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Current papers in developmental biology and gene function

Tuesday April 22st - Behavior

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>Easwaran, S., Montell, D. J. (2024). A genome-wide association study implicates the olfactory system in Drosophila melanogaster diapause-associated lifespan extension and fecundity. bioRxiv, PubMed ID: 39005458 Summary: The effects of environmental stress on animal life are gaining importance with climate change. Diapause is a dormancy program that occurs in response to an adverse environment, followed by resumption of development and reproduction upon the return of favorable conditions. Diapause is a complex trait, so this study leveraged the Drosophila genetic reference panel (DGRP) lines and conducted a Genome-Wide Association Study (GWAS) to characterize the genetic basis of diapause. Post-diapause and non-diapause fecundity was assessed pacross 193 DGRP lines. GWAS revealed 546 genetic variants, encompassing single nucleotide polymorphisms, insertions and deletions associated with post-diapause fecundity. 291 candidate diapause-associated genes were identified, 40 of which had previously been associated with diapause. 89 of the candidates were associated with more than one SNP. Gene network analysis indicated that the diapause-associated genes were primarily linked to neuronal and reproductive system development. Similarly, comparison with results from other fly GWAS revealed the greatest overlap with olfactory-behavior-associated and fecundity-and-lifespan-associated genes. An RNAi screen of the top candidates identified two neuronal genes, Dip-γ and Scribbler, to be required during recovery for post-diapause fecundity. The genetic analysis was complemented with a test of which neurons are required for successful diapause. Athough amputation of the antenna had little to no effect on non-diapause lifespan, it reduced diapause lifespan and postdiapause fecundity. It was further shown that olfactory receptor neurons and temperature-sensing neurons are required for successful recovery from diapause. These results provide insights into the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila .

Haustein, M., Blanke, A., Bockemuhl, T., Buschges, A. (2024). A leg model based on anatomical landmarks to study 3D joint kinematics of walking in Drosophila melanogaster. Frontiers in bioengineering and biotechnology, 12:1357598 PubMed ID: 38988867 Summary: fWalking is the most common form of how animals move on land. The model organism Drosophila melanogaster has become increasingly popular for studying how the nervous system controls behavior in general and walking in particular. Despite recent advances in tracking and modeling leg movements of walking Drosophila in 3D, there are still gaps in knowledge about the biomechanics of leg joints due to the tiny size of fruit flies. For instance, the natural alignment of joint rotational axes was largely neglected in previous kinematic analyses. This study therefore presents a detailed kinematic leg model in which not only the segment lengths but also the main rotational axes of the joints were derived from anatomical landmarks, namely, the joint condyles. The model with natural oblique joint axes is able to adapt to the 3D leg postures of straight and forward walking fruit flies with high accuracy. When the model was compared to an orthogonalized version, it was observed that the model showed a smaller error as well as differences in the used range of motion (ROM), highlighting the advantages of modeling natural rotational axes alignment for the study of joint kinematics. It was further found that the kinematic profiles of front, middle, and hind legs differed in the number of required degrees of freedom as well as their contributions to stepping, time courses of joint angles, and ROM. These findings provide deeper insights into the joint kinematics of walking in Drosophila, and, additionally, will help to develop dynamical, musculoskeletal, and neuromechanical simulations.

He, Y., Ding, Y., Gong, C., Zhou, J., Gong, Z. (2024). The tail segments are required by the performance but not the accomplishment of various modes of Drosophila larval locomotion. Behav Brain Res, 471:115074 PubMed ID: 38825023 Summary: The tail plays important roles in locomotion control in many animals. But in animals with multiple body segments, the roles of the hind body segments and corresponding innervating neurons in locomotion control are not clear. Using the Drosophila larva as the model animal, this study investigated the roles of the posterior terminal segments in various modes of locomotion and found that they participate in all of them. In forward crawling, paralysis of the larval tail by blocking the Abdb-Gal4 labeled neurons in the posterior segments of VNC led to a slower locomotion speed but did not prevent the initiation of forward peristalsis. In backward crawling, larvae with the Abdb-Gal4 neurons inhibited were unable to generate effective displacement although waves of backward peristalsis could be initiated and persist. In head swing where the movement of the tail is not obvious, disabling the larval tail by blocking Abdb-Gal4 neurons led to increased bending amplitude upon touching the head. In the case of larval lateral rolling, larval tail paralysis by inhibition of Abdb-Gal4 neurons did not prevent the accomplishment of rolling, but resulted in slower rolling speed. This work reveals that the contribution of Drosophila larval posterior VNC segments and corresponding body segments in the tail to locomotion is comprehensive but could be compensated at least partially by other body segments. It is suggested that the decentralization in locomotion control with respect to animal body parts helps to maintain the robustness of locomotion in multi-segment animals.

Canic, T., Lopez, J., Ortiz-Vega, N., Zhai, R. G., Syed, S. (2024). High-resolution, high-throughput analysis of Drosophila geotactic behavior. bioRxiv, PubMed ID: 38895419 Summary: Drosophila innate response to gravity, geotaxis, has been previously used to assess the impact of aging and disease on motor performance. Despite its rich history, fly geotaxis continues to be largely measured manually and assessed through simplistic metrics. The manual nature of this assay introduces substantial experimental variability while simplistic metrics provide limited analytic insights into the behavior. To address these shortcomings, this study has constructed a fully automated, programable apparatus, and developed a multi-object tracking software capable of following sub-second movements of individual flies, thus allowing reproducible, detailed, and quantitative analysis of geotactic behavior. The apparatus triggers and monitors geotaxis of 10 fly cohorts simultaneously, with each cohort consisting of up to 7 flies. The tracking program isolates cohorts and records individual fly coordinate outputs allowing for simultaneous multi-group, multi-fly tracks per experiment, greatly improving throughput and resolution. The algorithm tracks individual flies during the entire run with ~97% accuracy, yielding detailed climbing curve, speed, and movement direction with 1/30 second resolution. This tracking also allows the construction of multi-variable metrics and the detection of transitory movement phenotypes, such as slips and falls, which have thus far been neglected in geotaxis studies due to limited spatio-temporal resolution. Through a combination of automation and robust tracking, the platform is therefore poised to advance Drosophila geotaxis assay into a comprehensive assessment of locomotor behavior.

Pajusalu, M., Seager, S., Huang, J., Petkowski, J. J. (2024). A qualitative assessment of limits of active flight in low density atmospheres. Sci Rep, 14(1):13823 PubMed ID: 38879676 Summary: Exoplanet atmospheres are expected to vary significantly in thickness and chemical composition, leading to a continuum of differences in surface pressure and atmospheric density. This variability is exemplified within our Solar System, where the four rocky planets exhibit surface pressures ranging from 1 nPa on Mercury to 9.2 MPa on Venus. The direct effects and potential challenges of atmospheric pressure and density on life have rarely been discussed. For instance, atmospheric density directly affects the possibility of active flight in organisms, a critical factor since without it, dispersing across extensive and inhospitable terrains becomes a major limitation for the expansion of complex life. This paper proposes the existence of a critical atmospheric density threshold below which active flight is unfeasible, significantly impacting biosphere development. To qualitatively assess this threshold and differentiate it from energy availability constraints, the limits of active flight on Earth, using the common fruit fly, Drosophila melanogaster, as a model organism.Drosophila melanogaster was subjected to various atmospheric density scenarios, and rprevious data on flight limitations were reviewed. The observations show that flies in an N(2)-enriched environment recover active flying abilities more efficiently than those in a helium-enriched environment, highlighting behavioral differences attributable to atmospheric density vs. oxygen deprivation.

Rauscher, M. J., Fox, J. L. (2024). Asynchronous haltere input drives specific wing and head movements in Drosophila. Proceedings Biological sciences, 291(2024):20240311 PubMed ID: 38864337 Summary: Halteres are multifunctional mechanosensory organs unique to the true flies (Diptera). A set of reduced hindwings, the halteres beat at the same frequency as the lift-generating forewings and sense inertial forces via mechanosensory campaniform sensilla. Though haltere ablation makes stable flight impossible, the specific role of wing-synchronous input has not been established. Using small iron filings attached to the halteres of tethered flies and an alternating electromagnetic field, the wings and halteres of flying Drosophila were experimentally decoupled, and the resulting changes in wingbeat amplitude and head orientation were observed. Asynchronous haltere input results in fast amplitude changes in the wing (hitches), but does not appreciably move the head. In multi-modal experiments, wing and gaze optomotor responses were disrupted differently by asynchronous input. These effects of wing-asynchronous haltere input suggest that specific sensory information is necessary for maintaining wing amplitude stability and adaptive gaze control.

Monday April 21st - Adult Physiology and Metabolism

Brand, C. L., Oliver, G. T., Farkas, I. Z., Buszczak, M., Levine, M. T. (2024). Recurrent Duplication and Diversification of a Vital DNA Repair Gene Family Across Drosophila. Mol Biol Evol, 41(6) PubMed ID: 38865490 Summary: Maintaining genome integrity is vital for organismal survival and reproduction. Essential, broadly conserved DNA repair pathways actively preserve genome integrity. However, many DNA repair proteins evolve adaptively. Ecological forces like UV exposure are classically cited drivers of DNA repair evolution. Intrinsic forces like repetitive DNA, which also imperil genome integrity, have received less attention. We recently reported that a Drosophila melanogaster-specific DNA satellite array triggered species-specific, adaptive evolution of a DNA repair protein called Spartan>/?/MH. The Spartan family of proteases cleave hazardous, covalent crosslinks that form between DNA and proteins ("DNA-protein crosslink repair"). Appreciating that DNA satellites are both ubiquitous and universally fast-evolving, we hypothesized that satellite DNA turnover spurs adaptive evolution of DNA-protein crosslink repair beyond a single gene and beyond the D. melanogaster lineage. This hypothesis predicts pervasive Spartan gene family diversification across Drosophila species. To study the evolutionary history of the Drosophila Spartan gene family, we conducted population genetic, molecular evolution, phylogenomic, and tissue-specific expression analyses. We uncovered widespread signals of positive selection across multiple Spartan family genes and across multiple evolutionary timescales. We also detected recurrent Spartan family gene duplication, divergence, and gene loss. Finally, we found that ovary-enriched parent genes consistently birthed functionally diverged, testis-enriched daughter genes. To account for Spartan family diversification, we introduce a novel mechanistic model of antagonistic coevolution that links DNA satellite evolution and adaptive regulation of Spartan protease activity. This framework promises to accelerate our understanding of how DNA repeats drive recurrent evolutionary innovation to preserve genome integrity.

Kotov, A. A., Adashev, V. E., Kombarov, I. A., Bazylev, S. S., Shatskikh, A. S., Olenina, L. V. (2024). Molecular Insights into Female Hybrid Sterility in Interspecific Crosses between Drosophila melanogaster and Drosophila simulans. Int J Mol Sci, 25(11) PubMed ID: 38891872 Summary: Species of the genus Drosophila have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are under-investigated. This study performed an analysis of hybrid female sterility by crossing Drosophila melanogaster females and Drosophila simulans males. Using transcriptomic data analysis and molecular, cellular, and genetic approaches, differential gene expression, transposable element (TE) activity, piRNA biogenesis, and functional defects of oogenesis in hybrids were studied. Premature germline stem cell loss was the most prominent defect of oogenesis in hybrid ovaries. Because of the differential expression of genes encoding piRNA pathway components, rhino and deadlock, the functional RDC(mel) complex in hybrid ovaries was not assembled. However, the activity of the RDC(sim) complex was maintained in hybrids independent of the genomic origin of piRNA clusters. Despite the identification of a cohort of overexpressed TEs in hybrid ovaries, no evidence was found that their activity can be considered the main cause of hybrid sterility. A complicated pattern of Vasa protein expression was observed in the hybrid germline, including partial AT-chX piRNA targeting of the vasasim allele and a significant zygotic delay in vasamel/sup> expression. The conclusion was drawned that the hybrid sterility phenotype was caused by intricate multi-locus differences between the species.

Wiseglass, G., Rubinstein, R. (2024). Following the Evolutionary Paths of Dscam1 Proteins toward Highly Specific Homophilic Interactions. Mol Biol Evol, 41(7) PubMed ID: 38989909 Summary: Many adhesion proteins, evolutionarily related through gene duplication, exhibit distinct and precise interaction preferences and affinities crucial for cell patterning. Yet, the evolutionary paths by which these proteins acquire new specificities and prevent cross-interactions within their family members remain unknown. To bridge this gap, this study focuses on Drosophila Down syndrome cell adhesion molecule-1 (Dscam1) proteins, which are cell adhesion proteins that have undergone extensive gene duplication. Dscam1 evolved under strong selective pressure to achieve strict homophilic recognition, essential for neuronal self-avoidance and patterning. Through a combination of phylogenetic analyses, ancestral sequence reconstruction, and cell aggregation assays, we studied the evolutionary trajectory of Dscam1 exon 4 across various insect lineages. We demonstrated that recent Dscam1 duplications in the mosquito lineage bind with strict homophilic specificities without any cross-interactions. We found that ancestral and intermediate Dscam1 isoforms maintained their homophilic binding capabilities, with some intermediate isoforms also engaging in promiscuous interactions with other paralogs. Our results highlight the robust selective pressure for homophilic specificity integral to the Dscam1 function within the process of neuronal self-avoidance. Importantly, our study suggests that the path to achieving such selective specificity does not introduce disruptive mutations that prevent self-binding but includes evolutionary intermediates that demonstrate promiscuous heterophilic interactions. Overall, these results offer insights into evolutionary strategies that underlie adhesion protein interaction specificities.

Joyce, M., Falconio, F. A., Blackhurst, L., Prieto-Godino, L., French, A. S., Gilestro, G. F. (2024). Divergent evolution of sleep in Drosophila species. Nat Commun, 15(1):5091 PubMed ID: 38876988 Summary: Living organisms synchronize their biological activities with the earth's rotation through the circadian clock, a molecular mechanism that regulates biology and behavior daily. This synchronization factually maximizes positive activities (e.g., social interactions, feeding) during safe periods, and minimizes exposure to dangers (e.g., predation, darkness) typically at night. Beyond basic circadian regulation, some behaviors like sleep have an additional layer of homeostatic control, ensuring those essential activities are fulfilled. While sleep is predominantly governed by the circadian clock, a secondary homeostatic regulator, though not well-understood, ensures adherence to necessary sleep amounts and hints at a fundamental biological function of sleep beyond simple energy conservation and safety. This study explored sleep regulation across seven Drosophila species with diverse ecological niches, revealing that while circadian-driven sleep aspects are consistent, homeostatic regulation varies significantly. The findings suggest that in Drosophilids, sleep evolved primarily for circadian purposes. The more complex, homeostatically regulated functions of sleep appear to have evolved independently in a species-specific manner, and are not universally conserved. This laboratory model may reproduce and recapitulate primordial sleep evolution.

Roy, P. R., Castillo, D. M. (2024). The neurodevelopmental genes alan shepard and Neuroglian contribute to female mate preference in African Drosophila melanogaster. J Evol Biol, 37(8):877-890 PubMed ID: 38900077 Summary: Mate choice is a key trait that determines fitness for most sexually reproducing organisms, with females often being the choosy sex. Female preference often results in strong selection on male traits that can drive rapid divergence of traits and preferences between lineages, leading to reproductive isolation. Despite this fundamental property of female mate choice, very few loci have been identified that contribute to mate choice and reproductive isolation. This study used a combination of population genetics, quantitative complementation tests, and behavioural assays to demonstrate that alan shepard and Neuroglian contribute to female mate choice, and could contribute to partial reproductive isolation between populations of Drosophila melanogaster. This study is among the first to identify genes that contribute to female mate preference in this historically important system, where female preference is an active premating barrier to reproduction. The identification of loci that are primarily known for their roles in neurodevelopment provides intriguing questions of how female mate preference evolves in populations via changes in sensory system and higher learning brain centres.

Ridges, J. T., Bladen, J., King, T. D., Brown, N. C., Large, C. R. L., Cooper, J. C., Jones, A. J., Loppin, B., Dubruille, R., Phadnis, N. (2024). Overdrive is essential for targeted sperm elimination by Segregation Distorter. JbioRxiv, PubMed ID: 38895353 Summary: Intra-genomic conflict driven by selfish chromosomes is a powerful force that shapes the evolution of genomes and species. In the male germline, many selfish chromosomes bias transmission in their own favor by eliminating spermatids bearing the competing homologous chromosomes. However, the mechanisms of targeted gamete elimination remain mysterious. This study shows that Overdrive (Ovd), a gene required for both Responder spermatid elimination after the histone-to-protamine transition in the classical Segregation Distorter system. It is proposed that Ovd functions as a general spermatid quality checkpoint that is hijacked by independent selfish chromosomes to eliminate competing gametes.

Friday April 18th - Adult Physiology and Metabolism

Niosi, A., Vo, N. H., Sundaramurthy, P., ..., Jensen, M. H., Mulligan, K. (2024). Kismet/CHD7/CHD8 affects gut microbiota, mechanics, and the gut-brain axis in Drosophila melanogaster. Biophys J, PubMed ID: 38902926 Summary: The gut microbiome affects brain and neuronal development and may contribute to the pathophysiology of neurodevelopmental disorders. However, it is unclear how risk genes associated with such disorders affect gut physiology in a manner that could impact microbial colonization and how the mechanical properties of the gut tissue might play a role in gut-brain bidirectional communication. To address this, Drosophila melanogaster was used with a null mutation in the gene kismet, an ortholog of chromodomain helicase DNA-binding protein (CHD) family members CHD7 and CHD8. In humans, these are risk genes for neurodevelopmental disorders with co-occurring gastrointestinal symptoms. kismet mutant flies have a significant increase in gastrointestinal transit time, indicating the functional homology of kismet with CHD7/CHD8 in vertebrates. Rheological characterization of dissected gut tissue revealed significant changes in the mechanics of kismet mutant gut elasticity, strain stiffening behavior, and tensile strength. Using 16S rRNA metagenomic sequencing, it was also found that kismet mutants have reduced diversity and abundance of gut microbiota at every taxonomic level. To investigate the connection between the gut microbiome and behavior, gut microbiota were depleted in kismet mutant and control flies and quantified the flies' courtship behavior. Depletion of gut microbiota rescued courtship defects of kismet mutant flies, indicating a connection between gut microbiota and behavior. In striking contrast, depletion of the gut microbiome in the control strain reduced courtship activity, demonstrating that antibiotic treatment can have differential impacts on behavior and may depend on the status of microbial dysbiosis in the gut prior to depletion.

Ye, Y. Y., Liu, Z. H., Wang, H. L. (2024). Fat body-derived juvenile hormone acid methyltransferase functions to maintain iron homeostasis in Drosophila melanogaster. Faseb j, 38(14):e23805 PubMed ID: 39003630 Summary: Iron homeostasis is of critical importance to living organisms. Drosophila melanogaster has emerged as an excellent model to study iron homeostasis, while the regulatory mechanism of iron metabolism remains poorly understood. This study accidently found that knockdown of juvenile hormone (JH) acid methyltransferase (Jhamt) specifically in the fat body, a key rate-limiting enzyme for JH synthesis, led to iron accumulation locally, resulting in serious loss and dysfunction of fat body. Jhamt knockdown-induced phenotypes were mitigated by iron deprivation, antioxidant and Ferrostatin-1, a well-known inhibitor of ferroptosis, suggesting ferroptosis was involved in Jhamt knockdown-induced defects in the fat body. Further study demonstrated that upregulation of Tsf1 and Malvolio (Mvl, homolog of mammalian DMT1), two iron importers, accounted for Jhamt knockdown-induced iron accumulation and dysfunction of the fat body. Mechanistically, Kr-h1, a key transcription factor of JH, acts downstream of Jhamt inhibiting Tsf1 and Mvl transcriptionally. In summary, the findings indicated that fat body-derived Jhamt is required for the development of Drosophila by maintaining iron homeostasis in the fat body, providing unique insight into the regulatory mechanisms of iron metabolism in Drosophila.

Gera, J., Kumar, D., Chauhan, G., Choudhary, A., Rani, L., Mandal, L., Mandal, S. (2024). High sugar diet-induced fatty acid oxidation potentiates cytokine-dependent cardiac ECM remodeling. J Cell Biol, 223(9) PubMed ID: 38916917 Summary: Context-dependent physiological remodeling of the extracellular matrix (ECM) is essential for development and organ homeostasis. On the other hand, consumption of high-caloric diet leverages ECM remodeling to create pathological conditions that impede the functionality of different organs, including the heart. However, the mechanistic basis of high caloric diet-induced ECM remodeling has yet to be elucidated. Employing in vivo molecular genetic analyses in Drosophila, this study demonstrated that high dietary sugar triggers ROS-independent activation of JNK signaling to promote fatty acid oxidation (FAO) in the pericardial cells (nephrocytes). An elevated level of FAO, in turn, induces histone acetylation-dependent transcriptional upregulation of the cytokine Unpaired 3 (Upd3). Release of pericardial Upd3 augments fat body-specific expression of the cardiac ECM protein Pericardin, leading to progressive cardiac fibrosis. Importantly, this pathway is quite distinct from the ROS-Ask1-JNK/p38 axis that regulates Upd3 expression under normal physiological conditions. These results unravel an unknown physiological role of FAO in cytokine-dependent ECM remodeling, bearing implications in diabetic fibrosis.

Miyamoto, T., Hedjazi, S., Miyamoto, C., Amrein, H. (2024). Drosophila neuronal Glucose-6-Phosphatase is a modulator of neuropeptide release that regulates muscle glycogen stores via FMRFamide signaling. Proc Natl Acad Sci U S A, 121(30):e2319958121 PubMed ID: 39008673 Summary: Neuropeptides (NPs) and their cognate receptors are critical effectors of diverse physiological processes and behaviors. A noncanonical function of the Drosophila Glucose-6-Phosphatase (G6P) gene has ben recently reported in a subset of neurosecretory cells in the central nervous system that governs systemic glucose homeostasis in food-deprived flies. This study shows that G6P-expressing neurons define six groups of NP-secreting cells, four in the brain and two in the thoracic ganglion. Using the glucose homeostasis phenotype as a screening tool, it was found that neurons located in the thoracic ganglion expressing FMRFamide NPs (FMRFa(G6P) neurons) are necessary and sufficient to maintain systemic glucose homeostasis in starved flies. It was further shown that G6P is essential in FMRFa(G6P) neurons for attaining a prominent Golgi apparatus and secreting NPs efficiently. Finally, G6P-dependent FMRFa signaling was established as essential for the build-up of glycogen stores in the jump muscle which expresses the receptor for FMRFamides. A general model is proposed in which the main role of G6P is to counteract glycolysis in peptidergic neurons for the purpose of optimizing the intracellular environment best suited for the expansion of the Golgi apparatus, boosting release of NPs and enhancing signaling to respective target tissues expressing cognate receptors.

Pandey, P., Shrestha, B., Lee, Y. (2024). Avoiding alkaline taste through ionotropic receptors. iScience, 27(6):110087 PubMed ID: 38947501 Summary: Taste organsZ contain distinct gustatory receptors that help organisms differentiate between nourishing and potentially harmful foods. The detection of high pH levels plays a crucial role in food selection, but the specific gustatory receptors responsible for perceiving elevated pH in foods have remained unknown. By using Drosophila melanogaster as a model organism, this study has uncovered the involvement of ionotropic receptors (IRs) in avoiding high-pH foods. This study involved a combination of behavioral tests and electrophysiological analyses, which led to the identification of six Irs from bitter-sensing gustatory receptor neurons essential for rejecting food items with elevated pH levels. Using the same methodology, this study reevaluated the significance and OtopLa. The findings highlight that Alka, in conjunction with IRs, is crucial for detecting alkaline substances, whereas OtopLa does not contribute to this process. Overall, this study offers valuable insights into the intricate mechanisms governing taste perception in organisms.

Chu, S. Y., Lai, Y. W., Hsu, T. C., Lu, T. M., Yu, H. H. (2024). Isoforms of terminal selector Mamo control axon guidance during adult Drosophila memory center construction via Semaphorin-1a. Dev Biol, 515:1-6 PubMed ID: 38906235 Summary: In animals undergoing metamorphosis, the appearance of the nervous system is coincidently transformed by the morphogenesis of neurons. Such morphogenic alterations are exemplified in three types of intrinsic neurons in the Drosophila memory center. In contrast to the well-characterized remodeling of γ neurons, the morphogenesis of α/β and α'/β' neurons has not been adequately explored. This study showed that mamo a BTB-zinc finger transcription factor that acts as a terminal selector for α'/β' neurons, controls the formation of the correct axonal pattern of α'/β' neurons. Intriguingly, specific Mamo isoforms are preferentially expressed in α'/β' neurons to regulate the expression of axon guidance molecule Semaphorin-1a. This action directs proper axon guidance in α'/β' neurons, which is also crucial for wiring of α'/β' neurons with downstream neurons. Taken together, these results provide molecular insights into how neurons establish correct axonal patterns in circuitry assembly during adult memory center construction.

Wednesday April 16th - Synapse and Vesicles

Ferng, D., Sun, W., Shieh, B. H. (2024). Differential activation of rhodopsin triggers distinct endocytic trafficking and recycling in vivo via differential phosphorylation. PLoS One, 19(6):e0303882 PubMed ID: 38848405 Summary: Activated GPCRs are phosphorylated and internalized mostly via clathrin-mediated endocytosis (CME), and are then sorted for recycling or degradation. This study investigated how differential activation of the same GPCR affects its endocytic trafficking in vivo using rhodopsin as a model in pupal photoreceptors of flies expressing mCherry-tagged rhodopsin 1 (Rh1-mC) or GFP-tagged arrestin 1 (Arr1-GFP). Upon blue light stimulation, activated Rh1 recruited Arr1-GFP to the rhabdomere, which became co-internalized and accumulated in cytoplasmic vesicles of photoreceptors. This internalization was eliminated in shits1 mutants affecting dynamin. Moreover, it was blocked by either rdgA or rdgB mutations affecting the PIP2 biosynthesis. Together, the blue light-initiated internalization of Rh1 and Arr1 belongs to CME. Green light stimulation also triggered the internalization and accumulation of activated Rh1-mC in the cytoplasm but with faster kinetics. Importantly, Arr1-GFP was also recruited to the rhabdomere but not co-internalized with Rh1-mC. This endocytosis was not affected in shits1 nor rdgA mutants, indicating it is not CME. The fate of internalized Rh1-mC following CME was examined and it remained in cytoplasmic vesicles following 30 min of dark adaptation. In contrast, in the non-CME Rh1-mC appeared readily recycled back to the rhabdomere within five min of dark treatment. This faster recycling may be regulated by rhodopsin phosphatase, RdgC. Together, two distinct endocytic and recycling mechanisms of Rh1 were demonstrated via two light stimulations. It appears that each stimulation triggers a distinct conformation leading to different phosphorylation patterns of Rh1 capable of recruiting Arr1 to rhabdomeres. However, a more stable interaction leads to the co-internalization of Arr1 that orchestrates CME. A stronger Arr1 association appears to impede the recycling of the phosphorylated Rh1 by preventing the recruitment of RdgC. It is concluded that conformations of activated rhodopsin determine the downstream outputs upon phosphorylation that confers differential protein-protein interactions.

Madan, A., Kelly, K. P., Bahk, P., Sullivan, C. E., Poling, M. E., Brent, A. E., Alassaf, M., Dubrulle, J., Rajan, A. (2024). Atg8/LC3 controls systemic nutrient surplus signaling in flies and humans. Curr Biol, 34(15):3327-3341PubMed ID: 38955177 Summary: Organisms experience constant nutritional flux. Mechanisms at the interface of opposing nutritional states-scarcity and surplus-enable organismal energy homeostasis. Contingent on nutritional stores, adipocytes secrete adipokines, such as the fat hormone leptin, to signal nutrient status to the central brain. Increased leptin secretion underlies metabolic dysregulation during common obesity, but the molecular mechanisms regulating leptin secretion from human adipocytes are poorly understood. Here, we report that Atg8/LC3 family proteins, best known for their role in autophagy during nutrient scarcity, play an evolutionarily conserved role during nutrient surplus by promoting adipokine secretion. We show that in a well-fed state, Atg8/LC3 promotes the secretion of the Drosophila functional leptin ortholog unpaired 2 (Upd2) and leptin from human adipocytes. Proteomic analyses reveal that LC3 directs leptin to a secretory pathway in human cells. We identified LC3-dependent extracellular vesicle (EV) loading and secretion (LDELS) as a required step for leptin release, highlighting a unique secretory route adopted by leptin in human adipocytes. In Drosophila, mutations to Upd2's Atg8 interaction motif (AIM) result in constitutive adipokine retention. Atg8-mediated Upd2 retention alters lipid storage and hunger response and rewires the bulk organismal transcriptome in a manner conducive to starvation survival. Thus, Atg8/LC3's bidirectional role in nutrient sensing-conveying nutrient surplus and responding to nutrient deprivation-enables organisms to manage nutrient flux effectively. We posit that decoding how bidirectional molecular switches-such as Atg8/LC3-operate at the nexus of nutritional scarcity and surplus will inform therapeutic strategies to tackle chronic metabolic disorders.

Christophers, B., Leahy, S. N., Soffar, D. B., von Saucken, V. E., Broadie, K., Baylies, M. K. (2024). Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission. Development, 151(13) PubMed ID: 38869008 Summary: Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. It was hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, an RNA-sequencing analysis was conducted that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. DmCFL is enriched in the muscle postsynaptic compartment, and DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, no significant changes were found in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy.

Malis, Y., Armoza-Eilat, S., Nevo-Yassaf, I., Dukhovny, A., Sklan, E. H., Hirschberg, K. (2024). Rab1b facilitates lipid droplet growth by ER-to-lipid droplet targeting of DGAT2. Sci Adv, 10(22):eade7753 PubMed ID: 38809969 Summary: Lipid droplets (LDs) comprise a triglyceride core surrounded by a lipid monolayer enriched with proteins, many of which function in LD homeostasis. How proteins are targeted to the growing LD is still unclear. Rab1b (Drosophila homolog: Rab1), a GTPase regulating secretory transport, was recently associated with targeting proteins to LDs in a Drosophila RNAi screen. LD formation was prevented in human hepatoma cells overexpressing dominant-negative Rab1b. It was thus hypothesized that Rab1b recruits lipid-synthesizing enzymes, facilitating LD growth. Here, FRET between diacylglycerol acyltransferase 2 (DGAT2) and Rab1b and activity mutants of the latter demonstrated that Rab1b promotes DGAT2 ER to the LD surface redistribution. Last, alterations in LD metabolism and DGAT2 redistribution, consistent with Rab1b activity, were caused by mutations in the Rab1b-GTPase activating protein TBC1D20 in Warburg Micro syndrome (WARBM) model mice fibroblasts. These data contribute to understanding of the mechanism of Rab1b in LD homeostasis and WARBM, a devastating autosomal-recessive disorder caused by mutations in TBC1D20.

Han, T. H., Vicidomini, R., Ramos, C. I., Mayer, M., Serpe, M. (2024). Neto proteins differentially modulate the gating properties of Drosophila NMJ glutamate receptors. bioRxiv, PubMed ID: 38903091 Summary: The formation of functional synapses requires co-assembly of ion channels with their accessory proteins which controls where, when, and how neurotransmitter receptors function. The auxiliary protein Neto modulates the function of kainate-type glutamate receptors in vertebrates as well as at the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse widely used for genetic studies on synapse development. Previous work showed that Neto is essential for the synaptic recruitment and function of glutamate receptors. Using outside-out patch-clamp recordings and fast ligand application, this study examined the biophysical properties of recombinant Drosophila NMJ receptors expressed in HEK293T cells and compare them with native receptor complexes of genetically controlled composition. The two Neto isoforms, Neto-α and Neto-β, differentially modulate the gating properties of NMJ receptors. Surprisingly, it was found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of iGluR desensitization. The functional analyses of recombinant iGluRs that is reported in this study should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals.

Jay, T. R., Kang, Y., Ouellet-Massicotte, V., Micael, M. K. B., Kacouros-Perkins, V. L., Chen, J., Sheehan, A., Freeman, M. R. (2024). Developmental and age-related synapse elimination is mediated by glial Croquemort. bioRxiv, PubMed ID: 39026803 Summary: Neurons and glia work together to dynamically regulate neural circuit assembly and maintenance. This study shows Drosophila exhibit large-scale synapse formation and elimination as part of normal CNS circuit maturation, and that glia use conserved molecules to regulate these processes. Using a high throughput ELISA-based in vivo screening assay, new glial genes were identified that regulate synapse numbers in Drosophila in vivo, including the scavenger receptor ortholog Croquemort (Crq). Crq acts as an essential regulator of glial-dependent synapse elimination during development, with glial Crq loss leading to excess CNS synapses and progressive seizure susceptibility in adults. Loss of Crq in glia also prevents age-related synaptic loss in the adult brain. This work provides new insights into the cellular and molecular mechanisms that underlie synapse development and maintenance across the lifespan, and identifies glial Crq as a key regulator of these processes.

Tuesday April 15th - Signaling

Matakatsu, H., Fehon, R. G. (2024). Dachsous and Fat coordinately repress the Dachs-Dlish-Approximated complex to control growth. bioRxiv, PubMed ID: 38948705 Summary: Two protocadherins, Dachsous (Ds) and Fat (Ft), regulate organ growth in Drosophila via the Hippo pathway. Ds and Ft bind heterotypically to regulate the abundance and subcellular localization of a 'core complex' consisting of Dachs, Dlish and Approximated. This complex localizes to the junctional cortex where it promotes growth by repressing the pathway kinase Warts. Ds is believed to promote growth by recruiting and stabilizing the core complex at the junctional cortex, while Ft represses growth by promoting degradation of core complex components. This study examine the functions of intracellular domains of Ds and Ft and their relationship to the core complex. While Ds promotes accumulation of the core complex proteins in cortical puncta, it is not required for core complex assembly. Indeed, the core complex assembles maximally in the absence of both Ds and Ft. Furthermore, while Ds promotes growth in the presence of Ft, it represses growth in the absence of Ft by removing the core complex from the junctional cortex. Ft similarly recruits core complex components, however it normally promotes their degradation. These findings reveal that Ds and Ft constrain tissue growth by repressing the default 'on' state of the core complex.

Goins, L. M., Girard, J. R., Mondal, B. C., Buran, S., Su, C. C., Tang, R., Biswas, T., Kissi, J. A., Banerjee, U. (2024). Wnt signaling couples G2 phase control with differentiation during hematopoiesis in Drosophila. Dev Cell, PubMed ID: 38866012 Summary: During homeostasis, a critical balance is maintained between myeloid-like progenitors and their differentiated progeny, which function to mitigate stress and innate immune challenges. The molecular mechanisms that help achieve this balance are not fully understood. Using genetic dissection in Drosophila, this study showed that a /EGFR-signaling network simultaneously controls progenitor growth, proliferation, and differentiation. Unlike G1-quiescence of stem cells, hematopoietic progenitors are blocked in G2 phase by a β-catenin-independent (Wnt/STOP) Wnt6 pathway that restricts nuclear entry and promotes cell growth. Canonical β-catenin-dependent Wnt6 signaling is spatially confined to mature progenitors through localized activation of the tyrosine kinases EGFR and Abelson kinase (Abl), which promote nuclear entry of β-catenin and facilitate exit from G2. This strategy combines transcription-dependent and -independent forms of both Wnt6 and EGFR pathways to create a direct link between cell-cycle control and differentiation. This unique combinatorial strategy employing conserved components may underlie homeostatic balance and stress response in mammalian hematopoiesis.

Wu, J., Bala Tannan, N., Vuong, L. T., Koca, Y., Collu, G. M., Mlodzik, M. (2024). Par3/bazooka binds NICD and promotes notch signaling during Drosophila development. Dev Biol, 514:37-49 PubMed ID: 38885804 Summary: The conserved bazooka (baz/par3) gene acts as a key regulator of asymmetrical cell divisions across the animal kingdom. Associated Par3/Baz-Par6-aPKC protein complexes are also well known for their role in the establishment of apical/basal cell polarity in epithelial cells. Here we define a novel, positive function of Baz/Par3 in the Notch pathway. Using Drosophila wing and eye development, we demonstrate that Baz is required for Notch signaling activity and optimal transcriptional activation of Notch target genes. Baz appears to act independently of aPKC in these contexts, as knockdown of aPKC does not cause Notch loss-of-function phenotypes. Using transgenic Notch constructs, these data positions Baz activity downstream of activating Notch cleavage steps and upstream of Su(H)/CSL transcription factor complex activity on Notch target genes. A biochemical interaction was demonstrated between NICD and Baz, suggesting that Baz is required for NICD activity before NICD binds to Su(H). Taken together, these data define a novel role of the polarity protein Baz/Par3, as a positive and direct regulator of Notch signaling through its interaction with NICD.

Vuong, L. T., Mlodzik, M. (2024). Wg/Wnt-signaling-induced nuclear translocation of β-catenin is attenuated by a β-catenin peptide through its interference with the IFT-A complex. Cell Rep, 43(6):114362 PubMed ID: 38870008 Summary: Wnt/Wingless (Wg) signaling is critical in development and disease, including cancer. Canonical Wnt signaling is mediated by β-catenin/Armadillo (Arm in Drosophila) transducing signals to the nucleus, with IFT-A/Kinesin 2 complexes promoting nuclear translocation of β-catenin/Arm. This study demonstrates that a conserved small N-terminal Arm(34)(-)(87)/β-catenin peptide binds to IFT140, acting as a dominant interference tool to attenuate Wg/Wnt signaling in vivo. Arm(34)(-)(87) expression antagonizes endogenous Wnt/Wg signaling, resulting in the reduction of its target expression. Arm(34)(-)(87) inhibits Wg/Wnt signaling by interfering with nuclear translocation of endogenous Arm/β-catenin, and this can be modulated by levels of wild-type β-catenin or IFT140, with the Arm(34)(-)(87) effect being enhanced or suppressed. Importantly, this mechanism is conserved in mammals with the equivalent β-catenin(24)(-)(79) peptide blocking nuclear translocation and pathway activation, including in cancer cells. Our work indicates that Wnt signaling can be regulated by a defined N-terminal β-catenin peptide and thus might serve as an entry point for therapeutic applications to attenuate Wnt/β-catenin signaling.

Kamalesh, K., Segal, D., Avinoam, O., Schejter, E. D., Shilo, B. Z. (2024). Structured RhoGEF recruitment drives myosin II organization on large exocytic vesicles. J Cell Sci, 137(13) PubMed ID: 38899547 Summary: The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, the spatiotemporal regulation was followed of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern. Subsequently, the Rho1 activator RhoGEF2 distributes as an irregular meshwork on the vesicle membrane, activating Rho1 in a corresponding punctate pattern and driving local myosin II recruitment, resulting in vesicle constriction. Vesicle membrane buckling and subsequent crumpling occur at local sites of high myosin II concentrations. These findings indicate that distinct thresholds for activated Rho1 create a biphasic mode of actomyosin assembly, inducing anisotropic membrane crumpling during exocrine secretion.

Sayeesh, P. M., Iguchi, M., Inomata, K., Ikeya, T., Ito, Y. (2024). Structure and Dynamics of Drk-SH2 Domain and Its Site-Specific Interaction with Sev Receptor Tyrosine Kinase. Int J Mol Sci, 25(12) PubMed ID: 38928093 Summary: The Drosophila downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). This study presents the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three β strands imposed between two α helices. Additionally, the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide was interpreted through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2.

Monday April 14th - Genes, Enzymes and Protein expression, Evolution Structure and Function

Fisher, L. A. B., Carriqui-Madronal, B., Mulder, T., Huelsmann, S., Schock, F., Gonzalez-Morales, N. (2024). Filamin protects myofibrils from contractile damage through changes in its mechanosensory region. PLoS Genet, 20(6):e1011101 PubMed ID: 38905299 Summary: Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton> in a variety of shapes and tissues. In muscles, filamin crosslinks actin filaments from opposing sarcomeres, the smallest contractile units of muscles. This happens at the Z-disc, the actin-organizing center of sarcomeres. In flies and vertebrates, filamin mutations lead to fragile muscles that appear ruptured, suggesting filamin helps counteract muscle rupturing during muscle contractions by providing elastic support and/or through signaling. An elastic region at the C-terminus of filamin is called the mechanosensitive region and has been proposed to sense and counteract contractile damage. This study used molecularly defined mutants and microscopy analysis of the Drosophila indirect flight muscles to investigate the molecular details by which filamin provides cohesion to the Z-disc. Novel filamin mutations affecting the C-terminal region were made to interrogate the mechanosensitive region and detected three Z-disc phenotypes: dissociation of actin filaments, Z-disc rupture, and Z-disc enlargement. A constitutively closed filamin mutant was identified that prevents the elastic changes in the mechanosensitive region and results in ruptured Z-discs, and a constitutively open mutant which has the opposite elastic effect on the mechanosensitive region and gives rise to enlarged Z-discs. Finally, muscle contraction was shown to be required for Z-disc rupture. It is proposed that filamin senses myofibril damage by elastic changes in its mechanosensory region, stabilizes the Z-disc, and counteracts contractile damage at the Z-disc.

Landis, G. N., Bell, H. S., Peng, O. K., Fan, Y., Yan, K., Baybutt, B., Tower, J. (2024). Conditional Inhibition of Eip75B Eliminates the Effects of Mating and Mifepristone on Lifespan in Female Drosophila. Cells, 13(13) PubMed ID: 38994975 Summary: Mating in female Drosophila melanogaster causes midgut hypertrophy and reduced lifespan, and these effects are blocked by the drug mifepristone. Eip75B is a transcription factor previously reported to have pleiotropic effects on Drosophila lifespan. Because Eip75B null mutations are lethal, conditional systems and/or partial knock-down are needed to study Eip75B effects in adults. Previous studies showed that Eip75B is required for adult midgut cell proliferation in response to mating. To test the possible role of Eip75B in mediating the lifespan effects of mating and mifepristone, a tripartite FLP-recombinase-based conditional system was employed that provides controls for genetic background. Expression of a Hsp70-FLP transgene was induced in third instar larvae by a brief heat pulse. The FLP recombinase catalyzed the recombination and activation of an Actin5C-GAL4 transgene. The GAL4 transcription factor in turn activated expression of a UAS-Eip75B-RNAi transgene. Inhibition of Eip75B activity was confirmed by loss of midgut hypertrophy upon mating, and the lifespan effects of both mating and mifepristone were eliminated. In addition, the negative effects of mifepristone on egg production were eliminated. The data indicate that Eip75B mediates the effects of mating and mifepristone on female midgut hypertrophy, egg production, and lifespan.

Castillo-Mancho, V., Atienza-Manuel, A., Sarmiento-Jimenez, J., Ruiz-Gomez, M., Culi, J. (2024). Phospholipid scramblase 1: an essential component of the nephrocyte slit diaphragm.. Cell Mol Life Sci, 81(1):261 PubMed ID: 38878170 Summary: Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). By studying a homologous structure found in Drosophila nephrocytes, this study identified the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, polyhaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca(2+)-binding sites in Scramb1 are essential for its activity raising the possibility that Ca(2+) signaling may control the assembly of SDs by impacting on Scramb1 activity.

Xu, M., Wu, Z., Li, W., Xue, L. (2024). Gp93 inhibits unfolded protein response-mediated c-Jun N-terminal kinase activation and cell invasion. Journal of cellular physiology, 239(8):e31294 PubMed ID: 38922869 Summary: In eukaryotes, Hsp90B1 serves as a vital chaperonin, facilitating the accurate folding of proteins. Interestingly, Hsp90B1 exhibits contrasting roles in the development of various types of cancers, although the underlying reasons for this duality remain enigmatic. Through the utilization of the Drosophila model, this study unveils the functional significance of Gp93, the Drosophila ortholog of Hsp90B1, which hitherto had limited reported developmental functions. Employing the Drosophila cell invasion model, the pivotal role of Gp93 in regulating cell invasion and modulating c-Jun N-terminal kinase (JNK) activation were elucidated. Furthermore, this investigation highlights the involvement of the unfolded protein response-associated IRE1/XBP1 pathway in governing Gp93 depletion-induced, JNK-dependent cell invasion. Collectively, these findings not only uncover a novel molecular function of Gp93 in Drosophila, but also underscore a significant consideration pertaining to the testing of Hsp90B1 inhibitors in cancer therapy.

Murthy, S., Dey, U., Olymon, K., Abbas, E., Yella, V. R., Kumar, A. (2024). Discerning the Role of DNA Sequence, Shape, and Flexibility in Recognition by Drosophila Transcription Factors. ACS chemical biology, 19(7):1533-1543 PubMed ID: 38902964 Summary: The precise spatial and temporal orchestration of gene expression is crucial for the ontogeny of an organism and is mainly governed by transcription factors (TFs). The mechanism of recognition of cognate sites amid millions of base pairs in the genome by TFs is still incompletely understood. This study focuses on DNA sequence composition, shape, and flexibility preferences of 28 quintessential TFs from Drosophila melanogaster that are critical to development and body patterning mechanisms. This study finds that TFs exhibit distinct predilections for DNA shape, flexibility, and sequence compositions in the proximity of transcription factor binding sites (TFBSs). Notably, certain zinc finger proteins prefer GC-rich areas with less negative propeller twist, while homeodomains mainly seek AT-rich regions with a more negative propeller twist at their sites. Intriguingly, while numerous cofactors share similar binding site preferences and bind closer to each other in the genome, some cofactors that have different preferences bind farther apart. These findings shed light on TF DNA recognition and provide novel insights into possible cofactor binding and transcriptional regulation mechanisms.

Bordet, G., Tulin, A. V. (2024).. PARG Protein Regulation Roles in Drosophila Longevity Control. Int J Mol Sci, 25(11) PubMed ID: 38892377 Summary: Aging, marked by a gradual decline in physiological function and heightened vulnerability to age-related diseases, remains a complex biological process with multifaceted regulatory mechanisms. This study elucidates the critical role of poly(ADP-ribose) glycohydrolase (PARG), responsible for catabolizing poly(ADP-ribose) (pADPr) in the aging process by modulating the expression of age-related genes in Drosophila melanogaster. Specifically, the regulatory function was uncovered of the uncharacterized PARG C-terminal domain in controlling PARG activity. Flies lacking this domain exhibit a significantly reduced lifespan compared to wild-type counterparts. Furthermore, progressive dysregulation of age-related gene expression was observed during aging, accelerated in the absence of PARG activity, culminating in a premature aging phenotype. These findings reveal the critical involvement of the pADPr pathway as a key player in the aging process, highlighting its potential as a therapeutic target for mitigating age-related effects.

Friday, April 11th - RNAs, RNA interference, and Transposons

Chen, P., Pan, K. C., Park, E. H., Luo, Y., Lee, Y. C. G., Aravin, A. A. (2024). Escalation of genome defense capacity enables control of an expanding meiotic driver. bioRxiv, PubMed ID: 38915551 Summary: rom RNA interference to chromatin silencing, diverse genome defense pathways silence selfish genetic elements to safeguard genome integrity. Despite their diversity, different defense pathways share a modular organization, where numerous specificity factors identify diverse targets and common effectors silence them. In the PIWI-interacting RNA (piRNA) pathway, which controls selfish elements in the metazoan germline, diverse target RNAs are first identified by complementary base pairing with piRNAs and then silenced by PIWI-clade nucleases via enzymatic cleavage. Such a binary architecture allows the defense systems to be readily adaptable, where new targets can be captured via the innovation of new specificity factors. Thus, current understanding of genome defense against lineage-specific selfish genes has been largely limited to the evolution of specificity factors, while it remains poorly understood whether other types of innovations are required. This sttudy describes a new type of innovation, which escalates the defense capacity of the piRNA pathway to control a recently expanded selfish gene in Drosophila melanogaster. Through an in vivo RNAi screen for repressors of Stellate-a recently evolved and expanded selfish meiotic driver-a novel defense factor, Trailblazer, was discovered. Trailblazer is a transcription factor that promotes the expression of two PIWI-clade nucleases, Aub and AGO3, to match Stellate in abundance. Recent innovation in the DNA-binding domain of Trailblazer enabled it to drastically elevate Aub and AGO3 expression in the D. melanogaster lineage, thereby escalating the silencing capacity of the piRNA pathway to control expanded Stellate and safeguard fertility. As copy-number expansion is a recurrent feature of diverse selfish genes across the tree of life, it is envisioned that augmenting the defense capacity to quantitatively match selfish genes is likely a repeatedly employed defense strategy in evolution.

Menzies, J. A. C., Maia Chagas, A., Baden, T., Alonso, C. R. (2024). A microRNA that controls the emergence of embryonic movement. Elife, 13 PubMed ID: 38869942 Summary: Movement is a key feature of animal systems, yet its embryonic origins are not fully understood. This study investigated the genetic basis underlying the embryonic onset of movement in Drosophila focusing on the role played by small non-coding RNAs (microRNAs, miRNAs). To this end, a quantitative behavioural pipeline capable of tracking embryonic movement in large populations of fly embryos was developed, and uthis system discovered that the Drosophila miRNA miR-2b-1 plays a role in the emergence of movement. Through the combination of spectral analysis of embryonic motor patterns, cell sorting and RNA in situs, genetic reconstitution tests, and neural optical imaging it was found that miR-2b-1 influences the emergence of embryonic movement by exerting actions in the developing nervous system. Furthermore, through the combination of bioinformatics coupled to genetic manipulation of miRNA expression and phenocopy tests a previously uncharacterised (but evolutionarily conserved) chloride channel encoding gene was developed - which was termed Movement Modulator (Motor) - as a genetic target that mechanistically links miR-2b-11 to the onset of movement. Cell-specific genetic reconstitution of miR-2b-1 expression in a null miRNA mutant background, followed by behavioural assays and target gene analyses, suggest that miR-2b-1 affects the emergence of movement through effects in sensory elements of the embryonic circuitry, rather than in the motor domain. This work thus reports the first miRNA system capable of regulating embryonic movement, suggesting that other miRNAs are likely to play a role in this key developmental process in Drosophila as well as in other species.

Duan, Y., Ma, L., Zhao, T., Liu, J., Zheng, C., Song, F., Tian, L., Cai, W., Li, H. (2024). Conserved A-to-I RNA editing with non-conserved recoding expands the candidates of functional editing sites. Fly (Austin), 18(1):2367359 PubMed ID: 38889318 Summary: Adenosine-to-inosine (A-to-I) RNA editing recodes the genome and confers flexibility for the organisms to adapt to the environment. It is believed that RNA recoding sites are well suited for facilitating adaptive evolution by increasing the proteomic diversity in a temporal-spatial manner. The function and essentiality of a few conserved recoding sites are recognized. However, the experimentally discovered functional sites only make up a small corner of the total sites, and there is still the need to expand the repertoire of such functional sites with bioinformatic approaches. This study defines a new category of RNA editing sites termed 'conserved editing with non-conserved recoding' and systematically identify such sites in Drosophila editomes, figuring out their selection pressure and signals of adaptation at inter-species and intra-species levels. Surprisingly, conserved editing sites with non-conserved recoding are not suppressed and are even slightly overrepresented in Drosophila. DNA mutations leading to such cases are also favoured during evolution, suggesting that the function of those recoding events in different species might be diverged, specialized, and maintained. Finally, structural prediction suggests that such recoding in potassium channel Shab might increase ion permeability and compensate the effect of low temperature. In conclusion, conserved editing with non-conserved recoding might be functional as well. This study provides novel aspects in considering the adaptive evolution of RNA editing sites and meanwhile expands the candidates of functional recoding sites for future validation.

McQuarrie, D. W. J., Alizada, A., Nicholson, B. C., Soller, M. (2024). Rapid evolution of promoters from germline-specifically expressed genes including transposon silencing factors. BMC Genomics, 25(1):678 PubMed ID: 38977960 Summary: The piRNA pathway in animal gonads functions as an 'RNA-based immune system', serving to silence transposable elements and prevent inheritance of novel invaders. In Drosophila, this pathway relies on three gonad-specific Argonaute proteins (Argonaute-3, Aubergine and Piwi) that associate with 23-28 nucleotide piRNAs, directing the silencing of transposon-derived transcripts. Transposons constitute a primary driver of genome evolution, yet the evolution of piRNA pathway factors has not received in-depth exploration. Specifically, channel nuclear pore proteins, which impact piRNA processing, exhibit regions of rapid evolution in their promoters. Consequently, the question arises whether such a mode of evolution is a general feature of transposon silencing pathways. By employing genomic analysis of coding and promoter regions within genes that function in transposon silencing in Drosophila, this study demonstrated that the promoters of germ cell-specific piRNA factors are undergoing rapid evolution. These findings indicate that rapid promoter evolution is a common trait among piRNA factors engaged in germline silencing across insect species, potentially contributing to gene expression divergence in closely related taxa. Furthermore, it was observed that the promoters of genes exclusively expressed in germ cells generally exhibit rapid evolution, with some divergence in gene expression. These results suggest that increased germline promoter evolution, in partnership with other factors, could contribute to transposon silencing and evolution of species through differential expression of genes driven by invading transposons.

Teng, Z., Yang, L., Zhang, Q., Chen, Y., Wang, X., Zheng, Y., Tian, A., Tian, D., Lin, Z., Deng, W. M., Liu, H. (2024). Topoisomerase I is an evolutionarily conserved key regulator for satellite DNA transcription. Nat Commun, 15(1):5151 PubMed ID: 38886382 Summary: RNA Polymerase (RNAP) II transcription on non-coding repetitive satellite DNAs plays an important role in chromosome segregation, but a little is known about the regulation of satellite transcription. This study shows that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite DNAs on human centromeres. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation. Interestingly, in response to DNA double-stranded breaks (DSBs), α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner, and these DSB-induced α-satellite RNAs form into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, these findings reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.

Stewart, R. K., Nguyen, P., Laederach, A., Volkan, P. C., Sawyer, J. K., Fox, D. T. (2024). Orb2 enables rare-codon-enriched mRNA expression during Drosophila neuron differentiation. Nat Commun, 15(1):5270 PubMed ID: 38902233 Summary: Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, this study identified the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent mRNA stability in neurons. Using RNA sequencing, Orb2-upregulated mRNAs were revealed in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, rare-codon enrichment was demonstrated to be important for mRNA stability and social behavior function of the metabotropic glutamate receptor (mGluR). These findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA stability and protein expression.

Wednesday, April 9th - Adult neural structure, development and function

Medeiros, A. M., Hobbiss, A. F., Borges, G., Moita, M., Mendes, C. S. (2024). Mechanosensory bristles mediate avoidance behavior by triggering sustained local motor activity in Drosophila melanogaster. Curr Biol, 34(13):2812-2830. PubMed ID: 38861987 Summary: During locomotion, most vertebrates-and invertebrates such as Drosophila melanogaster-are able to quickly adapt to terrain irregularities or avoid physical threats by integrating sensory information along with motor commands. Key to this adaptability are leg mechanosensory structures, which assist in motor coordination by transmitting external cues and proprioceptive information to motor centers in the central nervous system. Nevertheless, how different mechanosensory structures engage these locomotor centers remains poorly understood. This study tested the role of mechanosensory structures in movement initiation by optogenetically stimulating specific classes of leg sensory structures. This study found that stimulation of leg mechanosensory bristles (MsBs) and the femoral chordotonal organ (ChO) is sufficient to initiate forward movement in immobile animals. While the stimulation of the ChO required brain centers to induce forward movement, unexpectedly, brief stimulation of leg MsBs triggered a fast response and sustained motor activity dependent only on the ventral nerve cord (VNC). Moreover, this leg-MsB-mediated movement lacked inter- and intra-leg coordination but preserved antagonistic muscle activity within joints. Finally, leg-MsB activation was shown to mediate strong avoidance behavior away from the stimulus source, which is preserved even in the absence of a central brain. Overall, these data show that mechanosensory stimulation can elicit a fast motor response, independently of central brain commands, to evade potentially harmful stimuli. In addition, it sheds light on how specific sensory circuits modulate motor control, including initiation of movement, allowing a better understanding of how different levels of coordination are controlled by the VNC and central brain locomotor circuits.

Smolin, N., Dombrovski, M., Hina, B. W., Moreno-Sanchez, A., Gossart, R., Carmona, C. R., Rehan, A., Hussein, R. H., Mirshahidi, P., Ausborn, J., Kurmangaliyev, Y. Z., von Reyn, C. R. (2024). Neuronal identity control at the resolution of a single transcription factor isoform. bioRxiv, PubMed ID: 38915533 Summary: The brain exhibits remarkable neuronal diversity which is critical for its functional integrity. From the sheer number of cell types emerging from extensive transcriptional, morphological, and connectome datasets, the question arises of how the brain is capable of generating so many unique identities. "Terminal selectors" are transcription factors hypothesized to determine the final identity characteristics in post-mitotic cells. Which transcription factors function as terminal selectors and the level of control they exert over different terminal characteristics are not well defined. This study establish a novel role for the transcription factor broad as a terminal selector in Drosophila melanogaster. This study capitalized on existing large sequencing and connectomics datasets and employ a comprehensive characterization of terminal characteristics including Perturb-seq and whole-cell electrophysiology. A single isoform Broad-z4 was found to serve as the switch between the identity of two visual projection neurons LPLC1 and LPLC2. Broad-z4 is natively expressed in LPLC1, and is capable of transforming the transcriptome, morphology, and functional connectivity of LPLC2 cells into LPLC1 cells when perturbed. This comprehensive work establishes a single isoform as the smallest unit underlying an identity switch, which may serve as a conserved strategy replicated across developmental programs.

Syed, D. S., Ravbar, P., Simpson, J. H. (2024). Inhibitory circuits generate rhythms for leg movements during Drosophila grooming. bioRxiv, PubMed ID: 38895414 Summary: Limbs execute diverse actions coordinated by the nervous system through multiple motor programs. The basic architecture of motor neurons that activate muscles that articulate joints for antagonistic flexion and extension movements is conserved from flies to vertebrates. While excitatory premotor circuits are expected to establish sets of leg motor neurons that work together, this study uncovered a new instructive role for inhibitory circuits: their ability to generate rhythmic leg movements. Using electron microscopy data for the Drosophila nerve cord, ~120 GABAergic inhibitory neurons were categorized from the 13A and 13B hemi-lineages into classes based on similarities in morphology and connectivity. By mapping their synaptic partners, pathways were uncovered for inhibiting specific groups of motor neurons, disinhibiting antagonistic counterparts, and inducing alternation between flexion and extension. The function of specific inhibitory neurons was tested through optogenetic activation and silencing, using an in-depth ethological analysis of leg movements during grooming. Anatomy and behavior analysis findings were combined to construct a computational model that can reproduce major aspects of the observed behavior, confirming the sufficiency of these premotor inhibitory circuits to generate rhythms.

Lesser, E., Azevedo, A. W., Phelps, J. S., Elabbady, L., Cook, A., Syed, D. S., Mark, B., Kuroda, S., Sustar, A., Moussa, A., Dallmann, C. J., Agrawal, S., Lee, S. J., Pratt, B., Skutt-Kakaria, K., Gerhard, S., Lu, R., Kemnitz, N., Lee, K., Halageri, A., Castro, M., Ih, D., Gager, J., Tammam, M., Dorkenwald, S., Collman, F., Schneider-Mizell, C., Brittain, D., Jordan, C. S., Macrina, T., Dickinson, M., Lee, W. A., Tuthill, J. C. (2024). Synaptic architecture of leg and wing premotor control networks in Drosophila. Nature, 631(8020):369-377 PubMed ID: 38926579 Summary: Animal movement is controlled by motor neurons (MNs), which project out of the central nervous system to activate muscles. MN activity is coordinated by complex premotor networks that facilitate the contribution of individual muscles to many different behaviours. This study used connectomics to analyse the wiring logic of premotor circuits controlling the Drosophila leg and wing. We find that both premotor networks cluster into modules that link MNs innervating muscles with related functions. Within most leg motor modules, the synaptic weights of each premotor neuron are proportional to the size of their target MNs, establishing a circuit basis for hierarchical MN recruitment. By contrast, wing premotor networks lack proportional synaptic connectivity, which may enable more flexible recruitment of wing steering muscles. Through comparison of the architecture of distinct motor control systems within the same animal, common principles were identified of premotor network organization and specializations that reflect the unique biomechanical constraints and evolutionary origins of leg and wing motor control.

Chu, S. Y., Lai, Y. W., Hsu, T. C., Lu, T. M., Yu, H. H. (2024). Isoforms of terminal selector Mamo control axon guidance during adult Drosophila memory center construction via Semaphorin-1a. Dev Biol, 515:1-6 PubMed ID: 38906235 Summary: In animals undergoing metamorphosis, the appearance of the nervous system is coincidently transformed by the morphogenesis of neurons. Such morphogenic alterations are exemplified in three types of intrinsic neurons in the Drosophila memory center. In contrast to the well-characterized remodeling of γ neurons, the morphogenesis of αβ and α'/β' neurons has not been adequately explored. Here, we show that mamo, a BTB-zinc finger transcription factor that acts as a terminal selector for α'/β' neurons, controls the formation of the correct axonal pattern of α'/β' neurons. Intriguingly, specific Mamo isoforms are preferentially expressed in α'/β' neurons to regulate the expression of axon guidance molecule Semaphorin-1a. This action directs proper axon guidance in α'/β' neurons, which is also crucial for wiring of α'/β' neurons with downstream neurons. Taken together, these results provide molecular insights into how neurons establish correct axonal patterns in circuitry assembly during adult memory center construction.

Turrel, O., Gao, L., Sigrist, S. J. (2024). Presynaptic regulators in memory formation. Learn Mem, 31(5) PubMed ID: 38862173 Summary: The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. This study investigated the roles of two pivotal presynaptic regulators, the small GTPase Rab3, enriched at synaptic vesicles, and the cell adhesion protein Neurexin-1, in the formation of distinct memory phases within the Drosophila mushroom body Kenyon cells. These findings suggest that both proteins play crucial roles in memory-supporting processes within the presynaptic terminal, operating within distinct plasticity modules. These modules likely encompass remodeling and maturation of existing active zones (AZs), as well as the formation of new AZs.

Monday, April 7th - Tumor Models in Drosophila

Church, S. J., Pulianmackal, A. J., Dixon, J. A., Loftus, L. V., Amend, S. R., Pienta, K., Cackowski, F. C., Buttitta, L. A. (2024). Oncogenic signaling in the adult Drosophila prostate-like accessory gland leads to activation of a conserved pro-tumorigenic program, in the absence of proliferation. bioRxiv, PubMed ID: 38853988 Summary: Drosophila models for tumorigenesis and metastasis have revealed conserved mechanisms of signaling that are also involved in mammalian cancer. Many of these models use the proliferating tissues of the larval stages of Drosophila development, when tissues are highly mitotically active, or stem cells are abundant. Fewer Drosophila tumorigenesis models use adult animals to initiate tumor formation when many tissues are largely terminally differentiated and postmitotic. The Drosophila accessory glands are prostate-like tissues and a model for some aspects of prostate tumorigenesis using this tissue has been explored. In this model, oncogenic signaling was induced during the proliferative stage of accessory gland development, raising the question of how oncogenic activity would impact the terminally differentiated and postmitotic adult tissue. Oncogenic signaling in the adult Drosophila accessory gland was shown to lead to activation of a conserved pro-tumorigenic program, similar to that observed in mitotic larval tissues, but in the absence of proliferation. Oncogenic signaling in the adult postmitotic gland leads to tissue hyperplasia with nuclear anaplasia and aneuploidy through endoreduplication, which increases polyploidy and occasionally results in non-mitotic neoplastic-like extrusions. Gene expression changes in the Drosophila model were compared with that of endocycling prostate cancer cells induced by chemotherapy, which potentially mediate tumor recurrence after treatment. Similar signaling pathways are activated in the Drosophila gland and endocycling cancer cells, suggesting the adult accessory glands provide a useful model for aspects of prostate cancer progression that do not involve cellular proliferation.

Pilesi, E., Tesoriere, G., Ferriero, A., Mascolo, E., Liguori, F., Argiro, L., Angioli, C., Tramonti, A., Contestabile, R., Volonte, C., Verni, F. (2024). Vitamin B6 deficiency cooperates with oncogenic Ras to induce malignant tumors in Drosophila. Cell Death Dis, 15(6):388 PubMed ID: 38830901 Summary: Vitamin B6 is a water-soluble vitamin which possesses antioxidant properties. Its catalytically active form, pyridoxal 5'-phosphate (PLP), is a crucial cofactor for DNA and amino acid metabolism. The inverse correlation between vitamin B6 and cancer risk has been observed in several studies, although dietary vitamin B6 intake sometimes failed to confirm this association. However, the molecular link between vitamin B6 and cancer remains elusive. Previous work has shown that vitamin B6 deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, suggesting that genome instability may correlate the lack of this vitamin to cancer. This study provides evidence in support of this hypothesis. Firstly, PLP deficiency, induced by the PLP antagonists 4-deoxypyridoxine (4DP) or ginkgotoxin (GT), was shown to promote tumorigenesis in eye larval discs transforming benign RasV12 tumors into aggressive forms. In contrast, PLP supplementation reduced the development of tumors. Low PLP levels, induced by 4DP or by silencing the sgll(PNPO) gene involved in PLP biosynthesis, worsened the tumor phenotype in another Drosophila cancer model generated by concomitantly activating RasV12 and downregulating Discs-large (Dlg) gene. Moreover, Ras(V12) eye discs from larvae reared on 4DP displayed CABs, reactive oxygen species (ROS) and low catalytic activity of serine hydroxymethyltransferase (SHMT), a PLP-dependent enzyme involved in thymidylate (dTMP) biosynthesis, in turnRRQrfrebbbgg required for DNA replication and repair. Feeding Ras(V12) 4DP-fed larvae with PLP or ascorbic acid (AA) plus dTMP, rescued both CABs and tumors. The same effect was produced by overexpressing catalase in Ras(V12) Dlg(RNAi) 4DP-fed larvae, thus allowing to establish a relationship between PLP deficiency, CABs, and cancer. Overall, these data provide the first in vivo demonstration that PLP deficiency can impact on cancer by increasing genome instability, which is in turn mediated by ROS and reduced dTMP levels.

Vialat, M., Baabdaty, E., Trousson, A., Kocer, A., Lobaccaro, J. A., Baron, S., Morel, L., de Joussineau, C. (2024). Cholesterol Dietary Intake and Tumor Cell Homeostasis Drive Early Epithelial Tumorigenesis: A Potential Modelization of Early Prostate Tumorigenesis. JCancers, 16(11) PubMed ID: 38893271 Summary: Epidemiological studies point to cholesterol as a possible key factor for both prostate cancer incidence and progression. It could represent a targetable metabolite as the most aggressive tumors also appear to be sensitive to therapies designed to decrease hypercholesterolemia, such as statins. However, it remains unknown whether and how cholesterol, through its dietary uptake and its metabolism, could be important for early tumorigenesis. Oncogene clonal induction in the Drosophila melanogaster accessory gland allows reproduction of tumorigenesis from initiation to early progression, where tumor cells undergo basal extrusion to form extra-epithelial tumors. These tumors accumulate lipids, and especially esterified cholesterol, as in human late carcinogenesis. Interestingly, a high-cholesterol diet has a limited effect on accessory gland tumorigenesis. On the contrary, cell-specific downregulation of cholesterol uptake, intracellular transport, or metabolic response impairs the formation of such tumors. Furthermore, in this context, a high-cholesterol diet suppresses this impairment. Interestingly, expression data from primary prostate cancer tissues indicate an early signature of redirection from cholesterol de novo synthesis to uptake. Taken together, these results reveal that during early tumorigenesis, tumor cells strongly increase their uptake and use of dietary cholesterol to specifically promote the step of basal extrusion. Hence, these results suggest the mechanism by which a reduction in dietary cholesterol could lower the risk and slow down the progression of prostate cancer.

Leung, N. Y., Xu, C., Li, J. S. S., Ganguly, A., Meyerhof, G. T., Regimbald-Dumas, Y., Lane, E. A., Breault, D. T., He, X., Perrimon, N., Montell, C. (2024). Gut tumors in flies alter the taste valence of an anti-tumorigenic bitter compound.. Curr Biol, 34(12):2623-2632 PubMed ID: 38823383 Summary: The sense of taste is essential for survival, as it allows animals to distinguish between foods that are nutritious from those that are toxic. However, innate responses to different tastants can be modulated or even reversed under pathological conditions. This study examined whether and how the internal status of an animal impacts taste valence by using Drosophila models of hyperproliferation in the gut. In all three models where proliferation-inducing transgenes were expressed in intestinal stem cells (ISCs), hyperproliferation of ISCs caused a tumor-like phenotype in the gut. While tumor-bearing flies had no deficiency in overall food intake, strikingly, they exhibited an increased gustatory preference for aristolochic acid (ARI), which is a bitter and normally aversive plant-derived chemical. ARI had anti-tumor effects in all three gut hyperproliferation models. For other aversive chemicals tested that are bitter but do not have anti-tumor effects, gut tumors did not affect avoidance behaviors. Bitter-sensing gustatory receptor neurons (GRNs) in tumor-bearing flies respond normally to ARI. Therefore, the internal pathology of gut hyperproliferation affects neural circuits that determine taste valence postsynaptic to GRNs rather than altering taste identity by GRNs. Overall, these data suggest that increased consumption of ARI may represent an attempt at self-medication. Finally, although ARI's potential use as a chemotherapeutic agent is limited by its known toxicity in the liver and kidney, these findings suggest that tumor-bearing flies might be a useful animal model to screen for novel anti-tumor drugs (Leung, 2024).

Rawal, C. C., Loubiere, V., Butova, N. L., Gracia, J., Parreno, V., Merigliano, C., Martinez, A. M., Cavalli, G., Chiolo, I. (2024). Sustained inactivation of the Polycomb PRC1 complex induces DNA repair defects and genomic instability in epigenetic tumors. Histochemistry and cell biology, 162(1-2):133-147 PubMed ID: 38888809 Summary: Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancer can also be driven purely by epigenetic alterations, without driver mutations. Specifically, a 24-h transient downregulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to induce epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 downregulation is performed for extended periods of time remains unclear. This study shows that prolonged depletion of PH, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad misregulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects accumulate during the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.

Parreno, V., Loubiere, V., Schuettengruber, B., Fritsch, L., Rawal, C. C., Erokhin, M., Gyorffy, B., Normanno, D., Di Stefano, M., Moreaux, J., Butova, N. L., Chiolo, I., Chetverina, D., Martinez, A. M., Cavalli, G. (2024). Transient loss of Polycomb components induces an epigenetic cancer fate. Nature, 629(8012):688-696 PubMed ID: 38658752 Summary: Although cancer initiation and progression are generally associated with the accumulation of somatic mutations, substantial epigenomic alterations underlie many aspects of tumorigenesis and cancer susceptibility, suggesting that genetic mechanisms might not be the only drivers of malignant transformation. However, whether purely non-genetic mechanisms are sufficient to initiate tumorigenesis irrespective of mutations has been unknown. This study shows that a transient perturbation of transcriptional silencing mediated by Polycomb group proteins is sufficient to induce an irreversible switch to a cancer cell fate in Drosophila. This is linked to the irreversible derepression of genes that can drive tumorigenesis, including members of the JAK-STAT signalling pathway and zfh1, the fly homologue of the ZEB1 oncogene, whose aberrant activation is required for Polycomb perturbation-induced tumorigenesis. These data show that a reversible depletion of Polycomb proteins can induce cancer in the absence of driver mutations, suggesting that tumours can emerge through epigenetic dysregulation leading to inheritance of altered cell fates (Parreno, 2024).

Friday, April 4th - Larval and Adult Development

Blackie, L., Gaspar, P., Mosleh, S., Lushchak, O., Kong, L., Jin, Y., Zielinska, A. P., Cao, B., Mineo, A., Silva, B., Ameku, T., Lim, S. E., Mao, Y., Prieto-Godino, L., Schoborg, T., Varela, M., Mahadevan, L., Miguel-Aliaga, I. (2024). The sex of organ geometry. Nature, 630(8016):392-400 PubMed ID: 38811741 Summary: Organs have a distinctive yet often overlooked spatial arrangement in the body. It is proposed that there is a logic to the shape of an organ and its proximitY to its neighbours. Here, by using volumetric scans of many Drosophila melanogaster flies, we develop methods to quantify three-dimensional features of organ shape, position and interindividual variability. Both the shapes of organs and their relative arrangement were consistent yet differ between the sexes, and identify unexpected interorgan adjacencies and left-right organ asymmetries. Focusing on the intestine, which traverses the entire body, this study investigate how sex differences in three-dimensional organ geometry arise. The configuration of the adult intestine is only partially determined by physical constraints imposed by adjacent organs; its sex-specific shape is actively maintained by mechanochemical crosstalk between gut muscles and vascular-like trachea. Indeed, sex-biased expression of a muscle-derived fibroblast growth factor-like ligand renders trachea sexually dimorphic. In turn, tracheal branches hold gut loops together into a male or female shape, with physiological consequences. Interorgan geometry represents a previously unrecognized level of biological complexity which might enable or confine communication across organs and could help explain sex or species differences in organ function.

Rigato, A., Meng, H., Chardes, C., Runions, A., Abouakil, F., Smith, R. S., LeGoff, L. (2024). A mechanical transition from tension to buckling underlies the jigsaw puzzle shape morphogenesis of histoblasts in the Drosophila epidermis. PLoS Biol, 22(6):e3002662 PubMed ID: 38870210 Summary: The polygonal shape of cells in proliferating epithelia is a result of the tensile forces of the cytoskeletal cortex and packing geometry set by the cell cycle. In the larval Drosophila epidermis, two cell populations, histoblasts and larval epithelial cells, compete for space as they grow on a limited body surface. They do so in the absence of cell divisions. A striking morphological transition of histoblasts was observed during larval development, where they change from a tensed network configuration with straight cell outlines at the level of adherens junctions to a highly folded morphology. The apical surface of histoblasts shrinks while their growing adherens junctions fold, forming deep lobules. Volume increase of growing histoblasts is accommodated basally, compensating for the shrinking apical area. The folded geometry of apical junctions resembles elastic buckling, and the imbalance between the shrinkage of the apical domain of histoblasts and the continuous growth of junctions triggers buckling. This model is supported by laser dissections and optical tweezer experiments together with computer simulations. This analysis pinpoints the ability of histoblasts to store mechanical energy to a much greater extent than most other epithelial cell types investigated so far, while retaining the ability to dissipate stress on the hours time scale. Finally, a possible mechanism is proposed for size regulation of histoblast apical size through the lateral pressure of the epidermis, driven by the growth of cells on a limited surface. Buckling effectively compacts histoblasts at their apical plane and may serve to avoid physical harm to these adult epidermis precursors during larval life. This work indicates that in growing nondividing cells, compressive forces, instead of tension, may drive cell morphology.

Petrosky, S. J., Williams, T. M., Rebeiz, M. (2024). A genetic screen of transcription factors in the Drosophila melanogaster abdomen identifies novel pigmentation genes. G3 (Bethesda), 14(9) PubMed ID: 38820091 Summary: Gene regulatory networks specify the gene expression patterns needed for traits to develop. Differences in these networks can result in phenotypic differences between organisms. Although loss-of-function genetic screens can identify genes necessary for trait formation, gain-of-function screens can overcome genetic redundancy and identify loci whose expression is sufficient to alter trait formation. Here, we leveraged transgenic lines from the Transgenic RNAi Project at Harvard Medical School to perform both gain- and loss-of-function CRISPR/Cas9 screens for abdominal pigmentation phenotypes. Measurable effects on pigmentation patterns in the Drosophila melanogaster abdomen were observed for 21 of 55 transcription factors in gain-of-function experiments and 7 of 16 tested by loss-of-function experiments. These included well-characterized pigmentation genes, such as bab1 and dsx, and transcription factors that had no known role in pigmentation, such as slp2. Finally, this screen was partially conducted by undergraduate students in a Genetics Laboratory course during the spring semesters of 2021 and 2022. This screen was found to be a successful model for student engagement in research in an undergraduate laboratory course that can be readily adapted to evaluate the effect of hundreds of genes on many different Drosophila traits, with minimal resources.

Kurogi, Y., Mizuno, Y., Kamiyama, T., Niwa, R. (2024). The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster. Sci Rep, 14(1):9631 PubMed ID: 38671036 Summary: Intestinal stem cells (ISCs) of the fruit fly, Drosophila melanogaster, offer an excellent genetic model to explore homeostatic roles of ISCs in animal physiology. Among available genetic tools, the escargot (esg)-GAL4 driver, expressing the yeast transcription factor gene, GAL4, under control of the esg gene promoter, has contributed significantly to ISC studies. This driver facilitates activation of genes of interest in proximity to a GAL4-binding element, Upstream Activating Sequence, in ISCs and progenitor enteroblasts (EBs). While esg-GAL4 has been considered an ISC/EB-specific driver, recent studies have shown that esg-GAL4 is also active in other tissues, such as neurons and ovaries. Therefore, the ISC/EB specificity of esg-GAL4 is questionable. This study reveals esg-GAL4 expression in the corpus allatum (CA), responsible for juvenile hormone (JH) production. When driving the oncogenic gene, Ras(V12), esg-GAL4 induces overgrowth in ISCs/EBs as reported, but also increases CA cell number and size. Consistent with this observation, animals alter expression of JH-response genes. These data show that esg-GAL4-driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of esg-GAL4 as a "specific" ISC/EB driver to examine ISC/EB-mediated animal physiology.

Raja, K. K. B., Yeung, K., Li, Y., Chen, R., Mardon, G. (2024). A single cell RNA sequence atlas of the early Drosophila larval eye. BMC Genomics, 25(1):616 PubMed ID: 38890587 Summary: The Drosophila eye has been an important model to understand principles of differentiation, proliferation, apoptosis and tissue morphogenesis. However, a single cell RNA sequence resource that captures gene expression dynamics from the initiation of differentiation to the specification of different cell types in the larval eye disc is lacking. This study reports transcriptomic data from 13,000 cells that cover six developmental stages of the larval eye. The data show cell clusters that correspond to all major cell types present in the eye disc ranging from the initiation of the morphogenetic furrow to the differentiation of each photoreceptor cell type as well as early cone cells. We identify dozens of cell type-specific genes whose function in different aspects of eye development have not been reported. These single cell data will greatly aid research groups studying different aspects of early eye development and will facilitate a deeper understanding of the larval eye as a model system.

Ray, A., Rai, Y., Inamdar, M. S. (2024). The Endosomal Sorting Complex, ESCRT, has diverse roles in blood progenitor maintenance, lineage choice and immune response. Biol Open, 13(6) PubMed ID: 38828842 Summary: Most hematological malignancies are associated with reduced expression of one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT). However, the roles of ESCRT in stem cell and progenitor maintenance are not resolved. Parsing signaling pathways in relation to the canonical role of ESCRT poses a challenge. The Drosophila hematopoietic organ, the larval lymph gland, provides a path to dissect the roles of cellular trafficking pathways such as ESCRT in blood development and maintenance. Drosophila has 13 core ESCRT components. Knockdown of individual ESCRTs showed that only Vps28 and Vp36 were required in all lymph gland progenitors. Using the well-conserved ESCRT-II complex as an example of the range of phenotypes seen upon ESCRT depletion, this study showed that ESCRTs have cell-autonomous as well as non-autonomous roles in progenitor maintenance and differentiation. ESCRT depletion also sensitized posterior lobe progenitors to respond to immunogenic wasp infestation. Key heterotypic roles for ESCRT have been found in position-dependent control of Notch activation to suppress crystal cell differentiation. This study shows that the cargo sorting machinery determines the identity of progenitors and their adaptability to the dynamic microenvironment. These mechanisms for control of cell fate may tailor developmental diversity in multiple contexts.

Thursday, April 3rd - Disease Models

Gunasekaran, M., Littel, H. R., Wells, N. M., Turner, J., Campos, G., Venigalla, S., Estrella, E. A., Ghosh, P. S., Daugherty, A. L., Stafki, S. A., Kunkel, L. M., Foley, A. R., Donkervoort, S., Bonnemann, C. G., Toledo-Bravo de Laguna, L., Nascimento, A., Benito, D. N., Draper, I., Bruels, C. C., Pacak, C. A., Kang, P. B. (2024). Effects of HMGCR deficiency on skeletal muscle development. bioRxiv, PubMed ID: 38903061 Summary: Pathogenic variants in HMGCR were recently linked to a limb-girdle muscular dystrophy (LGMD) phenotype. The protein product HMG CoA reductase (HMGCR) catalyzes a key component of the cholesterol synthesis pathway. The two other muscle diseases associated with HMGCR, statin-associated myopathy (SAM) and autoimmune anti-HMGCR myopathy, are not inherited in a Mendelian pattern. The mechanism linking pathogenic variants in HMGCR with skeletal muscle dysfunction is unclear. This study knocked down Hmgcr in mouse skeletal myoblasts, knocked down hmgcr in Drosophila, and expressed three pathogenic HMGCR variants (c.1327C>T, p.Arg443Trp; c.1522_1524delTCT, p.Ser508del; and c.1621G>A, p.Ala541Thr) in Hmgcr knockdown mouse myoblasts. Hmgcr deficiency was associated with decreased proliferation, increased apoptosis, and impaired myotube fusion. Transcriptome sequencing of Hmgcr knockdown versus control myoblasts revealed differential expression involving mitochondrial function, with corresponding differences in cellular oxygen consumption rates. Both ubiquitous and muscle-specific knockdown of hmgcr in Drosophila led to lethality. Overexpression of reference HMGCR cDNA rescued myotube fusion in knockdown cells, whereas overexpression of the pathogenic variants of HMGCR cDNA did not. These results suggest that the three HMGCR-related muscle diseases share disease mechanisms related to skeletal muscle development.

Zuniga, G., Katsumura, S., De Mange, J., Ramirez, P., Atrian, F., Morita, M., Frost, B. (2024). Pathogenic tau induces an adaptive elevation in mRNA translation rate at early stages of disease. Aging Cell:e14245 PubMed ID: 38932463 Summary: Alterations in the rate and accuracy of messenger RNA (mRNA) translation are associated with aging and several neurodegenerative disorders, including Alzheimer's disease and related tauopathies. Previous work showed that error-containing RNA that are normally cleared via nonsense-mediated mRNA decay (NMD), a key RNA surveillance mechanism, are translated in the adult brain of a Drosophila model of tauopathy. The current study found that newly-synthesized peptides and translation machinery accumulate within nuclear envelope invaginations that occur as a consequence of tau pathology, and that the rate of mRNA translation is globally elevated in early stages of disease in adult brains of Drosophila models of tauopathy. Polysome profiling from adult heads of tau transgenic Drosophila reveals the preferential translation of specific mRNA that have been previously linked to neurodegeneration. Unexpectedly, this study found that panneuronal elevation of NMD further elevates the global translation rate in tau transgenic Drosophila, as does treatment with rapamycin. As NMD activation and rapamycin both suppress tau-induced neurodegeneration, their shared effect on translation suggests that elevated rates of mRNA translation are an early adaptive mechanism to limit neurodegeneration. This work provides compelling evidence that tau-induced deficits in NMD reshape the tau translatome by increasing translation of RNA that are normally repressed in healthy cells.

Deb, W., Rosenfelt, C., Vignard, V., Papendorf, J. J., ...., McWalter, K., Lupski, J. R., Isidor, B., Bolduc, F. V., Bezieau, S., Kruger, E., Kury, S., Ebstein, F. (2024). PSMD11 loss-of-function variants correlate with a neurobehavioral phenotype, obesity, and increased interferon response. American journal of human genetics, 111(7):1352-1369 PubMed ID: 38866022 Summary: Primary proteasomopathies have recently emerged as a new class of rare early-onset neurodevelopmental disorders (NDDs) caused by pathogenic variants in the PSMB1, PSMC1, PSMC3, or PSMD12 proteasome genes. Proteasomes are large multi-subunit protein complexes that maintain cellular protein homeostasis by clearing ubiquitin-tagged damaged, misfolded, or unnecessary proteins. In this study, this study have identified PSMD11 as an additional proteasome gene in which pathogenic variation is associated with an NDD-causing proteasomopathy. PSMD11 loss-of-function variants caused early-onset syndromic intellectual disability and neurodevelopmental delay with recurrent obesity in 10 unrelated children. These findings demonstrate that the cognitive impairment observed in these individuals could be recapitulated in Drosophila melanogaster with depletion of the PMSD11 ortholog Rpn6, which compromised reversal learning. Investigations in subject samples further revealed that PSMD11 loss of function resulted in impaired 26S proteasome assembly and the acquisition of a persistent type I interferon (IFN) gene signature, mediated by the integrated stress response (ISR) protein kinase R (PKR). In summary, these data identify PSMD11 as an additional member of the growing family of genes associated with neurodevelopmental proteasomopathies and provide insights into proteasomal biology in human health.

Stojkovic, M., Petrovic, M., Capovilla, M., Milojevic, S., Makevic, V., Budimirovic, D. B., Corscadden, L., He, S., Protic, D. (2024). Using a Combination of Novel Research Tools to Understand Social Interaction in the Drosophila melanogaster Model for Fragile X Syndrome. Biology, 13(6) PubMed ID: 38927312 Summary: Fragile X syndrome (FXS), the most common monogenic cause of inherited intellectual disability and autism spectrum disorder, is caused by a full mutation (>200 CGG repeats) in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene. Individuals with FXS experience various challenges related to social interaction (SI). Animal models, such as the Drosophila melanogaster model for FXS where the only ortholog of human FMR1 (dFMR1) is mutated, have played a crucial role in the understanding of FXS. The aim of this study was to investigate SI in the dFMR1B55 mutants (the groups of flies of both sexes simultaneously) using the novel Drosophila Shallow Chamber and a Python data processing pipeline based on social network analysis (SNA). In comparison with wild-type flies (w(1118)), SNA analysis in dFMR1B55 mutants revealed hypoactivity, fewer connections in their networks, longer interaction duration, a lower ability to transmit information efficiently, fewer alternative pathways for information transmission, a higher variability in the number of interactions they achieved, and flies tended to stay near the boundaries of the testing chamber. These observed alterations indicate the presence of characteristic strain-dependent social networks in dFMR1B55 flies, commonly referred to as the group phenotype. Finally, combining novel research tools is a valuable method for SI research in fruit flies.

Sujkowski, A., Ranxhi, B., Bangash, Z. R., Chbihi, Z. M., Prifti, M. V., Qadri, Z., Alam, N., Todi, S. V., Tsou, W. L. (2024). Progressive degeneration in a new Drosophila model of spinocerebellar ataxia type 7. Sci Rep, 14(1):14332 PubMed ID: 38906973 Summary: Spinocerebellar ataxia type 7 (SCA7) is a progressive neurodegenerative disorder resulting from abnormal expansion of an uninterrupted polyglutamine (polyQ) repeat in its disease protein, ataxin-7 (ATXN7). ATXN7 is part of Spt-Ada-Gcn5 acetyltransferase (SAGA), an evolutionarily conserved transcriptional coactivation complex with critical roles in chromatin remodeling, cell signaling, neurodifferentiation, mitochondrial health and autophagy. SCA7 is dominantly inherited and characterized by genetic anticipation and high repeat-length instability. Patients with SCA7 experience progressive ataxia, atrophy, spasticity, and blindness. There is currently no cure for SCA7, and therapies are aimed at alleviating symptoms to increase quality of life. This studhy reports novel Drosophila lines of SCA7 with polyQ repeats in wild-type and human disease patient range. ATXN7 expression has age- and polyQ repeat length-dependent reduction in fruit fly survival and retinal instability, concomitant with increased ATXN7 protein aggregation. These new lines will provide important insight on disease progression that can be used in the future to identify therapeutic targets for SCA7 patients.

Sevegnani, M., Lama, A., Girardi, F., Hess, M. W., Castelo, M. P., Pichler, I., Biressi, S., Piccoli, G. (2024). Parkin R274W mutation affects muscle and mitochondrial physiology. Biochimica et biophysica acta Molecular basis of disease. 1870(7):167302 PubMed ID: 38878834 Summary: Recessive mutations in the Parkin gene (PRKN) are the most common cause of young-onset inherited parkinsonism. Parkin is a multifunctional E3 ubiquitin ligase that plays a variety of roles in the cell including the degradation of proteins and the maintenance of mitochondrial homeostasis, integrity, and biogenesis. In 2001, the R275W mutation in the PRKN gene was identified in two unrelated families with a multigenerational history of postural tremor, dystonia and parkinsonism. Drosophila models of Parkin R275W showed selective and progressive degeneration of dopaminergic neuronal clusters, mitochondrial abnormalities, and prominent climbing defects. In the Prkn mouse orthologue, the amino acid R274 corresponds to human R275. Here we described an age-related motor impairment and a muscle phenotype in R274W +/+ mice. In vitro, Parkin R274W mutation correlates with abnormal myoblast differentiation, mitochondrial defects, and alteration in mitochondrial mRNA and protein levels. These data suggest that the Parkin R274W mutation may impact mitochondrial physiology and eventually myoblast proliferation and differentiation.

Tuesday, April 1st - Signalling

Lee, G. G., Peterson, A. J., Kim, M. J., O'Connor, M. B., Park, J. H. (2024). Multiple isoforms of the Activin-like receptor baboon differentially regulate proliferation and conversion behaviors of neuroblasts and neuroepithelial cells in the Drosophila larval brain. PLoS One, 19(6):e0305696 PubMed ID: 38913612 Summary: In Drosophila coordinated proliferation of two neural stem cells, neuroblasts (NB) and neuroepithelial (NE) cells, is pivotal for proper larval brain growth that ultimately determines the final size and performance of an adult brain. The larval brain growth displays two phases based on behaviors of NB and NEs: the first one in early larval stages, influenced by nutritional status and the second one in the last larval stage, promoted by ecdysone signaling after critical weight checkpoint. Mutations of the baboon (babo) gene that produces three isoforms (BaboA-C), all acting as type-I receptors of Activin-type transforming growth factor β (TGF-β) signaling, cause a small brain phenotype due to severely reduced proliferation of the neural stem cells. This study show that loss of babo function severely affects proliferation of NBs and NEs as well as conversion of NEs from both phases. By analyzing babo-null and newly generated isoform-specific mutants by CRISPR mutagenesis as well as isoform-specific RNAi knockdowns in a cell- and stage-specific manner, the data support differential contributions of the isoforms for these cellular events with BaboA playing the major role. Stage-specific expression of EcR-B1 in the brain is also regulated primarily by BaboA along with function of the other isoforms. Blocking EcR function in both neural stem cells results in a small brain phenotype that is more severe than baboA-knockdown alone. In summary, this study proposes that the Babo-mediated signaling promotes proper behaviors of the neural stem cells in both phases and achieves this by acting upstream of EcR-B1 expression in the second phase.

Wodrich, A. P. K., Harris, B. T., Giniger, E. (2024). Changes in mitochondrial distribution occur at the axon initial segment in association with neurodegeneration in Drosophila. Biol Open, 13(7) PubMed ID: 38912559 Summary: Changes in mitochondrial distribution are a feature of numerous age-related neurodegenerative diseases. In Drosophila, reducing the activity of Cdk5 causes a neurodegenerative phenotype and is known to affect several mitochondrial properties. Therefore, this study investigated whether alterations of mitochondrial distribution are involved in Cdk5-associated neurodegeneration. Reducing Cdk5 activity did not alter the balance of mitochondrial localization to the somatodendritic versus axonal neuronal compartments of the mushroom body, the learning and memory center of the Drosophila brain. Changes in mitochondrial distribution were observed at the axon initial segment (AIS), a neuronal compartment located in the proximal axon involved in neuronal polarization and action potential initiation. Specifically, its was observed that mitochondria are partially excluded from the AIS in wild-type neurons, but that this exclusion is lost upon reduction of Cdk5 activity, concomitant with the shrinkage of the AIS domain that is known to occur in this condition. This mitochondrial redistribution into the AIS is not likely due to the shortening of the AIS domain itself but rather due to altered Cdk5 activity. Furthermore, mitochondrial redistribution into the AIS is unlikely to be an early driver of neurodegeneration in the context of reduced Cdk5 activity.

Sharma, V., Sachan, N., Sarkar, B., Mutsuddi, M., Mukherjee, A. (2024). E3 ubiquitin ligase Deltex facilitates the expansion of Wingless gradient and antagonizes Wingless signaling through a conserved mechanism of transcriptional effector Armadillo/β-catenin degradation. Elife, 12 PubMed ID: 38900140 Summary: The Wnt/Wg pathway controls myriads of biological phenomena throughout the development and adult life of all organisms across the phyla. Thus, an aberrant Wnt signaling is associated with a wide range of pathologies in humans. Tight regulation of Wnt/Wg signaling is required to maintain proper cellular homeostasis. This study reports a novel role of E3 ubiquitin ligase Deltex in Wg signaling regulation. Drosophila dx genetically interacts with wg and its pathway components. Furthermore, Dx LOF results in a reduced spreading of Wg while its over-expression expands the diffusion gradient of the morphogen. This change is attributed to change in Wg gradient to the endocytosis of Wg through Dx which directly affects the short- and long-range Wg targets. The role of Dx in regulating Wg effector Armadillo was demonstrated where Dx down-regulates Arm through proteasomal degradation. This study also showed the conservation of Dx function in the mammalian system where DTX1 is shown to bind with β-catenin and facilitates its proteolytic degradation, spotlighting a novel step that potentially modulates Wnt/Wg signaling cascade.

Wu, J., Bala Tannan, N., Vuong, L. T., Koca, Y., Collu, G. M., Mlodzik, M. (2024). Par3/bazooka binds NICD and promotes notch signaling during Drosophila development. Dev Biol, 514:37-49 PubMed ID: 38885804 Summary: The conserved bazooka (baz/par3) gene acts as a key regulator of asymmetrical cell divisions across the animal kingdom. Associated Par3/Baz-Par6-aPKC protein complexes are also well known for their role in the establishment of apical/basal cell polarity in epithelial cells. Here we define a novel, positive function of Baz/Par3 in the Notch pathway. Using Drosophila wing and eye development, we demonstrate that Baz is required for Notch signaling activity and optimal transcriptional activation of Notch target genes. Baz appears to act independently of aPKC in these contexts, as knockdown of aPKC does not cause Notch loss-of-function phenotypes. Using transgenic Notch constructs, these data positions Baz activity downstream of activating Notch cleavage steps and upstream of Su(H)/CSL transcription factor complex activity on Notch target genes. A biochemical interaction was demonstrated between NICD and Baz, suggesting that Baz is required for NICD activity before NICD binds to Su(H). Taken together, these data define a novel role of the polarity protein Baz/Par3, as a positive and direct regulator of Notch signaling through its interaction with NICD.

Singh, K., Das, S., Sutradhar, S., Howard, J., Ray, K. (2024). Insulin signaling accelerates the anterograde movement of Rab4 vesicles in axons through Klp98A/KIF16B recruitment via Vps34-PI3Kinase. bioRxiv, PubMed ID: 38895253 Summary: Rab4 GTPase organizes endosomal sorting essential for maintaining the balance between recycling and degradative pathways. Rab4 localizes to many cargos whose transport in neurons is critical for regulating neurotransmission and neuronal health. Furthermore, elevated Rab4 levels in the CNS are associated with synaptic atrophy and neurodegeneration in Drosophila and humans, respectively. However, how the transport of Rab4-associated vesicles is regulated in neurons remains unknown. Using in vivo time-lapse imaging of Drosophila larvae, this study showd that activation of insulin signaling via Dilp2 and dInR increases the anterograde velocity, run length, and flux of Rab4 vesicles in the axons. Molecularly, activation of neuronal insulin signaling further activates Vps34, elevates the levels of PI(3)P on Rab4-associated vesicles, recruits Klp98A (a PI(3)P-binding kinesin-3 motor) and activates their anterograde transport. Together, these observations delineate the role of insulin signaling in regulating axonal transport and synaptic homeostasis.

Malinauskas, T., Moore, G., Rudolf, A. F., Eggington, H., Belnoue-Davis, H. L., El Omari, K., Griffiths, S. C., Woolley, R. E., Duman, R., Wagner, A., Leedham, S. J., Baldock, C., Ashe, H. L., Siebold, C. (2024). Molecular mechanism of BMP signal control by Twisted gastrulation. Nat Commun, 15(1):4976 PubMed ID: 38862520 Summary: Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. This study reports crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. These studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.

Friday, March 29th - Adult Physiology and Metabolism

Nolan, R. B., Fan, J. Y., Price, J. L. (2024). Circadian rhythms in the Drosophila eye may regulate adaptation of vision to light intensity. Frontiers in neuroscience, 18:1401721 PubMed ID: 38872947 Summary: The sensitivity of the eye at night would lead to complete saturation of the eye during the day. Therefore, the sensitivity of the eye must be down-regulated during the day to maintain visual acuity. In the Drosophila eye, the opening of and TRPL channels leads to an influx of Ca(++) that triggers down-regulation of further responses to light, including the movement of the TRPL channel and G&alpha proteins out of signaling complexes found in actin-mediated microvillar extensions of the photoreceptor cells (the rhabdomere). The eye also exhibits a light entrained-circadian rhythm, and we have recently observed that one component of this rhythm (BDBT) becomes undetectable by antibodies after exposure to light even though immunoblot analyses still detect it in the eye. BDBT is necessary for normal circadian rhythms, and in several circadian and visual mutants this eye-specific oscillation of detection is lost. Many phototransduction signaling proteins (e.g., Rhodopsin, TRP channels and Gα) also become undetectable shortly after light exposure, most likely due to a light-induced compaction of the rhabdomeric microvilli. The circadian protein BDBT might be involved in light-induced changes in the rhabdomere, and if so this could indicate that circadian clocks contribute to the daily adaptations of the eye to light. Likewise, circadian oscillations of clock proteins are observed in photoreceptors of the mammalian eye and produce a circadian oscillation in the ERG. Disruption of circadian rhythms in the eyes of mammals causes neurodegeneration in the eye, demonstrating the importance of the rhythms for normal eye function.

Meyerhof, G. T., Easwaran, S., Bontempo, A. E., Montell, C., Montell, D. J. (2024). Altered circadian rhythm, sleep, and rhodopsin 7-dependent shade preference during diapause in Drosophila melanogaster. Proc Natl Acad Sci U S A, 121(27):e2400964121 PubMed ID: 38917005 Summary: To survive adverse environments, many animals enter a dormant state such as hibernation, dauer, or diapause. Various Drosophila species undergo adult reproductive diapause in response to cool temperatures and/or short day-length. While flies are less active during diapause, it is unclear how adverse environmental conditions affect circadian rhythms and sleep. This study shows that in diapause-inducing cool temperatures, Drosophila melanogaster exhibit altered circadian activity profiles, including severely reduced morning activity and an advanced evening activity peak. Consequently, the flies have a single activity peak at a time similar to when nondiapausing flies take a siesta. Temperatures ≤15 °C, rather than photoperiod, primarily drive this behavior. At cool temperatures, flies rapidly enter a deep-sleep state that lacks the sleep cycles of flies at higher temperatures and require high levels of stimulation for arousal. Furthermore, at 25 °C, flies prefer to siesta in the shade, a preference that is virtually eliminated at 10 °C. Resting in the shade is driven by an aversion to blue light that is sensed by Rhodopsin 7 outside of the eyes. Flies at 10 °C show neuronal markers of elevated sleep pressure, including increased expression of Bruchpilot and elevated Ca(2+) in the R5 ellipsoid body neurons. Therefore, sleep pressure might overcome blue light aversion. Thus, at the same temperatures that cause reproductive arrest, preserve germline stem cells, and extend lifespan, D. melanogaster are prone to deep sleep and exhibit dramatically altered, yet rhythmic, daily activity patterns.

Byrns, C. N., Perlegos, A. E., Miller, K. N., Jin, Z., Carranza, F. R., Manchandra, P., Beveridge, C. H., Randolph, C. E., Chaluvadi, V. S., Zhang, S. L., Srinivasan, A. R., Bennett, F. C., Sehgal, A., Adams, P. D., Chopra, G., Bonini, N. M. (2024). Senescent glia link mitochondrial dysfunction and lipid accumulation. Nature, 630(8016):475-483 PubMed ID: 38839958 Summary: Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species. Acutely, senescent cells promote wound healing and prevent tumour formation; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear. This study identified naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence, this study determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, these results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

Singh, A., Abhilasha, K. V., Acharya, K. R., Liu, H., Nirala, N. K., Parthibane, V., Kunduri, G., Abimannan, T., Tantalla, J., Zhu, L. J., Acharya, J. K., Acharya, U. R. (2024). A nutrient responsive lipase mediates gut-brain communication to regulate insulin secretion in Drosophila. Nat Commun, 15(1):4410 PubMed ID: 38782979 Summary: Pancreatic β cells secrete insulin in response to glucose elevation to maintain glucose homeostasis. A complex network of inter-organ communication operates to modulate insulin secretion and regulate glucose levels after a meal. Lipids obtained from diet or generated intracellularly are known to amplify glucose-stimulated insulin secretion, however, the underlying mechanisms are not completely understood. This study showed that a Drosophila secretory lipase, Vaha (CG8093), is synthesized in the midgut and moves to the brain where it concentrates in the insulin-producing cells in a process requiring Lipid Transfer Particle, a lipoprotein originating in the fat body. In response to dietary fat, Vaha stimulates insulin-like peptide release (ILP), and Vaha deficiency results in reduced circulatory ILP and diabetic features including hyperglycemia and hyperlipidemia. Our findings suggest Vaha functions as a diacylglycerol lipase physiologically, by being a molecular link between dietary fat and lipid amplified insulin secretion in a gut-brain axis.

Shin, M., Chang, E., Lee, D., Kim, N., Cho, B., Cha, N., Koranteng, F., Song, J. J., Shim, J. (2024). Drosophila immune cells transport oxygen through PPO2 protein phase transition. Nature, 631(8020):350-359 PubMed ID: 38926577 Summary: Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells. This study describes that Drosophila crystal cells-myeloid-like immune cells called haemocytes-control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, it id proposed that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.

Rai, M., Li, H., Policastro, R. A., Zentner, G. E., Nemkov, T., D'Alessandro, A., Tennessen, J. M. (2024). Glycolytic Disruption Triggers Interorgan Signaling to Nonautonomously Restrict Drosophila Larval Growth. bioRxiv, PubMed ID: 38895259 Summary: Drosophila larval growth requires efficient conversion of dietary nutrients into biomass. Lactate Dehydrogenase (Ldh) and Glycerol-3-phosphate dehydrogenase (Gpdh1) support larval biosynthetic metabolism by maintaining NAD(+)/NADH redox balance and promoting glycolytic flux. Consistent with the cooperative functions of Ldh and Gpdh1, the loss of both enzymes, but neither single enzyme, induces a developmental arrest. However, Ldh and Gpdh1 exhibit complex and often mutually exclusive expression patterns, suggesting that the Gpdh1; Ldh double mutant lethal phenotype could be mediated nonautonomously. This study find that the developmental arrest displayed by the double mutants extends beyond simple metabolic disruption and instead stems, in part, from changes in systemic growth factor signaling. Specifically, this synthetic lethality is linked to the upregulation of Upd3, a cytokine involved in the Jak/Stat signaling pathway. Moreover, either loss of the Upd3 or dietary administration of the steroid hormone 20-hydroxyecdysone (20E) rescue the synthetic lethal phenotype of Gpdh1; Ldh double mutants. Together, these findings demonstrate that metabolic disruptions within a single tissue can nonautonomously modulate interorgan signaling to ensure synchronous developmental growth.

Wednesday, March 26th - Adult neural structure, development, and function

Nguyen, P. K., Cheng, L. Y. (2024). Drosophila medulla neuroblast termination via apoptosis, differentiation, and gliogenic switch is scheduled by the depletion of the neuroepithelial stem cell pool. Elife, 13 PubMed ID: 38905123 Summary: The brain is consisted of diverse neurons arising from a limited number of neural stem cells. Drosophila neural stem cells called neuroblasts (NBs) produces specific neural lineages of various lineage sizes depending on their location in the brain. In the Drosophila visual processing centre - the optic lobes (OLs), medulla NBs derived from the neuroepithelium (NE) give rise to neurons and glia cells of the medulla cortex. The timing and the mechanisms responsible for the cessation of medulla NBs are so far not known. In this study, we show that the termination of medulla NBs during early pupal development is determined by the exhaustion of the NE stem cell pool. Hence, altering NE-NB transition during larval neurogenesis disrupts the timely termination of medulla NBs. Medulla NBs terminate neurogenesis via a combination of apoptosis, terminal symmetric division via Prospero, and a switch to gliogenesis via Glial Cell Missing (Gcm); however, these processes occur independently of each other. It waws also shown that temporal progression of the medulla NBs is mostly not required for their termination. As the Drosophila OL shares a similar mode of division with mammalian neurogenesis, understanding when and how these progenitors cease proliferation during development can have important implications for mammalian brain size determination and regulation of its overall function.

Sturner, T., Brooks, P., Capdevila, L. S., Morris, B. J., ...., Card, G. M., Costa, M., Jefferis, G., Eichler, K. (2024). Comparative connectomics of the descending and ascending neurons of the Drosophila nervous system: stereotypy and sexual dimorphism. bioRxiv, PubMed ID: 38895426 Summary: In most complex nervous systems there is a clear anatomical separation between the nerve cord, which contains most of the final motor outputs necessary for behaviour, and the brain. In insects, the neck connective is both a physical and information bottleneck connecting the brain and the ventral nerve cord (VNC, spinal cord analogue) and comprises diverse populations of descending (DN), ascending (AN) and sensory ascending neurons, which are crucial for sensorimotor signalling and control. Integrating three separate EM datasets, complete connectomic description is provided of the ascending and descending neurons of the female nervous system of Drosophila and compare them with neurons of the male nerve cord. Proofread neuronal reconstructions have been matched across hemispheres, datasets and sexes. Crucially, 51% of DN cell types was matched to light level data defining specific driver lines as well as classifying all ascending populations. We use these results to reveal the general architecture, tracts, neuropil innervation and connectivity of neck connective neurons. Cnnected chains of descending and ascending neurons were found spanning the neck, that may subserve motor sequences. A complete description of sexually dimorphic DN and AN populations is provided, with detailed analysis of circuits implicated in sex-related behaviours, including female ovipositor extrusion (DNp13), male courtship (DNa12/aSP22) and song production (AN hemilineage 08B). This work represents the first EM-level circuit analyses spanning the entire central nervous system of an adult animal.

Sen, E., El-Keredy, A., Jacob, N., Mancini, N., Asnaz, G., Widmann, A., Gerber, B., Thoener, J. (2024). Cognitive limits of larval Drosophila: testing for conditioned inhibition, sensory preconditioning, and second-order conditioning. Learn Mem, 31(5) PubMed ID: 38862170 Summary: Drosophila larvae are an established model system for studying the mechanisms of innate and simple forms of learned behavior. They have about 10 times fewer neurons than adult flies, and it was the low total number of their neurons that allowed for an electron microscopic reconstruction of their brain at synaptic resolution. Regarding the mushroom body, a central brain structure for many forms of associative learning in insects, it turned out that more than half of the classes of synaptic connection had previously escaped attention. Understanding the function of these circuit motifs, subsequently confirmed in adult flies, is an important current research topic. In this context, larval Drosophila were tested for their cognitive abilities in three tasks that are characteristically more complex than those previously studied. The data provide evidence for (i) conditioned inhibition, as has previously been reported for adult flies and honeybees. Unlike what is described for adult flies and honeybees, however, the data do not provide evidence for (ii) sensory preconditioning or (iii) second-order conditioning in Drosophila larvae. The methodological features of these experiments are discussed as well as four specific aspects of the organization of the larval brain that may explain why these two forms of learning are observed in adult flies and honeybees, but not in larval Drosophila.

Azevedo, A., Lesser, E., Phelps, J. S., Mark, B., Elabbady, L., Kuroda, S., Sustar, A., Moussa, A., Khandelwal, A., Dallmann, C. J., Agrawal, S., Lee, S. J., Pratt, B., Cook, A., Skutt-Kakaria, K., Gerhard, S., Lu, R., Kemnitz, N., Lee, K., Halageri, A., Castro, M., Ih, D., Gager, J., Tammam, M., Dorkenwald, S., Collman, F., Schneider-Mizell, C., Brittain, D., Jordan, C. S., Dickinson, M., Pacureanu, A., Seung, H. S., Macrina, T., Lee, W. A., Tuthill, J. C. (2024). Connectomic reconstruction of a female Drosophila ventral nerve cord. Nature, 631(8020):360-368 PubMed ID: 38926570 Summary: A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. This study applied automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC), which functions like the vertebrate spinal cord to sense and control the body. The fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, the muscle targets of leg and wing motor neurons were mapped using genetic driver lines and X-ray holographic nanotomography. With this motor neuron atlas, neural circuits were identified that coordinate leg and wing movements during take-off. This study provides the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.

Benchorin, G., Cho, R. J., Li, M. J., Molotkova, N., Kohwi, M. (2024). Dan forms condensates in neuroblasts and regulates nuclear architecture and progenitor competence in vivo. Nat Commun, 15(1):5097 PubMed ID: 38877037 Summary: Genome organization is thought to underlie cell type specific gene expression, yet how it is regulated in progenitors to produce cellular diversity is unknown. In Drosophila, a developmentally-timed genome reorganization in neural progenitors terminates competence to produce early-born neurons. These events require downregulation of Distal antenna (Dan), part of the conserved pipsqueak DNA-binding superfamily. Dan was found to form liquid-like condensates with high protein mobility, and whose size and subnuclear distribution are balanced with its DNA-binding. Further, a LARKS domain, a structural motif associated with condensate-forming proteins was identified. Deleting just 13 amino acids from LARKS abrogates Dan's ability to retain the early-born neural fate gene, hunchback, in the neuroblast nuclear interior and maintain competence in vivo. Conversely, domain-swapping with LARKS from known phase-separating proteins rescues Dan's effects on competence. Together, in vivo evidence is provided for condensate formation and the regulation of progenitor nuclear architecture underlying neuronal diversification.

Morano, N. C., Lopez, D. H., Meltzer, H., Sergeeva, A. P., Katsamba, P. S., Rostam, K. D., Gupta, H. P., Becker, J. E., Bornstein, B., Cosmanescu, F., Schuldiner, O., Honig, B., Mann, R. S., Shapiro, L. (2024). Cis inhibition of co-expressed DIPs and Dprs shapes neural development. bioRxiv, PubMed ID: 38895375 Summary: In Drosophila , two interacting adhesion protein families, Dprs and DIPs, coordinate the assembly of neural networks. While intercellular DIP/Dpr interactions have been well characterized, DIPs and Dprs are often co-expressed within the same cells, raising the question as to whether they also interact in cis . In cultured cells and in vivo, that DIP-α and DIP-δ can interact in cis with their ligands, Dpr6/10 and Dpr12, respectively. When co-expressed in cis with their cognate partners, these Dprs regulate the extent of trans binding, presumably through competitive cis interactions. The neurodevelopmental effects of cis inhibition in fly motor neurons and in the mushroom body are demonstrated. It was further shown that a long disordered region of DIP-&alpna; at the C-terminus is required for cis but not trans interactions, likely because it alleviates geometric constraints on cis binding. Thus, the balance between cis and trans interactions plays a role in controlling neural development.

Tuesday, March 25th - Genes and Protein

Zhou, T., Wu, W., Ma, S., Chen, J., Huang, J., Qiao, X. (2024). Effects of RDL GABA Receptor Point Mutants on Susceptibility to Meta-Diamide and Isoxazoline Insecticides in Drosophila melanogaster. Insects, 15(5) PubMed ID: 38786890 Summary: Ionotropic γ-aminobutyric acid (GABA) receptors in insects, specifically those composed of the RDL (resistant to dieldrin) subunit, serve as important targets for commonly used synthetic insecticides. These insecticides belong to various chemical classes, such as phenylpyrazoles, cyclodienes, meta-diamides, and isoxazolines, with the latter two potentially binding to the transmembrane inter-subunit pocket. However, the specific amino acid residues that contribute to the high sensitivity of insect RDL receptors to these novel insecticides remain elusive. This study investigated the susceptibility of seven distinct Drosophila melanogaster Rdl point mutants against four meta-diamide and isoxazoline insecticides: isocycloseram, fluxametamide, fluralaner, and broflanilide. The findings indicate that, despite exhibiting increased sensitivity to fluralaner in vitro, the Rdl(I276C) mutant showed resistance to isocycloseram and fluxametamide. Similarly, the double-points mutant Rdl(I276F+G279S) also showed decreased sensitivity to the tested isoxazolines. On the other hand, the Rdl(G335M) mutant displayed high levels of resistance to all tested insecticides. Molecular modeling and docking simulations further supported these findings, highlighting similar binding poses for these insecticides. In summary, this research provides robust in vivo evidence supporting the idea that the inter-subunit amino acids within transmembrane M1 and M3 domains form the binding site crucial for meta-diamide and isoxazoline insecticide interactions. This study highlights the complex interplay between mutations and insecticide susceptibility, paving the way for more targeted pest control strategies.

Monch, T. C., Smylla, T. K., Brandle, F., Preiss, A., Nagel, A. C. (2024). Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development. Genes, 15(5) PubMed ID: 38790181 Summary: Hairless (H) encodes the major antagonist in the Notch signaling pathway, which governs cellular differentiation of various tissues in Drosophila. By binding to the Notch signal transducer Suppressor of Hairless (Su(H)), H assembles repressor complexes onto Notch target genes. Using genome engineering, three new H alleles, H(FA), H(LLAA) and H(WA) were generated and a phenotypic series was established by several parameters, reflecting the residual H-Su(H) binding capacity. Occasionally, homozygous H(WA) flies develop to adulthood. They were compared with the likewise semi-viable H(NN) allele affecting H-Su(H) nuclear entry. The H homozygotes were short-lived, sterile and flightless, yet showed largely normal expression of several mitochondrial genes. Typical for H mutants, both H(WA) and H(NN) homozygous alleles displayed strong defects in wing venation and mechano-sensory bristle development. Strikingly, however, H(WA) displayed only a loss of bristles, whereas bristle organs of H(NN) flies showed a complete shaft-to-socket transformation. Apparently, the impact of H(WA) is restricted to lateral inhibition, whereas that of H(NN) also affects the respective cell type specification. Notably, reduction in Su(H) gene dosage only suppressed the H(NN) bristle phenotype, but amplified that of H(WA). These differences are interpreted as to the role of H regarding Su(H) stability and availability.

Zhang, L., Ge, R., Yang, Y., Chen, K., Li, C. (2024). The zona pellucida protein piopio regulates the metamorphosis and reproduction in Tribolium castaneum. Archives of insect biochemistry and physiology, 116(1):e22122 PubMed ID: 38783685 Summary: The zona pellucida domain protein Piopio (Pio) was reported to mediate only the adhesion of the apical epithelial surface and the overlying apical extracellular matrix in Drosophila melanogaster, but the developmental roles of Pio were poorly understood in insects. To address this issue, this study comprehensively analyzed the function of Pio in Tribolium castaneum. Phylogenetic analysis indicated that pio exhibited one-to-one orthologous relationship among insects. T. castaneum pio had a 1236-bp ORF and contained eight exons. During development pio was abundantly expressed from larva to adult and lowly expressed at the late stage of embryo and adult, while it had more transcripts in the head, epidermis, and gut but fewer in the fat body of late-stage larvae. Knockdown of pio inhibited the pupation, eclosion, and reproduction of T. castaneum. The expression of vitellogenin 1 (Vg1), Vg2, and Vg receptor (VgR) largely decreased in pio-silenced female adults. Silencing pio increased the 20-hydroxyecdysone titer by upregulating phantom and spo expression but decreased the juvenile hormone (JH) titer through downregulating JHAMT3 and promoting JHE, JHEH-r4, and JHDK transcription. These results suggested that Pio might regulate the metamorphosis and reproduction via modulating the ecdysone and JH metabolism in T. castaneum. This study found the novel roles of pio in insect metamorphosis and reproduction, and provided the new insights for analyzing other zona pellucida proteins functions in insects.

Bose, A., Schuster, K., Kodali, C., Sonam, S., Smith-Bolton, R. (2024). The pioneer transcription factor Zelda facilitates the exit from regeneration and restoration of patterning in Drosophila. bioRxiv, PubMed ID: 38854062 Summary: For a damaged tissue to regenerate, the injured site must repair the wound, proliferate, and restore the correct patterning and cell types. Zelda, a pioneer transcription factor largely known for its role in embryonic zygotic genome activation, is dispensable for normal wing development but crucial for wing disc patterning during regeneration. Impairing Zelda function during disc regeneration resulted in adult wings with a plethora of cell fate errors, affecting the veins, margins, and posterior compartment identity. Using CUT&RUN, this study identified and validated targets of Zelda including the cell fate genes cut, Delta and achaete, which failed to return to their normal expression patterns upon loss of Zelda. In addition, Zelda controls expression of factors previously established to preserve cell fate during regeneration like taranis and osa, which stabilizes engrailed expression during regeneration, thereby preserving posterior identity. Finally, Zelda ensures proper expression of the integrins encoded by multiple edematous wings and myospheroid during regeneration to prevent blisters in the resuting adult wing. Thus, Zelda is crucial for maintaining cell fate and structural architecture of the regenerating tissue.

Webb, J. A., Farrow, E., Cain, B., Yuan, Z., Yarawsky, A. E., Schoch, E., Gagliani, E. K., Herr, A. B., Gebelein, B., Kovall, R. A. (2024). Cooperative Gsx2-DNA binding requires DNA bending and a novel Gsx2 homeodomain interface. Nucleic Acids Res, 52(13):7987-8002 PubMed ID: 38874471 Summary: The conserved Gsx homeodomain (HD) transcription factors (see Ind) specify neural cell fates in animals from flies to mammals. Like many HD proteins, Gsx factors bind A/T-rich DNA sequences prompting the following question: How do HD factors that bind similar DNA sequences in vitro regulate specific target genes in vivo? Prior studies revealed that Gsx factors bind DNA both as a monomer on individual A/T-rich sites and as a cooperative homodimer to two sites spaced precisely 7 bp apart. However, the mechanistic basis for Gsx-DNA binding and cooperativity is poorly understood. This study used biochemical, biophysical, structural and modeling approaches to (i) show that Gsx factors are monomers in solution and require DNA for cooperative complex formation, (ii) define the affinity and thermodynamic binding parameters of Gsx2/DNA interactions, (iii) solve a high-resolution monomer/DNA structure that reveals that Gsx2 induces a 20° bend in DNA, (iv) identify a Gsx2 protein-protein interface required for cooperative DNA binding and (v) determine that flexible spacer DNA sequences enhance Gsx2 cooperativity on dimer sites. Altogether, these results provide a mechanistic basis for understanding the protein and DNA structural determinants that underlie cooperative DNA binding by Gsx factors.

Tikhonova, E. A., Georgiev, P. G., Maksimenko, O. G. (2024). Functional Role of C-terminal Domains in the MSL2 Protein of Drosophila melanogaster. Biochemistry (Mosc), 89(4):663-673 PubMed ID: 38831503 Summary: Dosage compensation complex (DCC), which consists of five proteins and two non-coding RNAs roX, specifically binds to the X chromosome in males, providing a higher level of gene expression necessary to compensate for the monosomy of the sex chromosome in male Drosophila compared to the two X chromosomes in females. The MSL2 protein contains the N-terminal RING domain, which acts as an E3 ligase in ubiquitination of proteins and is the only subunit of the complex expressed only in males. Functional role of the two C-terminal domains of the MSL2 protein, enriched with proline (P-domain) and basic amino acids (B-domain), was investigated. As a result, it was shown that the B-domain destabilizes the MSL2 protein, which is associated with the presence of two lysines ubiquitination of which is under control of the RING domain of MSL2. The unstructured proline-rich domain stimulates transcription of the roX2 gene, which is necessary for effective formation of the dosage compensation complex.

Monday, March 24th - RNAs

Petrauskas, A., Fortunati, D. L., Kandi, A. R., Pothapragada, S. S., Agrawal, K., Singh, A., Huelsmeier, J., Hillebrand, J., Brown, G., Chaturvedi, D., Lee, J., Lim, C., Auburger, G., VijayRaghavan, K., Ramaswami, M., Bakthavachalu, B. (2024). Structured and disordered regions of Ataxin-2 contribute differently to the specificity and efficiency of mRNP granule formation. PLoS Genet, 20(5):e1011251 PubMed ID: 38768217 Summary: Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. This study advances these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.

Makhijani, K., Mar, J., Gaziova, I., Bhat, K. M. (2024). Posttranscriptional regulation of the T-box gene midline via the 3'UTR in Drosophila is complex and cell- and tissue-dependent. Genetics, 227(4) PubMed ID: 38805187 Summary: The T-box (Tbx) proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. They are highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. This study reports posttranscriptional and translational regulation of midline (mid), a Tbx member in Drosophila. The 3'UTR of mid was found to have mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid-mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla luciferase reporter significantly reduced the activity of the Luciferase, whereas deleting only the degradation elements from the 3'UTR resulted in reduced activity, but not as much. Overexpression of mid in MP2, an embryonic neuroblast, showed no significant difference in the levels of mid-mRNA between the 2 transgenes, with and without the 3'UTR, indicating the absence of posttranscriptional regulation of mmid in MP2. Moreover, while elevated mid-RNA was detected in MP2 in nearly all hemisegments, only a fifth of those hemisegments had elevated levels of the protein. Overexpression of the 2 transgenes resulted in MP2-lineage defects at about the same frequency. These results indicate a translational/posttranslational regulation of mid in MP2. The regulation of ectopically expressed mmid in the wing imaginal disc was complex. In the wing disc, where b is not expressed, the ectopic expression of the transgene lacking the 3'UTR had a higher level of mid-RNA and the protein had a stronger phenotypic effect. These results indicate that the 3'UTR can subject mid-mRNA to degradation in a cell- and tissue-specific manner. A balancer-mediated transgenerational modifier effect was detected on the expression and gain of function effects of the 2 transgenes.

Crane, A. B., Jetti, S. K., Littleton, J. T. (2024). A stochastic RNA editing process targets a limited number of sites in individual Drosophila glutamatergic motoneurons. bioRxiv, PubMed ID: 38798345 Summary: RNA editing is a post-transcriptional source of protein diversity and occurs across the animal kingdom. Given the complete profile of mRNA targets and their editing rate in individual cells is unclear, this study analyzed single cell RNA transcriptomes from Drosophila larval tonic and phasic glutamatergic motoneuron subtypes to determine the most highly edited targets and identify cell-type specific editing. From ∼15,000 genes encoded in the genome, 316 high confidence A-to-I canonical RNA edit sites were identified, with 102 causing missense amino acid changes in proteins regulating membrane excitability, synaptic transmission, and cellular function. Some sites showed 100% editing in single neurons as observed with mRNAs encoding mammalian AMPA receptors. However, most sites were edited at lower levels and generated variable expression of edited and unedited mRNAs within individual neurons. Together, these data provide insights into how the RNA editing landscape alters protein function to modulate the properties of two well-characterized neuronal populations in Drosophila.

Tian, S., Asano, Y., Banerjee, T. D., Wee, J. L. Q., Lamb, A., Wang, Y., Murugesan, S. N., Ui-Tei, K., Wittkopp, P. J., Monteiro, A. (2024). A micro-RNA is the effector gene of a classic evolutionary hotspot locus. bioRxiv, PubMed ID: 38659873 Summary: In Lepidoptera (butterflies and moths), the genomic region around the gene cortex is a 'hotspot' locus, repeatedly used to generate intraspecific melanic wing color polymorphisms across 100-million-years of evolution. However, the identity of the effector gene regulating melanic wing color within this locus remains unknown. This study shows that none of the four candidate protein-coding genes within this locus, including cortex, serve as major effectors. Instead, a micro-RNA (miRNA), mir-193, serves as the major effector across three deeply diverged lineages of butterflies, and its function is conserved in Drosophila. In Lepidoptera, mir-193 is derived from a gigantic long non-coding RNA, ivory, and it functions by directly repressing multiple pigmentation genes. We show that a miRNA can drive repeated instances of adaptive evolution in animals.

Fingerhut, J. M., Lannes, R., Whitfield, T. W., Thiru, P., Yamashita, Y. M. (2024). Co-transcriptional splicing facilitates transcription of gigantic genes. PLoS Genet, 20(6):e1011241 PubMed ID: 38870220 Summary: Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. This study demonstrates that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. It is proposed that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. study reveals a novel biological significance of co-transcriptional splicing.

Zhang, Q., Fan, X., Fu, F., Zhu, Y., Luo, G., Chen, H. (2024). Adar Regulates Drosophila melanogaster Spermatogenesis via Modulation of BMP Signaling. Int J Mol Sci, 25(11) PubMed ID: 38891830 Summary: The dynamic process of Drosophila spermatogenesis involves asymmetric division, mitosis, and meiosis, which ultimately results in the production of mature spermatozoa. Disorders of spermatogenesis can lead to infertility in males. ADAR (Adenosine deaminase acting on RNA) mutations in Drosophila cause male infertility, yet the causative factors remain unclear. In this study, immunofluorescence staining was employed to visualize endogenous ADAR proteins and assess protein levels via fluorescence-intensity analysis. In addition, the early differentiation disorders and homeostatic alterations during early spermatogenesis in the testes were examined through quantification of transit-amplifying region length, counting the number of GSCs (germline stem cells), and fertility experiments. The findings suggest that deletion of ADAR causes testicular tip transit-amplifying cells to accumulate and become infertile in older male Drosophila. By overexpressing ADAR in early germline cells, male infertility can be partially rescued. Transcriptome analysis showed that ADAR maintained early spermatogenesis homeostasis through the bone-morphogenetic-protein (BMP) signaling pathway. Taken together, these findings have the potential to help explore the role of ADAR in early spermatogenesis.

Thursday, March 20th - Adult Neural Structure, Development and Function

Zhang, X., Sun, D., Wong, K., Salkini, A., Najafi, H., Kim, W. J. (2024). The astrocyte-enriched gene deathstar plays a crucial role in the development, locomotion, and lifespan of D. melanogaster. Fly (Austin), 18(1):2368336 PubMed ID: 38884422 Summary: The Drosophila melanogaster brain is a complex organ with various cell types, orchestrating the development, physiology, and behaviors of the fly. While each cell type in Drosophila brain is known to express a unique gene set, their complete genetic profile is still unknown. Advances in the RNA sequencing techniques at single-cell resolution facilitate identifying novel cell type markers and/or re-examining the specificity of the available ones. In this study, exploiting a single-cell RNA sequencing data of Drosophila optic lobe, the cells were wcategorized based on their expression pattern for known markers, then the genes with enriched expression in astrocytes were identified. CG11000 (Deathstar) was identified as a gene with a comparable expression profile to the Eaat1 gene, an astrocyte marker, in every individual cell inside the Drosophila optic lobe and midbrain, as well as in the entire Drosophila brain throughout its development. Consistent with these bioinformatics data, immunostaining of the brains dissected from transgenic adult flies showed co-expression of CG11000 with Eaat1 in a set of single cells corresponding to the astrocytes in the Drosophila brain. Physiologically, inhibiting CG11000 through RNA interference disrupted the normal development of male D. melanogaster, while having no impact on females. Expression suppression of CG11000 in adult flies led to decreased locomotion activity and also shortened lifespan specifically in astrocytes, indicating the gene's significance in astrocytes. This gene was designated as 'deathstar' due to its crucial role in maintaining the star-like shape of glial cells, astrocytes, throughout their development into adult stage.

Coban, B., Poppinga, H., Rachad, E. Y., Geurten, B., Vasmer, D., Rodriguez Jimenez, F. J., Gadgil, Y., Deimel, S. H., Alyagor, I., Schuldiner, O., Grunwald Kadow, I. C., Riemensperger, T. D., Widmann, A., Fiala, A. (2024). The caloric value of food intake structurally adjusts a neuronal mushroom body circuit mediating olfactory learning in Drosophila. JLearn Mem, 31(5) PubMed ID: 38862177 Summary: Associative learning enables the adaptive adjustment of behavioral decisions based on acquired, predicted outcomes. The valence of what is learned is influenced not only by the learned stimuli and their temporal relations, but also by prior experiences and internal states. This study used the fruit fly Drosophila melanogaster to demonstrate that neuronal circuits involved in associative olfactory learning undergo restructuring during extended periods of low-caloric food intake. Specifically, a decrease was observed in the connections between specific dopaminergic neurons (DANs) and Kenyon cells at distinct compartments of the mushroom body. This structural synaptic plasticity was contingent upon the presence of allatostatin A receptors in specific DANs and could be mimicked optogenetically by expressing a light-activated adenylate cyclase in exactly these DANs. Importantly, this rearrangement in synaptic connections was found to influence aversive, punishment-induced olfactory learning but did not impact appetitive, reward-based learning. Whether induced by prolonged low-caloric conditions or optogenetic manipulation of cAMP levels, this synaptic rearrangement resulted in a reduction of aversive associative learning. Consequently, the balance between positive and negative reinforcing signals shifted, diminishing the ability to learn to avoid odor cues signaling negative outcomes. These results exemplify how a neuronal circuit required for learning and memory undergoes structural plasticity dependent on prior experiences of the nutritional value of food.

Sukumar, S. K., Antonydhason, V., Molander, L., Sandakly, J., Kleit, M., Umapathy, G., Mendoza-Garcia, P., Masudi, T., Schlosser, A., Nässel, D. R., Wegener, C., Shirinian, M., Palmer, R. H. (2024). The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila central nervous system. Elife, 12 PubMed ID: 38904987 Summary: Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, this study employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, this study reports a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

Ehweiner, A., Duch, C., Brembs, B. (2024). Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila. F1000Research, 13:116 PubMed ID: 38779314 Summary: Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. This study presents novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. Wild type and transgenic Drosophila fruit flieswere operantly trained, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). This behavioral experiment was combined with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. Expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP was found to be necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. It takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. These experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and modulation of optomotor behavior was found after operant self-learning. aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

Lottes, E. N., Ciger, F., Bhattacharjee, S., Timmins, E. A., Tete, B., Tran, T., Matta, J., Patel, A. A., Cox, D. N. (2024). CCT and Cullin1 Regulate the TORC1 Pathway to Promote Dendritic Arborization in Health and Disease. Cells, 13(12) PubMed ID: 38920658 Summary: The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. This study examined the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, this study confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, this study examined CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, these data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.

Vaikakkara Chithran, A., Allan, D. W., O'Connor, T. P. (2024). Adult expression of the cell adhesion protein Fasciclin 3 is required for the maintenance of adult olfactory interneurons. J Cell Sci, 137(12) PubMed ID: 38934299 Summary: The proper functioning of the nervous system is dependent on the establishment and maintenance of intricate networks of neurons that form functional neural circuits. Once neural circuits are assembled during development, a distinct set of molecular programs is likely required to maintain their connectivity throughout the lifetime of the organism. This study demonstrates that Fasciclin 3 (Fas3), an axon guidance cell adhesion protein, is necessary for the maintenance of the olfactory circuit in adult Drosophila. The TARGET system was used to spatiotemporally knockdown Fas3 in selected populations of adult neurons. The findings show that Fas3 knockdown results in the death of olfactory circuit neurons and reduced survival of adults. It was also demonstrated that Fas3 knockdown activates caspase-3-mediated cell death in olfactory local interneurons, which can be rescued by overexpressing baculovirus p35, an anti-apoptotic protein. This work adds to the growing set of evidence indicating a crucial role for axon guidance proteins in the maintenance of neuronal circuits in adults.

Wednesday, March 19th - Disease Models

Au, W. H., Miller-Fleming, L., Sanchez-Martinez, A., Lee, J. A., Twyning, M. J., Prag, H. A., Raik, L., Allen, S. P., Shaw, P. J., Ferraiuolo, L., Mortiboys, H., Whitworth, A. J. (2024). Activation of the Keap1/Nrf2 pathway suppresses mitochondrial dysfunction, oxidative stress, and motor phenotypes in C9orf72 ALS/FTD models. Life science alliance, 7(9) PubMed ID: 38906677 Summary: Mitochondrial dysfunction is a common feature of C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD); however, it remains unclear whether this is a cause or consequence of the pathogenic process. Analysing multiple aspects of mitochondrial biology across several Drosophila models of C9orf72-ALS/FTD, this study found morphology, oxidative stress, and mitophagy are commonly affected, which correlated with progressive loss of locomotor performance. Notably, only genetic manipulations that reversed the oxidative stress levels were also able to rescue C9orf72 locomotor deficits, supporting a causative link between mitochondrial dysfunction, oxidative stress, and behavioural phenotypes. Targeting the key antioxidant Keap1/Nrf2 pathway, genetic reduction of Keap1 or pharmacological inhibition by dimethyl fumarate significantly was found to rescue the C9orf72-related oxidative stress and motor deficits. Finally, mitochondrial ROS levels were also elevated in C9orf72 patient-derived iNeurons and were effectively suppressed by dimethyl fumarate treatment. These results indicate that mitochondrial oxidative stress is an important mechanistic contributor to C9orf72 pathogenesis, affecting multiple aspects of mitochondrial function and turnover. Targeting the Keap1/Nrf2 signalling pathway to combat oxidative stress represents a therapeutic strategy for C9orf72-related ALS/FTD.

Zhou, C., Li, Z., Li, Y., Li, Y., Wang, W., Shang, W., Liu, J. P., Wang, L., Tong, C. (2024). TRABD modulates mitochondrial homeostasis and tissue integrity.. Cell Rep, 43(6):114304 PubMed ID: 38843396 Summary: High TRABD expression is associated with tau pathology in patients with Alzheimer's disease; however, the function of TRABD is unknown. Human TRABD encodes a mitochondrial outer-membrane protein. The loss of TRABD resulted in mitochondrial fragmentation, and TRABD overexpression led to mitochondrial clustering and fusion. The C-terminal tail of the TRABD anchored to the mitochondrial outer membrane and the TraB domain could form homocomplexes. Additionally, TRABD forms complexes with MFN2, MIGA2, and PLD6 to facilitate mitochondrial fusion. Flies lacking dTRABD are viable and have normal lifespans. However, aging flies exhibit reduced climbing ability and abnormal mitochondrial morphology in their muscles. The expression of dTRABD is increased in aged flies. dTRABD overexpression leads to neurodegeneration and enhances tau toxicity in fly eyes. The overexpression of dTRABD also increased reactive oxygen species (ROS), ATP production, and protein turnover in the mitochondria. This study suggested that TRABD-induced mitochondrial malfunctions contribute to age-related neurodegeneration.

Sidisky, J. M., Winters, A., Caratenuto, R., Babcock, D. T. (2024). Synaptic defects in a drosophila model of muscular dystrophy. Frontiers in cellular neuroscience, 18:1381112 PubMed ID: 38812789 Summary: Muscular dystrophies are a devastating class of diseases that result in a progressive loss of muscle integrity. Duchenne Muscular Dystrophy, the most prevalent form of Muscular Dystrophy, is due to the loss of functional Dystrophin. While much is known regarding destruction of muscle tissue in these diseases, much less is known regarding the synaptic defects that also occur in these diseases. Synaptic defects are also among the earliest hallmarks of neurodegenerative diseases, including the neuromuscular disease Amyotrophic Lateral Sclerosis (ALS). The current study investigates synaptic defects within adult muscle tissues as well as presynaptic motor neurons in Drosophila dystrophin mutants. The progressive, age-dependent loss of flight ability in dystrophin mutants is accompanied by disorganization of Neuromuscular Junctions (NMJs), including impaired localization of both presynaptic and postsynaptic markers. These synaptic defects, including presynaptic defects within motor neurons, are due to the loss of Dystrophin specifically within muscles. These results should help to better understand the early synaptic defects preceding cell loss in neuromuscular disorders.

Banerjee, S., Vernon, S., Ruchti, E., Limoni, G., Jiao, W., Asadzadeh, J., Van Campenhoudt, M., McCabe, B. D. (2024). Trio preserves motor synapses and prolongs motor ability during aging. Cell Rep, 43(6):114256 PubMed ID: 38795343 Summary: The decline of motor ability is a hallmark feature of aging and is accompanied by degeneration of motor synaptic terminals. Consistent with this, Drosophila motor synapses undergo characteristic age-dependent structural fragmentation co-incident with diminishing motor ability. Motor synapse levels of Trio, an evolutionarily conserved guanine nucleotide exchange factor (GEF), decline with age. Increasing Trio expression in adult Drosophila can abrogate age-dependent synaptic structural fragmentation, postpone the decline of motor ability, and maintain the capacity of motor synapses to sustain high-intensity neurotransmitter release. This preservative activity is conserved in transgenic human Trio, requires Trio Rac GEF function, and can also ameliorate synapse degeneration induced by depletion of miniature neurotransmission. These results support a paradigm where the structural dissolution of motor synapses precedes and promotes motor behavioral diminishment and where intervening in this process can postpone the decline of motor function during aging.

Zakharenko, L. P., Bobrovskikh, M. A., Gruntenko, N. E., Petrovskii, D. V., Verevkin, E. G., Putilov, A. A. (2024). Two Old Wild-Type Strains of Drosophila melanogaster Can Serve as an Animal Model of Faster and Slower Aging Processes. Insects, 15(5) PubMed ID: 38786885 Summary: Drosophila melanogaster provides a powerful platform to study the physiology and genetics of aging, i.e., the mechanisms underpinnings healthy aging, age-associated disorders, and acceleration of the aging process under adverse environmental conditions. This study tested the responses of daily rhythms to age-accelerated factors in two wild-type laboratory-adapted strains, Canton-S and Harwich. On the example of the 24 h patterns of locomotor activity and sleep, the responses of these two strains to such factors as aging, high temperature, carbohydrate diet, and diet were documented with different doses of caffeine-benzoate sodium. The strains demonstrated differential responses to these factors. Moreover, compared to Canton-S, Harwich showed a reduced locomotor activity, larger amount of sleep, faster rate of development, smaller body weight, lower concentrations of main sugars, lower fecundity, and shorter lifespan. It might be recommended to use at least two strains, one with a relatively fast and another with a relatively slow aging process, for the experimental elaboration of relationships between genes, environment, behavior, physiology, and health.

Li, M., Shou, H., Martínez Corrales, G., Svermova, T., Franco, A. V., Alic, N. (2024). Xbp1 targets canonical UPR(ER) and non-canonical pathways in separate tissues to promote longevity. iScience, 27(6):109962 PubMed ID: 38832022 Summary: Transcription factors can reprogram gene expression to promote longevity. Here, we investigate the role of Drosophila Xbp1. Xbp1 is activated by splicing of its primary transcript, Xbp1(u), to generate Xbp1(s), a key activator of the endoplasmic reticulum unfolded protein response (UPR(ER)). Xbp1(s) induces the conical UPR(ER) in the gut, promoting longevity from the resident stem cells. In contrast, in the fat body, Xbp1(s) does not appear to trigger UPR(ER) but alters metabolic gene expression and is still able to extend lifespan. In the fat body, Xbp1(s) and dFOXO impinge on the same target genes, including the PGC-1α orthologue Srl, and dfoxo requires Xbp1 to extend lifespan. Interestingly, unspliceable version of the Xbp1 mRNA, Xbp1(u) can also extend lifespan, hinting at roles in longevity for the poorly characterized Xbp1(u) transcription factor. These findings reveal the diverse functions of Xbp1 in longevity in the fruit fly.

Thursday, March 13th - Evolution

Barerra, T. S., Sattolo, M. L., Kwok, K. E., Agrawal, A. F., Rundle, H. D. (2024). Mating environments mediate the evolution of behavioral isolation during ecological speciation. Evolution letters, 8(3):448-454 PubMed ID: 38818417 Summary: The evolution of behavioral isolation is often the first step toward speciation. While past studies show that behavioral isolation will sometimes evolve as a by-product of divergent ecological selection, a more nuanced understanding of factors that may promote or hamper its evolution is lacking. The environment in which mating occurs may be important in mediating whether behavioral isolation evolves for two reasons. Ecological speciation could occur as a direct outcome of different sexual interactions being favored in different mating environments. Alternatively, mating environments may vary in the constraint they impose on traits underlying mating interactions, such that populations evolving in a "constraining" mating environment would be less likely to evolve behavioral isolation than populations evolving in a less constraining mating environment. In the latter, mating environment is not the direct cause of behavioral isolation but rather permits its evolution only if other drivers are present. These ideas were tested with a set of 28 experimental fly populations, each of which evolved under one of two mating environments and one of two larval environments. Counter to the prediction of ecological speciation by mating environment, behavioral isolation was not maximal between populations evolved in different mating environments. Nonetheless, mating environment was an important factor as behavioral isolation evolved among populations from one mating environment but not among populations from the other. Though one mating environment was conducive to the evolution of behavioral isolation, it was not sufficient: assortative mating only evolved between populations adapting to different-larval environments within that mating environment, indicating a role for ecological speciation. Intriguingly, the mating environment that promoted behavioral isolation is characterized by less sexual conflict compared to the other mating environment. These results suggest that mating environments play a key role in mediating ecological speciation via other axes of divergent selection.

Whittle, C. A., Extavour, C. G. (2024). Gene Protein Sequence Evolution Can Predict the Rapid Divergence of Ovariole Numbers in the Drosophila melanogaster Subgroup. Genome biology and evolution, 16(7) PubMed ID: 38848313 Summary: Ovaries play key roles in fitness and evolution: they are essential female reproductive structures that develop and house the eggs in sexually reproducing animals. In Drosophila, the mature ovary contains multiple tubular egg-producing structures known as ovarioles. Ovarioles arise from somatic cellular structures in the larval ovary called terminal filaments (TFs), formed by TF cells and subsequently enclosed by sheath (SH) cells. As in many other insects, ovariole number per female varies extensively in Drosophila. At present, however, there is a striking gap of information on genetic mechanisms and evolutionary forces that shape the well-documented rapid interspecies divergence of ovariole numbers. To address this gap, genes associated with Drosophila melanogaster ovariole number were studied for functions based on recent experimental and transcriptional datasets from larval ovaries, including TFs and SH cells, and their rates and patterns of molecular evolution were assessed in five closely related species of the melanogaster subgroup that exhibit species-specific differences in ovariole numbers. From comprehensive analyses of protein sequence evolution (dN/dS), branch-site positive selection, expression specificity (tau), and phylogenetic regressions (phylogenetic generalized least squares), evidence is reported of 42 genes that showed signs of playing roles in the genetic basis of interspecies evolutionary change of Drosophila ovariole number. These included the signaling genes upd2 and Ilp5 and extracellular matrix genes vkg and Col4a1, whose dN/dS predicted ovariole numbers among species. Together, we propose a model whereby a set of ovariole-involved gene proteins have an enhanced evolvability, including adaptive evolution, facilitating rapid shifts in ovariole number among Drosophila species.

Hsu, S. K., Lai, W. Y., Novak, J., Lehner, F., Jaksic, A. M., Versace, E., Schlotterer, C. (2024). Reproductive isolation arises during laboratory adaptation to a novel hot environment. Genome Biol, 25(1):141 PubMed ID: 38807159 Summary: Reproductive isolation can result from adaptive processes (e.g., ecological speciation and mutation-order speciation) or stochastic processes such as "system drift" model. Ecological speciation predicts barriers to gene flow between populations from different environments, but not among replicate populations from the same environment. In contrast, reproductive isolation among populations independently adapted to the same/similar environment can arise from both mutation-order speciation or system drift. In experimentally evolved populations adapting to a hot environment for over 100 generations, this study found evidence for pre- and postmating reproductive isolation. On one hand, an altered lipid metabolism and cuticular hydrocarbon composition pointed to possible premating barriers between the ancestral and replicate evolved populations. On the other hand, the pronounced gene expression differences in male reproductive genes may underlie the postmating isolation among replicate evolved populations adapting to the same environment with the same standing genetic variation. This study confirms that replicated evolution experiments provide valuable insights into the mechanisms of speciation. The rapid emergence of the premating reproductive isolation during temperature adaptation showcases incipient ecological speciation. The potential evidence of postmating reproductive isolation among replicates gave rise to two hypotheses: (1) mutation-order speciation through a common selection on early fecundity leading to an inherent inter-locus sexual conflict; (2) system drift with genetic drift along the neutral ridges.

Ribeiro, T. D. S., Lollar, M. J., Sprengelmeyer, Q. D., Huang, Y., Benson, D. M., Orr, M. S., Johnson, Z. C., Corbett-Detig, R. B., Pool, J. E. (2024). Recombinant inbred line panels inform the genetic architecture and interactions of adaptive traits in Drosophila melanogaster. bioRxiv, PubMed ID: 38798433 Summary: The distribution of allelic effects on traits, along with their gene-by-gene and gene-by-environment interactions, contributes to the phenotypes available for selection and the trajectories of adaptive variants. Nonetheless, uncertainty persists regarding the effect sizes underlying adaptations and the importance of genetic interactions. Herein, we aimed to investigate the genetic architecture and the epistatic and environmental interactions involving loci that contribute to multiple adaptive traits using two new panels of Drosophila melanogaster recombinant inbred lines (RILs). To better fit these data, functions from R/qtl were reimplemented using additive genetic models. We found 14 quantitative trait loci (QTL) underlying melanism, wing size, song pattern, and ethanol resistance. By combining our mapping results with population genetic statistics, potential new genes were identified related to these traits. None of the detected QTLs showed clear evidence of epistasis, and the power analysis indicated that at least one significant interaction was seen if sign epistasis or strong positive epistasis played a pervasive role in trait evolution. In contrast, roles for gene-by-environment interactions were found involving pigmentation traits. Overall, the data suggest that the genetic architecture of adaptive traits often involves alleles of detectable effect, that strong epistasis does not always play a role in adaptation, and that environmental interactions can modulate the effect size of adaptive alleles.

Bladen, J., Nam, H. J., Phadnis, N. (2024). Transformation of meiotic drive into hybrid sterility in Drosophila. bioRxiv, PubMed ID: 38798315 Summary: Hybrid male sterility is one of the fastest evolving intrinsic reproductive barriers between recently isolated populations. A leading explanation for the evolution of hybrid male sterility involves genomic conflicts with meiotic drivers in the male germline. There are, however, few examples directly linking meiotic drive to hybrid sterility. This study reports that the Sex-Ratio chromosome of Drosophila pseudoobscura, which causes X-chromosome drive within the USA subspecies, causes near complete male sterility when moved into the genetic background of the Bogota subspecies. In addition, this new form of sterility is genetically distinct from the sterility of F1 hybrid males in crosses between USA males and Bogota females. Our observations provide a tractable study system where non-cryptic drive within species is transformed into strong hybrid sterility between very young subspecies.

Li, X. C., Gandara, L., Ekelof, M., Richter, K., Alexandrov, T., Crocker, J. (2024). Rapid response of fly populations to gene dosage across development and generations. Nat Commun, 15(1):4551 PubMed ID: 38811562 Summary: Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, current understanding of how developmental programs react to these challenges remains limited. This study has examined the phenotypic consequences of disturbing the bicoid. regulatory network in early Dwrosophila embryos. Flies with two extra copies of bicoid, which causes a posterior shift of the network's regulatory outputs and a decrease in fitness. These flies were subjected to EMS mutagenesis, followed by experimental evolution. After only 8-15 generations, experimental populations have normalized patterns of gene expression and increased survival. Using a phenomics approach, this study found that populations were normalized through rapid increases in embryo size driven by maternal changes in metabolism and ovariole development. The results were extended to additional populations of flies, demonstrating predictability. Together, these w=== results necessitate a broader view of regulatory network evolution at the systems level.

Tuesday, March 11th - Adult Neural Development, Structure and Development

Kim, H., Zhong, Z., Cui, X., Sung, H., Agrawal, N., Jiang, T., Dus, M., Yapici, N. (2024). HisCl1 regulates gustatory habituation in sweet taste neurons and mediates sugar ingestion in Drosophila. bioRxiv, PubMed ID: 38765964 Summary: Similar to other animals, the fly, Drosophila melanogaster, reduces its responsiveness to tastants with repeated exposure, a phenomenon called gustatory habituation. Previous studies have focused on the circuit basis of gustatory habituation in the fly chemosensory system. However, gustatory neurons reduce their firing rate during repeated stimulation, suggesting that cell-autonomous mechanisms also contribute to habituation. This study used deep learning-based pose estimation and optogenetic stimulation to demonstrate that continuous activation of sweet taste neurons causes gustatory habituation in flies. A transgenic RNAi screen was conducted to identify genes involved in this process, and knocking down Histamine-gated chloride channel subunit 1 (HisCl1) in the sweet taste neurons was found to significantly reduced gustatory habituation. Anatomical analysis showed that HisCl1 is expressed in the sweet taste neurons of various chemosensory organs. Using single sensilla electrophysiology, this study showed that sweet taste neurons reduced their firing rate with prolonged exposure to sucrose. Knocking down HisCl1 in sweet taste neurons suppressed gustatory habituation by reducing the spike frequency adaptation observed in these neurons during high-concentration sucrose stimulation. Finally, flies lacking HisCl1 in sweet taste neurons were shown to increase their consumption of high-concentration sucrose solution at their first meal bout compared to control flies. Together, these results demonstrate that HisCl1 tunes spike frequency adaptation in sweet taste neurons and contributes to gustatory habituation and food intake regulation in flies. Since HisCl1 is highly conserved across many dipteran and hymenopteran species, these findings open a new direction in studying insect gustatory habituation.

Long, T., Mohapatra, P., Ballou, S., Menuz, K. (2024). Odorant receptor co-receptors affect expression of tuning receptors in Drosophila. Frontiers in cellular neuroscience, 18:1390557 PubMed ID: 38832356 Summary: Insects detect odorants using two large families of heteromeric receptors, the xOdorant Receptors (ORs) and Ionotropic Receptors (IRs). Most OR and IR genes encode odorant-binding "tuning" subunits, whereas four (Orco, Ir8a, Ir25a, andIr76b) encode co-receptor subunits required for receptor function. This study examined the role of co-receptors on olfactory neuron survival in Drosophila. Consistent with olfactory neuron degeneration, expression of many OR-family tuning receptors is decreased in Orco mutants relative to controls, and transcript loss is progressive with age. The effects of Orco are highly receptor-dependent, with expression of some receptor transcripts nearly eliminated and others unaffected. Surprisingly, further studies revealed that olfactory neuron classes with reduced tuning receptor expression generally survive in Orco mutant flies. Furthermore, there is little apoptosis or neuronal loss in the antenna of these flies. Yhe effects of IR family co-receptor mutants was investigated using similar approaches, and expression of IR tuning receptors was found to decrease in the absence of Ir8a and Ir25a, but not Ir76b. As in Orco mutants, Ir8a-dependent olfactory neurons mostly endure despite near-absent expression of associated tuning receptors. Finally, differential expression analysis was used to identify other antennal genes whose expression is changed in IR and OR co-receptor mutants. Taken together, these data indicate that odorant co-receptors are necessary for maintaining expression of many tuning receptors at the mRNA level. Further, most Drosophila olfactory neurons persist in OR and IR co-receptor mutants, suggesting that the impact of co-receptors on neuronal survival may vary across insect species.

Medeiros, A. M., Hobbiss, A. F., Borges, G., Moita, M., Mendes, C. S. (2024). Mechanosensory bristles mediate avoidance behavior by triggering sustained local motor activity in Drosophila melanogaster. Curr Biol, 34(13):2812-2830.e2815 PubMed ID: 38861987 Summary: During locomotion, most vertebrates-and invertebrates such as Drosophila melanogaster-are able to quickly adapt to terrain irregularities or avoid physical threats by integrating sensory information along with motor commands. Key to this adaptability are leg mechanosensory structures, which assist in motor coordination by transmitting external cues and proprioceptive information to motor centers in the central nervous system. Nevertheless, how different mechanosensory structures engage these locomotor centers remains poorly understood. This study tested the role of mechanosensory structures in movement initiation by optogenetically stimulating specific classes of leg sensory structures. timulation of leg mechanosensory bristles (MsBs) and the femoral chordotonal organ (ChO) is sufficient to initiate forward movement in immobile animals. While the stimulation of the ChO required brain centers to induce forward movement, unexpectedly, brief stimulation of leg MsBs triggered a fast response and sustained motor activity dependent only on the ventral nerve cord (VNC). Moreover, this leg-MsB-mediated movement lacked inter- and intra-leg coordination but preserved antagonistic muscle activity within joints. Finally, leg-MsB activation was shown to mediate strong avoidance behavior away from the stimulus source, which is preserved even in the absence of a central brain. Overall, these data show that mechanosensory stimulation can elicit a fast motor response, independently of central brain commands, to evade potentially harmful stimuli. In addition, it sheds light on how specific sensory circuits modulate motor control, including initiation of movement, allowing a better understanding of how different levels of coordination are controlled by the VNC and central brain locomotor circuits.

Schnaitmann, C., Pagni, M., Meyer, P. B., Steinhoff, L., Oberhauser, V., Reiff, D. F. (2024). Horizontal-cell like Dm9 neurons in Drosophila modulate photoreceptor output to supply multiple functions in early visual processing. Frontiers in molecular neuroscience, 17:1347540 PubMed ID: 38813436 Summary: Dm9 neurons in the Drosophila optic lobe have been proposed as functional homologs of horizontal cells in the outer retina of vertebrates. This study combined genetic dissection of neuronal circuit function, two-photon calcium imaging in Dm9 and inner photoreceptors, and immunohistochemical analysis to reveal novel insights into the functional role of Dm9 in early visual processing. These experiments show that Dm9 receive input from all four types of inner photoreceptors R7p, R7y, R8p, and R8y. Histamine released from all types R7/R8 directly inhibits Dm9 via the histamine receptor Ort, and outweighs simultaneous histamine-independent excitation of Dm9 by UV-sensitive R7. Dm9 in turn provides inhibitory feedback to all R7/R8, which is sufficient for color-opponent processing in R7 but not R8. Color opponent processing in R8 requires additional synaptic inhibition by R7 of the same ommatidium via axo-axonal synapses and the second Drosophila histamine receptor HisCl1. Notably, optogenetic inhibition of Dm9 prohibits color opponent processing in all types of R7/R8 and decreases intracellular calcium in photoreceptor terminals. The latter likely results from reduced release of excitatory glutamate from Dm9 and shifts overall photoreceptor sensitivity toward higher light intensities. In summary, these results underscore a key role of Dm9 in color opponent processing in Drosophila and suggest a second role of Dm9 in regulating light adaptation in inner photoreceptors. These novel findings on Dm9 are indeed reminiscent of the versatile functions of horizontal cells in the vertebrate retina.

Vilimelis Aceituno, P., Dall'Osto, D., Pisokas, I. (2024). Theoretical principles explain the structure of the insect head direction circuit. Elife, 13 PubMed ID: 38814703 Summary: To navigate their environment, insects need to keep track of their orientation. Previous work has shown that insects encode their head direction as a sinusoidal activity pattern around a ring of neurons arranged in an eight-column structure. However, it is unclear whether this sinusoidal encoding of head direction is just an evolutionary coincidence or if it offers a particular functional advantage. To address this question, the basic mathematical requirements were established for direction encoding and it was shown to be performed by many circuits, all with different activity patterns. Among these activity patterns, this study proved that the sinusoidal one is the most noise-resilient, but only when coupled with a sinusoidal connectivity pattern between the encoding neurons. This predicted optimal connectivity pattern was compared with anatomical data from the head direction circuits of the locust and the fruit fly, finding that the theory agrees with experimental evidence. Furthermore, the predicted circuit was shown to emerge using Hebbian plasticity, implying that the neural connectivity does not need to be explicitly encoded in the genetic program of the insect but rather can emerge during development. Finally, in this theory, the consistent presence of the eight-column organisation of head direction circuits across multiple insect species is not a chance artefact but instead can be explained by basic evolutionary principles.

Brown, M., Sciascia, E., Ning, K., Adam, W., Veraksa, A. (2024). Regulation of brain development by the Minibrain/Rala signaling network. bioRxiv, PubMed ID: 38766038 Summary: The human dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in the pathology of Down syndrome, microcephaly, and cancer, however the exact mechanism through which it functions is unknown. The role was studied of the Drosophila ortholog of DYRK1A, Minibrain (Mnb), in brain development. The neuroblasts (neural stem cells) that eventually give rise to differentiated neurons in the adult brain are formed from a specialized tissue in the larval optic lobe called the neuroepithelium, in a tightly regulated process. Molecular marker analysis of mnb mutants revealed alterations in the neuroepithelium and neuroblast regions of developing larval brains. Using affinity purification-mass spectrometry (AP-MS), the novel Mnb binding partners Ral interacting protein (Rlip) and RALBP1 associated Eps domain containing (Reps) were identified. Rlip and Reps physically and genetically interact with Mnb, and the three proteins may form a ternary complex. Mnb phosphorylates Reps, and human DYRK1A binds to the Reps orthologs REPS1 and REPS2. Furthermore, Mnb engages the small GTPase Ras-like protein A (Rala) to regulate brain and wing development. This work uncovers a previously unrecognized early role of Mnb in the neuroepithelium and defines the functions of the Mnb/Reps/Rlip/Rala signaling network in brain development.

Monday, March 10th - Chromatin

Salzler, H. R., Vandadi, V., Matera, A. G. (2024). Set2 and H3K36 regulate the Drosophila male X chromosome in a context-specific manner, independent from MSL complex spreading. bioRxiv, PubMed ID: 38766267 Summary: Dosage compensation in Drosophila involves upregulating male X-genes two-fold. This process is carried out by the MSL (male-specific lethal) complex, which binds high-affinity sites and spreads to surrounding genes. Current models of MSL spreading focus on interactions of MSL3 (male-specific lethal 3) with histone marks; in particular, Set2-dependent H3 lysine-36 trimethylation (H3K36me3). However, Set2 might affect DC via another target, or there could be redundancy between canonical H3.2 and variant H3.3 histones. Further, it is difficult to parse male-specific effects from those that are simply X-specific. To discriminate among these possibilities, we employed genomic approaches in H3K36 (residue) and Set2 (writer) mutants. The results confirm a role for Set2 in X-gene regulation, but show that expression trends in males are often mirrored in females. Instead of global male-specific reduction of X-genes in Set2/H3K36 mutants, the effects were heterogeneous. Cohorts of genes were identified whose expression was significantly altered following loss of H3K36 or Set2, but the changes were in opposite directions, suggesting that H3K36me states have reciprocal functions. In contrast to H4(K16R) controls, analysis of combined H3.2(K36R)/H3.3(K36R) mutants neither showed consistent reduction in X-gene expression, nor any correlation with MSL3 binding. Examination of other developmental stages/tissues revealed additional layers of context-dependence. These studies implicate BEAF-32 and other insulator proteins in Set2/H3K36-dependent regulation. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 directly recruits the MSL complex. It is proposed that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL but common to both sexes.

Snir, O., Elgart, M., Gnainsky, Y., Goldsmith, M., Ciabrelli, F., Dagan, S., Aviezer, I., Stoops, E., Cavalli, G., Soen, Y. (2024). Organ transformation by environmental disruption of protein integrity and epigenetic memory in Drosophila. PLoS Biol, 22(5):e3002629 PubMed ID: 38805504 Summary: Despite significant progress in understanding epigenetic reprogramming of cells, the mechanistic basis of "organ reprogramming" by (epi-)gene-environment interactions remained largely obscure. This study used the ether-induced haltere-to-wing transformations in Drosophila as a model for epigenetic "reprogramming" at the whole organism level. The findings support a mechanistic chain of events explaining why and how brief embryonic exposure to ether leads to haltere-to-wing transformations manifested at the larval stage and on. Ether interferes with protein integrity in the egg, leading to altered deployment of Hsp90 and widespread repression of Trithorax-mediated establishment of active H3K4me3 chromatin marks throughout the genome. Despite this global reduction, Ubx targets and wing development genes preferentially retain higher levels of H3K4me3 that predispose these genes for later up-regulation in the larval haltere disc, hence the wing-like outcome. Consistent with compromised protein integrity during the exposure, the penetrance of bithorax transformations increases by genetic or chemical reduction of Hsp90 function. Moreover, joint reduction in Hsp90 and trx gene dosage can cause bithorax transformations without exposure to ether, supporting an underlying epistasis between Hsp90 and trx loss-of-functions. These findings implicate environmental disruption of protein integrity at the onset of histone methylation with altered epigenetic regulation of developmental patterning genes. The emerging picture provides a unique example wherein the alleviation of the Hsp90 "capacitor function" by the environment drives a morphogenetic shift towards an ancestral-like body plan. The morphogenetic impact of chaperone response during a major setup of epigenetic patterns may be a general scheme for organ transformation by environmental cues.

Vorobyeva, N. E., Krasnov, A. N., Erokhin, M., Chetverina, D., Mazina, M. (2024). Su(Hw) interacts with Combgap to establish long-range chromatin contacts. Epigenetics & chromatin, 17(1):17 PubMed ID: 38773468 Summary: Insulator-binding proteins (IBPs) play a critical role in genome architecture by forming and maintaining contact domains. While the involvement of several IBPs in organising chromatin architecture in Drosophila has been described, the specific contribution of the Suppressor of Hairy wings (Su(Hw)) insulator-binding protein to genome topology remains unclear. This study provides evidence for the existence of long-range interactions between chromatin bound Su(Hw) and Combgap, which was first characterised as Polycomb response elements binding protein. Loss of Su(Hw) binding to chromatin results in the disappearance of Su(Hw)-Combgap long-range interactions and in a decrease in spatial self-interactions among a subset of Su(Hw)-bound genome sites. These findings suggest that Su(Hw)-Combgap long-range interactions are associated with active chromatin rather than Polycomb-directed repression. Furthermore, the majority of transcription start sites that are down-regulated upon loss of Su(Hw) binding to chromatin were observed to be located within 2 kb of Combgap peaks and exhibit Su(Hw)-dependent changes in Combgap and transcriptional regulators' binding. This study demonstrates that Su(Hw) insulator binding protein can form long-range interactions with Combgap, Polycomb response elements binding protein, and that these interactions are associated with active chromatin factors rather than with Polycomb dependent repression.

Sokolov, V., Kyrchanova, O., Klimenko, N., Fedotova, A., Ibragimov, A., Maksimenko, O., Georgiev, P. (2024). New Drosophila promoter-associated architectural protein Mzfp1 interacts with CP190 and is required for housekeeping gene expression and insulator activity. Nucleic Acids Res, 52(12):6886-6905 PubMed ID: 38769058 Summary: In Drosophila, a group of zinc finger architectural proteins recruits the CP190 protein to the chromatin, an interaction that is essential for the functional activity of promoters and insulators. This study describes a new architectural C2H2 protein called Madf and Zinc-Finger Protein 1 (Mzfp1) that interacts with CP190. Mzfp1 has an unusual structure that includes six C2H2 domains organized in a C-terminal cluster and two tandem MADF domains. Mzfp1 predominantly binds to housekeeping gene promoters located in both euchromatin and heterochromatin genome regions. In vivo mutagenesis studies showed that Mzfp1 is an essential protein, and both MADF domains and the CP190 interaction region are required for its functional activity. The C2H2 cluster is sufficient for the specific binding of Mzfp1 to regulatory elements, while the second MADF domain is required for Mzfp1 recruitment to heterochromatin. Mzfp1 binds to the proximal part of the Fub boundary that separates regulatory domains of the Ubx and abd-A genes in the Bithorax complex. Mzfp1 participates in Fub functions in cooperation with the architectural proteins Pita and Su(Hw). Thus, Mzfp1 is a new architectural C2H2 protein involved in the organization of active promoters and insulators in Drosophila.

Willnow, P., Teleman, A. A. (2024). Nuclear position and local acetyl-CoA production regulate chromatin state. Nature, 630(8016):466-474 PubMed ID: 38839952 Summary: Histone acetylation regulates gene expression, cell function and cell fate. The pattern of histone acetylation was studied in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid β-oxidation, which is also increased in the rim. Inhibition of fatty acid β-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.

Melnikova, L. S., Molodina, V. V., Georgiev, P. G., Golovnin, A. K. (2024). Role of Mod(mdg4)-67.2 Protein in Interactions between Su(Hw)-Dependent Complexes and Their Recruitment to Chromatin. Biochemistry (Mosc), 89(4):626-636 PubMed ID: 38831500 Summary: Su(Hw) belongs to the class of proteins that organize chromosome architecture, determine promoter activity, and participate in formation of the boundaries/insulators between the regulatory domains. This protein contains a cluster of 12 zinc fingers of the C2H2 type, some of which are responsible for binding to the consensus site. The Su(Hw) protein forms complex with the Mod(mdg4)-67.2 and the CP190 proteins, where the last one binds to all known Drosophila insulators. To further study functioning of the Su(Hw)-dependent complexes, the previously described su(Hw)(E8) mutation was used with inactive seventh zinc finger, which produces mutant protein that cannot bind to the consensus site. The present work shows that the Su(Hw)(E8) protein continues to directly interact with the CP190 and Mod(mdg4)-67.2 proteins. Through interaction with Mod(mdg4)-67.2, the Su(Hw)(E8) protein can be recruited into the Su(Hw)-dependent complexes formed on chromatin and enhance their insulator activity. These results demonstrate that the Su(Hw) dependent complexes without bound DNA can be recruited to the Su(Hw) binding sites through the specific protein-protein interactions that are stabilized by Mod(mdg4)-67.2.

Saturday, March 9th - Cell Cycle

Haseeb, M. A., Bernys, A. C., Dickert, E. E., Bickel, S. E. (2024). An RNAi screen to identify proteins required for cohesion rejuvenation during meiotic prophase in Drosophila oocytes. G3 (Bethesda), 14(8) PubMed ID: 38849129 Summary: Accurate chromosome segregation during meiosis requires the maintenance of sister chromatid cohesion, initially established during premeiotic S phase. In human oocytes, DNA replication and cohesion establishment occur decades before chromosome segregation and deterioration of meiotic cohesion is one factor that leads to increased segregation errors as women age. Previous work has led to a proposal that a cohesion rejuvenation program operates to establish new cohesive linkages during meiotic prophase in Drosophila oocytes and depends on the cohesin loader Nipped-B and the cohesion establishment factor Eco. In support of this model, recent work has demonstrated that chromosome-associated cohesin turns over extensively during meiotic prophase and failure to load cohesin onto chromosomes after premeiotic S phase results in arm cohesion defects in Drosophila oocytes. To identify proteins required for prophase cohesion rejuvenation but not S phase establishment, a Gal4-UAS inducible RNAi screen was conducted that utilized two distinct germline drivers. Using this strategy, 29 gene products were identifed for which hairpin expression during meiotic prophase, but not premeiotic S phase, significantly increased segregation errors. Prophase knockdown of Brahma or Pumilio, two positives with functional links to the cohesin loader, caused a significant elevation in the missegregation of recombinant homologs, a phenotype consistent with premature loss of arm cohesion. Moreover, fluorescence in situ hybridization confirmed that Brahma, Pumilio, and Nipped-B are required during meiotic prophase for the maintenance of arm cohesion. These data support the model that Brahma and Pumilio regulate Nipped-B-dependent cohesin loading during rejuvenation. Future analyses will better define the mechanism(s) that govern meiotic cohesion rejuvenation and whether additional prophase-specific positives function in this process.

Vicars, H., Karg, T., Mills, A., Sullivan, W. (2024). Acentric chromosome congression and alignment on the metaphase plate via kinetochore-independent forces in Drosophila.. bioRxiv, PubMed ID: 38798431 Summary: Chromosome congression and alignment on the metaphase plate involves lateral and microtubule plus-end interactions with the kinetochore. Here we take advantage of our ability to efficiently generate a GFP-marked acentric X chromosome fragment in Drosophila neuroblasts to identify forces acting on chromosome arms that drive congression and alignment. We find acentrics efficiently align on the metaphase plate, often more rapidly than kinetochore-bearing chromosomes. Unlike intact chromosomes, the paired sister acentrics oscillate as they move to and reside on the metaphase plate in a plane distinct and significantly further from the main mass of intact chromosomes. Consequently, at anaphase onset acentrics are oriented either parallel or perpendicular to the spindle. Parallel-oriented sisters separate by sliding while those oriented perpendicularly separate via unzipping. This oscillation, together with the fact that in monopolar spindles acentrics are rapidly shunted away from the poles, indicates that distributed plus-end directed forces are primarily responsible for acentric migration. This conclusion is supported by the observation that reduction of EB1 preferentially disrupts acentric alignment. In addition, reduction of http://Klp3a activity, a gene required for the establishment of pole-to-pole microtubules, preferentially disrupts acentric alignment. Taken together these studies suggest that plus-end forces mediated by the outer pole-to-pole microtubules are primarily responsible for acentric metaphase alignment. Surprisingly, this study found that a small fraction of sister acentrics are anti-parallel aligned indicating that the kinetochore is required to ensure parallel alignment of sister chromatids. Finally, induction of acentric chromosome fragments was found to result in a global reorganization of the congressed chromosomes into a torus configuration.

Marshall, W. F., Fung, J. C. (2024). Modeling homologous chromosome recognition via nonspecific interactions. Proc Natl Acad Sci U S A, 121(20):e2317373121 PubMed ID: 38722810 Summary: In many organisms, most notably Drosophila, homologous chromosomes associate in somatic cells, a phenomenon known as somatic pairing, which takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, mediated by different proteins that bind to these different regions. This study used computational modeling to evaluate an alternative "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. In this model, buttons are nonuniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a nonhomolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. By simulating randomly generated nonuniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. It is conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.

Hughes, S. E., Price, A., Briggs, S., Staber, C., James, M., Anderson, M., Hawley, R. S. (2024). A transcriptomics-based RNAi screen for regulators of meiosis and early stages of oocyte development in Drosophila melanogaster.G3 (Bethesda), 14(4) PubMed ID: 38333961 Summary: A properly regulated series of developmental and meiotic events must occur to ensure the successful production of gametes. In Drosophila melanogaster ovaries, these early developmental and meiotic events include the production of the 16-cell cyst, meiotic entry, synaptonemal complex (SC) formation, recombination, and oocyte specification. In order to identify additional genes involved in early oocyte development and meiosis, 3 published single-cell RNA-seq datasets from Drosophila ovaries were analyzed, using vasa (germline) together with c(3)G, cona, and corolla (SC) as markers. This analysis generated a 526 available RNAi lines containing shRNA constructs were in germline-compatible vectors representing 331 of the top 500 genes.Targeted ovaries were assessed for SC formation and maintenance, oocyte specification, cyst development, and double-strand break dynamics. Six uncharacterized genes exhibited early developmental defects. SC and developmental defects were observed for additional genes not well characterized in the early ovary. Interestingly, in some lines with developmental delays, meiotic events could still be completed once oocyte specificity occurred indicating plasticity in meiotic timing. These data indicate that a transcriptomics approach can be used to identify genes involved in functions in a specific cell type in the Drosophila ovary.

Vasavan, B., Das, N., Kahnamouei, P., Trombley, C., Swan, A. (2024). Skp2-Cyclin A Interaction Is Necessary for Mitotic Entry and Maintenance of Diploidy. J Mol Biol, 436(8):168505 PubMed ID: 38423454 Summary: Skp2, the substrate recognition component of the SCF(Skp2) ubiquitin ligase, has been implicated in the targeted destruction of a number of key cell cycle regulators and the promotion of S-phase. One of its critical targets is the Cyclin dependent kinase (Cdk) inhibitor p27 (Drosophila Dacapo), and indeed the overexpression of Skp2 in a number of cancers is directly correlated with the premature degradation of p27. Skp2 was first identified as a protein that interacts with Cyclin A in transformed cells, but its role in this complex has remained unclear. This paper demonstrates that Skp2 interacts with Cyclin A in Drosophila and is required to maintain Cyclin A levels and permit mitotic entry. Failure of mitotic entry in Skp2 mutant cells results in polyploidy. If these cells enter mitosis again they are unable to properly segregate their chromosomes, leading to checkpoint dependent cell cycle arrest or apoptosis. Thus, Skp2 is required for mitosis and for maintaining diploidy and genome stability.

Huang, Y. T., Hesting, L. L., Calvi, B. R. (2024). An unscheduled switch to endocycles induces a reversible senescent arrest that impairs growth of the Drosophila wing disc bioRxiv, PubMed ID: 38559130 Summary: A programmed developmental switch to G / S endocycles results in tissue growth through an increase in cell size. Unscheduled, induced endocycling cells (iECs) promote wound healing but also contribute to cancer. Much remains unknown, however, about how these iECs affect tissue growth. Using the D. melanogaster wing disc as model, this study found that populations of iECs initially increase in size but then subsequently undergo a heterogenous arrest that causes severe tissue undergrowth. iECs acquired DNA damage and activated a Jun N-terminal kinase (JNK) pathway, but, unlike other stressed cells, were apoptosis-resistant and not eliminated from the epithelium. Instead, iECs entered a JNK-dependent and reversible senescent-like arrest. Senescent iECs promoted division of diploid neighbors, but this compensatory proliferation did not rescue tissue growth. This study has uncovered unique attributes of iECs and their effects on tissue growth that have important implications for understanding their roles in wound healing and cancer.

Thursday, March 6th - Disease Models

Polet, S. S., de Koning, T. J., Lambrechts, R. A., Tijssen, M. A. J., Sibon, O. C. M., Gorter, J. A. (2024). Conventional and novel anti-seizure medications reveal a particular role for GABA(A) in a North Sea progressive myoclonus Epilepsy Drosophila model. Epilepsy research, 203:107380 PubMed ID: 38781737 Summary: North Sea Progressive Myoclonus Epilepsy (NS-PME) is a rare genetic disorder characterized by ataxia, myoclonus and seizures with a progressive course. Although the cause of NS-PME is known, namely a homozygous mutation in the GOSR2 gene (c.430 G>T; p. Gly144Trp), sufficient treatment is lacking. Despite combinations of on average 3-5 anti-seizure medications (ASMs), debilitating myoclonus and seizures persist. This study aimed to gain insight into the most effective anti-convulsive target in NS-PME by evaluating the individual effects of ASMs in a NS-PME Drosophila model. A previously generated Drosophila model for NS-PME was used displaying progressive heat-sensitive seizures. This model was used to test 1. a first-generation ASM (sodium barbital), 2. common ASMs used in NS-PME (clonazepam, valproic acid, levetiracetam, ethosuximide) and 3. a novel third-generation ASM (ganaxolone) with similar mode of action to sodium barbital. Compounds were administered by adding them to the food in a range of concentrations. After 7 days of treatment, the percentage of heat-induced seizures was determined and compared to non-treated but affected controls. As previously reported in the NS-PME Drosophila model, sodium barbital resulted in significant seizure suppression, with increasing effect at higher dosages. Of the commonly prescribed ASMs, clonazepam and ethosuximide resulted in significant seizure suppression, whereas both valproic acid and levetiracetam did not show any changes in seizures. Interestingly, ganaxolone did result in seizure suppression as well. Of the six drugs tested, three of the four that resulted in seizure suppression (sodium barbital, clonazepam, ganaxolone) are primary known for their direct effect on GABA(A) receptors. This suggests that GABA(A) could be a potentially important target in the treatment of NS-PME. Consequently, these findings add rationale to the exploration of the clinical effect of ganaxolone in NS-PME and other progressive myoclonus epilepsies.

Dubey, P. K., Singh, S., Khalil, H., Kommini, G. K., Bhat, K. M., Krishnamurthy, P. (2024). Obg-like ATPase 1 Genetic Deletion Leads to Dilated Cardiomyopathy in Mice and Structural Changes in Drosophila Heart. bioRxiv, PubMed ID: 38854005 Summary: Cardiomyopathy, disease of the heart muscle, is a significant contributor to heart failure. The pathogenesis of cardiomyopathy is multifactorial and involves genetic, environmental, and lifestyle factors. Identifying and characterizing novel genes that contribute to cardiac pathophysiology are crucial for understanding cardiomyopathy and effective therapies. This study investigated the role of a novel gene, Obg-like ATPase 1 ( Ola1 ), in cardiac pathophysiology using a cardiac-specific knockout mouse model as well as a Drosophila model (see CG1354). Previous work demonstrated that OLA1 modulates the hypertrophic response of cardiomyocytes through the GSK-beta/beta-catenin signaling pathway. Furthermore, recent studies have suggested that OLA1 plays a critical role in organismal growth and development. For example, Ola1 null mice exhibit increased heart size and growth retardation. It is not known, however, if loss of function for Ola1 leads to dilated cardiomyopathy. Cardiac-specific Ola1 knockout mice (OLA1-cKO) were generated to evaluate the role of OLA1 in cardiac pathophysiology. Ola1-cKO in mice leads to dilated cardiomyopathy (DCM) and left ventricular (LV) dysfunction. These mice developed severe LV dilatation, thinning of the LV wall, reduced LV function, and, in some cases, ventricular wall rupture and death. In Drosophila, RNAi-mediated knock-down specifically in developing heart cells led to the change in the structure of pericardial cells from round to elongated, and abnormal heart function. This also caused significant growth reduction and pupal lethality. Thus, these findings suggest that OLA1 is critical for cardiac homeostasis and that its deficiency leads to dilated cardiomyopathy and dysfunction. Furthermore, this study highlights the potential of the Ola1 gene as a therapeutic target for dilated cardiomyopathy and heart failure.

Lewis, S. A., Forstrom, J., Tavani, J., Schafer, R., Tiede, Z., Padilla-Lopez, S. R., Kruer, M. C. (2024). eIF2alpha phosphorylation evokes dystonia-like movements with D2-receptor and cholinergic origin and abnormal neuronal connectivity. bioRxiv, PubMed ID: 38798458 Summary: Dystonia is the 3(rd) most common movement disorder. Dystonia is acquired through either injury or genetic mutations, with poorly understood molecular and cellular mechanisms. Eukaryotic initiation factor alpha (eIF2α) controls cell state including neuronal plasticity via protein translation control and expression of ATF4. Dysregulated eIF2α phosphorylation (eIF2α-P) occurs in dystonia patients and models including DYT1 (Drosophila Torsin), but the consequences are unknown. This study increased/decreased eIF2α-P and tested motor control and neuronal properties in a Drosophila model. Bidirectionally altering eIF2α-P produced dystonia-like abnormal posturing and dyskinetic movements in flies. These movements were also observed with expression of the DYT1 risk allele. Cholinergic and D2-receptor neuroanatomical origins were identified of these dyskinetic movements caused by genetic manipulations to dystonia molecular candidates eIF2α-P, ATF4, or DYT1, with evidence for decreased cholinergic release. In vivo, increased and decreased eIF2α-P increase synaptic connectivity at the NMJ with increased terminal size and bouton synaptic release sites. Long-term treatment of elevated eIF2α-P with ISRIB restored adult longevity, but not performance in a motor assay. Disrupted eIF2α-P signaling may alter neuronal connectivity, change synaptic release, and drive motor circuit changes in dystonia.

Yuan, Y., Yu, L., Zhuang, X., Wen, D., He, J., Hong, J., Xie, J., Ling, S., Du, X., Chen, W., Wang, X. (2025). Drosophila models used to simulate human ATP1A1 gene mutations that cause Charcot-Marie-Tooth type 2 disease and refractory seizures. Neural Regen Res, 20(1):265-276 PubMed ID: 38767491 Summary: Certain amino acids changes in the human Na+/K+-ATPase pump, ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1), cause Charcot-Marie-Tooth disease type 2 (CMT2) disease and refractory seizures. To develop in vivo models to study the role of Na+/K+-ATPase in these diseases, the Drosophila gene homolog, Atpα, was modified to mimic the human ATP1A1 gene mutations that cause CMT2. Mutations located within the helical linker region of human ATP1A1 (I592T, A597T, P600T, and D601F) were simultaneously introduced into endogenous Drosophila Atpα by CRISPR/Cas9-mediated genome editing, generating the AtpαTTTF model. In addition, the same strategy was used to generate the corresponding single point mutations in flies (AtpαI571T, AtpαA576T, AtpαP579T, and AtpαD580F). Moreover, a deletion mutation (Atpαmut) that causes premature termination of translation was generated as a positive control. Of these alleles, two were found that could be maintained as homozygotes (AtpαI571T and AtpαP579T). Three alleles (AtpαA576T, AtpαP579 and AtpαD580F) can form heterozygotes with the Atpαmut allele. The Atpα allele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila. Flies heterozygous for AtpαTTTF mutations have motor performance defects, a reduced lifespan, seizures, and an abnormal neuronal morphology. These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.

Sodders, M. J., Avila-Pacheco, J., Okorie, E. C., Shen, M., Kumari, N., Marathi, A., Lakhani, M., Bullock, K., Pierce, K., Dennis, C., Jeanfavre, S., Sarkar, S., Scherzer, C. R., Clish, C., Olsen, A. L. (2024). Genetic screening and metabolomics identify glial adenosine metabolism as a therapeutic target in Parkinson's disease. bioRxiv, PubMed ID: 38798570 Summary: Parkinson's disease (PD) is the second most common neurodegenerative disorder and lacks disease-modifying therapies. A Drosophila model has been developed for identifying novel glial-based therapeutic targets for PD. Human alpha-synuclein is expressed in neurons and individual genes are independently knocked down in glia. A forward genetic screen was conducted, knocking down the entire Drosophila kinome in glia in alpha-synuclein expressing flies. Among the top hits were five genes (9Ak1, (Ak6, Adk1, Adk2, and awd) involved in adenosine metabolism. Knockdown of each gene improved locomotor dysfunction, rescued neurodegeneration, and increased brain adenosine levels. It was determined that the mechanism of neuroprotection involves adenosine itself, as opposed to a downstream metabolite. Deeper undersrtanding was obtained into the mechanism for one gene, Ak1, finding rescue of dopaminergic neuron loss, alpha-synuclein aggregation, and bioenergetic dysfunction after glial Ak1 knockdown. Metabolomics was performed in Drosophila and in human PD patients, allowing comprehensive characterization of changes in purine metabolism and potential biomarkers of dysfunctional adenosine metabolism were identified in people. These experiments support glial adenosine as a novel therapeutic target in PD.

Chauvin, S. D., Ando, S., Holley, J. A., ..., Onodera, O., Kato, T., Miner, J. J. (2024). Inherited C-terminal TREX1 variants disrupt homology-directed repair to cause senescence and DNA damage phenotypes in Drosophila, mice, and humans. Nat Commun, 15(1):4696 PubMed ID: 38824133 Summary: Age-related microangiopathy, also known as small vessel disease (SVD), causes damage to the brain, retina, liver, and kidney. Based on the DNA damage theory of aging, it was reasoned that genomic instability may underlie an SVD caused by dominant C-terminal variants in TREX1, the most abundant 3'-5' DNA exonuclease in mammals. C-terminal TREX1 variants cause an adult-onset SVD known as retinal vasculopathy with cerebral leukoencephalopathy (RVCL or RVCL-S). In RVCL, an aberrant, C-terminally truncated TREX1 mislocalizes to the nucleus due to deletion of its ER-anchoring domain. Since RVCL pathology mimics that of radiation injury, it was reasoned that nuclear TREX1 would cause DNA damage. This study shows that RVCL-associated TREX1 variants trigger DNA damage in humans, mice, and Drosophila, and that cells expressing RVCL mutant TREX1 are more vulnerable to DNA damage induced by chemotherapy and cytokines that up-regulate TREX1, leading to depletion of TREX1-high cells in RVCL mice. RVCL-associated TREX1 mutants inhibit homology-directed repair (HDR), causing DNA deletions and vulnerablility to PARP inhibitors. In women with RVCL, early-onset breast cancer was observed, similar to patients with BRCA1/2 variants. These results provide a mechanistic basis linking aberrant TREX1 activity to the DNA damage theory of aging, premature senescence, and microvascular disease.

Wednesday, March 5th - Adult physiology and metabolism

Wilkin, M. B., Whiteford, R., Akbar, T., Hosseini-Alghaderi, S., Revici, R., Carbery, A. M., Baron, M. (2024). The First Defined Null Allele of the Notch Regulator, a Suppressor of Deltex: Uncovering Its Novel Roles in Drosophila melanogaster Oogenesis. Biomolecules, 14(5) PubMed ID: 38785929 Summary: Suppressor of deltex (Su(dx)) is a Drosophila melanogaster member of the NEDD4 family of the HECT domain E3 ubiquitin ligases. Su(dx) acts as a regulator of Notch endocytic trafficking, promoting Notch lysosomal degradation and the down-regulation of both ligand-dependent and ligand-independent signalling, the latter involving trafficking through the endocytic pathway and activation of the endo/lysosomal membrane. Mutations of Su(dx) result in developmental phenotypes in the Drosophila wing that reflect increased Notch signalling, leading to gaps in the specification of the wing veins, and Su(dx) functions to provide the developmental robustness of Notch activity to environmental temperature shifts. The full developmental functions of Su(dx) are unclear; however, this is due to a lack of a clearly defined null allele. This study reports the first defined null mutation of Su(dx), generated by P-element excision, which removes the complete open reading frame. The mutation is recessive-viable, with the Notch gain of function phenotypes affecting wing vein and leg development. New roles were uncovered for Su(dx) in Drosophila oogenesis, where it regulates interfollicular stalk formation, egg chamber separation and germline cyst enwrapment by the follicle stem cells. Interestingly, while the null allele exhibited a gain in Notch activity during oogenesis, the previously described Su(dx)(SP) allele, which carries a seven amino acid in-frame deletion, displayed a Notch loss of function phenotypes and an increase in follicle stem cell turnover. This is despite both alleles displaying similar Notch gain of function in wing development. This unexpected context-dependent outcome of Su(dx)(sp) is thought to being due to the partial retention of function by the intact C2 and WW domain regions of the protein. These results extend understanding of the developmental role of Su(dx) in the tissue renewal and homeostasis of the Drosophila ovary and illustrate the importance of examining an allelic series of mutations to fully understand developmental functions.

Hemba-Waduge, R. U., Liu, M., Li, X., Sun, J. L., Budslick, E. A., Bondos, S. E., Ji, J. Y. (2024). Metabolic control by the Bithorax Complex-Wnt signaling crosstalk in Drosophila. bioRxiv, PubMed ID: 38853890 Summary: Adipocytes distributed throughout the body play crucial roles in lipid metabolism and energy homeostasis. Regional differences among adipocytes influence normal function and disease susceptibility, but the mechanisms driving this regional heterogeneity remain poorly understood. This study reports a genetic crosstalk between the Bithorax Complex ( BX-C ) genes and Wnt/Wingless signaling that orchestrates regional differences among adipocytes in Drosophila larvae. Abdominal adipocytes, characterized by the exclusive expression of abdominal A ( abd-A )and Abdominal B ( Abd-B ), exhibit distinct features compared to thoracic adipocytes, with Wnt signaling further amplifying these disparities. Depletion of BX-C genes in adipocytes reduces fat accumulation, delays larval-pupal transition, and eventually leads to pupal lethality. Depleting Abd-A or Abd-B reduces Wnt target gene expression, thereby attenuating Wnt signaling-induced lipid mobilization. Conversely, Wnt signaling stimulated abd-A transcription, suggesting a feedforward loop that amplifies the interplay between Wnt signaling and BX-C in adipocytes. These findings elucidate how the crosstalk between cell-autonomous BX-C gene expression and Wnt signaling define unique metabolic behaviors in adipocytes in different anatomical regions of fat body, delineating larval adipose tissue domains.

Zhang, T., Zhou, Q., Jusic, N., Lu, W., Pignoni, F., Neal, S. J. (2024). Mitf, with Yki and STRIPAK-PP2A, is a key determinant of form and fate in the progenitor epithelium of the Drosophila eye. European journal of cell biology, 103(2):151421 PubMed ID: 38776620 Summary: The Microphthalmia-associated Transcription Factor (MITF) governs numerous cellular and developmental processes. In mice, it promotes specification and differentiation of the retinal pigmented epithelium (RPE), and in humans, some mutations in MITF induce congenital eye malformations. This study explored the function and regulation of Mitf in Drosophila eye development, and two roles were uncovered. Knockdown of Mitf results in retinal displacement (RDis), a phenotype associated with abnormal eye formation. Mitf functions in the peripodial epithelium (PE), a retinal support tissue akin to the RPE, to suppress RDis, via the effector Yorkie (Yki). Yki physically interacts with Mitf and can modify its transcriptional activity in vitro. Severe loss of Mitf, instead, results in the de-repression of retinogenesis in the PE, precluding its development. This activity of Mitf requires the protein phosphatase 2 A holoenzyme STRIPAK-PP2A, but not Yki; Mitf transcriptional activity is potentiated by STRIPAK-PP2A in vitro and in vivo. Knockdown of STRIPAK-PP2A results in cytoplasmic retention of Mitf in vivo and in its decreased stability in vitro, highlighting two potential mechanisms for the control of Mitf function by STRIPAK-PP2A. Thus, Mitf functions in a context-dependent manner as a key determinant of form and fate in the Drosophila eye progenitor epithelium.

Zhai, C., Wang, Y., Qi, S., Yang, M., Wu, S. (2024). Yki stability and activity are regulated by Ca(2+)-calpains axis in Drosophila. J Genet Genomics, PubMed ID: 38663479 Summary: Yorkie (Yki) is a key effector of the Hippo pathway that activates the expression of targets by associating with the transcription factor Scalloped. Various upstream signals, such as cell polarity and mechanical cues, control transcriptional programs by regulating Yki activity. Searching for Yki regulatory factors has far-reaching significance for studying the Hippo pathway in animal development and human diseases. This study identified Calpain-A (CalpA) and Calpain-B (CalpB), two calcium (Ca(2+))-dependent modulatory proteases of the calpain family, as critical regulators of Yki in Drosophila that interact with Yki, respectively. Ca(2+) induces Yki cleavage in a CalpA/CalpB-dependent manner, and the protease activity of CalpA/CalpB is pivotal for the cleavage. Furthermore, overexpression of CalpA or CalpB in Drosophila partially restores the large wing phenotype caused by Yki overexpression, and F98 of Yki is an important cleavage site by the Ca(2+)-calpains axis. This study uncovers a unique mechanism whereby the Ca(2+)-calpain axis modulates Yki activity through protein cleavage.

Nuga, O., Richardson, K., Patel, N. C., Wang, X., Pagala, V., Stephan, A., Peng, J., Demontis, F., Todi, S. V. (2024). Linear poly-ubiquitin remodels the proteome and influences hundreds of regulators in Drosophila. G3 (Bethesda), 14(11) PubMed ID: 39325835 Summary: Ubiquitin controls many cellular processes via its post-translational conjugation onto substrates. Its use is highly variable due to its ability to form poly-ubiquitin with various topologies. Among them, linear chains have emerged as important regulators of immune responses and protein degradation. Previous studies in Drosophila melanogaster found that expression of linear poly-ubiquitin that cannot be dismantled into single moieties leads to their own ubiquitination and degradation or, alternatively, to their conjugation onto proteins. However, it remains largely unknown which proteins are sensitive to linear poly-ubiquitin. To address this question, this study expanded the toolkit to modulate linear chains and conducted ultra-deep coverage proteomics from flies that express non-cleavable, linear chains comprising 2, 4, or 6 moieties. These chains were found to regulate shared and distinct cellular processes in Drosophila by impacting hundreds of proteins. Theseresults provide key insight into the proteome subsets and cellular pathways that are influenced by linear poly-ubiquitin with distinct lengths and suggest that the ubiquitin system is exceedingly pliable.

Terry, D., Schweibenz, C., Moberg, K. (2024). Local Ecdysone synthesis in a wounded epithelium sustains developmental delay and promotes regeneration in Drosophila. Development, 151(12) PubMed ID: 38775023 Summary: Regenerative ability often declines as animals mature past embryonic and juvenile stages, suggesting that regeneration requires redirection of growth pathways that promote developmental growth. Intriguingly, the Drosophila larval epithelia require the hormone ecdysone (Ec) for growth but require a drop in circulating Ec levels to regenerate. Examining Ec dynamics more closely, this study found that transcriptional activity of the Ec-receptor (EcR) drops in uninjured regions of wing discs, but simultaneously rises in cells around the injury-induced blastema. In parallel, blastema depletion of genes encoding Ec biosynthesis enzymes blocks EcR activity and impairs regeneration but has no effect on uninjured wings. Local Ec/EcR signaling was found to be required for injury-induced pupariation delay following injury and that key regeneration regulators upd3 and Ets21c respond to Ec levels. Collectively, these data indicate that injury induces a local source of Ec within the wing blastema that sustains a transcriptional signature necessary for developmental delay and tissue repair.

Tuesday, March 4th - Spermatogenesis

Tian, S., Nguyen, H., Ye, Z., Rouskin, S., Thirumalai, D., Trcek, T. (2024). The Folding of Germ Granule mRNAs Controls Intermolecular Base Pairing in Germ Granules and Maintains Normal Fly Development. bioRxiv, PubMed ID: 38853845 Summary: Drosophila germ granules enrich mRNAs critical for fly development. Within germ granules, mRNAs form multi-transcript clusters marked by increased mRNA concentration, creating an elevated potential for intermolecular base pairing. However, the type and abundance of intermolecular base pairing in mRNA clusters is poorly characterized. Using single-molecule super-resolution microscopy, chemical probing for base accessibility, phase separation assays, and simulations, mRNAs were demonstrated t remain well-folded upon localization to germ granules. While most base pairing is intramolecular, mRNAs still display the ability for intermolecular base pairing, facilitating clustering without high sequence complementarity or significant melting of secondary structure. This base pairing among mRNAs is driven by scattered and discontinuous stretches of bases appearing on the surface of folded RNAs, providing multivalency to clustering but exhibits low probability for sustained interactions. Notably, engineered germ granule mRNAs with exposed GC-rich complementary sequences (CSs) presented within stable stem loops induce sustained base pairing in vitro and enhanced intermolecular interactions in vivo. However, the presence of these stem loops alone disrupts fly development, and the addition of GC-rich CSs exacerbates this phenotype. Although germ granule mRNAs contain numerous GC-rich CSs capable of stable intermolecular base pairing, they are primarily embedded by RNA folding. This study emphasizes the role of RNA folding in controlling the type and abundance of intermolecular base pairing, thereby preserving the functional integrity of mRNAs within the germ granules.

Chao, C. F., Pesch, Y. Y., Yu, H., Wang, C., Aristizabal, M. J., Huan, T., Tanentzapf, G., Rideout, E. (2024). An important role for triglyceride in regulating spermatogenesis. Elife, 12 PubMed ID: 38805376 Summary: Drosophila is a powerful model to study how lipids affect spermatogenesis. Yet, the contribution of neutral lipids, a major lipid group which resides in organelles called lipid droplets (LD), to sperm development is largely unknown. Emerging evidence suggests LD are present in the testis and that loss of neutral lipid- and LD-associated genes causes subfertility; however, key regulators of testis neutral lipids and LD remain unclear. This stuey shows LD are present in early-stage somatic and germline cells within the Drosophila testis. A role was identified for triglyceride lipase brummer (bmm) in regulating testis LD; whole-body loss of bmm leads to defects in sperm development. Importantly, these represent cell-autonomous roles for bmm in regulating testis LD and spermatogenesis. Because lipidomic analysis of bmm mutants revealed excess triglyceride accumulation, and spermatogenic defects in bmm mutants were rescued by genetically blocking triglyceride synthesis, these data suggest that bmm-mediated regulation of triglyceride influences sperm development. This identifies triglyceride as an important neutral lipid that contributes to Drosophila sperm development, and reveals a key role for bmm in regulating testis triglyceride levels during spermatogenesis.

Roach, T. V., Lenhart, K. F. (2024). Mating-induced Ecdysone in the testis disrupts soma-germline contacts and stem cell cytokinesis. Development, 151(11) PubMed ID: 38832826 Summary: Germline maintenance relies on adult stem cells to continually replenish lost gametes over a lifetime and respond to external cues altering the demands on the tissue. Mating worsens germline homeostasis over time, yet a negative impact on stem cell behavior has not been explored. Using extended live imaging of the Drosophila testis stem cell niche, short periods of mating in young males was found to disrupt cytokinesis in germline stem cells (GSCs). This defect leads to failure of abscission, preventing release of differentiating cells from the niche. GSC abscission failure is caused by increased Ecdysone hormone signaling induced upon mating, which leads to disrupted somatic encystment of the germline. Abscission failure is rescued by isolating males from females, but recurs with resumption of mating. Importantly, reiterative mating also leads to increased GSC loss, requiring increased restoration of stem cells via symmetric renewal and de-differentiation. Together, these results suggest a model whereby acute mating results in hormonal changes that negatively impact GSC cytokinesis but preserves the stem cell population.

He, L., Sun, F., Wu, Y., Li, Z., Fu, Y., Huang, Q., Li, J., Wang, Z., Cai, J., Feng, C., Deng, X., Gu, H., He, X., Yu, J., Sun, F. (2024). L(1)10Bb serves as a conservative determinant for soma-germline communications via cellular non-autonomous effects within the testicular stem cell niche. Molecular and cellular endocrinology, 591:112278 PubMed ID: 38795826 Summary: The testicular stem cell niche is the central regulator of spermatogenesis in Drosophila melanogaster. However, the underlying regulatory mechanisms arwe unclear. This study demonstrated the crucial role of lethal (1) 10Bb [l(1)10Bb] in regulating the testicular stem cell niche. Dysfunction of l(1)10Bb in early-stage cyst cells led to male fertility disorders and compromised cyst stem cell maintenance. Moreover, the dysfunction of l(1)10Bb in early-stage cyst cells exerted non-autonomous effects on germline stem cell differentiation, independently of hub signals. Notably, this study highlights the rescue of testicular defects through ectopic expression of L(1)10Bb and the human homologous protein BUD31 homolog (BUD31). In addition, l(1)10Bb dysfunction in early-stage cyst cells downregulated the expression of spliceosome subunits in the Sm and the precursor RNA processing complexes. Collectively, these findings established l(1)10Bb as a pivotal factor in the modulation of Drosophila soma-germline communications within the testicular stem cell niche.

Su, Q., Xu, B., Chen, X., Rokita, S. E. (2024). Misregulation of bromotyrosine compromises fertility in male Drosophila. Proc Natl Acad Sci U S A, 121(21):e2322501121 PubMed ID: 38748578 Summary: Biological regulation often depends on reversible reactions such as phosphorylation, acylation, methylation, and glycosylation, but rarely halogenation. A notable exception is the iodination and deiodination of thyroid hormones. This study reports detection of bromotyrosine and its subsequent debromination during Drosophila spermatogenesis. Bromotyrosine is not evident when Drosophila express a native flavin-dependent dehalogenase that is homologous to the enzyme responsible for iodide salvage from iodotyrosine in mammals. Deletion or suppression of the dehalogenase-encoding condet (cdt) gene in Drosophila allows bromotyrosine to accumulate with no detectable chloro- or iodotyrosine. The presence of bromotyrosine in the cdt mutant males disrupts sperm individualization and results in decreased fertility. Transgenic expression of the cdt gene in late-staged germ cells rescues this defect and enhances tolerance of male flies to bromotyrosine. These results are consistent with reversible halogenation affecting Drosophila spermatogenesis in a process that had previously eluded metabolomic, proteomic, and genomic analyses.

Shao, Z., Hu, J., Jandura, A., Wilk, R., Jachimowicz, M., Ma, L., Hu, C., Sundquist, A., Das, I., Samuel-Larbi, P., Brill, J. A., Krause, H. M. (2024). Spatially revealed roles for lncRNAs in Drosophila spermatogenesis, Y chromosome function and evolution. Nat Commun, 15(1):3806 PubMed ID: 38714658 Summary: Unlike coding genes, the number of lncRNA genes in organism genomes is relatively proportional to organism complexity. From plants to humans, the tissues with highest numbers and levels of lncRNA gene expression are the male reproductive organs. To learn why, a genome-wide analysis was initiated of Drosophila lncRNA spatial expression patterns in these tissues. The numbers of genes and levels of expression observed greatly exceed those previously reported, due largely to a preponderance of non-polyadenylated transcripts. In stark contrast to coding genes, the highest numbers of lncRNAs expressed are in post-meiotic spermatids. Correlations between expression levels, localization and previously performed genetic analyses indicate high levels of function and requirement. More focused analyses indicate that lncRNAs play major roles in evolution by controlling transposable element activities, Y chromosome gene expression and sperm construction. A new type of lncRNA-based particle found in seminal fluid may also contribute to reproductive outcomes.

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