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

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

Thursday, February 29th, 2024 - Adult Neural Structure, Development and Function

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Arican, C., Schmitt, F. J., Rossler, W., Strube-Bloss, M. F., Nawrot, M. P. (2023). The mushroom body output encodes behavioral decision during sensory-motor transformation. Curr Biol, 33(19):4217-4224. PubMed ID: 37657449
Summary:
Animals form a behavioral decision by evaluating sensory evidence on the background of past experiences and the momentary motivational state. In insects, it is understood of how and at which stage of the recurrent sensory-motor pathway behavioral decisions are formed is still lacking. The mushroom body (MB), a central brain structure in insects and crustaceans, integrates sensory input of different modalities with the internal state, the behavioral state, and external sensory context through a large number of recurrent, mostly neuromodulatory inputs, implicating a functional role for MBs in state-dependent sensory-motor transformation. A number of classical conditioning studies in honeybees and fruit flies have provided accumulated evidence that at its output, the MB encodes the valence of a sensory stimulus with respect to its behavioral relevance. Recent work has extended this notion of valence encoding to the context of innate behaviors. This study co-analyzed a defined feeding behavior and simultaneous extracellular single-unit recordings from MB output neurons (MBONs) in the cockroach in response to timed sensory stimulation with odors. Clear neuronal responses occurred almost exclusively during behaviorally responded trials. Early MBON responses to the sensory stimulus preceded the feeding behavior and predicted its occurrence or non-occurrence from the single-trial population activity. These results therefore suggest that at its output, the MB does not merely encode sensory stimulus valence. It is hypothesized instead that the MB output represents an integrated signal of internal state, momentary environmental conditions, and experience-dependent memory to encode a behavioral decision.

Singh, P., Aleman, A., Omoto, J. J., Nguyen, B. C., Kandimalla, P., Hartenstein, V., Donlea, J. M. (2023). Examining Sleep Modulation by Drosophila Ellipsoid Body Neurons. eNeuro, 10(9) PubMed ID: 37679041
Summary:
Recent work in Drosophila has uncovered several neighboring classes of sleep-regulatory neurons within the central complex. However, the logic of connectivity and network motifs remains limited by the incomplete examination of relevant cell types. Using a recent genetic-anatomic classification of ellipsoid body ring neurons, this study conducted a thermogenetic screen in female flies to assess sleep/wake behavior and identified two wake-promoting drivers that label ER3d neurons and two sleep-promoting drivers that express in ER3m cells. Intersectional genetics was used to refine driver expression patterns. Activation of ER3d cells shortened sleep bouts, suggesting a key role in sleep maintenance. While sleep-promoting drivers from the mini-screen label overlapping ER3m neurons, intersectional strategies cannot rule out sleep regulatory roles for additional neurons in their expression patterns. Suppressing GABA synthesis in ER3m neurons prevents postinjury sleep, and GABAergic ER3d cells are required for thermogenetically induced wakefulness. This study used an activity-dependent fluorescent reporter for putative synaptic contacts to embed these neurons within the known sleep-regulatory network. ER3m and ER3d neurons may receive connections from wake-active Helicon/ExR1 cells, and ER3m neurons likely inhibit ER3d neurons. Together, these data suggest a neural mechanism by which previously uncharacterized circuit elements stabilize sleep-wake states.

Kunin, A. B., Guo, J., Bassler, K. E., Pitkow, X., Josic, K. (2023). Hierarchical Modular Structure of the Drosophila Connectome. J Neurosci, 43(37):6384-6400 PubMed ID: 37591738
Summary:
This study applied novel community detection methods to analyze the synapse-level reconstruction of an adult female Drosophila melanogaster brain containing >20,000 neurons and 10 million synapses. Using a machine-learning algorithm, the most densely connected communities of neurons were found by maximizing a generalized modularity density measure. The community structure was resolved at a range of scales, from large (on the order of thousands of neurons) to small (on the order of tens of neurons). The network was found to be organized hierarchically, and larger-scale communities are composed of smaller-scale structures. These methods identify well-known features of the fly brain, including its sensory pathways. Moreover, focusing on specific brain regions,it was possible to identify subnetworks with distinct connectivity types. For example, manual efforts have identified layered structures in the fan-shaped body. Yhese methods not only automatically recover this layered structure, but also resolve finer connectivity patterns to downstream and upstream areas. A novel modular organization was found of the superior neuropil, with distinct clusters of upstream and downstream brain regions dividing the neuropil into several pathways. These methods show that the fine-scale, local network reconstruction made possible by modern experimental methods are sufficiently detailed to identify the organization of the brain across scales, and enable novel predictions about the structure and function of its parts.

Sizemore, T. R., Jonaitis, J., Dacks, A. M. (2023). Heterogeneous receptor expression underlies non-uniform peptidergic modulation of olfaction in Drosophila. Nat Commun, 14(1):5280 PubMed ID: 37644052
Summary:
Sensory systems are dynamically adjusted according to the animal's ongoing needs by neuromodulators, such as neuropeptides. Neuropeptides are often widely-distributed throughout sensory networks, but it is unclear whether such neuropeptides uniformly modulate network activity. This study leveraged the Drosophila antennal lobe (AL) to resolve whether myoinhibitory peptide (MIP) uniformly modulates AL processing. Despite being uniformly distributed across the AL, MIP decreases olfactory input to some glomeruli, while increasing olfactory input to other glomeruli. A heterogeneous ensemble of local interneurons (LNs) are the sole source of AL MIP; differential expression of the inhibitory MIP receptor across glomeruli allows MIP to act on distinct intraglomerular substrates. These findings demonstrate how even a seemingly simple case of modulation can have complex consequences on network processing by acting non-uniformly within different components of the overall network.

Corthals, K., Andersson, V., Churcher, A., Reimegard, J., Enjin, A. (2023). Genetic atlas of hygro-and thermosensory cells in the vinegar fly Drosophila melanogaster. Sci Rep, 13(1):15202 PubMed ID: 37709909
Summary:
The ability of animals to perceive and respond to sensory information is essential for their survival in diverse environments. While much progress has been made in understanding various sensory modalities, the sense of hygrosensation, which involves the detection and response to humidity, remains poorly understood. This study focused on the hygrosensory and closely related thermosensory systems in Drosophila to unravel the molecular profile of the cells of these senses. Using a transcriptomic analysis of over 37,000 nuclei, twelve distinct clusters of cells were identified corresponding to temperature-sensing arista neurons, humidity-sensing sacculus neurons, and support cells relating to these neurons. By examining the expression of known and novel marker genes, the identity of these clusters was validated, and their gene expression profiles identified. Each cell type could be characterized by a unique expression profile of ion channels, GPCR signaling molecules, synaptic vesicle cycle proteins, and cell adhesion molecules. These findings provide valuable insights into the molecular basis of hygro- and thermosensation. Understanding the mechanisms underlying hygro- and thermosensation may shed light on the broader understanding of sensory systems and their adaptation to different environmental conditions in animals.

Wednesday, February 28th - Oncogenesis and Growth

Yin, H., Wang, Z., Wang, D., Nuer, M., Han, M., Ren, P., Ma, S., Lin, C., Chen, J., Xian, H., Ai, D., Li, X., Ma, S., Lin, Z. and Pan, Y. (2023). TIMELESS promotes the proliferation and migration of lung adenocarcinoma cells by activating EGFR through AMPK and SPHK1 regulation. Eur J Pharmacol 955: 175883. PubMed ID: 37433364
Summary:
Lung adenocarcinoma (LUAD) has high morbidity and is prone to recurrence. TIMELESS (TIM), which regulates circadian rhythms in Drosophila, is highly expressed in various tumors. Tumor samples from patients with LUAD patient data from public databases were used to confirm the relationship of TIM expression with lung cancer. LUAD cell lines were used and siRNA of TIM was adopted to knock down TIM expression in LUAD cells, and further cell proliferation, migration and colony formation were analyzed. By using Western blot and qPCR, the influence was detected of TIM on epidermal growth factor receptor (EGFR), sphingosine kinase 1 (SPHK1) and AMP-activated protein kinase (AMPK). With proteomics analysis, this study comprehensively inspected the different changed proteins influenced by TIM, and global bioinformatic analysis was performed. TIM expression was found to be elevated in LUAD and that this high expression was positively correlated with more advanced tumor pathological stages and shorter overall and disease-free survival. TIM knockdown inhibited EGFR activation and also AKT/mTOR phosphorylation. This study also clarified that TIM regulated the activation of SPHK1 in LUAD cells. And with SPHK1 siRNA to knock down the expression level of SPHK1, it was found that EGFR activation were inhibited greatly too. Quantitative proteomics techniques combined with bioinformatics analysis clarified the global molecular mechanisms regulated by TIM in LUAD. The results of proteomics suggested that mitochondrial translation elongation and termination were altered, which were closely related to the process of mitochondrial oxidative phosphorylation. It was further confirmed that TIM knockdown reduced ATP content and promoted AMPK activation in LUAD cells. This study revealed that siTIM could inhibit EGFR activation through activating AMPK and inhibiting SPHK1 expression, as well as influencing mitochondrial function and altering the ATP level; TIM's high expression in LUAD is an important factor and a potential key target in LUAD.

Bertrand, M., Szeremeta, F., Hervouet-Coste, N., Sarou-Kanian, V., Landon, C., Morisset-Lopez, S., Decoville, M. (2023). An adult Drosophila glioma model to highlight metabolic dysfunctions and evaluate the role of the serotonin 5-HT(7) receptor as a potential therapeutic target. Faseb j, 37(11):e23230 PubMed ID: 37781977
Summary:
Gliomas account for 50% of brain cancers and are therefore the most common brain tumors. Molecular alterations involved in adult gliomas have been identified and mainly affect tyrosine kinase receptors with amplification and/or mutation of the epidermal growth factor receptor (EGFR) and its associated signaling pathways. Several targeted therapies have been developed, but current treatments remain ineffective for glioblastomas, the most severe forms. Thus, it is a priority to identify new pharmacological targets. Drosophila glioma models established in larvae and adults are useful to identify new genes and signaling pathways involved in glioma progression. This study used a Drosophila glioma model in adults, to characterize metabolic disturbances associated with glioma and assess the consequences of 5-HT(7) R expression on glioma development. First, by using in vivo magnetic resonance imaging, it was shown that expression of the constitutively active forms of EGFR and PI3K in adult glial cells induces brain enlargement. Then, altered cellular metabolism was exploited by using high-resolution magic angle spinning NMR and (1) H-(13) C heteronuclear single quantum coherence solution states. Discriminant metabolites identified highlight the rewiring of metabolic pathways in glioma and associated cachexia phenotypes. Finally, the expression of 5-HT(7) R in this adult model attenuates phenotypes associated with glioma development. Collectively, this whole-animal approach in Drosophila provides several rapid and robust phenotype readouts, such as enlarged brain volume and glioma-associated cachexia, as well as to determine the metabolic pathways involved in glioma genesis and finally to confirm the interest of the 5-HT(7) R in the treatment of glioma.

Kinoshita, Y., Shiratsuchi, N., Araki, M., Inoue, Y. H. (2023). Anti-Tumor Effect of Turandot Proteins Induced via the JAK/STAT Pathway in the mxc Hematopoietic Tumor Mutant in Drosophila. Cells, 12(16) PubMed ID: 37626857
Summary:
Several antimicrobial peptides suppress the growth of lymph gland (LG) tumors in Drosophila multi sex comb (mxc) mutant larvae. The activity of another family of polypeptides, called Turandots, is also induced via the JAK/STAT pathway after bacterial infection; however, their influence on Drosophila tumors remains unclear. The JAK/STAT pathway was activated in LG tumors, fat body, and circulating hemocytes of mutant larvae. The mRNA levels of Turandot (Tot) genes increased markedly in the mutant fat body and declined upon silencing Stat92E in the fat body, indicating the involvement of the JAK/STAT pathway. Furthermore, significantly enhanced tumor growth upon a fat-body-specific silencing of the mRNAs demonstrated the antitumor effects of these proteins. The proteins were found to be incorporated into small vesicles in mutant circulating hemocytes (as previously reported for several antimicrobial peptides) but not normal cells. In addition, more hemocytes containing these proteins were found to be associated with tumors. The mutant LGs contained activated effector caspases, and a fat-body-specific silencing of Tots inhibited apoptosis and increased the number of mitotic cells in the LG, thereby suggesting that the proteins inhibited tumor cell proliferation. Thus, Tot proteins possibly exhibit antitumor effects via the induction of apoptosis and inhibition of cell proliferation.

Voutyraki, C., Choromidis, A., Meligkounaki, A., Vlachopoulos, N. A., Theodorou, V., Grammenoudi, S., Athanasiadis, E., Monticelli, S., Giangrande, A., Delidakis, C. and Zacharioudaki, E. (2023). Growth deregulation and interaction with host hemocytes contribute to tumor progression in a Drosophila brain tumor model. Proc Natl Acad Sci U S A 120(33): e2221601120. PubMed ID: 37549261
Summary:
Tumors constantly interact with their microenvironment. This study presented data on a Notch-induced neural stem cell (NSC) tumor in Drosophila, which can be immortalized by serial transplantation in adult hosts. This tumor arises in the larva by virtue of the ability of Notch to suppress early differentiation-promoting factors in NSC progeny. Guided by transcriptome data, this study has addressed both tumor-intrinsic and microenvironment-specific factors and how they contribute to tumor growth and host demise. The growth promoting factors Myc, Imp, and Insulin receptor in the tumor cells are important for tumor expansion and killing of the host. From the host's side, hemocytes, professional phagocytic blood cells, are found associated with tumor cells. Phagocytic receptors, like NimC1, are needed in hemocytes to enable them to capture and engulf tumor cells, restricting their growth. In addition to their protective role, hemocytes may also increase the host's morbidity by their propensity to produce damaging extracellular reactive oxygen species.

Bosso, G., Cipressa, F., Tullo, L., Cenci, G. (2023). Co-amplification of CBX3 with EGFR or RAC1 in human cancers corroborated by a conserved genetic interaction among the genes. Cell Death Discov, 9(1):317 PubMed ID: 37633946
Summary:
Chromobox Protein 3 (CBX3; Drosophila homlog - HP1b) overexpression is a common event occurring in cancer, promotes cancer cell proliferation and represents a poor prognosis marker in a plethora of human cancers. This study describes that a wide spectrum of human cancers harbors a co-amplification of CBX3 gene with either EGFR or RAC1, which yields a statistically significant increase of both mRNA and protein levels of CBX3, EGFR and RAC1. It was also revealed that the simultaneous overexpression of CBX3, RAC1 and EGFR gene products correlates with a worse prognosis compared to the condition when CBX3, RAC1 and EGFR are singularly upregulated. Furthermore, this study also showed that a co-occurrence of low-grade amplification, in addition to high-grade amplification, between CBX3 and EGFR or RAC1 is associated with a reduced patient lifespan. Finally, CBX3 and RAC1/EGFR genetically interact in the model organism Drosophila melanogaster, suggesting that the simultaneous overexpression as well as well the co-occurrence of high- or low-grade copy number alterations in these genes is not accidental and could reflect evolutionarily conserved functional relationships.

Johannessen, J. A., Formica, M., Haukeland, A. L. C., Brathen, N. R., Al Outa, A., Aarsund, M., Therrien, M., Enserink, J. M., Knaevelsrud, H. (2023). The human leukemic oncogene MLL-AF4 promotes hyperplastic growth of hematopoietic tissues in Drosophila larvae. iScience, 26(10):107726 PubMed ID: 37720104
Summary:
MLL-rearranged (MLL-r) leukemias are among the leukemic subtypes with poorest survival, and treatment options have barely improved over the last decades. Despite increasing molecular understanding of the mechanisms behind these hematopoietic malignancies, this knowledge has had poor translation into the clinic. This study reports a Drosophila melanogaster model system to explore the pathways affected in MLL-r leukemia. Expression of the human leukemic oncogene MLL-AF4 in the Drosophila hematopoietic system resulted in increased levels of circulating hemocytes and an enlargement of the larval hematopoietic organ, the lymph gland. Strikingly, depletion of Drosophila orthologs of known interactors of MLL-AF4, such as DOT1L, rescued the leukemic phenotype. In agreement, treatment with small-molecule inhibitors of DOT1L also prevented the MLL-AF4-induced leukemia-like phenotype. Taken together, this model provides an in vivo system to unravel the genetic interactors involved in leukemogenesis and offers a system for improved biological understanding of MLL-r leukemia.

Tuesday, February 27th - Chromatin

Koury, S. A. (2023). Female meiotic drive shapes the distribution of rare inversion polymorphisms in Drosophila melanogaster. Genetics, 225(2) PubMed ID: 37616566
Summary:
In all species, new chromosomal inversions are constantly being formed by spontaneous rearrangement and then stochastically eliminated from natural populations. In Drosophila, when new chromosomal inversions overlap with a preexisting inversion in the population, their rate of elimination becomes a function of the relative size, position, and linkage phase of the gene rearrangements. These altered dynamics result from complex meiotic behavior wherein overlapping inversions generate asymmetric dyads that cause both meiotic drive/drag and segmental aneuploidy. In this context, patterns in rare inversion polymorphisms of a natural population can be modeled from the fundamental genetic processes of forming asymmetric dyads via crossing-over in meiosis I and preferential segregation from asymmetric dyads in meiosis II. Here, a mathematical model of crossover-dependent female meiotic drive is developed and parameterized with published experimental data from Drosophila melanogaster laboratory constructs. This mechanism is demonstrated to favor smaller, distal inversions and accelerate the elimination of larger, proximal inversions. Simulated sampling experiments indicate that the paracentric inversions directly observed in natural population surveys of D. melanogaster are a biased subset that both maximizes meiotic drive and minimizes the frequency of lethal zygotes caused by this cytogenetic mechanism. Incorporating this form of selection into a population genetic model accurately predicts the shift in relative size, position, and linkage phase for rare inversions found in this species. The model and analysis presented in this study suggest that this weak form of female meiotic drive is an important process influencing the genomic distribution of rare inversion polymorphisms.

Kingsley, G., Skagia, A., Passaretti, P., Fernandez-Cuesta, C., Reynolds-Winczura, A., Koscielniak, K., Gambus, A. (2023). DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation. Nucleic Acids Res, 51(18):9748-9763 PubMed ID: 37638758
Summary:
Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. This study shows that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients.

Shu, S., Jiang, M., Deng, X., Yue, W., Cao, X., Zhang, K., Wang, Z., He, H., Cui, J., Wang, Q., Qu, K., Fang, Y. (2023). Heterochromatic silencing of immune-related genes in glia is required for BBB integrity and normal lifespan in drosophila. Aging Cell, 22(10):e13947 PubMed ID: 37594178
Summary:
Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. This study reports that adult-onset downregulation of a Drosophila gene CG32529/GLAD led to shortened lifespan and age-dependent brain degeneration. This regulation exhibited cell type and subtype-specificity, involving mainly surface glia (comprising the BBB) and cortex glia (wrapping neuronal soma) in flies. In accordance, pan-glial knockdown of GLAD disrupted BBB integrity and the glial meshwork. GLAD expression in fly heads decreased with age, and the RNA-seq analysis revealed that the most affected transcriptional changes by RNAi-GLAD were associated with upregulation of immune-related genes. Furthermore, a series of lifespan rescue experiments was conducted and the results indicated that the profound upregulation of immune and related pathways was not the consequence but cause of the degenerative phenotypes of the RNAi-GLAD flies. Finally, this study showed that GLAD encoded a heterochromatin-associating protein that bound to the promoters of an array of immune-related genes and kept them silenced during the cell cycle. Together, these findings demonstrate a previously unappreciated role of heterochromatic gene silencing in repressing immunity in fly glia, which is required for maintaining BBB and brain integrity as well as normal lifespan.

Yang, J., Tang, R., Chen, S., Chen, Y., Yuan, K., Huang, R., Wang, L. (2023). Exposure to high-sugar diet induces transgenerational changes in sweet sensitivity and feeding behavior via H3K27me3 reprogramming. Elife, 12 PubMed ID: 37698486
Summary:
Human health is facing a host of new threats linked to unbalanced diets, including high-sugar diet (HSD), which contributes to the development of both metabolic and behavioral disorders. Studies have shown that diet-induced metabolic dysfunctions can be transmitted to multiple generations of offspring and exert long-lasting health burden. Meanwhile, whether and how diet-induced behavioral abnormalities can be transmitted to the offspring remains largely unclear. This study showed that ancestral HSD exposure suppressed sweet sensitivity and feeding behavior in the offspring in Drosophila. These behavioral deficits were transmitted through the maternal germline and companied by the enhancement of H3K27me3 modifications. PCL-PRC2 complex, a major driver of H3K27 trimethylation, was upregulated by ancestral HSD exposure, and disrupting its activity eliminated the transgenerational inheritance of sweet sensitivity and feeding behavior deficits. Elevated H3K27me3 inhibited the expression of a transcriptional factor Cad and suppressed sweet sensitivity of the sweet-sensing gustatory neurons, reshaping the sweet perception and feeding behavior of the offspring. Taken together, this study uncovered a novel molecular mechanism underlying behavioral abnormalities spanning multiple generations of offspring upon ancestral HSD exposure, which would contribute to the further understanding of long-term health risk of unbalanced diet.

Sarkar, K., Kotb, N. M., Lemus, A., Martin, E. T., McCarthy, A., Camacho, J., Iqbal, A., Valm, A. M., Sammons, M. A., Rangan, P. (2023). A feedback loop between heterochromatin and the nucleopore complex controls germ-cell-to-oocyte transition during Drosophila oogenesise. Dev Cell 58(22):2580-2596 PubMed ID: 37673064
Summary:
Germ cells differentiate into oocytes that launch the next generation upon fertilization. How the highly specialized oocyte acquires this distinct cell fate is poorly understood. During Drosophila oogenesis, H3K9me3 histone methyltransferase SETDB1 translocates from the cytoplasm to the nucleus of germ cells concurrently with oocyte specification. This study discovered that nuclear SETDB1 is required for silencing a cohort of differentiation-promoting genes by mediating their heterochromatinization. Intriguingly, SETDB1 is also required for upregulating 18 of the ∼30 nucleoporins (Nups) that compose the nucleopore complex (NPC), promoting NPC formation. NPCs anchor SETDB1-dependent heterochromatin at the nuclear periphery to maintain H3K9me3 and gene silencing in the egg chambers. Aberrant gene expression due to the loss of SETDB1 or Nups results in the loss of oocyte identity, cell death, and sterility. Thus, a feedback loop between heterochromatin and NPCs promotes transcriptional reprogramming at the onset of oocyte specification, which is critical for establishing oocyte identity.

Santos, A. S., Ramos, E. S., Valente-Gaiesky, V. L. S., de Melo Sene, F., Manfrin, M. H. (2023). Evidences of differential methylation in the genome during development in the cactophilic Drosophila species. Genesis:e23554 PubMed ID: 37750176
Summary:
DNA methylation with 5-methylcytosine (5mC) has been reported in the genome of several eukaryotes, with marked differences between vertebrates and invertebrates. DNA methylation is poorly understood as its role in evolution in insects. Drosophila gouveai (cluster Drosophila buzzatii) presents larvae that develop obligatorily in necrotic tissues of cacti in nature, with the distribution of populations in South America, and plasticity of phenotypes in insect-plant interaction. Organisms were characterized at developmental stages, and variations at multiple methylation-sensitive loci were analyzed in pupae, and adult flies using methylation sensitive amplification polymorphism. 326 loci with CCGG targets were obtained in the genome of D. gouveai. Genomic regions with molecular lengths from 100 to 700 pb were most informative about methylation states. Multiple loci show differences in methylation-sensitive sites (MSL) concerning developmental stages, such as in pupae (MSL = 40), female reproductive tissue (MSL = 76), and male reproductive tissues (MSL = 58). These results are the first evidence of genome-wide methylation in D. gouveai organisms.

Monday, February 26th - Adult Physiology and Metabolism

Dobson, A. J., Voigt, S., Kumpitsch, L., Langer, L., Voigt, E., Ibrahim, R., Dowling, D. K., Reinhardt, K. (2023). Mitonuclear interactions shape both direct and parental effects of diet on fitness and involve a SNP in mitoribosomal 16s rRNA. PLoS Biol, 21(8):e3002218 PubMed ID: 37603597
Summary:
Nutrition is a primary determinant of health, but responses to nutrition vary with genotype. Epistasis between mitochondrial and nuclear genomes may cause some of this variation, but which mitochondrial loci and nutrients participate in complex gene-by-gene-by-diet interactions? Furthermore, it remains unknown whether mitonuclear epistasis is involved only in the immediate responses to changes in diet, or whether mitonuclear genotype might modulate sensitivity to variation in parental nutrition, to shape intergenerational fitness responses. In Drosophila melanogaster this study shows that mitonuclear epistasis shapes fitness responses to variation in dietary lipids and amino acids. It was also shown that mitonuclear genotype modulates the parental effect of dietary lipid and amino acid variation on offspring fitness. Effect sizes for the interactions between diet, mitogenotype, and nucleogenotype were equal to or greater than the main effect of diet for some traits, suggesting that dietary impacts cannot be understood without first accounting for these interactions. Associating phenotype to mtDNA variation in a subset of populations implicated a C/T polymorphism in mt:lrRNA, which encodes the 16S rRNA of the mitochondrial ribosome. This association suggests that directionally different responses to dietary changes can result from variants on mtDNA that do not change protein coding sequence, dependent on epistatic interactions with variation in the nuclear genome.

Gruss, I., Twardowski, J., Samsel-Czekala, M., Beznosiuk, J., Wandzel, C., Twardowska, K., Wiglusz, R. J. (2023). The isothermal Boltzmann-Gibbs entropy reduction affects survival of the fruit fly Drosophila melanogaster. Sci Rep, 13(1):14166 PubMed ID: 37644276
Summary:
This study presents is the first experimental evidence of the effect of isothermal changes in entropy on a living organism. In greater detail, the effect of the reduction of the total Boltzmann-Gibbs entropy (S) of the aquatic environment on the survival rate and body mass of the fruit fly Drosophila melanogaster was investigated. The tests were carried out in standard thermodynamic states at room temperature of 296.15 K and ambient atmospheric pressure of 1 bar. Two variants of entropy reduction (ΔS) were tested for ΔS = 28.49 and 51.14 J K(-1) mol(-1) compared to the blind and control samples. The entropy level was experimentally changed, using the quantum system for isothermal entropy reduction. This system is based on quantum bound entanglement of phonons and the phenomenon of phonon resonance (interference of phonon modes) in condensed matter (Silicon dioxide (SiO(2)) and single crystals of Silicon (Si(0)), Aluminum (Al(0)) plates ("chips"), glass, and water). All studied organisms were of the same age (1 day). Mortality was observed daily until the natural death of the organisms. The investigations showed that changes in the Boltzmann-Gibbs entropy affected the survival and body mass of the fruit flies. On the one hand, the reduction in entropy under isothermal conditions in the aquatic environment for ΔS = 28.49 J K(-1) mol(-1) resulted in an extension of the lifespan and an increase in the body mass of female fruit flies. On the other hand, the almost twofold reduction in this entropy for ΔS = 51.14 J K(-1) mol(-1) shortened the lives of the males. Thus, the lifespan and body mass of flies turned out to be a specific reaction of metabolism related to changes in the entropy of the aquatic environment.

Kashio, S., Masuda, S., Miura, M. (2023). Involvement of neuronal tachykinin-like receptor at 86C in Drosophila disc repair via regulation of kynurenine metabolism. iScience, 26(9):107553 PubMed ID: 37636053
Summary:
Neurons contribute to the regeneration of projected tissues; however, it remains unclear whether they are involved in the non-innervated tissue regeneration. This study shows that a neuronal tachykinin-like receptor at 86C (TkR86C) is required for the repair of non-innervated wing discs in Drosophila. Using a genetic tissue repair system in Drosophila larvae, genetic screening was performed for G protein-coupled receptors to search for signal mediatory systems for remote tissue repair. An evolutionarily conserved neuroinflammatory receptor, TkR86C, was identified as the candidate receptor. Neuron-specific knockdown of TkR86C impaired disc repair without affecting normal development. The humoral metabolites of the kynurenine (Kyn) pathway regulated in the fat body were investigated because of their role as tissue repair-mediating factors. Neuronal knockdown of TkR86C hampered injury-dependent changes in the expression of vermillion in the fat body and humoral Kyn metabolites. These data indicate the involvement of TkR86C neurons upstream of Kyn metabolism in non-autonomous tissue regeneration.

Guilhot, R., Xuereb, A., Lagmairi, A., Olazcuaga, L., Fellous, S. (2023). Microbiota acquisition and transmission in Drosophila flies. iScience, 26(9):107656 PubMed ID: 37670792
Summary:
Understanding the ecological and evolutionary dynamics of host-microbiota associations notably involves exploring how members of the microbiota assemble and whether they are transmitted along host generations. This study investigated the larval acquisition of facultative bacterial and yeast symbionts of Drosophila melanogaster and Drosophila suzukii in ecologically realistic setups. Fly mothers and fruit were major sources of symbionts. Microorganisms associated with adult males also contributed to larval microbiota, mostly in D. melanogaster. Yeasts acquired at the larval stage maintained through metamorphosis, adult life, and were transmitted to offspring. All these observations varied widely among microbial strains, suggesting they have different transmission strategies among fruits and insects. This approach shows microbiota members of insects can be acquired from a diversity of sources and highlights the compound nature of microbiotas. Such microbial transmission events along generations should favor the evolution of mutualistic interactions and enable microbiota-mediated local adaptation of the insect host.

Socha, C., Pais, I. S., Lee, K. Z., Liu, J., Liegeois, S., Lestradet, M., Ferrandon, D. (2023). Fast drosophila enterocyte regrowth after infection involves a reverse metabolic flux driven by an amino acid transporter. iScience, 26(9):107490 PubMed ID: 37636057
Summary:
Upon exposure to a bacterial pore-forming toxin, enterocytes rapidly purge their apical cytoplasm into the gut lumen, resulting in a thin intestinal epithelium. The enterocytes regain their original shape and thickness within 16 h after the ingestion of the bacteria. This study shows that the regrowth of Drosophila enterocytes entails an inversion of metabolic fluxes from the organism back toward the intestine. A proton-assisted transporter, Arcus, was identified that is required for the reverse absorption of amino acids and the timely recovery of the intestinal epithelium. Arcus is required for a peak of amino acids appearing in the hemolymph shortly after infection. The regrowth of enterocytes involves the insulin signaling pathway and Myc. The purge decreases Myc mRNA levels, which subsequently remain at low levels in the arcus mutant. Interestingly, the action of arcus and Myc in the intestinal epithelium is not cell-autonomous, suggesting amino acid fluxes within the intestinal epithelium.

Mari, M., Voutyraki, C., Zacharioudaki, E., Delidakis, C., Filippidis, G. (2023). Lipid content evaluation of Drosophila tumour associated haemocytes through Third Harmonic Generation measurements. Journal of biophotonics, 16(12):e202300171 PubMed ID: 37643223
Summary:
Non-linear microscopy is a powerful imaging tool to examine structural properties and subcellular processes of various biological samples. The competence of Third Harmonic Generation (THG) includes the label free imaging with diffraction-limited resolution and three-dimensional visualization with negligible phototoxicity effects. In this study, THG records and quantifies the lipid content of Drosophila haemocytes, upon encountering normal or tumorigenic neural cells, in correlation with their shape or their state. The lipid accumulations of adult haemocytes were shown to be similar before and after encountering normal cells. In contrast, adult haemocytes prior to their interaction with cancer cells have a low lipid index, which increases while they are actively engaged in phagocytosis only to decrease again when haemocytes become exhausted. This dynamic change in the lipid accrual of haemocytes upon encountering tumour cells could potentially be a useful tool to assess the phagocytic capacity or activation state of tumour-associated haemocytes.

Friday, February 23rd - RNA and Transposons

Flynn, J. M., Ahmed-Braimah, Y. H., Long, M., Wing, R. A., Clark, A. G. (2023). High quality genome assemblies reveal evolutionary dynamics of repetitive DNA and structural rearrangements in the Drosophila virilis sub-group. bioRxiv, PubMed ID: 37645834
Summary:
High-quality genome assemblies across a range of non-traditional model organisms can accelerate the discovery of novel aspects of genome evolution. The Drosophila virilis group has several attributes that distinguish it from more highly studied species in the Drosophila genus, such as an unusual abundance of repetitive elements and extensive karyotype evolution, in addition to being an attractive model for speciation genetics. This study used long-read sequencing to assemble five genomes of three virilis group species and characterized sequence and structural divergence and repetitive DNA evolution. The contiguous genome assemblies allow characterization of chromosomal arrangements with ease and can facilitate analysis of inversion breakpoints. A small panel of resequenced strains was leveraged to explore the genomic pattern of divergence and polymorphism in this species and show that known demographic histories largely predicts the extent of genome-wide segregating polymorphism. It was further found that a neo-X chromosome in D. americana displays X-like levels of nucleotide diversity. Unusual repetitive elements were found to be responsible for much of the divergence in genome composition among species. Helitron-derived tandem repeats tripled in abundance on the Y chromosome in D. americana compared to D. novamexicana, accounting for most of the difference in repeat content between these sister species. Repeats with characteristics of both transposable elements and satellite DNAs expanded by three-fold, mostly in euchromatin, in both D. americana and D. novamexicana compared to D. virilis. These results represent a major advance in understanding of genome biology in this emerging model clade.

Meng, L. W., Yuan, G. R., Chen, M. L., Zheng, L. S., Dou, W., Peng, Y., Bai, W. J., Li, Z. Y., Vontas, J., Wang, J. J. (2023). Cuticular competing endogenous RNAs regulate insecticide penetration and resistance in a major agricultural pest. BMC Biol, 21(1):187 PubMed ID: 37667263
Summary:
The continuously developing pesticide resistance is a great threat to agriculture and human health. Understanding the mechanisms of insecticide resistance is a key step in dealing with the phenomenon. Insect cuticle is recently documented to delay xenobiotic penetration which breaks the previous stereotype that cuticle is useless in insecticide resistance, while the underlying mechanism remains scarce. This study found the integument contributes over 40.0% to insecticide resistance via different insecticide delivery strategies in oriental fruit fly. A negative relationship exists between cuticle thickening and insecticide penetration in resistant/susceptible, also in field strains of oriental fruit fly which is a reason for integument-mediated resistance. These investigations uncover a regulator of insecticide penetration that miR-994 mimic treatment causes cuticle thinning and increases susceptibility to malathion, whereas miR-994 inhibitor results in opposite phenotypes. The target of miR-994 is a most abundant cuticle protein (CPCFC) in resistant/susceptible integument expression profile, which possesses capability of chitin-binding and influences the cuticle thickness-mediated insecticide penetration. This analyses found an upstream transcriptional regulatory signal of miR-994 cascade, long noncoding RNA (lnc19419), that indirectly upregulates CPCFC in cuticle of the resistant strain by sponging miR-994. Thus, this study has elucidated the mechanism of cuticular competing endogenous RNAs for regulating insecticide penetration and demonstrate it also exists in field strain of oriental fruit fly. This study unveil a regulatory axis of lnc19419 ~ miR-994 ~ CPCFC on the cuticle thickness that leads to insecticide penetration resistance. These findings indicate that competing endogenous RNAs regulate insecticide resistance by modulating the cuticle thickness and provide insight into the resistance mechanism in insects.

Kim, C. J., Kim, H. H., Kim, H. K., Lee, S., Jang, D., Kim, C., Lim, D. H. (2023). MicroRNA miR-263b-5p Regulates Developmental Growth and Cell Association by Suppressing Laminin A in Drosophila. Biology, 12(8) PubMed ID: 37626982
Summary:
Basement membranes (BMs) play important roles under various physiological conditions in animals, including ecdysozoans. During development, BMs undergo alterations through diverse intrinsic and extrinsic regulatory mechanisms; however, the full complement of pathways controlling these changes remain unclear. This study found that fat body-overexpression of Drosophila miR-263b, which is highly expressed during the larval-to-pupal transition, resulted in a decrease in the overall size of the larval fat body, and ultimately, in a severe growth defect accompanied by a reduction in cell proliferation and cell size. Interestingly, it was further observed that a large proportion of the larval fat body cells were prematurely disassociated from each other. Moreover, evidence is presented that miR-263b-5p suppresses the main component of BMs, Laminin A (LanA). Through experiments using RNA interference (RNAi) of LanA, it was found that its depletion phenocopied the effects in miR-263b-overexpressing flies. Overall, these findings suggest a potential role for miR-263b in developmental growth and cell association by suppressing LanA expression in the Drosophila fat body.

Duan, Y., Xu, Y., Song, F., Tian, L., Cai, W., Li, H. (2023). Differential adaptive RNA editing signals between insects and plants revealed by a new measurement termed haplotype diversity. Biology direct, 18(1):47 PubMed ID: 37592344
Summary:
C-to-U RNA editing in plants is believed to confer its evolutionary adaptiveness by reversing unfavorable DNA mutations. This "restorative hypothesis" has not yet been tested genome-wide. In contrast, A-to-I RNA editing in insects like Drosophila and honeybee is already known to benefit the host by increasing proteomic diversity in a spatial-temporal manner (namely "diversifying hypothesis"). This study profiled the RNA editomes of multiple tissues of Arabidopsis thaliana, Drosophila melanogaster, and Apis melifera. The haplotype diversity (HD) of RNA molecules was unprecedentedly defined based on nonsynonymous editing events (recoding sites). Signals of adaptation is confirmed in Arabidopsis by observing higher frequencies and levels at nonsynonymous editing sites over synonymous sites. Compared to A-to-I recoding sites in Drosophila, the C-to-U recoding sites in Arabidopsis show significantly lower HD, presumably due to the stronger linkage between C-to-U events. It is concluded that C-to-U RNA editing in Arabidopsis is adaptive but it is not designed for diversifying the proteome like A-to-I editing in Drosophila. Instead, C-to-U recoding sites resemble DNA mutations. These observation supports the restorative hypothesis of plant C-to-U editing which claims that editing is used for fixing unfavorable genomic sequences.

Zhang, Z., Bae, B., Cuddleston, W. H., Miura, P. (2023). Coordination of alternative splicing and alternative polyadenylation revealed by targeted long read sequencing. Nat Commun, 14(1):5506 PubMed ID: 37679364
Summary:
Nervous system development is associated with extensive regulation of alternative splicing (AS) and alternative polyadenylation (APA). AS and APA have been extensively studied in isolation, but little is known about how these processes are coordinated. This study investigated the coordination of cassette exon (CE) splicing and APA in Drosophila using a targeted long-read sequencing approach called Pull-a-Long-Seq (PL-Seq). This cost-effective method uses cDNA pulldown and Nanopore sequencing combined with an analysis pipeline to quantify inclusion of alternative exons in connection with alternative 3' ends. Using PL-Seq, genes were identified that exhibit significant differences in CE splicing depending on connectivity to short versus long 3'UTRs. Genomic long 3'UTR deletion was found to alter upstream CE splicing in short 3'UTR isoforms and ELAV loss differentially affected CE splicing depending on connectivity to alternative 3'UTRs. This work highlights the importance of considering connectivity to alternative 3'UTRs when monitoring AS events.

Venkei, Z. G., Gainetdinov, I., Bagci, A., Starostik, M. R., Choi, C. P., Fingerhut, J. M., Chen, P., Balsara, C., Whitfield, T. W., Bell, G. W., Feng, S., Jacobsen, S. E., Aravin, A. A., Kim, J. K., Zamore, P. D., Yamashita, Y. M. (2023). A maternally programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila. Nat Cell Biol, 25(10):1495-1505 PubMed ID: 37723298
Summary:
In animals, PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In Drosophila and other species with a maternally specified germline, piRNAs deposited in the egg initiate piRNA biogenesis in the progeny. However, Y chromosome loci cannot participate in such a chain of intergenerational inheritance. How then can the biogenesis of Y-linked piRNAs be initiated? Using Suppressor of Stellate (Su(Ste)), a Y-linked Drosophila melanogaster piRNA locus as a model, this study showed that Su(Ste) piRNAs are made in the early male germline via 5'-to-3' phased piRNA biogenesis initiated by maternally deposited 1360/Hoppel transposon piRNAs. Notably, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, this study uncovers a developmentally programmed, intergenerational mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus.

Thursday, February 22nd - Gonads

Tu, R., Tang, X. A., Xu, R., Ping, Z., Yu, Z., Xie, T. (2023). Gap junction-transported cAMP from the niche controls stem cell progeny differentiation. Proc Natl Acad Sci U S A, 120(35):e2304168120 PubMed ID: 37603749
Summary:
The niche has been shown to control stem cell self-renewal in different tissue types and organisms. Recently, a separate niche has been proposed to control stem cell progeny differentiation, called the differentiation niche. However, it remains poorly understood whether and how the differentiation niche directly signals to stem cell progeny to control their differentiation. In the Drosophila ovary, inner germarial sheath (IGS) cells contribute to two separate niche compartments for controlling both germline stem cell (GSC) self-renewal and progeny differentiation. This study shows that IGS cells express Inx2 protein, which forms gap junctions (GJs) with germline-specific Zpg protein to control stepwise GSC lineage development, including GSC self-renewal, germline cyst formation, meiotic double-strand DNA break formation, and oocyte specification. Germline-specific Zpg and IGS-specific Inx2 knockdowns cause similar defects in stepwise GSC development. Additionally, secondary messenger cAMP is transported from IGS cells to GSCs and their progeny via GJs to activate PKA signaling for controlling stepwise GSC development. Therefore, this study demonstrates that the niche directly controls GSC progeny differentiation via the GJ-cAMP-PKA signaling axis, which provides important insights into niche control of stem cell differentiation and highlights the importance of GJ-transported cAMP in tissue regeneration. This may represent a general strategy for the niche to control adult stem cell development in various tissue types and organisms since GJs and cAMP are widely distributed.

Eichler, C. E., Li, H., Grunberg, M. E., Gavis, E. R. (2023). Localization of oskar mRNA by agglomeration in ribonucleoprotein granules. PLoS Genet, 19(8):e1010877. PubMed ID: 37624861
Summary:
Localization of oskar mRNA to the posterior of the Drosophila oocyte is essential for abdominal patterning and germline development. oskar localization is a multi-step process involving temporally and mechanistically distinct transport modes. Numerous cis-acting elements and trans-acting factors have been identified that mediate earlier motor-dependent transport steps leading to an initial accumulation of oskar at the posterior. Little is known, however, about the requirements for the later localization phase, which depends on cytoplasmic flows and results in the accumulation of large oskar ribonucleoprotein granules, called founder granules, by the end of oogenesis. Using super-resolution microscopy, this study showed that founder granules are agglomerates of smaller oskar transport particles. In contrast to the earlier kinesin-dependent oskar transport, late-phase localization depends on the sequence as well as on the structure of the spliced oskar localization element (SOLE), but not on the adjacent exon junction complex deposition. Late-phase localization also requires the oskar 3' untranslated region (3' UTR), which targets oskar to founder granules. Together, these results show that 3' UTR-mediated targeting together with SOLE-dependent agglomeration leads to accumulation of oskar in large founder granules at the posterior of the oocyte during late stages of oogenesis. In light of previous work showing that oskar transport particles are solid-like condensates, these findings indicate that founder granules form by a process distinct from that of well-characterized ribonucleoprotein granules like germ granules, P bodies, and stress granules. Additionally, they illustrate how an individual mRNA can be adapted to exploit different localization mechanisms depending on the cellular context.

Benner, L., Muron, S., Oliver, B. (2023). Female germline expression of OVO transcription factor bridges Drosophila generations. bioRxiv, PubMed ID: 37662231
Summary:
OVO is required for karyotypically female germ cell viability but has no known function in the male germline in Drosophila. ovo is autoregulated by two antagonistic isoforms, OVO-A and OVO-B. All ovo^- alleles were created as partial revertants of the antimorphic ovo(D1) allele. Creation of new targeted alleles in an ovo⁺ background indicated that disrupting the germline-specific exon extension of ovo-B leads to an arrested egg chamber phenotype, rather than germ cell death. RNA-seq analysis, including >1K full length cDNAs, indicates that ovo utilizes a number of unannotated splice variations in the extended exon and a minor population of ovo-B transcripts utilizes an alternative splice. This indicates that classical ovo alleles such as ovo^D1rv23, are not truly null for ovo, and are likely to be weak antimorphs. To generate bonafide nulls, the ovo-A and ovo-B promoters were deleted showing that only ovo-B is required for female germ cell viability and there is an early and polyphasic developmental requirement for ovo-B in the female germline. To visualize OVO expression and localization, endogenously tagged ovo was generated, and nuclear OVO was found in all differentiating female germ cells throughout oogenesis in adults. This study also found that OVO is maternally deposited into the embryo, where it showed nuclear localization in newly formed pole cells. Maternal OVO persisted in embryonic germ cells until zygotic OVO expression was detectable, suggesting that there is continuous nuclear OVO expression in the female germline in the transition from one generation to the next.

Emelyanov, A. V., Barcenilla-Merino, D., Loppin, B., Fyodorov, D. V. (2023). APOLLO, a testis-specific Drosophila ortholog of importin-4, mediates the loading of protamine-like protein Mst77F into sperm chromatin. J Biol Chem, 299(10):105212 PubMed ID: 37660905
Summary:
DNA in sperm is packed with small, charged proteins termed SNBPs (sperm nuclear basic proteins), including mammalian and Drosophila protamines. During spermiogenesis, somatic-type chromatin is taken apart and replaced with sperm chromatin in a multistep process leading to an extraordinary condensation of the genome. During fertilization, the ova face a similarly challenging task of SNBP eviction and reassembly of nucleosome-based chromatin. Despite its importance for the animal life cycle, sperm chromatin metabolism, including the biochemical machinery mediating the mutual replacement of histones and SNBPs, remains poorly studied. In Drosophila, Mst77F is one of the first SNBPs loaded into the spermatid nuclei. It persists in mature spermatozoa and is essential for sperm compaction and male fertility. By using in vitro biochemical assays this study identified chaperones that can mediate the eviction and loading of Mst77F on DNA, thus facilitating the interconversions of chromatin forms in the male gamete. Unlike NAP1 and TAP/p32 chaperones that disassemble Mst77F-DNA complexes, ARTEMIS and APOLLO, orthologs of mammalian importin-4 (IPO4), mediate the deposition of Mst77F on DNA or oligonucleosome templates, accompanied by the dissociation of histone-DNA complexes. In vivo, a mutation of testis-specific Apollo brings about a defect of Mst77F loading, abnormal sperm morphology, and male infertility. This study has identified IPO4 ortholog APOLLO as a critical component of sperm chromatin assembly apparatus in Drosophila. In addition to recognized roles in protein traffic, a nuclear transport receptor can function directly in chromatin remodeling as a dual, histone- and SNBP-specific, chaperone.

Lu, W., Lakonishok, M., Gelfand, V. I. (2023). The dynamic duo of microtubule polymerase Mini spindles/XMAP215 and cytoplasmic dynein is essential for maintaining Drosophila oocyte fate. Proc Natl Acad Sci U S A, 120(39):e2303376120 PubMed ID: 37722034
Summary:
In many species, only one oocyte is specified among a group of interconnected germline sister cells. In Drosophila melanogaster, 16 interconnected cells form a germline cyst, where one cell differentiates into an oocyte, while the rest become nurse cells that supply the oocyte with mRNAs, proteins, and organelles through intercellular cytoplasmic bridges named ring canals via microtubule-based transport. This study finds that a microtubule polymerase Mini spindles (Msps), the Drosophila homolog of XMAP215, is essential for maintenance of the oocyte specification. mRNA encoding Msps is transported and concentrated in the oocyte by dynein-dependent transport along microtubules. Translated Msps stimulates microtubule polymerization in the oocyte, causing more microtubule plus ends to grow from the oocyte through the ring canals into nurse cells, further enhancing nurse cell-to-oocyte transport by dynein. Knockdown of msps blocks the oocyte growth and causes gradual loss of oocyte determinants. Thus, the Msps-dynein duo creates a positive feedback loop, ensuring oocyte fate maintenance by promoting high microtubule polymerization activity in the oocyte, and enhancing dynein-dependent nurse cell-to-oocyte transport.

Kakino, K., Mon, H., Ebihara, T., Hino, M., Masuda, A., Lee, J. M., Kusakabe, T. (2023). Comprehensive Transcriptome Analysis in the Testis of the Silkworm, Bombyx mori. Insects, 14(8) PubMed ID: 37623394
Summary:
Spermatogenesis is an important process in reproduction and is conserved across species, but in Bombyx mori, it shows peculiarities, such as the maintenance of spermatogonia by apical cells and fertilization by dimorphic spermatozoa. This study attempted to characterize the genes expressed in the testis of B. mori, focusing on aspects of expression patterns and gene function by transcriptome comparisons between different tissues, internal testis regions, and Drosophila melanogaster. The transcriptome analysis of 12 tissues of B. mori, including those of testis, revealed the widespread gene expression of 20,962 genes and 1705 testis-specific genes. A comparative analysis of the stem region (SR) and differentiated regions (DR) of the testis revealed 4554 and 3980 specific-enriched genes, respectively. In addition, comparisons with D. melanogaster testis transcriptome revealed homologs of 1204 SR and 389 DR specific-enriched genes that were similarly expressed in equivalent regions of Drosophila testis. Moreover, gene ontology (GO) enrichment analysis was performed for SR-specific enriched genes and DR-specific enriched genes, and the GO terms of several biological processes were enriched, confirming previous findings. This study advances understanding of spermatogenesis in B. mori and provides an important basis for future research, filling a knowledge gap between fly and mammalian studies.

Wednesday, February 21st - Stress

Wen, D., Xie, J., Yuan, Y., Shen, L., Yang, Y., Chen, W. (2023). The endogenous antioxidant ability of royal jelly in Drosophila is independent of Keap1/Nrf2 by activating oxidoreductase activity. Insect Sci, PubMed ID: 37632209
Summary:
Royal jelly (RJ) is a biologically active substance secreted by the hypopharyngeal and mandibular glands of worker honeybees. It is widely claimed that RJ reduces oxidative stress. However, the antioxidant activity of RJ has mostly been determined by in vitro chemical detection methods or by external administration drugs that cause oxidative stress. Whether RJ can clear the endogenous production of reactive oxygen species (ROS) in cells remains largely unknown. This study systematically investigated the antioxidant properties of RJ using several endogenous oxidative stress models of Drosophila. RJ was found to enhanced sleep quality of aging Drosophila, which is decreased due to an increase of oxidative damage with age. RJ supplementation improved survival and suppressed ROS levels in gut cells of flies upon exposure to hydrogen peroxide or to the neurotoxic agent paraquat. Moreover, RJ supplementation moderated levels of ROS in endogenous gut cells and extended lifespan after exposure of flies to heat stress. Sleep deprivation leads to accumulation of ROS in the gut cells, and RJ attenuated the consequences of oxidative stress caused by sleep loss and prolonged lifespan. Mechanistically, RJ prevented cell oxidative damage caused by heat stress or sleep deprivation, with the antioxidant activity in vivo independent of Keap1/Nrf2 signaling. RJ supplementation activated oxidoreductase activity in the guts of flies, suggesting its ability to inhibit endogenous oxidative stress and maintain health, possibly in humans.

Wang, X., Zhou, P., Zhang, Z., Huang, Q., Chen, X., Ji, L., Cheng, X., Shi, Y., Yu, S., Tang, J., Sun, C., Zhao, X., Yu, J. (2023). A Drosophila model of gestational antimony exposure uncovers growth and developmental disorders caused by disrupting oxidative stress homeostasis. Free radical biology & medicine, 208:418-429 PubMed ID: 37666440
Summary:
The toxic heavy metal antimony (Sb) is ubiquitous. Various models have shown that Sb induces neuronal and reproductive toxicity. However, little is known about the developmental toxicity of Sb exposure during gestation and the underlying mechanisms. To study its effects on growth and development, Drosophila stages from eggs to pupae were exposed to different Sb concentrations (0, 0.3, 0.6 and 1.2 mg/mL Sb); RNA sequencing was used to identify the underlying mechanism. The model revealed that prenatal Sb exposure significantly reduced larval body size and weight, the pupation and eclosion rates, and the number of flies at all stages. With 1.2 mg/mL Sb exposure in 3rd instar larvae, 484 genes were upregulated and 694 downregulated compared to controls. Biological analysis showed that the disrupted transcripts were related to the oxidative stress pathway, as verified by reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and glutathione (GSH) intervention experiments. Sb exposure induced oxidative stress imbalance could be rectified by chelation and antioxidant effects of NAC/GSH. The Drosophila Schneider 2 (S2) model further demonstrated that NAC and GSH greatly ameliorated cell death induced by Sb exposure. In conclusion, gestational Sb exposure disrupted oxidative stress homeostasis, thereby impairing growth and development.

Hwang, R. D., Lu, Y. N., Tang, Q., Periz, G., Park, G., Li, X., Liu, Y., Zhang, T., Wang, J. (2023). DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment. bioRxiv, PubMed ID: 37745492
Summary:
Proteotoxic stress impairs cellular homeostasis and underlies the pathogeneses of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. This study reports a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of the proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in tissues from ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.

Reyes-Ramirez, A., Belgaidi, Z., Gibert, P., Pommier, T., Siberchicot, A., Mouton, L., Desouhant, E. (2023). Larval density in the invasive Drosophila suzukii: Immediate and delayed effects on life-history traits. Ecology and evolution, 13(8):e10433 PubMed ID: 37636864
Summary:
The effects of density are key in determining population dynamics, since they can positively or negatively affect the fitness of individuals. These effects have great relevance for polyphagous insects for which immature stages develop within a single site of finite feeding resources. Drosophila suzukii is a crop pest that induces severe economic losses for agricultural production; however, little is known about the effects of density on its life-history traits. This study, (i) investigated the egg distribution resulting from females' egg-laying strategy and (ii) tested the immediate (on immatures) and delayed (on adults) effects of larval density on emergence rate, development time, potential fecundity, and adult size. The density used varied in a range between 1 and 50 larvae. 44.27% of the blueberries used for the oviposition assay contained between 1 and 11 eggs in aggregates. The high experimental density (50 larvae) has no immediate effect in the emergence rate but has effect on larval developmental time. This trait was involved in a trade-off with adult life-history traits: The time of larval development was reduced as larval density increased, but smaller and less fertile females were produced. These results clearly highlight the consequences of larval crowding on the juveniles and adults of this fly.

Shui, K., Wang, C., Zhang, X., Ma, S., Li, Q., Ning, W., Zhang, W., Chen, M., Peng, D., Hu, H., Fang, Z., Guo, A., Gao, G., Ye, M., Zhang, L. and Xue, Y. (2023). Small-sample learning reveals propionylation in determining global protein homeostasis. Nat Commun 14(1): 2813. PubMed ID: 37198164
Summary:
Proteostasis is fundamental for maintaining organismal health. However, the mechanisms underlying its dynamic regulation and how its disruptions lead to diseases are largely unclear. This study conducted in-depth propionylomic profiling in Drosophila, and develop a small-sample learning framework to prioritize the propionylation at lysine 17 of H2B (H2BK17pr) to be functionally important. Mutating H2BK17 which eliminates propionylation leads to elevated total protein level in vivo. Further analyses reveal that H2BK17pr modulates the expression of 14.7-16.3% of genes in the proteostasis network, and determines global protein level by regulating the expression of genes involved in the ubiquitin-proteasome system. In addition, H2BK17pr exhibits daily oscillation, mediating the influences of feeding/fasting cycles to drive rhythmic expression of proteasomal genes. This study not only reveals a role of lysine propionylation in regulating proteostasis, but also implements a generally applicable method which can be extended to other issues with little prior knowledge.

Xu, X., An, H., Wu, C., Sang, R., Wu, L., Lou, Y., Yang, X. and Xi, Y. (2023). HR repair pathway plays a crucial role in maintaining neural stem cell fate under irradiation stress. Life Sci Alliance 6(8). PubMed ID: 37197982
Summary:
Environmental stress can cause mutation or genomic instability in stem cells which, in some cases, leads to tumorigenesis. Mechanisms to monitor and eliminate these mutant stem cells remain elusive. Using the Drosophila larval brain as a model, this study shows that X-ray irradiation (IR) at the early larval stage leads to accumulation of nuclear Prospero (Pros), resulting in premature differentiation of neural stem cells (neuroblasts, NBs). Through NB-specific RNAi screenings, the Mre11-Rad50-Nbs1 complex and the homologous recombination (HR) repair pathway, rather than non-homologous end-joining pathway that plays, a dominant role in the maintenance of NBs under IR stress. The DNA damage sensor ATR/mei-41 is shown to act to prevent IR-induced nuclear Pros in a WRNexo-dependent manner. The accumulation of nuclear Pros in NBs under IR stress, leads to NB cell fate termination, rather than resulting in mutant cell proliferation. This study reveals an emerging mechanism for the HR repair pathway in maintaining neural stem cell fate under irradiation stress.

Tuesday, February 20th - Disease Models

Clabough, E. B. D., Aspili, C., Fussy, W. S., ...., Venton, B. J., Hayes, D., Sipe, C. W. (2023). Huntingtin Plays a Role in the Physiological Response to Ethanol in Drosophila. Journal of Huntington's disease, 12(3):241-252 PubMed ID: 37661891
Summary:
Huntingtin (htt) protein is an essential regulator of nervous system function through its various neuroprotective and pro-survival functions, and loss of wild-type htt function is implicated in the etiology of Huntington's disease. While its pathological role is typically understood as a toxic gain-of-function, some neuronal phenotypes also result from htt loss. Therefore, it is important to understand possible roles for htt in other physiological circumstances. To elucidate the role of htt in the context of ethanol exposure, this study investigated how loss of htt impacts behavioral and physiological responses to ethanol in Drosophila. Flies lacking htt were tested for ethanol sensitivity and tolerance, preference for ethanol using capillary feeder assays, and recovery of mobility after intoxication. Levels of dopamine neurotransmitter and numbers of dopaminergic cells in brains lacking dhtt were also measured. dhtt-null flies were found to be both less sensitive and more tolerant to ethanol exposure in adulthood. Moreover, flies lacking dhtt are more averse to alcohol than controls, and they recover mobility faster following acute ethanol intoxication. dhtt was shown to mediate these effects at least in part through the dopaminergic system, as dhtt is required to maintain normal levels of dopamine in the brain and normal numbers of dopaminergic cells in the adult protocerebrum. These results demonstrate that htt regulates the physiological response to ethanol and indicate a novel neuroprotective role for htt in the dopaminergic system, raising the possibility that it may be involved more generally in the response to toxic stimuli.

Onkar, A., Sheshadri, D., Rai, A., Gupta, A. K., Gupta, N., Ganesh, S. (2023). Increase in brain glycogen levels ameliorates Huntington's disease phenotype and rescues neurodegeneration in Drosophila. Disease models & mechanisms, 16(10) PubMed ID: 37681238
Summary:
Under normal physiological conditions, the mammalian brain contains very little glycogen, most of which is stored in astrocytes. However, the aging brain and the subareas of the brain in patients with neurodegenerative disorders tend to accumulate glycogen, the cause and significance of which remain largely unexplored. Using cellular models, a neuroprotective role for neuronal glycogen and glycogen synthase has been recently demonstrated in the context of Huntington's disease. To gain insight into the role of brain glycogen in regulating proteotoxicity, a Drosophila model of Huntington's disease was used, in which glycogen synthase is either knocked down or expressed ectopically. Enhancing glycogen synthesis in the brains of flies with Huntington's disease decreased mutant Huntingtin aggregation and reduced oxidative stress by activating auto-lysosomal functions. Further, overexpression of glycogen synthase in the brain rescues photoreceptor degeneration, improves locomotor deficits and increases fitness traits in this Huntington's disease model. This study, thus, provides in vivo evidence for the neuroprotective functions of glycogen synthase and glycogen in neurodegenerative conditions, and their role in the neuronal autophagy process.

Zhang, F., Wang, L., Jin, J., Pang, Y., Shi, H., Fang, Z., Wang, H., Du, Y., Hu, Y., Zhang, Y., Ding, X., Zhu, Z. (2023). Insights into the genetic influences of the microbiota on the life span of a host. Frontiers in microbiology, 14:1138979 PubMed ID: 37601381
Summary:
Escherichia coli (E. coli) mutant strains have been reported to extend the life span of Caenorhabditis elegans (C. elegans). However, the specific mechanisms through which the genes and pathways affect aging are not yet clear. This study fed Drosophila melanogaster (fruit fly) various E. coli single-gene knockout strains to screen mutant strains with an extended lifespan. The results showed that D. melanogaster fed with E. coli purE had the longest mean lifespan, which was verified by C. elegans. RNA-sequencing and analysis of C. elegans fed with E. coli purE (a single-gene knockout mutant) was conducted to further explore the underlying molecular mechanism. Differential gene expression (DGE) analysis, enrichment analysis, and gene set enrichment analysis (GSEA) were ised to screen vital genes and modules with significant changes in overall expression. The results suggest that E. coli mutant strains may affect the host lifespan by regulating the protein synthesis rate (cfz-2) and ATP level (catp-4). To conclude, this study could provide new insights into the genetic influences of the microbiota on the life span of a host and a basis for developing anti-aging probiotics and drugs.

Tandon, S., Sarkar, S. (2023). Glutamine stimulates the S6K/4E-BP branch of insulin signalling pathway to mitigate human poly(Q) disorders in Drosophila disease models. Nutritional neuroscience:1-12 PubMed ID: 37658796
Summary:
Since, the S6K/4E-BP sub-pathway can be stimulated by various amino acids; this study examine if oral feeding of amino acids delivers rescue against human poly(Q) toxicity in Drosophila. Drosophila models of two different poly(Q) disorders were used to test this hypothesis. Glutamine was fed to the test flies orally mixed in the food. Control and treated flies were then tested for different parameters, such as formation of poly(Q) aggregates and neurodegeneration, to evaluate glutamine's proficiency in mitigating poly(Q) neurotoxicity. This study study, for the first time, reports that glutamine feeding stimulates the growth promoting S6K/4E-BP branch of insulin signalling pathway and restricts pathogenesis of poly(Q) disorders in Drosophila disease models. It is noted that glutamine treatment restricts the formation of neurotoxic poly(Q) aggregates and minimises neuronal deaths. Further, glutamine treatment re-establishes the chromatin architecture by improving the histone acetylation which is otherwise compromised in poly(Q) expressing neuronal cells. Since, the insulin signalling pathway as well as mechanism of action of glutamine are fairly conserved between human and Drosophila, this finding strongly suggests that glutamine holds immense potential to be developed as an intervention therapy against the incurable human poly(Q) disorders.

Ciampelli, C., Galleri, G., Puggioni, S., Fais, M., Iannotta, L., Galioto, M., Becciu, M., Greggio, E., Bernardoni, R., Crosio, C., Iaccarino, C. (2023). Inhibition of the Exocyst Complex Attenuates the LRRK2 Pathological Effects. Int J Mol Sci, 24(16) PubMed ID: 37628835
Summary:
Pathological mutations in leucine-rich repeat kinase 2 (LRRK2) gene are the major genetic cause of Parkinson's disease (PD). Multiple lines of evidence link LRRK2 to the control of vesicle dynamics through phosphorylation of a subset of RAB proteins. However, the molecular mechanisms underlying these processes are not fully elucidated. Previous work demonstrated that LRRK2 increases the exocyst complex assembly by Sec8 interaction, one of the eight members of the exocyst complex, and that Sec8 over-expression mitigates the LRRK2 pathological effect in PC12 cells. This analysis was extended using LRRK2 drosophila models and shows that the LRRK2-dependent exocyst complex assembly increase is downstream of RAB phosphorylation. Moreover, exocyst complex inhibition rescues mutant LRRK2 pathogenic phenotype in cellular and drosophila models. Finally, prolonged exocyst inhibition leads to a significant reduction in the LRRK2 protein level, overall supporting the role of the exocyst complex in the LRRK2 pathway. Taken together, this study suggests that modulation of the exocyst complex may represent a novel therapeutic target for PD.

Kim, H. S., Parker, D. J., Hardiman, M. M., Munkacsy, E., Jiang, N., Rogers, A. N., Bai, Y., Brent, C., Mobley, J. A., Austad, S. N., Pickering, A. M. (2023). Early-adulthood spike in protein translation drives aging via juvenile hormone/germline signaling. Nat Commun, 14(1):5021 PubMed ID: 37596266
Summary:
Protein translation (PT) declines with age in invertebrates, rodents, and humans. It has been assumed that elevated PT at young ages is beneficial to health and PT ends up dropping as a passive byproduct of aging. In Drosophila, this study shows that a transient elevation in PT during early-adulthood exerts long-lasting negative impacts on aging trajectories and proteostasis in later-life. Blocking the early-life PT elevation robustly improves life-/health-span and prevents age-related protein aggregation, whereas transiently inducing an early-life PT surge in long-lived fly strains abolishes their longevity/proteostasis benefits. The early-life PT elevation triggers proteostatic dysfunction, silences stress responses, and drives age-related functional decline via juvenile hormone-lipid transfer protein axis and germline signaling. These findings suggest that PT is adaptively suppressed after early-adulthood, alleviating later-life proteostatic burden, slowing down age-related functional decline, and improving lifespan. This work provides a theoretical framework for understanding how lifetime PT dynamics shape future aging trajectories.

Friday, February 16th - Evolution

Davies, N., Janicke, T., Morrow, E. H. (2023). Evidence for stronger sexual selection in males than in females using an adapted method of Bateman's classic study of Drosophila melanogaster. Evolution, 77(11):2420-2430 PubMed ID: 37624087
Summary:
Bateman's principles, originally a test of Darwin's theoretical ideas, have since become fundamental to sexual selection theory and vital to contextualizing the role of anisogamy in sex differences of precopulatory sexual selection. Despite this, Bateman's principles have received substantial criticism, and researchers have highlighted both statistical and methodological errors, suggesting that Bateman's original experiment contains too much sampling bias for there to be any evidence of sexual selection. This study uses Bateman's original method as a template, accounting for two fundamental flaws in his original experiments, (a) viability effects and (b) a lack of mating behavior observation. Experimental populations of Drosophila melanogaster consisted of wild-type focal individuals and nonfocal individuals established by backcrossing the brown eye (bw-) eye-color marker-thereby avoiding viability effects. Mating assays included direct observation of mating behavior and total number of offspring, to obtain measures of mating success, reproductive success, and standardized variance measures based on Bateman's principles. The results provide observational support for Bateman's principles, particularly that (a) males had significantly more variation in number of mates compared with females and (b) males had significantly more individual variation in total number of offspring. A significantly steeper Bateman gradient was found for males compared to females, suggesting that sexual selection is operating more intensely in males. However, female remating was limited, providing the opportunity for future study to further explore female reproductive success in correlation with higher levels of remating.

Lankinen, P., Kastally, C., Hoikkala, A. (2023). Clinal variation in the temperature and photoperiodic control of reproductive diapause in Drosophila montana females. J Insect Physiol, 150:104556 PubMed ID: 37598869
Summary:
Insect adaptation to climatic conditions at different latitudes has required changes in life-history traits linked with survival and reproduction. Several species, including Drosophila montana, show robust latitudinal variation in the critical day length (CDL), below which more than half of the emerging females enter reproductive diapause at a given temperature. This study used a novel approach to find out whether D. montana also shows latitudinal variation in the critical temperature (CTemp), above which the photoperiodic regulation of diapause is disturbed so that the females develop ovaries in daylengths that are far below their CDL. CTemp was estimated for 53 strains from different latitudes on 3 continents after measuring their diapause proportions at a range of temperatures in 12 h daylength (for 29 of the strains also in continuous darkness). In 12 h daylength, CTemp increased towards high latitudes alongside an increase in CDL, and in 3 high-latitude strains diapause proportion exceeded 50% in all temperatures. In continuous darkness, the diapause proportion was above 50% in the lowest temperature(s) in only 9 strains, all of which came from high latitudes. In the second part of the study, changes were measured in CTemp and CDL in a selection experiment favouring reproduction in short daylength (photoperiodic selection) and by exercising selection for females that reproduce in LD12:12 at low temperature (photoperiodic and temperature selection). In both experiments selection induced parallel changes in CDL and CTemp, confirming correlations seen between these traits along latitudinal clines. Overall, these findings suggest that selection towards strong photoperiodic diapause and long CDL at high latitudes has decreased the dependency of D. montana diapause on environmental temperature. Accordingly, the prevalence and timing of the diapause of D. montana is likely to be less vulnerable to climate warming in high- than low-latitude populations.

Ma, L., Zheng, C., Xu, S., Xu, Y., Song, F., Tian, L., Cai, W., Li, H., Duan, Y. (2023). A full repertoire of Hemiptera genomes reveals a multi-step evolutionary trajectory of auto-RNA editing site in insect Adar gene. RNA Biol, 20(1):703-714 PubMed ID: 37676051
Summary:
Adenosine-to-inosine (A-to-I) RNA editing, mediated by metazoan ADAR enzymes, is a prevalent post-transcriptional modification that diversifies the proteome and promotes adaptive evolution of organisms. The Drosophila Adar gene has an auto-recoding site (termed S>G site) that forms a negative-feedback loop and stabilizes the global editing activity. However, the evolutionary trajectory of Adar S>G site in many other insects remains largely unknown, preventing a deeper understanding on the significance of this auto-editing mechanism. This study retrieved the well-annotated genomes of 375 arthropod species including the five major insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera and Hemiptera) and several outgroup species. Comparative genomic analysis was performed on the Adar auto-recoding S>G site. The ancestral state of insect S>G site was found to be an uneditable serine codon (unSer); this state was largely maintained in Hymenoptera. The editable serine codon (edSer) appeared in the common ancestor of Lepidoptera, Diptera and Coleoptera and was almost fixed in the three orders. Interestingly, Hemiptera species possessed comparable numbers of unSer and edSer codons, and a few 'intermediate codons', demonstrating a multi-step evolutionary trace from unSer-to-edSer with non-synchronized mutations at three codon positions. It is argued that the evolution of Adar S>G site is the best genomic evidence supporting the 'proteomic diversifying hypothesis' of RNA editing. This work deepens understanding on the evolutionary significance of Adar auto-recoding site which stabilizes the global editing activity and controls transcriptomic diversity.

Blunk, S., Garcia-Verdugo, H., O'Sullivan, S., Camp, J., Haines, M., Coalter, T., Williams, T. A., Nagy, L. M. (2023). Functional Divergence of the Tribolium castaneum engrailed and invected Paralogs. Insects, 14(8) PubMed ID: 37623401
Summary:
engrailed (en) and invected (inv) encode paralogous transcription factors found as a closely linked tandem duplication within holometabolous insects. Drosophila en mutants segment normally, then fail to maintain their segments. Loss of Drosophila inv is viable, while loss of both genes results in asegmental larvae. Surprisingly, the knockdown of Oncopeltus inv can result in the loss or fusion of the entire abdomen and en knockdowns in Tribolium show variable degrees of segmental loss. The consequence of losing or knocking down both paralogs on embryogenesis has not been studied beyond Drosophila. To further investigate the relative functions of each paralog and the mechanism behind the segmental loss, Tribolium double and single knockdowns of en and inv were analyzed. The most common cuticular phenotype of the double knockdowns was small, limbless, and open dorsally, with all but a single, segmentally iterated row of bristles. Less severe knockdowns had fused segments and reduced appendages. The Tribolium paralogs appear to act synergistically: the knockdown of either Tribolium gene alone was typically less severe, with all limbs present, whereas the most extreme single knockdowns mimic the most severe double knockdown phenotype. Morphological abnormalities unique to either single gene knockdown were not found. inv expression was not affected in the Tribolium en knockdowns, but hh expression was unexpectedly increased midway through development. Thus, while the segmental expression of en/inv is broadly conserved within insects, the functions of en and inv are evolving independently in different lineages.

Sproul, J. S., Hotaling, S., Heckenhauer, J. ..., Pauls, S. U., Frandsen, P. B. (2023). Analyses of 600+ insect genomes reveal repetitive element dynamics and highlight biodiversity-scale repeat annotation challenges. Genome research, 33(10):1708-1717 PubMed ID: 37739812
Summary:
Repetitive elements (REs) are integral to the composition, structure, and function of eukaryotic genomes, yet remain understudied in most taxonomic groups. This study investigated REs across 601 insect species and report wide variation in RE dynamics across groups. Analysis of associations between REs and protein-coding genes revealed dynamic evolution at the interface between REs and coding regions across insects, including notably elevated RE-gene associations in lineages with abundant long interspersed nuclear elements (LINEs). This study leveraged this large, empirical data set to quantify impacts of long-read technology on RE detection and investigate fundamental challenges to RE annotation in diverse groups. In long-read assemblies, ∼36% more REs than in short-read assemblies, with long terminal repeats (LTRs) showing 162% increased detection, whereas DNA transposons and LINEs showed less respective technology-related bias. In most insect lineages, 25%-85% of repetitive sequences were "unclassified" following automated annotation, compared with only ∼13% in Drosophila species. Although the diversity of available insect genomes has rapidly expanded, this study shows the rate of community contributions to RE databases has not kept pace, preventing efficient annotation and high-resolution study of REs in most groups. This study highlight the tremendous opportunity and need for the biodiversity genomics field to embrace REs and suggest collective steps for making progress toward this goal.

Wang, Z., Pu, J., Richards, C., Giannetti, E., Cong, H., Lin, Z., Chung, H. (2023). Evolution of a fatty acyl-CoA elongase underlies desert adaptation in Drosophila. Sci Adv, 9(35):eadg0328 PubMed ID: 37647401
Summary:
Traits that allow species to survive in extreme environments such as hot-arid deserts have independently evolved in multiple taxa. However, the genetic and evolutionary mechanisms underlying these traits have thus far not been elucidated. This study shows that Drosophila mojavensis, a desert-adapted fruit fly species, has evolved high desiccation resistance by producing long-chain methyl-branched cuticular hydrocarbons (mbCHCs) that contribute to a cuticular lipid layer reducing water loss. The ability to synthesize these longer mbCHCs is due to evolutionary changes in a fatty acyl-CoA elongase (mElo). mElo knockout in D. mojavensis led to loss of longer mbCHCs and reduction of desiccation resistance at high temperatures but did not affect mortality at either high temperatures or desiccating conditions individually. Phylogenetic analysis showed that mElo is a Drosophila-specific gene, suggesting that while the physiological mechanisms underlying desert adaptation may be similar between species, the genes involved in these mechanisms may be species or lineage specific.

Thursday, February 15th - Synapse and Vesicles

Bertin, F., Jara-Wilde, J., Auer, B., Köhler-Solis, A., Gonzalez-Silva, C., Thomas, U., Sierralta, J. (2023). Drosophila Atlastin regulates synaptic vesicle mobilization independent of bone morphogenetic protein signaling. Biological research, 56(1):49 PubMed ID: 37710314
Summary:
The endoplasmic reticulum (ER). contacts endosomes in all parts of a motor neuron, including the axon and presynaptic terminal, to move structural proteins, proteins that send signals, and lipids over long distances. Atlastin (Atl), a large GTPase, is required for membrane fusion and the structural dynamics of the ER tubules. Atl mutations are the second most common cause of Hereditary Spastic Paraplegia (HSP), which causes spasticity in both sexes' lower extremities. Through an unknown mechanism, Atl mutations stimulate the BMP (bone morphogenetic protein) pathway in vertebrates and Drosophila. Synaptic defects are caused by atl mutations, which affect the abundance and distribution of synaptic vesicles (SV) in the bouton. It is hypothesized that BMP signaling, does not cause Atl-dependent SV abnormalities in Drosophila. This study shows that atl knockdown in motor neurons (Atl-KD) increases synaptic and satellite boutons in the same way that constitutively activating the BMP-receptor Tkv (thick veins) (Tkv-CA) increases the bouton number. The SV proteins Cysteine string protein (CSP) and vesicular glutamate transporter are reduced in Atl-KD and Tkv-CA larvae. Reducing the activity of the BMP receptor Wishful thinking (wit) can rescue both phenotypes. Unlike Tkv-CA larvae, Atl-KD larvae display altered activity-dependent distributions of CSP staining. Furthermore, Atl-KD larvae display an increased FM 1-43 unload than Control and Tkv-CA larvae. As decreasing wit function does not reduce the phenotype, the hypothesis that BMP signaling is not involved is supported. It was also found that Rab11/CSP colocalization increased in Atl-KD larvae, which supports the concept that late recycling endosomes regulate SV movements. These findings reveal that Atl modulates neurotransmitter release in motor neurons via SV distribution independently of BMP signaling, which could explain the observed SV accumulation and synaptic dysfunction. The data suggest that Atl is involved in membrane traffic as well as formation and/or recycling of the late endosome.

Yin, P., Cai, Y., Cui, T., Berg, A. J., Wang, T., Morency, D. T., Paganelli, P. M., Lok, C., Xue, Y., Vicini, S., Wang, T. (2023). Glial Sphingosine-Mediated Epigenetic Regulation Stabilizes Synaptic Function in Drosophila Models of Alzheimer's Disease. J Neurosci, 43(42):6954-6971 PubMed ID: 37669862
Summary:
Destabilization of neural activity caused by failures of homeostatic regulation has been hypothesized to drive the progression of Alzheimer's Disease (AD). However, the underpinning mechanisms that connect synaptic homeostasis and the disease etiology are yet to be fully understood. This study demonstrated that neuronal overexpression of amyloid β (Aβ) causes abnormal histone acetylation in peripheral glia and completely blocks presynaptic homeostatic potentiation (PHP) at the neuromuscular junction in Drosophila The synaptic deficits caused by Aβ overexpression in motoneurons are associated with motor function impairment at the adult stage. Moreover, a sphingosine analog drug, Fingolimod, was found to ameliorate synaptic homeostatic plasticity impairment, abnormal glial histone acetylation, and motor behavior defects in the Aβ models. It was further demonstrated that perineurial glial sphingosine kinase 2 (Sk2) is not only required for PHP, but also plays a beneficial role in modulating PHP in the Aβ models. Glial overexpression of Sk2 rescues PHP, glial histone acetylation, and motor function deficits that are associated with Aβ in Drosophila Finally, this study showed that glial overexpression of Sk2 restores PHP and glial histone acetylation in a genetic loss-of-function mutant of the Spt-Ada-Gcn5 Acetyltransferase complex, strongly suggesting that Sk2 modulates PHP through epigenetic regulation. Both male and female animals were used in the experiments and analyses in this study. Collectively, this study has provided genetic evidence demonstrating that abnormal glial epigenetic alterations in Aβ models in Drosophila are associated with the impairment of PHP and that the sphingosine signaling pathway displays protective activities in stabilizing synaptic physiology.

Srivastav, S., van der Graaf, K., Singh, P., Utama, A. B., Meyer, M. D., McNew, J. A., Stern, M. (2024). Atl (atlastin) regulates mTor signaling and autophagy in Drosophila muscle through alteration of the lysosomal network. Autophagy, 20(1):131-150 PubMed ID: 37649246
Summary:
The hereditary spastic paraplegias (HSPs) represent a family of genetic disorders comprising at least 72 different genes with the common pathology of progressive locomotor deficits and spasticity. ATL1/SPG3A (atlastin GTPase 1) encodes an ER fusion protein that controls ER morphology, which implicates ER structure as a causal factor in HSP. This study used Drosophila to study effects of decreased atl (atlastin) on properties of the larval body wall muscle. Muscle atl loss was shown to cause accumulation of aggregates containing polyubiquitin (polyUB), mostly bound to the autophagy receptor ref(2)P/SQSTM1/p62. Muscle atl loss also decreased volume and complexity of the endolysosomal network and decreased lysosome number. To determine effects of these lysosomal deficits on progression through the basal autophagy pathway, Atg8a tagged with both GFP and mCherry in a wild-type and atl mutant background. Numerous structures containing mCherry were found but not GFP fluorescence in wild type, indicating that Atg8a was found mostly in mature autolysosomes. In contrast, muscles lacking atl exhibited significant amounts of GFP signal, indicating failure of autophagosome maturation with acidic lysosomes. Many of these GFP-positive puncta contained the late-endosome marker Rab7 but not Lamp1, indicating that some autophagy cargo was accumulating within amphisomes. It was also found that this autophagy block was accompanied by an inability to activate the mTor kinase. These results provide mechanistic insights into the role of atl in maintaining proper function of the autophagy pathway and suggests that certain pathologies in patients with mutations in ATL1/SPG3A might result from altered MTOR signaling.

Hogan, C. A., Gratz, S. J., Dumouchel, J. L., Thakur, R. S., Delgado, A., Lentini, J. M., Madhwani, K. R., Fu, D., O'Connor-Giles, K. M. (2023). Expanded tRNA methyltransferase family member TRMT9B regulates synaptic growth and function. EMBO reports, 24(10):e56808 PubMed ID: 37642556
Summary:
Nervous system function rests on the formation of functional synapses between neurons. This study has identified TRMT9B as a new regulator of synapse formation and function in Drosophila. TRMT9B has been studied for its role as a tumor suppressor and is one of two metazoan homologs of yeast tRNA methyltransferase 9 (Trm9), which methylates tRNA wobble uridines. Whereas Trm9 homolog ALKBH8 is ubiquitously expressed, TRMT9B is enriched in the nervous system. However, in the absence of animal models, TRMT9B's role in the nervous system has remained unstudied. This study generate null alleles of TRMT9B and found it acts postsynaptically to regulate synaptogenesis and promote neurotransmission. Through liquid chromatography-mass spectrometry, it was found that ALKBH8 catalyzes canonical tRNA wobble uridine methylation, raising the question of whether TRMT9B is a methyltransferase. Structural modeling studies suggest TRMT9B retains methyltransferase function and, in vivo, disruption of key methyltransferase residues blocks TRMT9B's ability to rescue synaptic overgrowth, but not neurotransmitter release. These findings reveal distinct roles for TRMT9B in the nervous system and highlight the significance of tRNA methyltransferase family diversification in metazoans.

Meissner, J. M., Akhmetova, K., Szul, T., Viktorova, E. G., Sha, B., Bhatt, J. M., Lee, E. J., Kahn, R. A., Belov, G. A., Chesnokov, I., Sztul, E. (2023). The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi. Frontiers in cell and developmental biology, 11:1233272 PubMed ID: 37745300
Summary:
Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. This study identified the HDS3 domain as essential for GBF1 membrane association in mammalian cells and documents the critical role of HDS3 during the development of Drosophila melanogaster. Upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. GBF1 interdomain arrangements were illuminated by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on tese data, models of GBF1 engagement on the membrane are proposed to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function.

Boutet, A., Zeledon, C., Emery, G. (2023). ArfGAP1 regulates the endosomal sorting of guidance receptors to promote directed collective cell migration in vivo. iScience, 26(8):107467 PubMed ID: 37599820
Summary:
Chemotaxis drives diverse migrations important for development and involved in diseases, including cancer progression. Using border cells in the Drosophila egg chamber as a model for collective cell migration, this study characterized the role of ArfGAP1 in regulating chemotaxis during this process. ArfGAP1 is required for the maintenance of receptor tyrosine kinases, the guidance receptors, at the plasma membrane. In the absence of ArfGAP1, the level of active receptors is reduced at the plasma membrane and increased in late endosomes. Consequently, clusters with impaired ArfGAP1 activity lose directionality. Furthermore, it was found that the number and size of late endosomes and lysosomes are increased in the absence of ArfGAP1. Finally, genetic interactions suggest that ArfGAP1 acts on the kinase and GTPase Lrrk to regulate receptor sorting. Overall, the data indicate that ArfGAP1 is required to maintain guidance receptors at the plasma membrane and promote chemotaxis.

Wednesday, February 14th - Adult Neural Development, Structure and Function

Yoo, J., Dombrovski, M., Mirshahidi, P., Nern, A., LoCascio, S. A., Zipursky, S. L., Kurmangaliyev, Y. Z. (2023). Brain wiring determinants uncovered by integrating connectomes and transcriptomes. Curr Biol, 33(18):3998-4005. PubMed ID: 37647901
Summary:
Advances in brain connectomics have demonstrated the extraordinary complexity of neural circuits. Developing neurons encounter the axons and dendrites of many different neuron types and form synapses with only a subset of them. During circuit assembly, neurons express cell-type-specific repertoires comprising many cell adhesion molecules (CAMs) that can mediate interactions between developing neurites. Many CAM families have been shown to contribute to brain wiring in different ways. It has been challenging, however, to identify receptor-ligand pairs directly matching neurons with their synaptic targets. This study integrated the synapse-level connectome of the neural circuit with the developmental expression patterns and binding specificities of CAMs on pre- and postsynaptic neurons in the Drosophila visual system. To overcome the complexity of neural circuits, focus was placed on pairs of genetically related neurons that make differential wiring choices. In the motion detection circuit, closely related subtypes of T4/T5 neurons choose between alternative synaptic targets in adjacent layers of neuropil. This choice correlates with the matching expression in synaptic partners of different receptor-ligand pairs of the Beat and Side families of CAMs. Genetic analysis demonstrated that presynaptic Side-II and postsynaptic Beat-VI restrict synaptic partners to the same layer. Removal of this receptor-ligand pair disrupts layers and leads to inappropriate targeting of presynaptic sites and postsynaptic dendrites. It is proposde that different Side/Beat receptor-ligand pairs collaborate with other recognition molecules to determine wiring specificities in the fly brain. Combining transcriptomes, connectomes, and protein interactome maps allow unbiased identification of determinants of brain wiring.

Armour, E. M., Thomas, C. M., Greco, G., Bhatnagar, A., Elefant, F. (2023). Experience-dependent Tip60 nucleocytoplasmic transport is regulated by its NLS/NES sequences for neuroplasticity gene control. Molecular and cellular neurosciences, 127:103888 PubMed ID: 37598897
Summary:
Nucleocytoplasmic transport (NCT) in neurons is critical for enabling proteins to enter the nucleus and regulate plasticity genes in response to environmental cues. Such experience-dependent (ED) neural plasticity is central for establishing memory formation and cognitive function and can influence the severity of neurodegenerative disorders like Alzheimer's disease (AD). How histone acetyltransferases (HATs) respond to extracellular cues in the in vivo brain to drive HA-mediated activity-dependent gene control remains unclear. Previous work has demonstrated that extracellular stimulation of rat hippocampal neurons in vitro triggers Tip60 HAT nuclear import with concomitant synaptic gene induction. This study focussed on investigating Tip60 HAT subcellular localization and NCT specifically in neuronal activity-dependent gene control by using the learning and memory mushroom body (MB) region of the Drosophila brain as a powerful in vivo cognitive model system. Immunohistochemistry (IHC) was used to compare the subcellular localization of Tip60 HAT in the Drosophila brain under normal conditions and in response to stimulation of fly brain neurons in vivo either by genetically inducing potassium channels activation or by exposure to natural positive ED conditions. Furthermore, it was found that both inducible and ED condition-mediated neural induction triggered Tip60 nuclear import with concomitant induction of previously identified Tip60 target genes and that Tip60 levels in both the nucleus and cytoplasm were significantly decreased in the well-characterized Drosophila AD model. Mutagenesis of a putative nuclear localization signal (NLS) sequence and nuclear export signal (NES) sequence that were identified in the Drosophila Tip60 protein revealed that both are functionally required for appropriate Tip60 subcellular localization. These results support a model by which neuronal stimulation triggers Tip60 NCT via its NLS and NES sequences to promote induction of activity-dependent neuroplasticity gene transcription and that this process may be disrupted in AD.

Rui, M., Kong, W., Wang, W., Zheng, T., Wang, S., Xie, W. (2023). Droj2 Facilitates Somatosensory Neurite Sculpting via GTP-Binding Protein Arf102F in Drosophila. Int J Mol Sci, 24(17) PubMed ID: 37686022
Summary:
Developmental remodeling of neurite is crucial for the accurate wiring of neural circuits in the developing nervous system in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neuropsychiatric disorders, for instance, autism, Alzheimer's disease (AD), and schizophrenia. However, the molecular underpinnings underlying developmental remodeling are still not fully understood. This study has identified DnaJ-like-2 (Droj2), orthologous to human DNAJA1 and DNAJA4 that is predicted to be involved in protein refolding, as a developmental signal promoting dendrite sculpting of the class IV dendritic arborization (C4da) sensory neuron in Drosophila. It was further shown that Arf102F, a GTP-binding protein previously implicated in protein trafficking, serves downstream of Droj2 to govern neurite pruning of C4da sensory neurons. Intriguingly, these data consistently demonstrate that both Droj2 and Arf102F promote the downregulation of the conserved L1-type cell-adhesion molecule Neuroglian anterior to dendrite pruning. Mechanistically, Droj2 genetically interacts with Arf102F and promotes Neuroglian downregulation to initiate dendrite severing. Taken together, this systematic study sheds light on an unprecedented function of Droj2 and Arf102F in neuronal development.

Takakura, M., Lam, Y. H., Nakagawa, R., Ng, M. Y., Hu, X., Bhargava, P., Alia, A. G., Gu, Y., Wang, Z., Ota, T., Kimura, Y., Morimoto, N., Osakada, F., Lee, A. Y., Leung, D., Miyashita, T., Du, J., Okuno, H., Hirano, Y. (2023). Differential second messenger signaling via dopamine neurons bidirectionally regulates memory retention. PubMed ID: 37639608
Summary:
Memory formation and forgetting unnecessary memory must be balanced for adaptive animal behavior. While cyclic AMP (cAMP) signaling via dopamine neurons induces memory formation, this study reports that cyclic guanine monophosphate (cGMP) signaling via dopamine neurons launches forgetting of unconsolidated memory in Drosophila. Genetic screening and proteomic analyses showed that neural activation induces the complex formation of a histone H3K9 demethylase, Kdm4B, and a GMP synthetase, Bur, which is necessary and sufficient for forgetting unconsolidated memory. Kdm4B/Bur is activated by phosphorylation through NO-dependent cGMP signaling via dopamine neurons, inducing gene expression, including kek2 encoding a presynaptic protein. Accordingly, Kdm4B/Bur activation induced presynaptic changes. These data demonstrate a link between cGMP signaling and synapses via gene expression in forgetting, suggesting that the opposing functions of memory are orchestrated by distinct signaling via dopamine neurons, which affects synaptic integrity and thus balances animal behavior.

Ferreira, A. A. G., Desplan, C. (2023). An Atlas of the Developing Drosophila Visual System Glia and Subcellular mRNA Localization of Transcripts in Single Cells. bioRxiv, PubMed ID: 37609218
Summary:
Glial cells are essential for proper nervous system development and function. To understand glial development and function, glial cells were comprehensively annotated in a single-cell mRNA-sequencing (scRNAseq) atlas of the developing Drosophila visual system. This allowed study of their developmental trajectories, from larval to adult stages, and to understand how specific types of glia diversify during development. For example, neuropil glia that are initially transcriptionally similar in larvae, split into ensheathing and astrocyte-like glia during pupal stages. Other glial types, such as chiasm glia change gradually during development without splitting into two cell types. The analysis of scRNA-seq led to the discovery that the transcriptome of glial cell bodies can be distinguished from that of their broken processes. The processes contain distinct enriched mRNAs that were validated in vivo. Therefore, this study have identified most glial types in the developing optic lobe and devised a computational approach to identify mRNA species that are localized to cell bodies or cellular processes.

Poe, A. R., Zhu, L., Szuperak, M., McClanahan, P. D., Anafi, R. C., Scholl, B., Thum, A. S., Cavanaugh, D. J., Kayser, M. S. (2023). Developmental emergence of sleep rhythms enables long-term memory in Drosophila. Sci Adv, 9(36):eadh2301 PubMed ID: 37683005
Summary:
In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day. The mechanism through which sleep rhythms emerge, and consequent advantage conferred to a juvenile animal, is unknown. In the second-instar Drosophila larvae (L2), like in human infants, sleep is not under circadian control. This study identified the precise developmental time point when the clock begins to regulate sleep in Drosophila, leading to emergence of sleep rhythms in early third-instars (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousal-promoting Dh44 neurons, bringing arousal under clock control to drive emergence of circadian sleep. Last, this study demonstrated that L3 but not L2 larvae exhibit long-term memory (LTM) of aversive cues and that this LTM depends upon deep sleep generated once sleep rhythms begin. It is proposed that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.

Tuesday, February 13th - Chromatin and DNA

Yuan, Y., Chen, Q., Brovkina, M., Clowney, E. J., Yadlapalli, S. (2023). Clock-dependent chromatin accessibility rhythms regulate circadian transcription. bioRxiv, PubMed ID: 37645872
Summary:
Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) was employed on Drosophila clock neurons to assess genome-wide chromatin accessibility over the circadian cycle. Significant circadian oscillations were observed in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, a complete loss was observed of chromatin accessibility rhythms in per⁰¹ null mutants, with chromatin consistently accessible throughout the circadian cycle, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

Hodkinson, L. J., Smith, C., Comstra, H. S., ...., Zeng, H., Schmidt, C. A., Rieder, L. E. (2023). A bioinformatics screen reveals hox and chromatin remodeling factors at the Drosophila histone locus. BMC genomic data, 24(1):54 PubMed ID: 37735352
Summary:
Cells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation. To expand the list of known HLB factors, a candidate-based screen was performed by mapping 30 publicly available ChIP datasets of 27 unique factors to the Drosophila histone gene array. Novel transcription factor candidates were identified, including the Drosophila Hox proteins Ultrabithorax (Ubx), Abdominal-A (Abd-A), and Abdominal-B (Abd-B), suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, six other factors were identified that target the histone gene array: JIL-1, hormone-like receptor 78 (Hr78), the long isoform of female sterile homeotic (1) (fs(1)h) as well as the general transcription factors TBP associated factor 1 (TAF-1), Transcription Factor IIB (TFIIB), and Transcription Factor IIF (TFIIF). This foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, this study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.

Zhu, J. Y., Liu, C., Huang, X., van de Leemput, J., Lee, H. and Han, Z. (2023). H3K36 Di-Methylation Marks, Mediated by Ash1 in Complex with Caf1-55 and MRG15, Are Required during Drosophila Heart Development. J Cardiovasc Dev Dis 10(7). PubMed ID: 37504562
Summary:
Methyltransferases regulate transcriptome dynamics during development and aging, as well as in disease. Various methyltransferases have been linked to heart disease, through disrupted expression and activity, and genetic variants associated with congenital heart disease. However, in vivo functional data for many of the methyltransferases in the context of the heart are limited. This study used the Drosophila model system to investigate different histone 3 lysine 36 (H3K36) methyltransferases for their role in heart development. The data show that Drosophila Ash1 is the functional homolog of human ASH1L in the heart. Both Ash1 and Set2 H3K36 methyltransferases are required for heart structure and function during development. Furthermore, Ash1-mediated H3K36 methylation (H3K36me2) is essential for healthy heart function, which depends on both Ash1-complex components, Caf1-55 and MRG15, together. These findings provide in vivo functional data for Ash1 and its complex, and Set2, in the context of H3K36 methylation in the heart, and support a role for their mammalian homologs, ASH1L with RBBP4 and MORF4L1, and SETD2, during heart development and disease.

Miller, J. M., Prange, S., Ji, H., ...., Hanscom, T., McVey, M., Chiolo, I. (2023). Alternative end-joining results in smaller deletions in heterochromatin relative to euchromatin. bioRxiv, PubMed ID: 37645729
Summary:
Homologous recombination (HR) repair is uniquely regulated in the ericentromeric heterochromatin to enable 'safe' repair while preventing aberrant recombination. In Drosophila cells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR repair. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how alternative EJ (alt-EJ) pathways operate in heterochromatin remains largely uncharacterized. This study induced DSBs in single euchromatic and heterochromatic sites using a new system that combines the DR- white reporter and I-SceI expression in spermatogonia of flies. Using this approach, higher frequency of HR repair is detected in heterochromatin, relative to euchromatin. Further, sequencing of mutagenic repair junctions reveals the preferential use of different EJ pathways across distinct euchromatic and heterochromatic sites. Interestingly, synthesis-dependent microhomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin.

Zion, E. H., Ringwalt, D., Rinaldi, K., Kahney, E. W., Li, Y. and Chen, X. (2023). Old and newly synthesized histones are asymmetrically distributed in Drosophila intestinal stem cell divisions. EMBO Rep 24(7): e56404. PubMed ID: 37255015
Summary:
This study reports that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically partitioned during the division of Drosophila intestinal stem cells (ISCs). Furthermore, the inheritance patterns of old and new H3 and H4 in postmitotic cell pairs correlate with distinct expression patterns of Delta, an important cell fate gene. To understand the biological significance of this phenomenon, a mutant H3T3A was expressed to compromise asymmetric histone inheritance. Under this condition, an increase was observed in Delta-symmetric cell pairs and overpopulated ISC-like, Delta-positive cells. Single-cell RNA-seq assays further indicate that H3T3A expression compromises ISC differentiation. Together, these results indicate that asymmetric histone inheritance potentially contributes to establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings in Drosophila male germline stem cells.

Kleene, V., Corvaglia, V., Chacin, E., Forne, I., Konrad, D. B., Khosravani, P., Douat, C., Kurat, C. F., Huc, I., Imhof, A. (2023). DNA mimic foldamers affect chromatin composition and disturb cell cycle progression. Nucleic Acids Res, 51(18):9629-9642 PubMed ID: 37650653
Summary:
The use of synthetic chemicals to selectively interfere with chromatin and the chromatin-bound proteome represents a great opportunity for pharmacological intervention. Recently, synthetic foldamers that mimic the charge surface of double-stranded DNA have been shown to interfere with selected protein-DNA interactions. However, to better understand their pharmacological potential and to improve their specificity and selectivity, the effect of these molecules on complex chromatin needs to be investigated. Therefore this study systematically studied the influence of the DNA mimic foldamers on the chromatin-bound proteome using an in vitro chromatin assembly extract. These studies show that the foldamer efficiently interferes with the chromatin-association of the origin recognition complex in vitro and in vivo, which leads to a disturbance of cell cycle in cells treated with foldamers. This effect is mediated by a strong direct interaction between the foldamers and the origin recognition complex and results in a failure of the complex to organise chromatin around replication origins. Foldamers that mimic double-stranded nucleic acids thus emerge as a powerful tool with designable features to alter chromatin assembly and selectively interfere with biological mechanisms.

Monday, February 12th - Disease Models

Li, Q., Wang, L., Tang, C., Wang, X., Yu, Z., Ping, X., Ding, M., Zheng, L. (2024). Adipose Tissue Exosome circ_sxc Mediates the Modulatory of Adiposomes on Brain Aging by Inhibiting Brain dme-miR-87-3p. Molecular neurobiology, 61(1):224-238 PubMed ID: 37597108
Summary:
Aging of the brain usually leads to the decline of neurological processes and is a major risk factor for various neurodegenerative diseases, including sleep disturbances and cognitive decline. Adipose tissue exosomes, as adipocyte-derived vesicles, may mediate the regulatory processes of adipose tissue on other organs, including the brain; however, the regulatory mechanisms remain unclear. This study analyzed the sleep-wake behavior of young (10 days) and old (40 days) Drosophila and found that older Drosophila showed increased sleep fragmentation, which is similar to mammalian aging characteristics. To investigate the cross-tissue regulatory mechanisms of adiposity on brain aging, 10-day and 40-day Drosophila adipose tissue exosomes were extracted and circRNAs were identied with age-dependent expression differences by RNA-seq and differential analysis. Furthermore, by combining data from 3 datasets of the GEO database (GSE130158, GSE24992, and GSE184559), circ_sxc that was significantly downregulated with age was finally screened out. Moreover, dme-miR-87-3p, a conserved target of circ_sxc, accumulates in the brain with age and exhibits inhibitory effects in predicted binding relationships with neuroreceptor ligand genes. In summary, the current study showed that the Drosophila brain could obtain circ_sxc by uptake of adipose tissue exosomes which crossed the blood-brain barrier. And circ_sxc suppressed brain miR-87-3p expression through sponge adsorption, which in turn regulated the expression of neurological receptor ligand proteins (5-HT1B, GABA-B-R1, Rdl, Rh7, qvr, NaCP60E) and ensured brain neuronal synaptic signaling normal function of synaptic signaling. However, with aging, this regulatory mechanism is dysregulated by the downregulation of the adipose exosome circ_sxc, which contributes to the brain exhibiting sleep disturbances and other "aging" features.

Sung, H., Lloyd, T. E. (2024). Disrupted endoplasmic reticulum-mediated autophagosomal biogenesis in a Drosophila model of C9-ALS-FTD. Autophagy, 20(1):94-113 PubMed ID: 37599467
Summary:
Macroautophagy/autophagy is a major pathway for the clearance of protein aggregates and damaged organelles, and multiple intracellular organelles participate in the process of autophagy, from autophagosome formation to maturation and degradation. Dysregulation of the autophagy pathway has been implicated in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), however the mechanisms underlying autophagy impairment in these diseases are incompletely understood. Since the expansion of GGGGCC (G4C2) repeats in the first intron of the C9orf72 gene is the most common inherited cause of both ALS and FTD (C9-ALS-FTD), this study investigated autophagosome dynamics in Drosophila motor neurons expressing 30 G4C2 repeats (30 R). In vivo imaging demonstrates that expression of expanded G4C2 repeats markedly impairs biogenesis of autophagosomes at synaptic termini, whereas trafficking and maturation of axonal autophagosomes are unaffected. Motor neurons expressing 30 R display marked disruption in endoplasmic reticulum (ER) structure and dynamics in the soma, axons, and synapses. Disruption of ER morphology with mutations in Rtnl1 (Reticulon-like 1) or atl (atlastin) also impairs autophagosome formation in motor neurons, suggesting that ER integrity is critical for autophagosome formation. Furthermore, live imaging demonstrates that autophagosomes are generated from dynamic ER tubules at synaptic boutons, and this process fails to occur in a C9-ALS-FTD model. Together, these findings suggest that dynamic ER tubules are required for formation of autophagosomes at the neuromuscular junction, and that this process is disrupted by expanded G4C2 repeats that cause ALS-FTD.

Soustelle, L., Aimond, F., Lopez-Andres, C., Brugioti, V., Raoul, C., Layalle, S. (2023). ALS-Associated KIF5A Mutation Causes Locomotor Deficits Associated with Cytoplasmic Inclusions, Alterations of Neuromuscular Junctions, and Motor Neuron Loss. J Neurosci, 43(47):8058-8072 PubMed ID: 37748861
Summary:
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Recently, genome-wide association studies identified KIF5A as a new ALS-causing gene. KIF5A encodes a protein of the kinesin-1 family, allowing the anterograde transport of cargos along the microtubule rails in neurons. In ALS patients, mutations in the KIF5A gene induce exon 27 skipping, resulting in a mutated protein with a new C-terminal region (KIF5A Δ27). To understand how KIF5A Δ27 underpins the disease, this study developed an ALS-associated KIF5A Drosophila model. When selectively expressed in motor neurons, KIF5A Δ27 alters larval locomotion as well as morphology and synaptic transmission at neuromuscular junctions in both males and females. The distribution of mitochondria and synaptic vesicles was found to be profoundly disturbed by KIF5A Δ27 expression. That is consistent with the numerous KIF5A Δ27-containing inclusions observed in motor neuron soma and axons. Moreover, KIF5A Δ27 expression leads to motor neuron death and reduces life expectancy. This in vivo model reveals that a toxic gain of function underlies the pathogenicity of ALS-linked KIF5A mutant.

Cai, Y., Cui, T., Yin, P., Paganelli, P., Vicini, S., Wang, T. (2023). Dysregulated glial genes in Alzheimer's disease are essential for homeostatic plasticity: Evidence from integrative epigenetic and single cell analyses. Aging Cell, 22(11):e13989 PubMed ID: 37712202
Summary:
Synaptic homeostatic plasticity is a foundational regulatory mechanism that maintains the stability of synaptic and neural functions within the nervous system. Impairment of homeostatic regulation has been linked to synapse destabilization during the progression of Alzheimer's disease (AD). Various glial cell types play critical roles in modulating synaptic functions both during the aging process and in the context of AD. This study investigated the impact of glial dysregulation of histone acetylation and transcriptome in AD on synaptic homeostatic plasticity, using computational analysis combined with electrophysiological methods in Drosophila. By integrating snRNA-seq and H3K9ac ChIP-seq data from the same AD patient cohort, this study pinpointed cell type-specific signature genes that were transcriptionally altered by histone acetylation. The role of these glial genes in regulating presynaptic homeostatic potentiation was subsequently investigated in Drosophila. Remarkably, nine glial-specific genes, which were identified through computational methods as targets of H3K9ac and transcriptional dysregulation, were found to be crucial for the regulation of synaptic homeostatic plasticity at the NMJ in Drosophila. This genetic evidence connects abnormal glial transcriptomic changes in AD with the impairment of homeostatic plasticity in the nervous system. In summary, these integrative computational and genetic studies highlight specific glial genes as potential key players in the homeostatic imbalance observed in AD.

Koval, L. A., Proshkina, E. N., Zemskaya, N. V., Solovev, I. A., Schegoleva, E. V., Shaposhnikov, M. V., Moskalev, A. A. (2023). [Drosophila melanogaster Lifespan Is Regulated by nejire Gene Expression in Peripheral Tissues and Nervous System]. Molekuliarnaia biologiia, 57(5):833-852 PubMed ID: 37752649
Summary:
Histone acetyltransferases of the CBP/p300 family are involved in transcriptional regulation and many biological processes (cell proliferation and differentiation, development, and regulation of the stress response and metabolism). Overexpression and knockdown of the nejire (nej) gene (codes for an ortholog of human CBP/p300 proteins) in various tissues (the fat body, intestine, and nervous system) and at various stages of the life cycle (throughout all developmental stages or in adulthood only) were tested for effect on lifespan in the fruit fly Drosophila melanogaster. The activation of nej exerted a positive or a negative effect on the lifespan, depending on the induction mode and the sex. A 6-15% greater lifespan was observed in females with conditional overexpression of nej in the intestine and constitutive overexpression of nej in the nervous system. A decrease (to 44%) or lack of significant changes in lifespan was detected in all other cases observed. In addition, stress response genes (Sod1, Gadd45, Hsp27, Hsp68, and Hif1) were regulated by nej activation. nej knockdown caused a pronounced negative effect on the D. melanogaster lifespan in most variants of the experiment.

Dutta, D., Kanca, O., Byeon, S. K., Marcogliese, P. C., Zuo, Z., Shridharan, R. V., Park, J. H., Lin, G., Ge, M., Heimer, G., Kohler, J. N., Wheeler, M. T., Kaipparettu, B. A., Pandey, A., Bellen, H. J. (2023. A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels. Nature metabolism, 5(9):1595-1614 PubMed ID: 37653044
Summary:
In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. This study shows that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. A defect in Fe-S cluster biogenesis and increased iron levels were observed in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and this study shows that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.

Friday, February 9th - Cytoskeleton

Zhang, X. Q., Yang, R., Jin, L. and Li, G. Q. (2023). Requirement of Snakeskin for normal functions of midgut and Malpighian tubules in Henosepilachna vigintioctopunctata. Arch Insect Biochem Physiol 114(1): e22033. PubMed ID: 37401505
Summary:
Septate junctions (SJs) are located between epithelial cells and play crucial roles in epithelial barrier formation and epithelia cell homeostasis. Nevertheless, the molecular constituents, especially those related to smooth SJs (sSJs), have not been well explored in non-Drosophilid insects. A putative integral membrane protein Snakeskin (Ssk) was identified in a Coleoptera foliar pest Henosepilachna vigintioctopunctata. RNA interference-aided knockdown of Hvssk at the third-instar larval stage arrested larval development. Most resultant larvae failed to shed larval exuviae until their death. Silence of Hvssk at the fourth-instar larvae inhibited the growth and reduced foliage consumption. Dissection and microscopic observation revealed that compromised expression of Hvssk caused obvious phenotypic defects in the midgut. A great number of morphologically abnormal columnar epithelial cells accumulated throughout the midgut lumen. Moreover, numerous vesicles were observed in the malformed cells of the Malpighian tubules (Mt). All the Hvssk depleted larvae remained as prepupae; they gradually darkened and eventually died. Furthermore, depletion of Hvssk at the pupal stage suppressed adult feeding and shortened adult lifespan. These findings demonstrated that Ssk plays a vital role in the integrity and function of both midguts and Mt, and established the conservative roles of Ssk in the formation of epithelial barrier and the homeostasis of epithelial cells in H. vigintioctopunctata.

Salvador-Garcia, D., Jin, L., Hensley, A., Golcuk, M., Gallaud, E., Chaaban, S., Port, F., Vagnoni, A., Planelles-Herrero, V. J., McClintock, M. A., Derivery, E., Carter, A. P., Giet, R., Gur, M., Yildiz, A. and Bullock, S. L. (2023). A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression. bioRxiv. PubMed ID: 37577480
Summary:
The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate in vivo studies of its functions significantly. To address this issue, gene editing was used to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). Mutations associated with human neurological disease were shown to cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. A novel mutation in the microtubule-binding domain (MTBD) of Dhc is described that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. Mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, these data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, these work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.

Wippich, F., Vaishali, Hennrich, M. L. and Ephrussi, A. (2023). Nutritional stress-induced regulation of microtubule organization and mRNP transport by HDAC1 controlled α-tubulin acetylation. Commun Biol 6(1): 776. PubMed ID: 37491525
Summary:
In response to nutritional stress, microtubules in cells of the Drosophila female germline are depleted from the cytoplasm and accumulate cortically. This triggers aggregation of mRNPs into large processing bodies (P-bodies) and oogenesis arrest. This study shows that hyperacetylation of α-tubulin at lysine 40 (K40) alters microtubule dynamics and P-body formation. Depletion of histone deacetylase 1 (HDAC1) by RNAi phenocopies the nutritional stress. response, causing α-tubulin hyperacetylation and accumulation of maternally deposited mRNPs in P-bodies. Through in vitro and in vivo studies, this study identified HDAC1 as a direct regulator of α-tubulin K40 acetylation status. In well-fed flies, HDAC1 maintains low levels of α-tubulin acetylation, enabling the microtubule dynamics required for mRNP transport. Using quantitative phosphoproteomics, nutritional stress-induced changes were identified in protein phosphorylation that act upstream of α-tubulin acetylation, including phosphorylation of HDAC1 at S391, which reduces its ability to deacetylate α-tubulin. These results reveal that Drosophila HDAC1 senses and relays the nutritional status, which regulates germline development through modulation of cytoskeleton dynamics.

Zhao, H., Shi, L., Li, Z., Kong, R., Jia, L., Lu, S., Wang, J. H., Dong, M. Q., Guo, X., Li, Z. (2023). Diamond controls epithelial polarity through the dynactin-dynein complex. Traffic (Copenhagen, Denmark), 24(12):552-563 PubMed ID: 37642208
Summary:
Epithelial polarity is critical for proper functions of epithelial tissues, tumorigenesis, and metastasis. The evolutionarily conserved transmembrane protein Crumbs (Crb) is a key regulator of epithelial polarity. Both Crb protein and its transcripts are apically localized in epithelial cells. However, it remains not fully understood how they are targeted to the apical domain. Using Drosophila ovarian follicular epithelia as a model, it was show that epithelial polarity is lost and Crb protein is absent in the apical domain in follicular cells (FCs) in the absence of Diamond (Dind). Interestingly, Dind is found to associate with different components of the dynactin-dynein complex through co-IP-MS analysis. Dind stabilizes dynactin and depletion of dynactin results in almost identical defects as those observed in dind-defective FCs. Finally, both Dind and dynactin are also required for the apical localization of crb transcripts in FCs. Thus these data illustrate that Dind functions through dynactin/dynein-mediated transport of both Crb protein and its transcripts to the apical domain to control epithelial apico-basal (A/B) polarity.

Song, X., Cui, L., Wu, M., Wang, S., Song, Y., Liu, Z., Xue, Z., Chen, W., Zhang, Y., Li, H., Sun, L., Liang, X. (2023). DCX-EMAP is a core organizer for the ultrastructure of Drosophila mechanosensory organelles. J Cell Biol, 222(10). PubMed ID: 37651176
Summary:
Mechanoreceptor cells develop specialized mechanosensory organelles (MOs), where force-sensitive channels and supporting structures are organized in an orderly manner to detect forces. It is intriguing how MOs are formed. This issue was addressed by studying the MOs of fly ciliated mechanoreceptors. The main structure of the MOs is shown to be a compound cytoskeleton formed of short microtubules and electron-dense materials (EDMs). In a knock-out mutant of microtubule associated protein DCX-EMAP, this cytoskeleton is nearly absent, suggesting that DCX-EMAP is required for the formation of the MOs and in turn fly mechanotransduction. Further analysis reveals that DCX-EMAP expresses in fly ciliated mechanoreceptors and localizes to the MOs. Moreover, it plays dual roles by promoting the assembly/stabilization of the microtubules and the accumulation of the EDMs in the MOs. Therefore, DCX-EMAP serves as a core ultrastructural organizer of the MOs, and this finding provides novel molecular insights as to how fly MOs are formed.

Ventura Santos, C., Rogers, S. L., Carter, A. P. (2023). CryoET shows cofilactin filaments inside the microtubule lumen. EMBO reports, 24(11):e57264 PubMed ID: 37702953
Summary:
Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. This study used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin dephosphorylation, an activating modification, was observed in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

Thursday, February 8th - Behavior

Yu, J., Dancausse, S., Paz, M., Faderin, T., Gaviria, M., Shomar, J. W., Zucker, D., Venkatachalam, V. and Klein, M. (2023). Continuous, long-term crawling behavior characterized by a robotic transport system. Elife 12. PubMed ID: 37535068
Summary:
Detailed descriptions of behavior provide critical insight into the structure and function of nervous systems. In Drosophila larvae and many other systems, short behavioral experiments have been successful in characterizing rapid responses to a range of stimuli at the population level. However, the lack of long-term continuous observation makes it difficult to dissect comprehensive behavioral dynamics of individual animals and how behavior (and therefore the nervous system) develops over time. To allow for long-term continuous observations in individual fly larvae, a robotic instrument was engineered that automatically tracks and transports larvae throughout an arena. The flexibility and reliability of its design enables controlled stimulus delivery and continuous measurement over developmental time scales, yielding an unprecedented level of detailed locomotion data. This study utilized the new system's capabilities to perform continuous observation of exploratory search behavior over a duration of 6 hr with and without a thermal gradient present, and in a single larva for over 30 hr. Long-term free-roaming behavior and analogous short-term experiments show similar dynamics that take place at the beginning of each experiment. Finally, characterization of larval thermotaxis in individuals reveals a bimodal distribution in navigation efficiency, identifying distinct phenotypes that are obfuscated when only analyzing population averages.

Venkateswaran, V., Alali, I., Unni, A. P., Weissflog, J., Halitschke, R., Hansson, B. S., Knaden, M. (2023). Carbonyl products of ozone oxidation of volatile organic compounds can modulate olfactory choice behavior in insects. Environ Pollut, 337:122542 PubMed ID: 37717892
Summary:
Insects are a diverse group of organisms that provide important ecosystem services like pollination, pest control, and decomposition and rely on olfaction to perform these services. In the Anthropocene, increasing concentrations of oxidant pollutants such as ozone have been shown to corrupt odor-driven behavior in insects by chemically degrading e.g. flower signals or insect pheromones. The degradation, however, does not only result in a loss of signals, but also in a potential enrichment of oxidation products, predominantly small carbonyls. Whether and how these oxidation products affect insect olfactory perception remains unclear. This study examined the effects of ozone-generated small carbonyls on the olfactory behavior of the vinegar fly Drosophila melanogaster. A broad collection of neurophysiologically relevant odorants were compiled for the fly from databases and literature and predicted the formation of the types of stable small carbonyl products resulting from the odorant's oxidation by ozone. Based on these predictions, the olfactory detection and behavioral impact of the ten most frequently predicted carbonyl products were evaluated in the fly using single sensillum recordings (SSRs) and behavioral tests. The results demonstrate that the fly's olfactory system can detect the oxidation products, which then elicit either attractive or neutral behavioral responses, rather than repulsion. However, certain products alter behavioral choices to an attractive odor source of balsamic vinegar. These findings suggest that the enrichment of small carbonyl oxidation products due to increased ozone levels can affect olfactory guided insect behavior. This study underscores the implications for odor-guided foraging in insects and the essential ecosystem services they offer under carbonyl enriched environments.

Babin, A., Gatti, J. L., Poirie, M. (2023). Bacillus thuringiensis bioinsecticide influences Drosophila oviposition decision. R Soc Open Sci, 10(8):230565 PubMed ID: 37650056
Summary:
Behavioural avoidance has obvious benefits for animals facing environmental stressors such as pathogen-contaminated foods. Most current bioinsecticides are based on the environmental and opportunistic bacterium Bacillus thuringiensis (Bt) that kills targeted insect pests upon ingestion. While food and oviposition avoidance of Bt bioinsecticide by targeted insect species was reported, this remained to be addressed in non-target organisms, especially those affected by chronic exposure to Bt bioinsecticide such as Drosophila species. Here, using a two-choice oviposition test, it was shown that female flies of three Drosophila species (four strains of D. melanogaster, D. busckii and D. suzukii) avoided laying eggs in the presence of Bt var. kurstaki bioinsecticide, with potential benefits for the offspring and female's fitness. Avoidance occurred rapidly, regardless of the fraction of the bioinsecticide suspension (spores and toxin crystals versus soluble toxins/compounds) and independently of the female motivation for egg laying. These results suggest that, in addition to recent findings of developmental and physiological alterations upon chronic exposure to non-target Drosophila, this bioinsecticide may modify the competitive interactions between Drosophila species in treated areas and the interactions with their associated natural enemies.

Kobayashi, R., Nakane, S., Tomita, J., Funato, H., Yanagisawa, M., Kume, K. (2023). A phosphorylation-deficient mutant of Sik3, a homolog of Sleepy, alters circadian sleep regulation by PDF neurons in Drosophila. Frontiers in neuroscience, 17:1181555 PubMed ID: 37662102
Summary:
Sleep behavior has been observed from non-vertebrates to humans. Sleepy mutation in mice resulted in a notable increase in sleep and was identified as an exon-skipping mutation of the salt-inducible kinase 3 (Sik3) gene (See Drosophila Sik3), conserved among animals. The skipped exon includes a serine residue that is phosphorylated by protein kinase A. Overexpression of a mutant gene with the conversion of this serine into alanine (Sik3-SA) increased sleep in both mice and the fruit fly Drosophila melanogaster. However, the mechanism by which Sik3-SA increases sleep remains unclear. This study found that Sik3-SA overexpression in all neurons increased sleep under both light-dark (LD) conditions and constant dark (DD) conditions in Drosophila. Additionally, overexpression of Sik3-SA only in PDF neurons, which are a cluster of clock neurons regulating the circadian rhythm, increased sleep during subjective daytime while decreasing the amplitude of circadian rhythm. Furthermore, suppressing Sik3-SA overexpression specifically in PDF neurons in flies overexpressing Sik3-SA in all neurons reversed the sleep increase during subjective daytime. These results indicate that Sik3-SA alters the circadian function of PDF neurons and leads to an increase in sleep during subjective daytime under constant dark conditions.

McKenzie-Smith, G. C., Wolf, S. W., Ayroles, J. F., Shaevitz, J. W. (2023). Capturing continuous, long timescale behavioral changes in Drosophila melanogaster postural data. ArXiv, PubMed ID: 37731659
Summary:
Animal behavior spans many timescales, from short, seconds-scale actions to circadian rhythms over many hours to life-long changes during aging. Most quantitative behavior studies have focused on short-timescale behaviors such as locomotion and grooming. Analysis of these data suggests there exists a hierarchy of timescales; however, the limited duration of these experiments prevents the investigation of the full temporal structure. To access longer timescales of behavior, individual Drosophila melanogaster were continuously recorded at 100 frames per second for up to 7 days at a time in featureless arenas on sucrose-agarose media. The deep learning framework SLEAP was used to produce a full-body postural data set for 47 individuals resulting in nearly 2 billion pose instances. Stereotyped behaviors were identified such as grooming, proboscis extension, and locomotion, and the resulting ethograms were used to explore how the flies' behavior varies across time of day and days in the experiment. Distinct circadian patterns were observed all of the stereotyped behavior, and also changes were seen in behavior over the course of the experiment as the flies weaken and die.

Nguyen, M., Roman, G. W., Soibam, B. (2023). Drosophila genotypes can be predicted from their exploration locomotive trajectories using supervised machine learning. Behavioural processes, 212:104944 PubMed ID: 37717930
Summary:
This study employs supervised machine learning algorithms to test whether locomotive features during exploratory activity in open field arenas can serve as predictors for the genotype of fruit flies. Because of the nonlinearity in locomotive trajectories, traditional statistical methods that are used to compare exploratory activity between genotypes of fruit flies may not reveal all insights. 10-minute-long trajectories of four different genotypes of fruit flies in an open-field arena environment were captured. Turn angles and step size features extracted from the trajectories were used for training supervised learning models to predict the genotype of the fruit flies. Using the first five minute locomotive trajectories, an accuracy of 83% was achieved in differentiating wild-type flies from three other mutant genotypes. Using the final 5 min and the entire ten minute duration decreased the performance indicating that the most variations between the genotypes in their exploratory activity are exhibited in the first few minutes. Feature importance analysis revealed that turn angle is a better predictor than step size in predicting fruit fly genotype. Overall, this study demonstrates that features of trajectories can be used to predict the genotype of fruit flies through supervised machine learning methods.

Wednesday, February 7th - Adult physiology and metabolism

Strunov, A., Schoenherr, C. and Kapun, M. (2023). Wolbachia has subtle effects on thermal preference in highly inbred Drosophila melanogaster which vary with life stage and environmental conditions. Sci Rep 13(1): 13792. PubMed ID: 37612420
Summary:
Temperature fluctuations are challenging for ectotherms which are not able to regulate body temperature by physiological means and thus have to adjust their thermal environment via behavior. However, little is yet known about whether microbial symbionts influence thermal preference (T(p)) in ectotherms by modulating their physiology. Several recent studies have demonstrated substantial effects of Wolbachia infections on host T(p) in different Drosophila species. These data indicate that the direction and strength of thermal preference variation is strongly dependent on host and symbiont genotypes and highly variable among studies. By employing highly controlled experiments, this study investigated the impact of several environmental factors including humidity, food quality, light exposure, and experimental setup that may influence T(p) measurements in adult Drosophila melanogaster flies. Additionally, the effects of Wolbachia infection on T(p) of Drosophila was assessed at different developmental stages, which has not been done before. Only subtle effects were found of Wolbachia on host T(p) which are strongly affected by experimental variation in adult, but not during juvenile life stages. These in-depth analyses show that environmental variation has a substantial influence on T(p) which demonstrates the necessity of careful experimental design and cautious interpretations of T(p) measurements together with a thorough description of the methods and equipment used to conduct behavioral studies.

Ren, M., Xu, Y., Phoon, C. K. L., Erdjument-Bromage, H., Neubert, T. A., Schlame, M. (2023). Cardiolipin prolongs the lifetimes of respiratory proteins in Drosophila flight muscle. J Biol Chem, 299(10):105241 PubMed ID: 37690688
Summary:
Respiratory complexes and cardiolipins have exceptionally long lifetimes. The fact that they co-localize in mitochondrial cristae raises the question of whether their longevities have a common cause and whether the longevity of OXPHOS proteins is dependent on cardiolipin. To address these questions, a method was developed to measure side-by-side the half-lives of proteins and lipids in wild-type Drosophila and cardiolipin-deficient mutants. Adult flies were fed with stable isotope-labeled precursors ((13)C(6)(15)N(2)-lysine or (13)C(6)-glucose), and the relative abundance of heavy isotopomers in protein and lipid species was determined by mass spectrometry. To minimize the confounding effects of tissue regeneration, this analysis was restricted to the thorax, the bulk of which consists of post-mitotic flight muscles. Analysis of 680 protein and 45 lipid species showed that the subunits of respiratory complexes I-V and the carriers for phosphate and ADP/ATP were among the longest-lived proteins (average half-life of 48 ± 16 days) while the molecular species of cardiolipin were the longest-lived lipids (average half-life of 27 ± 6 days). The remarkable longevity of these crista residents was not shared by all mitochondrial proteins, especially not by those residing in the matrix and the inner boundary membrane. Ablation of cardiolipin synthase, which causes replacement of cardiolipin by phosphatidylglycerol, and ablation of tafazzin, which causes partial replacement of cardiolipin by monolyso-cardiolipin, decreased the lifetimes of the respiratory complexes. Ablation of tafazzin also decreased the lifetimes of the remaining cardiolipin species. These data suggest that an important function of cardiolipin in mitochondria is to protect respiratory complexes from degradation.

Bozkurt, B., Terlemez, G., Sezgin, E. (2023). Basidiomycota species in Drosophila gut are associated with host fat metabolism. Sci Rep, 13(1):13807 PubMed ID: 37612350
Summary:
The importance of bacterial microbiota on host metabolism and obesity risk is well documented. However, the role of fungal microbiota on host storage metabolite pools is largely unexplored. This study investigated the role of microbiota on D. melanogaster fat metabolism, and examine interrelatedness between fungal and bacterial microbiota, and major metabolic pools. Fungal and bacterial microbiota profiles, fat, glycogen, and trehalose metabolic pools are measured in a context of genetic variation represented by whole genome sequenced inbred Drosophila Genetic Reference Panel (DGRP) samples. Increasing Basidiomycota, Acetobacter persici, Acetobacter pomorum, and Lactobacillus brevis levels correlated with decreasing triglyceride levels. Host genes and biological pathways, identified via genome-wide scans, associated with Basidiomycota and triglyceride levels were different suggesting the effect of Basidiomycota on fat metabolism is independent of host biological pathways that control fungal microbiota or host fat metabolism. Although triglyceride, glycogen and trehalose levels were highly correlated, microorganisms' effect on triglyceride pool were independent of glycogen and trehalose levels. Multivariate analyses suggested positive interactions between Basidiomycota, A. persici, and L. brevis that collectively correlated negatively with fat and glycogen pools. In conclusion, fungal microbiota can be a major player in host fat metabolism. Interactions between fungal and bacterial microbiota may exert substantial control over host storage metabolite pools and influence obesity risk.

Dornan, A. J., Halberg, K. V., Beuter, L. K., Davies, S. A., Dow, J. A. T. (2023). Compromised junctional integrity phenocopies age-dependent renal dysfunction in Drosophila Snakeskin mutants. J Cell Sci, 136(19) PubMed ID: 37694602
Summary:
Transporting epithelia provide a protective barrier against pathogenic insults while allowing the controlled exchange of ions, solutes and water with the external environment. In invertebrates, these functions depend on formation and maintenance of 'tight' septate junctions (SJs). However, the mechanism by which SJs affect transport competence and tissue homeostasis, and how these are modulated by ageing, remain incompletely understood. This study demonstrated that the Drosophila renal (Malpighian) tubules undergo an age-dependent decline in secretory capacity, which correlates with mislocalisation of SJ proteins and progressive degeneration in cellular morphology and tissue homeostasis. Acute loss of the SJ protein Snakeskin in adult tubules induced progressive changes in cellular and tissue architecture, including altered expression and localisation of junctional proteins with concomitant loss of cell polarity and barrier integrity, demonstrating that compromised junctional integrity is sufficient to replicate these ageing-related phenotypes. Taken together, this work demonstrates a crucial link between epithelial barrier integrity, tubule transport competence, renal homeostasis and organismal viability, as well as providing novel insights into the mechanisms underpinning ageing and renal disease.

Williams-Simon, P. A., Oster, C., Moaton, J. A., Ghidey, R., Ng'oma, E., Middleton, K. M., Zars, T. and King, E. G. (2023). Naturally segregating genetic variants contribute to thermal tolerance in a D. melanogaster model system. bioRxiv. PubMed ID: 37461510
Summary:
Thermal tolerance is a fundamental physiological complex trait for survival in many species. This study took a multipronged approach to dissect the genetic architecture that controls thermal tolerance in natural populations using the Drosophila Synthetic Population Resource (DSPR) as a model system. First, quantitative genetics and Quantitative Trait Loci (QTL) mapping were used to identify major effect regions within the genome that influences thermal tolerance, then integrated RNA-sequencing to identify differences in gene expression, and lastly, the RNAi system was used to 1) alter tissue-specific gene expression and 2) functionally validate the findings. This powerful integration of approaches not only allows for the identification of the genetic basis of thermal tolerance but also the physiology of thermal tolerance in a natural population, which ultimately elucidates thermal tolerance through a fitness-associated lens.

Swope, S. D., Jones, T. W., Mellina, K. N., Nichols, S. J., DiAngelo, J. R. (2023). Arc1 : a regulator of triglyceride homeostasis in male Drosophila. Micro Publication Biology. PubMed ID: 37675078
Summary:
Achieving metabolic homeostasis is necessary for survival, and many genes are required to control organismal metabolism. A genetic screen in Drosophila larvae identified putative fat storage genes including Arc1. Arc1 has been shown to act in neurons to regulate larval lipid storage; however, whether Arc1 functions to regulate adult metabolism is unknown. Arc1^esm18 males store more fat than controls while both groups eat similar amounts. Arc1^esm18 flies express more brummer lipase and less of the glycolytic enzyme triose phosphate isomerase, which may contribute to excess fat observed in these mutants. These results suggest that Arc1 regulates adult Drosophila lipid homeostasis.

Tuesday, February 6th - Autophagy

Qin, B., Yu, S., Chen, Q., Jin, L. H. (2023). Atg2 Regulates Cellular and Humoral Immunity in Drosophila. Insects, 14(8) PubMed ID: 37623416
Summary:
Autophagy is a process that promotes the lysosomal degradation of cytoplasmic proteins and is highly conserved in eukaryotic organisms. Autophagy maintains homeostasis in organisms and regulates multiple developmental processes, and autophagy disruption is related to human diseases. However, the functional roles of autophagy in mediating innate immune responses are largely unknown. This study sought to understand how Atg2, an autophagy-related gene, functions in the innate immunity of Drosophila melanogaster. The results showed that a large number of melanotic nodules were produced upon inhibition of Atg2. In addition, inhibiting Atg2 suppressed the phagocytosis of latex beads, Staphylococcus aureus and Escherichia coli; the proportion of Nimrod C1 (one of the phagocytosis receptors)-positive hemocytes also decreased. Moreover, inhibiting Atg2 altered actin cytoskeleton patterns, showing longer filopodia but with decreased numbers of filopodia. The expression of AMP-encoding genes was altered by inhibiting Atg2. Drosomycin was upregulated, and the transcript levels of Attacin-A, Diptericin and Metchnikowin were decreased. Finally, the above alterations caused by the inhibition of Atg2 prevented flies from resisting invading pathogens, showing that flies with low expression of Atg2 were highly susceptible to Staphylococcus aureus and Erwinia carotovora carotovora 15 infections. In conclusion, Atg2 regulated both cellular and humoral innate immunity in Drosophila. This study has identified Atg2 as a crucial regulator in mediating the homeostasis of immunity, which further established the interactions between autophagy and innate immunity.

Szinyakovics, J., Keresztes, F., Kiss, E. A., Falcsik, G., Vellai, T. and Kovacs, T. (2023). Potent New Targets for Autophagy Enhancement to Delay Neuronal Ageing. Cells 12(13). PubMed ID: 37443788
Summary:
Autophagy is a lysosomal-dependent degradation process of eukaryotic cells responsible for breaking down unnecessary and damaged intracellular components. This study aimed to uncover new regulatory points where autophagy could be specifically activated and tested these potential drug targets in neurodegenerative disease models of Drosophila melanogaster. One possible way to activate autophagy is by enhancing autophagosome-lysosome fusion that creates the autolysosome in which the enzymatic degradation happens. The HOPS (homotypic fusion and protein sorting) and SNARE (Snap receptor) protein complexes regulate the fusion process. The HOPS complex forms a bridge between the lysosome and autophagosome with the assistance of small GTPase proteins. Thus, small GTPases are essential for autolysosome maturation, and among these proteins, Rab2 (Ras-associated binding 2), Rab7, and Arl8 (Arf-like 8) are required to degrade the autophagic cargo. For these experiments, Drosophila melanogaster was used as a model organism. Nerve-specific small GTPases were silenced and overexpressed. The effects were examined of these genetic interventions on lifespan, climbing ability, and autophagy. Finally, the activation of small GTPases was also studied in a Parkinson's disease model. The results revealed that GTP-locked, constitutively active Rab2 (Rab2-CA) and Arl8 (Arl8-CA) expression reduces the levels of the autophagic substrate p62/Ref(2)P in neurons, extends lifespan, and improves the climbing ability of animals during ageing. However, Rab7-CA expression dramatically shortens lifespan and inhibits autophagy. Rab2-CA expression also increases lifespan in a Parkinson's disease model fly strain overexpressing human mutant (A53T) α-synuclein protein. Data provided by this study suggests that Rab2 and Arl8 serve as potential targets for autophagy enhancement in the Drosophila nervous system.

Zhang, S., Yi, S., Wang, L., Li, S., Wang, H., Song, L., Ou, J., Zhang, M., Wang, R., Wang, M., Zheng, Y., Yang, K., Liu, T. and Ho, M. S. (2023). Cyclin-G-associated kinase GAK/dAux regulates autophagy initiation via ULK1/Atg1 in glia. Proc Natl Acad Sci U S A 120(29): e2301002120. PubMed ID: 37428930
Summary:
Autophagy is a major means for the elimination of protein inclusions in neurons in neurodegenerative diseases such as Parkinson's disease (PD). Yet, the mechanism of autophagy in the other brain cell type, glia, is less well characterized and remains largely unknown. This study presents evidence that the PD risk factor, Cyclin-G-associated kinase (GAK)/Drosophila homolog Auxilin (dAux), is a component in glial autophagy. The lack of GAK/dAux increases the autophagosome number and size in adult fly glia and mouse microglia, and generally up-regulates levels of components in the initiation and PI3K class III complexes. GAK/dAux interacts with the master initiation regulator UNC-51-like autophagy activating kinase 1/Atg1 via its uncoating domain and regulates the trafficking of Atg1 and Atg9 to autophagosomes, hence controlling the onset of glial autophagy. On the other hand, lack of GAK/dAux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/dAux might play additional roles. Importantly, dAux contributes to PD-like symptoms including dopaminergic neurodegeneration and locomotor function in flies. These findings identify an autophagy factor in glia; considering the pivotal role of glia under pathological conditions, targeting glial autophagy is potentially a therapeutic strategy for PD.

Bierlein, M., Charles, J., Polisuk-Balfour, T., Bretscher, H., Rice, M., Zvonar, J., Pohl, D., Winslow, L., Wasie, B., Deurloo, S., Van Wert, J., Williams, B., Ankney, G., Harmon, Z., Dann, E., Azuz, A., Guzman-Vargas, A., Kuhns, E., Neufeld, T. P., O'Connor, M. B., Amissah, F., Zhu, C. C. (2023). Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster). Genetics, 225(2) PubMed ID: >37594076
Summary:
Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. This report shows that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies but the lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Overexpression of wild-type Atg1 in adult fly muscle or adipocytes reduces the lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. This research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation.

Pino-Jimenez, B., Giannios, P. and Casanova, J. (2023). Polyploidy-associated autophagy promotes larval tracheal histolysis at Drosophila metamorphosis. Autophagy: 1-10. PubMed ID: 37424089
Summary:
Polyploidy is an extended phenomenon in biology. However, its physiological significance and whether it defines specific cell behaviors is not well understood. Polyploidy connection to macroautophagy/autophagy was examined, using the larval respiratory system of Drosophila as a model. This system comprises cells with the same function yet with notably different ploidy status, namely diploid progenitors and their polyploid larval counterparts, the latter destined to die during metamorphosis. An association was identified between polyploidy and autophagy and higher endoreplication status was found to correlate with elevated autophagy. Finally, it is reported that tissue histolysis in the trachea during Drosophila metamorphosis is mediated by autophagy, which triggers the apoptosis of polyploid cells.

Barrio, L., Gaspar, A. E., Muzzopappa, M., Ghosh, K., Romao, D., Clemente-Ruiz, M., Milan, M. (2023). Chromosomal instability-induced cell invasion through caspase-driven DNA damage. Curr Biol, 33(20):4446-4457.e4445 PubMed ID: 37751744
Summary:
Chromosomal instability (CIN), an increased rate of changes in chromosome structure and number, is observed in most sporadic human carcinomas with high metastatic activity. This study used a Drosophila epithelial model to show that DNA damage, as a result of the production of lagging chromosomes during mitosis and aneuploidy-induced replicative stress, contributes to CIN-induced invasiveness. A sub-lethal role of effector caspases in invasiveness was unraveled by enhancing CIN-induced DNA damage and identify the JAK/STAT signaling pathway as an activator of apoptotic caspases through transcriptional induction of pro-apoptotic genes. Evidence is provided that an autocrine feedforward amplification loop mediated by Upd3-a cytokine with homology to interleukin-6 and a ligand of the JAK/STAT signaling pathway-contributes to amplifying the activation levels of the apoptotic pathway in migrating cells, thus promoting CIN-induced invasiveness. This work sheds new light on the chromosome-signature-independent effects of CIN in metastasis.

Monday, February 5th - Splicing, RNAs, and RNAi

Wang, Y., Li, H., Liu, X., Gao, L., Fan, Y., Zhu, K. Y. and Zhang, J. (2023). Three alternative splicing variants of Loquacious play different roles in miRNA- and siRNA-mediated RNAi pathways in Locusta migratoria. RNA Biol 20(1): 323-333. PubMed ID: 37310197
Summary:
RNA interference (RNAi) is a specific post-transcriptional gene-silencing phenomenon, which plays an important role in the regulation of gene expression and the protection from transposable elements in eukaryotic organisms. In Drosophila melanogaster, RNAi can be induced by microRNA (miRNA), endogenous small interfering RNA (siRNA), or exogenous siRNA. However, the biogenesis of miRNA and siRNA in these RNAi pathways is aided by the double-stranded RNA binding proteins (dsRBPs) Loquacious (Loqs)-PB, Loqs-PD or R2D2. This studyidentified three alternative splicing variants of Loqs, namely Loqs-PA, -PB, and -PC in the orthopteran Locusta migratoria. in vitro and in vivo experiments were performed to study the roles of the three Loqs variants in the miRNA- and siRNA-mediated RNAi pathways. The results show that Loqs-PB assists the binding of pre-miRNA to Dicer-1 to lead to the cleavage of pre-miRNA to yield matured miRNA in the miRNA-mediated RNAi pathway. In contrast, different Loqs proteins participate in different siRNA-mediated RNAi pathways. In exogenous siRNA-mediated RNAi pathway, binding of Loqs-PA or LmLoqs-PB to exogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2, whereas in endogenous siRNA-mediated RNAi pathway, binding of Loqs-PB or Loqs-PC to endogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2. These findings provide new insights into the functional importance of different Loqs proteins derived from alternative splicing variants of Loqs in achieving high RNAi efficiency in different RNAi pathways in insects.

Yadav, A. K., Butler, C., Yamamoto, A., Patil, A. A., Lloyd, A. L. and Scott, M. J. (2023). CRISPR/Cas9-based split homing gene drive targeting doublesex for population suppression of the global fruit pest Drosophila suzukii. Proc Natl Acad Sci U S A 120(25): e2301525120. PubMed ID: 37307469
Summary:
Genetic-based methods offer environmentally friendly species-specific approaches for control of insect pests. One method, CRISPR homing gene drive that target genes essential for development, could provide very efficient and cost-effective control. While significant progress has been made in developing homing gene drives for mosquito disease vectors, little progress has been made with agricultural insect pests. This study reports the development and evaluation of split homing drives that target the doublesex (dsx) gene in Drosophila suzukii, an invasive pest of soft-skinned fruits. The drive component, consisting of dsx single guide RNA and DsRed genes, was introduced into the female-specific exon of dsx, which is essential for function in females but not males. However, in most strains, hemizygous females were sterile and produced the male dsx transcript. With a modified homing drive that included an optimal splice acceptor site, hemizygous females from each of the four independent lines were fertile. High transmission rates of the DsRed gene (94 to 99%) were observed with a line that expressed Cas9 with two nuclear localization sequences from the D. suzukii nanos promoter. Mutant alleles of dsx with small in-frame deletions near the Cas9 cut site were not functional and thus would not provide resistance to drive. Finally, mathematical modeling showed that the strains could be used for suppression of lab cage populations of D. suzukii with repeated releases at relatively low release ratios (1:4). These results indicate that the split CRISPR homing gene drive strains could potentially provide an effective means for control of D. suzukii populations.

Warden, M. S., DeRose, E. F., Tamayo, J. V., Mueller, G. A., Gavis, E. R. and Hall, T. M. T. (2023). The translational repressor Glorund uses interchangeable RNA recognition domains to recognize Drosophila nanos. Nucleic Acids Res. PubMed ID: 37427795
Summary:
The Drosophila melanogaster protein Glorund (Glo) represses nanos (nos) translation and uses its quasi-RNA recognition motifs (qRRMs) to recognize both G-tract and structured UA-rich motifs within the nos translational control element (TCE). It has been shown previously that each of the three qRRMs is multifunctional, capable of binding to G-tract and UA-rich motifs, yet if and how the qRRMs combine to recognize the nos TCE remained unclear. This study determined solution structures of a nos TCEI_III RNA containing the G-tract and UA-rich motifs. The RNA structure demonstrated that a single qRRM is physically incapable of recognizing both RNA elements simultaneously. In vivo experiments further indicated that any two qRRMs are sufficient to repress nos translation. interactions of Glo qRRMs were probed with TCEI_III RNA using NMR paramagnetic relaxation experiments. The in vitro and in vivo data support a model whereby tandem Glo qRRMs are indeed multifunctional and interchangeable for recognition of TCE G-tract or UA-rich motifs. This study illustrates how multiple RNA recognition modules within an RNA-binding protein may combine to diversify the RNAs that are recognized and regulated.

Zhai, R., Ruan, K., Perez, G. F., Kubat, M., Liu, J., Hofacker, I. and Wuchty, S. (2023). MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS): a global mechanism for the regulation of alternative splicing. Res Sq. PubMed ID: 37546804
Summary:
While RNA secondary structures are critical to regulate alternative splicing of long-range pre-mRNA, the factors that modulate RNA structure and interfere with the recognition of the splice sites are largely unknown. Previously, a small, non-coding microRNA was identified that sufficiently affects stable stem structure formation of Nmnat pre-mRNA to regulate the outcomes of alternative splicing. However, the fundamental question remains whether such microRNA-mediated interference with RNA secondary structures is a global molecular mechanism for regulating mRNA splicing. A bioinformatic pipeline was designed and refined to predict candidate microRNAs that potentially interfere with pre-mRNA stem-loop structures, and splicing predictions were experimentally verified of three different long-range pre-mRNAs in the Drosophila model system. Specifically, it was observed that microRNAs can either disrupt or stabilize stem-loop structures to influence splicing outcomes. Fhiz study suggests that MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing, increases the repertoire of microRNA function and further indicates cellular complexity of post-transcriptional regulation.

Lee, S., Kim, N., Jang, D., Kim, H. K., Kim, J., Jeon, J. W., Lim, D. H. (2023). Ecdysone-induced microRNA miR-276a-3p controls developmental growth by targeting the insulin-like receptor in Drosophila. Insect Mol Biol, 32(6):703-715 PubMed ID: 37702106
Summary:
Animal growth is controlled by a variety of external and internal factors during development. The steroid hormone ecdysone plays a critical role in insect development by regulating the expression of various genes. In this study, it was found that fat body-specific expression of miR-276a, an ecdysone-responsive microRNA (miRNA), led to a decrease in the total mass of the larval fat body, resulting in significant growth reduction in Drosophila. Changes in miR-276a expression also affected the proliferation of Drosophila S2 cells. Furthermore, it was found that the insulin-like receptor (InR) is a biologically relevant target gene regulated by miR-276a-3p. In addition, its was found that miR-276a-3p is upregulated by the canonical ecdysone signalling pathway involving the ecdysone receptor and broad complex. A reduction in cell proliferation caused by ecdysone was compromised by blocking miR-276a-3p activity. Thus, these results suggest that miR-276a-3p is involved in ecdysone-mediated growth reduction by controlling InR expression in the insulin signalling pathway.

Wilinski, D. and Dus, M. (2023). N(6)-adenosine methylation controls the translation of insulin mRNA. Nat Struct Mol Biol. PubMed ID: 37488356
Summary:
Control of insulin mRNA translation is crucial for energy homeostasis, but the mechanisms remain largely unknown. This study discovered that insulin mRNAs across invertebrates, vertebrates and mammals feature the modified base N(6)-methyladenosine (m(6)A). In flies, this RNA modification enhances insulin mRNA translation by promoting the association of the transcript with polysomes. Depleting m(6)A in Drosophila melanogaster insulin 2 mRNA (dilp2) directly through specific 3' untranslated region (UTR) mutations, or indirectly by mutating the m(6)A writer Mettl3, decreases dilp2 protein production, leading to aberrant energy homeostasis and diabetic-like phenotypes. Together, these findings reveal adenosine mRNA methylation as a key regulator of insulin protein synthesis with notable implications for energy balance and metabolic disease.

Friday, February 2nd - Adult Neural Development and Function

Garcia-Vaquero, M. L., Heim, M., Flix, B., Pereira, M., Palin, L., Marques, T. M., Pinto, F. R., de Las Rivas, J., Voigt, A., Besse, F., Gama-Carvalho, M. (2023). Analysis of asymptomatic Drosophila models for ALS and SMA reveals convergent impact on functional protein complexes linked to neuro-muscular degeneration. BMC Genomics, 24(1):576 PubMed ID: 37759179
Summary:
Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 (TBPH) and FUS. There is currently no model to explain the resulting motor neuron dysfunction. This study generated parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, a computational approach was devised based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown. Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through the integrative approach it was possible to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes. These results show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings.

Upadhyay, A., Chhangani, D., Rao, N. R., Kofler, J., Vassar, R., Rincon-Limas, D. E., Savas, J. N. (2023). Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity. Molecular neurodegeneration, 18(1):61 PubMed ID: 37710351
Summary:
The accumulation of amyloid beta (β) peptides in fibrils is prerequisite for Alzheimer's disease (AD). understanding of the proteins that promote Aβ (see Drosophila jmuller/appl1.htm) fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. To investigate how amyloid fibrils form and cause neurotoxicity in AD brain, this study developed a robust biochemical strategy. The success of these purifications was benchmarked using electron microscopy, amyloid dyes, and a large panel of Aβ immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed comparison oc amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic Aβ42 flies, and Aβ42 seeded cultured neurons. Amyloid fibrils were found to be primarily composed by Aβ42 and unexpectedly harbor Aβ38 but generally lacked Aβ40 peptides. Multidimensional quantitative proteomics allowed redefinition of the fibril proteome by identifying 20 new amyloid-associated proteins. Notably, 57 previously reported plaque-associated proteins were identified. A panel of these proteins was validated as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 was found to be tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, a transgenic Aβ42 fly model was used to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Here, it was possible to functionally validate 20 genes as modifiers of Aβ42 toxicity in vivo. These discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.

Stewart, T. M., Foley, J. R., Holbert, C. E., Khomutov, M., Rastkari, N., Tao, X., Khomutov, A. R., Zhai, R. G., Casero, R. A. (2023). Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome. EMBO molecular medicine, 15(11):e17833 PubMed ID: 37702369
Summary:
Snyder-Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. The current studies aimed to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. The repurposing of 2-difluoromethylornithine (DFMO), an FDA-approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells is reported. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.

Luedke, K. P., Yoshino, J., Yin, C., Jiang, N., Huang, J. M., Huynh, K., Parrish, J. Z. (2023). Dendrite intercalation between epidermal cells tunes nociceptor sensitivity to mechanical stimuli in Drosophila larvae. bioRxiv PubMed ID: 37745567
Summary:
An animal's skin provides a first point of contact with the sensory environment, including noxious cues that elicit protective behavioral responses. Nociceptive somatosensory neurons densely innervate and intimately interact with epidermal cells to receive these cues. This study identified a role for dendrite intercalation between epidermal cells in tuning sensitivity of Drosophila larvae to noxious mechanical stimuli. In wild-type larvae, dendrites of nociceptive class IV da neurons intercalate between epidermal cells at apodemes, which function as body wall muscle attachment sites. From a genetic screen miR-14 was identified as a regulator of dendrite positioning in the epidermis: miR-14 is expressed broadly in the epidermis but not in apodemes, and miR-14 inactivation leads to excessive apical dendrite intercalation between epidermal cells. miR-14 regulates expression and distribution of the epidermal Innexins ogre and Inx2 and that these epidermal gap junction proteins restrict epidermal dendrite intercalation. Finally, it was found that altering the extent of epidermal dendrite intercalation had corresponding effects on nociception: increasing epidermal intercalation sensitized larvae to noxious mechanical inputs and increased mechanically evoked calcium responses in nociceptive neurons, whereas reducing epidermal dendrite intercalation had the opposite effects. Altogether, these studies identify epidermal dendrite intercalation as a mechanism for mechanical coupling of nociceptive neurons to the epidermis, with nociceptive sensitivity tuned by the extent of intercalation.

Zhu, B. and Liu, S. (2023). Preservation of ∼12-hour ultradian rhythms of gene expression of mRNA and protein metabolism in the absence of canonical circadian clock. bioRxiv PubMed ID: 37205336
Summary:
Besides the ∼24-hour circadian rhythms, ∼12-hour ultradian rhythms of gene expression, metabolism and behaviors exist in animals ranging from crustaceans to mammals. Three major hypotheses were proposed on the origin and mechanisms of regulation of ∼12-hour rhythms, namely that they are not cell-autonomous and controlled by a combination of the circadian clock and environmental cues, that they are regulated by two anti-phase circadian transcriptional factors in a cell-autonomous manner, or that they are established by a cell-autonomous ∼12-hour oscillator. To distinguish among these possibilities, a post-hoc analysis was performed of two high temporal resolution transcriptome dataset in animals and cells lacking the canonical circadian clock. In both the liver of BMAL1 knockout mice and Drosophila S2 cells, robust and prevalent ∼12-hour rhythms of gene expression were observed, enriched in fundamental processes of mRNA and protein metabolism that show large convergence with those identified in wild-type mice liver. Bioinformatics analysis further predicted ELF1 (Drosophila Ecdysone-induced protein 74EF) and ATF6B (Drosophila Atf6) as putative transcription factors regulating the ∼12-hour rhythms of gene expression independently of the circadian clock in both fly and mice. These findings provide additional evidence to support the existence of an evolutionarily conserved 12-hour oscillator that controls ∼12-hour rhythms of gene expression of protein and mRNA metabolism in multiple species.

Zsindely, N., Nagy, G., Siagi, F., Farkas, A. and Bodai, L. (2023). Dysregulated miRNA and mRNA Expression Affect Overlapping Pathways in a Huntington's Disease Model. Int J Mol Sci 24(15). PubMed ID: 37569316
Summary:
Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the Huntingtin gene. Transcriptional dysregulation is one of the main cellular processes affected by mutant Huntingtin (mHtt). This study investigated the alterations in miRNA and mRNA expression levels in a Drosophila model of HD by RNA sequencing and assessed the functional effects of misregulated miRNAs in vivo. in head samples of HD flies, the level of 32 miRNAs were found to change significantly; half of these were upregulated, while the other half were downregulated. After comparing miRNA and mRNA expression data, similarities were discovered in the impacted molecular pathways. Additionally, it was observed that the putative targets of almost all dysregulated miRNAs were overrepresented were tested among the upregulated mRNAs. The effects were tested of overexpression of five misregulated miRNAs in the HD model, and it was found that while mir-10 and mir-219 enhanced, mir-137, mir-305, and mir-1010 ameliorated mHtt-induced phenotypes. Based on these results, it is proposed that while altered expression of mir-10, mir-137, and mir-1010 might be part of HD pathology, the upregulation of mir-305 might serve as a compensatory mechanism as a response to mHtt-induced transcriptional dysregulation.

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