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Friday, December 29th, 2023 - RNA and Transposons

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Ruan, K., Perez, G. F., Liu, J., Kubat, M., Hofacker, I., Wuchty, S. and Zhai, R. G. (2023). MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS): a global mechanism for the regulation of alternative splicing. bioRxiv. PubMed ID: 37425843
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. This study designed and refined a bioinformatic pipeline to predict candidate microRNAs that potentially interfere with pre-mRNA stem-loop structures, and experimentally verified splicing predictions 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. This 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. It is concluded that MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing.
Perrotta, M. M., Lucibelli, F., Mazzucchiello, S. M., Fucci, N., Hay Mele, B., Giordano, E., Salvemini, M., Ruggiero, A., Vitagliano, L., Aceto, S. and Saccone, G. (2023). Female Sex Determination Factors in Ceratitis capitata: Molecular and Structural Basis of TRA and TRA2 Recognition. Insects 14(7). PubMed ID: 37504611
In the model system for genetics, Drosophila melanogaster, sexual differentiation and male courtship behavior are controlled by sex-specific splicing of doublesex (dsx) and fruitless (fru). In vitro and in vivo studies showed that female-specific Transformer (TRA) and the non-sex-specific Transformer 2 (TRA2) splicing factors interact, forming a complex promoting dsx and fru female-specific splicing. TRA/TRA2 complex binds to 13 nt long sequence repeats in their pre-mRNAs. In the Mediterranean fruitfly Ceratitis capitata (Medfly), a major agricultural pest, which shares with Drosophila a ~120 million years old ancestor, Cctra and Cctra2 genes seem to promote female-specific splicing of Ccdsx and Ccfru, which contain conserved TRA/TRA2 binding repeats. Unlike Drosophila tra, Cctra autoregulates its female-specific splicing through these putative regulatory repeats. In Ceratitis, a yeast two-hybrid assay shows that CcTRA interacts with CcTRA2, despite its high amino acid divergence compared to Drosophila TRA. Interestingly, CcTRA2 interacts with itself, as also observed for Drosophila TRA2. A three-dimensional model was generated of the complex formed by CcTRA and CcTRA2 using predictive approaches based on Artificial Intelligence. This structure also identified an evolutionary and highly conserved putative TRA2 recognition motif in the TRA sequence. The Y2H approach, combined with powerful predictive tools of three-dimensional protein structures, could use helpful also in this and other insect species to understand the potential links between different upstream proteins acting as primary sex-determining signals and the conserved TRA and TRA2 transducers.
Sokolova, O., Morgunova, V., Sizova, T. V., Komarov, P. A., Olenkina, O. M., Babaev, D. S., Mikhaleva, E. A., Kwon, D. A., Erokhin, M. and Kalmykova, A. (2023). The insulator BEAF32 controls the spatial-temporal expression profile of the telomeric retrotransposon TART in the Drosophila germline. Development 150(12). PubMed ID: 37317951
Insulators are architectural elements implicated in the organization of higher-order chromatin structures and transcriptional regulation. However, it is still unknown how insulators contribute to Drosophila telomere maintenance. Although the Drosophila telomeric retrotransposons HeT-A and TART occupy a common genomic niche, they are regulated independently. TART elements are believed to provide reverse transcriptase activity, whereas HeT-A transcripts serve as a template for telomere elongation. Thia study reporta that insulator complexes associate with TART and contribute to its transcriptional regulation in the Drosophila germline. Chromatin immunoprecipitation revealed that the insulator complex containing BEAF32, Chriz, and DREF proteins occupy the TART promoter. BEAF32 depletion causes derepression and chromatin changes at TART in ovaries. Moreover, an expansion of TART copy number was observed in the genome of the BEAF32 mutant strain. BEAF32 localizes between the TART enhancer and promoter, suggesting that it blocks enhancer-promoter interactions. This study found that TART repression is released in the germ cysts as a result of the normal reduction of BEAF32 expression at this developmental stage. It is suggested that coordinated expression of telomeric repeats during development underlies telomere elongation control.
Titus, M. B., Chang, A. W., Popitsch, N., Ebmeier, C. C., Bono, J. M. and Olesnicky, E. C. (2023). The identification of protein and RNA interactors of the splicing factor Caper in the adult Drosophila nervous system. Front Mol Neurosci 16: 1114857. PubMed ID: 37435576
Post-transcriptional gene regulation is a fundamental mechanism that helps regulate the development and healthy aging of the nervous system. Mutations that disrupt the function of RNA-binding proteins (RBPs), which regulate post-transcriptional gene regulation, have increasingly been implicated in neurological disorders including amyotrophic lateral sclerosis, Fragile X Syndrome, and spinal muscular atrophy. Interestingly, although the majority of RBPs are expressed widely within diverse tissue types, the nervous system is often particularly sensitive to their dysfunction. It is therefore critical to elucidate how aberrant RNA regulation that results from the dysfunction of ubiquitously expressed RBPs leads to tissue specific pathologies that underlie neurological diseases. The highly conserved RBP and alternative splicing factor Caper is widely expressed throughout development and is required for the development of Drosophila sensory and motor neurons. Furthermore, caper dysfunction results in larval and adult locomotor deficits. Nonetheless, little is known about which proteins interact with Caper, and which RNAs are regulated by Caper. This study identified proteins that interact with Caper in both neural and muscle tissue, along with neural specific Caper target RNAs. Furthermore, it was shown that a subset of these Caper-interacting proteins and RNAs genetically interact with caper to regulate Drosophila gravitaxis behavior.
Nelson, J. O., Slicko, A. and Yamashita, Y. M. (2023). The retrotransposon R2 maintains Drosophila ribosomal DNA repeats. Proc Natl Acad Sci U S A 120(23): e2221613120. PubMed ID: 37252996
Ribosomal DNA (rDNA) loci contain hundreds of tandemly repeated copies of ribosomal RNA genes needed to support cellular viability. This repetitiveness makes it highly susceptible to copy number (CN) loss due to intrachromatid recombination between rDNA copies, threatening multigenerational maintenance of rDNA. How this threat is counteracted to avoid extinction of the lineage has remained unclear. This study shows that the rDNA-specific retrotransposon R2 is essential for restorative rDNA CN expansion to maintain rDNA loci in the Drosophila male germline. The depletion of R2 led to defective rDNA CN maintenance, causing a decline in fecundity over generations and eventual extinction. Double-stranded DNA breaks created by the R2 endonuclease, a feature of R2's rDNA-specific retrotransposition, initiate the process of rDNA CN recovery, which relies on homology-dependent repair of the DNA break at rDNA copies. This study reveals that an active retrotransposon provides an essential function for its host, contrary to transposable elements' reputation as entirely selfish. These findings suggest that benefiting host fitness can be an effective selective advantage for transposable elements to offset their threat to the host, which may contribute to retrotransposons' widespread success throughout taxa.
Ray, M., Conard, A. M., Urban, J., Mahableshwarkar, P., Aguilera, J., Huang, A., Vaidyanathan, S. and Larschan, E. (2023). Sex-specific splicing occurs genome-wide during early Drosophila embryogenesis. Elife 12. PubMed ID: 37466240
Sex-specific splicing is an essential process that regulates sex determination and drives sexual dimorphism. Yet, how early in development widespread sex-specific transcript diversity occurs was unknown because it had yet to be studied at the genome-wide level. This study used the powerful Drosophila model to show that widespread sex-specific transcript diversity occurs early in development, concurrent with zygotic genome activation. A new pipeline is presented called time2Splice to quantify changes in alternative splicing over time. Furthermore, it was determined that one of the consequences of losing an essential maternally deposited pioneer factor called CLAMP (chromatin-linked adapter for MSL proteins) is altered sex-specific splicing of genes involved in diverse biological processes that drive development. Overall, this study shows that sex-specific differences in transcript diversity exist even at the earliest stages of development.

Thursday, December 28th - Evolution

Shen, R., Wenzel, M., Messer, P. W. and Aquadro, C. F. (2023). Evolution under a model of functionally buffered deleterious mutations can lead to positive selection in protein-coding genes. Evolution. PubMed ID: 37464886
Selective pressures on DNA sequences often result in departures from neutral evolution that can be captured by the McDonald-Kreitman (MK) test. However, the nature of such selective forces often remains unknown to experimentalists. Amino acid fixations driven by natural selection in protein coding genes are commonly associated with a genetic arms race or changing biological purposes, leading to proteins with new functionality. This study evaluated the expectations of population genetic patterns under a buffering mechanism driving selective amino acids to fixation, which is motivated by an observed phenotypic rescue of otherwise deleterious nonsynonymous substitutions at bag of marbles (bam) and Sex lethal (Sxl) in Drosophila melanogaster. These two genes were shown to experience strong episodic bursts of natural selection potentially due to infections of the endosymbiotic bacteria Wolbachia observed among multiple Drosophila species. Using simulations to implement and evaluate the evolutionary dynamics of a Wolbachia buffering model, it was demonstrated that selectively fixed amino acid replacements will occur, but that the proportion of adaptive amino acid fixations and the statistical power of the MK test to detect the departure from an equilibrium neutral model are both significantly lower than seen for an arms race/change-in-function model that favors proteins with diversified amino acids. The observed selection pattern at bam in a natural population of D. melanogaster was found to be more consistent with an arms race model than with the buffering model.
Srivastav, S., Feschotte, C. and Clark, A. G. (2023). Rapid evolution of piRNA clusters in the Drosophila melanogaster ovary. bioRxiv. PubMed ID: 37214865
Animal genomes are parasitized by a horde of transposable elements (TEs) whose mutagenic activity can have catastrophic consequences. The piRNA pathway is a conserved mechanism to repress TE activity in the germline via a specialized class of small RNAs associated with effector Piwi proteins called piwi-associated RNAs (piRNAs). piRNAs are produced from discrete genomic regions called piRNA clusters (piCs). To investigate piC evolution, a population genomics approach was used to compare piC activity and sequence composition across 8 geographically distant strains of D. melanogaster. Extensive annotations were performed of ovary piCs and TE content in each strain, and predictions of two proposed models of piC evolution were tested. The 'de novo' model posits that individual TE insertions can spontaneously attain the status of a small piC to generate piRNAs silencing the entire TE family. The 'trap' model envisions large and evolutionary stable genomic clusters where TEs tend to accumulate and serves as a long-term "memory" of ancient TE invasions and produce a great variety of piRNAs protecting against related TEs entering the genome. It remains unclear which model best describes the evolution of piCs. This analysis uncovers extensive variation in piC activity across strains and signatures of rapid birth and death of piCs in natural populations. Most TE families inferred to be recently or currently active show an enrichment of strain-specific insertions into large piCs, consistent with the trap model. By contrast, only a small subset of active LTR retrotransposon families is enriched for the formation of strain-specific piCs, suggesting that these families have an inherent proclivity to form de novo piCs. Thus, these findings support aspects of both 'de novo' and 'trap' models of piC evolution. It is proposed that these two models represent two extreme stages along an evolutionary continuum, which begins with the emergence of piCs de novo from a few specific LTR retrotransposon insertions that subsequently expand by accretion of other TE insertions during evolution to form larger 'trap' clusters. This study shows that piCs are evolutionarily labile and that TEs themselves are the major force driving the formation and evolution of piCs.
Thorholludottir, D. A. V., Nolte, V. and Schlotterer, C. (2023). Temperature driven gene expression evolution in natural and laboratory populations highlights the crucial role of correlated fitness effects for polygenic adaptation. Evolution. PubMed ID: 37455661
The influence of pleiotropy on adaptive responses is a highly controversial topic, with limited empirical evidence available. Recognizing the pivotal role of the correlation of fitness effects, an experiment was designed to compare the adaptive gene expression evolution of natural and experimental populations. To test this,the evolution of gene expression was studied in response to temperature in two Drosophila species on a natural temperature cline in North America and replicated populations evolving in hot and cold temperature regimes. If fitness effects of affected traits are independent, pleiotropy is expected to constrain the adaptive response in both settings. However, when fitness effects are more correlated in natural populations, adaptation in the wild will be facilitated by pleiotropy. Remarkably, evidence was found for both predicted effects. In both settings, genes with strong pleiotropic effects contribute less to adaptation, indicating that the majority of fitness effects are not correlated. In addition, this study also discovered that genes involved in adaptation exhibited more pleiotropic effects in natural populations. It is proposed that this pattern can be explained by a stronger correlation of fitness effects in nature. More insights into the dual role of pleiotropy will be crucial for the understanding of polygenic adaptation.
Scott, J. G., Norris, R. H., Mertz, R. W., Dressel, A. E. and Loeb, G. (2023). Selection and characterization of spinetoram resistance in field collected Drosophila melanogaster. Pestic Biochem Physiol 194: 105508. PubMed ID: 37532361
Insecticides are commonly employed in vineyards to control vinegar flies and limit sour rot disease. Widespread resistance to available insecticides is having a negative impact on managing Drosophila melanogaster populations, rendering control of sour rot more difficult. An insecticide registered for use in vineyards to which resistance is not yet widespread (at least in New York and Missouri) is spinetoram. Spinetoram targets the nicotinic acetylcholine receptor α6, and mutations in α6 have been associated with resistance in some insects. The goals of this study were to select for a spinetoram resistant strain of D. melanogaster (starting with field collected populations), characterize the resistance, and identify the mutation responsible. After five selections a strain (SpinR) with >190-fold resistance was obtained. Resistance could not be overcome by insecticide synergists, suggesting an altered target site was involved. The α6 allele from the spinetoram resistant strain was cloned and sequenced and Ωa mutation was cloned and sequenced causing a glycine to alanine change at amino acid 301 (equivalent position to the G275E mutation found in some spinosad/spinetoram resistant insects). This mutation was found at low levels in field populations, but increased with each selection until it became homozygous in SpinR. How the identification of the spinetoram resistance mutation can be used for resistance management is discussed.
Titova, A. V., Kau, B. E., Tibor, S., Mach, J., Vo-Doan, T. T., Wittlinger, M. and Straw, A. D. (2023). Displacement experiments provide evidence for path integration in Drosophila. J Exp Biol 226(12). PubMed ID: 37226998
Like many other animals, insects are capable of returning to previously visited locations using path integration, which is a memory of travelled direction and distance. Recent studies suggest that Drosophila can also use path integration to return to a food reward. However, the existing experimental evidence for path integration in Drosophila has a potential confound: pheromones deposited at the site of reward might enable flies to find previously rewarding locations even without memory. This study shows that pheromones can indeed cause naive flies to accumulate where previous flies had been rewarded in a navigation task. Therefore, an experiment was designed to determine if flies can use path integration memory despite potential pheromonal cues by displacing the flies shortly after an optogenetic reward. It was found that rewarded flies returned to the location predicted by a memory-based model. Several analyses are consistent with path integration as the mechanism by which flies returned to the reward. It is concluded that although pheromones are often important in fly navigation and must be carefully controlled for in future experiments, Drosophila may indeed be capable of performing path integration.
Singh, A., Hasan, A. and Agrawal, A. F. (2023). An investigation of the sex-specific genetic architecture of fitness in Drosophila melanogaster. Evolution. PubMed ID: 37329263
In dioecious populations, the sexes employ divergent reproductive strategies to maximize fitness and, as a result, genetic variants can affect fitness differently in males and females. Moreover, recent studies have highlighted an important role of the mating environment in shaping the strength and direction of sex-specific selection. This study measured adult fitness for each sex of 357 lines from the Drosophila Synthetic Population Resource (DSPR) in two different mating environments. The data was analyzed using three different approaches to gain insight into the sex-specific genetic architecture for fitness: classical quantitative genetics, genomic associations, and a mutational burden approach. The quantitative genetics analysis finds that, on average segregating genetic variation in this population has concordant fitness effects both across the sexes and across mating environments. Specific genomic regions with strong associations with either sexually antagonistic (SA) or sexually concordant (SC) fitness effects were not found, yet there is modest evidence of an excess of genomic regions with weak associations, both with SA and SC fitness effects. This examination of mutational burden indicates stronger selection against indels and loss-of-function variants in females than males.

Wednesday, December 27th - Adult Neural Structure, Development, and Function

Vijayan, V., Wang, F., Wang, K., Chakravorty, A., Adachi, A., Akhlaghpour, H., Dickson, B. J. and Maimon, G. (2023). A rise-to-threshold process for a relative-value decision. Nature 619(7970): 563-571. PubMed ID: 37407812
Whereas progress has been made in the identification of neural signals related to rapid, cued decisions, less is known about how brains guide and terminate more ethologically relevant decisions in which an animal's own behaviour governs the options experienced over minutes. Drosophila search for many seconds to minutes for egg-laying sites with high relative value and have neurons, called oviDNs, whose activity fulfills necessity and sufficiency criteria for initiating the egg-deposition motor programme This study shows that oviDNs express a calcium signal that (1) dips when an egg is internally prepared (ovulated), (2) drifts up and down over seconds to minutes-in a manner influenced by the relative value of substrates-as a fly determines whether to lay an egg and (3) reaches a consistent peak level just before the abdomen bend for egg deposition. This signal is apparent in the cell bodies of oviDNs in the brain and it probably reflects a behaviourally relevant rise-to-threshold process in the ventral nerve cord, where the synaptic terminals of oviDNs are located and where their output can influence behaviour. Perturbational evidence is provided that the egg-deposition motor programme is initiated once this process hits a threshold and that subthreshold variation in this process regulates the time spent considering options and, ultimately, the choice taken. Finally, a small recurrent circuit was found that feeds into oviDNs, and that activity in each of its constituent cell types was shown to be required for laying an egg. These results argue that a rise-to-threshold process regulates a relative-value, self-paced decision and provide initial insight into the underlying circuit mechanism for building this process.
Shiu, P. K., Sterne, G. R., Spiller, N., Franconville, R., Sandoval, A., Zhou, J., Simha, N., Kang, C. H., Yu, S., Kim, J. S., Dorkenwald, S., Matsliah, A., Schlegel, P., Yu, S. C., McKellar, C. E., Sterling, A., Costa, M., Eichler, K., Jefferis, G., Murthy, M., Bates, A. S., Eckstein, N., Funke, J., Bidaye, S. S., Hampel, S., Seeds, A. M. and Scott, K. (2023). A leaky integrate-and-fire computational model based on the connectome of the entire adult Drosophila brain reveals insights into sensorimotor processing. bioRxiv. PubMed ID: 37205514
This study describes a leaky integrate-and-fire computational model of the entire Drosophila brain, based on neural connectivity and neurotransmitter identity, to study circuit properties of feeding and grooming behaviors. Activation of sugar-sensing or water-sensing gustatory neurons in the computational model accurately predicts neurons that respond to tastes and are required for feeding initiation. Computational activation of neurons in the feeding region of the Drosophila brain predicts those that elicit motor neuron firing, a testable hypothesis that this study validated by optogenetic activation and behavioral studies. Moreover, computational activation of different classes of gustatory neurons makes accurate predictions of how multiple taste modalities interact, providing circuit-level insight into aversive and appetitive taste processing. This computational model predicts that the sugar and water pathways form a partially shared appetitive feeding initiation pathway, which calcium imaging and behavioral experiments confirmed. Additionally, this model was applied to mechanosensory circuits and found that computational activation of mechanosensory neurons predicts activation of a small set of neurons comprising the antennal grooming circuit that do not overlap with gustatory circuits, and accurately describes the circuit response upon activation of different mechanosensory subtypes. These results demonstrate that modeling brain circuits purely from connectivity and predicted neurotransmitter identity generates experimentally testable hypotheses and can accurately describe complete sensorimotor transformations.
Stone, M. C., Mauger, A. S. and Rolls, M. M. (2023). Ciliated sensory neurons can regenerate axons after complete axon removal. J Exp Biol 226(12). PubMed ID: 37212026
Axon regeneration helps maintain lifelong function of neurons in many animals. Depending on the site of injury, new axons can grow either from the axon stump (after distal injury) or from the tip of a dendrite (after proximal injury). However, some neuron types do not have dendrites to be converted to a regenerating axon after proximal injury. For example, many sensory neurons receive information from a specialized sensory cilium rather than a branched dendrite arbor. It was hypothesized that the lack of traditional dendrites would limit the ability of ciliated sensory neurons to respond to proximal axon injury. This hypothesis was tested by performing laser microsurgery on ciliated lch1 neurons in Drosophila larvae and tracking cells over time. These cells survived proximal axon injury as well as distal axon injury, and, like many other neurons, initiated growth from the axon stump after distal injury. After proximal injury, neurites regrew in a surprisingly flexible manner. Most cells initiated outgrowth directly from the cell body, but neurite growth could also emerge from the short axon stump or base of the cilium. New neurites were often branched. Although outgrowth after proximal axotomy was variable, it depended on the core DLK axon injury signaling pathway. Moreover, each cell had at least one new neurite specified as an axon based on microtubule polarity and accumulation of the endoplasmic reticulum. It is concluded that ciliated sensory neurons are not intrinsically limited in their ability to grow a new axon after proximal axon removal.
Tatarko, A. R., Leonard, A. S. and Mathew, D. (2023). A neonicotinoid pesticide alters Drosophila olfactory processing. Sci Rep 13(1): 10606. PubMed ID: 37391495
Neonicotinoid pesticides are well-known for their sublethal effects on insect behavior and physiology. Recent work suggests neonicotinoids can impair insect olfactory processing, with potential downstream effects on behavior and possibly survival. However, it is unclear whether impairment occurs during peripheral olfactory detection, during information processing in central brain regions, or in both contexts. This study used Drosophila melanogaster to explore the potential for neonicotinoids to disrupt olfaction by conducting electrophysiological analyses of single neurons and whole antennae of flies exposed to varying concentrations of the neonicotinoid imidacloprid (IMD) that were shown to cause relative differences in fly survival. The results demonstrated that IMD exposure significantly reduced the activity of a single focal olfactory neuron and delayed the return to baseline activity of the whole antenna. To determine if IMD also impacts olfactory-guided behavior, flies' relative preference for odor sources varying in ethanol content were compared. Flies exposed to IMD had a greater relative preference for ethanol-laced pineapple juice than control flies, demonstrating that neuronal shifts induced by IMD that were observed are associated with changes in relative preference. Given the interest in the sensory impacts of agrochemical exposure on wild insect behavior and physiology, this study highlights the potential of Drosophila as a tractable model for investigating the effects of pesticides at scales ranging from single-neuron physiology to olfactory-guided behavior.
Shekhar, S., Moehlman, A. T., Park, B., Ewnetu, M., Tracy, C., Titos, I., Pawlowski, K., Tagliabracci, V. S. and Kramer, H. (2023). Allnighter pseudokinase-mediated feedback links proteostasis and sleep in Drosophila. Nat Commun 14(1): 2932. PubMed ID: 37217484
In nervous systems, retrograde signals are key for organizing circuit activity and maintaining neuronal homeostasis. This study identified the conserved Allnighter (Aln) pseudokinase as a cell non-autonomous regulator of proteostasis responses necessary for normal sleep and structural plasticity of Drosophila photoreceptors. In aln mutants exposed to extended ambient light, proteostasis is dysregulated and photoreceptors develop striking, but reversible, dysmorphology. The aln gene is widely expressed in different neurons, but not photoreceptors. However, secreted Aln protein is retrogradely endocytosed by photoreceptors. Inhibition of photoreceptor synaptic release reduces Aln levels in lamina neurons, consistent with secreted Aln acting in a feedback loop. In addition, aln mutants exhibit reduced night time sleep, providing a molecular link between dysregulated proteostasis and sleep, two characteristics of ageing and neurodegenerative diseases.
Taisz, I., Dona, E., Munch, D., Bailey, S. N., Morris, B. J., Meechan, K. I., Stevens, K. M., Varela-Martinez, I., Gkantia, M., Schlegel, P., Ribeiro, C., Jefferis, G. and Galili, D. S. (2023). Generating parallel representations of position and identity in the olfactory system. Cell 186(12): 2556-2573. PubMed ID: 37236194
In Drosophila, a dedicated olfactory channel senses a male pheromone, cis-vaccenyl acetate (cVA), promoting female courtship while repelling males. This study shows that separate cVA-processing streams extract qualitative and positional information. cVA sensory neurons respond to concentration differences in a 5-mm range around a male. Second-order projection neurons encode the angular position of a male by detecting inter-antennal differences in cVA concentration, which are amplified through contralateral inhibition. At the third circuit layer, 47 cell types were identified with diverse input-output connectivity. One population responds tonically to male flies, a second is tuned to olfactory looming, while a third integrates cVA and taste to coincidentally promote female mating. The separation of olfactory features resembles the mammalian what and where visual streams; together with multisensory integration, this enables behavioral responses appropriate to specific ethological contexts.

Tuesday, December 27th - Behavior

Rohrbach, E. W., Knapp, E. M., Deshpande, S. A. and Krantz, D. E. (2023). Drosophila cells that express octopamine receptors can either inhibit or promote oviposition. bioRxiv. PubMed ID: 37205438
Adrenergic signaling is known to play a critical role in regulating female reproductive processes in both mammals and insects. In Drosophila, the ortholog of noradrenaline, octopamine (Oa), is required for ovulation as well as several other female reproductive processes. Loss of function studies using mutant alleles of receptors, transporters, and biosynthetic enzymes for Oa have led to a model in which disruption of octopaminergic pathways reduces egg laying. However, neither the complete expression pattern in the reproductive tract nor the role of most octopamine receptors in oviposition is known. This study shows that all six known Oa receptors are expressed in peripheral neurons at multiple sites within in the female fly reproductive tract as well as in non-neuronal cells within the sperm storage organs. The complex pattern of Oa receptor expression in the reproductive tract suggests the potential for influencing multiple regulatory pathways, including those known to inhibit egg-laying in unmated flies. Indeed, activation of some neurons that express Oa receptors inhibits oviposition, and neurons that express different subtypes of Oa receptor can affect different stages of egg laying. Stimulation of some Oa receptor expressing neurons (OaRNs) also induces contractions in lateral oviduct muscle and activation of non-neuronal cells in the sperm storage organs by Oa generates OAMB-dependent intracellular calcium release. These results are consistent with a model in which adrenergic pathways play a variety of complex roles in the fly reproductive tract that includes both the stimulation and inhibition of oviposition.
Pantalia, M., Lin, Z., Tener, S. J., Qiao, B., Tang, G., Ulgherait, M., O'Connor, R., Delventhal, R., Volpi, J., Syed, S., Itzhak, N., Canman, J. C., Fernandez, M. P. and Shirasu-Hiza, M. (2023). Drosophila mutants lacking the glial neurotransmitter-modifying enzyme Ebony exhibit low neurotransmitter levels and altered behavior. Sci Rep 13(1): 10411. PubMed ID: 37369755
Inhibitors of enzymes that inactivate amine neurotransmitters (dopamine, serotonin), such as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), are thought to increase neurotransmitter levels and are widely used to treat Parkinson's disease and psychiatric disorders, yet the role of these enzymes in regulating behavior remains unclear. This study investigated the genetic loss of a similar enzyme in the model organism Drosophila melanogaster. Because the enzyme Ebony modifies and inactivates amine neurotransmitters, its loss is assumed to increase neurotransmitter levels, increasing behaviors such as aggression and courtship and decreasing sleep. This study quantitatively confirmed that ebony mutants exhibited increased aggressive behaviors such as boxing but also decreased courtship behaviors and increased sleep. Through tissue-specific knockdown, the role of ebony in these behaviors was found to be specific to glia. Unexpectedly, direct measurement of amine neurotransmitters in ebony brains revealed that their levels were not increased but reduced. Thus, increased aggression is the anomalous behavior for this neurotransmitter profile. It was further found that ebony mutants exhibited increased aggression only when fighting each other, not when fighting wild-type controls. Moreover, fights between ebony mutants were less likely to end with a clear winner than fights between controls or fights between ebony mutants and controls. In ebony vs. control fights, ebony mutants were more likely to win. Together, these results suggest that ebony mutants exhibit prolonged aggressive behavior only in a specific context, with an equally dominant opponent.
Rodrigues, N. R., Macedo, G. E., Martins, I. K., Vieira, P. B., Kich, K. G., Posser, T. and Franco, J. L. (2023). Sleep disturbance induces a modulation of clock gene expression and alters metabolism regulation in Drosophila. Physiol Behav: 114334. PubMed ID: 37595818
Sleep disorders are catching attention worldwide as they can induce dyshomeostasis and health issues in all animals, including humans. Circadian rhythms are biological 24-hour cycles that influence physiology and behavior in all living organisms. Sleep is a crucial resting state for survival and is under the control of circadian rhythms. Studies have shown the influence of sleep on various pathological conditions, including metabolic diseases; however, the biological mechanisms involving the circadian clock, sleep, and metabolism regulation are not well understood. Previous work standardized a sleep disturbance protocol and, observed that short-time sleep deprivation and sleep-pattern alteration induce homeostatic sleep regulation, locomotor deficits, and increase oxidative stress. This study investigated the relationship between these alterations with the circadian clock and energetic metabolism. This study evaluated the expression of the circadian clock and Drosophila insulin-like peptides (DILPs) genes and metabolic markers glucose, triglycerides, and glycogen in fruit flies subjected to short-term sleep disruption protocols. The sleep disturbance altered the expression of clock genes and DILP gene expression, and modulated glucose, triglycerides, and glycogen levels. Moreover, changes were demonstrated in mTor/dFoxo genes, AKT phosphorylation, and dopamine levels in nocturnal light-exposed flies. Thus, these results suggest a connection between clock genes and metabolism disruption as a consequence of sleep disruption, demonstrating the importance of sleep quality in health maintenance.
Silva-Lopez, J., Godoy, P., Jara, L. and Godoy-Herrera, R. (2023). Interaction and integration among behaviors of adult Drosophila in nature. PLoS One 18(7): e0278427. PubMed ID: 37440503
Living in environments whose ecologies vary in periods as short as 24 h is a challenge for animals as Drosophila species that inhabit pear and apple orchards. These orchards have sunny and shady sections. The size and shape of these habitats change daily according to the position of the sun in the sky. Sunny areas are related to dryness and water loss, and shady places have lower temperatures and higher humidity. The presence of heterospecific flies may lead to competition for space and food. In sunny habitats adult Drosophila were not found. In shady sections conspecific groups D. melanogaster, D. simulans, D. immigrans, D. subobscura, and the Chilean endemic D. pavani were found perched on grasses and herbs at 8-10 cm from fruits that had fallen on the ground. In the fruits, 99% of the adults were females and they were not grouped. The way in which daily changes in the size and shape of shady habitats together with the presence of heterospecific adults influence the selection of places to live is poorly understood in Drosophila. These experiments show that adults of the five species prefer dark areas. The experimental results show that the odors of each species: 1) influence conspecifics to select similar perch sites and decrease mobility, and 2) increase mobility in heterospecific adults and modify their perch site preferences. Attractions between conspecifics, the repulsions between species, and preferences for shaded areas matter in choosing a place to live in the five Drosophila species. These behaviors seem to have evolved as coordinated routines, contributing to the coexistence of the five Drosophila species in the apple and pear orchards examined.
Souto-Maior, C., Serrano Negron, Y. L. and Harbison, S. T. (2023). Nonlinear expression patterns and multiple shifts in gene network interactions underlie robust phenotypic change in Drosophila melanogaster selected for night sleep duration. PLoS Comput Biol 19(8): e1011389. PubMed ID: 37561813
All but the simplest phenotypes are believed to result from interactions between two or more genes forming complex networks of gene regulation. Sleep is a complex trait known to depend on the system of feedback loops of the circadian clock, and on many other genes; however, the main components regulating the phenotype and how they interact remain an unsolved puzzle. Genomic and transcriptomic data may well provide part of the answer, but a full account requires a suitable quantitative framework. An artificial selection experiment was conducted for sleep duration with RNA-seq data acquired each generation. The phenotypic results are robust across replicates and previous experiments, and the transcription data provides a high-resolution, time-course data set for the evolution of sleep-related gene expression. In addition to a Hierarchical Generalized Linear Model analysis of differential expression that accounts for experimental replicates a flexible Gaussian Process model was constructed that estimates interactions between genes. 145 gene pairs are found to have interactions that are different from controls. This method appears to be not only more specific than standard correlation metrics but also more sensitive, finding correlations not significant by other methods. Statistical predictions were compared to experimental data from public databases on gene interactions. Mutations of candidate genes implicated by these results affected night sleep, and gene expression profiles largely met predicted gene-gene interactions.
Takayanagi-Kiya, S., Shioya, N., Nishiuchi, T., Iwami, M. and Kiya, T. (2023). Cell assembly analysis of neural circuits for innate behavior in Drosophila melanogaster using an immediate early gene stripe/egr-1. Proc Natl Acad Sci U S A 120(33): e2303318120. PubMed ID: 37549285
Innate behavior, such as courtship behavior, is controlled by a genetically defined set of neurons. To date, it remains challenging to visualize and artificially control the neural population that is active during innate behavior in a whole-brain scale. Immediate early genes (IEGs), whose expression is induced by neural activity, can serve as powerful tools to map neural activity in the animal brain. IEGs were screened for in the vinegar fly Drosophila melanogaster and stripe/egr-1 was identified as a potent neural activity marker. Focusing on male courtship as a model of innate behavior, it was demonstrated that stripe-GAL4-mediated reporter expression can label fruitless (fru)-expressing neurons involved in courtship in an activity (experience)-dependent manner. Optogenetic reactivation of the labeled neurons elicited sexual behavior in males, whereas silencing of the labeled neurons suppressed courtship and copulation. Further, by combining stripe-GAL4-mediated reporter expression and detection of endogenous Stripe expression, methods were establised that can label neurons activated under different contexts in separate time windows in the same animal. The cell assembly analysis of fru neural population in males revealed that distinct groups of neurons are activated during interactions with a female or another male. These methods will contribute to building a deeper understanding of neural circuit mechanisms underlying innate insect behavior.

Friday, December 22nd - Tumors, Cancer and Growth

Takarada, K., Kinoshita, J. and Inoue, Y. H. (2023). Ectopic expression of matrix metalloproteinases and filopodia extension via JNK activation are involved in the invasion of blood tumor cells in Drosophila mxc mutant. Genes Cells. PubMed ID: 37615261
Drosophila mxc(mbn1) mutant exhibits severe hyperplasia in larval hematopoietic tissue called the lymph glands (LGs). However, the malignant nature of these cells remains unknown. This study aimed to identify if mxc(mbn1) LG cells behave as malignant tumor cells and uncover the mechanism(s) underlying the malignancy of the mutant hemocytes. When mutant LG cells were allografted into normal adult abdomens, they continued to proliferate; however, normal LG cells did not proliferate. Mutant circulating hemocytes also attached to the larval central nervous system (CNS), where the basement membrane was disrupted. The mutant hemocytes displayed higher expression of matrix metalloproteinase (MMP) 1 and MMP2 and higher activation of the c-Jun N-terminal kinase (JNK) pathway than normal hemocytes. Depletion of MMPs or JNK mRNAs in LGs resulted in reduced numbers of hemocytes attached to the CNS, suggesting that the invasive phenotype involved elevated expression of MMPs via hyperactivation of the JNK pathway. Moreover, hemocytes with elongated filopodia and extra lamellipodia were frequently observed in the mutant hemolymph, which also depended on JNK signaling. Thus, the MMP upregulation and overextension of actin-based cell protrusions were also involved in hemocyte invasion in mxc(mbn1) larvae. These findings contribute to the understanding of molecular mechanisms underlying mammalian leukemic invasion.
Sekar, A., Leiblich, A., Wainwright, S. M., Mendes, C. C., Sarma, D., Hellberg, J., Gandy, C., Goberdhan, D. C. I., Hamdy, F. C. and Wilson, C. (2023). Rbf/E2F1 control growth and endoreplication via steroid-independent Ecdysone Receptor signalling in Drosophila prostate-like secondary cells. PLoS Genet 19(6): e1010815. PubMed ID: 37363926
In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. It has been shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This study tested whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, it was found that excess Rbf activity reversibly suppresses binucleation in adult SCs. It was also demonstrated that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, it was shown that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, this work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females.
Kim, H. K., Kim, C. J., Jang, D. and Lim, D. H. (2023). MicroRNA miR-274-5p supresses Found-in-Neurons associated with Melanotic mass Formation and Developmental Growth in Drosophila.. Insects 14(8). PubMed ID: 37623419
The hematopoietic system plays a crucial role in immune defense response and normal development, and it is regulated by various factors from other tissues. The dysregulation of hematopoiesis is associated with melanotic mass formation; however, the molecular mechanisms underlying this process are poorly understood. This study observed that the overexpression of miR-274 in the fat body resulted in the formation of melanotic masses. Moreover, abnormal activation of the and JAK/STAT signaling pathways was linked to these consequences. In addition to this defect, miR-274 overexpression in the larval fat body decreased the total tissue size, leading to a reduction in body weight. miR-274-5p was found to directly supress the expression of found-in-neurons (fne), which encodes an RNA-binding protein. Similar to the effects of miR-274 overexpression, fne depletion led to melanotic mass formation and growth reduction. Collectively, miR-274 plays a regulatory role in the fne-JNK signaling axis in melanotic mass formation and growth control.
Kinoshita, Y., Shiratsuchi, N., Araki, M. and 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
Several antimicrobial peptides supress 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.
Martinez-Abarca Millan, A., Soler Beatty, J., Valencia Exposito, A. and Martin-Bermudo, M. D. (2023). Drosophila as Model System to Study Ras-Mediated Oncogenesis. The Case of the Tensin Family of Proteins. Genes (Basel) 14(7). PubMed ID: 37510408
Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, tissue growth induced by oncogenic Ras is restrained by the induction of cellular senescence, and additional mutations are required to induce tumor progression. Therefore, identifying cooperating cancer genes is of paramount importance. Recently, the tensin family of focal adhesion proteins, TNS1-4, have emerged as regulators of carcinogenesis, yet their role in cancer appears somewhat controversial. Around 90% of human cancers are of epithelial origin. This study used the Drosophila wing imaginal disc epithelium as a model system to gain insight into the roles of two orthologs of human TNS2 and 4, blistery (by) and PVRAP, in epithelial cancer progression. This study has generated null mutations in PVRAP and found that, as is the case for by and mammalian tensins, PVRAP mutants are viable. This study also found that elimination of either PVRAP or by potentiates Ras(V12)-mediated wing disc hyperplasia. Furthermore, the results have unraveled a mechanism by which tensins may limit Ras oncogenic capacity, the regulation of cell shape and growth. These results demonstrate that Drosophila tensins behave as supressors of Ras-driven tissue hyperplasia, suggesting that the roles of tensins as modulators of cancer progression might be evolutionarily conserved.
Sekiya, S., Fukuda, J., Yamamura, R., Ooshio, T., Satoh, Y., Kosuge, S., Sato, R., Hatanaka, K. C., Hatanaka, Y., Mitsuhashi, T., Nakamura, T., Matsuno, Y., Hirano, S. and Sonoshita, M. (2023). Drosophila Screening Identifies Dual Inhibition of MEK and AURKB as an Effective Therapy for Pancreatic Ductal Adenocarcinoma. Cancer Res 83(16): 2704-2715. PubMed ID: 37378549
Significant progress has been made in understanding the pathogenesis of pancreatic ductal adenocarcinoma (PDAC) by generating and using murine models. To accelerate drug discovery by identifying novel therapeutic targets on a systemic level, this study generated a Drosophila model mimicking the genetic signature in PDAC (KRAS, TP53, CDKN2A, and SMAD4 alterations), which is associated with the worst prognosis in patients. The '4-hit' flies displayed epithelial transformation and decreased survival. Comprehensive genetic screening of their entire kinome revealed kinases including MEK and AURKB as therapeutic targets. Consistently, a combination of the MEK inhibitor trametinib and the AURKB inhibitor BI-831266 suppressed the growth of human PDAC xenografts in mice. In patients with PDAC, the activity of AURKB was associated with poor prognosis. This fly-based platform provides an efficient whole-body approach that complements current methods for identifying therapeutic targets in PDAC. Development of a Drosophila model mimicking genetic alterations in human pancreatic ductal adenocarcinoma provides a tool for genetic screening that identifies MEK and AURKB inhibition as a potential treatment strategy.

Thursday, December 21th - Apoptosis and Autophagy

Park, J. E., Lee, J., Ok, S., Byun, S., Chang, E. J., Yoon, S. E., Kim, Y. J. and Kang, M. J. (2023). Wg/Wnt1 and Erasp link ER stress to proapoptotic signaling in an autosomal dominant retinitis pigmentosa model. Exp Mol Med 55(7): 1544-1555. PubMed ID: 37464094
The endoplasmic reticulum (ER) is a subcellular organelle essential for cellular homeostasis. Perturbation of ER functions due to various conditions can induce apoptosis. Chronic ER stress has been implicated in a wide range of diseases, including autosomal dominant retinitis pigmentosa (ADRP), which is characterized by age-dependent retinal degeneration caused by mutant rhodopsin alleles. However, the signaling pathways that mediate apoptosis in response to ER stress remain poorly understood. In this study, an unbiased in vivo RNAi screen was performed with a Drosophila ADRP model and found that Wg/Wnt1 mediated apoptosis. Subsequent transcriptome analysis revealed that ER stress-associated serine protease (Erasp), which has been predicted to show serine-type endopeptidase activity, was a downstream target of Wg/Wnt1 during ER stress. Furthermore, knocking down Erasp via RNAi suppressed apoptosis induced by mutant rhodopsin-1 (Rh-1(P37H)) toxicity, alleviating retinal degeneration in the Drosophila ADRP model. In contrast, overexpression of Erasp resulted in enhanced caspase activity in Drosophila S2 cells treated with apoptotic inducers and the stabilization of the initiator caspase Dronc (Death regulator Nedd2-like caspase) by stimulating DIAP1 (Drosophila inhibitor of apoptosis protein 1) degradation. These findings helped identify a novel cell death signaling pathway involved in retinal degeneration in an autosomal dominant retinitis pigmentosa model.
Qin, B., Yu, S., Chen, Q. and Jin, L. H. (2023). Atg2 Regulates Cellular and Humoral Immunity in Drosophila. Insects 14(8). PubMed ID: 37623416
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.
Walker, D., Schmid, E., Schinaman, J. and Williams, K. (2023). Accumulation of F-actin drives brain aging and limits healthspan in Drosophila. Res Sq. PubMed ID: 37577708
The actin cytoskeleton is a key determinant of cell and tissue homeostasis. However, tissue-specific roles for actin dynamics in aging, notably brain aging, are not understood. This study shows that there is an age-related increase in filamentous actin (F-actin) in Drosophila brains, which is counteracted by prolongevity interventions. Critically, modulating F-actin levels in aging neurons prevents age-onset cognitive decline and extends organismal healthspan. Mechanistically, autophagy, a recycling process required for neuronal homeostasis, was shown to be disabled upon actin dysregulation in the aged brain. Remarkably, disrupting actin polymerization in aged animals with cytoskeletal drugs restores brain autophagy to youthful levels and reverses cellular hallmarks of brain aging. Finally, reducing F-actin levels in aging neurons slows brain aging and promotes healthspan in an autophagy-dependent manner. These data identify excess actin polymerization as a hallmark of brain aging, which can be targeted to reverse brain aging phenotypes and prolong healthspan.
Nakano, S., Kashio, S., Nishimura, K., Takeishi, A., Kosakamoto, H., Obata, F., Kuranaga, E., Chihara, T., Yamauchi, Y., Isobe, T. and Miura, M. (2023). Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies. PLoS Genet 19(6): e1010761. PubMed ID: 37319131
The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spatzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, it was revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, it was found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage (Nakano, 2023).
Szabo, A., Vincze, V., Chhatre, A. S., Jipa, A., Bognar, S., Varga, K. E., Banik, P., Harmatos-Urmosi, A., Neukomm, L. J. and Juhasz, G. (2023). LC3-associated phagocytosis promotes glial degradation of axon debris after injury in Drosophila models. Nat Commun 14(1): 3077. PubMed ID: 37248218:
Glial engulfment of neuron-derived debris after trauma, during development, and in neurodegenerative diseases supports nervous system functions. However, mechanisms governing the efficiency of debris degradation in glia have remained largely unexplored. This study showed that LC3-associated phagocytosis (LAP), an engulfment pathway assisted by certain autophagy factors, promotes glial phagosome maturation in the Drosophila wing nerve. A LAP-specific subset of autophagy-related genes is required in glia for axon debris clearance, encoding members of the Atg8a (LC3) conjugation system and the Vps34 lipid kinase complex including UVRAG and Rubicon. Phagosomal Rubicon and Atg16 WD40 domain-dependent conjugation of Atg8a mediate proper breakdown of internalized axon fragments, and Rubicon overexpression in glia accelerates debris elimination. Finally, LAP promotes survival following traumatic brain injury. These results reveal a role of glial LAP in the clearance of neuronal debris in vivo, with potential implications for the recovery of the injured nervous system.
Villalobos, T. V., Ghosh, B., DeLeo, K. R., Alam, S., Ricaurte-Perez, C., Wang, A., Mercola, B. M., Butsch, T. J., Ramos, C. D., Das, S., Eymard, E. D., Bohnert, K. A. and Johnson, A. E. (2023). Tubular lysosome induction couples animal starvation to healthy aging. Nat Aging. PubMed ID: 37580394
Dietary restriction promotes longevity in several species via autophagy activation. However, changes to lysosomes underlying this effect remain unclear. In this study using the nematode Caenorhabditis elegans, This study showed that the induction of autophagic tubular lysosomes (TLs), which occurs upon dietary restriction or mechanistic target of rapamycin inhibition, is a critical event linking reduced food intake to lifespan extension. Starvation was found to induce TLs not only in affected individuals but also in well-fed descendants, and the presence of gut TLs in well-fed progeny is predictive of enhanced lifespan. Furthermore, this study demonstrated that expression of Drosophila small VCP-interacting protein, a TL activator in flies, artificially induces TLs in well-fed worms and improves C. elegans health in old age. These findings identify TLs as a new class of lysosomes that couples starvation to healthy aging.

Tuesday, December 19th - Immune Response

Li, J., Lyu, B., Bi, J., Shan, R., Stanley, D., Feng, Q. and Song, Q. (2023). Partner of neuropeptide bursicon homodimer pburs mediates a novel antimicrobial peptide Ten3LP via Dif/Dorsal2 in Tribolium castaneum. Int J Biol Macromol 247: 125840. PubMed ID: 37454995
Bursicon is a cystine knot family neuropeptide, composed of two subunits, bursicon (burs) and partner of burs (pburs). The subunits can form heterodimers to regulate cuticle tanning and wing maturation and homodimers to signal different biological functions in innate immunity, midgut stem cell proliferation and energy homeostasis, and reproductive physiology in the model insects Drosophila melanogaster or Tribolium castaneum. This study reports on the role of the pburs homodimer in signaling innate immunity in T. castaneum larvae. Through transcriptome analysis a set of immune-related genes were identified that respond to pburs RNAi. Treating larvae with recombinant-pburs protein led to up-regulation of antimicrobial peptide (AMP) genes in vivo and in vitro. The upregulation of most AMP genes was dependent on the NF-κB transcription factor Relish. Most importantly, a novel AMP was discovered, Tenecin 3-like peptide (Ten3LP), regulated by pburs via NF-kappaB transcription factor Dorsal-related immunity factor (Dif)/Dorsal2, but not Relish. Ten3LP RNAi was conduction, recombinant Ten3LP protein was synthesized for microbial inhibition assays, and Ten3LP was functionally characterized as an AMP specific for fungi and Gram-positive bacteria. Expression of Ten3LP is activated by pburs via the Toll pathway. These findings identify new molecular targets for development of potential antibiotics for treating microbial infections and perhaps for RNAi based pest management technology.
Mouawad, C., Awad, M. K., Liegeois, S., Ferrandon, D., Sanchis-Borja, V. and El Chamy, L. (2023). The NF-kappaB factor Relish is essential for the epithelial defenses protecting against delta-endotoxin dependent effects of Bacillus thuringiensis israelensis infection in the Drosophila model. Res Microbiol 174(6): 104089. PubMed ID: 37348743
Bacillus thuringiensis israelensis is largely regarded as the most selective, safe and ecofriendly biopesticide used for the control of insect vectors of human diseases. Bti enthomopathogenicity relies on the Cry and Cyt δ-endotoxins, produced as crystalline inclusions during sporulation. Insecticidal selectivity of Bti is mainly ascribed to the binding of the Cry toxins to receptors in the gut of target insects. However, the contribution of epithelial defenses in limiting Bti side effects in non-target species remains largely unexplored. This study took advantage of the genetically tractable Drosophila melanogaster model and its amenability for deciphering highly conserved innate immune defenses, a central role was unraveled of the NF-kappaB factor Relish in the protection against the effects of ingested Bti spores in a non-susceptible host. Intriguingly, the data indicate that the Bti-induced Relish response is independent of its canonical activation downstream of peptidoglycan sensing and does not involve its longstanding role in the regulation of antimicrobial peptides encoding genes. In contrast, the data highlight a novel enterocyte specific function of Relish that is essential for preventing general septicemia following Bti oral infections strictly when producing δ-endotoxins. Altogether, these data provide novel insights into Bti-hosts interactions of prominent interest for the optimization and sustainability of insects' biocontrol strategies.
Odnokoz, O., Earland, N., Badinloo, M., Klichko, V. I., Benes, J., Orr, W. C. and Radyuk, S. N. (2023). Peroxiredoxins Play an Important Role in the Regulation of Immunity and Aging in Drosophila. Antioxidants (Basel) 12(8). PubMed ID: 37627611
Aberrant immune responses and chronic inflammation can impose significant health risks and promote premature aging. Pro-inflammatory responses are largely mediated via reactive oxygen species (ROS) and reduction-oxidation reactions. A pivotal role in maintaining cellular redox homeostasis and the proper control of redox-sensitive signaling belongs to a family of antioxidant and redox-regulating thiol-related peroxidases designated as peroxiredoxins (Prx). Recent studies in Drosophila have shown that Prxs play a critical role in aging and immunity. This study identified two important 'hubs', the endoplasmic reticulum (ER) and mitochondria, where extracellular and intracellular stress signals are transformed into pro-inflammatory responses that are modulated by the activity of the Prxs residing in these cellular organelles. This study found that mitochondrial Prx activity in the intestinal epithelium is required to prevent the development of intestinal barrier dysfunction, which can drive systemic inflammation and premature aging. Using a redox-negative mutant, it ws demonstrated that Prx acts in a redox-dependent manner in regulating the age-related immune response. The hyperactive immune response observed in flies under-expressing mitochondrial Prxs is due to a response to abiotic signals but not to changes in the bacterial content. This hyperactive response, but not reduced lifespan phenotype, can be rescued by the ER-localized Prx.
Shi, X. Z., Yang, M. C., Kang, X. L., Li, Y. X., Hong, P. P., Zhao, X. F., Vasta, G. R. and Wang, J. X. (2023). Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans. Proc Natl Acad Sci U S A 120(24): e2216574120. PubMed ID: 37276415
The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. This study identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.
Li, Y., Slavik, K. M., Toyoda, H. C., Morehouse, B. R., de Oliveira Mann, C. C., Elek, A., Levy, S., Wang, Z., Mears, K. S., Liu, J., Kashin, D., Guo, X., Maβ, T., Sebe-Pedros, A., Schwede, F. and Kranzusch, P. J. (2023). cGLRs are a diverse family of pattern recognition receptors in innate immunity. Cell 186(15): 3261-3276. PubMed ID: 37379839
Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates STING-dependent downstream immunity. This study discovered that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, >3,000 cGLRs present in nearly all metazoan phyla were identified. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways is explained. The results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.
Radhika, R. and Lazzaro, B. P. (2023). No evidence for trans-generational immune priming in Drosophila melanogaster. PLoS One 18(7): e0288342. PubMed ID: 37440541
Most organisms are under constant and repeated exposure to pathogens, leading to perpetual natural selection for more effective ways to fight-off infections. This could include the evolution of memory-based immunity to increase protection from repeatedly-encountered pathogens both within and across generations. There is mixed evidence for intra- and trans-generational priming in non-vertebrates, which lack the antibody-mediated acquired immunity characteristic of vertebrates. This work tested for trans-generational immune priming in adult offspring of the fruit fly, Drosophila melanogaster, after maternal challenge with 10 different bacterial pathogens. Focus was placed on natural opportunistic pathogens of Drosophila spanning a range of virulence from 10% to 100% host mortality. Mothers were infected via septic injury and tested for enhanced resistance to infection in their adult offspring, measured as the ability to suppress bacterial proliferation and survive infection. The mothers were categorized into four classes for each bacterium tested: those that survived infection, those that succumbed to infection, sterile-injury controls, and uninjured controls. No evidence was found for trans-generational priming by any class of mother in response to any of the bacteria.

Monday, December 18th - Larval and Adult Neural Development, Structure, and Function

Schlegel, P., Yin, Y., Bates, A. S., ..., Hartenstein, V., Bock, D. D. and Jefferis, G. (2023). Whole-brain annotation and multi-connectome cell typing quantifies circuit stereotypy in Drosophila. bioRxiv. PubMed ID: 37425808
The fruit fly combines surprisingly sophisticated behaviour with a highly tractable nervous system. As presented in a FlyWire companion paper, available resources include the first full brain connectome of an adult animal. This study reports the systematic and hierarchical annotation of this ~130,000-neuron connectome including neuronal classes, cell types and developmental units (hemilineages). Crucially, this resource includes 4,552 cell types. 3,094 are rigorous consensus validations of cell types previously proposed in the hemibrain connectome. In addition, 1,458 new cell types are proposed, arising mostly from the fact that the FlyWire connectome spans the whole brain, whereas the hemibrain derives from a subvolume. Comparison of FlyWire and the hemibrain showed that cell type counts and strong connections were largely stable, but connection weights were surprisingly variable within and across animals. Further analysis defined simple heuristics for connectome interpretation: connections stronger than 10 unitary synapses or providing >1% of the input to a target cell are highly conserved. Some cell types showed increased variability across connectomes: the most common cell type in the mushroom body, required for learning and memory, is almost twice as numerous in FlyWire as the hemibrain. Evidence was found for functional homeostasis through adjustments of the absolute amount of excitatory input while maintaining the excitation-inhibition ratio. Finally, and surprisingly, about one third of the cell types proposed in the hemibrain connectome could not yet be reliably identified in the FlyWire connectome. It is therefore suggested that cell types should be defined to be robust to inter-individual variation, namely as groups of cells that are quantitatively more similar to cells in a different brain than to any other cell in the same brain. Joint analysis of the FlyWire and hemibrain connectomes demonstrates the viability and utility of this new definition. This work defines a consensus cell type atlas for the fly brain and provides both an intellectual framework and open source toolchain for brain-scale comparative connectomics.
Roach, S. T., Ford, M. C., Simhambhatla, V., Loutrianakis, V., Farah, H., Li, Z., Periandri, E. M., Abdalla, D., Huang, I., Kalra, A. and Shaw, P. J. (2023). Sleep deprivation, sleep fragmentation, and social jet lag increase temperature preference in Drosophila. Front Neurosci 17: 1175478. PubMed ID: 37274220
Despite the fact that sleep deprivation substantially affects the way animals regulate their body temperature, the specific mechanisms behind this phenomenon are not well understood. In both mammals and flies, neural circuits regulating sleep and thermoregulation overlap, suggesting an interdependence that may be relevant for sleep function. To investigate this relationship further, flies were exposed to 12 h of sleep deprivation, or 48 h of sleep fragmentation, and temperature preference was evaluated in a thermal gradient. Flies exposed to 12 h of sleep deprivation chose warmer temperatures after sleep deprivation. Importantly, sleep fragmentation, which prevents flies from entering deeper stages of sleep, but does not activate sleep homeostatic mechanisms nor induce impairments in short-term memory also resulted in flies choosing warmer temperatures. To identify the underlying neuronal circuits, RNAi was used to knock down the receptor for Pigment dispersing factor, a peptide that influences circadian rhythms, temperature preference and sleep. Expressing UAS-Pdfr(RNAi) in subsets of clock neurons prevented sleep fragmentation from increasing temperature preference. Finally, temperature preference was evaluated after flies had undergone a social jet lag protocol which is known to disrupt clock neurons. In this protocol, flies experience a 3 h light phase delay on Friday followed by a 3 h light advance on Sunday evening. Flies exposed to social jet lag exhibited an increase in temperature preference which persisted for several days. These findings identify specific clock neurons that are modulated by sleep disruption to increase temperature preference. Moreover, the data indicate that temperature preference may be a more sensitive indicator of sleep disruption than learning and memory.
Richhariya, S., Shin, D., Le, J. Q. and Rosbash, M. (2023). Dissecting neuron-specific functions of circadian genes using modified cell-specific CRISPR approaches. Proc Natl Acad Sci U S A 120(29): e2303779120. PubMed ID: 37428902
Circadian behavioral rhythms in Drosophila melanogaster are regulated by about 75 pairs of brain neurons. They all express the core clock genes but have distinct functions and gene expression profiles. To understand the importance of these distinct molecular programs, neuron-specific gene manipulations are essential. Although RNAi based methods are standard to manipulate gene expression in a cell-specific manner, they are often ineffective, especially in assays involving smaller numbers of neurons or weaker Gal4 drivers. A neuron-specific CRISPR-based method has been exploited to mutagenize genes within circadian neurons. This study further explores this approach to mutagenize three well-studied clock genes: the transcription factor gene vrille, the photoreceptor gene Cryptochrome (cry), and the neuropeptide gene Pdf (pigment dispersing factor). The CRISPR-based strategy not only reproduced their known phenotypes but also assigned cry function for different light-mediated phenotypes to discrete, different subsets of clock neurons. Two recently published methods for temporal regulation in adult neurons were tested, inducible Cas9 and the auxin-inducible gene expression system. The results were not identical, but both approaches successfully showed that the adult-specific knockout of the neuropeptide Pdf reproduces the canonical loss-of-function mutant phenotypes. In summary, a CRISPR-based strategy is a highly effective, reliable, and general method to temporally manipulate gene function in specific adult neurons.
Rey, S., Ohm, H., Moschref, F., Zeuschner, D., Praetz, M. and Klambt, C. (2023). Glial-dependent clustering of voltage-gated ion channels in Drosophila precedes myelin formation. Elife 12. PubMed ID: 37278291
Neuronal information conductance often involves the transmission of action potentials. The spreading of action potentials along the axonal process of a neuron is based on three physical parameters: the axial resistance of the axon, the axonal insulation by glial membranes, and the positioning of voltage-gated ion channels. In vertebrates, myelin and channel clustering allow fast saltatory conductance. This study shows that in Drosophila melanogaster voltage-gated sodium and potassium channels, Para and Shal, co-localize and cluster in an area resembling the axon initial segment. The local enrichment of Para but not of Shal localization depends on the presence of peripheral wrapping glial cells. In larvae, relatively low levels of Para channels are needed to allow proper signal transduction and nerves are simply wrapped by glial cells. In adults, the concentration of Para increases and is prominently found at the axon initial segment of motor neurons. Concomitantly, these axon domains are covered by a mesh of glial processes forming a lacunar structure that possibly serves as an ion reservoir. Directly flanking this domain glial processes forming the lacunar area appear to collapse and closely apposed stacks of glial cell processes can be detected, resembling a myelin-like insulation. Thus, Drosophila development may reflect the evolution of myelin which forms in response to increased levels of clustered voltage-gated ion channels.
Schwartz, S., Wilson, S. J., Hale, T. K. and Fitzsimons, H. L. (2023). Ankyrin2 is essential for neuronal morphogenesis and long-term courtship memory in Drosophila. Mol Brain 16(1): 42. PubMed ID: 37194019
Dysregulation of HDAC4 expression and/or nucleocytoplasmic shuttling results in impaired neuronal morphogenesis and long-term memory in Drosophila melanogaster. A recent genetic screen for genes that interact in the same molecular pathway as HDAC4 identified the cytoskeletal adapter Ankyrin2 (Ank2). This study sought to investigate the role of Ank2 in neuronal morphogenesis, learning and memory. Ank2 is expressed widely throughout the Drosophila brain where it localizes predominantly to axon tracts. Pan-neuronal knockdown of Ank2 in the mushroom body, a region critical for memory formation, resulted in defects in axon morphogenesis. Similarly, reduction of Ank2 in lobular plate tangential neurons of the optic lobe disrupted dendritic branching and arborization. Conditional knockdown of Ank2 in the mushroom body of adult Drosophila significantly impaired long-term memory (LTM) of courtship suppression, and its expression was essential in the γ neurons of the mushroom body for normal LTM. In summary, this study provides the first characterization of the expression pattern of Ank2 in the adult Drosophila brain and demonstrates that Ank2 is critical for morphogenesis of the mushroom body and for the molecular processes required in the adult brain for the formation of long-term memories.
Sgammeglia, N., Widmer, Y. F., Kaldun, J. C., Fritsch, C., Bruggmann, R. and Sprecher, S. G. (2023). Memory phase-specific genes in the Mushroom Bodies identified using CrebB-target DamID. PLoS Genet 19(6): e1010802. PubMed ID: 37307281
The formation of long-term memories requires changes in the transcriptional program and de novo protein synthesis. One of the critical regulators for long-term memory (LTM) formation and maintenance is the transcription factor CREB. Genetic studies have dissected the requirement of CREB activity within memory circuits, however less is known about the genetic mechanisms acting downstream of CREB and how they may contribute defining LTM phases. To better understand the downstream mechanisms, a targeted DamID approach (TaDa) was used. A CREB-Dam fusion protein was generated using the fruit fly Drosophila melanogaster as model. Expressing CREB-Dam in the mushroom bodies (MBs), a brain center implicated in olfactory memory formation, identified genes that are differentially expressed between paired and unpaired appetitive training paradigm. Of those genes candidates were selected for an RNAi screen in which genes were identified causing increased or decreased LTM.

Friday, December 15th - Adult Physiology and Metabolism

Sato, M., Ota, R., Kobayashi, S., Yamakawa-Kobayashi, K., Miura, T., Ido, A. and Ohhara, Y. (2023). Bioproduction of n-3 polyunsaturated fatty acids by nematode fatty acid desaturases and elongase in Drosophila melanogaster. Transgenic Res. PubMed ID: 37615877
n-3 polyunsaturated fatty acids (n-3 PUFAs), including α-linolenic acid and eicosapentaenoic acid (EPA), are essential nutrients for vertebrates including humans. Vertebrates are n-3 PUFA-auxotrophic; hence, dietary intake of n-3 PUFAs is required for their normal physiology and development. Although fish meal and oil have been utilized as primary sources of n-3 PUFAs by humans and aquaculture, these traditional n-3 PUFA sources are expected to be exhausted because of the increasing consumption requirements of humans. Hence, it is necessary to establish alternative n-3 PUFA sources to reduce the gap between the supply and demand of n-3 PUFAs. This study investigated whether insects, which are considered as a novel source of essential nutrients, could store n-3 PUFAs by the forced expression of n-3 PUFA biosynthetic enzymes. Drosophila was used as an insect model to generate transgenic strains expressing Caenorhabditis elegans PUFA biosynthetic enzymes, and their effects on the proportion of fatty acids was examined. The ubiquitous expression of methyl-end desaturase FAT-1 prominently enhanced the proportions of α-linolenic acid, indicating that FAT-1 is useful for metabolic engineering to fortify α-linolenic acid in insect. Furthermore, the ubiquitous expression of nematode front-end desaturases (FAT-3 and FAT-4), PUFA elongase (ELO-1), and FAT-1 led to EPA bioproduction. Hence, nematode PUFA biosynthetic genes may serve as powerful genetic tools for enhancing the proportion of EPA in insects. This study represents the first step toward the establishment of n-3 PUFA-producing insects.
Pradhan, R. N., Shrestha, B. and Lee, Y. (2023). Molecular Basis of Hexanoic Acid Taste in Drosophila melanogaster. Mol Cells 46(7): 451-460. PubMed ID: 37202372
Animals generally prefer nutrients and avoid toxic and harmful chemicals. Recent behavioral and physiological studies have identified that sweet-sensing gustatory receptor neurons (GRNs) in Drosophila melanogaster mediate appetitive behaviors toward fatty acids. Sweet-sensing GRN activation requires the function of the ionotropic receptors IR25a, IR56d, and IR76b, as well as the gustatory receptor GR64e. However, this study reveals that hexanoic acid (HA) is toxic rather than nutritious to D. melanogaster. HA is one of the major components of the fruit Morinda citrifolia (noni). Thus, the gustatory responses to one of major noni fatty acids, HA, were examined via electrophysiology and proboscis extension response (PER) assay. Electrophysiological tests show this is reminiscent of arginine-mediated neuronal responses. It was determined that a low concentration of HA induced attraction, which was mediated by sweet-sensing GRNs, and a high concentration of HA induced aversion, which was mediated by bitter-sensing GRNs. It was also demonstrated that a low concentration of HA elicits attraction mainly mediated by GR64d and IR56d expressed by sweet-sensing GRNs, but a high concentration of HA activates three gustatory receptors (GR32a, GR33a, and GR66a) expressed by bitter-sensing GRNs. The mechanism of sensing HA is biphasic in a dose dependent manner. Furthermore, HA inhibit sugar-mediated activation like other bitter compounds. Taken together, this study discovered a binary HA-sensing mechanism that may be evolutionarily meaningful in the foraging niche of insects.
Saini, S., Rani, L., Shukla, N., Thakur, R. S., Patel, D. K., Ansari, M. S., Banerjee, M. and Gautam, N. K. (2023). Hsp27 over expression protect against cadmium induced nephrotoxicity in Drosophila melanogaster. Comp Biochem Physiol C Toxicol Pharmacol: 109716. PubMed ID: 37586579
Cadmium (Cd) exposure to the animals including humans is reported as nephrotoxic compounds i.e., disturbing redox status (increase oxidative stress), mitochondrial dysfunction, renal cell death and altered transporters in the renal system. Hsp27 (a small heat shock protein) has been shown as one of the modulators in the renal dysfunction and increased against the Cd induced toxicity. However, no studies are reported on the genetic modulation of stress protein against the Cd-induced nephrotoxicity. The current study aimed to examine the protective role of hsp27 overexpression against the Cd-induced nephrotoxicity using Drosophila melanogaster as an animal model. D. melanogaster renal system includes nephrocytes and Malpighian tubules (MTs) that show the functional similarity with mammalian kidney nephron. Overexpression of the hsp27 was found to reduce the Cd induced oxidative stress, rescue cell death in MTs of Cd exposed D. melanogaster larvae. The rescued GSH (Glutathione) level, NADPH level and glucose 6 phosphate dehydrogenase (G6PD) activity were also observed in the MTs of the Cd exposed organism. Function (efflux activity and fluid secretion rate) of the MTs was restored in Cd exposed hsp27 overexpressed larvae. Further, results were confirmed by restored brush border microvilli density and reduced uric acid level. Tissue specific knockdown of hsp27 developed Cd like phenotypes in MTs and the phenotypes enhanced in Cd exposed condition. The present study clearly shows the role of hsp27 overexpression in restoration of the MTs function and protection against the Cd induced renal toxicity.
Santos-Cruz, L. F., Sigrist-Flores, S. C., Castaneda-Partida, L., Heres-Pulido, M. E., Duenas-García, I. E., Piedra-Ibarra, E., Ponciano-Gomez, A., Jimenez-Flores, R. and Campos-Aguilar, M. (2023). Effects of Fructose and Palmitic Acid on Gene Expression in Drosophila melanogaster Larvae: Implications for Neurodegenerative Diseases. Int J Mol Sci 24(12). PubMed ID: 37373426
One of the largest health problems worldwide is the development of chronic noncommunicable diseases due to the consumption of hypercaloric diets. Among the most common alterations are cardiovascular diseases, and a high correlation between overnutrition and neurodegenerative diseases has also been found. The urgency in the study of specific damage to tissues such as the brain and intestine led this study to use Drosophila melanogaster to examine the metabolic effects caused by the consumption of fructose and palmitic acid in specific tissues. Thus, third instar larvae (96 ± 4 h) of the wild Canton-S strain of D. melanogaster were used to perform transcriptomic profiling in brain and midgut tissues to test for the potential metabolic effects of a diet supplemented with fructose and palmitic acid. The data suggest that this diet can alter the biosynthesis of proteins at the mRNA level that participate in the synthesis of amino acids, as well as fundamental enzymes for the dopaminergic and GABAergic systems in the midgut and brain. These also demonstrated alterations in the tissues of flies that may help explain the development of various reported human diseases associated with the consumption of fructose and palmitic acid in humans. These studies will not only help to better understand the mechanisms by which the consumption of these alimentary products is related to the development of neuronal diseases but may also contribute to the prevention of these conditions.
Roussel, D., Janillon, S., Teulier, L. and Pichaud, N. (2023). Succinate oxidation rescues mitochondrial ATP synthesis at high temperature in Drosophila melanogaster. FEBS Lett. PubMed ID: 37463836
Decreased NADH-induced and increased reduced FADH(2) -induced respiration rates at high temperatures are associated with thermal tolerance in Drosophila. This study determined whether this change was associated with adjustments of adenosine triphosphate (ATP) production rate and coupling efficiency (ATP/O) in Drosophila melanogaster. Decreased pyruvate + malate oxidation at 35°C was shown to be associated with a collapse of ATP synthesis and a drop in ATP/O ratio. However, adding succinate triggered a full compensation of both oxygen consumption and ATP synthesis rates at this high temperature. Addition of glycerol-3-phosphate (G3P) led to a huge increase in respiration with no further advantage in terms of ATP production. It is concluded that succinate is the only alternative substrate able to compensate both oxygen consumption and ATP production rates during oxidative phosphorylation at high temperature, which has important implications for thermal adaptation.
Saavedra, P., Dumesic, P. A., Hu, Y., Filine, E., Jouandin, P., Binari, R., Wilensky, S. E., Rodiger, J., Wang, H., Chen, W., Liu, Y., Spiegelman, B. M. and Perrimon, N. (2023). REPTOR and CREBRF encode key regulators of muscle energy metabolism. Nat Commun 14(1): 4943. PubMed ID: 37582831
Metabolic flexibility of muscle tissue describes the adaptive capacity to use different energy substrates according to their availability. The disruption of this ability associates with metabolic disease. Using a Drosophila model of systemic metabolic dysfunction triggered by yorkie-induced gut tumors, this study showed that the transcription factor REPTOR is an important regulator of energy metabolism in muscles. Evidence is presented that REPTOR is activated in muscles of adult flies with gut yorkie-tumors, where it modulates glucose metabolism. Further, in vivo studies indicate that sustained activity of REPTOR is sufficient in wildtype muscles to repress glycolysis and increase tricarboxylic acid (TCA) cycle metabolites. Consistent with the fly studies, higher levels of CREBRF, the mammalian ortholog of REPTOR, reduce glycolysis in mouse myotubes while promoting oxidative metabolism. Altogether, these results define a conserved function for REPTOR and CREBRF as key regulators of muscle energy metabolism.

Thursday, December 14th - Disease Models

Servettini, I., Talani, G., Megaro, A., Setzu, M. D., Biggio, F., Briffa, M., Guglielmi, L., Savalli, N., Binda, F., Delicata, F., Bru-Mercier, G., Vassallo, N., Maglione, V., Cauchi, R. J., Di Pardo, A., Collu, M., Imbrici, P., Catacuzzeno, L., D'Adamo, M. C., Olcese, R. and Pessia, M. (2023). An activator of voltage-gated K(+) channels Kv1.1 as a therapeutic candidate for episodic ataxia type 1. Proc Natl Acad Sci U S A 120(31): e2207978120. PubMed ID: 37487086
Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. This study uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. Through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5)s. By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.
Rossi, S., Di Salvio, M., Balì, M., De Simone, A., Apolloni, S., D'Ambrosi, N., Arisi, I., Cipressa, F., Cozzolino, M. and Cestra, G. (2023). C9orf72 Toxic Species Affect ArfGAP-1 Function. Cells 12(15). PubMed ID: 37566088
Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs. It has been reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in the cell nuclei. To further characterize the process, this study set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS RNA repeats. Interestingly, pathway analysis showed that the mRNAs involved in membrane trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicated that C9orf72 toxic species affect the membrane trafficking route regulated by ADP-Ribosylation Factor 1 GTPase Activating Protein (ArfGAP-1), which exerts its GTPase-activating function on the small GTPase ADP-ribosylation factor 1 to dissociate coat proteins from Golgi-derived vesicles. This study demonstrated that the function of ArfGAP-1 is specifically affected by expanded C9orf72 RNA repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), indicating the retrograde Golgi-to-ER vesicle-mediated transport as a target of C9orf72 toxicity.
Rozich, E., Randolph, L. K. and Insolera, R. (2023). An optimized temporally controlled Gal4 system in Drosophila reveals degeneration caused by adult-onset neuronal Vps13D knockdown. Front Neurosci 17: 1204068. PubMed ID: 37457002
Mutations in the human gene VPS13D cause the adult-onset neurodegenerative disease ataxia. Previous work showed that disruptions in the Vps13D gene in Drosophila neurons causes mitochondrial defects. However, developmental lethality caused by Vps13D loss limited understanding of the long-term physiological effects of Vps13D perturbation in neurons. This study optimized a previously generated system to temporally knock down Vps13D expression precisely in adult Drosophila neurons using a modification to the Gal4/UAS system. Adult-onset activation of Gal4 was enacted using the chemically-inducible tool which fuses a destabilization-domain to the Gal4 repressor Gal80 (Gal80-DD). Optimization of the Gal80-DD tool shows that feeding animals the DD-stabilizing drug trimethoprim (TMP) during development and rearing at a reduced temperature maximally represses Gal4 activity. Temperature shift and removal of TMP from the food after eclosion robustly activates Gal4 expression in adult neurons. Using the optimized Gal80-DD system, this study found that adult-onset Vps13D RNAi expression in neurons causes the accumulation of mitophagy intermediates, progressive deficits in locomotor activity, early lethality, and brain vacuolization characteristic of neurodegeneration. The development of this optimized system allowed more precise examination the degenerative phenotypes caused by Vps13D disruption and can likely be utilized in the future for other genes associated with neurological diseases whose manipulation causes developmental lethality in Drosophila.
Rundell, T. B., Brunelli, M., Alvi, A., Safian, G., Capobianco, C., Tu, W., Subedi, S., Fiumera, A. and Musselman, L. P. (2023). Polygenic adaptation to overnutrition reveals a role for cholinergic signaling in longevity. bioRxiv. PubMed ID: 37398379
Overnutrition by high-sugar (HS) feeding reduces both the lifespan and healthspan across taxa. Pressuring organisms to adapt to overnutrition can highlight genes and pathways important for the healthspan in stressful environments. This study used an experimental evolution approach to adapt four replicate, outbred population pairs of Drosophila melanogaster to a HS or control diet. Sexes were separated and aged on either diet until mid-life, then mated to produce the next generation, allowing enrichment for protective alleles over time. All HS-selected populations increased their lifespan and were therefore used as a platform to compare allele frequencies and gene expression. Pathways functioning in the nervous system were overrepresented in the genomic data and showed evidence for parallel evolution, although very few genes were the same across replicates. Acetylcholine-related genes, including the muscarinic receptor mAChR-A, showed significant changes in allele frequency in multiple selected populations and differential expression on a HS diet. Using genetic and pharmacological approaches, it was shown that cholinergic signaling affects Drosophila feeding in a sugar-specific fashion. Together, these results suggest that adaptation produces changes in allele frequencies that benefit animals under conditions of overnutrition and that it is repeatable at the pathway level.
Sanchez-Martinez, A., Martinez, A. and Whitworth, A. J. (2023). FBXO7/ntc and USP30 antagonistically set the ubiquitination threshold for basal mitophagy and provide a target for Pink1 phosphorylation in vivo. PLoS Biol 21(8): e3002244. PubMed ID: 37535686
Functional analyses of genes linked to heritable forms of Parkinson's disease (PD) have revealed fundamental insights into the biological processes underpinning pathogenic mechanisms. Mutations in PARK15/FBXO7 cause autosomal recessive PD and FBXO7 has been shown to regulate mitochondrial homeostasis. This study investigated the extent to which FBXO7 and its Drosophila orthologue, ntc, share functional homology and explored its role in mitophagy in vivo. ntc mutants partially phenocopy Pink1 and parkin mutants and ntc overexpression supresses parkin phenotypes. Furthermore, ntc can modulate basal mitophagy in a Pink1- and parkin-independent manner by promoting the ubiquitination of mitochondrial proteins, a mechanism that is opposed by the deubiquitinase USP30. This basal ubiquitination serves as the substrate for Pink1-mediated phosphorylation that triggers stress-induced mitophagy. It is proposed that FBXO7/ntc works in equilibrium with USP30 to provide a checkpoint for mitochondrial quality control in basal conditions in vivo and presents a new avenue for therapeutic approaches.
Sanz, F. J., Martinez-Carrion, G., Solana-Manrique, C. and Paricio, N. (2023). Evaluation of type 1 diabetes mellitus as a risk factor of Parkinson's disease in a Drosophila model. J Exp Zool A Ecol Integr Physiol. PubMed ID: 37381093
Diabetes mellitus (DM) is a chronic metabolic disease characterized by high blood glucose levels, resulting from insulin dysregulation. Parkinson's disease (PD) is the most common neurodegenerative motor disorder caused by the selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. DM and PD are both age-associated diseases that are turning into epidemics worldwide. Previous studies have indicated that type 2 DM might be a risk factor of developing PD. However, scarce information about the link between type 1 DM (T1DM) and PD does exist. This study generated a Drosophila model of T1DM based on insulin deficiency to evaluate if T1DM could be a risk factor to trigger PD onset. As expected, model flies exhibited T1DM-related phenotypes such as insulin deficiency, increased content of carbohydrates and glycogen, and reduced activity of insulin signaling. Interestingly, the results also demonstrated that T1DM model flies presented locomotor defects as well as reduced levels of tyrosine hydroxylase (a marker of DA neurons) in brains, which are typical PD-related phenotypes. In addition, T1DM model flies showed elevated oxidative stress levels, which could be causative of DA neurodegeneration. Therefore, these results indicate that T1DM might be a risk factor of developing PD, and encourage further studies to shed light into the exact link between both diseases.

Wednesday, December 13th - Evolution

Peng, J. and Zhao, L. (2023). The origin and structural evolution of de novo genes in Drosophila. bioRxiv. PubMed ID: 37425675
Although previously thought to be unlikely, recent studies have shown that de novo gene origination from previously non-genic sequences is a relatively common mechanism for gene innovation in many species and taxa. These young genes provide a unique set of candidates to study the structural and functional origination of proteins. However, understanding of their protein structures and how these structures originate and evolve are still limited, due to a lack of systematic studies. This study combined high-quality base-level whole genome alignments, bioinformatic analysis, and computational structure modeling to study the origination, evolution, and protein structure of lineage-specific de novo genes. 555 de novo gene candidates were identified in D. melanogaster that originated within the Drosophilinae lineage. A gradual shift was found in sequence composition, evolutionary rates, and expression patterns with their gene ages, which indicates possible gradual shifts or adaptations of their functions. Surprisingly, little overall protein structural changes were found for de novo genes in the Drosophilinae lineage. Using Alphafold2, ESMFold, and molecular dynamics, a number of de novo gene candidates were identified with protein products that are potentially well-folded, many of which are more likely to contain transmembrane and signal proteins compared to other annotated protein-coding genes. Using ancestral sequence reconstruction, it was found that most potentially well-folded proteins are often born folded. Interestingly, one case was observed where disordered ancestral proteins become ordered within a relatively short evolutionary time. Single-cell RNA-seq analysis in testis showed that although most de novo genes are enriched in spermatocytes, several young de novo genes are biased in the early spermatogenesis stage, indicating potentially important but less emphasized roles of early germline cells in the de novo gene origination in testis. This study provides a systematic overview of the origin, evolution, and structural changes of Drosophilinae -specific de novo genes.
Perez-Pereira, N., Quesada, H. and Caballero, A. (2023). An empirical evaluation of the estimation of inbreeding depression from molecular markers under suboptimal conditions. Evol Appl 16(7): 1302-1315. PubMed ID: 37492144
Inbreeding depression (ID), the reduction in fitness due to inbreeding, is typically measured by the regression of the phenotypic values of individuals for a particular trait on their corresponding inbreeding coefficients (F). While genealogical records can provide these coefficients, they may be unavailable or incomplete, making molecular markers a useful alternative. The power to detect ID and its accuracy depend on the variation of F values of individuals, the sample sizes available, and the accuracy in the estimation of individual fitness traits and F values. This study used Drosophila melanogaster to evaluate the effectiveness of molecular markers in estimating ID under suboptimal conditions. Two sets of 100 pairs of unrelated individuals were generated from a large panmictic population, and they were mated for two generations to produce non-inbred and unrelated individuals (F = 0) and inbred individuals (full-sib progeny; F = 0.25). Using these expected genealogical F values, inbreeding depression was calculated for two fitness-related traits, pupae productivity and competitive fitness.The males from 17 non-inbred pairs and 17 inbred pairs were sequenced to obtain their genomic inbreeding coefficients and estimate ID for the two traits. The scenario assumed was rather restrictive in terms of estimation of ID because: (1) the individuals belonged to the same generation of a large panmictic population, leading to low variation in individual F coefficients; (2) the sample sizes were small; and (3) the traits measured depended on both males and females while only males were sequenced. Despite the challenging conditions of this study, it was found that molecular markers provided estimates of ID that were comparable to those obtained from simple pedigree estimations with larger sample sizes. The results therefore suggest that genomic measures of inbreeding are useful to provide estimates of inbreeding depression even under very challenging scenarios.
Polak, M., Bose, J., Benoit, J. B. and Singh, H. (2023). Heritability and pre-adult survivorship costs of ectoparasite resistance in the naturally occurring Drosophila-Gamasodes mite system. Evolution. PubMed ID: 37393947
Our understanding of the evolutionary significance of ectoparasites in natural communities is limited by a paucity of information concerning the mechanisms and heritability of resistance to this ubiquitous group of organisms. This study reports the results of artificial selection for increasing ectoparasite resistance in replicate lines of Drosophila melanogaster derived from a field-fresh population. Resistance, as ability to avoid infestation by naturally co-occurring Gamasodes queenslandicus mites, increased significantly in response to selection, and realized heritability (s.e.) was estimated to be 0.11 (0.0090). Deployment of energetically expensive bursts of flight from the substrate was a main mechanism of host resistance that responded to selection, aligning with previously documented metabolic costs of fly behavioral defenses. Host body size, which affects parasitism rate in some fly-mite systems, was not shifted by selection. In contrast, resistant lines expressed significant reductions in larva-to-adult survivorship with increasing toxic (ammonia) stress, identifying an environmentally modulated pre-adult cost of resistance. Flies selected for resistance to G. queenslandicus were also more resistant to a different mite, Macrocheles subbadius, suggesting that genetic variation and a pleiotropic cost of broad-spectrum behavioral immunity against ectoparasites was documented. The results demonstrate significant evolutionary potential of resistance to an ecologically important class of parasites.
Santos, M. A., Antunes, M. A., Grandela, A., Quina, A. S., Santos, M., Matos, M. and Simoes, P. (2023). Slow and population specific evolutionary response to a warming environment. Sci Rep 13(1): 9700. PubMed ID: 37322066
Current rising temperatures are threatening biodiversity. It is therefore crucial to understand how climate change impacts male and female fertility and whether evolutionary responses can help in coping with heat stress can help in coping with he. Experimental evolution was used to study male and female fertility during the real-time evolution of two historically differentiated populations of Drosophila subobscura under different thermal selection regimes for 23 generations. This study aimed to (a) tease apart sex-specific differences in fertility after exposure to warming conditions during development, (b) test whether thermal selection can enhance fertility under thermal stress, and (c) address the role of historically distinct genetic backgrounds. Contrary to expectations, heat stress during development had a higher negative impact on female fertility than on male fertility. No clear evidence was found for enhanced fertility in males or females evolving under warming conditions. Population history had a clear impact on fertility response under thermal stress, particularly in males with those from lower latitude presenting better performance than their higher latitude counterparts. This study shows that the impact of thermal stress on fertility varies between traits, sexes, and genetic backgrounds. Incorporating these several levels of variation is crucial for a deeper understanding of how fertility evolves under climate change.
Santos, M. A., Antunes, M. A., Grandela, A., Quina, A. S., Santos, M., Matos, M. and Simoes, P. (2023). Slow and population specific evolutionary response to a warming environment. Sci Rep 13(1): 9700. PubMed ID: 37322066
Adaptation to increasingly warmer environments may be critical to avoid extinction. Whether and how these adaptive responses can arise is under debate. Though several studies have tackled evolutionary responses under different thermal selective regimes, very few have specifically addressed the underlying patterns of thermal adaptation under scenarios of progressive warming conditions. Also, considering how much past history affects such evolutionary response is critical. This study reports a long-term experimental evolution study addressing the adaptive response of Drosophila subobscura populations with distinct biogeographical history to two thermal regimes. The results showed clear differences between the historically differentiated populations, with adaptation to the warming conditions only evident in the low latitude populations. Furthermore, this adaptation was only detected after more than 30 generations of thermal evolution. These findings show some evolutionary potential of Drosophila populations to respond to a warming environment, but the response was slow and population specific, emphasizing limitations to the ability of ectotherms to adapt to rapid thermal shifts.
Robinson, C. E., Thyagarajan, H. and Chippindale, A. K. (2023). Evolution of reproductive isolation in a long-term evolution experiment with Drosophila melanogaster: 30 years of divergent life-history selection. Evolution 77(8): 1756-1768. PubMed ID: 37256776
This study asked if three decades and over 1,500 generations of divergent life-history selection on age at reproduction has resulted in the evolution of reproductive isolation (RI) between laboratory populations of Drosophila melanogaster. Tests were performed for premating, postmating-prezygotic, and postzygotic reproductive isolation between three replicate population pairs. Large, evolved differences in body size between selection treatments suggested the potential for prezygotic barriers driven by sexual selection or physical incompatibilities between the sexes. Although a simple prediction would be preference for larger size, creating directional isolation, the results from individual mate choice trials indicate that populations from both selection treatments show a marked bias towards homotypic mate choice; indicative of prezygotic RI driven by sexual selection or sexual conflict. Hybridization between the focal populations resulted in the production of viable adult flies with intermediate size and developmental traits. A suggestive but statistically nonsignificant trend of fitness decline was observed in the F2 generation of hybrids, but no significant evidence suggesting the evolution of postmating-prezygotic or postzygotic RI. These findings are in accord with extant literature that posits that premating RI evolves before postmating forms of RI.

Tuesday, December 12th - Adult Neural Structure, Development and Function

Raicu, A. M., Suresh, M. and Arnosti, D. N. (2023). A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor. bioRxiv. PubMed ID: 37292674
The C-terminal Binding Protein (CtBP) is a transcriptional corepressor that plays critical roles in development, tumorigenesis, and cell fate. CtBP proteins are structurally similar to alpha hydroxyacid dehydrogenases and additionally feature an unstructured C-terminal domain (CTD). The role of a possible dehydrogenase activity has been postulated for the corepressor, although in vivo substrates are unknown, but the functional significance of the CTD is unclear. In the mammalian system, CtBP proteins lacking the CTD are able to function as transcriptional regulators and oligomerize, putting into question the significance of the CTD for gene regulation. Yet, the presence of an unstructured CTD of ∼100 residues, including some short motifs, is conserved across Bilateria, indicating the importance of this domain. To study the in vivo functional significance of the CTD, the Drosophila melanogaster system was used. This system naturally expresses isoforms with the CTD (CtBP(L)), and isoforms lacking the CTD (CtBP(S)). The CRISPRi system was used to test dCas9-CtBP(S) and dCas9-CtBP(L) on diverse endogenous genes, to directly compare their transcriptional impacts in vivo. Interestingly, CtBP(S) was able to significantly repress transcription of the E2F2 and Mpp6 genes, while CtBP(L) had minimal impact, suggesting that the long CTD modulates CtBP's repression activity. In contrast, in cell culture, the isoforms behaved similarly on a transfected Mpp6 reporter. Thus, this study has identified context-specific effects of these two developmentally-regulated isoforms and proposes that differential expression of CtBP(S) and CtBP(L) may provide a spectrum of repression activity suitable for developmental programs.
Peebles, K. E., LaFever, K. S., Page-McCaw, P. S., Colon, S., Wang, D., Stricker, A. M., Ferrell, N., Bhave, G. and Page-McCaw, A. (2023). Analysis of Drosophila and mouse mutants reveals that Peroxidasin is required for tissue mechanics and full viability. bioRxiv. PubMed ID: 37503104
Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membrane that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved, indicating they are essential, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, mutants were analyzed in Drosophila, including a newly generated catalytic null, and found that homozygotes were mostly lethal with 13% viable escapers. A Mendelian analysis of mouse mutants shows a similar pattern, with homozygotes displaying ~50% lethality and ~50% escapers. Despite the strong mutations, the homozygous escapers had low but detectable levels of Col4 crosslinking, indicating that inefficient alternative mechanisms exist and that are probably responsible for the viable escapers. Further, fly mutants have phenotypes consistent with a decrease in stiffness. Interestingly, we found that even after adult basement membranes are assembled and crosslinked, Peroxidasin is still required to maintain stiffness. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.
Mattedi, F., Lloyd-Morris, E., Hirth, F. and Vagnoni, A. (2023). Optogenetic cleavage of the Miro GTPase reveals the direct consequences of real-time loss of function in Drosophila. PLoS Biol 21(8): e3002273. PubMed ID: 37590319
Miro GTPases control mitochondrial morphology, calcium homeostasis, and regulate mitochondrial distribution by mediating their attachment to the kinesin and dynein motor complex. It is not clear, however, how Miro proteins spatially and temporally integrate their function as acute disruption of protein function has not been performed. To address this issue, an optogenetic loss of function "Split-Miro" allele was developed for precise control of Miro-dependent mitochondrial functions in Drosophila. Rapid optogenetic cleavage of Split-Miro leads to a striking rearrangement of the mitochondrial network, which is mediated by mitochondrial interaction with the microtubules. Unexpectedly, this treatment did not impact the ability of mitochondria to buffer calcium or their association with the endoplasmic reticulum. While Split-Miro overexpression is sufficient to augment mitochondrial motility, sustained photocleavage shows that Split-Miro is surprisingly dispensable to maintain elevated mitochondrial processivity. In adult fly neurons in vivo, Split-Miro photocleavage affects both mitochondrial trafficking and neuronal activity. Furthermore, functional replacement of endogenous Miro with Split-Miro identifies its essential role in the regulation of locomotor activity in adult flies, demonstrating the feasibility of tuning animal behaviour by real-time loss of protein function.
Rhimi, M., Da Lage, J. L., Haser, R., Feller, G. and Aghajari, N. (2023). Structural and Functional Characterization of Drosophila melanogaster alpha-Amylase. Molecules 28(14). PubMed ID: 37513201
Insects rely on carbohydrates such as starch and glycogen as an energy supply for growth of larvae and for longevity. In this sense α-amylases have essential roles under extreme conditions, e.g., during nutritional or temperature stress, thereby contributing to survival of the insect. This makes them interesting targets for combating insect pests. Drosophila melanogaster α-amylase, DMA, which belongs to the glycoside hydrolase family 13, sub family 15, has been studied from an evolutionary, biochemical, and structural point of view. These studies revealed that the DMA enzyme is active over a broad temperature and pH range, which is in agreement with the fluctuating environmental changes with which the insect is confronted. Crystal structures disclosed a new nearly fully solvated metal ion, only coordinated to the protein via Gln263. This residue is only conserved in the subgroup of D. melanogaster and may thus contribute to the enzyme adaptive response to large temperature variations. Studies of the effect of plant inhibitors and the pseudo-tetrasaccharide inhibitor acarbose on DMA activity, underlined the important role of the so-called flexible loop on activity/inhibition, but also to suggest that the inhibition modes of the wheat inhibitors WI-1 and WI-3 on DMA, are likely different.
Nayak, S. R., Joseph, D., Hofner, G., Dakua, A., Athreya, A., Wanner, K. T., Kanner, B. I. and Penmatsa, A. (2023). Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism. Nat Struct Mol Biol 30(7): 1023-1032. PubMed ID: 37400654
The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) is presented at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, this study will enable the rational design of specific antiepileptics.
Murari, A., Rhooms, S. K., Vimal, D., Hossain, K. F. B., Saini, S., Villanueva, M., Schlame, M. and Owusu-Ansah, E. (2023). Phospholipids can regulate complex I assembly independent of their role in maintaining mitochondrial membrane integrity. Cell Rep 42(8): 112846. PubMed ID: 37516961
Several phospholipid (PL) molecules are intertwined with some mitochondrial complex I (CI) subunits in the membrane domain of CI, but their function is unclear. This study reports that when the Drosophila melanogaster ortholog of the intramitochondrial PL transporter, STARD7 (CG6565), is severely disrupted, assembly of the oxidative phosphorylation (OXPHOS) system is impaired, and the biogenesis of several CI subcomplexes is hampered. However, intriguingly, a restrained knockdown of STARD7 impairs the incorporation of NDUFS5 and NDUFA1 into the proximal part of the CI membrane domain without directly affecting the incorporation of subunits in the distal part of the membrane domain, OXPHOS complexes already assembled, or mitochondrial cristae integrity. Importantly, the restrained knockdown of STARD7 appears to induce a modest amount of cardiolipin remodeling, indicating that there could be some alteration in the composition of the mitochondrial phospholipidome. It is concluded that PLs can regulate CI biogenesis independent of their role in maintaining mitochondrial membrane integrity.

Tuesday, December 12th - Enzymes and Protein Expression, Evolution, Structure, and Function

Patel, A. A., Cardona, A. and Cox, D. N. (2023). Neural substrates of cold nociception in Drosophila larva. bioRxiv. PubMed ID: 37577520
Metazoans detect and differentiate between innocuous (non-painful) and/or noxious (harmful) environmental cues using primary sensory neurons, which serve as the first node in a neural network that computes stimulus specific behaviors to either navigate away from injury-causing conditions or to perform protective behaviors that mitigate extensive injury. The ability of an animal to detect and respond to various sensory stimuli depends upon molecular diversity in the primary sensors and the underlying neural circuitry responsible for the relevant behavioral action selection. Recent studies in Drosophila larvae have revealed that somatosensory class III multidendritic (CIII md) neurons function as multimodal sensors regulating distinct behavioral responses to innocuous mechanical and nociceptive thermal stimuli. Recent advances in circuit bases of behavior have identified and functionally validated Drosophila larval somatosensory circuitry involved in innocuous (mechanical) and noxious (heat and mechanical) cues. However, central processing of cold nociceptive cues remained unexplored. This study implicates multisensory integrators (Basins), premotor (Down-and-Back) and projection (A09e and TePns) neurons as neural substrates required for cold-evoked behavioral and calcium responses. Neural silencing of cell types downstream of CIII md neurons led to significant reductions in cold-evoked behaviors and neural co-activation of CIII md neurons plus additional cell types facilitated larval contraction (CT) responses. It was further demonstrated that optogenetic activation of CIII md neurons evokes calcium increases in these neurons. Collectively, this study demonstrates how Drosophila larvae process cold stimuli through functionally diverse somatosensory circuitry responsible for generating stimulus specific behaviors.
Prokhorenko, M. A. and Smyth, J. T. (2023). Astrocyte store-operated calcium entry is required for centrally mediated neuropathic pain. bioRxiv. PubMed ID: 37333230
Central sensitization is defined by nociceptive and somatosensory circuitry changes in the spinal cord leading to dysfunction of antinociceptive gamma-aminobutyric acid (GABA)ergic cells, amplification of ascending nociceptive signals, and hypersensitivity. Astrocytes are key mediators of the neurocircuitry changes that underlie central sensitization and neuropathic pain, and astrocytes respond to and regulate neuronal function through complex Ca (2+) signaling mechanisms. Ca (2+) release from astrocyte endoplasmic reticulum (ER) Ca (2+) stores via the inositol 1,4,5-trisphosphate receptor (IP(3)]R) is required for centrally mediated neuropathic pain; however recent evidence suggests the involvement of additional astrocyte Ca (2+) signaling mechanisms. Therefore this study investigated the role of astrocyte store-operated Ca (2+) entry (SOCE), which mediates Ca (2+) influx in response to ER Ca (2+) store depletion. Using an adult Drosophila melanogaster model of central sensitization based on thermal allodynia in response to leg amputation nerve injury, it was shown that astrocytes exhibit SOCE-dependent Ca (2+) signaling events three to four days following nerve injury. Astrocyte-specific suppression of Stim and Orai, the key mediators of SOCE Ca (2+) influx, completely inhibited the development of thermal allodynia seven days following injury, and also inhibited the loss of ventral nerve cord (VNC) GABAergic neurons that is required for central sensitization in flies. It was lastly shown that constitutive SOCE in astrocytes results in thermal allodynia even in the absence of nerve injury. These results collectively demonstrate that astrocyte SOCE is necessary and sufficient for central sensitization and development of hypersensitivity in Drosophila, adding key new understanding to the astrocyte Ca (2+) signaling mechanisms involved in chronic pain.
Patop, I. L., Anduaga, A. M., Bussi, I. L., Ceriani, M. F. and Kadener, S. (2023). Organismal landscape of clock cells and circadian gene expression in Drosophila. bioRxiv. PubMed ID: 37292867
Circadian rhythms time physiological and behavioral processes to 24-hour cycles. It is generally assumed that most cells contain self-sustained circadian clocks that drive circadian rhythms in gene expression that ultimately generating circadian rhythms in physiology. While those clocks supposedly act cell autonomously, current work suggests that in Drosophila some of them can be adjusted by the brain circadian pacemaker through neuropeptides, like the Pigment Dispersing Factor (PDF). Despite these findings and the ample knowledge of the molecular clockwork, it is still unknown how circadian gene expression in Drosophila is achieved across the body. This study used single-cell and bulk RNAseq data to identify cells within the fly that express core-clock components. Surprisingly, it was found that less than a third of the cell types in the fly express core-clock genes. Moreover, Lamina wild field (Lawf) and Ponx-neuro positive (Poxn) neurons were identified as putative new circadian neurons. In addition, several cell types were found that do not express core clock components but are highly enriched for cyclically expressed mRNAs. Strikingly, these cell types express the PDF receptor (Pdfr), suggesting that PDF drives rhythmic gene expression in many cell types in flies. Other cell types express both core circadian clock components and Pdfr, suggesting that in these cells, PDF regulates the phase of rhythmic gene expression. Together, these data suggest three different mechanisms generate cyclic daily gene expression in cells and tissues: canonical endogenous canonical molecular clock, PDF signaling-driven expression, or a combination of both.
Ravenscroft, T. A., Jacobs, A., Gu, M., Eberl, D. F. and Bellen, H. J. (2023). The Voltage-Gated Sodium Channel in Drosophila, Para, Localizes to Dendrites As Well As Axons in Mechanosensitive Chordotonal Neurons. eNeuro 10(6). PubMed ID: 37328295
The fruit fly Drosophila melanogaster has provided important insights into how sensory information is transduced by transient receptor potential (TRP) channels in the peripheral nervous system (PNS). However, TRP channels alone have not been able to completely model mechanosensitive transduction in mechanoreceptive chordotonal neurons (CNs). This study shows that, in addition to TRP channels, the sole voltage-gated sodium channel (Na(V)) in Drosophila, Para, is localized to the dendrites of CNs. Para is localized to the distal tip of the dendrites in all CNs, from embryos to adults, and is colocalized with the mechanosensitive TRP channels No mechanoreceptor potential C (NompC) and Inactive/Nanchung (Iav/Nan). Para localization also demarcates spike initiation zones (SIZs) in axons and the dendritic localization of Para is indicative of a likely dendritic SIZ in fly CNs. Para is not present in the dendrites of other peripheral sensory neurons. In both multipolar and bipolar neurons in the PNS, Para is present in a proximal region of the axon, comparable to the axonal initial segment (AIS) in vertebrates, 40-60 μm from the soma in multipolar neurons and 20-40 μm in bipolar neurons. Whole-cell reduction of para expression using RNAi in CNs of the adult Johnston's organ (JO) severely affects sound-evoked potentials (SEPs). However, the duality of Para localization in the CN dendrites and axons identifies a need to develop resources to study compartment-specific roles of proteins that will enable better understanding of Para's role in mechanosensitive transduction.
Pirogova, N. and Borst, A. (2023). Contrast normalization affects response time-course of visual interneurons. PLoS One 18(6): e0285686. PubMed ID: 37294743
In natural environments, light intensities and visual contrasts vary widely, yet neurons have a limited response range for encoding them. Neurons accomplish that by flexibly adjusting their dynamic range to the statistics of the environment via contrast normalization. The effect of contrast normalization is usually measured as a reduction of neural signal amplitudes, but whether it influences response dynamics is unknown. This study shows that contrast normalization in visual interneurons of the Drosophila melanogaster optic lobe not only suppresses the amplitude but also alters the dynamics of responses when a dynamic surround is present. A simple model is presented that qualitatively reproduces the simultaneous effect of the visual surround on the response amplitude and temporal dynamics by altering the cells' input resistance and, thus, their membrane time constant. In conclusion, single-cell filtering properties as derived from artificial stimulus protocols like white-noise stimulation cannot be transferred one-to-one to predict responses under natural conditions.
Puri, P., Wu, S. T., Su, C. Y. and Aljadeff, J. (2023). Shallow networks run deep: Peripheral preprocessing facilitates odor classification. bioRxiv. PubMed ID: 37546820
The mammalian brain implements sophisticated sensory processing algorithms along multilayered ('deep') neural-networks. Strategies that insects use to meet similar computational demands, while relying on smaller nervous systems with shallow architectures, remain elusive. Using Drosophila as a model, this study uncovered the algorithmic role of odor preprocessing by a shallow network of compartmentalized olfactory receptor neurons. Each compartment operates as a ratiometric unit for specific odor-mixtures. This computation arises from a simple mechanism: electrical coupling between two differently-sized neurons. Downstream synaptic connectivity is shaped to optimally leverage amplification of a hedonic value signal in the periphery. Furthermore, peripheral preprocessing is shown to markedly improve novel odor classification in a higher brain center. Together, this work highlights a far-reaching functional role of the sensory periphery for downstream processing. By elucidating the implementation of powerful computations by a shallow network, insights into general principles of efficient sensory processing algorithms were provided.

Friday, December 8th - Adult Development

Hopkins, B. R., Barmina, O. and Kopp, A. (2023). A single-cell atlas of the sexually dimorphic Drosophila foreleg and its sensory organs during development. PLoS Biol 21(6): e3002148. PubMed ID: 37379332
To respond to the world around them, animals rely on the input of a network of sensory organs distributed throughout the body. Distinct classes of sensory organs are specialized for the detection of specific stimuli such as strain, pressure, or taste. The features that underlie this specialization relate both to the neurons that innervate sensory organs and the accessory cells they comprise. To understand the genetic basis of this diversity of cell types, both within and between sensory organs, single-cell RNA sequencing was performed on the first tarsal segment of the male Drosophila melanogaster foreleg during pupal development. This tissue displays a wide variety of functionally and structurally distinct sensory organs, including campaniform sensilla, mechanosensory bristles, and chemosensory taste bristles, as well as the sex comb, a recently evolved male-specific structure. This study characterized the cellular landscape in which the sensory organs reside, identify a novel cell type that contributes to the construction of the neural lamella and resolved the transcriptomic differences among support cells within and between sensory organs. The genes that distinguish between mechanosensory and chemosensory neurons were identified, a combinatorial transcription factor code was resolved that defines 4 distinct classes of gustatory neurons and several types of mechanosensory neurons, and the expression of sensory receptor genes were matched to specific neuron classes. Collectively, this work identifies core genetic features of a variety of sensory organs and provides a rich, annotated resource for studying their development and function.
Rathore, S., Stahl, A., Benoit, J. B. and Buschbeck, E. K. (2023). Exploring the molecular makeup of support cells in insect camera eyes. bioRxiv. PubMed ID: 37503285
Animals generally have either compound eyes, which have evolved repeatedly in different invertebrates, or camera eyes, which have evolved many times across the animal kingdom. Both eye types include two important kinds of cells: photoreceptor cells, which can be excited by light, and non-neuronal support cells (SupCs), which provide essential support to photoreceptors. Only a handful of studies, primarily on the compound eyes of Drosophila melanogaster, have demonstrated molecular similarities in SupCs. D. melanogaster SupCs (Semper cells and primary pigment cells) are specialized eye glia that share several molecular similarities with certain vertebrate eye glia, including Müller glia. This led to speculation as to whether there are conserved molecular signatures of SupCs, even in functionally different eyes such as the image-forming larval camera eyes of the sunburst diving beetle Thermonectus marmoratus. To investigate this possibility, an in-depth comparative whole-tissue transcriptomics approach was used. Specifically, the larval principal camera eyes were dissected into SupC- and retina-containing regions and the respective transcriptomes were generated. This analysis revealed several conserved features of SupCs including enrichment of genes that are important for glial function (e.g. gap junction proteins such as innexin 3), glycogen production (glycogenin), and energy metabolism (glutamine synthetase 1 and 2). To evaluate the extent of conservation, the transcriptomes were compared with those of fly (Semper cells) and vertebrate (Müller glia) eye glia as well as respective retinas. T. marmoratus SupCs were found to have distinct genetic overlap with both fly and vertebrate eye glia. These results provide molecular evidence for the deep conservation of SupCs in addition to photoreceptor cells, raising essential questions about the evolutionary origin of eye-specific glia in animals.
Meyer, C., Drechsler, M., Meyer, H. and Paululat, A. (2023). Differentiation and function of cardiac valves in the adult Drosophila heart. J Exp Biol 226(13). PubMed ID: 37306013
Drosophila, like all insects, has an open circulatory system for the distribution of haemolymph and its components. The circulation of the haemolymph is essentially driven by the pumping activity of the linear heart. The heart is constructed as a tube into which the haemolymph is sucked and pumped forward by rhythmic contractions running from the posterior to the anterior, where it leaves the heart tube. The heart harbours cardiac valves to regulate flow directionality, with a single heart valve differentiating during larval development to separate the heart tube into two chambers. During metamorphosis, the heart is partially restructured, with the linear heart tube with one terminal wide-lumen heart chamber being converted into a linear four-chambered heart tube with three valves. As in all metazoan circulatory systems, the cardiac valves play an essential role in regulating the direction of blood flow. This study provides evidence that the valves in adult flies arise via transdifferentiation, converting lumen-forming contractile cardiomyocytes into differently structured valve cells. Interestingly, adult cardiac valves exhibit a similar morphology to their larval counterparts, but act differently upon heart beating. Applying calcium imaging in living specimens to analyse activity in valve cells, adult cardiac valves were shown to operate owing to muscle contraction. However, valve cell shape dynamics are altered compared with larval valves, which led the proposal of the current model of the opening and closing mechanisms in the fly heart.
Klipa, O., El Gammal, M. and Hamaratoglu, F. (2023). Elimination of aberrantly specified cell clones is independent of interfacial Myosin II accumulation. J Cell Sci 136(13). PubMed ID: 37309190
Spatial organization within an organ is essential and needs to be maintained during development. This is largely implemented via compartment boundaries that serve as barriers between distinct cell types. Biased accumulation of junctional non-muscle Myosin II along the interface between differently fated groups of cells contributes to boundary integrity and maintains its shape via increased tension. Using the Drosophila wing imaginal disc, whether interfacial tension driven by accumulation of Myosin is responsible for the elimination of aberrantly specified cells that would otherwise compromise compartment organization was tested. To this end, Myosin II levels were genetically reduced in three different patterns: in both wild-type and misspecified cells, only in misspecified cells, and specifically at the interface between wild-type and aberrantly specified cells. The recognition and elimination of aberrantly specified cells do not strictly rely on tensile forces driven by interfacial Myosin cables. Moreover, apical constriction of misspecified cells and their separation from wild-type neighbours occurred even when Myosin levels were greatly reduced. Thus, it is concluded that the forces that drive elimination of aberrantly specified cells are largely independent of Myosin II accumulation.
Ramezani, A., Britton, S., Zandi, R., Alber, M., Nematbakhsh, A. and Chen, W. (2023). A multiscale chemical-mechanical model predicts impact of morphogen spreading on tissue growth. NPJ Syst Biol Appl 9(1): 16. PubMed ID: 37210381
The exact mechanism controlling cell growth remains a grand challenge in developmental biology and regenerative medicine. The Drosophila wing disc tissue serves as an ideal biological model to study mechanisms involved in growth regulation. Most existing computational models for studying tissue growth focus specifically on either chemical signals or mechanical forces. This study developed a multiscale chemical-mechanical model to investigate the growth regulation mechanism based on the dynamics of a morphogen gradient. By comparing the spatial distribution of dividing cells and the overall tissue shape obtained in model simulations with experimental data of the wing disc, it is shown that the size of the domain of the Dpp morphogen is critical in determining tissue size and shape. A larger tissue size with a faster growth rate and more symmetric shape can be achieved if the Dpp gradient spreads in a larger domain. Together with Dpp absorbance at the peripheral zone, the feedback regulation that downregulates Dpp receptors on the cell membrane allows for further spreading of the morphogen away from its source region, resulting in prolonged tissue growth at a more spatially homogeneous growth rate.
Houtman, A., Gruber, S., Reisert, H., Amini, M., Fiore, C., Gonzalez, P., Han, V., Jazic, A., Kusupholnand, M., Miller, M., Nam, J., Wang, Z., Yu, Y., Dong, P., Oak, A. S. W., Sharma, A. and Spana, E. P. (2023). Characterization of the tilt (tt) phenotype in Drosophila melanogaster. MicroPubl Biol 2023. PubMed ID: 37193546
In the early 20th century, Calvin Bridges and Thomas Morgan identified a number of spontaneous mutations that displayed visible phenotypes in adult flies and subsequent analysis of these mutations over the past century have provided fundamental insights into subdisciplines of biology such as genetics, developmental, and cell biology. One of the mutations they identified in 1915 was named tilt (tt) and was described by Bridges and Morgan as having two visible phenotype characteristics in the wing. The wings were "held out at a wider angle from the body" and had a break in wing vein L3. Subsequent analysis of the tilt phenotype identified another phenotype: the wings were missing a varying number of campaniform sensilla on L3. Though Bridges and Morgan provided an ink drawing of the wing posture phenotype, only the vein and campaniform sensilla loss images have been published. This study confirmed and documented the tilt phenotypes that have been previously described. Yhe penetrance of these phenotypes was also shown: the vein break and the distinct outward wing posture have decreased since its discovery.

Thursday, December 7th - Disease Models

Rai, P., Ratnaparkhi, A. and Roy, J. K. (2023). Rab11 rescues muscle degeneration and synaptic morphology in the park(13)/+ Parkinson model of Drosophila melanogaster. Brain Res 1816: 148442. PubMed ID: 37302569
Mutation in parkin and pink1 is associated with Parkinson's disease (PD), the most common movement disorder characterized by muscular dysfunction. In a previous study, it was observed that Rab11, a member of the small Ras GTPase family, regulates the mitophagy pathway mediated by Parkin and Pink1 in the larval brain of the Drosophila PD model. sThis study describes that the expression and interaction of Rab11 in the PD model of Drosophila is highly conserved across different phylogenic groups. The loss of function in these two proteins, i.e., Parkin and Pink1, leads to mitochondrial aggregation. Rab11 loss of function results in muscle degeneration, movement disorder and synaptic morphological defects. Overexpression of Rab11 in park13 heterozygous mutant improves muscle and synaptic organization by reducing mitochondrial aggregations and improving cytoskeleton structural organization. The functional relationship was also shown between Rab11 and Brp, a pre-synaptic scaffolding protein, required for synaptic neurotransmission. Using park13 heterozygous mutant and pink1RNAi lines, this study showed reduced expression of Brp and consequently, there were synaptic dysfunctions including impaired synaptic transmission, decreased bouton size, increase in the bouton numbers, and the length of axonal innervations at the larval neuromuscular junction (NMJ). These synaptic alterations were rescued with the over-expression of Rab11 in the park13 heterozygous mutants. In conclusion, this work emphasizes the importance of Rab11 in rescuing muscle degeneration, movement dysfunction and synaptic morphology by preserving mitochondrial function in the PD model of Drosophila.
Molina-Mateo, D., Valderrama, B. P., Zarate, R. V., Hidalgo, S., Tamayo-Leiva, J., Soto-Gonzalez, A., Guerra-Ayala, S., Arriagada-Vera, V., Oliva, C., Diez, B. and Campusano, J. M. (2023). Kanamycin treatment in the pre-symptomatic stage of a Drosophila PD model prevents the onset of non-motor alterations. Neuropharmacology 236: 109573. PubMed ID: 37196855
Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor alterations, which is preceded by a prodromal stage where non-motor symptoms are observed. Over recent years, it has become evident that this disorder involves other organs that communicate with the brain like the gut. Importantly, the microbial community that lives in the gut plays a key role in this communication, the so-called microbiota-gut-brain axis. Alterations in this axis have been associated to several disorders including PD. This study proposed that the gut microbiota is different in the presymptomatic stage of a Drosophila model for PD, the Pink1B9 mutant fly, as compared to that observed in control animals. The results show this is the case: there is basal dysbiosis in mutant animals evidenced by substantial difference in the composition of midgut microbiotaz in 8-9 days old Pink1B9 mutant flies as compared with control animals. Further, young adult control and mutant flies were fed kanamycin, and motor and non-motor behavioral parameters were analyzed in these animals. Data show that kanamycin treatment induces the recovery of some of the non-motor parameters altered in the pre-motor stage of the PD fly model, while there is no substantial change in locomotor parameters recorded at this stage. On the other hand, the results show that feeding young animals the antibiotic, results in a long-lasting improvement of locomotion in control flies. These data support that manipulations of gut microbiota in young animals could have beneficial effects on PD progression and age-dependent motor impairments. This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".
Myers, R. R., John, A., Zhang, W., Zou, W. Q., Cembran, A. and Fernandez-Funez, P. (2023). Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila. J Biol Chem 299(7): 104881. PubMed ID: 37269948
Prion protein (PrP) misfolding is the key trigger in the devastating prion diseases. Yet the sequence and structural determinants of PrP conformation and toxicity are not known in detail. This study describes the impact of replacing Y225 in human PrP with A225 from rabbit PrP, an animal highly resistant to prion diseases. First, human PrP-Y225A was examined by molecular dynamics simulations. Next, human PrP was introduced in Drosophila, and the toxicity of human PrP-WT and Y225A was compared in the eye and in brain neurons. Y225A stabilizes the β2-α2 loop into a 3(10)-helix from six different conformations identified in WT and lowers hydrophobic exposure. Transgenic flies expressing PrP-Y225A exhibit less toxicity in the eye and in brain neurons and less accumulation of insoluble PrP. Overall, this study determined that Y225A lowers toxicity in Drosophila assays by promoting a structured loop conformation that increases the stability of the globular domain. These findings are significant because they shed light on the key role of distal α-helix 3 on the dynamics of the loop and the entire globular domain.
Patel, N., Alam, N., Libohova, K., Dulay, R., Todi, S. V. and Sujkowski, A. (2023). Phenotypic defects from the expression of wild-type and pathogenic TATA-Binding Proteins in new Drosophila models of Spinocerebellar Ataxia Type 17. G3 (Bethesda). PubMed ID: 37551423
Spinocerebellar Ataxia Type 17 (SCA17) is the most recently identified member of the polyglutamine (polyQ) family of disorders, resulting from abnormal CAG/CAA expansion in the TATA box binding protein (TBP), an initiation factor essential for of all eukaryotic transcription. A largely autosomal dominant inherited disease, SCA17 is unique in both its heterogeneous clinical presentation and low incidence of genetic anticipation, the phenomenon in which subsequent generations inherit longer polyQ expansions that yield earlier and more severe symptom onset. Like other polyQ disease family members, SCA17 patients experience progressive ataxia and dementia, and treatments are limited to preventing symptoms and increasing quality of life. This study reports two new Drosophila models that express human TBP with polyQ repeats in either wild-type or SCA17 patient range. TBP expression was found to have age- and tissue-specific effects on neurodegeneration, with polyQ-expanded SCA17 protein expression generally having more severe effects. In addition, SCA17 model flies accumulate more aggregation-prone TBP, with a greater proportion localizing to the nucleus. These new lines provide a new resource for the biochemical characterization of SCA17 pathology and the future identification of therapeutic targets.
Moron-Oset, J., Fischer, L. K., Carcole, M., Giblin, A., Zhang, P., Isaacs, A. M., Gronke, S. and Partridge, L. (2023). Toxicity of C9orf72-associated dipeptide repeat peptides is modified by commonly used protein tags. Life Sci Alliance 6(9). PubMed ID: 37308278
Hexanucleotide repeat expansions in the C9orf72 gene are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts of the expansions are translated into toxic dipeptide repeat (DPR) proteins. Most preclinical studies in cell and animal models have used protein-tagged polyDPR constructs to investigate DPR toxicity but the effects of tags on DPR toxicity have not been systematically explored. This study used Drosophila to assess the influence of protein tags on DPR toxicity. Tagging of 36 but not 100 arginine-rich DPRs with mCherry increased toxicity, whereas adding mCherry or GFP to GA100 completely abolished toxicity. FLAG tagging also reduced GA100 toxicity but less than the longer fluorescent tags. Expression of untagged but not GFP- or mCherry-tagged GA100 caused DNA damage and increased p62 levels. Fluorescent tags also affected GA100 stability and degradation. In summary, protein tags affect DPR toxicity in a tag- and DPR-dependent manner, and GA toxicity might be underestimated in studies using tagged GA proteins. Thus, including untagged DPRs as controls is important when assessing DPR toxicity in preclinical models.
Pandey, P., Wall, P., Lopez, S., Dubuisson, O., Zunica, E., Dantas, W., Kirwan, J., Axelrod, C. and Johnson, A. (2023). A familial natural short sleep mutation promotes healthy aging and extends lifespan in Drosophila. Res Sq. PubMed ID: 37398097
Sleep loss typically imposes negative effects on animal health. However, humans with a rare genetic mutation in the dec2 gene (dec2P384R) present an exception; these individuals sleep less without the usual effects associated with sleep deprivation. Thus, it has been suggested that the dec2P384R mutation activates compensatory mechanisms that allows these individuals to thrive with less sleep. To test this directly, a Drosophila model was used to study the effects of the dec2P384R mutation on animal health. Expression of human dec2P384R in fly sleep neurons was sufficient to mimic the short sleep phenotype and, remarkably, dec2P384R mutants lived significantly longer with improved health despite sleeping less. The improved physiological effects were enabled, in part, by enhanced mitochondrial fitness and upregulation of multiple stress response pathways. Moreover, evidence is provided that upregulation of pro-health pathways also contributes to the short sleep phenotype, and this phenomenon may extend to other pro-longevity models.

Wednesday, December 6th - Evolutionary Homologs of Drosophila Proteins

Mio, C., Baldan, F. and Damante, G. (2023). NK2 homeobox gene cluster: Functions and roles in human diseases. Genes Dis 10(5): 2038-2048. PubMed ID: 37492711
NK2 genes (NKX2 gene cluster in humans) encode for homeodomain-containing transcription factors that are conserved along the phylogeny. According to the most detailed classifications, vertebrate NKX2 genes are classified into two distinct families, NK2.1 and NK2.2. The former is constituted by NKX2-1 and NKX2-4 genes, which are homologous to the Drosophila scro gene; the latter includes NKX2-2 and NKX2-8 genes, which are homologous to the Drosophila vnd gene. Conservation of these genes is not only related to molecular structure and expression, but also to biological functions. In Drosophila and vertebrates, NK2 genes share roles in the development of ventral regions of the central nervous system. In vertebrates, NKX2 genes have a relevant role in the development of several other organs such as the thyroid, lung, and pancreas. Loss-of-function mutations in NKX2-1 and NKX2-2 are the monogenic cause of the brain-lung-thyroid syndrome and neonatal diabetes, respectively. Alterations in NKX2-4 and NKX2-8 genes may play a role in multifactorial diseases, autism spectrum disorder, and neural tube defects, respectively. NKX2-1, NKX2-2, and NKX2-8 are expressed in various cancer types as either oncogenes or tumor supressor genes. Several data indicate that evaluation of their expression in tumors has diagnostic and/or prognostic value.
Pintor, S., Lopez, A., Flores, D., Lozoya, B., Soti, B., Pokhrel, R., Negrete, J., Persans, M. W., Gilkerson, R., Gunn, B. and Keniry, M. (2023). FOXO1 promotes the expression of canonical WNT target genes in examined basal-like breast and glioblastoma multiforme cancer cells. FEBS Open Bio. PubMed ID: 37584250
Basal-like breast cancer (BBC) and glioblastoma multiforme (GBM) are aggressive cancers associated with poor prognosis. BBC and GBM have stem-cell-like gene expression signatures, which are in part driven by forkhead box O (FOXO) transcription factors. To gain further insight into the impact of FOXO1 in BBC, BT549 cells were treated with AS1842856 and RNA sequencing was performed. AS1842856 binds to unphosphorylated FOXO1 and inhibits its ability to directly bind to DNA. Gene Set Enrichment Analysis (GSEA) indicated that a set of WNT pathway target genes, including lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 7 (TCF7), were robustly induced after AS1842856 treatment. These same genes were also induced in GBM cell lines U87MG, LN18, LN229, A172 and DBTRG upon AS1842856 treatment. In contrast, follow-up RNA interference (RNAi) targeting of FOXO1 led to reduced LEF1 and TCF7 gene expression in BT549 and U87MG cells. In agreement with RNAi experiments, CRISPR Cas9-mediated FOXO1 disruption reduced the expression of canonical WNT genes LEF1 and TCF7 in U87MG cells. The loss of TCF7 gene expression in FOXO1 disruption mutants was restored by exogenous expression of the DNA-binding-deficient FOXO1-H215R. Therefore, FOXO1 induces TCF7 in a DNA-binding-independent manner, similar to other published FOXO1-activated genes such as TCF4 and hes family bHLH transcription factor 1 (HES1). This work demonstrates that FOXO1 promotes canonical WNT gene expression in examined BBC and GBM cells, similar to results found in Drosophila melanogaster, T-cell development and murine acute myeloid leukemia (AML) models.
Kushwaha, A. and Thakur, M. K. (2023). Suv39h1 Silencing Recovers Memory Decline in Scopolamine-Induced Amnesic Mouse Model. Mol Neurobiol. PubMed ID: 37626270
Histone post-translational modifications play an important role in the regulation of long-term memory and modulation of expression of neuronal immediate early genes (IEGs). The lysine methyltransferase KMT1A/ SU (VAR) 3-9) aids in the methylation of histone H3 at lysine 9. It was previously reported that age-related memory decline is associated with an increase in Suv39h1 expression in the hippocampus of male mice. The scopolamine-induced amnesic mouse model is a well-known animal model of memory impairment. The current study made an attempt to find a link between the changes in the H3K9 trimethylation pattern and memory decline during scopolamine-induced amnesia. It was followed by checking the effect of siRNA-mediated silencing of hippocampal Suv39h1 on memory and expression of neuronal IEGs. Scopolamine treatment significantly increased global levels of H3K9me3 and Suv39h1 in the amnesic hippocampus. Suv39h1 silencing in amnesic mice reduced H3K9me3 levels at the neuronal IEGs (Arc and BDNF) promoter, increased the expression of Arc and BDNF in the hippocampus, and improved recognition memory. Thus, these findings suggest that the silencing of Suv39h1 alone or in combination with other epigenetic drugs might be effective for treating memory decline during amnesia.
Radaszkiewicz, K. A., Sulcova, M., Kohoutkova, E. and Harnos, J. (2023). The role of prickle proteins in vertebrate development and pathology. Mol Cell Biochem. PubMed ID: 37358815
Prickle is an evolutionarily conserved family of proteins exclusively associated with planar cell polarity (PCP) signalling. This signalling pathway provides directional and positional cues to eukaryotic cells along the plane of an epithelial sheet, orthogonal to both apicobasal and left-right axes. Through studies in the fruit fly Drosophila, PCP signalling was learned to be manifested by the spatial segregation of two protein complexes, namely Prickle/Vangl and Frizzled/Dishevelled. While Vangl, Frizzled, and Dishevelled proteins have been extensively studied, Prickle has been largely neglected. This is likely because its role in vertebrate development and pathologies is still being explored and is not yet fully understood. The current review aims to address this gap by summarizing current knowledge on vertebrate Prickle proteins and to cover their broad versatility. Accumulating evidence suggests that Prickle is involved in many developmental events, contributes to homeostasis, and can cause diseases when its expression and signalling properties are deregulated. This review highlights the importance of Prickle in vertebrate development, discusses the implications of Prickle-dependent signalling in pathology, and points out the blind spots or potential links regarding Prickle, which could be studied further.
Lee, S., Abini-Agbomson, S., Perry, D. S., Goodman, A., Rao, B., Huang, M. Y., Diedrich, J. K., Moresco, J. J., Yates, J. R., 3rd, Armache, K. J. and Madhani, H. D. (2023). Intrinsic mesoscale properties of a Polycomb protein underpin heterochromatin fidelity. Nat Struct Mol Biol 30(7): 891-901. PubMed ID: 37217653
Little is understood about how the two major types of heterochromatin domains (HP1 and Polycomb) are kept separate. In the yeast Cryptococcus neoformans, the Polycomb-like protein Ccc1 prevents deposition of H3K27me3 at HP1 domains. This study shows that phase separation propensity underpins Ccc1 function. Mutations of the two basic clusters in the intrinsically disordered region or deletion of the coiled-coil dimerization domain alter phase separation behavior of Ccc1 in vitro and have commensurate effects on formation of Ccc1 condensates in vivo, which are enriched for PRC2. Notably, mutations that alter phase separation trigger ectopic H3K27me3 at HP1 domains. Supporting a direct condensate-driven mechanism for fidelity, Ccc1 droplets efficiently concentrate recombinant C. neoformans PRC2 in vitro whereas HP1 droplets do so only weakly. These studies establish a biochemical basis for chromatin regulation in which mesoscale biophysical properties play a key functional role.
Maseko, S. B., Brammerloo, Y., Van Molle, I., Sogues, A., Martin, C., Gorgulla, C., Plant, E., Olivet, J., Blavier, J., Ntombela, T., Delvigne, F., Arthanari, H., El Hajj, H., Bazarbachi, A., Van Lint, C., Salehi-Ashtiani, K., Remaut, H., Ballet, S., Volkov, A. N. and Twizere, J. C. (2023). Identification of small molecule antivirals against HTLV-1 by targeting the hDLG1-Tax-1 protein-protein interaction. Antiviral Res 217: 105675. PubMed ID: 37481039
Human T-cell leukemia virus type-1 (HTLV-1) is the first pathogenic retrovirus discovered in human. Although HTLV-1-induced diseases are well-characterized and linked to the encoded Tax-1 oncoprotein, there is currently no strategy to target Tax-1 functions with small molecules. This study analyzed the binding of Tax-1 to the human homolog of the drosophila discs large tumor supressor (hDLG1/SAP97), a multi-domain scaffolding protein involved in Tax-1-transformation ability. This study solved the structures of the PDZ binding motif (PBM) of Tax-1 in complex with the PDZ1 and PDZ2 domains of hDLG1 and assessed the binding of 10 million molecules by virtual screening. Among the 19 experimentally confirmed compounds, one systematically inhibited the Tax-1-hDLG1 interaction in different biophysical and cellular assays, as well as HTLV-1 cell-to-cell transmission in a T-cell model. Thus, this work demonstrates that interactions involving Tax-1 PDZ-domains are amenable to small-molecule inhibition, which provides a framework for the design of targeted therapies for HTLV-1-induced diseases.

Tuesday, December 5th - Enhancers and Transcriptional Regulation

Melo, D., Pallares, L. F. and Ayroles, J. F. (2023). Reassessing the modularity of gene co-expression networks using the Stochastic Block Model. bioRxiv. PubMed ID: 37398186
Finding communities in gene co-expression networks is a common first step toward extracting biological insight from such complex datasets. Most community detection algorithms expect genes to be organized into assortative modules, that is, groups of genes that are more associated with each other than with genes in other groups. While it is reasonable to expect that these modules exist, using methods that assume they exist a priori is risky, as it guarantees that alternative organizations of gene interactions will be ignored. This study asked: can meaningful communities be found without imposing a modular organization on gene co-expression networks, and how modular are these communities? For this, a recently developed community detection method was used, the weighted degree corrected stochastic block model (SBM), that does not assume that assortative modules exist. Instead, the SBM attempts to efficiently use all information contained in the co-expression network to separate the genes into hierarchically organized blocks of genes. Using RNA-seq gene expression data measured in two tissues derived from an outbred population of Drosophila melanogaster, this study shows that (a) the SBM is able to find ten times as many groups as competing methods, that (b) several of those gene groups are not modular, and that (c) the functional enrichment for non-modular groups is as strong as for modular communities. These results show that the transcriptome is structured in more complex ways than traditionally thought and that the long-standing assumption that modularity is the main driver of the structuring of gene co-expression networks should be revisited.
Puixeu, G., Macon, A. and Vicoso, B. (2023). Sex-specific estimation of cis and trans regulation of gene expression in heads and gonads of Drosophila melanogaster. G3 (Bethesda) 13(8). PubMed ID: 37259621
The regulatory architecture of gene expression is known to differ substantially between sexes in Drosophila, but most studies performed so far used whole-body data and only single crosses, which may have limited their scope to detect patterns that are robust across tissues and biological replicates. This study used allele-specific gene expression of parental and reciprocal hybrid crosses between 6 Drosophila melanogaster inbred lines to quantify cis- and trans-regulatory variation in heads and gonads of both sexes separately across 3 replicate crosses. The results suggest that female and male heads, as well as ovaries, have a similar regulatory architecture. On the other hand, testes display more and substantially different cis-regulatory effects, suggesting that sex differences in the regulatory architecture that have been previously observed may largely derive from testis-specific effects. The difference in cis-regulatory variation of genes across different levels of sex bias in gonads and heads was also examined. Consistent with the idea that intersex correlations constrain expression and can lead to sexual antagonism, this study found more cis variation in unbiased and moderately biased genes in heads. In ovaries, reduced cis variation is observed for male-biased genes, suggesting that cis variants acting on these genes in males do not lead to changes in ovary expression. Finally, the dominance patterns were examined of gene expression, and it was found that sex- and tissue-specific patterns of inheritance as well as trans-regulatory variation are highly variable across biological crosses, although these were performed in highly controlled experimental conditions. This highlights the importance of using various genetic backgrounds to infer generalizable patterns.
Kyrchanova, O., Sokolov, V., Tikhonov, M., Manukyan, G., Schedl, P. and Georgiev, P. (2023). Transcriptional Readthrough Interrupts Boundary Function in Drosophila. Int J Mol Sci 24(14). PubMed ID: 37511131
In higher eukaryotes, distance enhancer-promoter interactions are organized by topologically associated domains, tethering elements, and chromatin insulators/boundaries. While insulators/boundaries play a central role in chromosome organization, the mechanisms regulating their functions are largely unknown. This study has taken advantage of the well-characterized Drosophila bithorax complex (BX-C) to study one potential mechanism for controlling boundary function. The regulatory domains of BX-C are flanked by boundaries, which block crosstalk with their neighboring domains and also support long-distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, this study asked whether readthrough transcription (RT) can impact boundary function. For this purpose, advantage was taken of two BX-C boundary replacement platforms, Fab-7(attP50) and F2(attP), in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. Boundary elements, promoters, and polyadenylation signals arranged in different combinations were introduced and then assayed for boundary function. The results show that RT can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is therefore possible that RT may be a widely used mechanism to alter boundary function and regulation of gene expression.
McQuarrie, D. W. J., Read, A. M., Stephens, F. H. S., Civetta, A. and Soller, M. (2023). Indel driven rapid evolution of core nuclear pore protein gene promoters. Sci Rep 13(1): 8035. PubMed ID: 37198214
Nuclear pore proteins (Nups) prominently are among the few genes linked to speciation from hybrid incompatibility in Drosophila. These studies have focused on coding sequence evolution of Nup96 and Nup160 and show evidence of positive selection driving nucleoporin evolution. Intriguingly, channel Nup54 functionality is required for neuronal wiring underlying the female post-mating response induced by male-derived sex-peptide. A region of rapid evolution in the core promoter of Nup54 suggests a critical role for general transcriptional regulatory elements at the onset of speciation, but whether this is a general feature of Nup genes has not been determined. Consistent with findings for Nup54, additional channel Nup58 and Nup62 promoters also rapidly accumulate insertions/deletions (indels). Comprehensive examination of Nup upstream regions reveals that core Nup complex gene promoters accumulate indels rapidly. Since changes in promoters can drive changes in expression, these results indicate an evolutionary mechanism driven by indel accumulation in core Nup promoters. Compensation of such gene expression changes could lead to altered neuronal wiring, rapid fixation of traits caused by promoter changes and subsequently the rise of new species. Hence, the nuclear pore complex may act as a nexus for species-specific changes via nucleo-cytoplasmic transport regulated gene expression.
Nowling, R. J., Njoya, K., Peters, J. G. and Riehle, M. M. (2023). Prediction accuracy of regulatory elements from sequence varies by functional sequencing technique. Front Cell Infect Microbiol 13: 1182567. PubMed ID: 37600946
In this study machine learning models were employed to evaluate the accuracy with which cis-regulatory elements identified by various commonly used sequencing techniques can be predicted by their underlying sequence alone to distinguish between cis-regulatory activity that is reflective of sequence content versus secondary processes. Models trained and evaluated on D. melanogaster sequences identified through DNase-seq and STARR-seq are significantly more accurate than models trained on sequences identified by H3K4me1, H3K4me3, and H3K27ac ChIP-seq, FAIRE-seq, and ATAC-seq. These results suggest that the activity detected by DNase-seq and STARR-seq can be largely explained by underlying DNA sequence, independent of secondary processes. Experimentally, a subset of DNase-seq and H3K4me1 ChIP-seq sequences were tested for enhancer activity using luciferase assays and compared with previous tests performed on STARR-seq sequences. The experimental data indicated that STARR-seq sequences are substantially enriched for enhancer-specific activity, while the DNase-seq and H3K4me1 ChIP-seq sequences are not. Taken together, these results indicate that the DNase-seq approach identifies a broad class of regulatory elements of which enhancers are a subset and the associated data are appropriate for training models for detecting regulatory activity from sequence alone, STARR-seq data are best for training enhancer-specific sequence models, and H3K4me1 ChIP-seq data are not well suited for training and evaluating sequence-based models for cis-regulatory element prediction.
Mohana, G., Dorier, J., Li, X., Mouginot, M., Smith, R. C., Malek, H., Leleu, M., Rodriguez, D., Khadka, J., Rosa, P., Cousin, P., Iseli, C., Restrepo, S., Guex, N., McCabe, B. D., Jankowski, A., Levine, M. S. and Gambetta, M. C. (2023). Chromosome-level organization of the regulatory genome in the Drosophila nervous system. sCell. PubMed ID: 37536338
Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. This study presents evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. Evidence is presented that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations.

Monday, December 4th - Embryonic Development

Niloy, R. A., Holcomb, M. C., Thomas, J. H. and Blawzdziewicz, J. (2023). The mechanics of cephalic furrow formation in the Drosophila embryo. Biophys J. PubMed ID: 37571824
Cephalic furrow formation (CFF) is a major morphogenetic movement during gastrulation in Drosophila melanogaster embryos that gives rise to a deep, transitory epithelial invagination. Recent studies have identified the individual cell shape changes that drive the initiation and progression phases of CFF; however, the underlying mechanics are not yet well understood. During the progression phase, the furrow deepens as columnar cells from both the anterior and posterior directions fold inwards rotating by 90°. To analyze the mechanics of this process, this study has developed an advanced two-dimensional lateral vertex model that includes multinode representation of cellular membranes and allows capturing of the membrane curvature associated with pressure variation. These investigations reveal some key potential mechanical features of CFF, as follows. When cells begin to roll over the cephalic furrow cleft, they become wedge shaped as their apical cortices and overlying membranes expand, lateral cortices and overlying membranes release tension, internal pressures drop, and basal cortices and membranes contract. Then, cells reverse this process by shortening apical cortices and membranes, increasing lateral tension, and causing internal pressures to rise. Since the basal membranes expand, the cells recover their rotated columnar shape once in the furrow. Interestingly, these findings indicate that the basal membranes may be passively reactive throughout the progression phase. It was also found that the smooth rolling of cells over the cephalic furrow cleft necessitates that internalized cells provide a solid base through high levels of membrane tension and internal pressure, which allows the transmission of tensile force that pulls new cells into the furrow. These results led to the suggestion that CFF helps to establish a baseline tension across the apical surface of the embryo to facilitate cellular coordination of other morphogenetic movements via mechanical stress feedback mechanisms.
Ogneva, I. V. (2023). The Mechanoreception in Drosophila melanogaster Oocyte under Modeling Micro- and Hypergravity. Cells 12(14). PubMed ID: 37508484
The hypothesis about the role of the cortical cytoskeleton as the primary mechanosensor was tested. Drosophila melanogaster oocytes were exposed to simulated microgravity (by 3D clinorotation in random directions with 4 rotations per minute-sμg group) and hypergravity at the 2 g level (by centrifugal force from one axis rotation-hg group) for 30, 90, and 210 min without and with cytochalasin B, colchicine, acrylamide, and calyculin A. Cell stiffness was measured by atomic force microscopy, protein content in the membrane and cytoplasmic fractions by Western blotting, and cellular respiration by polarography. The obtained results indicate that the stiffness of the cortical cytoskeleton of Drosophila melanogaster oocytes decreases in simulated micro- (after 90 min) and hypergravity (after 30 min), possibly due to intermediate filaments. The cell stiffness recovered after 210 min in the hg group, but intact microtubules were required for this. Already after 30 min of exposure to sμg, the cross-sectional area of oocytes decreased, which indicates deformation, and the singed protein, which organizes microfilaments into longitudinal bundles, diffused from the cortical cytoskeleton into the cytoplasm. Under hg, after 30 min, the cross-sectional area of the oocytes increased, and the proteins that organize filament networks, alpha-actinin and spectrin, diffused from the cortical cytoskeleton.
Ho, E. K., Oatman, H. R., McFann, S. E., Yang, L., Johnson, H. E., Shvartsman, S. Y. and Toettcher, J. E. (2023). Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo. Development. PubMed ID: 37602510
Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. This study use optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. High or low doses of ERK signaling produce transient or sustained byn expression, respectively. While tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. It was further shown that "blurring" of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, this study provides a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.
Brauns, F., Claussen, N. H., Wieschaus, E. F. and Shraiman, B. I. (2023). Epithelial flow by controlled transformation of internal force-balance geometry. bioRxiv. PubMed ID: 37398061
Shape changes of epithelia during animal development, such as convergent extension, are achieved through concerted mechanical activity of individual cells. While much is known about the corresponding large scale tissue flow and its genetic drivers, the question of cell-scale coordination remains open. It is proposed that this coordination can be understood in terms of mechanical interactions and instantaneous force balance within the tissue. Using whole embryo imaging data for Drosophila gastrulation, this study exploited the relation between balance of local cortical tension forces and cell geometry. This unveils how local positive feedback on active tension and passive global deformations account for coordinated cell rearrangements. A model was developed that bridges the cell and tissue scale dynamics and predicts the dependence of total tissue extension on initial anisotropy and hexagonal order of the cell packing. This study provides general insight into the encoding of global tissue shape in local cell-scale activity.
Huang, X., Fu, Y., Lee, H., Zhao, Y., Yang, W., van de Leemput, J. and Han, Z. (2023). Single-cell profiling of the developing embryonic heart in Drosophila. Development 150(16). PubMed ID: 37526610
Drosophila is an important model for studying heart development and disease. Yet, single-cell transcriptomic data of its developing heart have not been performed. This study reports single-cell profiling of the entire fly heart using ~3000 Hand-GFP embryos collected at five consecutive developmental stages, ranging from bilateral migrating rows of cardiac progenitors to a fused heart tube. The data revealed six distinct cardiac cell types in the embryonic fly heart: cardioblasts, both Svp+ and Tin+ subtypes; and five types of pericardial cell (PC) that can be distinguished by four key transcription factors (Eve, Odd, Ct and Tin) and include the newly described end of the line PC. Notably, the embryonic fly heart combines transcriptional signatures of the mammalian first and second heart fields. Using unique markers for each heart cell type, this study defined their number and location during heart development to build a comprehensive 3D cell map. These data provide a resource to track the expression of any gene in the developing fly heart, which can serve as a reference to study genetic perturbations and cardiac diseases.
Jiang, J. and Aegerter, C. M. (2023). An integrated vertex model of the mesoderm invagination during the embryonic development of Drosophila. J Theor Biol 572: 111581. PubMed ID: 37481232
The mesoderm invagination of the Drosophila embryo is known as an archetypal morphogenic process. To explore the roles of the active cellular forces and the regulation of these forces, this study developed an integrated vertex model that combines the regulation of morphogen expression with cell movements and tissue mechanics. The results suggest that a successful furrow formation requires an apical tension gradient, decreased basal tension, and increased lateral tension, which corresponds to apical constriction, basal expansion, and apicobasal shortening respectively. The model also considers the mechanical feedback which leads to an ectopic twist expression with external compression as observed in experiments.The model predicts that ectopic invagination could happen if an external compressive gradient is applied.

Friday, December 1st - Adult Physiology and Metabolism

Nozawa, N., Noguchi, M., Shinno, K., Saito, T., Asada, A., Ishii, T., Takahashi, K., Ishizuka, M. and Ando, K. (2023). 5-Aminolevulinic acid bypasses mitochondrial complex I deficiency and corrects physiological dysfunctions in Drosophila. Hum Mol Genet 32(16): 2611-2622. PubMed ID: 37364055
Complex I (CI) deficiency in mitochondrial oxidative phosphorylation (OXPHOS) is the most common cause of mitochondrial diseases, and limited evidence-based treatment options exist. Although CI provides the most electrons to OXPHOS, complex II (CII) is another entry point of electrons. Enhancement of this pathway may compensate for a loss of CI; however, the effects of boosting CII activity on CI deficiency are unclear at the animal level. 5-Aminolevulinic acid (5-ALA) is a crucial precursor of heme, which is essential for CII, complex III, complex IV (CIV) and cytochrome c activities. This study shows that feeding a combination of 5-ALA hydrochloride and sodium ferrous citrate (5-ALA-HCl + SFC) increases ATP production and suppresses defective phenotypes in Drosophila with CI deficiency. Knockdown of sicily, a Drosophila homolog of the critical CI assembly protein NDUFAF6, caused CI deficiency, accumulation of lactate and pyruvate and detrimental phenotypes such as abnormal neuromuscular junction development, locomotor dysfunctions and premature death. 5-ALA-HCl + SFC feeding increased ATP levels without recovery of CI activity. The activities of CII and CIV were upregulated, and accumulation of lactate and pyruvate was suppressed. 5-ALA-HCl + SFC feeding improved neuromuscular junction development and locomotor functions in sicily-knockdown flies. These results suggest that 5-ALA-HCl + SFC shifts metabolic programs to cope with CI deficiency. Bullet outline 5-Aminolevulinic acid (5-ALA-HCl + SFC) increases ATP production in flies with complex I deficiency.5-ALA-HCl + SFC increases the activities of complexes II and IV.5-ALA-HCl + SFC corrects metabolic abnormalities and suppresses the detrimental phenotypes caused by complex I deficiency.
Poliacikova, G., Barthez, M., Rival, T., Aouane, A., Luis, N. M., Richard, F., Daian, F., Brouilly, N., Schnorrer, F., Maurel-Zaffran, C., Graba, Y. and Saurin, A. J. (2023). M1BP is an essential transcriptional activator of oxidative metabolism during Drosophila development. Nat Commun 14(1): 3187. PubMed ID: 37268614
Oxidative metabolism is the predominant energy source for aerobic muscle contraction in adult animals. How the cellular and molecular components that support aerobic muscle physiology are put in place during development through their transcriptional regulation is not well understood. Using the Drosophila flight muscle model, this study shows that the formation of mitochondria cristae harbouring the respiratory chain is concomitant with a large-scale transcriptional upregulation of genes linked with oxidative phosphorylation (OXPHOS) during specific stages of flight muscle development. It was further demonstrated, using high-resolution imaging, transcriptomic and biochemical analyses, that Motif-1-binding protein (M1BP) transcriptionally regulates the expression of genes encoding critical components for OXPHOS complex assembly and integrity. In the absence of M1BP function, the quantity of assembled mitochondrial respiratory complexes is reduced and OXPHOS proteins aggregate in the mitochondrial matrix, triggering a strong protein quality control response. This results in isolation of the aggregate from the rest of the matrix by multiple layers of the inner mitochondrial membrane, representing a previously undocumented mitochondrial stress response mechanism. Together, this study provides mechanistic insight into the transcriptional regulation of oxidative metabolism during Drosophila development and identifies M1BP as a critical player in this process.
Oliveras-Canellas, N., Castells-Nobau, A., de la Vega-Correa, L., Latorre-Luque, J., Motger-Alberti, A., Arnoriaga-Rodriguez, M., Garre-Olmo, J., Zapata-Tona, C., Coll-Martínez, C., Ramio-Torrenta, L., Moreno-Navarrete, J. M., Puig, J., Villarroya, F., Ramos, R., Casado-Anguera, V., Martin-Garcia, E., Maldonado, R., Mayneris-Perxachs, J. and Fernandez-Real, J. M. (2023). Adipose tissue coregulates cognitive function. Sci Adv 9(32): eadg4017. PubMed ID: 37566655
Obesity is associated with cognitive decline. Recent observations in mice propose an adipose tissue (AT)-brain axis. This study identified 188 genes from RNA sequencing of AT in three cohorts that were associated with performance in different cognitive domains. These genes were mostly involved in synaptic function, phosphatidylinositol metabolism, the complement cascade, anti-inflammatory signaling, and vitamin metabolism. These findings were translated into the plasma metabolome. The circulating blood expression levels of most of these genes were also associated with several cognitive domains in a cohort of 816 participants. Targeted misexpression of candidate gene ortholog in the Drosophila fat body significantly altered flies memory and learning. Among them, down-regulation of the neurotransmitter release cycle-associated gene SLC18A2 improved cognitive abilities in Drosophila and in mice. Up-regulation of RIMS1 in Drosophila fat body enhanced cognitive abilities. Current results show previously unidentified connections between AT transcriptome and brain function in humans, providing unprecedented diagnostic/therapeutic targets in AT.
Oh, Y. and Suh, G. S. B. (2023). Starvation-induced sleep suppression requires the Drosophila brain nutrient sensor. J Neurogenet 37(1-2): 70-77. PubMed ID: 37267057
Animals increase their locomotion activity and reduce sleep duration under starved conditions. This suggests that sleep and metabolic status are closely interconnected. The nutrient and hunger sensors in the Drosophila brain, including diuretic hormone 44 (DH44)-, CN-, and cupcake-expressing neurons, detect circulating glucose levels in the internal milieu, regulate the insulin and glucagon secretion and promote food consumption. Food deprivation is known to reduce sleep duration, but a potential role mediated by the nutrient and hunger sensors in regulating sleep and locomotion activity remains unclear. This study shows that DH44 neurons are involved in regulating starvation-induced sleep suppression, but CN neurons or cupcake neurons may not be involved in regulating starvation-induced sleep suppression or baseline sleep patterns. Inactivation of DH44 neurons resulted in normal daily sleep durations and patterns under fed conditions, whereas it ablated sleep reduction under starved conditions. Inactivation of CN neurons or cupcake neurons, which were proposed to be nutrient and hunger sensors in the fly brain, did not affect sleep patterns under both fed and starved conditions. It is proposed that the glucose-sensing DH44 neurons play an important role in mediating starvation-induced sleep reduction.
Medeiros, M. J., Seo, L., Macias, A., Price, D. K. and Yew, J. Y. (2023). Bacterial and fungal components of the gut microbiome have distinct, sex-specific roles in Hawaiian Drosophila reproduction. bioRxiv. PubMed ID: 37503295
Gut microbiomes provide numerous physiological benefits for host animals. The role of bacterial members of microbiomes in host physiology is well-documented. However, much less is known about the contributions and interactions of fungal members of the microbiome even though fungi are significant components of many microbiomes, including those of humans and insects. This study used antibacterial and antifungal drugs to manipulate the gut microbiome of a Hawaiian picture-wing Drosophila species, D. grimshawi, and identified distinct, sex-specific roles for the bacteria and fungi in microbiome community stability and reproduction. Female oogenesis, fecundity and mating drive were significantly diminished when fungal communities were suppressed. By contrast, male fecundity was more strongly affected by bacterial but not fungal populations. For males and females, suppression of both bacteria and fungi severely reduced fecundity and altered fatty acid levels and composition, implicating the importance of interkingdom interactions on reproduction and lipid metabolism. Overall, these results reveal that bacteria and fungi have distinct, sexually-dimorphic effects on host physiology and interkingdom dynamics in the gut help to maintain microbiome community stability and enhance reproduction.
Pham, T. C. P., Dollet, L., Ali, M. S., Raun, S. H., Møller, L. L. V., Jafari, A., Ditzel, N., Andersen, N. R., Fritzen, A. M., Gerhart-Hines, Z., Kiens, B., Suomalainen, A., Simpson, S. J., Salling Olsen, M., Kieser, A., Schjerling, P., Nieminen, A. I., Richter, E. A., Havula, E. and Sylow, L. (2023. TNIK is a conserved regulator of glucose and lipid metabolism in obesity. Sci Adv 9(32): eadf7119. PubMed ID: 37556547
Obesity and type 2 diabetes (T2D) are growing health challenges with unmet treatment needs. Traf2- and NCK-interacting protein kinase (TNIK) is a recently identified obesity- and T2D-associated gene with unknown functions. This study shows that TNIK governs lipid and glucose homeostasis in Drosophila and mice. Loss of the Drosophila ortholog of TNIK, misshapen, altered the metabolite profiles and impaired de novo lipogenesis in high sugar-fed larvae. Tnik knockout mice exhibited hyperlocomotor activity and were protected against diet-induced fat expansion, insulin resistance, and hepatic steatosis. The improved lipid profile of Tnik knockout mice was accompanied by enhanced skeletal muscle and adipose tissue insulin-stimulated glucose uptake and glucose and lipid handling. Using the T2D Knowledge Portal and the UK Biobank, associations were observed of TNIK variants with blood glucose, HbA1c, body mass index, body fat percentage, and feeding behavior. These results define an untapped paradigm of TNIK-controlled glucose and lipid metabolism.
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