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Current papers in developmental biology and gene function


Tuesday, February 28th, 2023 - Adult physiology and metabolism

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Signore, S. J. D., Mitzner, M. G., Silveira, A. M., Fai, T. G. and Rodal, A. A. (2022). An approach for quantitative mapping of synaptic periactive zone architecture and organization. Mol Biol Cell: mbcE22080372. PubMed ID: 36542486
Following exocytosis at active zones, synaptic vesicle membranes and membrane-bound proteins must be recycled. The endocytic machinery that drives this recycling accumulates in the periactive zone (PAZ), a region of the synapse adjacent to active zones, but the organization of this machinery within the PAZ, and how PAZ composition relates to active zone release properties remains unknown. The PAZ is also enriched for cell adhesion proteins, but their function at these sites is poorly understood. In this study, using Airyscan and STED imaging of Drosophila synapses, a quantitative framework was developed describing the organization and ultrastructure of the PAZ. Different endocytic proteins localize to distinct regions of the PAZ, suggesting that sub-domains are specialized for distinct biochemical activities, stages of membrane remodeling, or synaptic functions. It was found that the accumulation and distribution of endocytic but not adhesion PAZ proteins correlate with the abundance of the scaffolding protein Bruchpilot at active zones - a structural correlate of release probability. These data suggest that endocytic and exocytic activities are spatially correlated. Taken together, these results identify novel relationships between the exocytic and endocytic apparatus at the synapse and provide a new conceptual framework to quantify synaptic architecture.
Sobrido-Camean, D., Oswald, M. C. W., Bailey, D. M. D., Mukherjee, A. and Landgraf, M. (2022). Activity-regulated growth of motoneurons at the neuromuscular junction is mediated by NADPH oxidases. Front Cell Neurosci 16: 1106593. PubMed ID: 36713781
Neurons respond to changes in the levels of activity they experience in a variety of ways, including structural changes at pre- and postsynaptic terminals. An essential plasticity signal required for such activity-regulated structural adjustments are reactive oxygen species (ROS). To identify sources of activity-regulated ROS required for structural plasticity in vivo the Drosophila larval neuromuscular junction as a highly tractable experimental model system. For adjustments of presynaptic motor terminals, a requirement was found for both NADPH oxidases, Nox and dual oxidase (Duox), that are encoded in the Drosophila genome. This contrasts with the postsynaptic dendrites from which Nox is excluded. NADPH oxidases generate ROS to the extracellular space. This study shows that two aquaporins, Bib and Drip, are necessary ROS conduits in the presynaptic motoneuron for activity regulated, NADPH oxidase dependent changes in presynaptic motoneuron terminal growth. These data further suggest that different aspects of neuronal activity-regulated structural changes might be regulated by different ROS sources: changes in bouton number require both NADPH oxidases, while activity-regulated changes in the number of active zones might be modulated by other sources of ROS. Overall, these results show NADPH oxidases as important enzymes for mediating activity-regulated plasticity adjustments in neurons.
Dannhauser, S., Mrestani, A., Gundelach, F., Pauli, M., Komma, F., Kollmannsberger, P., Sauer, M., Heckmann, M. and Paul, M. M. (2022). Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation. Front Cell Neurosci 16: 1074304. PubMed ID: 36589286
Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. This study employed minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13 (GFSTF) 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13(GFSTF), Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13(GFSTF) subclusters that move toward the AZ center during PHP with unaltered Unc-13(GFSTF) protein levels.
Dyson, A., Ryan, M., Garg, S., Evans, D. G. and Baines, R. A. (2022). Loss of NF1 in Drosophila Larvae Causes Tactile Hypersensitivity and Impaired Synaptic Transmission at the Neuromuscular Junction. J Neurosci 42(50): 9450-9472. PubMed ID: 36344265
Autism spectrum disorder (ASD) is a neurodevelopmental condition in which the mechanisms underlying its core symptomatology are largely unknown. Studying animal models of monogenic syndromes associated with ASD, such as neurofibromatosis type 1 (NF1), can offer insights into its etiology. This study shows that loss of function of the Drosophila NF1 ortholog results in tactile hypersensitivity following brief mechanical stimulation in the larva (mixed sexes), paralleling the sensory abnormalities observed in individuals with ASD. Mutant larvae also exhibit synaptic transmission deficits at the glutamatergic neuromuscular junction (NMJ), with increased spontaneous but reduced evoked release. While the latter is homeostatically compensated for by a postsynaptic increase in input resistance, the former is consistent with neuronal hyperexcitability. Indeed, diminished expression of NF1 specifically within central cholinergic neurons induces both excessive neuronal firing and tactile hypersensitivity, suggesting the two may be linked. Furthermore, both impaired synaptic transmission and behavioral deficits are fully rescued via knock-down of Ras proteins. These findings validate NF1 (-/-) Drosophila as a tractable model of ASD with the potential to elucidate important pathophysiological mechanisms.
Fruin, A. M., Leon, K. E. and DiAngelo, J. R. (2022). The ESCRT-III Protein Chmp1 Regulates Lipid Storage in the Drosophila Fat Body. Med Sci (Basel) 11(1). PubMed ID: 36649042
Defects in how excess nutrients are stored as triglycerides can result in several diseases including obesity, heart disease, and diabetes. Understanding the genes responsible for normal lipid homeostasis will help understand the pathogenesis of these diseases. RNAi screens performed in Drosophila cells identified genes involved in vesicle formation and protein sorting as important for the formation of lipid droplets; however, all of the vesicular trafficking proteins that regulate lipid storage are unknown. This study characterized the function of the Drosophila Charged multivesicular protein 1 (Chmp1) gene in regulating fat storage. Chmp1 is a member of the ESCRT-III complex that targets membrane localized signaling receptors to intralumenal vesicles in the multivesicular body of the endosome and then ultimately to the lysosome for degradation. When Chmp1 levels are decreased specifically in the fly fat body, triglyceride accumulates while fat-body-specific Chmp1 overexpression decreases triglycerides. Chmp1 controls triglyceride storage by regulating the number and size of fat body cells produced and not by altering food consumption or lipid metabolic enzyme gene expression. Together, these data uncover a novel function for Chmp1 in controlling lipid storage in Drosophila and supports the role of the endomembrane system in regulating metabolic homeostasis.
Perry, S., Han, Y., Qiu, C., Chien, C., Goel, P., Nishimura, S., Sajnani, M., Schmid, A., Sigrist, S. J. and Dickman, D. (2022). A glutamate receptor C-tail recruits CaMKII to suppress retrograde homeostatic signaling. Nat Commun 13(1): 7656. PubMed ID: 36496500
Presynaptic homeostatic plasticity (PHP) adaptively enhances neurotransmitter release following diminished postsynaptic glutamate receptor (GluR) functionality to maintain synaptic strength. While much is known about PHP expression mechanisms, postsynaptic induction remains enigmatic. For over 20 years, diminished postsynaptic Ca(2+) influx was hypothesized to reduce CaMKII activity and enable retrograde PHP signaling at the Drosophila neuromuscular junction. This study has interrogated inductive signaling, and active CaMKII was found to colocalizes with and requires the GluRIIA receptor subunit. Next, Ca(2+)-impermeable GluRs were generated to reveal that both CaMKII activity and PHP induction are Ca(2+)-insensitive. Rather, a GluRIIA C-tail domain is necessary and sufficient to recruit active CaMKII. Finally, chimeric receptors demonstrate that the GluRIIA tail constitutively occludes retrograde homeostatic signaling by stabilizing active CaMKII. Thus, the physical loss of the GluRIIA tail is sensed, rather than reduced Ca(2+), to enable retrograde PHP signaling, highlighting a unique, Ca(2+)-independent control mechanism for CaMKII in gating homeostatic plasticity.

Monday, February 27th - Adult physiology and metabolism

Sadasivan, J., Vlok, M., Wang, X., Nayak, A., Andino, R. and Jan, E. (2022). Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation. PLoS Pathog 18(12): e1010598. PubMed ID: 36455064
Viruses have evolved mechanisms to modulate cellular pathways to facilitate infection. One such pathway is the formation of stress granules (SG), which are ribonucleoprotein complexes that assemble during translation inhibition following cellular stress. Inhibition of SG assembly has been observed under numerous virus infections across species, suggesting a conserved fundamental viral strategy. However, the significance of SG modulation during virus infection is not fully understood. The 1A protein encoded by the model dicistrovirus, Cricket paralysis virus (CrPV), is a multifunctional protein that can bind to and degrade Ago-2 in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway and inhibit SG formation. Moreover, the R146 residue of 1A is necessary for SG inhibition and CrPV infection in both Drosophila S2 cells and adult flies. This study uncoupled CrPV-1A's functions and provides insight into its underlying mechanism for SG inhibition. CrPV-1A mediated inhibition of SGs requires the E3 ubiquitin-ligase binding domain and the R146 residue, but not the Ago-2 binding domain. Wild-type but not mutant CrPV-1A R146A localizes to the nuclear membrane which correlates with nuclear enrichment of poly(A)+ RNA. Transcriptome changes in CrPV-infected cells are dependent on the R146 residue. Finally, Nup358/RanBP2 is targeted and degraded in CrPV-infected cells in an R146-dependent manner and the depletion of Nup358 blocks SG formation. It is proposed that CrPV utilizes a multiprong strategy whereby the CrPV-1A protein interferes with a nuclear event that contributes to SG inhibition in order to promote infection.
Sella, Y., Broderick, N. A., Stouffer, K., McEwan, D. L., Ausubel, F. M., Casadevall, A. and Bergman, A. (2022). Chaotic signatures in host-microbe interactions. bioRxiv. PubMed ID: 36561184
Host-microbe interactions constitute dynamical systems that can be represented by mathematical formulations that determine their dynamic nature. Dynamical interactions can be categorized as deterministic, stochastic, or chaotic. Knowing the type of dynamical interaction is essential for understanding the system under study. Very little experimental work has been done to determine the dynamical characteristics of host-microbe interactions and its study poses significant challenges. The most straightforward experimental outcome involves an observation of time to death upon infection. However, in measuring this outcome, the internal parameters and dynamics of each host-microbe interaction are hidden from the experimentalist. To investigate whether a time-to-death (time to event) dataset provides adequate information for searching for chaotic signatures, the ability to detect chaos in simulated data sets of time-to-event measurements was determined and the time-to-event distribution of a chaotic process was successfully distinguished from a comparable stochastic one. To do so, an inversion measure was introduced to test for a chaotic signature in time-to-event distributions. Next, chaos was sought in time-to-death of Caenorhabditis elegans and Drosophila melanogaster infected with Pseudomonas aeruginosa or Pseudomonas entomophila, respectively. Suggestions were found of chaotic signatures in both systems, but it is cautioned that the results are still preliminary and highlight the need for more fine-grained and larger data sets in determining dynamical characteristics. If validated, the occurrence of chaos in host-microbe interactions would have important implications for the occurrence and outcome of infectious diseases, the reproducibility of experiments in the field of microbial pathogenesis and the prediction of future microbial threats.
Liessem, S., Held, M., Bisen, R. S., Haberkern, H., Lacin, H., Bockemuhl, T. and Ache, J. M. (2022). Behavioral state-dependent modulation of insulin-producing cells in Drosophila. Curr Biol. PubMed ID: 36580915
Insulin signaling plays a pivotal role in metabolic control and aging, and insulin accordingly is a key factor in several human diseases. Despite this importance, the in vivo activity dynamics of insulin-producing cells (IPCs) are poorly understood. This study characterized the effects of locomotion on the activity of IPCs in Drosophila. Using in vivo electrophysiology and calcium imaging, it was found that IPCs were strongly inhibited during walking and flight and that their activity rebounded and overshot after cessation of locomotion. Moreover, IPC activity changed rapidly during behavioral transitions, revealing that IPCs are modulated on fast timescales in behaving animals. Optogenetic activation of locomotor networks ex vivo, in the absence of actual locomotion or changes in hemolymph sugar levels, was sufficient to inhibit IPCs. This demonstrates that the behavioral state-dependent inhibition of IPCs is actively controlled by neuronal pathways and is independent of changes in glucose concentration. By contrast, the overshoot in IPC activity after locomotion was absent ex vivo and after starvation, indicating that it was not purely driven by feedforward signals but additionally required feedback derived from changes in hemolymph sugar concentration. It is hypothesized that IPC inhibition during locomotion supports mobilization of fuel stores during metabolically demanding behaviors, while the rebound in IPC activity after locomotion contributes to replenishing muscle glycogen stores. In addition, the rapid dynamics of IPC modulation support a potential role of insulin in the state-dependent modulation of sensorimotor processing.
Ramesh, R., Skog, S., Orkenby, L., Kugelberg, U., Natt, D. and Ost, A. (2022). Dietary sugar shifts mitochondrial metabolism and small RNA biogenesis in sperm. Antioxid Redox Signal. PubMed ID: 36509450
Increasing concentrations of dietary sugar results in a linear accumulation of triglycerides in male Drosophila, while inducing a U-shaped obesity response in their offspring. Using a combination of proteomics and small RNA (sRNA) sequencing, this study aimed to understand the molecular underpinning in sperm for such plasticity. Proteomic analysis of seminal vesicles revealed that increasing concentrations of dietary sugar resulted in a bell-shaped induction of proteins involved in metabolic/redox regulation. Using stains and in vivo redox reporter flies, this pattern could be explained by changes in sperm production of reactive oxygen species (ROS), more exactly mitochondria-derived H2O2. By quenching ROS with the antioxidant N acetyl cysteine (NAC) and performing sRNA-seq on sperm, it was found that sperm miRNA is increased in response to ROS. Moreover, sperm mitosRNA was found to be increased in high sugar diet conditions (independent of ROS). Reanalyzing previously published data revealed a similar global upregulation of human sperm mitosRNA in response to a high-sugar diet, suggesting evolutionary conserved mechanisms. This work highlights a fast response to dietary sugar in mitochondria produced H2O2. These data supports a model where changes in the sperm mitochondria in response to dietary sugar is the primary event, and changes in redox homoeostasis are secondary to mitochondrial ROS production. This data provides multiple candidates for paternal intergenerational metabolic responses as well as potential biomarkers for human male fertility.
Eickelberg, V., Rimbach, G., Seidler, Y., Hasler, M., Staats, S. and Luersen, K. (2022). Fat Quality Impacts the Effect of a High-Fat Diet on the Fatty Acid Profile, Life History Traits and Gene Expression in Drosophila melanogaster. Cells 11(24). PubMed ID: 36552807
Feeding a high-fat diet (HFD) has been shown to alter phenotypic and metabolic parameters in Drosophila melanogaster. However, the impact of fat quantity and quality remains uncertain. Butterfat (BF) was first used as an example to investigate the effects of increasing dietary fat content (3-12%) on male and female fruit flies. Although body weight and body composition were not altered by any BF concentration, health parameters, such as lifespan, fecundity and larval development, were negatively affected in a dose-dependent manner. When fruit flies were fed various 12% HFDs (BF, sunflower oil, olive oil, linseed oil, fish oil), their fatty acid profiles shifted according to the dietary fat qualities. Moreover, fat quality was found to determine the effect size of the response to an HFD for traits, such as lifespan, climbing activity, or fertility. Consistently, a highly fat quality-specific transcriptional response to three exemplary HFD qualities was also found with a small overlap of only 30 differentially expressed genes associated with the immune/stress response and fatty acid metabolism. In conclusion, these data indicate that not only the fat content but also the fat quality is a crucial factor in terms of life-history traits when applying an HFD in D. melanogaster.
Inomata, N., Miyazaki, M., Noguchi, M. and Itoh, M. (2022). A comparative study of natural variation in hemolymph glucose levels under different dietary sugar conditions in Drosophila melanogaster and D. simulans. Genes Genet Syst. PubMed ID: 36464279
Physiological responses to environmental changes play important roles in adaptive evolution. In particular, homeostatic regulatory systems that maintain constant circulating glucose levels are crucial in animals. However, variation in circulating glucose levels and the genetic effects on phenotypic variation in natural populations remain to be clarified. This study investigated the hemolymph glucose levels in natural populations of Drosophila melanogaster and its sibling species, D. simulans, in Japan. Hemolymph glucose concentrations were quantified in third instar larvae of 27 lines for each species, which were reared on either glucose-free or glucose-rich food. In both species, genetic variation was not a major component of phenotypic variation on either glucose-free or glucose-rich food. The hemolymph glucose concentrations were much higher in D. simulans than in D. melanogaster. Genetic variance was larger in D. simulans than in D. melanogaster. The observed differences between the two species may be associated with the much more recent colonization history of D. simulans populations in Japan and/or the tolerance to environmental stresses. These findings suggest that natural selection acting on hemolymph glucose levels in D. melanogaster is different from that in D. simulans.

Friday, February 24th - Chromatin, DNA Replication, and Chromosome Dynamics

Messina, G., Celauro, E., Marsano, R. M., Prozzillo, Y. and Dimitri, P. (2022). Epigenetic Silencing of P-Element Reporter Genes Induced by Transcriptionally Active Domains of Constitutive Heterochromatin in Drosophila melanogaster. Genes (Basel) 14(1). PubMed ID: 36672753
Reporter genes inserted via P-element integration into different locations of the Drosophila melanogaster genome have been routinely used to monitor the functional state of chromatin domains. It is commonly thought that P-element-derived reporter genes are subjected to position effect variegation (PEV) when transposed into constitutive heterochromatin because they acquire heterochromatin-like epigenetic modifications that promote silencing. However, sequencing and annotation of the D. melanogaster genome have shown that constitutive heterochromatin is a genetically and molecularly heterogeneous compartment. In fact, in addition to repetitive DNAs, it harbors hundreds of functional genes, together accounting for a significant fraction of its entire genomic territory. Notably, most of these genes are actively transcribed in different developmental stages and tissues, irrespective of their location in heterochromatin. An open question in the genetic and molecular studies on PEV in D. melanogaster is whether functional heterochromatin domains, i.e., heterochromatin harboring active genes, are able to silence reporter genes therein transposed or, on the contrary, can drive their expression. This work provides experimental evidence showing that strong silencing of the Pw(+) reporters is induced even when they are integrated within or near actively transcribed loci in the pericentric regions of chromosome 2. Interestingly, some Pw(+) reporters were found insensitive to the action of a known PEV suppressor. Two of them are inserted within Yeti, a gene expressed in the deep heterochromatin of chromosome 2 that carries active chromatin marks. The difference sensitivity to suppressors-exhibited Pw(+) reporters supports the view that different epigenetic regulators or mechanisms control different regions of heterochromatin. Together, these results suggest that there may be more complexity regarding the molecular mechanisms underlying PEV.
Poikela, N., Laetsch, D. R., Kankare, M., Hoikkala, A. and Lohse, K. (2022). Experimental introgression in Drosophila: Asymmetric postzygotic isolation associated with chromosomal inversions and an incompatibility locus on the X chromosome. Mol Ecol. PubMed ID: 36461113
Interspecific gene flow (introgression) is an important source of new genetic variation, but selection against it can reinforce reproductive barriers between interbreeding species. This study used an experimental approach to trace the role of chromosomal inversions and incompatibility genes in preventing introgression between two partly sympatric Drosophila virilis group species, D. flavomontana and D. montana. F(1) hybrid females from a cross between D. flavomontana female and D. montana male with the males of the parental species were backcrossed for two generations, and pools of parental strains and their reciprocal second generation backcross (BC(2) mon and BC(2) fla) females were sequenced. Contrasting the observed amount of introgression (mean hybrid index, HI) in BC(2) female pools along the genome to simulations under different scenarios allowed identification of chromosomal regions of restricted and increased introgression. No deviation was found from the HI expected under a neutral null model for any chromosome for the BC(2) mon pool, suggesting no evidence for genetic incompatibilities in backcrosses towards D. montana. In contrast, the BC(2) fla pool showed high variation in the observed HI between different chromosomes, and massive reduction of introgression on the X chromosome (large X-effect). This observation is compatible with reduced recombination combined with at least one dominant incompatibility locus residing within the X inversion(s). Overall, this study suggests that genetic incompatibilities arising within chromosomal inversions can play an important role in speciation.
Lama, J., Srivastav, S., Tasnim, S., Hubbard, D., Hadjipanteli, S., Smith, B. R., Macdonald, S. J., Green, L. and Kelleher, E. S. (2022). Genetic variation in P-element dysgenic sterility is associated with double-strand break repair and alternative splicing of TE transcripts. PLoS Genet 18(12): e1010080. PubMed ID: 36477699
The germline mobilization of transposable elements (TEs) by small RNA mediated silencing pathways is conserved across eukaryotes and critical for ensuring the integrity of gamete genomes. However, genomes are recurrently invaded by novel TEs through horizontal transfer. These invading TEs are not targeted by host small RNAs, and their unregulated activity can cause DNA damage in germline cells and ultimately lead to sterility. This study used hybrid dysgenesis-a sterility syndrome of Drosophila caused by transposition of invading P-element DNA transposons-to uncover host genetic variants that modulate dysgenic sterility. Using a panel of highly recombinant inbred lines of Drosophila melanogaster, two linked quantitative trait loci (QTL) were identified that determine the severity of dysgenic sterility in young and old females, respectively. Ovaries of fertile genotypes exhibit increased expression of splicing factors that suppress the production of transposase encoding transcripts, which likely reduces the transposition rate and associated DNA damage. It was also shown that fertile alleles are associated with decreased sensitivity to double-stranded breaks and enhanced DNA repair, explaining their ability to withstand high germline transposition rates. Together, this work reveals a diversity of mechanisms whereby host genotype modulates the cost of an invading TE, and points to genetic variants that were likely beneficial during the P-element invasion.
Morgunova, V. V., Sokolova, O. A., Sizova, T. V., Malaev, L. G., Babaev, D. S., Kwon, D. A. and Kalmykova, A. I. (2022). Dysfunction of Lamin B and Physiological Aging Cause Telomere Instability in Drosophila Germline. Biochemistry (Mosc) 87(12): 1600-1610. PubMed ID: 36717449
Chromatin spatial organization in the nucleus is essential for the genome functioning and regulation of gene activity. The nuclear lamina and lamina-associated proteins, lamins, play a key role in this process. Lamin dysfunction leads to the decompaction and transcriptional activation of heterochromatin, which is associated with the premature aging syndrome. In many cell types, telomeres are located at the nuclear periphery, where their replication and stability are ensured by the nuclear lamina. Moreover, diseases associated with defects in lamins and telomeres have similar manifestations and resemble physiological aging. Understanding molecular changes associated with aging at the organismal level is especially important. This study compared the effects caused by the mutation in lamin B and physiological aging in the germline of the model organism Drosophila melanogaster. The impaired localization of lamin B was shown to lead to the heterochromatin decompaction and transcriptional activation of some transposable elements and telomeric repeats. Both DNA damage and activation of homologous recombination in the telomeres were observed in the germ cells of lamin B mutants. The instability of repeat-enriched heterochromatin can be directly related to the genome destabilization, germ cell death, and sterility observed in lamin B mutants. Similar processes were observed in Drosophila germline in the course of physiological aging, which indicates a close link between the maintenance of the heterochromatin stability at the nuclear periphery and mechanisms of aging.
Gandara, L., Tsai, A., Ekelof, M., Galupa, R., Preger-Ben Noon, E., Alexandrov, T. and Crocker, J. (2022). Developmental phenomics suggests that H3K4 monomethylation confers multi-level phenotypic robustness. Cell Rep 41(11): 111832. PubMed ID: 36516782
How histone modifications affect animal development remains difficult to ascertain. Despite the prevalence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on phenotype and viability. This study genetically reduce H3K4me1 deposition in Drosophila melanogaster and found that hypomethylation reduces transcription factor enrichment in nuclear microenvironments, disrupts gene expression, and reduces phenotypic robustness. Using a developmental phenomics approach, changes were found in morphology, metabolism, behavior, and offspring production. However, many phenotypic changes are only detected when hypomethylated flies develop outside of standard laboratory environments or with specific genetic backgrounds. Therefore, quantitative phenomics measurements can unravel how pleiotropic modulators of gene expression affect developmental robustness under conditions resembling the natural environments of a species.
Herman, N., Kadener, S. and Shifman, S. (2022). The chromatin factor ROW cooperates with BEAF-32 in regulating long-range inducible genes. EMBO Rep 23(12): e54720. PubMed ID: 36245419
Insulator proteins located at the boundaries of topological associated domains (TAD) are involved in higher-order chromatin organization and transcription regulation. However, it is still not clear how long-range contacts contribute to transcriptional regulation. This study shows that relative-of-WOC (ROW) is essential for the long-range transcription regulation mediated by the boundary element-associated factor of 32kD (BEAF-32). ROW physically interacts with heterochromatin proteins (HP1b and HP1c) and the insulator protein BEAF-32. These proteins interact at TAD boundaries where ROW, through its AT-hook motifs, binds AT-rich sequences flanked by BEAF-32-binding sites and motifs. Knockdown of row downregulates genes that are long-range targets of BEAF-32 and bound indirectly by ROW (without binding motif). Analyses of high-throughput chromosome conformation capture (Hi-C) data reveal long-range interactions between promoters of housekeeping genes bound directly by ROW and promoters of developmental genes bound indirectly by ROW. Thus, these results show cooperation between BEAF-32 and the ROW complex, including HP1 proteins, to regulate the transcription of developmental and inducible genes through long-range interactions.

Thursday, February 23rd - Apoptosis

Periasamy, A., Mitchell, N., Zaytseva, O., Chahal, A. S., Zhao, J., Colman, P. M., Quinn, L. M. and Gulbis, J. M. (2022). An increase in mitochondrial TOM activates apoptosis to drive retinal neurodegeneration. Sci Rep 12(1): 21634. PubMed ID: 36517509
Intronic polymorphic TOMM40 variants increasing TOMM40 mRNA expression are strongly correlated to late onset Alzheimer's Disease. The gene product, hTomm40, encoded in the APOE gene cluster, is a core component of TOM, the translocase that imports nascent proteins across the mitochondrial outer membrane. This study used Drosophila melanogaster eyes as an in vivo model to investigate the relationship between elevated Tom40 (the Drosophila homologue of hTomm40) expression and neurodegeneration. Evidence is provided that an overabundance of Tom40 in mitochondria invokes caspase-dependent cell death in a dose-dependent manner, leading to degeneration of the primarily neuronal eye tissue. Degeneration is contingent on the availability of co-assembling TOM components, indicating that an increase in assembled TOM is the factor that triggers apoptosis and degeneration in a neural setting. Eye death is not contingent on inner membrane translocase components, suggesting it is unlikely to be a direct consequence of impaired import. Another effect of heightened Tom40 expression is upregulation and co-association of a mitochondrial oxidative stress biomarker, DmHsp22, implicated in extension of lifespan, providing new insight into the balance between cell survival and death. Activation of regulated death pathways, culminating in eye degeneration, suggests a possible causal route from TOMM40 polymorphisms to neurodegenerative disease.
Farrell, L., Puig-Barbe, A., Haque, M. I., Amcheslavsky, A., Yu, M., Bergmann, A. and Fan, Y. (2022). Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation. PLoS Genet 18(12): e1010533. PubMed ID: 36469525
Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. This study identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP.
Hargitai, D., Kenez, L., Al-Lami, M., Szenczi, G., Lorincz, P. and Juhasz, G. (2022). Autophagy controls Wolbachia infection upon bacterial damage and in aging Drosophila. Front Cell Dev Biol 10: 976882. PubMed ID: 36299486
Autophagy is a conserved catabolic process in eukaryotic cells that degrades intracellular components in lysosomes, often in an organelle-specific selective manner (mitophagy, ERphagy, etc). Cells also use autophagy as a defense mechanism, eliminating intracellular pathogens via selective degradation known as xenophagy. Wolbachia pipientis is a Gram-negative intracellular bacterium, which is one of the most common parasites on Earth affecting approximately half of terrestrial arthropods. Interestingly, infection grants the host resistance against other pathogens and modulates lifespan, so this bacterium resembles an endosymbiont. This study demonstrates that Drosophila somatic cells normally degrade a subset of these bacterial cells, and autophagy is required for selective elimination of Wolbachia upon antibiotic damage. In line with these, Wolbachia overpopulates in autophagy-compromised animals during aging while its presence fails to affect host lifespan unlike in case of control flies. The autophagic degradation of Wolbachia thus represents a novel antibacterial mechanism that controls the propagation of this unique bacterium, behaving both as parasite and endosymbiont at the same time.
Xu, L., Qiu, Y., Wang, X., Shang, W., Bai, J., Shi, K., Liu, H., Liu, J. P., Wang, L. and Tong, C. (2022). ER-mitochondrial contact protein Miga regulates autophagy through Atg14 and Uvrag. Cell Rep 41(5): 111583. PubMed ID: 36323251
Mitochondrial malfunction and autophagy defects are often concurrent phenomena associated with neurodegeneration. This study shows that Miga, a mitochondrial outer-membrane protein that regulates endoplasmic reticulum-mitochondrial contact sites (ERMCSs), is required for autophagy. Loss of Miga results in an accumulation of autophagy markers and substrates, whereas PI3P and Syx17 levels are reduced. Further experiments indicated that the fusion between autophagosomes and lysosomes is defective in Miga mutants. Miga binds to Atg14 and Uvrag; concordantly, Miga overexpression results in Atg14 and Uvrag recruitment to mitochondria. The heightened PI3K activity induced by Miga requires Uvrag, whereas Miga-mediated stabilization of Syx17 is dependent on Atg14. Miga-regulated ERMCSs are critical for PI3P formation but are not essential for the stabilization of Syx17. In summary, this study identified a mitochondrial protein that regulates autophagy by recruiting two alternative components of the PI3K complex present at the ERMCSs.
Ohta, T., Tanimura, T. and Kimura, K. I. (2022). A gain-of-function mutation in head involution defective, Wrinkled, causes precocious cell death of wing epidermal cells in Drosophila. MicroPubl Biol 2022. PubMed ID: 36606079
In Drosophila, wing epidermal cells undergo programmed cell death as the last step of metamorphosis. The aim of this study was to evaluate the role of hid, particularly the Wrinkled mutation (hidW), an allele of hid, in the cell death. The wing epithelial cell death is suppressed by loss-of-function mutation of hid, indicating that the death is governed by a cascade involving hid. Examination of the cell death in hidW showed that precocious death started at G stage, 3 h before eclosion. Thus, mutated-HID in the hidW mutant was activated at G stage, supporting the gain-of-function effect of hidW mutation.
Guo, X., Ma, X. and Xue, L. (2022). A conserved interplay between FOXO and SNAI/snail in autophagy. Autophagy 18(11): 2759-2760. PubMed ID: 35422194
Dysfunction of macroautophagy/autophagy has been implicated in homeostasis maintenance and contributes to various diseases. Yet the mechanisms that regulate autophagy have not been fully understood. In a recent study, a coherent FOXO3-SNAI2 feed-forward regulatory loop in mammals was uncovered that reinforces autophagy gene induction upon energy stress. Strikingly, a foxo-sna (snail) feed-forward circuit also exists in Drosophila, suggesting this regulating loop is evolutionarily conserved. Moreover, these results highlight that binding of FOXO3 to the DNA appears to be both necessary and sufficient to antagonize CRM1-dependent nuclear export, illustrating a critical role of DNA in regulating protein nuclear localization.

Wednesday, February 22nd - Adult Neural Development and Function

Munroe, J. A., Syed, M. H. and Doe, C. Q. (2022). Imp is required for timely exit from quiescence in Drosophila type II neuroblasts. PLoS One 17(12): e0272177. PubMed ID: 36520944
Stem cells must balance proliferation and quiescence, with excess proliferation favoring tumor formation, and premature quiescence preventing proper organogenesis. Drosophila brain neuroblasts are a model for investigating neural stem cell entry and exit from quiescence. Neuroblasts begin proliferating during embryogenesis, enter quiescence prior to larval hatching, and resume proliferation 12-30h after larval hatching. This study focussed on the mechanism used to exit quiescence, focusing on "type II" neuroblasts. There are 16 type II neuroblasts in the brain, and they undergo the same cycle of embryonic proliferation, quiescence, and proliferation as do most other brain neuroblasts. This study focussed on type II neuroblasts due to their similar lineage as outer radial glia in primates (both have extended lineages with intermediate neural progenitors), and because of the availability of specific markers for type II neuroblasts and their progeny. This study characterized the role of Insulin-like growth factor II mRNA-binding protein (Imp) in type II neuroblast proliferation and quiescence. Imp has previously been shown to promote proliferation in type II neuroblasts, in part by acting antagonistically to another RNA-binding protein called Syncrip (Syp). This study shows that reducing Imp levels delays exit from quiescence in type II neuroblasts, acting independently of Syp, with Syp levels remaining low in both quiescent and newly proliferating type II neuroblasts. It is concluded that Imp promotes exit from quiescence, a function closely related to its known role in promoting neuroblast proliferation.
Marquis, M. and Wilson, R. I. (2022). Locomotor and olfactory responses in dopamine neurons of the Drosophila superior-lateral brain. Curr Biol 32(24): 5406-5414.e5405. PubMed ID: 36450284
The Drosophila brain contains about 50 distinct morphological types of dopamine neurons. Physiological studies of Drosophila dopamine neurons have been largely limited to one brain region, the mushroom body, where they are implicated in learning. By comparison, little is known about the physiology of other Drosophila dopamine neurons. Interestingly, a recent whole-brain imaging study found that dopamine neuron activity in several fly brain regions is correlated with locomotion. This is notable because many dopamine neurons in the rodent brain are also correlated with locomotion or other movements; however, most rodent studies have focused on learned and rewarded behaviors, and few have investigated dopamine neuron activity during spontaneous (self-timed) movements. This study monitored dopamine neurons in the Drosophila brain during self-timed locomotor movements, focusing on several previously uncharacterized cell types that arborize in the superior-lateral brain, specifically the lateral horn and superior-lateral protocerebrum. It was found that activity of all of these dopamine neurons correlated with spontaneous fluctuations in walking speed, with different cell types showing different speed correlations. Some dopamine neurons also responded to odors, but these responses were suppressed by repeated odor encounters. Finally, the same identifiable dopamine neuron encode different combinations of locomotion and odor in different individuals. If these dopamine neurons promote synaptic plasticity-like the dopamine neurons of the mushroom body-then, their tuning profiles would imply that plasticity depends on a flexible integration of sensory signals, motor signals, and recent experience.
Lin, H. W., Chen, C. C., Jhang, R. Y., Chen, L., de Belle, J. S., Tully, T. and Chiang, A. S. (2022). CREBB repression of protein synthesis in mushroom body gates long-term memory formation in Drosophila. Proc Natl Acad Sci U S A 119(50): e2211308119. PubMed ID: 36469774
Learned experiences are not necessarily consolidated into long-term memory (LTM) unless they are periodic and meaningful. LTM depends on de novo protein synthesis mediated by cyclic AMP response element-binding protein (CREB) activity. In Drosophila, two creb genes (crebA, crebB) and multiple CREB isoforms have reported influences on aversive olfactory LTM in response to multiple cycles of spaced conditioning. How CREB isoforms regulate LTM effector genes in various neural elements of the memory circuit is unclear, especially in the mushroom body (MB), a prominent associative center in the fly brain that has been shown to participate in LTM formation. This study reports that 1) spaced training induces crebB expression in MB α-lobe neurons and 2) elevating specific CREBB isoform levels in the early α/β subpopulation of MB neurons enhances LTM formation. By contrast, learning from weak training 3) induces 5-HT1A serotonin receptor synthesis, 4) activates 5-HT1A in early α/β neurons, and 5) inhibits LTM formation. 6) LTM is enhanced when this inhibitory effect is relieved by down-regulating 5-HT1A or overexpressing CREBB. These findings show that spaced training-induced CREBB antagonizes learning-induced 5-HT1A in early α/β MB neurons to modulate LTM consolidation.
Llobet Rosell, A., Paglione, M., Gilley, J., Kocia, M., Perillo, G., Gasparrini, M., Cialabrini, L., Raffaelli, N., Angeletti, C., Orsomando, G., Wu, P. H., Coleman, M. P., Loreto, A. and Neukomm, L. J. (2022). The NAD(+) precursor NMN activates dSarm to trigger axon degeneration in Drosophila. Elife 11. PubMed ID: 36476387
Axon degeneration contributes to the disruption of neuronal circuit function in diseased and injured nervous systems. Severed axons degenerate following the activation of an evolutionarily conserved signaling pathway, which culminates in the activation of SARM1 in mammals to execute the pathological depletion of the metabolite NAD(+). SARM1 NADase activity is activated by the NAD(+) precursor nicotinamide mononucleotide (NMN). In mammals, keeping NMN levels low potently preserves axons after injury. However, it remains unclear whether NMN is also a key mediator of axon degeneration and dSarm activation in flies. This study demonstrates that lowering NMN levels in Drosophila through the expression of a newly generated prokaryotic NMN-Deamidase (NMN-D) preserves severed axons for months and keeps them circuit-integrated for weeks. NMN-D alters the NAD(+) metabolic flux by lowering NMN, while NAD(+) remains unchanged in vivo. Increased NMN synthesis by the expression of mouse nicotinamide phosphoribosyltransferase (mNAMPT) leads to faster axon degeneration after injury. It was also shown that NMN-induced activation of dSarm mediates axon degeneration in vivo. Finally, NMN-D delays neurodegeneration caused by loss of the sole NMN-consuming and NAD(+)-synthesizing enzyme dNmnat. These results reveal a critical role for NMN in neurodegeneration in the fly, which extends beyond axonal injury. The potent neuroprotection by reducing NMN levels is similar to the interference with other essential mediators of axon degeneration in Drosophila.
Ozel, M. N., Gibbs, C. S., Holguera, I., Soliman, M., Bonneau, R. and Desplan, C. (2022). Coordinated control of neuronal differentiation and wiring by sustained transcription factors. Science 378(6626): eadd1884. PubMed ID: 36480601
The large diversity of cell types in nervous systems presents a challenge in identifying the genetic mechanisms that encode it. This study reports that nearly 200 distinct neurons in the Drosophila visual system can each be defined by unique combinations of on average 10 continuously expressed transcription factors. Targeted modifications of this terminal selector code induce predictable conversions of neuronal fates that appear morphologically and transcriptionally complete. Cis-regulatory analysis of open chromatin links one of these genes to an upstream patterning factor that specifies neuronal fates in stem cells. Experimentally validated network models describe the synergistic regulation of downstream effectors by terminal selectors and ecdysone signaling during brain wiring. These results provide a generalizable framework of how specific fates are implemented in postmitotic neurons.
Deere, J. U., Sarkissian, A. A., Yang, M., Uttley, H. A., Martinez Santana, N., Nguyen, L., Ravi, K. and Devineni, A. V. (2023). Selective integration of diverse taste inputs within a single taste modality. Elife 12. PubMed ID: 36692370
This study investigated sensory processing in the Drosophila bitter taste system, which contains diverse bitter-sensing cells residing in different taste organs. First, subsets of bitter neurons were optogenetically activated within each organ. These subsets elicited broad and highly overlapping behavioral effects, suggesting that they converge onto common downstream pathways, but behavioral differences were observed that argue for biased convergence. Consistent with these results, transsynaptic tracing revealed that bitter neurons in different organs connect to overlapping downstream pathways with biased connectivity. Taste processing was investigated in one type of downstream bitter neuron that projects to the higher brain. These neurons integrate input from multiple organs and regulate specific taste-related behaviors. Downstream circuits were traced, providing the first glimpse into taste processing in the higher brain. Together, these results reveal that different bitter inputs are selectively integrated early in the circuit, enabling the pooling of information, while the circuit then diverges into multiple pathways that may have different roles.

Tuesday, February 21st - Evolution

Li, D., Gandhi, D., Kumon, T. and Yamashita, Y. M. (2022). Ribosomal DNA Instability as a Potential Cause of Karyotype Evolution. Mol Biol Evol 39(11). PubMed ID: 36223491
Karyotype refers to the configuration of the genome into a set of chromosomes. The karyotype difference between species is expected to impede various biological processes, such as chromosome segregation and meiotic chromosome pairing, potentially contributing to incompatibility. Karyotypes can rapidly change between closely related species and even among populations of the same species. However, the forces driving karyotype evolution are poorly understood. This study describes a unique karyotype of a Drosophila melanogaster strain isolated from the Seychelles archipelago. This strain has lost the ribosomal DNA (rDNA) locus on the X chromosome. Because the Y chromosome is the only other rDNA-bearing chromosome, all females carry at least one Y chromosome as the source of rDNA. Interestingly, it was found that the strain also carries a truncated Y chromosome (YS) that is stably maintained in the population despite its inability to support male fertility. Modeling and cytological analysis suggest that the Y chromosome has a larger negative impact on female fitness than the YS chromosome. Moreover, an independent strain was generated that lacks X rDNA and has a karyotype of XXY females and XY males. This strain quickly evolved multiple karyotypes: two new truncated Y chromosomes (similar to YS), as well as two independent X chromosome fusions that contain the Y-derived rDNA fragment, eliminating females' dependence on the Y chromosome. Considering that Robertsonian fusions frequently occur at rDNA loci in humans, it is proposed that rDNA loci instability may be one of driving forces of karyotype evolution.
Torosin, N. S., Golla, T. R., Lawlor, M. A., Cao, W. and Ellison, C. E. (2022). Mode and Tempo of 3D Genome Evolution in Drosophila. Mol Biol Evol 39(11). PubMed ID: 36201625
Topologically associating domains (TADs) are thought to play an important role in preventing gene misexpression by spatially constraining enhancer-promoter contacts. The deleterious nature of gene misexpression implies that TADs should, therefore, be conserved among related species. Several early studies comparing chromosome conformation between species reported high levels of TAD conservation; however, more recent studies have questioned these results. Furthermore, recent work suggests that TAD reorganization is not associated with extensive changes in gene expression.This study investigated the evolutionary conservation of TADs among 11 species of Drosophila. Hi-C data was used to identify TADs in each species and employ a comparative phylogenetic approach to derive empirical estimates of the rate of TAD evolution. Surprisingly, it was found that TADs evolve rapidly. However, it was also found that the rate of evolution depends on the chromatin state of the TAD, with TADs enriched for developmentally regulated chromatin evolving significantly slower than TADs enriched for broadly expressed, active chromatin. It was also found that, after controlling for differences in chromatin state, highly conserved TADs do not exhibit higher levels of gene expression constraint. These results suggest that, in general, most TADs evolve rapidly and their divergence is not associated with widespread changes in gene expression. However, higher levels of evolutionary conservation and gene expression constraints in TADs enriched for developmentally regulated chromatin suggest that these TAD subtypes may be more important for regulating gene expression, likely due to the larger number of long-distance enhancer-promoter contacts associated with developmental genes.
Morimoto, J. (2022). Uric acid metabolism modulates diet-dependent responses to intraspecific competition in Drosophila larvae. iScience 25(12): 105598. PubMed ID: 36458254
Intraspecific competition drives ecological specialization, and niche expansion. In holometabolous insects, larval intraspecific competition ("crowding") has lasting fitness consequences for individuals and shapes adaptive responses. However, understanding of the molecular profile of larval crowding responses remains allusive. This study used a genetic construct in Drosophila melanogaster with disrupted uric acid metabolism (Uro-) to demonstrate the role of the uric acid metabolism-and its interactive effects with larval crowding, diet, and urea concentration-on female oviposition and larval development. Uro- larvae developed faster in sugar-rich diets. However, Uro- larvae pupated at lower heights in protein- and sugar-rich diets in crowded larval conditions and showed slower development in protein-rich mixed-genotype environments. Uro- did not affect female oviposition nor larval pupation success. Overall, this study provides the first step toward an integrated understanding of the molecular pathways underpinning the responses to intraspecific competition in holometabolous insects.
Coughlan, J. M., Dagilis, A. J., Serrato-Capuchina, A., Elias, H., Peede, D., Isbell, K., Castillo, D. M., Cooper, B. S. and Matute, D. R. (2022). Patterns of Population Structure and Introgression Among Recently Differentiated Drosophila melanogaster Populations. Mol Biol Evol 39(11). PubMed ID: 36251862
This study examined patterns of population genetic structure, admixture, and the spatial structuring of candidate incompatibility alleles across a global sample, including 223 new accessions, predominantly from remote regions in Southern Africa. Nine major ancestries were identified, six that primarily occur in Africa and one that has not been previously described. Evidence was found for both contemporary and historical admixture between ancestries, with admixture rates varying both within and between continents. For example, while previous work has highlighted an admixture zone between broadly defined African and European ancestries in the Caribbean and southeastern USA, West African ancestry was identified as the most likely African contributor. Moreover, loci showing the strongest signal of introgression between West Africa and the Caribbean/southeastern USA include several genes relating to neurological development and male courtship behavior, in line with previous work showing shared mating behaviors between these regions. Finally, while it was hypothesized that potential incompatibility loci may contribute to population genetic structure across the range of D. melanogaster; these loci are, on average, not highly differentiated between ancestries. This work contributes to understanding of the evolutionary history of a key model system, and provides insight into the partitioning of diversity across its range.
Raja, K. K. B., Shittu, M. O., Nouhan, P. M. E., Steenwinkel, T. E., Bachman, E. A., Kokate, P. P., McQueeney, A., Mundell, E. A., Armentrout, A. A., Nugent, A. and Werner, T. (2022). The regulation of a pigmentation gene in the formation of complex color patterns in Drosophila abdomens. PLoS One 17(12): e0279061. PubMed ID: 36534652
Changes in the control of developmental gene expression patterns have been implicated in the evolution of animal morphology. However, the genetic mechanisms underlying complex morphological traits remain largely unknown. This study investigated the molecular mechanisms that induce the pigmentation gene yellow in a complex color pattern on the abdomen of Drosophila guttifera. At least five developmental genes may collectively activate one cis-regulatory module of yellow in distinct spot rows and a dark shade to assemble the complete abdominal pigment pattern of Drosophila guttifera. One of these genes, wingless, may play a conserved role in the early phase of spot pattern development in several species of the quinaria group. These findings shed light on the evolution of complex animal color patterns through modular changes of gene expression patterns.
De Lisle, S. P. (2022). Rapid evolution of ecological sexual dimorphism driven by resource competition. Ecol Lett. PubMed ID: 36366784
Sex differences in ecologically important traits are common in animals and plants, and prompted Darwin to first propose an ecological cause of sexual dimorphism. Despite theoretical plausibility and Darwin's original notion, a role for ecological resource competition in the evolution of sexual dimorphism has never been directly demonstrated and remains controversial. This study used experimental evolution in Drosophila melanogaster to test the hypothesis that resource competition can drive the evolution of sex differences in diet. Following just three generations of adaptation, offspring from flies evolved in low-resource, high-competition environments show elevated sexual dimorphism in diet preference compared to both the ancestor and populations evolved on high-resource availability. This increased sexual dimorphism was the result of divergence in male sucrose intake and female yeast intake consistent with the differential nutritional requirements of the sexes. These results provide the first real-time direct evidence for evolution of sexual dimorphism driven by resource competition.

Monday, February 20th - Larval and Adult Development

Chen, K., Yu, Y., Zhang, Z., Hu, B., Liu, X. and Tan, A. (2022). The morphogen Hedgehog is essential for proper adult morphogenesis in Bombyx mori. Insect Biochem Mol Biol 153: 103906. PubMed ID: 36587810
The well-known morphogen Hedgehog (Hh) is indispensable for embryo patterning and organ development from invertebrates to vertebrates. The role of Hh signaling pathway has been extensively investigated in the model organism Drosophila melanogaster, whereas its biological functions are still poorly understood in non-drosophilid insects. This study describes comprehensive investigation of Hh biological roles in the model lepidopteran insect Bombyx mori by using both CRISPR/Cas9-mediated gene ablation and Gal4/UAS-mediated ectopic expression. Direct injection of Cas9 protein and Hh-specific sgRNAs into preblastoderm embryos induced complete lethality. In contrast, Hh mutants obtained by the binary transgenic CRISPR/Cas9 system showed no deleterious phenotypes during embryonic and larval stages. However, mutants showed abnormalities from the pupal stage and most of adult body appendages exhibited severe developmental defects. Molecular analysis focused on wing development reveal that Hh signaling, Imd signaling and Wnt signaling pathways were distorted in Hh mutant wings. Ectopic expression by using the binary Gal4/UAS system induce early larval lethality. On contrary, moderate overexpression of Hh by using a unitary transgenic system resulted in severe defects in adult leg and antenna development. These data directly provide genetic evidence that Hh plays vital roles in imaginal discs development and proper adult morphogenesis in B. mori.
Chen, J., Zou, X., Zhu, W., Duan, Y., Merzendorfer, H., Zhao, Z. and Yang, Q. (2022). Fatty acid binding protein is required for chitin biosynthesis in the wing of Drosophila melanogaster. Insect Biochem Mol Biol 149: 103845. PubMed ID: 36165873
Chitin, the major structural polysaccharide in arthropods such as insects and mites, is a linear polymer of N-acetylglucosamine units. The growth and development of insects are intimately coupled with chitin biosynthesis. The membrane-bound β-glycosyltransferase chitin synthase is known to catalyze the key polymerization step of N-acetylglucosamine. However, the additional proteins that might assist chitin synthase during chitin biosynthesis are not well understood. Recently, fatty acid binding protein (Fabp) has been suggested as a candidate that interacts with the chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster. Using split-ubiquitin membrane yeast two-hybrid and pull-down assays, this study has demonstrated that the Fabp-B splice variant physically interacts with Kkv in vitro. The global knockdown of Fabp in D. melanogaster using RNA interference (RNAi) induced lethality at the larval stage. Moreover, in tissue-specific RNAi experiments, silenced Fabp expression in the epidermis and tracheal system caused a lethal larval phenotype. Fabp knockdown in the wings resulted in an abnormal wing development and uneven cuticular surface. In addition to reducing the chitin content in the first longitudinal vein of wings, Fabp silencing also caused the loss of procuticle laminate structures. This study revealed that Fabp plays an important role in chitin synthesis and contributes to a comprehensive understanding of the complex insect chitin biosynthesis.
Meyer, C., Breitsprecher, L., Bataille, L., Vincent, A. J. M., Drechsler, M., Meyer, H. and Paululat, A. (2022). Formation and function of a highly specialised type of organelle in cardiac valve cells. Development 149(19). PubMed ID: 36189830
Within a cell, vesicles play a crucial role in the transport of membrane material and proteins to a given target membrane, and thus regulate a variety of cellular functions. Vesicular transport occurs by means of, among others, endocytosis, where cargoes are taken up by the cell and are processed further upon vesicular trafficking, i.e. transported back to the plasma membrane via recycling endosomes or the degraded by fusion of the vesicles with lysosomes. During evolution, a variety of vesicles with individual functions arose, with some of them building up highly specialised subcellular compartments. This study analysed the biosynthesis of a new vesicular compartment present in the valve cells of Drosophila melanogaster. The compartment is formed by invaginations of the plasma membrane and grows via re-routing of the recycling endosomal pathway. This is achieved by inactivation of other membrane-consuming pathways and a plasma membrane-like molecular signature of the compartment in these highly specialised heart cells.
Meyer, C., Bataille, L., Drechsler, M. and Paululat, A. (2022). Tailup expression in Drosophila larval and adult cardiac valve cells. Genesis: e23506. PubMed ID: 36546531
n Drosophila larvae, the direction of blood flow within the heart tube, as well as the diastolic filling of the posterior heart chamber, is regulated by a single cardiac valve. This valve is sufficient to close the heart tube at the junction of the ventricle and the aorta and is formed by only two cells; both are integral parts of the heart tube. The valve cells regulate hemolymph flow by oscillating between a spherical and a flattened cell shape during heartbeats. At the spherical stage, the opposing valve cells close the heart lumen. The dynamic cell shape changes of valve cells are supported by a dense, criss-cross orientation of myofibrils and the presence of the valvosomal compartment, a large intracellular cavity. Both structures are essential for the valve cells' function. In a screen for factors specifically expressed in cardiac valve cells, the transcription factor Tailup was identified. Knockdown of tailup causes abnormal orientation and differentiation of cardiac muscle fibers in the larval aorta and inhibits the formation of the ventral longitudinal muscle layer located underneath the heart tube in the adult fly and affects myofibrillar orientation of valve cells. Furthermore, this study has identified regulatory sequences of tup that control the expression of tailup in the larval and adult valve cells.
Doerksen, A., Mulder, M., Ingram, S., Nelson, D., Defehr, J., Reimer, E., Atallah, J. and Malagon, J. N. (2022). Dynamics of changes in apical cell area during sex comb rotation in Drosophila melanogaster. MicroPubl Biol 2022. PubMed ID: 36606080
Epithelia are highly dynamic tissues displaying various types of tissue rearrangements. This study describes the dynamics of changes in apical cell area (ACA) in an epithelial system displaying tissue rearrangement resulting in sex comb rotation on the forelegs of male Drosophila melanogaster. The sex comb is a row of leg bristles which rotates during morphogenesis. The ACA was quantified in the region proximal to the developing sex comb by tracing apical cell boundaries using ImageJ in pupal first leg imaginal discs. Cells display intricate irregular oscillations in size as the comb rotates. However, the net changes in ACA within most of the cells studied are subtle, only 0 to +/-15%. The current working hypothesis suggests these irregular oscillations confer flexibility during tissue rearrangement and can be an important mechanism for tissue homeostasis.
Nagy, A., Szenci, G., Boda, A., Al-Lami, M., Csizmadia, T., Lorincz, P., Juhasz, G. and Low, P. (2022). Ecdysone receptor isoform specific regulation of secretory granule acidification in the larval Drosophila salivary gland. Eur J Cell Biol 101(4): 151279. PubMed ID: 36306596
Bulk production and release of glue containing secretory granules takes place in the larval salivary gland during Drosophila development in order to attach the metamorphosing animal to a dry surface. These granules undergo a maturation process to prepare glue for exocytosis, which includes homotypic fusions to increase the size of granules, vesicle acidification and ion uptake. The steroid hormone 20-hydroxyecdysone is known to be required for the first and last steps of this process: glue synthesis and secretion, respectively. This study shows that the B1 isoform of Ecdysone receptor (EcR), together with its binding partner Ultraspiracle, are also necessary for the maturation of glue granules by promoting their acidification via regulation of Vha55 expression, which encodes an essential subunit of the V-ATPase proton pump. This is antagonized by the EcR-A isoform, overexpression of which decreases EcR-B1 and Vha55 expression and glue granule acidification. These data shed light on a previously unknown, ecdysone receptor isoform-specific regulation of glue granule maturation.

Friday, February 17th - Disease Models

Huber, R. G., Pandey, S., Chhangani, D., Rincon-Limas, D. E., Staff, N. P. and Yeo, C. J. J. (2022). Identification of potential pathways and biomarkers linked to progression in ALS. Ann Clin Transl Neurol. PubMed ID: 36533811
To identify potential diagnostic and prognostic biomarkers for clinical management and clinical trials in amyotrophic lateral sclerosis. Proteomics data of ALS patient-induced pluripotent stem cell-derived motor neurons available through the AnswerALS consortium were analyzed. After stratifying patients using clinical ALSFRS-R and ALS-CBS scales, differentially expressed proteins indicative of ALS disease severity and progression rate were identified as candidate ALS-related and prognostic biomarkers. Pathway analysis for identified proteins was performed using STITCH. Protein sets were correlated with the effects of drugs using the Connectivity Map tool to identify compounds likely to affect similar pathways. RNAi screening was performed in a Drosophila TDP-43 ALS model to validate pathological relevance. A statistical classification machine learning model was constructed using ridge regression that uses proteomics data to differentiate ALS patients from controls. This study identified 76, 21, 71 and 1 candidate ALS-related biomarkers and 22, 41, 27 and 64 candidate prognostic biomarkers from patients stratified by ALSFRS-R baseline, ALSFRS-R progression slope, ALS-CBS baseline and ALS-CBS progression slope, respectively. Nineteen proteins enhanced or suppressed pathogenic eye phenotypes in the ALS fly model. Nutraceuticals, dopamine pathway modulators, statins, anti-inflammatories and antimicrobials were predicted starting points for drug repurposing using the connectivity map tool. Ten diagnostic biomarker proteins were predicted by machine learning to identify ALS patients with high accuracy and sensitivity. This study showcases the powerful approach of iPSC-motor neuron proteomics combined with machine learning and biological confirmation in the prediction of novel mechanisms and diagnostic and predictive biomarkers in ALS.
Khalil, B., Chhangani, D., .., Rincon-Limas, D. E. and Rossoll, W. (2022). Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy. Mol Neurodegener 17(1): 80. PubMed ID: 36482422
Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. This study examined the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. Several members of the nuclear import receptor protein family were able to reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, its activity was found to depend on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. These results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. hese findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration.
Kim, J., de Haro, M., Al-Ramahi, I., Garaicoechea, L. L., Jeong, H. H., Sonn, J. Y., Tadros, B., Liu, Z., Botas, J. and Zoghbi, H. Y. (2022). Evolutionarily conserved regulators of tau identify targets for new therapies. Neuron. PubMed ID: 36610398
Tauopathies are neurodegenerative diseases that involve the pathological accumulation of tau proteins; in this family are Alzheimer disease, corticobasal degeneration, and chronic traumatic encephalopathy, among others. Hypothesizing that reducing this accumulation could mitigate pathogenesis, a cross-species genetic screen was performed targeting 6,600 potentially druggable genes in human cells and Drosophila. 83 hits were found and validated in cells and 11 hits were further validated in the mouse brain. Three of these hits (USP7, RNF130, and RNF149) converge on the C terminus of Hsc70-interacting protein (CHIP) to regulate tau levels, highlighting the role of CHIP in maintaining tau proteostasis in the brain. Knockdown of each of these three genes in adult tauopathy mice reduced tau levels and rescued the disease phenotypes. This study thus identifies several points of intervention to reduce tau levels and demonstrates that reduction of tau levels via regulation of this pathway is a viable therapeutic strategy for Alzheimer disease and other tauopathies.
Mandik, F., Kanana, Y., Rody, J., Misera, S., Wilken, B., Laabs von Holt, B. H., Klein, C. and Vos, M. (2022). A new model for fatty acid hydroxylase-associated neurodegeneration reveals mitochondrial and autophagy abnormalities. Front Cell Dev Biol 10: 1000553. PubMed ID: 36589738
Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a rare disease that exhibits brain modifications and motor dysfunctions in early childhood. The condition is caused by a homozygous or compound heterozygous mutation in fatty acid 2 hydroxylase (FA2H), whose encoded protein synthesizes 2-hydroxysphingolipids and 2-hydroxyglycosphingolipids and is therefore involved in sphingolipid metabolism. Drosophila is an excellent model for many neurodegenerative disorders; hence, this study has characterized and validated the first FAHN Drosophila model. The investigation of loss of dfa2h lines revealed behavioral abnormalities, including motor impairment and flying disability, in addition to a shortened lifespan. Furthermore, alterations in mitochondrial dynamics, and autophagy were identified. Analyses of patient-derived fibroblasts, and rescue experiments with human FA2H, indicated that these defects are evolutionarily conserved. This study thus presents a FAHN Drosophila model organism that provides new insights into the cellular mechanism of FAHN.
Kodani, A., Yamaguchi, M., Itoh, R., Huynh, M. A. and Yoshida, H. (2022). A Drosophila model of the neurological symptoms in Mpv17-related diseases. Sci Rep 12(1): 22632. PubMed ID: 36587049
Mutations in the Mpv17 gene are responsible for MPV17-related hepatocerebral mitochondrial DNA depletion syndrome and Charcot-Marie-Tooth (CMT) disease. CG11077 (Drosophila Mpv17; dMpv17), an ortholog of human MPV17, was knocked down in the nervous system in Drosophila melanogaster and the behavioral and cellular phenotypes were investigated. The resulting dMpv17 knockdown larvae showed impaired locomotor activity and learning ability consistent with mitochondrial defects suggested by the reductions in mitochondrial DNA and ATP production and the increases in the levels of lactate and reactive oxygen species. Furthermore, an abnormal morphology of the neuromuscular junction, at the presynaptic terminal, was observed in dMpv17 knockdown larvae. These results reproduce well the symptoms of human diseases and partially reproduce the phenotypes of Mpv17-deficient model organisms. Therefore, it is suggested that neuron-specific dMpv17 knockdown in Drosophila is a useful model for investigation of MPV17-related hepatocerebral mitochondrial DNA depletion syndrome and CMT caused by Mpv17 dysfunction.
Krausel, V., Pund, L., Nusse, H., Bachir, H., Ricker, A., Klingauf, J., Weide, T., Pavenstadt, H., Krahn, M. P. and Braun, D. A. (2022). The transcription factor ATF4 mediates endoplasmic reticulum stress-related podocyte injury and slit diaphragm defects. Kidney Int. PubMed ID: 36587794
Mutations in OSGEP and four other genes that encode subunits of the KEOPS complex cause Galloway-Mowat syndrome, a severe, inherited kidney-neurological disease. The complex catalyzes an essential posttranscriptional modification of tRNA and its loss of function induces endoplasmic reticulum (ER) stress. Using Drosophila garland nephrocytes and cultured human podocytes, this study aimed to elucidate the molecular pathogenic mechanisms of KEOPS-related glomerular disease and to test pharmacological inhibition of ER stress-related signaling as a therapeutic principle. ATF4, an ER stress-mediating transcription factor, or its fly orthologue Crc, were upregulated in both fly nephrocytes and human podocytes. Knockdown of Tcs3, a fly orthologue of OSGEP, caused slit diaphragm defects, recapitulating the human kidney phenotype. OSGEP cDNA with mutations found in patients lacked the capacity for rescue. Genetic interaction studies in Tcs3-deficient nephrocytes revealed that Crc mediates not only cell injury, but surprisingly also slit diaphragm defects, and that genetic or pharmacological inhibition of Crc activation attenuates both phenotypes. These findings are conserved in human podocytes where ATF4 inhibition improved the viability of podocytes with OSGEP knockdown, with chemically induced ER stress, and where ATF4 target genes and pro-apoptotic gene clusters are upregulated upon OSGEP knockdown. Thus, these data identify ATF4-mediated signaling as a molecular link among ER stress, slit diaphragm defects, and podocyte injury, and the data suggest that modulation of ATF4 signaling may be a potential therapeutic target for certain podocyte diseases.

Thursday, February 16th - Adult neural development and function

Zhuravlev, A. V., Ivanova, P. N., Makaveeva, K. A., Zakharov, G. A., Nikitina, E. A. and Savvateeva-Popova, E. V. (2022). cd(1) Mutation in Drosophila Affects Phenoxazinone Synthase Catalytic Site and Impairs Long-Term Memory. Int J Mol Sci 23(20). PubMed ID: 36293213
Being involved in development of Huntington's, Parkinson's and Alzheimer's diseases, kynurenine pathway (KP) of tryptophan metabolism plays a significant role in modulation of neuropathology. Accumulation of a prooxidant 3-hydroxykynurenine (3-HOK) leads to oxidative stress and neuronal cell apoptosis. Drosophila mutant cardinal (cd1 with 3-HOK excess shows age-dependent neurodegeneration and short-term memory impairments, thereby presenting a model for senile dementia. Although cd gene for phenoxazinone synthase (PHS) catalyzing 3-HOK dimerization has been presumed to harbor the cd1 mutation, its molecular nature remained obscure. Using next generation sequencing, this study has shown that the cd gene in cd1 carries a long deletion leading to PHS active site destruction. Contrary to the wild type Canton-S (CS), cd1 males showed defective long-term memory (LTM) in conditioned courtship suppression paradigm (CCSP) at days 5-29 after eclosion. The number of dopaminergic neurons (DAN) regulating fly locomotor activity showed an age-dependent tendency to decrease in cd1 relative to CS. Thus, in accordance with the concept "from the gene to behavior" proclaimed by S. Benzer, this study has shown that the aberrant PHS sequence in cd1 provokes drastic LTM impairments and DAN alterations.
Yamagata, N., Imanishi, Y., Wu, H., Kondo, S., Sano, H. and Tanimoto, H. (2022). Nutrient responding peptide hormone CCHamide-2 consolidates appetitive memory. Front Behav Neurosci 16: 986064. PubMed ID: 36338876
CCHamide-2 (CCHa2) is a protostome excitatory peptide ortholog known for various arthropod species. In fruit flies, CCHa2 plays a crucial role in the endocrine system, allowing peripheral tissue to communicate with the central nervous system to ensure proper development and the maintenance of energy homeostasis. Since the formation of odor-sugar associative long-term memory (LTM) depends on the nutrient status in an animal, CCHa2 may play an essential role in linking memory and metabolic systems. This study shows that CCHa2 signals are important for consolidating appetitive memory by acting on the rewarding dopamine neurons. Genetic disruption of CCHa2 using mutant strains abolished appetitive LTM but not short-term memory (STM). A post-learning thermal suppression of CCHa2 expressing cells impaired LTM. In contrast, a post-learning thermal activation of CCHa2 cells stabilized STM induced by non-nutritious sugar into LTM. The receptor of CCHa2, CCHa2-R, was expressed in a subset of dopamine neurons that mediate reward for LTM. In accordance, the receptor expression in these dopamine neurons was required for LTM specifically. It is thus concluded that CCHa2 conveys a sugar nutrient signal to the dopamine neurons for memory consolidation. This finding establishes a direct interplay between brain reward and the putative endocrine system for long-term energy homeostasis.
Chen, X., Li, J., Gao, Z., Yang, Y., Kuang, W., Dong, Y., Chua, G. H., Huang, X., Jiang, B., Tian, H., Wang, Y., Huang, X., Li, Y., Lam, S. M. and Shui, G. (2022). Endogenous ceramide phosphoethanolamine modulates circadian rhythm via neural-glial coupling in Drosophila. Natl Sci Rev 9(12): nwac148. PubMed ID: 36713590
While endogenous lipids are known to exhibit rhythmic oscillations, less is known about how specific lipids modulate circadian behavior. Through a series of loss-of-function and gain-of-function experiments on ceramide phosphoethanolamine (CPE) synthase of Drosophila, it was demonstrated that pan-glial-specific deficiency in membrane CPE, the structural analog of mammalian sphingomyelin (SM), leads to arrhythmic locomotor behavior and shortens lifespan, while the reverse is true for increasing CPE. Comparative proteomics uncovered dysregulated synaptic glutamate utilization and transport in CPE-deficient flies. An extensive genetic screen was conducted to verify the role of differentially expressed proteins in circadian regulation. Arrhythmic locomotion under cpes(1) mutant background was rescued only by restoring endogenous CPE or SM through expressing their respective synthases. These results underscore the essential role of CPE in maintaining synaptic glutamate homeostasis and modulating circadian behavior in Drosophila. The findings suggest that region-specific elevations of functional membrane lipids can benefit circadian regulation.
Zocchi, D., Ye, E. S., Hauser, V., O'Connell, T. F. and Hong, E. J. (2022). Parallel encoding of CO(2) in attractive and aversive glomeruli by selective lateral signaling between olfactory afferents. Curr Biol 32(19): 4225-4239. PubMed ID: 36070776
This study describes a novel form of selective crosstalk between specific classes of primary olfactory receptor neurons (ORNs) in the Drosophila antennal lobe. Neurotransmitter release from ORNs is driven by two distinct sources of excitation: direct activity derived from the odorant receptor and stimulus-selective lateral signals originating from stereotypic subsets of other ORNs. Consequently, the level of presynaptic neurotransmitter release from an ORN can be significantly dissociated from its firing rate. Stimulus-selective lateral signaling results in the distributed representation of CO(2)-a behaviorally important environmental cue that directly excites a single ORN class-in multiple olfactory glomeruli, each with distinct response dynamics. CO(2)-sensitive glomeruli coupled to behavioral attraction respond preferentially to fast changes in CO(2) concentration, whereas those coupled to behavioral aversion more closely follow absolute levels of CO(2). Behavioral responses to CO(2) also depend on the temporal structure of the stimulus: flies walk upwind to fluctuating, but not sustained, pulses of CO(2). Stimulus-selective lateral signaling generalizes to additional odors and glomeruli, revealing a subnetwork of lateral interactions between ORNs that reshapes the spatial and temporal structure of odor representations in a stimulus-specific manner.
Lee, Y., Wang, M., Imamura, K. and Sato, M. (2022). Quantitative analysis of the roles of IRM cell adhesion molecules in column formation in the fly brain. Dev Growth Differ. PubMed ID: 36534021
The Drosophila visual center shows columnar structures, basic structural and functional units of the brain, that are shared with the mammalian cerebral cortex. Visual information received in the ommatidia in the compound eye is transmitted to the columns in the brain. However, the developmental mechanisms of column formation are largely unknown. The Irre Cell Recognition Module (IRM) proteins are a family of immunoglobulin cell adhesion molecules. The four Drosophila IRM proteins are localized to the developing columns, the structure of which is affected in IRM mutants, suggesting that IRM proteins are essential for column formation. Since IRM proteins are cell adhesion molecules, they may regulate cell adhesion between columnar neurons. To test this possibility, this study specifically knocked down IRM genes in columnar neurons and examined the defects in column formation. A system was developed that automatically extracts the individual column images and quantifies the column shape. Using this system, it was demonstrated that IRM genes play critical roles in regulating column shape in a core columnar neuron, Mi1. It was also shown that their expression in the other columnar neurons, Mi4 and T4/5, is essential, suggesting that the interactions between IRM proteins and multiple neurons shape the columns in the fly brain.
Fisher, Y. E., Marquis, M., D'Alessandro, I. and Wilson, R. I. (2022). Dopamine promotes head direction plasticity during orienting movements. Nature 612(7939): 316-322. PubMed ID: 36450986
In neural networks that store information in their connection weights, there is a tradeoff between sensitivity and stability. Connections must be plastic to incorporate new information, but if they are too plastic, stored information can be corrupted. A potential solution is to allow plasticity only during epochs when task-specific information is rich, on the basis of a 'when-to-learn' signal. It was reasoned that dopamine provides a when-to-learn signal that allows the brain's spatial maps to update when new spatial information is available-that is, when an animal is moving. This study shows that the dopamine neurons innervating the Drosophila head direction network are specifically active when the fly turns to change its head direction. Moreover, their activity scales with moment-to-moment fluctuations in rotational speed. Pairing dopamine release with a visual cue persistently strengthens the cue's influence on head direction cells. Conversely, inhibiting these dopamine neurons decreases the influence of the cue. This mechanism should accelerate learning during moments when orienting movements are providing a rich stream of head direction information, allowing learning rates to be low at other times to protect stored information. These results show how spatial learning in the brain can be compressed into discrete epochs in which high learning rates are matched to high rates of information intake.

Wednesday, February 15th - Disease Models

Ding, M., Li, Q. F., Peng, T. H., Wang, T. Q., Yan, H. H., Tang, C., Wang, X. Y., Guo, Y. and Zheng, L. (2022). Early life exercise training and inhibition of apoLpp mRNA expression to improve age-related arrhythmias and prolong the average lifespan in Drosophila melanogaster. Aging (Albany NY) 14(24): 9908-9923. PubMed ID: 36470666
Cardiovascular disease (CVD) places a heavy burden on older patients and the global healthcare system. A large body of evidence suggests that exercise training is essential in preventing and treating cardiovascular disease, but the underlying mechanisms are not well understood. This study used the Drosophila melanogaster animal model to study the effects of early-life exercise training (Exercise) on the aging heart and lifespan. It was found in flies that age-induced arrhythmias are conserved across different genetic backgrounds. The fat body is the primary source of circulating lipoproteins in flies. Inhibition of fat body apoLpp (Drosophila apoB homolog) demonstrated that low expression of apoLpp reduced the development of arrhythmias in aged flies but did not affect average lifespan. At the same time, exercise can also reduce the expression of apoLpp mRNA in aged flies and have a protective effect on the heart, which is similar to the inhibition of apoLpp mRNA. Although treatment of UAS-apoLpp(RNAi) and exercise alone had no significant effect on lifespan, the combination of UAS-apoLpp(RNAi) and exercise extended the average lifespan of flies. Therefore, it is concluded that UAS-apoLpp(RNAi) and exercise are sufficient to resist age-induced arrhythmias, which may be related to the decreased expression of apoLpp mRNA, and that UAS-apoLpp(RNAi) and exercise have a combined effect on prolonging the average lifespan.
Gandini, A., Goncalves, A. E., Strocchi, S., Albertini, C., Janockova, J., Tramarin, A., Grifoni, D., Poeta, E., Soukup, O., Munoz-Torrero, D., Monti, B., Sabate, R., Bartolini, M., Legname, G. and Bolognesi, M. L. (2022). Discovery of Dual Abeta/Tau Inhibitors and Evaluation of Their Therapeutic Effect on a Drosophila Model of Alzheimer's Disease. ACS Chem Neurosci 13(23): 3314-3329. PubMed ID: 36445009
Alzheimer's disease (AD), the most common type of dementia, currently represents an extremely challenging and unmet medical need worldwide. Amyloid-β (Aβ) and Tau proteins are prototypical AD hallmarks, as well as validated drug targets. Accumulating evidence now suggests that they synergistically contribute to disease pathogenesis. This could not only help explain negative results from anti-Aβ clinical trials but also indicate that therapies solely directed at one of them may have to be reconsidered. This study describes the development of a focused library of 2,4-thiazolidinedione (TZD)-based bivalent derivatives as dual Aβ and Tau aggregation inhibitors. The aggregating activity of the 24 synthesized derivatives was tested in intact Escherichia coli cells overexpressing Aβ(42) and Tau proteins. Their neuronal toxicity and ability to cross the blood-brain barrier (BBB) were evaluated, together with the in vitro interaction with the two isolated proteins. Finally, the most promising (most active, nontoxic, and BBB-permeable) compounds 22 and 23 were tested in vivo, in a Drosophila melanogaster model of AD. The carbazole derivative 22 (20 &mi;M) showed extremely encouraging results, being able to improve both the lifespan and the climbing abilities of Aβ(42) expressing flies and generating a better outcome than doxycycline (50 &mi;M). Moreover, 22 proved to be able to decrease Aβ(42) aggregates in the brains of the flies. It is concluded that bivalent small molecules based on 22 deserve further attention as hits for dual Aβ/Tau aggregation inhibition in AD.
Asadi, P., Milev, M. P., Saint-Dic, D., Gamberi, C. and Sacher, M. (2022). Vitamin B5, a coenzyme A precursor, rescues TANGO2 deficiency disease-associated defects in Drosophila and human cells. J Inherit Metab Dis. PubMed ID: 36502486
Mutations in the Transport and Golgi Organization 2 (TANGO2) gene are associated with intellectual deficit, neurodevelopmental delay and regression. Individuals can also present with an acute metabolic crisis that includes rhabdomyolysis, cardiomyopathy, and cardiac arrhythmias, the latter of which are potentially lethal. While preventing metabolic crises has the potential to reduce mortality, no treatments currently exist for this condition. The function of TANGO2 remains unknown but is suspected to be involved in some aspect of lipid metabolism. This study describes a model of TANGO2-related disease in the fruit fly Drosophila melanogaster that recapitulates crucial disease traits. Pairing a new fly model with human cells, the effects were examined of vitamin B5, a coenzyme A (CoA) precursor, on alleviating the cellular and organismal defects associated with TANGO2 deficiency. It was demonstrated that vitamin B5 specifically improves multiple defects associated with TANGO2 loss-of-function in Drosophila and rescues membrane trafficking defects in human cells. A partial rescue was observed of one of the fly defects by vitamin B3, though to a lesser extent than vitamin B5. These data suggest that a B complex supplement containing vitamin B5/pantothenate may have therapeutic benefits in individuals with TANGO2-deficiency disease. Possible mechanisms for the rescue are discussed that may include restoration of lipid homeostasis.
Farago, A., Zsindely, N., Farkas, A., Neller, A., Siagi, F., Szabo, M. R., Csont, T. and Bodai, L. (2022). Acetylation State of Lysine 14 of Histone H3.3 Affects Mutant Huntingtin Induced Pathogenesis. Int J Mol Sci 23(23). PubMed ID: 36499499
Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by the expansion of a polyglutamine-coding CAG repeat in the Huntingtin gene. One of the main causes of neurodegeneration in HD is transcriptional dysregulation that, in part, is caused by the inhibition of histone acetyltransferase (HAT) enzymes. HD pathology can be alleviated by increasing the activity of specific HATs or by inhibiting histone deacetylase (HDAC) enzymes. To determine which histone's post-translational modifications (PTMs) might play crucial roles in HD pathology, this study investigated the phenotype-modifying effects of PTM mimetic mutations of variant histone H3.3 in a Drosophila model of HD. Specifically, the mutations (K→Q: acetylated; K→R: non-modified; and K→M: methylated) of lysine residues K9, K14, and K27 of transgenic H3.3 was studied. In the case of H3.3K14Q modification, the amelioration was observed of all tested phenotypes (viability, longevity, neurodegeneration, motor activity, and circadian rhythm defects), while H3.3K14R had the opposite effect. H3.3K14Q expression prevented the negative effects of reduced Gcn5 (a HAT acting on H3K14) on HD pathology, while it only partially hindered the positive effects of heterozygous Sirt1 (an HDAC acting on H3K14). Thus, it is concluded that the Gcn5-dependent acetylation of H3.3K14 might be an important epigenetic contributor to HD pathology.
Hernandez, S. J., Lim, R. G., Onur, T., Dane, M. A., Smith, R., Wang, K., Jean, G. E., Devlin, K., Miramontes, R., Wu, J., Casale, M., Kilburn, D., Heiser, L. M., Korkola, J. E., Van Vactor, D., Botas, J., Thompson-Peer, K. L. and Thompson, L. M. (2022). An altered extracellular matrix-integrin interface contributes to Huntington's disease-associated CNS dysfunction in glial and vascular cells. Hum Mol Genet. PubMed ID: 36547263
Astrocytes and brain endothelial cells are components of the neurovascular unit that comprises the blood brain barrier (BBB) and their dysfunction contributes to pathogenesis in Huntington's disease (HD). Defining the contribution of these cells to disease can inform cell-type specific effects and uncover new disease-modifying therapeutic targets. These cells express integrin (ITG) adhesion receptors that anchor the cells to the extracellular matrix (ECM) to maintain the integrity of the BBB. HD patient-derived induced pluripotent stem cell (iPSC) modeling was used to study the ECM-ITG interface in astrocytes and brain microvascular endothelial cells (BMECs) and found ECM-ITG dysregulation in human iPSC-derived cells that may contribute to dysfunction of the BBB in HD. This disruption has functional consequences since reducing ITG expression in glia in an HD Drosophila model suppressed disease-associated CNS dysfunction. Since ITGs can be targeted therapeutically and manipulating ITG signaling prevents neurodegeneration in other diseases, defining the role of ITGs in HD may provide a novel strategy of intervention to slow CNS pathophysiology to treat HD.
Sinha Ray, S., Dutta, D., Dennys, C., Powers, S., Roussel, F., Lisowski, P., Glažar, P., Zhang, X., Biswas, P., Caporale, J. R., Rajewsky, N., Bickle, M., Wein, N., Bellen, H. J., Likhite, S., Marcogliese, P. C. and Meyer, K. C. (2022). Mechanisms of IRF2BPL-related disorders and identification of a potential therapeutic strategy. Cell Rep 41(10): 111751. PubMed ID: 36476864
The recently discovered neurological disorder NEDAMSS is caused by heterozygous truncations in the transcriptional regulator IRF2BPL. This study reprogram patient skin fibroblasts to astrocytes and neurons to study mechanisms of this newly described disease. While full-length IRF2BPL primarily localizes to the nucleus, truncated patient variants sequester the wild-type protein to the cytoplasm and cause aggregation. Moreover, patient astrocytes fail to support neuronal survival in coculture and exhibit aberrant mitochondria and respiratory dysfunction. Treatment with the small molecule copper ATSM (CuATSM) rescues neuronal survival and restores mitochondrial function. Importantly, the in vitro findings are recapitulated in vivo, where co-expression of full-length and truncated IRF2BPL in Drosophila results in cytoplasmic accumulation of full-length IRF2BPL. Moreover, flies harboring heterozygous truncations of the IRF2BPL ortholog (Pits) display progressive motor defects that are ameliorated by CuATSM treatment. These findings provide insights into mechanisms involved in NEDAMSS and reveal a promising treatment for this severe disorder.

Tuesday, February 14th - Enzymes and protein expression, evolution, structure, and function

Cooper, R. L. and Krall, R. M. (2022). Hyperpolarization Induced by Lipopolysaccharides but Not by Chloroform Is Inhibited by Doxapram, an Inhibitor of Two-P-Domain K(+) Channel (K2P). Int J Mol Sci 23(24). PubMed ID: 36555429
Bacterial septicemia is commonly induced by Gram-negative bacteria. The immune response is triggered in part by the secretion of bacterial endotoxin lipopolysaccharide (LPS). LPS induces the subsequent release of inflammatory cytokines which can result in pathological conditions. There is no known blocker to the receptors of LPS. The Drosophila larval muscle is an amendable model to rapidly screen various compounds that affect membrane potential and synaptic transmission such as LPS. LPS induces a rapid hyperpolarization in the body wall muscles and depolarization of motor neurons. These actions are blocked by the compound doxapram (10 mM), which is known to inhibit a subtype of the two-P-domain K+ channel (K2P channels). However, the K2P channel blocker PK-THPP had no effect on the Drosophila larval muscle at 1 and 10 mM. These channels are activated by chloroform, which also induces a rapid hyperpolarization of these muscles, but the channels are not blocked by doxapram. Likewise, chloroform does not block the depolarization induced by doxapram. LPS blocks the postsynaptic glutamate receptors on Drosophila muscle. Pre-exposure to doxapram reduces the LPS block of these ionotropic glutamate receptors. Given that the larval Drosophila body wall muscles are depolarized by doxapram and hyperpolarized by chloroform, they offer a model to begin pharmacological profiling of the K2P subtype channels with the potential of identifying blockers for the receptors to mitigate the actions of the Gram-negative endotoxin LPS.
Nandigrami, P., Szczepaniak, F., Boughter, C. T., Dehez, F., Chipot, C. and Roux, B. (2022). Computational Assessment of Protein-Protein Binding Specificity within a Family of Synaptic Surface Receptors. J Phys Chem B 126(39): 7510-7527. PubMed ID: 35787023
Atomic-level information is essential to explain the formation of specific protein complexes in terms of structure and dynamics. The set of Dpr and DIP proteins, which play a key role in the neuromorphogenesis in the nervous system of Drosophila melanogaster, offer a rich paradigm to learn about protein-protein recognition. Many members of the DIP subfamily cross-react with several members of the Dpr family and vice versa. While there exists a total of 231 possible Dpr-DIP heterodimer complexes from the 21 Dpr and 11 DIP proteins, only 57 "cognate" pairs have been detected by surface plasmon resonance (SPR) experiments, suggesting that the remaining 174 pairs have low or unreliable binding affinity. The goal of this study was to assess the performance of computational approaches to characterize the global set of interactions between Dpr and DIP proteins and identify the specificity of binding between each DIP with their corresponding Dpr binding partners. In addition, this study aimed to characterize how mutations influence the specificity of the binding interaction. In this work, a wide range of knowledge-based and physics-based approaches are utilized, including mutual information, linear discriminant analysis, homology modeling, molecular dynamics simulations, Poisson-Boltzmann continuum electrostatics calculations, and alchemical free energy perturbation to decipher the origin of binding specificity of the Dpr-DIP complexes examined. Ultimately, the results show that those two broad strategies are complementary, with different strengths and limitations. Biological inter-relations are more clearly revealed through knowledge-based approaches combining evolutionary and structural features, the molecular determinants controlling binding specificity can be predicted accurately with physics-based approaches based on atomic models.
Takayama, K., Ito, R., Yamamoto, H., Otsubo, S., Matsumoto, R., Ojima, H., Komori, Y., Matsuda, K. and Ihara, M. (2022). Effects of cofactors RIC-3, TMX3 and UNC-50, together with distinct subunit ratios on the agonist actions of imidacloprid on Drosophila melanogaster Dalpha1/Dbeta1 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. Pestic Biochem Physiol 187: 105177. PubMed ID: 36127041
Insect nicotinic acetylcholine receptors (nAChRs) require cofactors for functional heterologous expression. A previous study revealed that TMX3 was crucial for the functional expression of Drosophila melanogaster Dα1/Dβ1 nAChRs in Xenopus laevis oocytes, while UNC-50 and RIC-3 enhanced the acetylcholine (ACh)-induced responses of the nAChRs. However, it is unclear whether the coexpression of UNC-50 and RIC-3 with TMX3 and the subunit stoichiometry affect pharmacology of Dα1/Dβ1 nAChRs when expressed in X. laevis oocytes. This study investigated the effects of coexpressing UNC-50 and RIC-3 with TMX3 as well as changing the subunit stoichiometry on the agonist activity of ACh and imidacloprid on the Dα1/Dβ1 nAChRs. UNC-50 and RIC-3 hardly affected the agonist affinity of ACh and imidacloprid for the Dα1/Dβ1 nAChRs formed by injecting into X. laevis oocytes with an equal amount mixture of the subunit cRNAs, but enhanced current amplitude of the ACh-induced response. Imidacloprid showed higher affinity for the Dβ1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 1/5) nAChRs than the Dα1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 5/1) nAChRs, suggesting that imidacloprid prefers the Dα1/Dβ1 orthosteric site over the Dα1/Dβ1 orthosteric site.
de Freitas Fernandes, A., Leonardo, D. A., Cavini, I. A., Rosa, H. V. D., Vargas, J. A., D'Muniz Pereira, H., Nascimento, A. S. and Garratt, R. C. (2022). Conservation and divergence of the G-interfaces of Drosophila melanogaster septins. Cytoskeleton (Hoboken). PubMed ID: 36576069
Septins possess a conserved guanine nucleotide-binding (G) domain that participates in the stabilization of organized hetero-oligomeric complexes which assemble into filaments, rings and network-like structures. The fruit fly, Drosophila melanogaster, has five such septin genes encoding Sep1, Sep2, Sep4, Sep5 and Pnut. This study reports the crystal structure of the heterodimer formed between the G-domains of Sep1 and Sep2, the first from an insect to be described to date. A G-interface stabilizes the dimer (in agreement with the expected arrangement for the Drosophila hexameric particle) and this bears significant resemblance to its human counterparts, even down to the level of individual amino acid interactions. On the other hand, a model for the G-interface formed between the two copies of Pnut which occupy the centre of the hexamer, shows important structural differences, including the loss of a highly favourable bifurcated salt-bridge network. Whereas wild-type Pnut purifies as a monomer, the reintroduction of the salt-bridge network results in stabilizing the dimeric interface in solution as shown by size exclusion chromatography and thermal stability measurements. Adaptive steered molecular dynamics reveals an unzipping mechanism for dimer dissociation which initiates at a point of electrostatic repulsion within the switch II region. Overall, the data contribute to a better understanding of the molecular interactions involved in septin assembly/disassembly.
Yamamoto-Matsuda, H., Miyoshi, K., Moritoh, M., Yoshitane, H., Fukada, Y., Saito, K., Yamanaka, S. and Siomi, M. C. (2022). Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain. EMBO Rep 23(10): e53813. PubMed ID: 35993198
Loss-of-function mutations in Drosophila lethal(3)malignant brain tumor [l(3)mbt] cause ectopic expression of germline genes and brain tumors. Loss of L(3)mbt function in ovarian somatic cells (OSCs) aberrantly activates germ-specific piRNA amplification and leads to infertility. However, the underlying mechanism remains unclear. In this study, ChIP-seq for L(3)mbt in cultured OSCs and RNA-seq before and after L(3)mbt depletion shows that L(3)mbt genomic binding is not necessarily linked to gene regulation and that L(3)mbt controls piRNA pathway genes in multiple ways. Lack of known L(3)mbt co-repressors, such as Lint-1, has little effect on the levels of piRNA amplifiers. Identification of L(3)mbt interactors in OSCs and subsequent analysis reveals CG2662 as a novel co-regulator of L(3)mbt, termed "L(3)mbt interactor in OSCs" (Lint-O). Most of the L(3)mbt-bound piRNA amplifier genes are also bound by Lint-O in a similar fashion. Loss of Lint-O impacts the levels of piRNA amplifiers, similar to the lack of L(3)mbt. The lint-O-deficient flies exhibit female sterility and tumorous brains. Thus, L(3)mbt and its novel co-suppressor Lint-O cooperate in suppressing target genes to maintain homeostasis in the ovary and brain.
Miao, T., Kim, J., Kang, P., Fujiwara, H., Hsu, F. F. and Bai, H. (2022). Acetyl-CoA-mediated autoacetylation of fatty acid synthase as a metabolic switch of de novo lipogenesis in Drosophila. Proc Natl Acad Sci U S A 119(49): e2212220119. PubMed ID: 36459649
De novo lipogenesis is a highly regulated metabolic process, which is known to be activated through transcriptional regulation of lipogenic genes, including fatty acid synthase (FASN). Unexpectedly, this study found that the expression of FASN protein remains unchanged during Drosophila larval development from the second to the third instar larval stages (L2 to L3) when lipogenesis is hyperactive. Instead, acetylation of FASN is significantly upregulated in fast-growing larvae. This study further showed that lysine K813 residue is highly acetylated in developing larvae, and its acetylation is required for elevated FASN activity, body fat accumulation, and normal development. Intriguingly, K813 is autoacetylated by acetyl-CoA (AcCoA) in a dosage-dependent manner independent of acetyltransferases. Mechanistically, the autoacetylation of K813 is mediated by a novel P-loop-like motif (N-xx-G-x-A). Lastly, this study found that K813 is deacetylated by Sirt1, which brings FASN activity to baseline level. In summary, this work uncovers a previously unappreciated role of FASN acetylation in developmental lipogenesis and a novel mechanism for protein autoacetylation, through which Drosophila larvae control metabolic homeostasis by linking AcCoA, lysine acetylation, and de novo lipogenesis.

Monday, February 13 - Immune Response

Giordani, G., Cattabriga, G., Becchimanzi, A., Di Lelio, I., De Leva, G., Gigliotti, S., Pennacchio, F., Gargiulo, G. and Cavaliere, V. (2022). Role of neuronal and non-neuronal acetylcholine signaling in Drosophila humoral immunity. Insect Biochem Mol Biol 153: 103899. PubMed ID: 36596348
Acetylcholine (ACh) is one the major neurotransmitters in insects, whose role in mediating synaptic interactions between neurons in the central nervous system is well characterized. It also plays largely unexplored regulatory functions in non-neuronal tissues. This study demonstrates that ACh signaling is involved in the modulation of the innate immune response of Drosophila melanogaster. Knockdown of ACh synthesis or ACh vesicular transport in neurons reduced the activation of drosomycin (drs), a gene encoding an antimicrobial peptide, in adult flies infected with a Gram-positive bacterium. drs transcription was similarly affected in Drosophila α7 nicotinic acetylcholine receptor, nAChRalpha7 (D&al;pha;7) mutants, as well as in flies expressing in the nervous system a dominant negative form (Dα7(DN)) of this specific receptor subunit. Interestingly, Dα7(DN) elicited a comparable response when it was expressed in non-neuronal tissues and even when it was specifically produced in the hemocytes. Consistently, full activation of the drs gene required Dα7 expression in these cells. Moreover, knockdown of ACh synthesis in non-neuronal cells affected drs expression. Overall, these findings uncover neural and non-neural cholinergic signals that modulate insect immune defenses and shed light on the role of hemocytes in the regulation of the humoral immune response.
Shrivastava, N. K., Chauhan, N. and Shakarad, M. N. (2022). Heightened immune surveillance in Drosophila melanogaster populations selected for faster development and extended longevity. Heliyon 8(12): e12090. PubMed ID: 36544838
Maximization of life-history traits is under constraints due to both, limitations of resource acquisition and the restricted pathways of resource allocation. Drosophila melanogaster has served as an excellent model organism to not only unravel various trade-offs among life history traits but also numerous aspects of host immune response. Drosophila larvae are semi-aquatic that live, feed and excrete inside the food source-often over-ripe fruits and vegetables that are rich in both commensal and pathogenic microbiota that can impact the larval survival. This study used six populations of D. melanogaster, three of which are selected for faster pre-adult development and extended adult longevity, and their three ancestral controls, to explore the impact of selection on the basal immune activity in the larval stage. The larvae from selected populations had nearly significantly upregulated plasmatocyte density, significantly higher percent phagocytosis, phagocytic index and higher transcript levels of Tep3, eater and NimC1. Selected populations also had significantly upregulated crystal cell number along with higher transcript of PPO2. Out of seven tested AMPs level, Drosomycin was significantly upregulated in selected populations while Drosocin was significantly higher in control populations. ROS levels were comparable in the selected and control populations. The results strongly suggest that enhanced basal immune activity during larval stage manages the faster development and could be responsible for comparable larval survival of selected and control populations.
Kim, Y., Goh, G. and Kim, Y. H. (2022). Expression of antimicrobial peptides associated with different susceptibilities to environmental chemicals in Drosophila suzukii and Drosophila melanogaster. Pestic Biochem Physiol 187: 105210. PubMed ID: 36127054
Drosophila suzukii is a serious agricultural pest. The evolved morphology of the female D. suzukii assists in penetrating the surface of fresh fruit and spawns eggs with its unique ovipositor. Conversely, Drosophila melanogaster, a taxonomically close species with D. suzukii, largely inhabits decaying and fermenting fruits and is consistently exposed to extensive environmental chemicals, such as 2-phenylethanol, ethanol, and acetic acid, produced by microorganisms. Considering the distinct habitats of the two flies, D. suzukii is thought to be more susceptible to environmental chemicals than D. melanogaster. This study investigated the significantly higher survival rate of D. melanogaster following exposure to 2-phenylethanol, ethanol, and acetic acid. A comparison of the expression of antimicrobial peptides (AMPs) between the two flies treated with chemicals established that AMPs were generally more abundantly induced in D. melanogaster than in D. suzukii, particularly in the gut and fat body. Among the AMPs, the induction of genes (Diptericin A, Diptericin B, and Metchnikowin), which are regulated by the immune deficiency (IMD) pathway, was significantly higher than that of Drosomycin, which belongs to the Toll pathway in chemical-treated D. melanogaster. A transgenic RNAi fly (D. melanogaster) with silenced expression of AMPs and Relish, a transcription factor of the IMD pathway, exhibited significantly reduced survival rates than the control fly. These results suggest that AMPs regulated by the IMD pathway play an important role in the chemical tolerance of D. melanogaster, and these flies are adapted to their habitats by physiological response.
Xu, Y., Viswanatha, R., Sitsel, O., Roderer, D., Zhao, H., Ashwood, C., Voelcker, C., Tian, S., Raunser, S., Perrimon, N. and Dong, M. (2022). CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor. Nature 610(7931): 349-355. PubMed ID: 36171290
Entomopathogenic nematodes are widely used as biopesticides. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors. No protein receptors are known for any Tc toxins, which limits understanding of their specificity and pathogenesis. This study used genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. These findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.
Cai, Q., Guo, H., Fang, R., Hua, Y., Zhu, Y., Zheng, X., Yan, J., Wang, J., Hu, Y., Zhang, C., Zhang, C., Duan, R., Kong, F., Zhang, S., Chen, D. and Ji, S. (2022). A Toll-dependent Bre1/Rad6-cact feedback loop in controlling host innate immune response. Cell Rep 41(11): 111795. PubMed ID: 36516751
The Toll signaling pathway was initially identified for its involvement in the control of early embryogenesis. It was later shown to be also part of a major innate immune pathway controlling the expression of anti-microbial peptides in many eukaryotes including humans; cactus, the essential negative regulator of this pathway in flies, was found to be induced in parallel to the Toll-dependent activation process during immune defenses. This study was interested in the mechanisms of this dual effect and provides evidence that upon pathogenic stimuli, Dorsal, one of the transcription factors of the fly Toll pathway, can induce the expression of the E3 ligase Bre1. It was further shown that Bre1 complexes with the E2 Rad6 to mono-ubiquitinate histone H2B and to promote the transcription of cactus to achieve homeostasis of the Toll immune response. These studies characterize a Toll signal-dependent regulatory machinery in governing the Toll pathway in Drosophila.
Pavlidaki, A., Panic, R., Monticelli, S., Riet, C., Yuasa, Y., Cattenoz, P. B., Nait-Oumesmar, B. and Giangrande, A. (2022). An anti-inflammatory transcriptional cascade conserved from flies to humans. Cell Rep 41(3): 111506. PubMed ID: 36261018
Innate immunity is an ancestral process that can induce pro- and anti-inflammatory states. A major challenge is to characterize transcriptional cascades that modulate the response to inflammation. Since the Drosophila glial cells missing (Gcm) transcription factor has an anti-inflammatory role, this study explored its regulation and evolutionary conservation. The murine Gcm2 (mGcm2) gene was shown to be expressed in a subpopulation of aged microglia (chronic inflammation) and upon lysophosphatidylcholine (LPC)-induced central nervous system (CNS) demyelination (acute inflammation). Moreover, mGcm2 conditional knockout mice show an increased inflammatory phenotype upon aging or LPC injection, and hGCM2 is expressed in active demyelinating lesions of patients with multiple sclerosis. Finally, Drosophila Gcm expression is induced upon aging and acute challenge, and its overexpression decreases the inflammatory phenotype. Altogether, these data indicate that the inducible Gcm cascade is conserved from flies to humans and represents a potential therapeutic target in the control of the inflammatory response.

Friday, February 10th - Embryonic Development

Nazar, A. P., Delgado, M. J. and Lavore, A. (2022). Empty-spiracles is maternally expressed and essential for neurodevelopment and early embryo determination in Rhodnius prolixus. Dev Biol 490: 144-154. PubMed ID: 35988717
Since empty-spiracles (ems) was identified and characterized in Drosophila melanogaster as a head-gap gene, several studies have been carried out in other insect orders to confirm its evolutionary conserved function. Using the blood-sucking bug Rhodnius prolixus as biological model, this study found an ems transcript with three highly conserved regions: Box-A, Box-B, and the homeodomain. R. prolixus embryos silenced by parental RNAi for two of these ems conserved regions showed both maternal and zygotic defects. Rp-emsB fragment results in early lethal embryogenesis, with eggs without any embryonic structure inside. Rp-emsB expression pattern is only maternally expressed and localized in the ovary tropharium, follicular cells, and in the unfertilized female pronucleus. Rp-emsA fragment is zygotically expressed during early blastoderm formation until late developmental stages in two main patterns: anterior in the antennal segment, and in a segmentary in the neuroblast and tracheal pits. R. prolixus knockdown embryos for Rp-emsA showed an incomplete larval hatching, reduced heads, and severe neuromotor defects. Furthermore, in situ hybridization revealed a spatial and temporal expression pattern that highly correlates with Rp-ems observed function. In this study Rp-ems function in R. prolixus development was validated, showing that empty-spiracles does not act as a true head-gap gene, but it is necessary for proper head development and crucial for early embryo determination and neurodevelopment.
Schroeder, A. M., Nielsen, T., Lynott, M., Vogler, G., Colas, A. R. and Bodmer, R. (2022). Nascent polypeptide-Associated Complex and Signal Recognition Particle have cardiac-specific roles in heart development and remodeling. PLoS Genet 18(10): e1010448. PubMed ID: 36240221
Establishing a catalog of Congenital Heart Disease (CHD) genes and identifying functional networks would improve understanding of its oligogenic underpinnings. The current studies identified protein biogenesis cofactors Nascent polypeptide-Associated Complex (NAC) and Signal-Recognition-Particle (SRP) as disease candidates and novel regulators of cardiac differentiation and morphogenesis. Knockdown (KD) of the α-subunit (Nacα) or beta-subunit (bicaudal, bic) of NAC in the developing Drosophila heart disrupted cardiac developmental remodeling resulting in a fly with no heart. Heart loss was rescued by combined KD of Nacα with the posterior patterning Hox gene Abd-B. Consistent with a central role for this interaction in cardiogenesis, KD of Nacα in cardiac progenitors derived from human iPSCs impaired cardiac differentiation while co-KD with human HOXC12 and HOXD12 rescued this phenotype. These data suggest that Nacα KD preprograms cardioblasts in the embryo for abortive remodeling later during metamorphosis, as Nacα KD during translation-intensive larval growth or pupal remodeling only causes moderate heart defects. KD of SRP subunits in the developing fly heart produced phenotypes that targeted specific segments and cell types, again suggesting cardiac-specific and spatially regulated activities. Together, this study demonstrated directed function for NAC and SRP in heart development, and that regulation of NAC function depends on Hox genes.
Symonenko, A. V., Roshina, N. V., Krementsova, A. V., Rybina, O. Y. and Pasyukova, E. G. (2022). Shuttle craft Gene Affects Lifespan of Drosophila melanogaster by Controlling Early Development and Modifying Aging Program. Biochemistry (Mosc) 87(12): 1611-1621. PubMed ID: 36717450
Fundamental mechanisms underlying genetic control of lifespan are intensively studied and discussed due to the increasing importance of extending healthy human life. The stc gene of the model organism Drosophila melanogaster encodes a transcription factor, homolog of the human transcription factor NF-X1, involved in regulation of neuronal development and other processes, as well as in control of lifespan. This work demonstrates that the stc knockdown in embryonic and nerve cells leads to changes in lifespan, with the nature of changes depending on the cell type and sex of individuals. Based on these results, it is suggested that stc gene is involved in transcription regulation throughout life, and, as a result, also affects a complex integral trait, lifespan. At the same time, it was shown that the reduction of stc expression in neurons can alleviate the negative effect of glutamate on longevity, possibly preventing development of glutamate excitotoxicity, thus modifying the cell death program and preventing death of individuals due to phenoptosis.
Sharrock, T. E., Evans, J., Blanchard, G. B. and Sanson, B. (2022). Different temporal requirements for tartan and wingless in the formation of contractile interfaces at compartmental boundaries. Development 149(21). PubMed ID: 36178136
Compartmental boundaries physically separate developing tissues into distinct regions, which is fundamental for the organisation of the body plan in both insects and vertebrates. In many examples, this physical segregation is caused by a regulated increase in contractility of the actomyosin cortex at boundary cell-cell interfaces, a property important in developmental morphogenesis beyond compartmental boundary formation. This study performed an unbiased screening approach to identify cell surface receptors required for actomyosin enrichment and polarisation at parasegmental boundaries (PSBs) in early Drosophila embryos, from the start of germband extension at gastrulation and throughout the germband extended stages (stages 6 to 11). First, it was found that Tartan is required during germband extension for actomyosin enrichment at PSBs, confirming an earlier report. Next, by following in real time the dynamics of loss of boundary straightness in tartan mutant embryos compared with wild-type and ftz mutant embryos, it was shown that Tartan is required during germband extension but not beyond. Candidate genes were identified that could take over from Tartan at PSBs, and it was confirmed that at germband extended stages, actomyosin enrichment at PSBs requires Wingless signalling.
Calderon, D., Blecher-Gonen, R., Huang, X., Secchia, S., Kentro, J., Daza, R. M., Martin, B., Dulja, A., Schaub, C., Trapnell, C., Larschan, E., O'Connor-Giles, K. M., Furlong, E. E. M. and Shendure, J. (2022). The continuum of Drosophila embryonic development at single-cell resolution. Science 377(6606): eabn5800. PubMed ID: 35926038
Drosophila melanogaster is a powerful, long-standing model for metazoan development and gene regulation. This study profiled chromatin accessibility in almost 1 million and gene expression in half a million nuclei from overlapping windows spanning the entirety of embryogenesis. Leveraging developmental asynchronicity within embryo collections, deep neural networks were applied to infer the age of each nucleus, resulting in continuous, multimodal views of molecular and cellular transitions in absolute time. Cell lineages were determined; their developmental relationships were infered; and dynamic changes in enhancer usage, transcription factor (TF) expression, and the accessibility of TFs' cognate motifs were linked. With these data, the dynamics of enhancer usage and gene expression can be explored within and across lineages at the scale of minutes, including for precise transitions like zygotic genome activation.
Clark, E., Battistara, M. and Benton, M. A. (2022). A timer gene network is spatially regulated by the terminal system in the Drosophila embryo. Elife 11. PubMed ID: 36524728
In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the 'timer' genes caudal, Dichaete and odd-paired, whose expression dynamics correlate with the mode of segmentation. In Drosophila, the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small 'tail' region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless, but beyond this the regulation of the timer genes is poorly understood. This study used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. It is proposed that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation.

Thursday, February 9th - Behavior

Banu, A., Gowda, S. B. M., Salim, S. and Mohammad, F. (2022). Serotonergic control of feeding microstructure in Drosophila. Front Behav Neurosci 16: 1105579. PubMed ID: 36733453
To survive, animals maintain energy homeostasis by seeking out food. Compared to freely feeding animals, food-deprived animals may choose different strategies to balance both energy and nutrition demands, per the metabolic state of the animal. Serotonin mediates internal states, modifies existing neural circuits, and regulates animal feeding behavior, including in humans and fruit flies. However, an in-depth study on the neuromodulatory effects of serotonin on feeding microstructure has been held back for several technical reasons. Firstly, most feeding assays lack the precision of manipulating neuronal activity only when animals start feeding, which does not separate neuronal effects on feeding from foraging and locomotion. Secondly, despite the availability of optogenetic tools, feeding in adult fruit flies has primarily been studied using thermogenetic systems, which are confounded with heat. Thirdly, most feeding assays have used food intake as a measurement, which has a low temporal resolution to dissect feeding at the microstructure level. To circumvent these problems, OptoPAD assay, which provides the precision of optogenetics to control neural activity contingent on the ongoing feeding behavior, was utilized. Manipulating the serotonin circuit optogenetically affects multiple feeding parameters state-dependently. Food-deprived flies with optogenetically activated and suppressed serotonin systems feed with shorter and longer sip durations and longer and shorter inter-sip intervals, respectively. It was further shown that serotonin suppresses and enhances feeding via 5-HT1B and 5-HT7 receptors, respectively.
Fowler, E. K., Leigh, S., Rostant, W. G., Thomas, A., Bretman, A. and Chapman, T. (2022). Memory of social experience affects female fecundity via perception of fly deposits. BMC Biol 20(1): 244. PubMed ID: 36310170
Animals can exhibit remarkable reproductive plasticity in response  to their social surroundings, with profound fitness consequences. The presence of same-sex conspecifics can signal current or future expected competition for resources or mates. Plastic responses to elevated sexual competition caused by exposure to same-sex individuals have been well-studied in males. However, much less is known about such plastic responses in females, whether this represents sexual or resource competition, or if it leads to changes in investment in mating behaviour and/or reproduction. Drosophila melanogaster to measure the impact of experimentally varying female exposure to other females prior to mating on fecundity before and after mating. Then physical and genetic methods were deployed to manipulate the perception of different social cues and sensory pathways and reveal the potential mechanisms involved. The results showed that females maintained in social isolation prior to mating were significantly more likely to retain unfertilised eggs before mating, but to show the opposite and lay significantly more fertilised eggs in the 24h after mating. More than 48h of exposure to other females was necessary for this social memory response to be expressed. Neither olfactory nor visual cues were involved in mediating fecundity plasticity-instead, the relevant cues were perceived through direct contact with the non-egg deposits left behind by other females. The results demonstrate that females show reproductive plasticity in response to their social surroundings and can carry this memory of their social experience forward through mating. Comparisons of these results with previous work show that the nature of female plastic reproductive responses and the cues they use differ markedly from those of males. The results emphasise the deep divergence in how each sex realises its reproductive success.
Mishra, S., Sharma, N., Singh, S. K. and Lone, S. R. (2022). Peculiar sleep features in sympatric species may contribute to the temporal segregation. J Comp Physiol B. PubMed ID: 36271924
Sleep is conserved in the animal kingdom and plays a pivotal role in the adaptation of species. Sleep in Drosophila melanogaster is defined as any continuous 5 min of quiescence, shows a prominent siesta, and consolidated nighttime sleep. This study analyzed the sleep of two other species D. malerkotliana (DMK) and D. ananassae (DA), and compared it with D. melanogaster (DM). The DMK males and females have siesta like DM. However, unlike DM, flies continue to sleep beyond siesta till the evening. DA has a less prominent siesta compared to DM and DMK. In the morning, DA took a longer time to respond to the lights ON and continued to sleep for at least half an hour. The nighttime sleep of the DA flies is higher than the other two species. Average length of sleep episode is three times more than that of DM and DMK with few wake episodes. Thus, the nighttime sleep of DA males and females is deep and needs exposure to more potent stimuli to wake up relative to the other two species. DA males and females show higher sleep rebound than the other two species, suggesting the robustness of sleep homeostasis. Although total sleep of DMK and DA is similar, DA is a day-active species with highly consolidated night sleep. DMK, like DM, is a crepuscular species with a midday siesta. Thus, these results suggest that temporal partitioning of sleep, in sympatric species may contribute to temporal segregation.
Koch, R. E. and Dowling, D. K. (2022). Effects of mitochondrial haplotype on pre-copulatory mating success in male fruit flies (Drosophila melanogaster). J Evol Biol 35(10): 1396-1402. PubMed ID: 35988150
While mitochondria have long been understood to be critical to cellular function, questions remain as to how genetic variation within mitochondria may underlie variation in general metrics of organismal function. To date, studies investigating links between mitochondrial genotype and phenotype have largely focused on differences in expression of genes and physiological and life-history traits across haplotypes. Mating display behaviours may also be sensitive to mitochondrial functionality and so may also be affected by sequence variation in mitochondrial DNA, with consequences for sexual selection and fitness. This study tested whether the pre-copulatory mating success of male fruit flies (Drosophila melanogaster) varies across six different mitochondrial haplotypes expressed alongside a common nuclear genetic background. A significant effect of mitochondrial haplotype was found on measuring of competitive mating success, driven largely by the relatively poor performance of males with one particular haplotype. This haplotype, termed 'Brownsville', has previously been shown to have complex and sex-specific effects, most notably including depressed fertility in males but not females. This study extends this disproportionate effect on male reproductive success to pre-copulatory aspects of reproduction. The results demonstrate that mutations in mitochondrial DNA can plausibly affect pre-copulatory mating success, with implications for future study into the subcellular underpinnings of such behaviours and the information they may communicate.
Vijayan, V., Wang, Z., Chandra, V., Chakravorty, A., Li, R., Sarbanes, S. L., Akhlaghpour, H. and Maimon, G. (2022). An internal expectation guides Drosophila egg-laying decisions. Sci Adv 8(43): eabn3852. PubMed ID: 36306348
To better understand how animals make ethologically relevant decisions, egg-laying substrate choice was studied in Drosophila. Flies were found to dynamically increase or decrease their egg-laying rates while exploring substrates so as to target eggs to the best, recently visited option. Visiting the best option typically yielded inhibition of egg laying on other substrates for many minutes. The data support a model in which flies compare the current substrate's value with an internally constructed expectation on the value of available options to regulate the likelihood of laying an egg. Dopamine neuron activity is critical for learning and/or expressing this expectation, similar to its role in certain tasks in vertebrates. Integrating sensory experiences over minutes to generate an estimate of the quality of available options allows flies to use a dynamic reference point for judging the current substrate and might be a general way in which decisions are made.
Love, C. R., Gautam, S., Lama, C., Le, N. H. and Dauwalder, B. (2023). The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship. Genes Brain Behav 22(1): e12836. PubMed ID: 36636829
The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. This study shows that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. These findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.

Wednesday, February 8th - Synapse and Junctions

Armstrong, N. S. and Frank, C. A. (2022). The calcineurin regulator Sarah enables distinct forms of homeostatic plasticity at the Drosophila neuromuscular junction. Front Synaptic Neurosci 14: 1033743. PubMed ID: 36685082
The ability of synapses to maintain physiological levels of evoked neurotransmission is essential for neuronal stability. A variety of perturbations can disrupt neurotransmission, but synapses often compensate for disruptions and work to stabilize activity levels, using forms of homeostatic synaptic plasticity. Presynaptic homeostatic potentiation (PHP) is one such mechanism. PHP is expressed at the Drosophila melanogaster larval neuromuscular junction (NMJ) synapse, as well as other NMJs. In PHP, presynaptic neurotransmitter release increases to offset the effects of impairing muscle transmitter receptors. Prior Drosophila work has studied PHP using different ways to perturb muscle receptor function-either acutely (using pharmacology) or chronically (using genetics). Some previous data suggested that cytoplasmic calcium signaling was important for expression of PHP after genetic impairment of glutamate receptors. This study followed up on that observation. This study used a combination of transgenic Drosophila RNA interference and overexpression lines, along with NMJ electrophysiology, synapse imaging, and pharmacology to test if regulators of the calcium/calmodulin-dependent protein phosphatase calcineurin are necessary for the normal expression of PHP. It was found that either pre- or postsynaptic dysregulation of a Drosophila gene regulating calcineurin, sarah (sra), blocks PHP. Tissue-specific manipulations showed that either increases or decreases in sra expression are detrimental to PHP. Additionally, pharmacologically and genetically induced forms of expression of PHP are functionally separable depending entirely upon which sra genetic manipulation is used. Surprisingly, dual-tissue pre- and postsynaptic sra knockdown or overexpression can ameliorate PHP blocks revealed in single-tissue experiments. Pharmacological and genetic inhibition of calcineurin corroborated this latter finding. These results suggest tight calcineurin regulation is needed across multiple tissue types to stabilize peripheral synaptic outputs.
BabiSova, K., Mentelova, L., Geisseova, T. K., Benova-Liszekova, D., Beno, M., Chase, B. A. and Farka, R. (2022). Apocrine secretion in the salivary glands of Drosophilidae and other dipterans is evolutionarily conserved. Front Cell Dev Biol 10: 1088055. PubMed ID: 36712974
Apocrine secretion is a transport and secretory mechanism that remains only partially characterized, even though it is evolutionarily conserved among all metazoans, including humans. The excellent genetic model organism Drosophila melanogaster holds promise for elucidating the molecular mechanisms regulating this fundamental metazoan process. Two prerequisites for such investigations are to clearly define an experimental system to investigate apocrine secretion and to understand the evolutionarily and functional contexts in which apocrine secretion arose in that system. To this end, it was recently demonstrated that, in D. melanogaster, the prepupal salivary glands utilize apocrine secretion prior to pupation to deliver innate immune and defense components to the exuvial fluid that lies between the metamorphosing pupae and its chitinous case. This finding provided a unique opportunity to appraise how this novel non-canonical and non-vesicular transport and secretory mechanism is employed in different developmental and evolutionary contexts. This study demonstrates that this apocrine secretion, which is mechanistically and temporarily separated from the exocytotic mechanism used to produce the massive salivary glue secretion (Sgs), is shared across Drosophilidae and two unrelated dipteran species. Screening more than 30 species of Drosophila from divergent habitats across the globe revealed that apocrine secretion is a widespread and evolutionarily conserved cellular mechanism used to produce exuvial fluid. Species with longer larval and prepupal development than D. melanogaster activate apocrine secretion later, while smaller and more rapidly developing species activate it earlier. In some species, apocrine secretion occurs after the secretory material is first concentrated in cytoplasmic structures of unknown origin that were named "collectors." Strikingly, in contrast to the widespread use of apocrine secretion to provide exuvial fluid, not all species use exocytosis to produce the viscid salivary glue secretion that is seen in D. melanogaster. Thus, apocrine secretion is the conserved mechanism used to realize the major function of the salivary gland in fruitflies and related species: it produces the pupal exuvial fluid that provides an active defense against microbial invasion during pupal metamorphosis.
Dyson, A., Ryan, M., Garg, S., Evans, D. G. and Baines, R. A. (2022). Loss of NF1 in Drosophila larvae causes tactile hypersensitivity and impaired synaptic transmission at the neuromuscular junction. J Neurosci. PubMed ID: 36344265
Autism Spectrum Disorder (ASD) is a neurodevelopmental condition in which the mechanisms underlying its core symptomatology are largely unknown. Studying animal models of monogenic syndromes associated with ASD, such as neurofibromatosis type 1 (NF1), can offer insights into its aetiology. This study shows that loss of function of the Drosophila NF1 ortholog results in tactile hypersensitivity following brief mechanical stimulation in the larva (mixed sexes), paralleling the sensory abnormalities observed in individuals with ASD. Mutant larvae also exhibit synaptic transmission deficits at the glutamatergic neuromuscular junction (NMJ), with increased spontaneous but reduced evoked release. While the latter is homeostatically compensated for by a postsynaptic increase in input resistance, the former is consistent with neuronal hyperexcitability. Indeed, diminished expression of NF1 specifically within central cholinergic neurons induces both excessive neuronal firing and tactile hypersensitivity, suggesting the two may be linked. Furthermore, both impaired synaptic transmission and behavioural deficits are fully rescued via knockdown of Ras proteins. These findings validate NF1(-/-) Drosophila as a tractable model of ASD with the potential to elucidate important pathophysiological mechanisms.
Vacassenno, R. M., Haddad, C. N. and Cooper, R. L. (2023). The effects of doxapram (blocker of K2p channels) on resting membrane potential and synaptic transmission at the Drosophila neuromuscular junction. Comp Biochem Physiol C Toxicol Pharmacol 263: 109497. PubMed ID: 36306997
The resting membrane potential of most cells is maintained by potassium K2p channels. The pharmacological profile and distribution of various K2p channel subtypes in organisms are still being investigated. The Drosophila genome contains 11 subtypes; however, their function and expression profiles have not yet been determined. Doxapram is clinically used to enhance respiration in humans and blocks the acid-sensitive K2p TASK subtype in mammals. The resting membrane potential of larval Drosophila muscle and synaptic transmission at the neuromuscular junction are pH sensitive. The present study investigated the effects of doxapram on membrane potential and synaptic transmission using intracellular recordings of larval Drosophila muscles. Doxapram (1 mM and 10 mM) depolarizes the muscle and appears to depolarize motor neurons, causing an increase in the frequency of spontaneous quantal events and evoked excitatory junction potentials. Verapamil (1 and 10 mM) paralleled the action of doxapram. These changes were matched by an extracellular increase in KCl (50 mM) and blocked by Cd(2+). It is assumed that the motor nerve depolarizes to open voltage-gated Ca(2+) channels in presynaptic nerve terminals because of exposure to doxapram. These findings are significant for building models to better understand the function of pharmacological agents that affect K2p channels and how K2p channels contribute to the physiology of tissues. Drosophila offers a genetically amenable model that can alter the tissue-specific expression of K2p channel subtypes to simulate known human diseases related to this family of channels.
Choudhury, S. D., Dwivedi, M. K., Pippadpally, S., Patnaik, A., Mishra, S., Padinjat, R. and Kumar, V. (2022). AP2 Regulates Thickveins Trafficking to Attenuate NMJ Growth Signaling in Drosophila. eNeuro 9(5). PubMed ID: 36180220
Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. This study shows that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad (components of BMP signaling) to control neuromuscular junction (NMJ) growth in Drosophila Ultrastructural analysis of σ2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. Mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of σ2-adaptin mutations. A model is proposed in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth.
Zhou, L., Xue, X., Yang, K., Feng, Z., Liu, M. and Pastor-Pareja, J. C. (2023). Convergence of secretory, endosomal, and autophagic routes in trans-Golgi-associated lysosomes. J Cell Biol 222(1). PubMed ID: 36239631
At the trans-Golgi, complex traffic connections exist to the endolysosomal system additional to the main Golgi-to-plasma membrane secretory route. This study investigated three hits in a Drosophila screen displaying secretory cargo accumulation in autophagic vesicles: ESCRT-III component Vps20, SNARE-binding Rop, and lysosomal pump subunit VhaPPA1-1. Vps20, Rop, and lysosomal markers were found to localize near the trans-Golgi. Furthermore, this study documented that the vicinity of the trans-Golgi is the main cellular location for lysosomes and that early, late, and recycling endosomes associate as well with a trans-Golgi-associated degradative compartment where basal microautophagy of secretory cargo and other materials occurs. Disruption of this compartment causes cargo accumulation in the hits, including Munc18 homolog Rop, required with Syx1 and Syx4 for Rab11-mediated endosomal recycling. Finally, besides basal microautophagy, it was shown that the trans-Golgi-associated degradative compartment contributes to the growth of autophagic vesicles in developmental and starvation-induced macroautophagy. These results argue that the fly trans-Golgi is the gravitational center of the whole endomembrane system.

Tuesday, February 7th - Chromatin

Feijao, T., Marques, B., Silva, R. D., Carvalho, C., Sobral, D., Matos, R., Tan, T., Pereira, A., Morais-de-Sa, E., Maiato, H., DeLuca, S. Z. and Martinho, R. G. (2022). Polycomb group (PcG) proteins prevent the assembly of abnormal synaptonemal complex structures during meiosis. Proc Natl Acad Sci U S A 119(42): e2204701119. PubMed ID: 36215502
The synaptonemal complex (SC) is a proteinaceous scaffold that is assembled between paired homologous chromosomes during the onset of meiosis. Timely expression of SC coding genes is essential for SC assembly and successful meiosis. However, SC components have an intrinsic tendency to self-organize into abnormal repetitive structures, which are not assembled between the paired homologs and whose formation is potentially deleterious for meiosis and gametogenesis. This creates an interesting conundrum, where SC genes need to be robustly expressed during meiosis, but their expression must be carefully regulated to prevent the formation of anomalous SC structures. This manuscript showa that the Polycomb group protein Sfmbt, the Drosophila ortholog of human MBTD1 and L3MBTL2, is required to avoid excessive expression of SC genes during prophase I. Although SC assembly is normal after Sfmbt depletion, SC disassembly is abnormal with the formation of multiple synaptonemal complexes (polycomplexes) within the oocyte. Overexpression of the SC gene corona and depletion of other Polycomb group proteins are similarly associated with polycomplex formation during SC disassembly. These polycomplexes are highly dynamic and have a well-defined periodic structure. Further confirming the importance of Sfmbt, germ line depletion of this protein is associated with significant metaphase I defects and a reduction in female fertility. Since transcription of SC genes mostly occurs during early prophase I, these results suggest a role of Sfmbt and other Polycomb group proteins in downregulating the expression of these and other early prophase I genes during later stages of meiosis.
Andreyeva, E. N., Emelyanov, A. V., Nevil, M., Sun, L., Vershilova, E., Hill, C. A., Keogh, M. C., Duronio, R. J., Skoultchi, A. I. and Fyodorov, D. V. (2022). Drosophila SUMM4 complex couples insulator function and DNA replication control. Elife 11. PubMed ID: 36458689
Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underrelicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. This study developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. This findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.
Simmons, J. R., An, R., Amankwaa, B., Zayac, S., Kemp, J. and Labrador, M. (2022). Phosphorylated histone variant gammaH2Av is associated with chromatin insulators in Drosophila. PLoS Genet 18(10): e1010396. PubMed ID: 36197938
Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). This study demonstrates an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components su(Hw) and Cp190 results in a significant reduction in γH2Av levels in chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2AvH2Av localization in insulator mutants. It was also shown that γH2Av, but not H2Av, γH2A is a component of insulator bodies, which are protein condensates that form during osmotic stress. Phosphatase activity is required for insulator body dissolution after stress recovery. Together, these results implicate the H2A variant with a novel mechanism of insulator function and boundary formation.
Herman, N., Kadener, S. and Shifman, S. (2022). The chromatin factor ROW cooperates with BEAF-32 in regulating long-range inducible genes. EMBO Rep: e54720. PubMed ID: 36245419
Insulator proteins located at the boundaries of topological associated domains (TAD) are involved in higher-order chromatin organization and transcription regulation. However, it is still not clear how long-range contacts contribute to transcriptional regulation. This study shows that relative-of-WOC (ROW) is essential for the long-range transcription regulation mediated by the boundary element-associated factor of 32kD (BEAF-32). ROA physically interacts with heterochromatin proteins (HP1b and HP1c) and the insulator protein (BEAF-32). These proteins interact at TAD boundaries where ROW, through its AT-hook motifs, binds AT-rich sequences flanked by BEAF-32-binding sites and motifs. Knockdown of row downregulates genes that are long-range targets of BEAF-32 and bound indirectly by ROW (without binding motif). Analyses of high-throughput chromosome conformation capture (Hi-C) data reveal long-range interactions between promoters of housekeeping genes bound directly by ROW and promoters of developmental genes bound indirectly by ROW. Thus, the results show cooperation between BEAF-32 and the ROW complex, including HP1 proteins, to regulate the transcription of developmental and inducible genes through long-range interactions.
Hunt, G., Boija, A. and Mannervik, M. (2022). p300/CBP sustains Polycomb silencing by non-enzymatic functions. Mol Cell 82(19): 3580-3597.e3589. PubMed ID: 36206738
Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb-group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. This study shows that the p300/CREB-binding protein (CBP) co-activator is associated with two-thirds of PcG regions and required for PcG occupancy at many of these in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG-bound repressive sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states.
Ertl, H. A., Hill, M. S. and Wittkopp, P. J. (2022). Differential Grainy head binding correlates with variation in chromatin structure and gene expression in Drosophila melanogaster. BMC Genomics 23(1): 854. PubMed ID: 36575386
Phenotypic evolution is often caused by variation in gene expression resulting from altered gene regulatory mechanisms. Genetic variation affecting chromatin remodeling has been identified as a potential source of variable gene expression; however, the roles of specific chromatin remodeling factors remain unclear. This study addresses this knowledge gap by examining the relationship between variation in gene expression, variation in chromatin structure, and variation in binding of the pioneer factor Grainy head between imaginal wing discs of two divergent strains of Drosophila melanogaster and their F(1) hybrid. This study found that (1) variation in Grainy head binding is mostly due to sequence changes that act in cis but are located outside of the canonical Grainy head binding motif, (2) variation in Grainy head binding correlates with changes in chromatin accessibility, and (3) this variation in chromatin accessibility, coupled with variation in Grainy head binding, correlates with variation in gene expression in some cases but not others. Interactions among these three molecular layers is complex, but these results suggest that genetic variation affecting the binding of pioneer factors contributes to variation in chromatin remodeling and the evolution of gene expression.

Monday, February 6th - Adult Physiology and Metabolism

Andersen, M. K., Robertson, R. M. and MacMillan, H. A. (2022). Plasticity in Na+/K+-ATPase thermal kinetics drives variation in the temperature of cold-induced neural shutdown of adult Drosophila melanogaster. J Exp Biol 225(24). PubMed ID: 36477887
Most insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum, a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. This study focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated Drosophila melanogaster. After first confirming cold acclimation altered SD onset, the dependency of the SD event on Na+/K+-ATPase activity was investigated by injecting the inhibitor ouabain into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, it was found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.
Andreatta, G., Montagnese, S. and Costa, R. (2022). Natural alleles of the clock gene timeless differentially affect life-history traits in Drosophila. Front Physiol 13: 1092951. PubMed ID: 36703932
Circadian clocks orchestrate a variety of physiological and behavioural functions within the 24-h day. These timekeeping systems have also been implicated in developmental and reproductive processes that span more (or less) than 24 h. Whether natural alleles of cardinal clock genes affect entire sets of life-history traits (i.e., reproductive arrest, developmental time, fecundity), thus providing a wider substrate for seasonal adaptation, remains unclear. This study shows that natural alleles of the timeless (tim) gene of Drosophila melanogaster, previously shown to modulate flies' propensity to enter reproductive dormancy, differentially affect correlated traits such as early-life fecundity and developmental time. Homozygous flies expressing the shorter TIM isoform (encoded by the s-tim allele) not only show a lower dormancy incidence compared to those homozygous for ls-tim (which produce both the short and an N-terminal additional 23-residues longer TIM isoform), but also higher fecundity in the first 12 days of adult life. Moreover, s-tim homozygous flies develop faster than ls-tim homozygous flies at both warm (25°C) and cold (15°C) temperatures, with the gap being larger at 15°C. In summary, this phenotypic analysis shows that natural variants of tim affect a set of life-history traits associated with reproductive dormancy in Drosophila. It is speculated that this provides further adaptive advantage in temperate regions (with seasonal changes); it is proposed that the underlying mechanisms might not be exclusively dependent on photoperiod, as previously suggested.
Wang, J., Zhu, Y., Zhang, C., Duan, R., Kong, F., Zheng, X. and Hua, Y. (2022). A conserved role of bam in maintaining metabolic homeostasis via regulating intestinal microbiota in Drosophila. PeerJ 10: e14145. PubMed ID: 36248714
Previous studies have proven that bag-of-marbles (bam) plays a pivotal role in promoting early germ cell differentiation in Drosophila ovary. However, whether it functions in regulating the metabolic state of the host remains largely unknown. This study utilized GC-MS, qPCR, and some classical kits to examine various metabolic profiles and gut microbial composition in bam loss-of-function mutants and age-paired controls. Genetic manipulations were performed to explore the tissue/organ-specific role of bam in regulating energy metabolism in Drosophila. The DSS-induced mouse colitis was generated to identify the role of Gm114, the mammalian homolog of bam, in modulating intestinal homeostasis. It was shown that loss of bam leads to an increased storage of energy in Drosophila. Silence of bam in intestines results in commensal microbial dysbiosis and metabolic dysfunction of the host. Moreover, recovery of bam expression in guts almost rescues the obese phenotype in bam loss-of-function mutants. Further examinations of mammalian Gm114 imply a similar biological function in regulating the intestinal homeostasis and energy storage with its Drosophila homolog bam. These studies uncover a novel biological function of bam/Gm114 in regulating the host lipid homeostasis.
Zabihihesari, A., Parand, S., Coulthard, A. B., Molnar, A., Hilliker, A. J. and Rezai, P. (2022). An in-vivo microfluidic assay reveals cardiac toxicity of heavy metals and the protective effect of metal responsive transcription factor (MTF-1) in Drosophila model. 3 Biotech 12(10): 279. PubMed ID: 36275358
Previous toxicity assessments of heavy metals on Drosophila are limited to investigating the survival, development rate, and climbing behaviour by oral administration while cardiac toxicity of these elements have not been investigated. This study utilized a microfluidic device to inject known dosages of zinc (Zn) or cadmium (Cd) into the larvae's hemolymph to expose their heart directly and study their heart rate and arrhythmicity. The effect of heart-specific overexpression of metal responsive transcription factor (MTF-1) on different heartbeat parameters and survival of Drosophila larvae was investigated. The heart rate of wild-type larvae decreased by 24.8% or increased by 11.9%, 15 min after injection of 40 nL of 100 mM Zn or 10 mM Cd solution, respectively. The arrhythmicity index of wild-type larvae increased by 58.2% or 76.8%, after injection of Zn or Cd, respectively. MTF-1 heart overexpression ameliorated these effects completely. Moreover, it increased larvae's survival to pupal and adulthood stages and prolonged the longevity of flies injected with Zn and Cd. The microfluidic-based cardiac toxicity assay illustrated that heart is an acute target of heavy metals toxicity, and MTF-1 overexpression in this tissue can ameliorate cardiac toxicity of Zn and Cd. The method can be used for cardiotoxicity assays with other pollutants in the future.
Shinhmar, H., Hoh Kam, J., Mitrofanis, J., Hogg, C. and Jeffery, G. (2022). Shifting patterns of cellular energy production (adenosine triphosphate) over the day and key timings for the effect of optical manipulation. J Biophotonics 15(10): e202200093. PubMed ID: 35860879
Mitochondria are optically responsive organelles producing energy for cell function via adenosine triphosphate (ATP). But ATP production appears to vary over the day. This study used Drosophila melanogaster to reveal daily shifts in whole animal ATP production in a tight 24 hours' time series. A marked production peak in the morning was shown to declines around midday and remains low through afternoon and night. ATP production can be improved with long wavelengths (>660 nm), but apparently not at all times. Hence, flies were treated with 670 nm light to reveal optimum times. Exposures at 670 nm resulted in a significant ATP increases and a shift in the ATP/adenosine diphosphate (ADP) ratio at 8.00 and 11.00, whilst application at other time points had no effect. Hence, light-induced ATP increases appear limited to periods when natural production is high. In summary, long wavelength influences on mitochondria are conserved across species from fly to human. Determining times for their administration to improve function in ageing and disease are of key importance. This study progresses this problem.
Shaible, T. M. and Matzkin, L. M. (2022). Physiological and life history changes associated with seasonal adaptation in the cactophilic Drosophila mojavensis. Biol Open 11(10). PubMed ID: 36285699
Many insects inhabiting temperate climates are faced with changing environmental conditions throughout the year. Depending on the species, these environmental fluctuations can be experienced within a single generation or across multiple generations. Strategies for dealing with these seasonal changes vary across populations. Drosophila mojavensis is a cactophilic Drosophila species endemic to the Sonoran Desert. The Sonoran Desert regularly reaches temperatures of 50°C in the summer months. As individuals of this population are rare to collect in the summer months, this study simulated the cycling temperatures experienced by D. mojavensis in the Sonoran Desert from April to July (four generations) in a temperature- and light-controlled chamber, to understand the physiological and life history changes that allow this population to withstand these conditions. In contrast to the hypothesis of a summer aestivation, it was found that D. mojavensis continue to reproduce during the summer months, albeit with lower viability, but the adult survivorship of the population is highly reduced during this period. As expected, stress resistance increased during the summer months in both the adult and the larval stages. This study examines several strategies for withstanding the Sonoran Desert summer conditions which may be informative in the study of other desert endemic species.

Friday, February 3rd - Tumors, cancer, and growth

Tabata, J., Nakaoku, T., Araki, M., Yoshino, R., Kohsaka, S., Otsuka, A., Ikegami, M., Ui, A., Kanno, S. I., Miyoshi, K., Matsumoto, S., Sagae, Y., Yasui, A., Sekijima, M., Mano, H., Okuno, Y., Okamoto, A. and Kohno, T. (2022). Novel Calcium-Binding Ablating Mutations Induce Constitutive RET Activity and Drive Tumorigenesis. Cancer Res 82(20): 3751-3762. PubMed ID: 36166639
Distinguishing oncogenic mutations from variants of unknown significance (VUS) is critical for precision cancer medicine. In this study, computational modeling of 71,756 RET variants for positive selection together with functional assays of 110 representative variants identified a three-dimensional cluster of VUSs carried by multiple human cancers that cause amino acid substitutions in the calmodulin-like motif (CaLM) of RET. Molecular dynamics simulations indicated that CaLM mutations decrease interactions between Ca2+ and its surrounding residues and induce conformational distortion of the RET cysteine-rich domain containing the CaLM. RET-CaLM mutations caused ligand-independent constitutive activation of RET kinase by homodimerization mediated by illegitimate disulfide bond formation. RET-CaLM mutants possessed oncogenic and tumorigenic activities that could be suppressed by tyrosine kinase inhibitors targeting RET. This study identifies calcium-binding ablating mutations as a novel type of oncogenic mutation of RET and indicates that in silico-driven annotation of VUSs of druggable oncogenes is a promising strategy to identify targetable driver mutations.
Yang, S., Jiang, H., Bian, W., Xu, W., Guo, Y., Song, S., Zheng, J., Kuang, X., Wu, C., Ding, X., Guo, X., Xue, L., Yu, Z., Zhang, Y., Ryoo, H. D., Li, X. and Ma, X. (2022). Bip-Yorkie interaction determines oncogenic and tumor-suppressive roles of Ire1/Xbp1s activation. Proc Natl Acad Sci U S A 119(42): e2202133119. PubMed ID: 36215479
Unfolded protein response (UPR) is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. This study reports that the Ire1/Xbp1s pathway has surprisingly oncogenic and tumor-suppressive roles in a context-dependent manner. Activation of Ire1/Xbp1s up-regulates their downstream target Bip, which sequesters Yorkie (Yki), a Hippo pathway transducer, in the cytoplasm to restrict Yki transcriptional output. This regulation provides an endogenous defensive mechanism in organ size control, intestinal homeostasis, and regeneration. Unexpectedly, Xbp1 ablation promotes tumor overgrowth but suppresses invasiveness in a Drosophila cancer model. Mechanistically, hyperactivated Ire1/Xbp1s signaling in turn induces JNK-dependent developmental and oncogenic cell migration and epithelial-mesenchymal transition (EMT) via repression of Yki. In humans, a negative correlation between XBP1 and YAP (Yki ortholog) target gene expression specifically exists in triple-negative breast cancers (TNBCs), and those with high XBP1 or HSPA5 (Bip ortholog) expression have better clinical outcomes. In human TNBC cell lines and xenograft models, ectopic XBP1s or HSPA5 expression alleviates tumor growth but aggravates cell migration and invasion. These findings uncover a conserved crosstalk between the Ire1/Xbp1s and Hippo signaling pathways under physiological settings, as well as a crucial role of Bip-Yki interaction in tumorigenesis that is shared from Drosophila to humans.
Chatterjee, D., Costa, C. A. M., Wang, X. F., Jevitt, A., Huang, Y. C. and Deng, W. M. (2022). Single-cell transcriptomics identifies Keap1-Nrf2 regulated collective invasion in a Drosophila tumor model. Elife 11. PubMed ID: 36321803
Apicobasal cell-polarity loss is a founding event in Epithelial-Mesenchymal Transition (EMT) and epithelial tumorigenesis, yet how pathological polarity loss links to plasticity remains largely unknown. To understand the mechanisms and mediators regulating plasticity upon polarity loss, single-cell RNA sequencing was performed of Drosophila ovaries, where inducing polarity-gene l(2)gl-knockdown (Lgl-KD) causes invasive multilayering of the follicular epithelia. Analyzing the integrated Lgl-KD and wildtype transcriptomes, it was discovered the cells specific to the various discernible phenotypes and characterized the underlying gene expression. A genetic requirement of Keap1-Nrf2 signaling in promoting multilayer formation of Lgl-KD cells was further identified. Ectopic expression of Keap1 increased the volume of delaminated follicle cells that showed enhanced invasive behavior with significant changes to the cytoskeleton. Overall, these findings describe the comprehensive transcriptome of cells within the follicle-cell tumor model at the single-cell resolution and identify a previously unappreciated link between Keap1-Nrf2 signaling and cell plasticity at early tumorigenesis.
Kumar, A. and Baker, N. E. (2022). The CRL4 E3 ligase Mahjong/DCAF1 controls cell competition through the transcription factor Xrp1, independently of polarity genes. Development 149(22). PubMed ID: 36278853
Cell competition, the elimination of cells surrounded by more fit neighbors, is proposed to suppress tumorigenesis. Mahjong (Mahj), a ubiquitin E3 ligase substrate receptor, has been thought to mediate competition of cells mutated for lethal giant larvae (lgl), a neoplastic tumor suppressor that defines apical-basal polarity of epithelial cells. This study shows that Drosophila cells mutated for mahjong, but not for lgl [l(2)gl], are competed because they express the bZip-domain transcription factor Xrp1, already known to eliminate cells heterozygous for ribosomal protein gene mutations (Rp/+ cells). Xrp1 expression in mahj mutant cells results in activation of JNK signaling, autophagosome accumulation, eIF2α phosphorylation and lower translation, just as in Rp/+ cells. Cells mutated for damage DNA binding-protein 1 (ddb1; pic) or cullin 4 (cul4), which encode E3 ligase partners of Mahj, also display Xrp1-dependent phenotypes, as does knockdown of proteasome subunits. These data suggest a new model of mahj-mediated cell competition that is independent of apical-basal polarity and couples Xrp1 to protein turnover.
Enomoto, M. and Igaki, T. (2022). Cell-cell interactions that drive tumorigenesis in Drosophila. Fly (Austin) 16(1): 367-381. PubMed ID: 36413374
Cell-cell interactions within tumour microenvironment play crucial roles in tumorigenesis. Genetic mosaic techniques available in Drosophila have provided a powerful platform to study the basic principles of tumour growth and progression via cell-cell communications. This led to the identification of oncogenic cell-cell interactions triggered by endocytic dysregulation, mitochondrial dysfunction, cell polarity defects, or Src activation in Drosophila imaginal epithelia. Such oncogenic cooperations can be caused by interactions among epithelial cells, mesenchymal cells, and immune cells. Moreover, microenvironmental factors such as nutrients, local tissue structures, and endogenous growth signalling activities critically affect tumorigenesis. Dissecting various types of oncogenic cell-cell interactions at the single-cell level in Drosophila will greatly increase understanding of how tumours progress in living animals.
Wang, Z., Xia, X., Li, J. and Igaki, T. (2022). Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling. Elife 11. PubMed ID: 36222503
JNK signaling plays a critical role in both tumor promotion and tumor suppression. This study identified clustered microRNAs (miRNAs) miR-306 and miR-79 as novel tumor-suppressor miRNAs that specifically eliminate JNK-activated tumors in Drosophila. While showing only a slight effect on normal tissue growth, miR-306 and miR-79 strongly suppressed growth of multiple tumor models, including malignant tumors caused by Ras activation and cell polarity defects. Mechanistically, these miRNAs commonly target the mRNA of an E3 ubiquitin ligase ring finger protein 146 (RNF146). RNF146 promotes degradation of tankyrase (Tnks), an ADP-ribose polymerase that promotes JNK activation in a noncanonical manner. Thus, downregulation of RNF146 by miR-306 and miR-79 leads to hyper-enhancement of JNK activation. These data show that, while JNK activity is essential for tumor growth, elevation of miR-306 or miR-79 overactivate JNK signaling to the lethal level via noncanonical JNK pathway and thus eliminate tumors, providing a new miRNA-based strategy against cancer.

Thursday, February 2nd - Disease Models

Su, M. T., Lu, C. W., Wu, W. J., Jheng, Y. S., Yang, S. Y., Chuang, W. C., Lee, M. C. and Wu, C. H. (2022). Applications of Immunomagnetic Reduction Technology as a Biosensor in Therapeutic Evaluation of Chinese Herbal Medicine in Tauopathy Alleviation of an AD Drosophila Model. Biosensors (Basel) 12(10). PubMed ID: 36291020
Alzheimer's disease (AD) is the most common form of dementia. The most convincing biomarkers in the blood for AD are currently β-amyloid (Aβ) and Tau protein because amyloid plaques and neurofibrillary tangles are pathological hallmarks in the brains of patients with AD. The development of assay technologies in diagnosing early-stage AD is very important. The study of human AD subjects is hindered by ethical and technical limitations. Thus, many studies have therefore turned to AD animal models, such as Drosophila melanogaster, to explore AD pathology. However, AD biomarkers such as Aβ and p-Tau protein in Drosophila melanogaster occur at extremely low levels and are difficult to detect precisely. This study applied the immunomagnetic reduction (IMR) technology of nanoparticles for the detection of p-Tau expressions in hTau(R406W) flies, an AD Drosophila model. Furthermore, IMR technology was used as a biosensor in the therapeutic evaluation of Chinese herbal medicines in hTau(R406W) flies with Tau-induced toxicity. To uncover the pathogenic pathway and identify therapeutic interventions of Chinese herbal medicines in Tau-induced toxicity, tauopathy was modeled in the notum of hTau(R406W) flies. IMR data showed that the selected Chinese herbal medicines can significantly reduce p-Tau expressions in hTau(R406W) flies. Using evidence of notal bristle quantification and Western blotting analysis, the IMR data was confirmed and validated. Thus, it is suggested that IMR can serve as a new tool for measuring tauopathy and therapeutic evaluation of Chinese herbal medicine in an AD Drosophila model.
Wang, X., Rimal, S., Tantray, I., Geng, J., Bhurtel, S., Khaket, T. P., Li, W., Han, Z. and Lu, B. (2022). Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2-encoded Nsp1. Proc Natl Acad Sci U S A 119(42): e2202322119. PubMed ID: 36170200
An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. This study shows that overexpression of SARS-CoV-2-encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.
Usher, J. L., Sanchez-Martinez, A., Terriente-Felix, A., Chen, P. L., Lee, J. J., Chen, C. H. and Whitworth, A. J. (2022). Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila. EMBO Rep: e202153552. PubMed ID: 36250243
Parkinson's disease-related proteins, PINK1 and Parkin, act in a common pathway to maintain mitochondrial quality control. While the PINK1-Parkin pathway can promote autophagic mitochondrial turnover (mitophagy) following mitochondrial toxification in cell culture, alternative quality control pathways are suggested. To analyse the mechanisms by which the PINK1-Parkin pathway operates in vivo, methods were developed to detect Ser65-phosphorylated ubiquitin (pS65-Ub) in Drosophila. Exposure to the oxidant paraquat led to robust, Pink1-dependent pS65-Ub production, while pS65-Ub accumulates in unstimulated parkin-null flies, consistent with blocked degradation. Additionally, it was shown that pS65-Ub specifically accumulates on disrupted mitochondria in vivo. Depletion of the core autophagy proteins Atg1, Atg5 and Atg8a did not cause pS65-Ub accumulation to the same extent as loss of parkin, and overexpression of parkin promoted turnover of both basal and paraquat-induced pS65-Ub in an Atg5-null background. Thus, this study has established that pS65-Ub immunodetection can be used to analyse Pink1-Parkin function in vivo as an alternative to reporter constructs. Moreover, the findings suggest that the Pink1-Parkin pathway can promote mitochondrial turnover independently of canonical autophagy in vivo.
Yu, J., Liufu, T., Zheng, Y., Xu, J., Meng, L., Zhang, W., Yuan, Y., Hong, D., Charlet-Berguerand, N., Wang, Z. and Deng, J. (2022). CGG repeat expansion in NOTCH2NLC causes mitochondrial dysfunction and progressive neurodegeneration in Drosophila model. Proc Natl Acad Sci U S A 119(41): e2208649119. PubMed ID: 36191230
Neuronal intranuclear inclusion disease (NIID) is a neuromuscular/neurodegenerative disease caused by the expansion of CGG repeats in the 5' untranslated region (UTR) of the NOTCH2NLC gene. These repeats can be translated into a polyglycine-containing protein, uN2CpolyG, which forms protein inclusions and is toxic in cell models, albeit through an unknown mechanism. This study established a transgenic Drosophila model expressing uN2CpolyG in multiple systems, which resulted in progressive neuronal cell loss, locomotor deficiency, and shortened lifespan. Interestingly, electron microscopy revealed mitochondrial swelling both in transgenic flies and in muscle biopsies of individuals with NIID. Immunofluorescence and immunoelectron microscopy showed colocalization of uN2CpolyG with mitochondria in cell and patient samples, while biochemical analysis revealed that uN2CpolyG interacted with a mitochondrial RNA binding protein, LRPPRC (leucine-rich pentatricopeptide repeat motif-containing protein). Furthermore, RNA sequencing (RNA-seq) analysis and functional assays showed down-regulated mitochondrial oxidative phosphorylation in uN2CpolyG-expressing flies and NIID muscle biopsies. Finally, idebenone treatment restored mitochondrial function and alleviated neurodegenerative phenotypes in transgenic flies. Overall, these results indicate that transgenic flies expressing uN2CpolyG recapitulate key features of NIID and that reversing mitochondrial dysfunction might provide a potential therapeutic approach for this disorder.
Riccardi, C., D'Aria, F., Fasano, D., Digilio, F. A., Carillo, M. R., Amato, J., De Rosa, L., Paladino, S., Melone, M. A. B., Montesarchio, D. and Giancola, C. (2022). Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!. Int J Mol Sci 23(20). PubMed ID: 36293267
Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been designed and evaluated in this study for their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington's disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer.
Siddique, Y. H., Rahul, Ara, G., Afzal, M., Varshney, H., Gaur, K., Subhan, I., Mantasha, I. and Shahid, M. (2022). Beneficial effects of apigenin on the transgenic Drosophila model of Alzheimer's disease. Chem Biol Interact 366: 110120. PubMed ID: 36027948
Alzheimer's disease (AD) is a progressive neurodegenerative disorder. The available drugs improve the symptoms but do not play role in modifying disease effects. Currently, the treatment strategies focus on inhibiting the production of Aβ-42 aggregates and tau filaments. In this context the natural plant products could act as a potent candidate. Therefore, the effect was studied of apigenin, a free-radical scavenger and antioxidant, on the transgenic Drosophila model of AD i.e., expressing Aβ-42 in the neurons. The AD flies were allowed to feed on the diet having 25, 50, 75 and 100 μM of apigenin for 30 days. The exposure of AD flies to apigenin showed a dose dependent significant decrease in the oxidative stress and delay in the loss of climbing ability. Apigenin also inhibits the activity of acetylcholinesterase. The immunostaining and molecular docking studies suggest that apigenin inhibits the formation of Aβ-42 aggregates. Apigenin is potent in reducing the AD symptoms being mimicked in the transgenic Drosophila model of AD.

Wednesday, February 1st - Adult Neural Development and Function

Chockley, A. S., Dinges, G. F., Di Cristina, G., Ratican, S., Bockemuhl, T. and Buschges, A. (2022). Subsets of leg proprioceptors influence leg kinematics but not interleg coordination in Drosophila melanogaster walking. J Exp Biol 225(20). PubMed ID: 36268799
Legged locomotion in terrestrial animals is often essential for mating and survival, and locomotor behavior must be robust and adaptable to be successful. This adaptability is largely provided by proprioceptors monitoring positions and movements of body parts and providing feedback to other components of locomotor networks. In insects, proprioceptive chordotonal organs span joints and encode parameters of relative movement between segments. Previous studies have used whole-organ ablation, reduced preparations or broad physiological manipulations to impair the function of the femoral chordotonal organ (fCO), which monitors the femur-tibia joint, and have demonstrated its contribution to interleg coordination and walking behavior. The fCO in Drosophila melanogaster comprises groups of neurons that differ in their morphology and encoding properties (club, hook, claw); sub-population-level manipulations of fCO function have not been methodologically accessible. This study took advantage of the genetic toolkit available in D. melanogaster to identify sub-populations of fCO neurons and used transient optogenetic inhibition to investigate their roles in locomotor coordination. The findings demonstrate that optogenetic inhibition of a subset of club and hook neurons replicates the effects of inhibiting the whole fCO; when inhibited alone, however, the individual subset types did not strongly affect spatial aspects of single-leg kinematics. Moreover, fCO subsets seem to play only a minor role in interleg temporal coordination. Thus, the fCO contains functionally distinct subgroups, and this functional classification may differ from those based on anatomy and encoding properties; this should be investigated in future studies of proprioceptors and their involvement in locomotor networks.
Scalzotto, M., Ng, R., Cruchet, S., Saina, M., Armida, J., Su, C. Y. and Benton, R. (2022). Pheromone sensing in Drosophila requires support cell-expressed Osiris 8. BMC Biol 20(1): 230. PubMed ID: 36217142
The nose of most animals comprises multiple sensory subsystems, which are defined by the expression of different olfactory receptor families. Drosophila melanogaster antennae contain two morphologically and functionally distinct subsystems that express odorant receptors (Ors) or ionotropic receptors (Irs). Although these receptors have been thoroughly characterized in this species, the subsystem-specific expression and roles of other genes are much less well-understood. This study generate subsystem-specific transcriptomic datasets to identify hundreds of genes, encoding diverse protein classes, that are selectively enriched in either Or or Ir subsystems. Using single-cell antennal transcriptomic data and RNA in situ hybridization, this study found that most neuronal genes-other than sensory receptor genes-are broadly expressed within the subsystems. By contrast, many non-neuronal genes were identified that exhibit highly selective expression, revealing substantial molecular heterogeneity in the non-neuronal cellular components of the olfactory subsystems. One Or subsystem-specific non-neuronal molecule, Osiris 8 (Osi8), a conserved member of a large, insect-specific family of transmembrane proteins. Osi8 is expressed in the membranes of tormogen support cells of pheromone-sensing trichoid sensilla. Loss of Osi8 does not have obvious impact on trichoid sensillar development or basal neuronal activity, but abolishes high sensitivity responses to pheromone ligands. This work identifies a new protein required for insect pheromone detection, emphasizes the importance of support cells in neuronal sensory functions, and provides a resource for future characterization of other olfactory subsystem-specific genes.
Liu, Z., Wu, M. H., Wang, Q. X., Lin, S. Z., Feng, X. Q., Li, B. and Liang, X. (2022). Drosophila mechanical nociceptors preferentially sense localized poking. Elife 11. PubMed ID: 36200757
Mechanical nociception is an evolutionarily conserved sensory process required for the survival of living organisms. Previous studies have revealed much about the neural circuits and sensory molecules in mechanical nociception, but the cellular mechanisms adopted by nociceptors in force detection remain elusive. To address this issue, the mechanosensation of a fly larval nociceptor (class IV da neurons, c4da) was studied using a customized mechanical device. C4da were found to be sensitive to mN-scale forces and make uniform responses to the forces applied at different dendritic regions. Moreover, c4da showed a greater sensitivity to localized forces, consistent with them being able to detect the poking of sharp objects, such as wasp ovipositor. Further analysis reveals that high morphological complexity, mechanosensitivity to lateral tension and possibly also active signal propagation in dendrites contribute to the sensory features of c4da. In particular, it was discovered that Piezo and Ppk1/Ppk26, two key mechanosensory molecules, make differential but additive contributions to the mechanosensitivity of c4da. In all, these results provide updates into understanding how c4da process mechanical signals at the cellular level and reveal the contributions of key molecules.
Vaughen, J. P., Theisen, E., Rivas-Serna, I. M., Berger, A. B., Kalakuntla, P., Anreiter, I., Mazurak, V. C., Rodriguez, T. P., Mast, J. D., Hartl, T., Perlstein, E. O., Reimer, R. J., Clandinin, M. T. and Clandinin, T. R. (2022). Glial control of sphingolipid levels sculpts diurnal remodeling in a circadian circuit. Neuron 110(19): 3186-3205. PubMed ID: 35961319
Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. This study shows that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior.
Mohandasan, R., Thakare, M., Sunke, S., Iqbal, F. M., Sridharan, M. and Das, G. (2022). Enhanced olfactory memory detection in trap-design Y-mazes allows the study of imperceptible memory traces in Drosophila. Learn Mem 29(10): 355-366. PubMed ID: 36180129
The neural basis of behavior is identified by systematically manipulating the activity of specific neurons and screening for loss or gain of phenotype. Therefore, robust, high-scoring behavioral assays are necessary for determining the neural circuits of novel behaviors. This study reports a simple Y-maze design for Drosophila olfactory learning and memory assay. Memory scores in the Y-mazes are considerably better and longer-lasting than scores obtained with commonly used T-mazes. The results suggest that trapping flies to an odor choice in a Y-maze could improve scores. It is postulated that the improved scores could reveal previously undetectable memory traces, enabling the study of underlying neural mechanisms. Indeed, unreported protein synthesis-dependent long-term memories (LTMs) were identified, reinforced by ingestion of (1) an aversive compound and (2) a sweet but nonnutritious sugar, both 24 h after training. Y-mazes were used to probe how using a greater reward may change memory dynamics. These findings predict that a greater sugar reward may extend existing memory traces or reinforce additional novel ones.
Pribbenow, C., Chen, Y. C., Heim, M. M., Laber, D., Reubold, S., Reynolds, E., Balles, I., FernAndez, D. V. A. T., Suárez-Grimalt, R., Scheunemann, L., Rauch, C., Matkovic, T., Rösner, J., Lichtner, G., Jagannathan, S. R. and Owald, D. (2022). Postsynaptic plasticity of cholinergic synapses underlies the induction and expression of appetitive and familiarity memories in Drosophila. Elife 11. PubMed ID: 36250621
In vertebrates, several forms of memory-relevant synaptic plasticity involve postsynaptic rearrangements of glutamate receptors. In contrast, previous work indicates that Drosophila and other invertebrates store memories using presynaptic plasticity of cholinergic synapses. This study provides evidence for postsynaptic plasticity at cholinergic output synapses from the Drosophila mushroom bodies (MBs). The nicotinic acetylcholine receptor (nAChR) subunit α5 is required within specific MB output neurons (MBONs) for appetitive memory induction, but is dispensable for aversive memories. In addition, nAChR α2 subunits mediate memory expression and likely function downstream of α5 and the postsynaptic scaffold protein Dlg. This study shows that ostsynaptic plasticity traces can be induced independently of the presynapse, and that in vivo dynamics of α2 nAChR subunits are changed both in the context of associative and non-associative (familiarity) memory formation, underlying different plasticity rules. Therefore, regardless of neurotransmitter identity, key principles of postsynaptic plasticity support memory storage across phyla.
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