P., Quagliariello, A., Maritan, E., Silva-Soares, N. F., Battistolli, M., Beisel, C. L. and Martino, M. E. (2022). Beneficial commensal bacteria promote Drosophila growth by downregulating the expression of peptidoglycan recognition proteins. iScience 25(6): 104357. PubMed ID: 35601912
Commensal bacteria are known to promote host growth. Such effect partly relies on the capacity of microbes to regulate the host's transcriptional response. However, these evidences mainly come from comparing the transcriptional response caused by commensal bacteria with that of axenic animals, making it difficult to identify the animal genes that are specifically regulated by beneficial microbes. This study employed Drosophila melanogaster associated with Lactiplantibacillus plantarum to understand the host genetic pathways regulated by beneficial bacteria and leading to improved host growth. It was shown that microbial benefit to the host relies on the downregulation of peptidoglycan-recognition proteins. Specifically, it is reported that bacterial proliferation triggers the lower expression of PGRP-SC1 in larval midgut, which ultimately leads to improved host growth and development. This study helps elucidate the mechanisms underlying the beneficial effect exerted by commensal bacteria, defining the role of immune effectors in the relationship between Drosophila and its gut microbes.

Monday, October 3rd - Behavior

Lobato-Rios, V., Ramalingasetty, S. T., Ozdil, P. G., Arreguit, J., Ijspeert, A. J. and Ramdya, P. (2022). NeuroMechFly, a neuromechanical model of adult Drosophila melanogaster. Nat Methods 19(5): 620-627. PubMed ID: 35545713
Animal behavior emerges from an interaction between neural network dynamics, musculoskeletal properties and the physical environment. Accessing and understanding the interplay between these elements requires the development of integrative and morphologically realistic neuromechanical simulations. This study presents NeuroMechFly, a data-driven model of the widely studied organism, Drosophila melanogaster. NeuroMechFly combines four independent computational modules: a physics-based simulation environment, a biomechanical exoskeleton, muscle models and neural network controllers. To enable use cases, the minimum degrees of freedom of the leg was defined from real three-dimensional kinematic measurements during walking and grooming. Then, it was shown how, by replaying these behaviors in the simulator, one can predict otherwise unmeasured torques and contact forces. Finally, NeuroMechFly's full neuromechanical capacity was leveraged to discover neural networks and muscle parameters that drive locomotor gaits optimized for speed and stability. Thus, NeuroMechFly can increase understanding of how behaviors emerge from interactions between complex neuromechanical systems and their physical surroundings.
Cellini, B., Salem, W. and Mongeau, J. M. (2022). Complementary feedback control enables effective gaze stabilization in animals. Proc Natl Acad Sci U S A 119(19): e2121660119. PubMed ID: 35503912
Visually active animals coordinate vision and movement to achieve spectacular tasks. An essential prerequisite to guide agile locomotion is to keep gaze level and stable. Since the eyes, head and body can move independently to control gaze, how does the brain effectively coordinate these distinct motor outputs? To address this questions, gaze control was studied in flying fruit flies (Drosophila) using a paradigm which permitted direct measurement of head and body movements. By combining experiments with mathematical modeling, this study shows that body movements are sensitive to the speed of visual motion whereas head movements are sensitive to its acceleration. This complementary tuning of the head and body permitted flies to stabilize a broader range of visual motion frequencies. It was discovered that flies implement proportional-derivative (PD) control, but unlike classical engineering control systems, relay the proportional and derivative signals in parallel to two distinct motor outputs. This scheme, although derived from flies, recapitulated classic primate vision responses thus suggesting convergent mechanisms across phyla. By applying scaling laws, it was quantified that animals as diverse as flies, mice, and humans as well as bio-inspired robots can benefit energetically by having a high ratio between head, body, and eye inertias. These results provide insights into the mechanical constraints that may have shaped the evolution of active vision and present testable neural control hypotheses for visually guided behavior across phyla.
Wang, T., Jing, B., Deng, B., Shi, K., Li, J., Ma, B., Wu, F. and Zhou, C. (2022). Drosulfakinin signaling modulates female sexual receptivity in Drosophila. Elife 11. PubMed ID: 35475782
Female sexual behavior as an innate behavior is of prominent biological importance for survival and reproduction. However, molecular and circuit mechanisms underlying female sexual behavior is not well understood. This study identified the Cholecystokinin-like peptide Drosulfakinin (DSK) to promote female sexual behavior in Drosophila. Loss of DSK function reduces female receptivity while overexpressing DSK enhances female receptivity. Two pairs of Dsk-expressing neurons in the central brain were found to promote female receptivity. DSK peptide acts through one of its receptors, CCKLR-17D3, to modulate female receptivity. Manipulation of CCKLR-17D3 and its expressing neurons alters female receptivity. It was further revealed that the two pairs of Dsk-expressing neurons receive input signal from pC1 neurons that integrate sex-related cues and mating status. These results demonstrate how a neuropeptide pathway interacts with a central neural node in the female sex circuitry to modulate sexual receptivity.
Chechi, T. S., Narasimhan, A., Biswas, B. and Prasad, N. G. (2022). Male mating success evolves in response to increased levels of male-male competition. Evolution. PubMed ID: 35598115
Male-biased operational sex ratios can increase male-male competition and can potentially select for both increased pre- and postcopulatory male success. In the present study, using populations of Drosophila melanogaster evolved under male-biased (M) or female-biased (F) sex ratios, it was asked whether (a) male mating success can evolve, (b) males are better at mating females that they have coevolved with, (c) males mating success is affected by female mating status, and (d) male mating success is correlated with their courtship effort. M and F males were directly competed for mating with (a) virgin ancestral (common) females, (b) virgin females from the M and F populations, and (c) singly mated females from the M and F populations. The courtship frequency was assessed of the males when paired with mated M or F females. The results show that M males, evolving under an increased level of male-male competition, have higher mating success than F males irrespective of the female evolutionary history. However, the difference in mating success is more pronounced if the females had mated before. M males also have a higher courtship frequency than F males, but no correlation was found between mating success and courtship frequency.
Vijendravarma, R. K., Narasimha, S., Steinfath, E., Clemens, J. and Leopold, P. (2022). Drosophila females have an acoustic preference for symmetric males. Proc Natl Acad Sci U S A 119(13): e2116136119. PubMed ID: 35312357
Theoretically, symmetry in bilateral animals is subject to sexual selection, since it can serve as a proxy for genetic quality of competing mates during mate choice. This study reports female preference for symmetric males in Drosophila, using a mate-choice paradigm where males with environmentally or genetically induced wing asymmetry were competed. Analysis of courtship songs revealed that males with asymmetric wings produced songs with asymmetric features that served as acoustic cues, facilitating this female preference. Females experimentally evolved in the absence of mate choice lost this preference for symmetry, suggesting that it is maintained by sexual selection.
Zurl, M., Poehn, B., Rieger, D., Krishnan, S., Rokvic, D., Veedin Rajan, V. B., Gerrard, E., Schlichting, M., Orel, L., Coric, A., Lucas, R. J., Wolf, E., Helfrich-Forster, C., Raible, F. and Tessmar-Raible, K. (2022). Two light sensors decode moonlight versus sunlight to adjust a plastic circadian/circalunidian clock to moon phase. Proc Natl Acad Sci U S A 119(22): e2115725119. PubMed ID: 35622889
The moon provides highly reliable time information to organisms. Whereas sunlight is known to set daily animal timing systems, mechanistic insight into the impact of moonlight on such systems remains scarce. This study establish that the marine bristleworm Platynereis dumerilii times the precise hours of mass spawning by integrating lunar light information into a plastic daily timing system able to run with circadian (∼24 h) or circalunidian (∼24.8 h) periodicity. The correct interpretation of moonlight is mediated by the interplay of two light sensors: a cryptochrome and a melanopsin ortholog provide information on light valence and moonrise time, respectively. Besides its ecological relevance, this work provides a plausible explanation for long-standing observations of light intensity-dependent differences in circadian clock periods.
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