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Current papers in developmental biology and gene function


Thursday, November 30th, 2023 - Signaling

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Ozakman, Y., Raval, D. and Eleftherianos, I. (2023). Drosophila melanogaster Imd signaling interacts with insulin signaling and alters feeding rate upon parasitic nematode infection. Heliyon 9(5): e16139. PubMed ID: 37251825
Significant progress has been made in recent years on exploring immunometabolism, a field that integrates two processes essential for maintaining tissue and organismal homeostasis, immunity and metabolism. The nematode parasite Heterorhabditis gerrardi, its mutualistic bacteria Photorhabdus asymbiotica, and the fruit fly Drosophila melanogaster constitute a unique system to investigate the molecular basis of host immunometabolic response to nematode-bacterial complexes. This study explored the contribution of the two major immune signaling pathways, Toll and Imd, to sugar metabolism in D. melanogaster larvae during infection with H. gerrardi nematodes. Toll or Imd signaling loss-of-function mutant larvae were infected with H. gerrardi nematodes and larval survival ability, feeding rate, and sugar metabolism were assessed. No significant differences were found in the survival ability or levels of sugar metabolites in any of the mutant larvae when responding to H. gerrardi infection. However, the Imd mutant larvae have higher feeding rate than controls during the early stages of infection. In addition, feeding rates are lower in Imd mutants relative to the control larvae as the infection progresses. It was further shown that Dilp2 and Dilp3 gene expression increases in Imd mutants compared to controls early in the infection, but their expression levels decrease at later times. These findings indicate that Imd signaling activity regulates the feeding rate and Dilp2 and Dilp3 expression in D. melanogaster larvae infected with H. gerrardi. Results from this study facilitate understanding of the link between host innate immunity and sugar metabolism in the context of infectious diseases caused by parasitic nematodes.
Messer, C. L. and McDonald, J. A. (2023). Rap1 promotes epithelial integrity and cell viability in a growing tissue. Dev Biol 501: 1-19. PubMed ID: 37269969
Having intact epithelial tissues is critical for embryonic development and adult homeostasis. How epithelia respond to damaging insults or tissue growth while still maintaining intercellular connections and barrier integrity during development is poorly understood. The conserved small GTPase Rap1 is critical for establishing cell polarity and regulating cadherin-catenin cell junctions. This study identified a new role for Rap1 in maintaining epithelial integrity and tissue shape during Drosophila oogenesis. loss of Rap1 activity disrupted the follicle cell epithelium and the shape of egg chambers during a period of major growth. Rap1 was required for proper E-Cadherin localization in the anterior epithelium and for epithelial cell survival. Both Myo-II and the adherens junction-cytoskeletal linker protein α-Catenin were required for normal egg chamber shape but did not strongly affect cell viability. Blocking the apoptotic cascade failed to rescue the cell shape defects caused by Rap1 inhibition. One consequence of increased cell death caused by Rap1 inhibition was the loss of polar cells and other follicle cells, which later in development led to fewer cells forming a migrating border cell cluster. These results thus indicate dual roles for Rap1 in maintaining epithelia and cell survival in a growing tissue during development.
Ninova, M., Holmes, H., Lomenick, B., Fejes Toth, K. and Aravin, A. A. (2023). Pervasive SUMOylation of heterochromatin and piRNA pathway proteins. Cell Genom 3(7): 100329. PubMed ID: 37492097
Genome regulation involves complex protein interactions that are often mediated through post-translational modifications (PTMs). SUMOylation-modification by the small ubiquitin-like modifier (SUMO)-has been implicated in numerous essential processes in eukaryotes. In Drosophila, SUMO is required for viability and fertility, with its depletion from ovaries leading to heterochromatin loss and ectopic transposon and gene activation. This study developed a proteomics-based strategy to uncover the Drosophila ovarian "SUMOylome," which revealed that SUMOylation is widespread among proteins involved in heterochromatin regulation and different aspects of the Piwi-interacting small RNA (piRNA) pathway that represses transposons. Furthermore, it was shown that SUMOylation of several piRNA pathway proteins occurs in a Piwi-dependent manner. Together, these data highlight broad implications of protein SUMOylation in epigenetic regulation and indicate novel roles of this modification in the cellular defense against genomic parasites. Finally, this work provides a resource for the study of SUMOylation in other biological contexts in the Drosophila model.
Philpott, J. M., Freeberg, A. M., Park, J., Lee, K., Ricci, C. G., Hunt, S. R., Narasimamurthy, R., Segal, D. H., Robles, R., Cai, Y., Tripathi, S., McCammon, J. A., Virshup, D. M., Chiu, J. C., Lee, C. and Partch, C. L. (2023). PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period. Mol Cell 83(10): 1677-1692.e1678. PubMed ID: 37207626
PERIOD (PER) and Casein Kinase 1&delta regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1Δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. This study shows that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. This study found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.
Montembault, E., Deduyer, I., Claverie, M. C., Bouit, L., Tourasse, N. J., Dupuy, D., McCusker, D. and Royou, A. (2023). Two RhoGEF isoforms with distinct localisation control furrow position during asymmetric cell division. Nat Commun 14(1): 3209. PubMed ID: 37268622
Cytokinesis partitions cellular content between daughter cells. It relies on the formation of an acto-myosin contractile ring, whose constriction induces the ingression of the cleavage furrow between the segregated chromatids. Rho1 GTPase and its RhoGEF (Pbl) are essential for this process. However, how Rho1 is regulated to sustain furrow ingression while maintaining correct furrow position remains poorly defined. This study showed that during asymmetric division of Drosophila neuroblasts, Rho1 is controlled by two Pbl isoforms with distinct localisation. Spindle midzone- and furrow-enriched Pbl-A focuses Rho1 at the furrow to sustain efficient ingression, while Pbl-B pan-plasma membrane localization promotes the broadening of Rho1 activity and the subsequent enrichment of myosin on the entire cortex. This enlarged zone of Rho1 activity is critical to adjust furrow position, thereby preserving correct daughter cell size asymmetry. This work highlights how the use of isoforms with distinct localisation makes an essential process more robust.
Nag, S., Szederkenyi, K., Gorbenko, O., Tyrrell, H., Yip, C. M. and McQuibban, G. A. (2023). PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics. Cell Rep 42(8): 112895. PubMed ID: 37498743
Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. This study showed that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, this study demonstrates that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. These results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.

Wednesday, November 29th - Evolution

Pelaez, J. N., Gloss, A. D., Goldman-Huertas, B., Kim, B., Lapoint, R. T., Pimentel-Solorio, G., Verster, K. I., Aguilar, J. M., Nelson Dittrich, A. C., Singhal, M., Suzuki, H. C., Matsunaga, T., Armstrong, E. E., Charboneau, J. L. M., Groen, S. C., Hembry, D. H., Ochoa, C. J., O'Connor, T. K., Prost, S., Zaaijer, S., Nabity, P. D., Wang, J., Rodas, E., Liang, I. and Whiteman, N. K. (2023). Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae. G3 (Bethesda) 13(8). PubMed ID: 37317982
Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genomic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families-genes directly mediating interactions with plant chemical defenses-underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many insect lineages are ancient (>150 million years ago (mya)), obscuring genomic evolutionary patterns. This study characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (>15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several nonherbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza has among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant-binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on living plants (bitter or electrophilic phytotoxins) or their ancestral diet (fermenting plant volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight gene candidates that have also been linked to other dietary transitions in Drosophila.
Lankinen, P., Kastally, C. and Hoikkala, A. (2023). Clinal variation in the temperature and photoperiodic control of reproductive diapause in Drosophila montana females. J Insect Physiol: 104556. PubMed ID: 37598869
Insect adaptation to climatic conditions at different latitudes has required changes in life-history traits linked with survival and reproduction. Several species, including Drosophila montana, show robust latitudinal variation in the critical day length (CDL), below which more than half of the emerging females enter reproductive diapause at a given temperature. This study used a novel approach to find out whether D. montana also shows latitudinal variation in the critical temperature (CTemp), above which the photoperiodic regulation of diapause is disturbed so that the females develop ovaries in daylengths that are far below their CDL. CTemp was estimated for 53 strains from different latitudes on 3 continents after measuring their diapause proportions at a range of temperatures in 12 h daylength (for 29 of the strains also in continuous darkness). In 12 h daylength, CTemp increased towards high latitudes alongside an increase in CDL, and in 3 high-latitude strains diapause proportion exceeded 50 % in all temperatures. In continuous darkness, the diapause proportion was above 50 % in the lowest temperature(s) in only 9 strains, all of which came from high latitudes. In the second part of the study, changes were measured in CTemp and CDL in a selection experiment favouring reproduction in short daylength (photoperiodic selection) and by exercising selection for females that reproduce in LD12:12 at low temperature (photoperiodic and temperature selection). In both experiments selection induced parallel changes in CDL and CTemp, confirming correlations seen between these traits along latitudinal clines. Overall, the findings suggest that selection towards strong photoperiodic diapause and long CDL at high latitudes has decreased the dependency of D. montana diapause on environmental temperature. Accordingly, the prevalence and timing of the diapause of D. montana is likely to be leβ vulnerable to climate warming in high- than low-latitude populations.
Lombardo, K. D., Sheehy, H. K., Cridland, J. M. and Begun, D. J. (2023). Identifying candidate de novo genes expressed in the somatic female reproductive tract of Drosophila melanogaster. G3 (Bethesda) 13(8). PubMed ID: 37259569
Most eukaryotic genes have been vertically transmitted to the present from distant ancestors. However, variable gene number across species indicates that gene gain and loss also occurs. While new genes typically originate as products of duplications and rearrangements of preexisting genes, putative de novo genes-genes born out of ancestrally nongenic sequence-have been identified. Previous studies of de novo genes in Drosophila have provided evidence that expression in male reproductive tissues is common. However, no studies have focused on female reproductive tissues. This study begins addressing this gap in the literature by analyzing the transcriptomes of 3 female reproductive tract organs (spermatheca, seminal receptacle, and parovaria) in 3 species-the focal species, Drosophila melanogaster and 2 closely related species, Drosophila simulans and Drosophila yakuba, with the goal of identifying putative D. melanogaster-specific de novo genes expressed in these tissues. Several candidate genes, located in sequence annotated as intergenic, were discovered. Consistent with the literature, these genes tend to be short, single exon, and lowly expressed. Evidence was also found evidence that some of these genes are expressed in other D. melanogaster tissues and both sexes. The relatively small number of intergenic candidate genes discovered here is similar to that observed in the accessory gland, but substantially fewer than that observed in the testis.
Khatib, L., Subasi, B. S., Fishman, B., Kapun, M. and Tauber, E. (2023). Unveiling Subtle Geographical Clines: Phenotypic Effects and Dynamics of Circadian Clock Gene Polymorphisms. Biology (Basel) 12(6). PubMed ID: 37372143
Understanding of the gene regulatory network that constitutes the circadian clock has greatly increased in recent decades, notably due to the use of Drosophila as a model system. In contrast, the analysis of natural genetic variation that enables the robust function of the clock under a broad range of environments has developed more slowly. The current study analyzed comprehensive genome sequencing data from wild European populations of Drosophila, which were densely sampled through time and space. Hundreds of single nucleotide polymorphisms (SNPs) were identified in nine genes associated with the clock, 276 of which exhibited a latitudinal cline in their allele frequencies. While the effect sizes of these clinal patterns were small, indicating subtle adaptations driven by natural selection, they provided important insights into the genetic dynamics of circadian rhythms in natural populations. Nine SNPs in different genes were chosen and their impact on circadian and seasonal phenotypes was assessed by reconstructing outbred populations fixed for either of the SNP alleles, from inbred DGRP strains. The circadian free-running period of the locomotor activity rhythm was affected by an SNP in doubletime (dbt) and eyes absent (Eya). The SNPs in Clock (Clk), Shaggy (Sgg), period (per), and timeless (tim) affected the acrophase. the time period in a cycle during which the cycle crests or peaks. The alleles of the SNP in Eya conferred different levels of diapause and the chill coma recovery response.
Langmuller, A. M., Nolte, V., Dolezal, M. and Schlotterer, C. (2023). The genomic distribution of transposable elements is driven by spatially variable purifying selection. Nucleic Acids Res. PubMed ID: 37560917
It is widely accepted that the genomic distribution of transposable elements (TEs) mainly reflects the outcome of purifying selection and insertion bias (1). Nevertheless, the relative importance of these two evolutionary forces could not be tested thoroughly. This study introduced an experimental system, which allows separating purifying selection from TE insertion bias. Experimental evolution was used to study the TE insertion patterns in Drosophila simulans founder populations harboring 1040 insertions of an active P-element. After 10 generations at a large population size, strong selection was detected against P-element insertions. The exception were P-element insertions in genomic regions for which a strong insertion bias has been proposed. Because recurrent P-element insertions cannot explain this pattern, it is concluded that purifying selection, with variable strength along the chromosomes, is the major determinant of the genomic distribution of P-elements. Genomic regions with relaxed purifying selection against P-element insertions exhibit normal levels of purifying selection against base substitutions. This suggests that different types of purifying selection operate on base substitutions and P-element insertions. These results highlight the power of experimental evolution to understand basic evolutionary processes, which are difficult to infer from patterns of natural variation alone.
Lai, W. Y., Otte, K. A. and Schlotterer, C. (2023). Evolution of Metabolome and Transcriptome Supports a Hierarchical Organization of Adaptive Traits. Genome Biol Evol 15(6). PubMed ID: 37232360
Most organismal phenotypes have a polygenic basis, which enables adaptive phenotypic responses on ecological time scales. While adaptive phenotypic changes are highly parallel in replicate populations, this does not apply to the contributing loci. In particular for small populations, the same phenotypic shift can be fueled by different sets of alleles at alternative loci (genetic redundancy). Although this phenomenon is empirically well supported, the molecular basis of the genetic redundancy is not yet understood. To fill this gap, this study compared the heterogeneity of the evolutionary transcriptomic and metabolomic response in ten Drosophila simulans populations which evolved parallel high-level phenotypic changes in a novel temperature environment but used different allelic combinations of alternative loci. The metabolome was shown to evolved more parallel than the transcriptome, confirming a hierarchical organization of molecular phenotypes. Different sets of genes responded in each evolved population but led to the enrichment of similar biological functions and a consistent metabolic profile. Since even the metabolomic response was still highly heterogeneous across evolved populations, it is propose that selection may operate on pathways/networks.

Tuesday, November 28th - Gonads

Oramas, R., Knapp, E. M., Zeng, B. and Sun, J. (2023). The bHLH-PAS transcriptional complex Sim:Tgo plays active roles in late oogenesis to promote follicle maturation and ovulation. Development 150(12). PubMed ID: 37218521
Across species, ovulation is a process induced by a myriad of signaling cascades that ultimately leads to the release of encapsulated oocytes from follicles. Follicles first need to mature and gain ovulatory competency before ovulation; however, the signaling pathways regulating follicle maturation are incompletely understood in Drosophila and other species. Previous work has shown that the bHLH-PAS transcription factor Single-minded (Sim) plays important roles in follicle maturation downstream of the nuclear receptor Ftz-f1 in Drosophila. This study demonstrates that Tango (Tgo), another bHLH-PAS protein, acts as a co-factor of Sim to promote follicle cell differentiation from stages 10 to 12. In addition, this study discovered that re-upregulation of Sim in stage-14 follicle cells is also essential to promote ovulatory competency by upregulating octopamine receptor in mushroom body (OAMB), matrix metalloproteinase 2 (Mmp2) and NADPH oxidase (NOX), either independently of or in conjunction with the zinc-finger protein Hindsight (Hnt). All these factors are crucial for successful ovulation. Together, this work indicates that the transcriptional complex Sim:Tgo plays multiple roles in late-stage follicle cells to promote follicle maturation and ovulation.
Neiswender, H., Baker, F. C., Veeranan-Karmegam, R., Allen, P. and Gonsalvez, G. B. (2023). dTtc1, a conserved tetratricopeptide repeat protein, is required for maturation of Drosophila egg chambers via its role in stabilizing electron transport chain components. Front Cell Dev Biol 11: 1148773. PubMed ID: 37333987
The Drosophila ortholog of TTC1 (dTtc1) has been identified as an interacting partner of Egalitarian, an RNA adaptor of the Dynein motor. In order to better understand the function of this relatively uncharacterized protein, dTtc1 was deleted in the Drosophila female germline. Depletion of dTtc1 resulted in defective oogenesis and no mature eggs were produced. A closer examination revealed that mRNA cargoes normally transported by Dynein were relatively unaffected. However, mitochondria in dTtc1 depleted egg chambers displayed an extremely swollen phenotype. Ultrastructural analysis revealed a lack of cristae. These phenotypes were not observed upon disruption of Dynein. Thus, this function of dTtc1 is likely to be Dynein independent. Consistent with a role for dTtc1 in mitochondrial biology, a published proteomics screen revealed that dTtc1 interacts with numerous components of electron transport chain (ETC) complexes. These results indicate that the expression level of several of these ETC components was significantly reduced upon depletion of dTtc1. Importantly, this phenotype was completely rescued upon expression of wild-type GFP-dTtc1 in the depleted background. Lastly, this study demonstrated that the mitochondrial phenotype caused by a lack of dTtc1 is not restricted to the germline but is also observed in somatic tissues. This model suggests that dTtc1, likely in combination with cytoplasmic chaperones, is required for stabilizing ETC components.
Loh, M., Bernard, F. and Guichet, A. (2023). Kinesin-1 promotes centrosome clustering and nuclear migration in the Drosophila oocyte. Development 150(13). PubMed ID: 37334771
Microtubules and their associated motors are important players in nucleus positioning. Although nuclear migration in Drosophila oocytes is controlled by microtubules, a precise role for microtubule-associated molecular motors in nuclear migration has yet to be reported. This study characterized novel landmarks that allow a precise description of the pre-migratory stages. Using these newly defined stages, it is reported that, before migration, the nucleus moves from the oocyte anterior side toward the center and concomitantly the centrosomes cluster at the posterior of the nucleus. In the absence of Kinesin-1, centrosome clustering is impaired and the nucleus fails to position and migrate properly. The maintenance of a high level of Polo-kinase at centrosomes prevents centrosome clustering and impairs nuclear positioning. In the absence of Kinesin-1, SPD-2, an essential component of the pericentriolar material, is increased at the centrosomes, suggesting that Kinesin-1-associated defects result from a failure to reduce centrosome activity. Consistently, depleting centrosomes rescues the nuclear migration defects induced by Kinesin-1 inactivation. These results suggest that Kinesin-1 controls nuclear migration in the oocyte by modulating centrosome activity.
Mellentine, S. Q., Ramsey, A. S., Li, J., Brown, H. N. and Tootle, T. L. (2023). Specific prostaglandins are produced in the migratory cells and the surrounding substrate to promote Drosophila border cell migration. bioRxiv. PubMed ID: 37425965
A key regulator of collective cell migration is prostaglandin (PG) signaling. However, it remains largely unclear whether PGs act within the migratory cells or their microenvironment to promote migration. This study used Drosophila border cell migration as a model to uncover the cell-specific roles of two PGs in collective migration. Prior work shows PG signaling is required for on-time migration and cluster cohesion. The PGE (2) synthase cPGES is required in the substrate, while the PGF (2α) synthase Akr1B is required in the border cells for on-time migration. Akr1B acts in both the border cells and their substrate to regulate cluster cohesion. One means by which Akr1B regulates border cell migration is by promoting integrin-based adhesions. Additionally, Akr1B limits myosin activity, and thereby cellular stiffneβ, in the border cells, whereas cPGES limits myosin activity in both the border cells and their substrate. Together these data reveal that two PGs, PGE (2) and PGF (2α), produced in different locations, play key roles in promoting border cell migration. These PGs likely have similar migratory versus microenvironment roles in other collective cell migrations.
Molina Lopez, E., Kabanova, A., Winkel, A., Franze, K., Palacios, I. M. and Martín-Bermudo, M. D. (2023). Constriction imposed by basement membrane regulates developmental cell migration. PLoS Biol 21(6): e3002172. PubMed ID: 37379333
The basement membrane (BM) is a specialized extracellular matrix (ECM), which underlies or encases developing tissues. Mechanical properties of encasing BMs have been shown to profoundly influence the shaping of associated tissues. This study used the migration of the border cells (BCs) of the Drosophila egg chamber to unravel a new role of encasing BMs in cell migration. BCs move between a group of cells, the nurse cells (NCs), that are enclosed by a monolayer of follicle cells (FCs), which is, in turn, surrounded by a BM, the follicle BM. Increasing or reducing the stiffness of the follicle BM, by altering laminins or type IV collagen levels, conversely affects BC migration speed and alters migration mode and dynamics. Follicle BM stiffness also controls pairwise NC and FC cortical tension. It is proposed that constraints imposed by the follicle BM influence NC and FC cortical tension, which, in turn, regulate BC migration. Encasing BMs emerge as key players in the regulation of collective cell migration during morphogenesis.
Mukherjee, A., Schuppe, M. and Renault, A. D. (2023). The Lipid Phosphate Phosphatase Wunen Promotes Eggshell Formation and Is Essential for Fertility in Drosophila. Biology (Basel) 12(7). PubMed ID: 37508432
The eggshell that surrounds insect eggs acts as a barrier, protecting against biotic factors and desiccation. The eggshell is a multi-layered structure which is synthesised by the somatic follicle cells that surround the developing oocyte. Although the temporal order of expression of the protein eggshell components goes someway to explaining how the different layers are built up, but how the precise three-dimensional structure is achieved and how lipid components responsible for desiccation resistance are incorporated are poorly understood. This paper demonstrates that wunen, which encodes a lipid phosphate phosphatase, is necessary for fertility in Drosophila females. Compared to sibling controls, females null for wunen lay fewer eggs which subsequently collapse such that no larvae emerge. This is due to a requirement for wunen in the ovarian follicle cells which is needed to produce an ordered and functional eggshell. Knockdown of a septate junction component also results in collapsed eggs, supporting the idea that similar to its role in embryonic tracheal development, Wunen in follicle cells also promotes septate junction function.

Monday, November 27th - Adult Neural Structure, Development, and Function

Oliveira, A. C. and Homem, C. C. F. (2023). Opposing effects of ecdysone signaling regulate neuroblast proliferation to ensure coordination of brain and organism development. Dev Biol 503: 53-67. PubMed ID: 37549863
Growth regulation must be robust to ensure correct final size, but also adaptative to adjust to less favorable environmental conditions. Developmental coordination between whole-organism and the brain is particularly important, as the brain is a critical organ with little adaptability. Brain growth mainly depends on neural stem cell (NSC) proliferation to generate differentiated neural cells, it is however unclear how organism developmental progression is coordinated with NSCs. This study demonstrates that the steroid hormone ecdysone plays a multi-step, stage specific role in regulating Drosophila NSCs, the neuroblasts. Animals were used that are unable to synthesize ecdysone, to show that the developmental milestone called 'critical weight peak', the peak that informs the body has reached minimum viable weight to survive metamorphosis, acts a checkpoint necessary to set neuroblast cell cycle pace during larval neurogenesis. The peaks of ecdysone that occur post-critical weight are no longer required to maintain neuroblast division rate. It was additionally shown that in a second stage, at the onset of pupariation, ecdysone is instead required to trigger neuroblast's proliferation exit and consequently the end of neurogenesis. It was demonstrated that, without this signal from ecdysone, neuroblasts lose their ability to exit proliferation. Interestingly, although these neuroblasts proliferate for a longer period, the number of differentiated neurons is smaller compared to wild-type brains, suggesting a role for ecdysone in neuron maintenance. This study provides insights into how neural stem cells coordinate their division rate with the pace of body growth, identifying a novel coordination mechanism between animal development and NSC proliferation.
Mohamed, A., Malekou, I., Sim, T., O'Kane, C. J., Maait, Y., Scullion, B. and Masuda-Nakagawa, L. M. (2023). Mushroom body output neurons MBON-a1/a2 define an odor intensity channel that regulates behavioral odor discrimination learning in larval Drosophila. Front Physiol 14: 1111244. PubMed ID: 37256074
The sensitivity of animals to sensory input must be regulated to ensure that signals are detected and also discriminable. However, how circuits regulate the dynamic range of sensitivity to sensory stimuli is not well understood. A given odor is represented in the insect mushroom bodies (MBs) by sparse combinatorial coding by Kenyon cells (KCs), forming an odor quality representation. To address how intensity of sensory stimuli is processed at the level of the MB input region, the calyx, this study characterized a set of novel mushroom body output neurons that respond preferentially to high odor concentrations. A pair of MB calyx output neurons, MBON-a1/2, were shown to be postsynaptic in the MB calyx, where they receive extensive synaptic inputs from KC dendrites, the inhibitory feedback neuron APL, and octopaminergic sVUM1 neurons, but relatively few inputs from projection neurons. This pattern is broadly consistent in the third-instar larva as well as in the first instar connectome. MBON-a1/a2 presynaptic terminals innervate a region immediately surrounding the MB medial lobe output region in the ipsilateral and contralateral brain hemispheres. By monitoring calcium activity using jRCamP1b, it was found that MBON-a1/a2 responses are odor-concentration dependent, responding only to ethyl acetate (EA) concentrations higher than a 200-fold dilution, in contrast to MB neurons which are more concentration-invariant and respond to EA dilutions as low as 10-4. Optogenetic activation of the calyx-innervating sVUM1 modulatory neurons originating in the SEZ (Subesophageal zone), did not show a detectable effect on MBON-a1/a2 odor responses. Optogenetic activation of MBON-a1/a2 using CsChrimson impaired odor discrimination learning compared to controls. It is proposed that MBON-a1/a2 form an output channel of the calyx, summing convergent sensory and modulatory input, firing preferentially to high odor concentration, and might affect the activity of downstream MB targets.
Mehta, K., Goldin, R. F. and Ascoli, G. A. (2023). Circuit analysis of the Drosophila brain using connectivity-based neuronal classification reveals organization of key communication pathways. Netw Neurosci 7(1): 269-298. PubMed ID: 37339321
This study presents a functionally relevant, quantitative characterization of the neural circuitry of Drosophila melanogaster at the mesoscopic level of neuron types as classified exclusively based on potential network connectivity. Starting from a large neuron-to-neuron brain-wide connectome of the fruit fly, stochastic block modeling and spectral graph clustering were used to group neurons together into a common 'cell class' if they connect to neurons of other classes according to the same probability distributions. Then the connectivity-based cell classes were characterized with standard neuronal biomarkers, including neurotransmitters, developmental birthtimes, morphological features, spatial embedding, and functional anatomy. Mutual information indicates that connectivity-based classification reveals aspects of neurons that are not adequately captured by traditional classification schemes. Next, using graph theoretic and random walk analyses to identify neuron classes as hubs, sources, or destinations, pathways and patterns of directional connectivity were detected that potentially underpin specific functional interactions in the Drosophila brain. A core of highly interconnected dopaminergic cell classes functioning as the backbone communication pathway for multisensory integration. Additional predicted pathways pertain to the facilitation of circadian rhythmic activity, spatial orientation, fight-or-flight response, and olfactory learning. This analysis provides experimentally testable hypotheses critically deconstructing complex brain function from organized connectomic architecture.
Oikawa, I., Kondo, S., Hashimoto, K., Yoshida, A., Hamajima, M., Tanimoto, H., Furukubo-Tokunaga, K. and Honjo, K. (2023). A descending inhibitory mechanism of nociception mediated by an evolutionarily conserved neuropeptide system in Drosophila. Elife 12. PubMed ID: 37310871
Nociception is a neural process that animals have developed to avoid potentially tissue-damaging stimuli. While nociception is triggered in the peripheral nervous system, its modulation by the central nervous system is a critical process in mammals, whose dysfunction has been extensively implicated in chronic pain pathogenesis. The peripheral mechanisms of nociception are largely conserved across the animal kingdom. However, it is unclear whether the brain-mediated modulation is also conserved in non-mammalian species. This study shows that Drosophila has a descending inhibitory mechanism of nociception from the brain, mediated by the neuropeptide Drosulfakinin (DSK), a homolog of cholecystokinin (CCK) that plays an important role in the descending control of nociception in mammals. Mutants lacking dsk or its receptors are hypersensitive to noxious heat. Through a combination of genetic, behavioral, histological, and Ca(2+) imaging analyses, neurons involved in DSK-mediated nociceptive regulation were subsequently revealed at a single-cell resolution, and a DSKergic descending neuronal pathway was identified that inhibits nociception. This study provides the first evidence for a descending modulatory mechanism of nociception from the brain in a non-mammalian species that is mediated by the evolutionarily conserved CCK system, raising the possibility that the descending inhibition is an ancient mechanism to regulate nociception.
Nguyen, Y. D. H., Yoshida, H., Tran, T. M. and Kamei, K. (2023). Lipin knockdown in pan-neuron of Drosophila induces reduction of lifespan, deficient locomotive behavior, and abnormal morphology of motor neuron. Neuroreport 34(12): 629-637. PubMed ID: 37470742
The Lipin family is evolutionarily conserved among insects and mammals, and its crucial roles in lipid synthesis and homeostatic control of energy balance have been well documented. This study investigated the function of Lipin in neuronal function and neurodegeneration. The GAL4/UAS system was used to knock down Lipin in the nervous system of Drosophila and investigate its behavioral and cellular phenotypes. The neuromuscular junction (NMJ) morphology was detected by immunostaining. Moreover, triacylglycerol and ATP levels were analyzed by using assay Kit. This study found that Lipin is localized almost in the cytoplasm of neurons in the brain lobe and ventral nerve cord, which are part of the central nervous system (CNS) of Drosophila melanogaster. Lipin knockdown larvae exhibit decreased locomotor activity, aberrant morphology of motor nerve terminals at NMJs, and reduced number and size of lipid droplets in the CNS. Furthermore, neuron-specific knockdown of Lipin leads to locomotor defects and a shortened lifespan, accompanied by a reduction in ATP levels in the adult stage. These results indicate that Lipin plays a crucial role in the CNS of Drosophila.
Modi, M. N., Rajagopalan, A. E., Rouault, H., Aso, Y. and Turner, G. C. (2023). Flexible specificity of memory in Drosophila depends on a comparison between choices. Elife 12. PubMed ID: 37318123
Memory guides behavior across widely varying environments and must therefore be both sufficiently specific and general. A memory too specific will be useless in even a slightly different environment, while an overly general memory may lead to suboptimal choices. Animals successfully learn to both distinguish between very similar stimuli and generalize across cues. Rather than forming memories that strike a balance between specificity and generality, Drosophila can flexibly categorize a given stimulus into different groups depending on the options available. This study asked how this flexibility manifests itself in the well-characterized learning and memory pathways of the fruit fly. It was shown that flexible categorization in neuronal activity as well as behavior depends on the order and identity of the perceived stimuli. These results identify the neural correlates of flexible stimulus-categorization in the fruit fly.

Wednesday, November 22nd - Cytoskeleton and Junctions

O'Neill, R. S., Sodeinde, A. K., Welsh, F. C., Fagerstrom, C. J., Galletta, B. J. and Rusan, N. M. (2023). Spd-2 gene duplication reveals cell-type-specific pericentriolar material regulation. Curr Biol 33(14): 3031-3040.e3036. PubMed ID: 37379844
Centrosomes are multi-protein organelles that function as microtubule (MT) organizing centers (MTOCs), ensuring spindle formation and chromosome segregation during cell division. Centrosome structure includes core centrioles that recruit pericentriolar material (PCM) that anchors γ-tubulin to nucleate MTs. In Drosophila melanogaster, PCM organization depends on proper regulation of proteins like Spd-2, which dynamically localizes to centrosomes and is required for PCM, γ-tubulin, and MTOC activity in brain neuroblast (NB) mitosis and male spermatocyte (SC) meiosis. Some cells have distinct requirements for MTOC activity due to differences in characteristics like cell size or whether they are mitotic or meiotic. How centrosome proteins achieve cell-type-specific functional differences is poorly understood. Previous work identified alternative splicing and binding partners as contributors to cell-type-specific differences in centrosome function. Gene duplication, which can generate paralogs with specialized functions, is also implicated in centrosome gene evolution, including cell-type-specific centrosome genes. To gain insight into cell-type-specific differences in centrosome protein function and regulation, this study investigated a duplication of Spd-2 in Drosophila willistoni, which has Spd-2A (ancestral) and Spd-2B (derived). Spd-2A functions in NB mitosis, whereas Spd-2B functions in SC meiosis. Ectopically expressed Spd-2B accumulates and functions in mitotic NBs, but ectopically expressed Spd-2A failed to accumulate in meiotic SCs, suggesting cell-type-specific differences in translation or protein stability. This failure to accumulate and function in meiosis was mapped to the C-terminal tail domain of Spd-2A, revealing a novel regulatory mechanism that can potentially achieve differences in PCM function across cell types.
Rothenberg, K. E., Chen, Y., McDonald, J. A. and Fernandez-Gonzalez, R. (2023). Rap1 coordinates cell-cell adhesion and cytoskeletal reorganization to drive collective cell migration in vivo. Curr Biol 33(13): 2587-2601. PubMed ID: 37244252
Collective cell movements contribute to tissue development and repair and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of adherens junctions and the actomyosin cytoskeleton. However, the mechanisms that coordinate cell-cell adhesion and cytoskeletal remodeling during collective cell migration in vivo are unclear. This study investigated the mechanisms of collective cell migration during epidermal wound healing in Drosophila embryos. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein non-muscle myosin II to form a supracellular cable around the wound that coordinates cell movements. The cable anchors at former tricellular junctions (TCJs) along the wound edge, and TCJs are reinforced during wound closure. The small GTPase Rap1 was necessary and sufficient for rapid wound repair. Rap1 promoted myosin polarization to the wound edge and E-cadherin accumulation at TCJs. Using embryos expressing a mutant form of the Rap1 effector Canoe/Afadin that cannot bind Rap1, it was found that Rap1 signals through Canoe for adherens junction remodeling, but not for actomyosin cable assembly. Instead, Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. The RhoGEF Ephexin localized to the wound edge in a Rap1-dependent manner, and Ephexin was necessary for myosin polarization and rapid wound repair, but not for E-cadherin redistribution. Together, these data show that Rap1 coordinates the molecular rearrangements that drive embryonic wound healing, promoting actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration in vivo.
Ibar, C., Chinthalapudi, K., Heiβler, S. M. and Irvine, K. D. (2023). Competition between myosin II and β(H)-spectrin regulates cytoskeletal tension. Elife 12. PubMed ID: 37367948
Spectrins are membrane cytoskeletal proteins generally thought to function as heterotetramers comprising two α-spectrins and two β-spectrins. They influence cell shape and Hippo signaling, but the mechanism by which they influence Hippo signaling has remained unclear. this study has investigated the role and regulation of the Drosophila β-heavy spectrin (β(H)-spectrin, encoded by the karst gene) in wing imaginal discs. The results establish that β(H)-spectrin regulates Hippo signaling through the Jub biomechanical pathway due to its influence on cytoskeletal tension. While it was found that α-spectrin also regulates Hippo signaling through Jub, unexpectedly, it was found that β(H)-spectrin localizes and functions independently of α-spectrin. Instead, β(H)-spectrin co-localizes with and reciprocally regulates and is regulated by myosin. In vivo and in vitro experiments support a model in which β(H)-spectrin and myosin directly compete for binding to apical F-actin. This competition can explain the influence of β(H)-spectrin on cytoskeletal tension and myosin accumulation. It also provides new insight into how β(H)-spectrin participates in ratcheting mechanisms associated with cell shape change.
Nithianantham, S., Iwanski, M. K., Gaska, I., Pandey, H., Bodrug, T., Inagaki, S., Major, J., Brouhard, G. J., Gheber, L., Rosenfeld, S. S., Forth, S., Hendricks, A. G. and Al-Bassam, J. (2023). The kinesin-5 tail and bipolar miniflament domains are the origin of its microtubule crosslinking and sliding activity. Mol Biol Cell: mbcE23070287. PubMed ID: 37610838
Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, a minimal kinesin-5 tetramer (mini-tetramer) was constructed. The X-ray structure of the minimal, 34-nm BASS domain was determined. Guided by these structural studies, active bipolar kinesin-5 mini-tetramer motors from Drosophila and human orthologs were constructe the are half the length of native kinesin-5. These kinesin-5 mini-tetramers were used to examine the role of two unique structural adaptations of kinesin-5: the length and flexibility of the tetramer, and the C-terminal tails which interact with the motor domains to coordinate their ATPase activity. The C-terminal domain causes frequent pausing and clustering of kinesin-5. By comparing microtubule crosslinking and sliding by mini-tetramer and full-length kinesin-5, it was found that both the length and flexibility of kinesin-5 and the C-terminal tails govern its ability to crosslink microtubules. Once crosslinked, stiffer mini-tetramers slide antiparallel microtubules more efficiently than full-length motors.
Hodge, R. A., Ghannam, M., Edmond, E., de la Torre, F., D'Alterio, C., Kaya, N. H., Resnik-Docampo, M., Reiff, T. and Jones, D. L. (2023). The septate junction component bark beetle is required for Drosophila intestinal barrier function and homeostasis. iScience 26(6): 106901. PubMed ID: 37332603
Age-related loss of intestinal barrier function has been documented across species, but the causes remain unknown. The intestinal barrier is maintained by tight junctions (TJs) in mammals and septate junctions (SJs) in insects. Specialized TJs/SJs, called tricellular junctions (TCJs), are located at the nexus of three adjacent cells, and this study have shown that aging results in changes to TCJs in intestines of adult Drosophila melanogaster. This study now demonstrates that localization of the TCJ protein bark beetle (Bark) decreases in aged flies. Depletion of bark from enterocytes in young flies led to hallmarks of intestinal aging and shortened lifespan, whereas depletion of bark in progenitor cells reduced Notch activity, biasing differentiation toward the secretory lineage. These data implicate Bark in EC maturation and maintenance of intestinal barrier integrity. Understanding the assembly and maintenance of TCJs to ensure barrier integrity may lead to strategies to improve tissue integrity when function is compromised.
Jejina, A., Ayala, Y., Hernandez, G. and Suter, B. (2023). Role of BicDR in bristle shaft construction, tracheal development, and support of BicD functions. bioRxiv. PubMed ID: 37398393
Cell polarization requires asymmetric localization of numerous mRNAs, proteins, and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors, which function as multiprotein complexes. In the dynein/dynactinBicaudal-D (DDB) transport machinery, Bicaudal-D (BicD) links the cargo to the motor. This study focused on the role of BicD-related (BicDR) and its contribution to microtubule-dependent transport processes. Drosophila BicDR is required for the normal development of bristles and dorsal trunk tracheae. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft and the localization of Spn-F and Rab6 at the distal tip. BicDR supports the function of BicD in bristle development and the results suggest that BicDR transports cargo more locally whereas BicD is more responsible for delivering functional cargo over the long distance to the distal tip. This study identified the proteins that interact with BicDR and appear to be BicDR cargo in embryonic tissues. For one of them, EF1γ, this study showed that EF1γ genetically interacts with BicD and BicDR in the construction of the bristles.

Tuesday, November 21st - Behavior

Morimoto, J., McDonald, G. C. and Wigby, S. (2023). Social group composition modulates the role of last male sperm precedence in post-copulatory sexual selection. J Evol Biol 36(8): 1102-1115. PubMed ID: 37341163
In many species, the order in which males mate with a female explains much of the variation in paternity arising from post-copulatory sexual selection. Research in Drosophila suggests that mating order may account for the majority of the variance in male reproductive success. However, the effects of mating order on paternity bias might not be static but could potentially vary with social or environmental factors. To test this idea, an existing dataset was used, collated from an experiment that was previously published (Morimoto et al., PLoS One, 11, 2016, e0154468), with the addition of unpublished data from the same experiment. These previous experiments manipulated larval density in Drosophila melanogaster which generated variation in male and female body size, assembled groups of individuals of different sizes, and measured the mating success and paternity share of focal males. The data presented in this study provides information on each focal male's mating order and the frequency in which focal males remated with same females ('repetitive matings'). This information was combined with previously reported focal male reproductive success to partition variance in paternity into male mating order and repetitive matings across groups that differed in the body size composition of males and females. It was found, as expected, that male mating order explained a considerable portion of the variance in male paternity. However, it was also found that the impact of male mating order on male paternity was influenced by the body size composition of groups. Specifically, males that tended to mate last had a greater paternity advantage, and displayed lower variance, in groups containing a heterogenous mixture male body sizes than in groups with a single male body size. Repetitive mating only had a minor contribution to the variance in male paternity share across all experiments. Overall, these findings contribute to the growing body of research showing that post-copulatory sexual selection is subject to socio-ecological influences.
Malik, M. Z., Dashti, M., Fatima, Y., Channanath, A., John, S. E., Singh, R. K. B., Al-Mulla, F. and Thanaraj, T. A. (2023). Disruption in the regulation of casein kinase 2 in circadian rhythm leads to pathological states: cancer, diabetes and neurodegenerative disorders. Front Mol Neurosci 16: 1217992. PubMed ID: 37475884
Circadian rhythm maintains the sleep-wake cycle in biological systems. Various biological activities are regulated and modulated by the circadian rhythm, disruption of which can result in onset of diseases. Robust rhythms of phosphorylation profiles and abundances of PERIOD (PER) proteins are thought to be the master keys that drive circadian clock functions. The role of casein kinase 2 (CK2) in circadian rhythm via its direct interactions with the PER protein has been extensively studied; however, the exact mechanism by which it affects circadian rhythms at the molecular level is not known. This study proposes an extended circadian rhythm model in Drosophila that incorporates the crosstalk between the PER protein and CK2. The regulatory role of CK2 was studied in the dynamics of PER proteins involved in circadian rhythm using the stochastic simulation algorithm. It was observed that variations in the concentration of CK2 in the circadian rhythm model modulates the PER protein dynamics at different cellular states, namely, active, weakly active, and rhythmic death. These oscillatory states may correspond to distinct pathological cellular states of the living system. Molecular noise was found at the expression level of CK2 to switch normal circadian rhythm to any of the three above-mentioned circadian oscillatory states. The results suggest that the concentration levels of CK2 in the system has a strong impact on its dynamics, which is reflected in the time evolution of PER protein. It is believed that these findings can contribute towards understanding the molecular mechanisms of circadian dysregulation in pathways driven by the PER mutant genes and their pathological states, including cancer, obesity, diabetes, neurodegenerative disorders, and socio-psychological disease.
Liu, Y., Hasegawa, E., Nose, A., Zwart, M. F. and Kohsaka, H. (2023). Synchronous multi-segmental activity between metachronal waves controls locomotion speed in Drosophila larvae. Elife 12. PubMed ID: 37551094
The ability to adjust the speed of locomotion is essential for survival. In limbed animals, the frequency of locomotion is modulated primarily by changing the duration of the stance phase. The underlying neural mechanisms of this selective modulation remain an open question. This study reports a neural circuit controlling a similarly selective adjustment of locomotion frequency in Drosophila larvae. Drosophila larvae crawl using peristaltic waves of muscle contractions. This study found that larvae adjust the frequency of locomotion mostly by varying the time between consecutive contraction waves, reminiscent of limbed locomotion. A specific set of muscles, the lateral transverse (LT) muscles, co-contract in all segments during this phase, the duration of which sets the duration of the interwave phase. Two types of GABAergic interneurons were identified in the LT neural network, premotor neuron A26f and its presynaptic partner A31c, which exhibit segmentally synchronized activity and control locomotor frequency by setting the amplitude and duration of LT muscle contractions. Altogether, these results reveal an inhibitory central circuit that sets the frequency of locomotion by controlling the duration of the period in between peristaltic waves. Further analysis of the descending inputs onto this circuit will help understand the higher control of this selective modulation.
Lee, S. G., Sun, D., Miao, H., Wu, Z., Kang, C., Saad, B., Nguyen, K. H., Guerra-Phalen, A., Bui, D., Abbas, A. H., Trinh, B., Malik, A., Zeghal, M., Auge, A. C., Islam, M. E., Wong, K., Stern, T., Lebedev, E., Sherratt, T. N. and Kim, W. J. (2023). Taste and pheromonal inputs govern the regulation of time investment for mating by sexual experience in male Drosophila melanogaster. PLoS Genet 19(5): e1010753. PubMed ID: 37216404
Males have finite resources to spend on reproduction. Thus, males rely on a 'time investment strategy' to maximize their reproductive success. For example, male Drosophila melanogaster extends their mating duration when surrounded by conditions enriched with rivals. This study reports a different form of behavioral plasticity whereby male fruit flies exhibit a shortened duration of mating when they are sexually experienced; this plasticity is referred to as 'shorter-mating-duration (SMD)'. SMD is a plastic behavior and requires sexually dimorphic taste neurons. Several neurons were identified in the male foreleg and midleg that express specific sugar and pheromone receptors. Using a cost-benefit model and behavioral experiments, it was further shown that SMD behavior exhibits adaptive behavioral plasticity in male flies. Thus, this study delineates the molecular and cellular basis of the sensory inputs required for SMD; this represents a plastic interval timing behavior that could serve as a model system to study how multisensory inputs converge to modify interval timing behavior for improved adaptation.
Lev, A. and Pischedda, A. (2023). Male size does not affect the strength of male mate choice for high-quality females in Drosophila melanogaster. J Evol Biol. PubMed ID: 37534751
Theory predicts that the strength of male mate choice should vary depending on male quality when higher-quality males receive greater fitness benefits from being choosy. This pattern extends to differences in male body size, with larger males often having stronger pre- and post-copulatory preferences than smaller males. This study sought to determine whether large males and small males differ in the strength (or direction) of their preference for large, high-fecundity females using the fruit fly, Drosophila melanogaster. Male courtship preferences and mating duration were measured to show that male body size had no impact on the strength of male mate choice; all males, regardless of their size, had equally strong preferences for large females. To understand the selective pressures shaping male mate choice in males of different sizes, the fitness benefits associated with preferring large females for both large and small males were also measured. Male body size did not affect the benefits that males received: large and small males were equally successful at mating with large females, received the same direct fitness benefits from mating with large females, and showed similar competitive fertilization success with large females. These findings provide insight into why the strength of male mate choice was not affected by male body size in this system. This study highlights the importance of evaluating the benefits and costs of male mate choice across multiple males to predict when differences in male mate choice should occur.
Manoli, G., Zandawala, M., Yoshii, T. and Helfrich-Forster, C. (2023). Characterization of clock-related proteins and neuropeptides in Drosophila littoralis and their putative role in diapause. J Comp Neurol 531(15): 1525-1549. PubMed ID: 37493077
Insects from high latitudes spend the winter in a state of overwintering diapause, which is characterized by arrested reproduction, reduced food intake and metabolism, and increased life span. The main trigger to enter diapause is the decreasing day length in summer-autumn. It is thus assumed that the circadian clock acts as an internal sensor for measuring photoperiod and orchestrates appropriate seasonal changes in physiology and metabolism through various neurohormones. However, little is known about the neuronal organization of the circadian clock network and the neurosecretory system that controls diapause in high-latitude insects. This was addressed by mapping the expression of clock proteins and neuropeptides/neurohormones in the high-latitude fly Drosophila littoralis. The principal organization of both systems was found to be similar to that in Drosophila melanogaster, but with some striking differences in neuropeptide expression levels and patterns. The small ventrolateral clock neurons that express pigment-dispersing factor (PDF) and short neuropeptide F (sNPF) and are most important for robust circadian rhythmicity in D. melanogaster virtually lack PDF and sNPF expression in D. littoralis. In contrast, dorsolateral clock neurons that express ion transport peptide in D. melanogaster additionally express allatostatin-C and appear suited to transfer day-length information to the neurosecretory system of D. littoralis. The lateral neurosecretory cells of D. littoralis contain more neuropeptides than D. melanogaster. Among them, the cells that coexpress corazonin, PDF, and diuretic hormone 44 appear most suited to control diapause. This work sets the stage to investigate the roles of these diverse neuropeptides in regulating insect diapause.

Monday November 20th - Disease Models

Mirzoyan, Z., Valenza, A., Zola, S., Bonfanti, C., Arnaboldi, L., Ferrari, N., Pollard, J., Lupi, V., Caβinelli, M., Frattaroli, M., Sahin, M., Pasini, M. E. and Bellosta, P. (2023). A Novel Drosophila Model to Investigate Adipose tissue Macrophage Infiltration (ATM) and Obesity highlights the Therapeutic Potential of Attenuating Eiger/TNFα Signaling to Ameliorate Insulin Resistance and ATM. bioRxiv. PubMed ID: 37461586
Tnis study presents a novel Drosophila model to investigate the mechanisms underlying adipose tissue macrophage(ATM) infiltration. This study demonstrated the therapeutic potential of attenuating Eiger/TNFα signaling to ameliorate insulin resistance and ATM. To study ATM infiltration and its consequences, a novel Drosophila model (OBL) was developed that mimics key aspects of human adipose tissue. Genetic manipulation was used to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic, and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy, and high levels of circulating glucose. Moreover, a significant infiltration of immune cells (hemocytes) in the fat bodies was observed, accompanied by insulin resistance and systemic metabolic dysregulation. Furthermore, it was found that attenuation of Eiger/TNFα signaling and using metformin and anti-oxidant bio-products like anthocyanins led to a reduction in ATM infiltration and improved insulin sensitivity. These data suggest that the key mechanisms that trigger immune cell infiltration into adipose tissue are evolutionarily conserved and may provide the opportunity to develop Drosophila models to better understand pathways critical for immune cell recruitment into adipose tissue, in relation to the development of insulin resistance in metabolic diseases such as obesity and type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). This OBL model can also be a valuable tool and provide a platform either to perform genetic screens or to test the efficacy and safety of novel therapeutic interventions for these diseases.
Moehlman, A. T., Kanfer, G. and Youle, R. J. (2023). Loss of STING in parkin mutant flies supresses muscle defects and mitochondria damage. PLoS Genet 19(7): e1010828. PubMed ID: 37440574
The early pathogenesis and underlying molecular causes of motor neuron degeneration in Parkinson's Disease (PD) remains unresolved. In the model organism Drosophila melanogaster, loss of the early-onset PD gene parkin (the ortholog of human PRKN) results in impaired climbing ability, damage to the indirect flight muscles, and mitochondrial fragmentation with swelling. These stressed mitochondria have been proposed to activate innate immune pathways through release of damage associated molecular patterns (DAMPs). Parkin-mediated mitophagy is hypothesized to supress mitochondrial damage and subsequent activation of the cGAS/STING innate immunity pathway, but the relevance of this interaction in the fly remains unresolved. Using a combination of genetics, immunoassays, and RNA sequencing, this study investigated a potential role for STING in the onset of parkin-null phenotypes. The findings demonstrate that loss of Drosophila STING in flies rescues the thorax muscle defects and the climbing ability of parkin-/- mutants. loss of STING also supresses the disrupted mitochondrial morphology in parkin-/- flight muscles, suggesting unexpected feedback of STING on mitochondria integrity or activation of a compensatory mitochondrial pathway. In the animals lacking both parkin and sting, PINK1 is activated and cell death pathways are surpressed. These findings support a unique, non-canonical role for Drosophila STING in the cellular and organismal response to mitochondria streβ.
Maurya, C. K. and Tapadia, M. G. (2023). Expanded polyQ aggregates interact with sarco-endoplasmic reticulum calcium ATPase and Drosophila inhibitor of apoptosis protein1 to regulate polyQ mediated neurodegeneration in Drosophila. Mol Cell Neurosci 126: 103886. PubMed ID: 37567489
Polyglutamine (polyQ) induced neurodegeneration is one of the leading causes of progreβive neurodegenerative disorders characterized clinically by deteriorating movement defects, psychiatric disability, and dementia. Calcium [Ca(2+)] homeostasis, which is essential for the functioning of neuronal cells, is disrupted under these pathological conditions. Huntington's disease phenotype was simulated in the neuronal cells of the Drosophila eye, and [Ca(2+)] pump, sarco-endoplasmic reticulum calcium ATPase (SERCA), was identified as one of the genetic modifiers of the neurodegenerative phenotype. This paper shows genetic and molecular interaction between polyglutamine (polyQ) aggregates, SERCA and DIAP1. Evidence is presented that polyQ aggregates interact with SERCA and alter its dynamics, resulting in a decrease in cytosolic [Ca(2+)] and an increase in ER [Ca(2+)], and thus toxicity. Downregulating SERCA lowers the enhanced calcium levels in the ER and rescues, morphological and functional defects caused due to expanded polyQ repeats. Cell proliferation markers such as Yorkie (Yki), Scalloped (Sd), and phosphatidylinositol 3 kinases/protein kinase B (PI3K/Akt), also respond to varying levels of calcium due to genetic manipulations, adding to the amelioration of degeneration. These results imply that neurodegeneration due to expanded polyQ repeats is sensitive to SERCA activity, and its manipulation can be an important step toward its therapeutic measures.
Migunova, E., Rajamani, S., Bonanni, S., Wang, F., Zhou, C. and Dubrovsky, E. B. (2023). Cardiac RNase Z edited via CRISPR-Cas9 drives heart hypertrophy in Drosophila. PLoS One 18(5): e0286214. PubMed ID: 37228086
Cardiomyopathy (CM) is a group of diseases distinguished by morphological and functional abnormalities in the myocardium. It is etiologically heterogeneous and may develop via cell autonomous and/or non-autonomous mechanisms. One of the most severe forms of CM has been linked to the deficiency of the ubiquitously expressed RNase Z endoribonuclease. RNase Z cleaves off the 3'-trailer of both nuclear and mitochondrial primary tRNA (pre-tRNA) transcripts. Cells mutant for RNase Z accumulate unprocessed pre-tRNA molecules. Patients carrying RNase Z variants with reduced enzymatic activity display a plethora of symptoms including muscular hypotonia, microcephaly and severe heart hypertrophy; still, they die primarily due to acute heart decompensation. Determining whether the underlying mechanism of heart malfunction is cell autonomous or not will provide an opportunity to develop novel strategies of more efficient treatments for these patients. This study used CRISPR-TRiM technology to create Drosophila models that carry cardiomyopathy-linked alleles of RNase Z only in the cardiomyocytes. This modification is sufficient for flies to develop heart hypertrophy and systolic dysfunction. These observations support the idea that the RNase Z linked CM is driven by cell autonomous mechanisms.
Mann, J. R., McKenna, E. D., Mawrie, D., Papakis, V., Aleβandrini, F., Anderson, E. N., Mayers, R., Ball, H. E., Kaspi, E., Lubinski, K., Baron, D. M., Tellez, L., Landers, J. E., Pandey, U. B. and Kiskinis, E. (2023). Loss of function of the ALS-associated NEK1 kinase disrupts microtubule homeostasis and nuclear import. Sci Adv 9(33): eadi5548. PubMed ID: 37585529
Loss-of-function variants in NIMA-related kinase 1 (NEK1) constitute a major genetic cause of amyotrophic lateral sclerosis (ALS), accounting for 2 to 3% of all cases. However, how NEK1 mutations cause motor neuron (MN) dysfunction is unknown. Using maβ spectrometry analyses for NEK1 interactors and NEK1-dependent expreβion changes, this study found functional enrichment for proteins involved in the microtubule cytoskeleton and nucleocytoplasmic transport. α-tubulin and importin-β1, two key proteins involved in these proceβes, are phosphorylated by NEK1 in vitro. NEK1 is eβential for motor control and survival in Drosophila models in vivo, while using several induced pluripotent stem cell (iPSC)-MN models, including NEK1 knockdown, kinase inhibition, and a patient mutation, evidence was foundfor disruptions in microtubule homeostasis and nuclear import. Notably, stabilizing microtubules with two distinct claβes of drugs restored NEK1-dependent deficits in both pathways. The capacity of NEK1 to modulate these proceβes that are critically involved in ALS pathophysiology renders this kinase a formidable therapeutic candidate.
Maor, G., Dubreuil, R. R. and Feany, M. B. (2023). alpha-synuclein promotes neuronal dysfunction and death by disrupting the binding of ankyrin to β-spectrin. bioRxiv. PubMed ID: 37333277
α-synuclein plays a key role in the pathogenesis of Parkinson's disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. This study shows that α-synuclein binds directly to β-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders this study demonstrated that β-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of β-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na (+) /K (+) ATPase, is mislocalized when human α-synuclein is expressed in Drosophila. Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. The same pathway was examined in human neurons, and it was found that Parkinson's disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na (+) /K (+) ATPase, and membrane potential depolarization. These findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson's disease and related α-synucleinopathies leads to neuronal dysfunction and death.

Friday, November 17th - Apoptosis and Autophagy

Metaxakis, A., Pavlidis, M. and Tavernarakis, N. (2023). Neuronal atg1 Coordinates Autophagy Induction and Physiological Adaptations to Balance mTORC1 Signalling. Cells 12(16). PubMed ID: 37626835
The mTORC1 nutrient-sensing pathway integrates metabolic and endocrine signals into the brain to evoke physiological responses to food deprivation, such as autophagy. Nevertheless, the impact of neuronal mTORC1 activity on neuronal circuits and organismal metabolism remains obscure. This study shows that mTORC1 inhibition acutely perturbs serotonergic neurotransmission via proteostatic alterations evoked by the autophagy inducer Atg1. Neuronal ATG1 alters the intracellular localization of the serotonin transporter, which increases the extracellular serotonin and stimulates the 5HTR7 postsynaptic receptor. 5HTR7 enhances food-searching behaviour and ecdysone-induced catabolism in Drosophila. Along similar lines, the pharmacological inhibition of mTORC1 in zebrafish also stimulates food-searching behaviour via serotonergic activity. These effects occur in parallel with neuronal autophagy induction, irrespective of the autophagic activity and the protein synthesis reduction. In addition, ectopic neuronal atg1 expression enhances catabolism via insulin pathway downregulation, impedes peptidergic secretion, and activates non-cell autonomous cAMP/PKA. The above exert diverse systemic effects on organismal metabolism, development, melanisation, and longevity. It is concluded that neuronal atg1 aligns neuronal autophagy induction with distinct physiological modulations, to orchestrate a coordinated physiological response against reduced mTORC1 activity.
Prasad, D., Illek, K., Fischer, F., Holstein, K. and Classen, A. K. (2023). Bilateral JNK activation is a hallmark of interface surveillance and promotes elimination of aberrant cells. Elife 12. PubMed ID: 36744859
Tissue-intrinsic defense mechanisms eliminate aberrant cells from epithelia and thereby maintain the health of developing tissues or adult organisms. 'Interface surveillance' comprises one such distinct mechanism that specifically guards against aberrant cells which undergo inappropriate cell fate and differentiation programs. The cellular mechanisms which facilitate detection and elimination of these aberrant cells are currently unknown. This study findd that in Drosophila imaginal discs, clones of cells with inappropriate activation of cell fate programs induce bilateral JNK activation at clonal interfaces, where wild type and aberrant cells make contact. JNK activation is required to drive apoptotic elimination of interface cells. Importantly, JNK activity and apoptosis are highest in interface cells within small aberrant clones, which likely supports the successful elimination of aberrant cells when they arise. These findings are consistent with a model where clone size affects the topology of interface contacts and thereby the strength of JNK activation in wild type and aberrant interface cells. Bilateral JNK activation is unique to 'interface surveillance' and is not observed in other tissue-intrinsic defense mechanisms, such as classical 'cell-cell competition'. Thus, bilateral JNK interface signaling provides an independent tissue-level mechanism to eliminate cells with inappropriate developmental fate but normal cellular fitness. Finally, oncogenic Ras-expressing clones activate 'interface surveillance' but evade elimination by bilateral JNK activation. Combined, this work establishes bilateral JNK interface signaling and interface apoptosis as a new hallmark of interface surveillance and highlights how oncogenic mutations evade tumor suppressor function encoded by this tissue-intrinsic surveillance system.
Li, Y., Zhang, W., Ye, Y., Sun, Y., Yang, L., Chen, G., Chen, K., Smith, S. and Zhou, J. (2023). Atg4b Overexpression Extends Lifespan and Healthspan in Drosophila melanogaster. Int J Mol Sci 24(12). PubMed ID: 37373039 ID:
Autophagy plays important but complex roles in aging, affecting health and longevity. In the general population, the levels of ATG4B and ATG4D were found to decrease during aging, yet they are upregulated in centenarians, suggesting that overexpression of ATG4 members could be positive for healthspan and lifespan. Therefore this study analyzed the effect of overexpressing Atg4b (a homolog of human ATG4D) in Drosophila and found that, indeed, Atg4b overexpression increased stress, desiccation stress and fitness as measured by climbing ability. The overexpression induced since mid-life increased lifespan. Transcriptome analysis of Drosophila subjected to desiccation stress revealed that Atg4b overexpression increased stress response pathways. In addition, overexpression of ATG4B delayed cellular senescence, and improved cell proliferation. These results suggest that ATG4B have contributed to a slowdown in cellular senescence, and in Drosophila, Atg4b overexpression may have led to improved healthspan and lifespan by promoting a stronger stress response. Overall, this study suggests that ATG4D and ATG4B have the potential to become targets for health and lifespan interventions.
Long, S., Cao, W., Qiu, Y., Deng, R., Liu, J., Zhang, L., Dong, R., Liu, F., Li, S., Zhao, H., Li, N. and Li, K. (2023). The appearance of cytoplasmic cytochrome C precedes apoptosis during Drosophila salivary gland degradation. Insect Sci. PubMed ID: 37370257
Apoptosis is an important process for organism development that functions to eliminate cell damage, maintain homeostasis, and remove obsolete tissues during morphogenesis. In mammals, apoptosis is accompanied by the release of cytochrome C (Cyt-c) from mitochondria to the cytoplasm. However, whether this process is conserved in the fruit fly, Drosophila melanogaster, remains controversial. This study discovered that during the degradation of Drosophila salivary gland, the transcription of mitochondria apoptosis factors (MAPFs), Cyt-c, and death-associated APAF1-related killer (Dark) encoding genes are all upregulated antecedent to initiator and effector caspases encoding genes. The proteins Cyt-c and the active caspase 3 appear gradually in the cytoplasm during salivary gland degradation. Meanwhile, the Cyt-c protein colocates with mito-GFP, the marker indicating cytoplasmic mitochondria, and the change in mitochondrial membrane potential coincides with the appearance of Cyt-c in the cytoplasm. Moreover, impeding or promoting 20E-induced transcription factor E93 suppresses or enhances the staining of Cyt-c and the active caspase 3 in the cytoplasm of salivary gland, and accordingly decreases or increases the mitochondrial membrane potential, respectively.This research provides evidence that cytoplasmic Cyt-c appears before apoptosis during Drosophila salivary gland degradation, shedding light on partial conserved mechanism in apoptosis between insects and mammals.
Bierlein, M., Charles, J., Polisuk-Balfour, T., Bretscher, H., Rice, M., Zvonar, J., Pohl, D., Winslow, L., Wasie, B., Deurloo, S., Van Wert, J., Williams, B., Ankney, G., Harmon, Z., Dann, E., Azuz, A., Guzman-Vargas, A., Kuhns, E., Neufeld, T. P., O'Connor, M. B., Amissah, F. and Zhu, C. C. (2023). Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster). Genetics. PubMed ID: 37594076
Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. This report shows that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies, but lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Over-expression of wild type Atg1 in adult fly muscle or adipocytes reduces lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. These research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation.
Kira, A., Tatsutomi, I., Saito, K., Murata, M., Hattori, I., Kajita, H., Muraki, N., Oda, Y., Satoh, S., Tsukamoto, Y., Kimura, S., Onoue, K., Yonemura, S., Arakawa, S., Kato, H., Hirashima, T. and Kawane, K. (2023). Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion. Dev Cell 58(14): 1282-1298. PubMed ID: 37315563
Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. This study reports a conserved execution mechanism of apoptotic cell extrusion. Extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells was found at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.

Thursday, November 16th - Adult neural structure, development, and function

Liu, J., Liu, W., Thakur, D., Mack, J., Spina, A. and Montell, C. (2023). Alleviation of thermal nociception depends on heat-sensitive neurons and a TRP channel in the brain. Curr Biol 33(12): 2397-2406. PubMed ID: 37201520
Acute avoidance of dangerous temperatures is critical for animals to prevent or minimize injury. Therefore, surface receptors have evolved to endow neurons with the capacity to detect noxious heat so that animals can initiate escape behaviors. Animals including humans have evolved intrinsic pain-suppressing systems to attenuate nociception under some circumstances. Using Drosophila melanogaster, this study uncovered a new mechanism through which thermal nociception is suppressed. A single descending neuron was identified in each brain hemisphere, which is the center for suppression of thermal nociception. These Epi neurons, for Epione-the goddess of soothing of pain-express a nociception-suppressing neuropeptide Allatostatin C (AstC), which is related to a mammalian anti-nociceptive peptide, somatostatin. Epi neurons are direct sensors for noxious heat, and when activated they release AstC, which diminishes nociception. Epi neurons also express the heat-activated TRP channel, Painless (Pain), and thermal activation of Epi neurons and the subsequent suppression of thermal nociception depend on Pain. Thus, while TRP channels are well known to sense noxious temperatures to promote avoidance behavior, this work reveals the first role for a TRP channel for detecting noxious temperatures for the purpose of suppressing rather than enhancing nociception behavior in response to hot thermal stimuli.
Megwa, O. F., Pascual, L. M., Gunay, C., Pulver, S. R. and Prinz, A. A. (2023). Temporal dynamics of Na/K pump mediated memory traces: insights from conductance-based models of Drosophila neurons. Front Neurosci 17: 1154549. PubMed ID: 37284663
Sodium potassium ATPases (Na/K pumps) mediate long-lasting, dynamic cellular memories that can last tens of seconds. The mechanisms controlling the dynamics of this type of cellular memory are not well understood and can be counterintuitive. This study used computational modeling to examine how Na/K pumps and the ion concentration dynamics they influence shape cellular excitability. In a Drosophila larval motor neuron model, a Na/K pump, a dynamic intracellular Na(+) concentration, and a dynamic Na(+) reversal potential were incorporated. Neuronal excitability was probed with a variety of stimuli, including step currents, ramp currents, and zap currents, then the sub- and suprathreshold voltage responses were monitored on a range of time scales. The interactions of a Na(+)-dependent pump current with a dynamic Na(+) concentration and reversal potential were found to endow the neuron with rich response properties that are absent when the role of the pump is reduced to the maintenance of constant ion concentration gradients. In particular, these dynamic pump-Na(+) interactions contribute to spike rate adaptation and result in long-lasting excitability changes after spiking and even after sub-threshold voltage fluctuations on multiple time scales. It was further shown that modulation of pump properties can profoundly alter a neuron's spontaneous activity and response to stimuli by providing a mechanism for bursting oscillations. This work has implications for experimental studies and computational modeling of the role of Na/K pumps in neuronal activity, information processing in neural circuits, and the neural control of animal behavior.
Lottes, E. N., Ciger, F. H., Bhattacharjee, S., Timmins-Wilde, E. A., Tete, B., Tran, T., Matta, J., Patel, A. A. and Cox, D. N. (2023). CCT and Cullin1 regulate the TORC1 pathway to promote dendritic arborization in health and disease. bioRxiv. PubMed ID: 37577581
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 homeostatic dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multidendritic 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 has confirmed that LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases dendritic complexity in CIV neurons. Both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus CIV dendritic development was examined in disease conditions as well. Expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by TORC1 disinhibition via Cullin1 LOF. Together, these data suggest that Cullin1 and CCT influence dendritic arborization through regulation of TORC1 in both health and disease.
Mamiya, A., Sustar, A., Siwanowicz, I., Qi, Y., Lu, T. C., Gurung, P., Chen, C., Phelps, J. S., Kuan, A. T., Pacureanu, A., Lee, W. A., Li, H., Mhatre, N. and Tuthill, J. C. (2023). Biomechanical origins of proprioceptor feature selectivity and topographic maps in the Drosophila leg. Neuron. PubMed ID: 37562405
The ability to sense and move one's body relies on proprioceptors, sensory neurons that detect mechanical forces within the body. Different subtypes of proprioceptors detect different kinematic features, such as joint position, movement, and vibration, but the mechanisms that underlie proprioceptor feature selectivity remain poorly understood. Using single-nucleus RNA sequencing (RNA-seq), this study found that proprioceptor subtypes in the Drosophila leg lack differential expression of mechanosensitive ion channels. However, anatomical reconstruction of the proprioceptors and connected tendons revealed major biomechanical differences between subtypes. A model was built of the proprioceptors and tendons that identified a biomechanical mechanism for joint angle selectivity and predicted the existence of a topographic map of joint angle, which was confirmed using calcium imaging. These findings suggest that biomechanical specialization is a key determinant of proprioceptor feature selectivity in Drosophila. More broadly, the discovery of proprioceptive maps reveals common organizational principles between proprioception and other topographically organized sensory systems.
Marques, G. S., Teles-Reis, J., Konstantinides, N., Brito, P. H. and Homem, C. C. F. (2023). Asynchronous transcription and translation of neurotransmitter-related genes characterize the initial stages of neuronal maturation in Drosophila. PLoS Biol 21(5): e3002115. PubMed ID: 37205703
Neuron specification and maturation are essential for proper central nervous system development. However, the precise mechanisms that govern neuronal maturation, essential to shape and maintain neuronal circuitry, remain poorly understood. This study analysed early-born secondary neurons in the Drosophila larval brain, revealing that the early maturation of secondary neurons goes through 3 consecutive phases: (1) Immediately after birth, neurons express pan-neuronal markers but do not transcribe terminal differentiation genes; (2) Transcription of terminal differentiation genes, such as neurotransmitter-related genes VGlut, ChAT, or Gad1, starts shortly after neuron birth, but these transcripts are, however, not translated; (3) Translation of neurotransmitter-related genes only begins several hours later in mid-pupa stages in a coordinated manner with animal developmental stage, albeit in an ecdysone-independent manner. These results support a model where temporal regulation of transcription and translation of neurotransmitter-related genes is an important mechanism to coordinate neuron maturation with brain development.
McMullen, E., Hertenstein, H., Strassburger, K., Deharde, L., Brankatschk, M. and Schirmeier, S. (2023). Glycolytically impaired Drosophila glial cells fuel neural metabolism via β-oxidation. Nat Commun 14(1): 2996. PubMed ID: 37225684
Neuronal function is highly energy demanding and thus requires efficient and constant metabolite delivery by glia. Drosophila glia are highly glycolytic and provide lactate to fuel neuronal metabolism. Flies are able to survive for several weeks in the absence of glial glycolysis. This study examined how Drosophila glial cells maintain sufficient nutrient supply to neurons under conditions of impaired glycolysis. Glycolytically impaired glia were shown to rely on mitochondrial fatty acid breakdown and ketone body production to nourish neurons, suggesting that ketone bodies serve as an alternate neuronal fuel to prevent neurodegeneration. In times of long-term starvation, glial degradation of absorbed fatty acids is essential to ensure survival of the fly. Further, it was shown that Drosophila glial cells act as a metabolic sensor and can induce mobilization of peripheral lipid stores to preserve brain metabolic homeostasis. This study gives evidence of the importance of glial fatty acid degradation for brain function, and survival, under adverse conditions in Drosophila.

Wednesday, November 15th - Enzymes and Proteins - Evolution, Structure and Function

Lee, G. G., Zeng, K., Duffy, C. M., Sriharsha, Y., Yoo, S. and Park, J. H. (2023). In vivo characterization of the maturation steps of PDF neuropeptide precursor in the Drosophila circadian pacemaker neurons. Genetics. PubMed ID: 37364299
Pigment Dispersing Factor (PDF), is a key signaling molecule coordinating the neuronal network associated with the circadian rhythms in Drosophila. The precursor (proPDF) of the mature PDF (mPDF) consists of two motifs, a larger PDF-associated peptide (PAP) and PDF. Through cleavage and amidation, the proPDF is predicted to produce cleaved-PAP (cPAP) and mPDF. To delve into the in vivo mechanisms underlying proPDF maturation, various mutations were generated that eliminate putative processing sites, and the effect of each mutation on the production of cPAP and mPDF was then analyzed by four different antibodies in both ectopic and endogenous conditions. The knockdown effects of processing enzymes on the proPDF maturation was also studied. At the functional level, circadian phenotypes were measured for all mutants and knockdown lines. As results, the roles of key enzymes and their target residues was confirmed: Amontillado (Amon) for the cleavage at the consensus dibasic KR site, Silver (Svr) for the removal of C-terminal basic residues from the intermediates, PAP-KR and PDF-GK, derived from proPDF, and PHM (Peptidylglycine-α-hydroxylating monooxygenase) for the amidation of PDF. The results suggest that the C-terminal amidation occurs independently of proPDF cleavage. Moreover, PAP domain is important for the proPDF trafficking into the secretory vesicles and a close association between cPAP and mPDF following cleavage seems required for their stability within the vesicles. These studies highlight the biological significance of individual processing steps and the roles of the PAP for the stability and function of mPDF which is essential for the circadian clockworks.
Mallik, B., Brusich, D. J., Heyrman, G. and Frank, C. A. (2023). Precise mapping of one classic and three novel GluRIIA mutants in Drosophila melanogaster. MicroPubl Biol 2023. PubMed ID: 37334199
Mutation of the Drosophila melanogaster GluRIIA gene or pharmacological agents targeting it are commonly used to assess homeostatic synaptic function at the larval neuromuscular junction (NMJ). The commonly used mutation, GluRIIA (SP16), is a null allele created by a large and imprecise excision of a P-element which affects GluRIIA and multiple upstream genes. This study mapped the exact bounds of the GluRIIA (SP16) allele, refined a multiplex PCR strategy for positive identification of GluRIIA (SP16) in homozygous or heterozygous backgrounds, and sequenced and characterized three new CRISPR-generated GluRIIA mutants. The three new GluRIIA alleles are apparent nulls that lack GluRIIA immunofluorescence signal at the 3 (rd) instar larval NMJ and are predicted to cause premature truncations at the genetic level. Further, these new mutants have similar electrophysiological outcomes as GluRIIA (SP16), including reduced miniature excitatory postsynaptic potential (mEPSP) amplitude and frequency compared to controls, and they express robust homeostatic compensation as evidenced by normal excitatory postsynaptic potential (EPSP) amplitude and elevated quantal content. These findings and new tools extend the capacity of the D. melanogaster NMJ for assessment of synaptic function.
Li, J., Cao, Y., Yang, Y., Ma, H., Zhao, J., Zhang, Y. and Liu, N. (2023). Quantitative Acetylomics Reveals Substrates of Lysine Acetyltransferase GCN5 in Adult and Aging Drosophila. J Proteome Res. PubMed ID: 37545086

Protein lysine acetylation is a dynamic post-translational modification (PTM) that regulates a wide spectrum of cellular events including aging. General control nonderepressible 5 (GCN5) is a highly conserved lysine acetyltransferase (KAT). However, the acetylation substrates of GCN5 in vivo remain poorly studied, and moreover, how lysine acetylation changes with age and the contribution of KATs to aging remain to be addressed. Useing Drosophila, this study performed label-free quantitative acetylomic analysis, identifying new substrates of GCN5 in the adult and aging process. The dynamics were further characterize of protein acetylation with age, that exhibits a trend of increase. Since the expression of endogenous fly Gcn5 progressively increases during aging, it was reason that, by combining the substrate analysis, the increase in acetylation with age is triggered, at least in part, by GCN5. Collectively, this study substantially expands the atlas of GCN5 substrates in vivo, provides a resource of protein acetylation that naturally occurs with age, and demonstrates how individual KAT contributes to the aging acetylome.

Kara, E., McCambridge, A., Proffer, M., Dilts, C., Pumnea, B., Eshak, J., Smith, K. A., Fielder, I., Doyle, D. A., Ortega, B. M., Mukatash, Y., Malik, N., Mohammed, A. R., Govani, D., Niepielko, M. G. and Gao, M. (2023). Mutational analysis of the functional motifs of the DEAD-box RNA helicase Me31B/DDX6 in Drosophila germline development. FEBS Lett 597(14): 1848-1867. PubMed ID: 37235728
Me31B/DDX6 is a DEAD-box family RNA helicase playing roles in post-transcriptional RNA regulation in different cell types and species. Despite the known motifs/domains of Me31B, the in vivo functions of the motifs remain unclear. This study used the Drosophila germline as a model and used CRISPR to mutate the key Me31B motifs/domains: helicase domain, N-terminal domain, C-terminal domain and FDF-binding motif. Then, screening characterization was performed on the mutants, and the effects of the mutations on the Drosophila germline, on processes such as fertility, oogenesis, embryo patterning, germline mRNA regulation and Me31B protein expression were reported. The study indicates that the Me31B motifs contribute different functions to the protein and are needed for proper germline development, providing insights into the in vivo working mechanism of the helicase.
Kubíkova, J., Ubartaite, G., Metz, J. and Jeske, M. (2023). Structural basis for binding of Drosophila Smaug to the GPCR Smoothened and to the germline inducer Oskar. Proc Natl Acad Sci U S A 120(32): e2304385120. PubMed ID: 37523566
Drosophila Smaug and its orthologs comprise a family of mRNA repressor proteins. Smaug proteins contain a characteristic RNA-binding sterile-α motif (SAM) domain and a conserved but uncharacterized N-terminal domain (NTD). This study resolved the crystal structure of the NTD of the human SAM domain-containing protein 4A (SAMD4A, a.k.a. Smaug1) to 1.6 Å resolution, which revealed its composition of a homodimerization D subdomain and a subdomain with similarity to a pseudo-HEAT-repeat analogous topology (PHAT) domain. Drosophila Smaug directly interacts with the Drosophila germline inducer Oskar and with the Hedgehog signaling transducer Smoothened through its NTD. The crystal structure of the NTD of Smaug was determined in complex with a Smoothened α-helical peptide to 2.0 Å: resolution. The peptide binds within a groove that is formed by both the D and PHAT subdomains. Structural modeling supported by experimental data suggested that an α-helix within the disordered region of Oskar binds to the NTD of Smaug in a mode similar to Smoothened. Together, these data uncover the NTD of Smaug as a peptide-binding domain.
Levdansky, Y., Raisch, T., Deme, J. C., Pekovic, F., Elmlund, H., Lea, S. M. and Valkov, E. (2023). Structure and assembly of the NOT10:11 module of the CCR4-NOT complex. Commun Biol 6(1): 739. PubMed ID: 37460791
NOT1, NOT10, and NOT11 form a conserved module in the CCR4-NOT complex, critical for post-transcriptional regulation in eukaryotes, but how this module contributes to the functions of the CCR4-NOT remains poorly understood. This study presents cryo-EM structures of human and chicken NOT1:NOT10:NOT11 ternary complexes to sub-3 Å resolution, revealing an evolutionarily conserved, flexible structure. Through biochemical dissection studies, which include the Drosophila orthologs, we show that the module assembly is hierarchical, with NOT11 binding to NOT10, which then organizes it for binding to NOT1. A short proline-rich motif in NOT11 stabilizes the entire module assembly.

Tuesday, November 14th - Disease Models

Lin, Y. C., Zhang, M., Chang, Y. J. and Kuo, T. H. (2023). Comparisons of lifespan and stress resistance between sexes in Drosophila melanogaster. Heliyon 9(8): e18178. PubMed ID: 37576293
Animals exhibit different extents of sexual dimorphism in a variety of phenotypes. Sex differences in longevity, one of the most complex life history traits, have also been reported. Although lifespan regulation has been studied extensively in the fruit fly, Drosophila melanogaster, the sex differences in lifespan have not been consistent in previous studies. To explore this issue, this question was revisited by examining the lifespan and stress resistance of both sexes among 15 inbred strains. Positive correlations was found between males and females from the same strain in terms of lifespan and resistance to starvation and desiccation stress. Although the lifespan difference between male and female flies varied greatly depending on the strain, males across all strains collectively had a longer lifespan. In contrast, females showed better resistance to starvation and desiccation stress. Greater variation in lifespan and resistance to starvation and desiccation stress was observed in females. Unexpectedly, there was no notable correlation observed between lifespan and the three types of stress resistance in either males or females. Overall, this study provides new data regarding sexual dimorphism in fly lifespan and stress resistance; this information may promote the investigation of mechanisms underlying longevity in future research.
Lo Piccolo, L., Umegawachi, T., Yeewa, R., Potikanond, S., Nimlamool, W., Prachayasittikul, V., Gotoh, Y., Yoshida, H., Yamaguchi, M. and Jantrapirom, S. (2023). A Novel Drosophila-based Drug Repurposing Platform Identified Fingolimod As a Potential Therapeutic for TDP-43 Proteinopathy. Neurotherapeutics. PubMed ID: 37493896
Pathogenic changes to TAR DNA-binding protein 43 (TDP-43) leading to alteration of its homeostasis are a common feature shared by several progressive neurodegenerative diseases for which there is no effective therapy. Here, we developed Drosophila lines expressing either wild type TDP-43 (WT) or that carrying an Amyotrophic Lateral Sclerosis/Frontotemporal Lobar Degeneration-associating G384C mutation that recapitulate several aspects of the TDP-43 pathology. To identify potential therapeutics for TDP-43-related diseases, a drug repurposing strategy was utilized that involved three consecutive steps. Firstly, the improvement of eclosion rate was evaluated, followed by the assessment of locomotive functions at early and late developmental stages. Through this approach, fingolimod, was successfully identified as a promising candidate for modulating TDP-43 toxicity. Fingolimod exhibited several beneficial effects in both WT and mutant models of TDP-43 pathology, including post-transcriptional reduction of TDP-43 levels, rescue of pupal lethality, and improvement of locomotor dysfunctions. These findings provide compelling evidence for the therapeutic potential of fingolimod in addressing TDP-43 pathology, thereby strengthening the rationale for further investigation and consideration of clinical trials. Furthermore, this study demonstrates the utility of a Drosophila-based screening pipeline in identifying novel therapeutics for TDP-43-related diseases. These findings encourage further scale-up screening endeavors using this platform to discover additional compounds with therapeutic potential for TDP-43 pathology.
Lovato, T. L., Blotz, B., Bileckyj, C., Johnston, C. A. and Cripps, R. M. (2023). Using Drosophila to model a variant of unknown significance in the human cardiogenic gene Nkx2.5. bioRxiv. PubMed ID: 37425758
Sequencing of human genome samples has unearthed genetic variants for which functional testing is necessary to validate their clinical significance. This study used the Drosophila system to analyze a variant of unknown significance in the human congenital heart disease gene, Nkx2.5. An R321N allele of the Nkx2.5 ortholog tinman (tin) was generated to model a human K158N variant and tested its function in vitro and in vivo. The R321N Tin isoform bound poorly to DNA in vitro and was deficient in activating a Tin-dependent enhancer in tissue culture. Mutant Tin also showed a significantly reduced interaction with a Drosophila Tbox cardiac factor named Dorsocross1. A tin (R321N) allele was generated using CRISPR/Cas9, for which homozygotes were viable and had normal heart specification, but showed defects in the differentiation of the adult heart that were exacerbated by further loss of tin function. It is concluded that the human K158N mutation is likely pathogenic through causing both a deficiency in DNA binding and a reduced ability to interact with a cardiac cofactor, and that cardiac defects might arise later in development or adult life.
Liu, Y., Dantas, E., Ferrer, M., Liu, Y., Comjean, A., Davidson, E. E., Hu, Y., Goncalves, M. D., Janowitz, T. and Perrimon, N. (2023). Tumor Cytokine-Induced Hepatic Gluconeogenesis Contributes to Cancer Cachexia: Insights from Full Body Single Nuclei Sequencing. bioRxiv. PubMed ID: 37292804
A primary cause of death in cancer patients is cachexia, a wasting syndrome attributed to tumor-induced metabolic dysregulation. Despite the major impact of cachexia on the treatment, quality of life, and survival of cancer patients, relatively little is known about the underlying pathogenic mechanisms. Hyperglycemia detected in glucose tolerance test is one of the earliest metabolic abnormalities observed in cancer patients; however, the pathogenesis by which tumors influence blood sugar levels remains poorly understood. In this study, utilizing a Drosophila model, it was demonstrated that the tumor secreted interleukin-like cytokine Upd3 induces fat body expression of Pepck1 and Pdk, two key regulatory enzymes of gluconeogenesis, contributing to hyperglycemia. These data further indicate a conserved regulation of these genes by IL-6/JAK STAT signaling in mouse models. Importantly, in both fly and mouse cancer cachexia models, elevated gluconeogenesis gene levels are associated with poor prognosis. Altogether, this study uncovers a conserved role of Upd3/IL-6/JAK-STAT signaling in inducing tumor-associated hyperglycemia, which provides insights into the pathogenesis of IL-6 signaling in cancer cachexia.
Lu, T. C., Brbic, M., Park, Y. J., Jackson, T., Chen, J., Kolluru, S. S., Qi, Y., Katheder, N. S., Cai, X. T., Lee, S., Chen, Y. C., Auld, N., Liang, C. Y., Ding, S. H., Welsch, D., D'Souza, S., Pisco, A. O., Jones, R. C., Leskovec, J., Lai, E. C., Bellen, H. J., Luo, L., Jasper, H., Quake, S. R. and Li, H. (2023). Aging Fly Cell Atlas identifies exhaustive aging features at cellular resolution. Science 380(6650): eadg0934. PubMed ID: 37319212

Aging is characterized by a decline in tissue function, but the underlying changes at cellular resolution across the organism remain unclear. This study presents the Aging Fly Cell Atlas, a single-nucleus transcriptomic map of the whole aging Drosophila. 163 distinct cell types were characterized, and an in-depth analysis was performed of changes in tissue cell composition, gene expression, and cell identities. Aging clock models were developed to predict fly age, and ribosomal gene expression was found to be a conserved predictive factor for age. Combining all aging features, distinctive cell type-specific aging patterns were found. This atlas provides a valuable resource for studying fundamental principles of aging in complex organisms.

Malik, I., Tseng, Y. J., Wieland, C. M., Green, K. M., Zheng, K., Calleja, K. and Todd, P. K. (2023). Dissecting the roles of EIF4G homologs reveals DAP5 as a modifier of CGG repeat-associated toxicity in a Drosophila model of FXTAS. Neurobiol Dis 184: 106212. PubMed ID: 37352983
Neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS) is caused by a CGG trinucleotide repeat expansion in the 5' UTR of FMR1. Expanded CGG repeat RNAs form stable secondary structures, which in turn support repeat-associated non-AUG (RAN) translation to produce toxic peptides. The parameters that impact RAN translation initiation efficiency are not well understood. This study used a Drosophila melanogaster model of FXTAS to evaluate the role of the eIF4G family of eukaryotic translation initiation factors (EIF4G1, EIF4GII and EIF4G2/DAP5) in modulating RAN translation and CGG repeat-associated toxicity. DAP5 knockdown robustly suppressed CGG repeat-associated toxicity and inhibited RAN translation. Furthermore, knockdown of initiation factors that preferentially associate with DAP5 (such as E , EIF3F and EIF3G) also selectively suppressed CGG repeat-induced eye degeneration. In mammalian cellular reporter assays, DAP5 knockdown exhibited modest and cell-type specific effects on RAN translation. Taken together, these data support a role for DAP5 in CGG repeat associated toxicity possibly through modulation of RAN translation.

Monday, November 13th - Adult Physiology and Metabolism

Kumar, M., Has, C., Lam-Kamath, K., Ayciriex, S., Dewett, D., Bashir, M., Poupault, C., Schuhmann, K., Knittelfelder, O., Raghuraman, B. K., Ahrends, R., Rister, J. and Shevchenko, A. (2023). Lipidome unsaturation affects the morphology and proteome of the Drosophila eye. bioRxiv. PubMed ID: 37214967
While the proteome of an organism is largely determined by the genome, the lipidome is shaped by a poorly understood interplay of environmental factors and metabolic processes. To gain insights into the underlying mechanisms, this study analyzed the impacts of dietary lipid manipulations on the ocular proteome of Drosophila melanogaster. The lipidome was manipulated with synthetic food media that differed in the supplementation of an equal amount of saturated or polyunsaturated triacylglycerols. This allowed generation of flies whose eyes had a highly contrasting length and unsaturation of glycerophospholipids, the major lipid class of biological membranes, while the abundance of other membrane lipid classes remained unchanged. By bioinformatically comparing the resulting ocular proteomic trends and contrasting them with the impacts of vitamin A deficiency, ocular proteins were identified whose abundances are differentially affected by lipid saturation and unsaturation. For instance, a group of proteins was unexpectedly identified that have muscle-related functions and increase their abundances in the eye upon lipidome unsaturation but are unaffected by lipidome saturation. Moreover, two differentially lipid-responsive proteins involved in stress responses were identified, Turandot A and Smg5, whose abundances decrease with lipid unsaturation. Lastly, it was discovered that the ocular lipid class composition is robust to dietary changes, and it is proposed that this may be a general homeostatic feature of the organization of eukaryotic tissues, while the length and unsaturation of fatty acid moieties is more variable to compensate environmental challenges. It is anticipated that these insights into the molecular responses of the Drosophila eye proteome to specific lipid manipulations will guide the genetic dissection of the mechanisms that maintain visual function when the eye is exposed to dietary challenges.
Kilwein, M. D., Dao, T. K. and Welte, M. A. (2023). Drosophila embryos allocate lipid droplets to specific lineages to ensure punctual development and redox homeostasis. PLoS Genet 19(8): e1010875. PubMed ID: 37578970
Lipid droplets (LDs) are ubiquitous organelles that facilitate neutral lipid storage in cells, including energy-dense triglycerides. They are found in all investigated metazoan embryos where they are thought to provide energy for development. Intriguingly, early embryos of diverse metazoan species asymmetrically allocate LDs amongst cellular lineages, a process which can involve massive intracellular redistribution of LDs. However, the biological reason for asymmetric lineage allocation is unknown. To address this issue, we utilize the Drosophila embryo where the cytoskeletal mechanisms that drive allocation are well characterized. We disrupt allocation by two different means: Loss of the LD protein Jabba results in LDs adhering inappropriately to glycogen granules; loss of Klar alters the activities of the microtubule motors that move LDs. Both mutants cause the same dramatic change in LD tissue inheritance, shifting allocation of the majority of LDs to the yolk cell instead of the incipient epithelium. Embryos with such mislocalized LDs do not fully consume their LDs and are delayed in hatching. Through use of a dPLIN2 mutant, which appropriately localizes a smaller pool of LDs, we find that failed LD transport and a smaller LD pool affect embryogenesis in a similar manner. Embryos of all three mutants display overlapping changes in their transcriptome and proteome, suggesting that lipid deprivation results in a shared embryonic response and a widespread change in metabolism. Excitingly, we find abundant changes related to redox homeostasis, with many proteins related to glutathione metabolism upregulated. LD deprived embryos have an increase in peroxidized lipids and rely on increased utilization of glutathione-related proteins for survival. Thus, embryos are apparently able to mount a beneficial response upon lipid stress, rewiring their metabolism to survive. In summary, we demonstrate that early embryos allocate LDs into specific lineages for subsequent optimal utilization, thus protecting against oxidative stress and ensuring punctual development.
Li, Y., Zhou, X., Cheng, C., Ding, G., Zhao, P., Tan, K., Chen, L., Perrimon, N., Veenstra, J. A., Zhang, L. and Song, W. (2023). Gut AstA mediates sleep deprivation-induced energy wasting in Drosophila. Cell Discov 9(1): 49. PubMed ID: 37221172
Severe sleep deprivation (SD) has been highly associated with systemic energy wasting, such as lipid loss and glycogen depletion. Despite immune dysregulation and neurotoxicity observed in SD animals, whether and how the gut-secreted hormones participate in SD-induced disruption of energy homeostasis remains largely unknown. Using Drosophila as a conserved model organism, this study characterize that production of intestinal Allatostatin A (AstA), a major gut-peptide hormone, is robustly increased in adult flies bearing severe SD. Interestingly, the removal of AstA production in the gut using specific drivers significantly improves lipid loss and glycogen depletion in SD flies without affecting sleep homeostasis. The molecular mechanisms were revealed whereby gut AstA promotes the release of an adipokinetic hormone (Akh), an insulin counter-regulatory hormone functionally equivalent to mammalian glucagon, to mobilize systemic energy reserves by remotely targeting its receptor AstA-R2 in Akh-producing cells. Similar regulation of glucagon secretion and energy wasting by AstA/galanin is also observed in SD mice. Further, integrating single-cell RNA sequencing and genetic validation, this study uncovered that severe SD results in ROS accumulation in the gut to augment AstA production via TrpA1. Altogether, these results demonstrate the essential roles of the gut-peptide hormone AstA in mediating SD-associated energy wasting.
Luo, J., Sun, A., Yu, Y., Pei, Y., Zuo, Y. and Hu, Z. (2023). Periplocoside P affects synaptic transmission at the neuromuscular junction and reduces synaptic excitability in Drosophila melanogaster by inhibiting V-ATPase. Pest Manag Sci. PubMed ID: 37556562
Periplocoside P (PSP) is a major component of Periploca sepium Bunge known for its potent insecticidal activity. V-Type adenosine triphosphatase (V-ATPase), which is widely distributed in the cytoplasmic membranes and organelles of eukaryotic cells, plays a crucial role in synaptic excitability conduction. Previous research has shown that PSP targets the apical membrane of goblet cells in the insect midgut. However, the effects of PSP on synaptic transmission at the neuromuscular junction are often overlooked.The bioassay revealed that Drosophila adults with different genetic backgrounds showed varying levels of susceptibility to PSP in the order: parats1  > parats1 ;DSC1-/-  ≊ w1118  > DSC1-/-. Intracellular electrode recording demonstrated that PSP, similar to bafilomycin A1, had an impact on the amplitude of the excitatory junction potential (EJP) and accelerated excitability decay. Furthermore, the alteration in EJP amplitude is concentration-dependent. Another surprising discovery was that the knockout DSC1 channel showed insensitivity to PSP. These findings confirm that PSP can influence synaptic transmission at the neuromuscular junction of Drosophila larvae by targeting V-ATPase. These results provide a basis for investigating the mechanism of action of PSP and its potential application in designing novel insecticides.
Luersen, K., Jockel, T., Chin, D., Demetrowitsch, T., Schwarz, K. and Rimbach, G. (2023). Reduced iron and cobalt levels in response to curcumin supplementation are not responsible for the prolonged larval development and do not affect the oxidative stress tolerance and polyamine status of D. melanogaster. Biofactors. PubMed ID: 37597249
Recent reports indicated that the phytochemical curcumin possesses iron-chelating activity. By employing the fruit fly Drosophila melanogaster, this study conducted feeding studies supplementing curcumin or, as a control, the iron chelator bathophenanthroline (BPA). First, the absorption and further metabolization of dietary curcuminoids were proved by metabolomics analyses. Next, it was found that 0.2% dietary curcumin, similar to BPA, lowered the iron but also the cobalt content, and to a lesser extent affected the manganese and zinc status. Supplementation during larval stages was required and sufficient for both compounds to elicit these alterations in adult animals. However, curcumin-induced retarded larval development was not attributable to the changed trace metal status. In addition, a reduction in the iron content of up to 70% by curcumin or BPA supplementation did not reduce heme-dependent catalase activity and tolerance toward H(2) O(2) in D. melanogaster. Moreover, polyamines were not influenced by curcumin treatment and decreased iron levels. This was confirmed for selected organs from 0.2% curcumin-treated mice, except for the spleen. Here, elevated spermidine level and concomitant upregulation of genes involved in polyamine production were associated with a putatively anemia-derived increased spleen mass. These data underline that the metal-chelating property of curcumin needs to be considered in feeding studies.
Humphreys, J. M., Teixeira, L. R., Akella, R., He, H., Kannangara, A. R., Sekulski, K., Pleinis, J., Liwocha, J., Jiou, J., Servage, K. A., Orth, K., Joachimiak, L., Rizo, J., Cobb, M. H., Brautigam, C. A., Rodan, A. R. and Goldsmith, E. J. (2023). Hydrostatic Pressure Sensing by WNK kinases. Mol Biol Cell: mbcE23030113. PubMed ID: 37585288
Previous work has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N(2) gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N(2) gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

Friday, November 10th - Signaling

Katarachia, S. A., Markaki, S. P., Velentzas, A. D. and Stravopodis, D. J. (2023). Genetic Targeting of dSAMTOR. A Negative dTORC1 Regulator, during Drosophila Aging: A Tissue-Specific Pathology. Int J Mol Sci 24(11). PubMed ID: 37298625
mTORC1 regulates mammalian cell metabolism and growth in response to diverse environmental stimuli. Nutrient signals control the localization of mTORC1 onto lysosome surface scaffolds that are critically implicated in its amino acid-dependent activation. Arginine, leucine and S-adenosyl-methionine (SAM) can serve as major mTORC1-signaling activators, with SAM binding to SAMTOR (SAM + TOR), a fundamental SAM sensor, preventing the protein's (SAMTOR's) inhibitory action(s) against mTORC1, thereby triggering its (mTORC1) kinase activity. Given the lack of knowledge regarding the role of SAMTOR in invertebrates, this study has identified the Drosophila SAMTOR homologue (dSAMTOR) in silico and have genetically targeted it through the utilization of the GAL4/UAS transgenic tool. Survival profiles and negative geotaxis patterns were examined in both control and dSAMTOR-downregulated adult flies during aging. One of the two gene-targeted schemes resulted in lethal phenotypes, whereas the other one caused rather moderate pathologies in most tissues. The screening of head-specific kinase activities, via PamGene technology application, unveiled the significant upregulation of several kinases, including the dTORC1 characteristic substrate dp70S6K, in dSAMTOR-downregulated flies, thus strongly supporting the inhibitory dSAMTOR action(s) upon the dTORC1/dp70S6K signaling axis in Drosophila brain settings. Importantly, genetic targeting of the Drosophila BHMT bioinformatics counterpart (dBHMT), an enzyme that catabolizes betaine to produce methionine (the SAM precursor), led to severe compromises in terms of fly longevity, with glia-, motor neuron- and muscle-specific dBHMT downregulations exhibiting the strongest effects. Abnormalities in wing vein architectures were also detected in dBHMT-targeted flies, thereby justifying their notably reduced negative geotaxis capacities herein observed mainly in the brain-(mid)gut axis. In vivo adult fly exposure to clinically relevant doses of methionine revealed the mechanistic synergism of decreased dSAMTOR and increased methionine levels in pathogenic longevity, thus rendering (d)SAMTOR an important component in methionine-associated disorders, including homocystinuria(s).
Jaiswal, J., Egert, J., Engesser, R., Peyrotón, A. A., Nogay, L., Weichselberger, V., Crucianelli, C., Grass, I., Kreutz, C., Timmer, J. and Classen, A. K. (2023). Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration. PLoS Biol 21(5): e3001665. PubMed ID: 37252939
Epithelial repair relies on the activation of stress signaling pathways to coordinate tissue repair. Their deregulation is implicated in chronic wound and cancer pathologies. Using TNF-α/Eiger-mediated inflammatory damage to Drosophila imaginal discs, this study investigate how spatial patterns of signaling pathways and repair behaviors arise. Eiger expression, which drives JNK/AP-1 signaling, was found to transiently arrest proliferation of cells in the wound center and is associated with activation of a senescence program. This includes production of the mitogenic ligands of the Upd family, which allows JNK/AP-1-signaling cells to act as paracrine organizers of regeneration. Surprisingly, JNK/AP-1 cell-autonomously suppress activation of Upd signaling via Ptp61F and Socs36E, both negative regulators of JAK/STAT signaling. As mitogenic JAK/STAT signaling is suppressed in JNK/AP-1-signaling cells at the center of tissue damage, compensatory proliferation occurs by paracrine activation of JAK/STAT in the wound periphery. Mathematical modelling suggests that cell-autonomous mutual repression between JNK/AP-1 and JAK/STAT is at the core of a regulatory network essential to spatially separate JNK/AP-1 and JAK/STAT signaling into bistable spatial domains associated with distinct cellular tasks. Such spatial stratification is essential for proper tissue repair, as coactivation of JNK/AP-1 and JAK/STAT in the same cells creates conflicting signals for cell cycle progression, leading to excess apoptosis of senescently stalled JNK/AP-1-signaling cells that organize the spatial field. Finally, this study demonstrated that bistable separation of JNK/AP-1 and JAK/STAT drives bistable separation of senescent signaling and proliferative behaviors not only upon tissue damage, but also in RasV12, scrib tumors. Revealing this previously uncharacterized regulatory network between JNK/AP-1, JAK/STAT, and associated cell behaviors has important implications for conceptual understanding of tissue repair, chronic wound pathologies, and tumor microenvironments.
Loudhaief, R., Jneid, R., Christensen, C. F., Mackay, D. J., Andersen, D. S. and Colombani, J. (2023). The Drosophila tumor necrosis factor receptor, Wengen, couples energy expenditure with gut immunity. Sci Adv 9(23): eadd4977. PubMed ID: 37294765
It is well established that tumor necrosis factor (TNF) plays an instrumental role in orchestrating the metabolic disorders associated with late stages of cancers. However, it is not clear whether TNF/TNF receptor (TNFR) signaling controls energy homeostasis in healthy individuals. This study shows that the highly conserved Drosophila TNFR, Wengen (Wgn), is required in the enterocytes (ECs) of the adult gut to restrict lipid catabolism, suppress immune activity, and maintain tissue homeostasis. Wgn limits autophagy-dependent lipolysis by restricting cytoplasmic levels of the TNFR effector, TNFR-associated factor 3 (dTRAF3), while it suppresses immune processes through inhibition of the dTAK1/TAK1-Relish/NF-κB pathway in a dTRAF2-dependent manner. Knocking down dTRAF3 or overexpressing dTRAF2 is sufficient to suppress infection-induced lipid depletion and immune activation, respectively, showing that Wgn/TNFR functions as an intersection between metabolism and immunity allowing pathogen-induced metabolic reprogramming to fuel the energetically costly task of combatting an infection.
Liu, G. Y., Jouandin, P., Bahng, R. E., Perrimon, N. and Sabatini, D. M. (2023). An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway. bioRxiv. PubMed ID: 37292894
Animals must sense and respond to nutrient availability in their local niche. This task is coordinated in part by the mTOR complex 1 (mTORC1) pathway, which regulates growth and metabolism in response to nutrients. In mammals, mTORC1 senses specific amino acids through specialized sensors that act through the upstream GATOR1/2 signaling hub. To reconcile the conserved architecture of the mTORC1 pathway with the diversity of environments that animals can occupy, this study hypothesized that the pathway might maintain plasticity by evolving distinct nutrient sensors in different metazoan phyla. This study identified the Drosophila protein Unmet expectations (Unmet, as a species-restricted nutrient sensor and traced its incorporation into the mTORC1 pathway. Upon methionine starvation, Unmet binds to the fly GATOR2 complex to inhibit dTORC1. S -adenosylmethionine (SAM), a proxy for methionine availability, directly relieves this inhibition. Unmet expression is elevated in the ovary, a methionine-sensitive niche, and flies lacking Unmet fail to maintain the integrity of the female germline under methionine restriction. By monitoring the evolutionary history of the Unmet-GATOR2 interaction, this study shows that the GATOR2 complex evolved rapidly in Dipterans to recruit and repurpose an independent methyltransferase as a SAM sensor. Thus, the modular architecture of the mTORC1 pathway allows it to co-opt preexisting enzymes and expand its nutrient sensing capabilities, revealing a mechanism for conferring evolvability on an otherwise highly conserved system.
Jiang, Z., Qu, L., Cui, G. and Zhong, G. (2023). Smoothened antagonist sonidegib affects the development of D. melanogaster larvae via suppression of epidermis formation. Pestic Biochem Physiol 194: 105462. PubMed ID: 37532307
Hedgehog (Hh) signaling is essential for the regulation of embryonic growth and development, the maintenance of stem cell autostasis, and tissue formation, whether in vertebrates or invertebrates. However, exploration into the Hh pathway antagonists in Drosophila or other pests of agricultural importance has been scant. In order to gain a better understanding of the potential utility of the antagonists in insect investigations, a conventional Hh antagonist, sonidegib, was used to evaluate the effects on the development of Drosophila larvae. The results showed that early instar larvae exposed to sonidegib exhibited new epidermal abnormalities and decreased motility after molting. Transcriptome analysis revealed that Sonidegib had a profound effect on chitin-based cuticle development throughout all stages of larvae. Physiological experiments revealed that sonidegib suppressed the epidermis formation and decreased the chitin content. The results of this study shed new light on the potential use of Hh antagonists in agricultural pest management.
Lockyer, J., Reading, A., Vicenzi, S., Delandre, C., Marshall, O., Gasperini, R., Foa, L. and Lin, J. Y. (2023). Optogenetic inhibition of Gα signalling alters and regulates circuit functionality and early circuit formation. bioRxiv. PubMed ID: 37214843
Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. This work develop and validate a new optogenetic tool that disrupt Gαq signaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach, P hoto- i nduced M odulation of G α protein - I nhibition of G&slphs; q (PiGM-Iq), exhibited potent and selective inhibition of Gα q signaling. The behavior of C. elegans and Drosophila was altered with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, it was also shown that this approach is amenable to Gαi signaling. Wednesday, November 8th - Synapse and Vesicles
Jusyte, M., Blaum, N., Bohme, M. A., Berns, M. M. M., Bonard, A. E., Vámosi Á, B., Pushpalatha, K. V., Kobbersmed, J. R. L. and Walter, A. M. (2023). Unc13A dynamically stabilizes vesicle priming at synaptic release sites for short-term facilitation and homeostatic potentiation. Cell Rep 42(6): 112541. PubMed ID: 37243591
Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic homeostatic potentiation (PHP) of NT release stabilizes transmission over minutes. Despite different timescales of STF and PHP, analysis of Drosophila neuromuscular junctions reveals functional overlap and shared molecular dependence on the release-site protein Unc13A. Mutating Unc13A's calmodulin binding domain (CaM-domain) increases baseline transmission while blocking STF and PHP. Mathematical modeling suggests that Ca(2+)/calmodulin/Unc13A interaction plastically stabilizes vesicle priming at release sites and that CaM-domain mutation causes constitutive stabilization, thereby blocking plasticity. Labeling the functionally essential Unc13A MUN domain reveals higher STED microscopy signals closer to release sites following CaM-domain mutation. Acute phorbol ester treatment similarly enhances NT release and blocks STF/PHP in synapses expressing wild-type Unc13A, while CaM-domain mutation occludes this, indicating common downstream effects. Thus, Unc13A regulatory domains integrate signals across timescales to switch release-site participation for synaptic plasticity.
Lewis, S. A., Bakhtiari, S., Forstrom, J., Bayat, A., Bilan, F., Le Guyader, G., Alkhunaizi, E., Vernon, H., Padilla-Lopez, S. R. and Kruer, M. C. (2023). AGAP1-associated endolysosomal trafficking abnormalities link gene-environment interactions in a neurodevelopmental disorder. Dis Model Mech. PubMed ID: 37470098
AGAP1 is an Arf1 GAP that regulates endolysosomal trafficking. Damaging variants have been linked to cerebral palsy and autism. This study reports three new individuals with microdeletion variants in AGAP1. Affected individuals have intellectual disability (3/3), autism (3/3), dystonia with axial hypotonia (1/3), abnormalities of brain maturation (1/3), growth impairment (2/3) and facial dysmorphism (2/3). Mechanisms potentially underlying AGAP1 neurodevelopmental impairments were investigated using the Drosophila ortholog, CenG1a. Reduced axon terminal size, increased neuronal endosome abundance, and elevated autophagy were discovered at baseline. Given potential incomplete penetrance, gene-environment interactions were assessed. Basal elevation was found in phosphorylation of the integrated stress-response protein eIF2α and inability to further increase eIF2α-P with subsequent cytotoxic stressors. CenG1a-mutant flies have increased lethality from exposure to environmental insults. A model is proposed wherein disruption of AGAP1 function impairs endolysosomal trafficking, chronically activating the integrated stress response, and leaving AGAP1-deficient cells susceptible to a variety of second hit cytotoxic stressors. This model may have broader applicability beyond AGAP1 in instances where both genetic and environmental insults co-occur in individuals with neurodevelopmental disorders.
Lee, P. H., Anaya, M., Ladinsky, M. S., Reitsma, J. M. and Zinn, K. (2023). An extracellular vesicle targeting ligand that binds to Arc proteins and facilitates Arc transport in vivo. Elife 12. PubMed ID: 37326306
Communication between distant cells can be mediated by extracellular vesicles (EVs) that deliver proteins and RNAs to recipient cells. Little is known about how EVs are targeted to specific cell types. This study identified the Drosophila cell-surface protein Stranded at second (Sas) as a targeting ligand for EVs. Full-length Sas is present in EV preparations from transfected Drosophila Schneider 2 (S2) cells. Sas is a binding partner for the Ptp10D receptor tyrosine phosphatase, and Sas-bearing EVs preferentially target to cells expressing Ptp10D. Co-immunoprecipitation and peptide binding to show that the cytoplasmic domain (ICD) of Sas binds to dArc1 and mammalian Arc. dArc1 and Arc are related to retrotransposon Gag proteins. They form virus-like capsids which encapsulate Arc and other mRNAs and are transported between cells via EVs. The Sas ICD contains a motif required for dArc1 binding that is shared by the mammalian and Drosophila amyloid precursor protein (APP) orthologs, and the APP ICD also binds to mammalian Arc. Sas facilitates delivery of dArc1 capsids bearing dArc1 mRNA into distant Ptp10D-expressing recipient cells in vivo.
Jetti, S. K., Crane, A. B., Akbergenova, Y., Aponte-Santiago, N. A., Cunningham, K. L., Whittaker, C. A. and Littleton, J. T. (2023). Molecular logic of synaptic diversity between Drosophila tonic and phasic motoneurons. Neuron. PubMed ID: 37611584
Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features are poorly understood. To identify molecular pathways that contribute to synaptic diversity, single-neuron Patch-seq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated that synaptic active zones in phasic motoneurons are more compact and display enhanced Ca(2+) influx compared with their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications, and intracellular Ca(2+) buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.
Kozlov, E. N., Deev, R. V., Tokmatcheva, E. V., Tvorogova, A., Kachaev, Z. M., Gilmutdinov, R. A., Zhukova, M., Savvateeva-Popova, E. V., Schedl, P. and Shidlovskii, Y. V. (2023). 3'UTR of mRNA Encoding CPEB Protein Orb2 Plays an Essential Role in Intracellular Transport in Neurons. Cells 12(13). PubMed ID: 37443751
Intracellular trafficking plays a critical role in the functioning of highly polarized cells, such as neurons. Transport of mRNAs, proteins, and other molecules to synaptic terminals maintains contact between neurons and ensures the transmission of nerve impulses. Cytoplasmic polyadenylation element binding (CPEB) proteins play an essential role in long-term memory (LTM) formation by regulating local translation in synapses. This study shows that the 3'UTR of the Drosophila CPEB gene orb2 is required for targeting the orb2 mRNA and protein to synapses and that this localization is important for LTM formation. When the orb2 3'UTR is deleted, the orb2 mRNAs and proteins fail to localize in synaptic fractions, and pronounced LTM deficits arise.WYhe phenotypic effects of the orb2 3'UTR deletion were rescued by introducing the 3'UTR from the orb, another Drosophila CPEB gene. In contrast, the phenotypic effects of the 3'UTR deletion were not rescued by the 3'UTR from one of the Drosophila α-tubulin genes. These results show that the orb2 mRNAs must be targeted to the correct locations in neurons and that proper targeting depends upon sequences in the 3'UTR.
Liu, H., Shao, W., Liu, W., Shang, W., Liu, J. P., Wang, L. and Tong, C. (2023). PtdIns4P exchange at endoplasmic reticulum-autolysosome contacts is essential for autophagy and neuronal homeostasis. Autophagy: 1-20. PubMed ID: 37289040
Inter-organelle contacts enable crosstalk among organelles, facilitating the exchange of materials and coordination of cellular events. This study demonstrated that, upon starvation, autolysosomes recruit Pi4KIIα (Phosphatidylinositol 4-kinase II α) to generate phosphatidylinositol-4-phosphate (PtdIns4P) on their surface and establish endoplasmic reticulum (ER)-autolysosome contacts through PtdIns4P binding proteins Osbp (Oxysterol binding protein) and cert (ceramide transfer protein). Sac1 (Sac1 phosphatase), Osbp, and cert proteins are required for the reduction of PtdIns4P on autolysosomes. Loss of any of these proteins leads to defective macroautophagy/autophagy and neurodegeneration. Osbp, cert, and Sac1 are required for ER-Golgi contacts in fed cells. These data establishes a new mode of organelle contact formation - the ER-Golgi contact machinery can be reused by ER-autolysosome contacts by re-locating PtdIns4P from the Golgi apparatus to autolysosomes when faced withstarvation.

Tuesday, November 7th - Development, Structure and Function of the Adult Brain

Ketkar, M. D., Shao, S., Gjorgjieva, J. and Silies, M. (2023). Multifaceted luminance gain control beyond photoreceptors in Drosophila. Curr Biol 33(13): 2632-2645. PubMed ID: 37285845
Animals navigating in natural environments must handle vast changes in their sensory input. Visual systems, for example, handle changes in luminance at many timescales, from slow changes across the day to rapid changes during active behavior. To maintain luminance-invariant perception, visual systems must adapt their sensitivity to changing luminance at different timescales. This study demonstrates that luminance gain control in photoreceptors alone is insufficient to explain luminance invariance at both fast and slow timescales and reveal the algorithms that adjust gain past photoreceptors in the fly eye. Imaging and behavioral experiments were combined with computational modeling to show that downstream of photoreceptors, circuitry taking input from the single luminance-sensitive neuron type L3 implements gain control at fast and slow timescales. This computation is bidirectional in that it prevents the underestimation of contrasts in low luminance and overestimation in high luminance. An algorithmic model disentangles these multifaceted contributions and shows that the bidirectional gain control occurs at both timescales. The model implements a nonlinear interaction of luminance and contrast to achieve gain correction at fast timescales and a dark-sensitive channel to improve the detection of dim stimuli at slow timescales. Together, this work demonstrates how a single neuronal channel performs diverse computations to implement gain control at multiple timescales that are together important for navigation in natural environments.
Lesser, E., Azevedo, A. W., Phelps, J. S., Elabbady, L., Cook, A., 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., Seung, H. S., Macrina, T., Dickinson, M., Lee, W. A. and Tuthill, J. C. (2023). Synaptic architecture of leg and wing motor control networks in Drosophila. bioRxiv. PubMed ID: 37398440
Animal movement is controlled by motor neurons (MNs), which project out of the central nervous system to activate muscles. Because individual muscles may be used in many different behaviors, MN activity must be flexibly coordinated by dedicated premotor circuitry, the organization of which remains largely unknown. This study used comprehensive reconstruction of neuron anatomy and synaptic connectivity from volumetric electron microscopy (i.e., connectomics) to analyze the wiring logic of motor circuits controlling the Drosophila leg and wing. Both leg and wing premotor networks were found to be organized into modules that link MNs innervating muscles with related functions. However, the connectivity patterns within leg and wing motor modules are distinct. Leg premotor neurons exhibit proportional gradients of synaptic input onto MNs within each module, revealing a novel circuit basis for hierarchical MN recruitment. In comparison, wing premotor neurons lack proportional synaptic connectivity, which may allow muscles to be recruited in different combinations or with different relative timing. By comparing the architecture of distinct limb motor control systems within the same animal, this study identified common principles of premotor network organization and specializations that reflect the unique biomechanical constraints and evolutionary origins of leg and wing motor control.
Kunin, A. B., Guo, J., Bassler, K. E., Pitkow, X. and Josic, K. (2023). Hierarchical Modular Structure of the Drosophila Connectome. J Neurosci. PubMed ID: 37591738
This study applied community detection methods to analyze the synapse-level reconstruction of an adult female Drosophila brain containing over 20 thousand neurons and 10 million synapses. Using a machine-learning algorithm, the most densely connected communities of neurons were found by maximizing a generalized modularity density measure. The community structure was resolved at a range of scales, from large (on the order of thousands of neurons) to small (on the order of tens of neurons). The network was found to be is organized hierarchically and larger-scale communities are composed of smaller-scale structures. These methods identify well-known features of the fly brain, including its sensory pathways. Moreover, focusing on specific brain regions, it was possible to identify subnetworks with distinct connectivity types, including the fan-shaped body and the superior neuropil, with distinct clusters of upstream and downstream brain regions dividing the neuropil into several pathways. These methods show that the fine-scale, local network reconstruction made possible by modern experimental methods are sufficiently detailed to identify the organization of the brain across scales, and enable novel predictions about the structure and function of its parts.
Kurogi, Y., Imura, E., Mizuno, Y., Hoshino, R., Nouzova, M., Matsuyama, S., Mizoguchi, A., Kondo, S., Tanimoto, H., Noriega, F. G. and Niwa, R. (2023). Female reproductive dormancy in Drosophila is regulated by DH31-producing neurons projecting into the corpus allatum. Development 150(10). PubMed ID: 37218457
Female insects can enter reproductive diapause, a state of suspended egg development, to conserve energy under adverse environments. In many insects, including the fruit fly, Drosophila melanogaster, reproductive diapause, also frequently called reproductive dormancy, is induced under low-temperature and short-day conditions by the downregulation of juvenile hormone (JH) biosynthesis in the corpus allatum (CA). This study demonstrates that neuropeptide Diuretic hormone 31 (DH31) produced by brain neurons that project into the CA plays an essential role in regulating reproductive dormancy by suppressing JH biosynthesis in adult D. melanogaster. The CA expresses the gene encoding the DH31 receptor, which is required for DH31-triggered elevation of intracellular cAMP in the CA. Knocking down Dh31 in these CA-projecting neurons or DH31 receptor in the CA suppresses the decrease of JH titer, normally observed under dormancy-inducing conditions, leading to abnormal yolk accumulation in the ovaries. These findings provide the first molecular genetic evidence demonstrating that CA-projecting peptidergic neurons play an essential role in regulating reproductive dormancy by suppressing JH biosynthesis.
Li, K., Tsukasa, Y., Kurio, M., Maeta, K., Tsumadori, A., Baba, S., Nishimura, R., Murakami, A., Onodera, K., Morimoto, T., Uemura, T. and Usui, T. (2023). Belly roll, a GPI-anchored Ly6 protein, regulates Drosophila melanogaster escape behaviors by modulating the excitability of nociceptive peptidergic interneurons. Elife 12. PubMed ID: 37309249
Appropriate modulation of escape behaviors in response to potentially damaging stimuli is essential for survival. Although nociceptive circuitry has been studied, it is poorly understood how genetic contexts affect relevant escape responses. Using an unbiased genome-wide association analysis, this study identified an Ly6/α-neurotoxin family protein, Belly roll (Bero), which negatively regulates Drosophila nociceptive escape behavior. Bero is expressed in abdominal leucokinin-producing neurons (ABLK neurons) and bero knockdown in ABLK neurons resulted in enhanced escape behavior. Furthermore, it was demonstrated that ABLK neurons responded to activation of nociceptors and initiated the behavior. Notably, bero knockdown reduced persistent neuronal activity and increased evoked nociceptive responses in ABLK neurons. These findings reveal that Bero modulates an escape response by regulating distinct neuronal activities in ABLK neurons.
Lin, A., Yang, R., Dorkenwald, S., Matsliah, A., Sterling, A. R., Schlegel, P., Yu, S. C., McKellar, C. E., Costa, M., Eichler, K., Bates, A. S., Eckstein, N., Funke, J., Jefferis, G. and Murthy, M. (2023). Network Statistics of the Whole-Brain Connectome of Drosophila. bioRxiv. PubMed ID: 37547019
Animal brains are complex organs composed of thousands of interconnected neurons. Characterizing the network properties of these brains is a requisite step towards understanding mechanisms of computation and information flow. With the completion of the Flywire project, access is available to the connectome of a complete adult Drosophila brain, containing 130,000 neurons and millions of connections. This study presents a statistical summary and data products of the Flywire connectome, delving into its network properties and topological features. To gain insights into local connectivity, the prevalence of two- and three-node network motifs was computed, their strengths and neurotransmitter compositions were examined, and these topological metrics were compared with wiring diagrams of other animals. A population of highly connected neurons was uncovered known as the "rich club" and identified subsets of neurons that may serve as integrators or broadcasters of signals. Finally, subnetworks were examined based on 78 anatomically defined brain regions. The freely available data and neuron populations presented in this study will serve as a foundation for models and experiments exploring the relationship between neural activity and anatomical structure.

Monday November 6th - Disease Models

Li, Y., Chang, P., Sankaran, S., Jang, H., Nie, Y., Zeng, A., Hussain, S., Wu, J. Y., Chen, X. and Shi, L. (2023). Bioorthogonal Stimulated Raman Scattering Imaging Uncovers Lipid Metabolic Dynamics in Drosophila Brain During Aging. GEN Biotechnol 2(3): 247-261. PubMed ID: 37363411
Studies have shown that brain lipid metabolism is associated with biological aging and influenced by dietary and genetic manipulations; however, the underlying mechanisms are elusive. High-resolution imaging techniques propose a novel and potent approach to understanding lipid metabolic dynamics in situ. Applying deuterium water (D(2)O) probing with stimulated Raman scattering (DO-SRS) microscopy, it was revealed that lipid metabolic activity in Drosophila brain decreased with aging in a sex-dependent manner. Female flies showed an earlier occurrence of lipid turnover decrease than males. Dietary restriction (DR) and downregulation of insulin/IGF-1 signaling (IIS) pathway, two scenarios for lifespan extension, led to significant enhancements of brain lipid turnover in old flies. Combining SRS imaging with deuterated bioorthogonal probes (deuterated glucose and deuterated acetate), it was discovered that, under DR treatment and downregulation of IIS pathway, brain metabolism shifted to use acetate as a major carbon source for lipid synthesis. For the first time, this study directly visualizes and quantifies spatiotemporal alterations of lipid turnover in Drosophila brain at the single organelle (lipid droplet) level. This study not only demonstrates a new approach for studying brain lipid metabolic activity in situ but also illuminates the interconnection of aging, dietary, and genetic manipulations on brain lipid metabolic regulation.
Li, Q., Wang, L., Tang, C., Wang, X., Yu, Z., Ping, X., Ding, M. and Zheng, L. (2023). Adipose Tissue Exosome circ_sxc Mediates the Modulatory of Adiposomes on Brain Aging by Inhibiting Brain dme-miR-87-3p. Mol Neurobiol. PubMed ID: 37597108
Aging of the brain usually leads to the decline of neurological processes. Adipose tissue exosomes, as adipocyte-derived vesicles, may mediate the regulatory processes of adipose tissue on other organs, including the brain; however, the regulatory mechanisms remain unclear. This study analyzed the sleep-wake behavior of young (10 days) and old (40 days) Drosophila and found that older Drosophila showed increased sleep fragmentation, which is similar to mammalian aging characteristics. To investigate the cross-tissue regulatory mechanisms of adiposity on brain aging, 10-day and 40-day Drosophila adipose tissue exosomes were isolated and circRNAs were identified with age-dependent expression differences by RNA-seq and differential analysis. Furthermore, by combining data from 3 datasets of the GEO database, circ_sxc that was significantly downregulated with age was finally screened out. Moreover, dme-miR-87-3p, a conserved target of circ_sxc, accumulates in the brain with age and exhibits inhibitory effects in predicted binding relationships with neuroreceptor ligand genes. In summary, the current study showed that the Drosophila brain could obtain circ_sxc by uptake of adipose tissue exosomes which crossed the blood-brain barrier. And circ_sxc suppressed brain miR-87-3p expression through sponge adsorption, which in turn regulated the expression of neurological receptor ligand proteins (5-HT1B, GABA-B-R1, Rdl, Rh7, qvr, NaCP60E) and ensured brain neuronal synaptic signaling normal function of synaptic signaling. However, with aging, this regulatory mechanism is dysregulated by the downregulation of the adipose exosome circ_sxc, which contributes to the brain exhibiting sleep disturbances and other "aging" features.
Landis, J. E., Sungu, K., Sipe, H. and Copeland, J. M. (2023). RNAi of Complex I and V of the electron transport chain in glutamate neurons extends life span, increases sleep, and decreases locomotor activity in Drosophila melanogaster. PLoS One 18(6): e0286828. PubMed ID: 37319260
RNAi targeting the electron transport chain has been proven to prolong life span in many different species, and experiments specifically with Drosophila melanogaster and Caenorhabditis elegans have shown a distinct role for neurons. To determine which subset of neurons is implicated in this life span extension, the GAL4/UAS system was used to activate RNAi against genes of Complex I and Complex V. Life span extension of 18-24% with two glutamate neuron (D42 and VGlut) GAL4 lines. We used the GAL80 system to determine if the overlapping set of glutamate neurons in these two GAL4 lines imparts the life span extension. Limiting GAL4 activity to non-VGlut glutamate neurons in the D42 background failed to extend life span, suggesting that glutamate neurons have an important role in aging. Interestingly, RNAi of the electron transport chain in D42 glutamate neurons also caused an increase in daytime and nighttime sleep and a decrease in nighttime locomotor activity. Changes to sleep patterns and prolonged life span were not accompanied by any changes in female fertility or response to starvation. These findings demonstrate that a small subset of neurons can control life span, and further studies can look into the contributions made by glutamate neurons.
Li, Y., Chen, W. and Wang, D. (2023). Promotion of mitochondrial fragmentation suppresses the formation of mitochondrial spherical compartmentation in PINK1(B9)Drosophila melanogaster. Biochem Biophys Res Commun 676: 48-57. PubMed ID: 37481943
Mitochondria undergo structural changes reflective of functional statuses. Ultrastructural characterizing of mitochondria is valuable for understanding mitochondrial dysfunction in various pathological conditions. PINK1, a Parkinson's disease (PD) associated gene, plays key roles in maintaining mitochondrial function and integrity. In Drosophila melanogaster, deficiency of PINK1 results in PD-like pathologies due to mitochondrial abnormalities. This study reports the existence of a new type of mitochondrial-membrane deformity, mitochondrial spherical compartmentation (MSC), caused by PINK1 deficiency in Drosophila. The MSC is a three-dimensional spheroid-like mitochondrial membrane structure encompassing nonselective contents. Upregulation of dDrp1, downregulation of dMarf, and upregulation of dArgK1-A-all resulting in mitochondrial fragmentation-were able to suppress the formation of MSC. Furthermore, arginine kinase, only when localizing to the vicinity of mitochondria, induced mitochondrial fragmentation and reversed the MSC phenotype. In summary, this study demonstrates that loss of dPINK1 leads to the formation of mitochondrial-membrane deformity MSC, which responds to mitochondrial dynamics. In addition, these data suggest a new perspective of how phosphagen energy-buffer system might regulate mitochondrial dynamics.
Li, M., Luo, S., Li, Y., Li, Y., Ma, B., Liu, F., Wang, H., Guo, J. and Ling, L. (2023). Dyclonine relieves the Parkinson's disease progression in rotenone-induced Drosophila model. Behav Brain Res 452: 114561. PubMed ID: 37394123
It has been estimated that there will be 930 million Parkinson's disease (PD) patients in 2030 in the whole world. However, no therapy has been effective for PD until now. Only levodopa is the available primary drug for the treatment of motor symptoms. Therefore, it is an urgent task to develop new drugs to inhibit the progression of PD and improve the quality of the patient's life. Dyclonine which was found to have antioxidant activity and would benefit patients with Friedreich's ataxia, is a commonly used local anesthetic. This study reports that dyclonine improved the motor ability and loss of dopaminergic neurons in the rotenone-induced Drosophila PD model for the first time. Furthermore, dyclonine upregulated the Nrf2/HO pathway, decreased the ROS and MDA levels, and inhibited the apoptosis of neurons in the brain of PD model flies. Hence, dyclonine might be an attractive FDA-approved drug for the exploration of effective PD therapy.
Li, Y., Xu, S., Wang, L., Shi, H., Wang, H., Fang, Z., Hu, Y., Jin, J., Du, Y., Deng, M., Wang, L. and Zhu, Z. (2023). Gut microbial genetic variation modulates host lifespan, sleep, and motor performance. Isme j. PubMed ID: 37550381
Recent studies have shown that gut microorganisms can modulate host lifespan and activities, including sleep quality and motor performance. However, the role of gut microbial genetic variation in regulating host phenotypes remains unclear. This study investigated the links between gut microbial genetic variation and host phenotypes using Saccharomyces cerevisiae and Drosophila melanogaster as research models. The results suggested a novel role for peroxisome-related genes in yeast in regulating host lifespan and activities by modulating gut oxidative stress. Specifically, it was found that deficiency in catalase A (CTA1) in yeast reduced both the sleep duration and lifespan of fruit flies significantly. Furthermore, this research also expanded understanding of the relationship between sleep and longevity. Using a large sample size and excluding individual genetic background differences, this study found that lifespan is associated with sleep duration, but not sleep fragmentation or motor performance. Overall, this study provides novel insights into the role of gut microbial genetic variation in regulating host phenotypes and offers potential new avenues for improving health and longevity.

Friday, November 3rd - Gonads

Hu, Q., Xiao, Y., Wei, R., Tang, T., Wen, L., Lu, Y. and Yu, X. Q. (2023). Identification and functional analysis of CG3526 in spermatogenesis of Drosophila melanogaster. Insect Sci. PubMed ID: 37465843
Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. This study identified a testis-specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C(2) H(2) type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin-protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587-Gal4 > CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, it was found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92EF06346 mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, these results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila.
Li, P., Messina, G. and Lehner, C. F. (2023). Nuclear elongation during spermiogenesis depends on physical linkage of nuclear pore complexes to bundled microtubules by Drosophila Mst27D. PLoS Genet 19(7): e1010837. PubMed ID: 37428798
Spermatozoa in animal species are usually highly elongated cells with a long motile tail attached to a head that contains the haploid genome in a compact and often elongated nucleus. In Drosophila melanogaster, the nucleus is compacted two hundred-fold in volume during spermiogenesis and re-modeled into a needle that is thirty-fold longer than its diameter. Nuclear elongation is preceded by a striking relocalization of nuclear pore complexes (NPCs). While NPCs are initially located throughout the nuclear envelope (NE) around the spherical nucleus of early round spermatids, they are later confined to one hemisphere. In the cytoplasm adjacent to this NPC-containing NE, the so-called dense complex with a strong bundle of microtubules is assembled. Functional characterization of the spermatid specific Mst27D protein now resolves this deficit. Mst27D establishes physical linkage between NPC-NE and dense complex. The C-terminal region of Mst27D binds to the nuclear pore protein Nup358. The N-terminal CH domain of Mst27D, which is similar to that of EB1 family proteins, binds to microtubules. At high expression levels, Mst27D promotes bundling of microtubules in cultured cells. Microscopic analyses indicated co-localization of Mst27D with Nup358 and with the microtubule bundles of the dense complex. Time-lapse imaging revealed that nuclear elongation is accompanied by a progressive bundling of microtubules into a single elongated bundle. In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal. Thus, it is proposed that Mst27D permits normal nuclear elongation by promoting the attachment of the NPC-NE to the microtubules of the dense complex, as well as the progressive bundling of these microtubules.
Gabbert, A. M., Campanale, J. P., Mondo, J. A., Mitchell, N. P., Myers, A., Streichan, S. J., Miolane, N. and Montell, D. J. (2023). Septins regulate border cell surface geometry, shape, and motility downstream of Rho in Drosophila. Dev Cell 58(15): 1399-1413.e1395. PubMed ID: 37329886
Septins (see Drosophila Peanut) self-assemble into polymers that bind and deform membranes in vitro and regulate diverse cell behaviors in vivo. How their in vitro properties relate to their in vivo functions is under active investigation. This study uncovered requirements for septins in detachment and motility of border cell clusters in the Drosophila ovary. Septins and myosin colocalize dynamically at the cluster periphery and share phenotypes but, surprisingly, do not impact each other. Instead, Rho independently regulates myosin activity and septin localization. Active Rho recruits septins to membranes, whereas inactive Rho sequesters septins in the cytoplasm. Mathematical analyses identify how manipulating septin expression levels alters cluster surface texture and shape. This study shows that the level of septin expression differentially regulates surface properties at different scales. This work suggests that downstream of Rho, septins tune surface deformability while myosin controls contractility, the combination of which governs cluster shape and movement.
Galletta, B. J., Varadarajan, R., Fagerstrom, C. J., Yang, B., Haase, K. P., McJunkin, K. and Rusan, N. M. (2023). The E3 ligase Poe promotes Pericentrin degradation. Mol Biol Cell 34(9): br15. PubMed ID: 37342879
Centrosomes are essential parts of diverse cellular processes, and precise regulation of the levels of their constituent proteins is critical for their function. One such protein is Pericentrin (PCNT) in humans and Pericentrin-like protein (PLP) in Drosophila. Increased PCNT expression and its protein accumulation are linked to clinical conditions including cancer, mental disorders, and ciliopathies. However, the mechanisms by which PCNT levels are regulated remain underexplored. A previous study demonstrated that PLP levels are sharply down-regulated during early spermatogenesis and this regulation is essential to spatially position PLP on the proximal end of centrioles. It was hypothesized that the sharp drop in PLP protein was a result of rapid protein degradation during the male germ line premeiotic G2 phase. This study shows that PLP is subject to ubiquitin-mediated degradation and identified multiple proteins that promote the reduction of PLP levels in spermatocytes, including the UBR box containing E3 ligase Poe (UBR4), which was shown to bind to PLP. Although protein sequences governing posttranslational regulation of PLP are not restricted to a single region of the protein, this study identified a region that is required for Poe-mediated degradation. Experimentally stabilizing PLP, via internal PLP deletions or loss of Poe, leads to PLP accumulation in spermatocytes, its mispositioning along centrioles, and defects in centriole docking in spermatids.
Giedt, M. S., Thomalla, J. M., White, R. P., Johnson, M. R., Lai, Z. W., Tootle, T. L. and Welte, M. A. (2023). Adipose triglyceride lipase promotes prostaglandin-dependent actin remodeling by regulating substrate release from lipid droplets. Development 150(20). PubMed ID: 37306387
Lipid droplets (LDs), crucial regulators of lipid metabolism, accumulate during oocyte development. However, their roles in fertility remain largely unknown. During Drosophila oogenesis, LD accumulation coincides with the actin remodeling necessary for follicle development. Loss of the LD-associated Adipose Triglyceride Lipase (ATGL) disrupts both actin bundle formation and cortical actin integrity, an unusual phenotype also seen when the prostaglandin (PG) synthase Pxt is missing. Dominant genetic interactions and PG treatment of follicles indicate that ATGL acts upstream of Pxt to regulate actin remodeling. The data suggest that ATGL releases arachidonic acid (AA) from LDs to serve as the substrate for PG synthesis. Lipidomic analysis detects AA-containing triglycerides in ovaries, and these are increased when ATGL is lost. High levels of exogenous AA block follicle development; this is enhanced by impairing LD formation and suppressed by reducing ATGL. Together, these data support the model that AA stored in LD triglycerides is released by ATGL to drive the production of PGs, which promote the actin remodeling necessary for follicle development. It is speculated that this pathway is conserved across organisms to regulate oocyte development and promote fertility.
Li, A. Y. Z., Di, Y., Rathore, S., Chiang, A. C., Jezek, J. and Ma, H. (2023). Milton assembles large mitochondrial clusters, mitoballs, to sustain spermatogenesis. Proc Natl Acad Sci U S A 120(34): e2306073120. PubMed ID: 37579146
Mitochondria are dynamic organelles that undergo frequent remodeling to accommodate developmental needs. This study describes a striking organization of mitochondria into a large ball-like structure adjacent to the nucleus in premeiotic Drosophila melanogaster spermatocytes, which we term "mitoball". Mitoballs are transient structures that colocalize with the endoplasmic reticulum, Golgi bodies, and the fusome. Similar premeiotic mitochondrial clusters were observed in a wide range of insect species, including mosquitos and cockroaches. Through a genetic screen, Milton, an adaptor protein that links mitochondria to microtubule-based motors, mediates mitoball formation. Flies lacking a 54 amino acid region in the C terminus of Milton completely lacked mitoballs, had swollen mitochondria in their spermatocytes, and showed reduced male fertility. It is suggested that the premeiotic mitochondrial clustering is a conserved feature of insect spermatogenesis that supports sperm development.

Thursday, November 2nd - Enhancers and Transcriptional Regulation

Mau, C., Rudolf, H., Strobl, F., Schmid, B., Regensburger, T., Palmisano, R., Stelzer, E. H. K., Taher, L. and El-Sherif, E. (2023). How enhancers regulate wavelike gene expression patterns. Elife 12. PubMed ID: 37432987
A key problem in development is to understand how genes turn on or off at the right place and right time during embryogenesis. Such decisions are made by non-coding sequences called 'enhancers.' Many models of how enhancers work rely on the assumption that genes are activated de novo as stable domains across embryonic tissues. Such a view has been strengthened by the intensive landmark studies of the early patterning of the anterior-posterior (AP) axis of the Drosophila embryo, where indeed gene expression domains seem to arise more or less stably. However, careful analysis of gene expression patterns in other model systems (including the AP patterning in vertebrates and short-germ insects like the beetle Tribolium castaneum) painted a different, very dynamic view of gene regulation, where genes are oftentimes expressed in a wavelike fashion. How such gene expression waves are mediated at the enhancer level is so far unclear. This study establish the AP patterning of the short-germ beetle Tribolium as a model system to study dynamic and temporal pattern formation at the enhancer level. To that end, an enhancer prediction system was estalished in Tribolium based on time- and tissue-specific ATAC-seq and an enhancer live reporter system based on MS2 tagging. Using this experimental framework, several Tribolium enhancers were discovered, and the spatiotemporal activities of some of them in live embryos was assessed assessed. The data was found to be consistent with a model in which the timing of gene expression during embryonic pattern formation is mediated by a balancing act between enhancers that induce rapid changes in gene expression patterns (that are called 'dynamic enhancers') and enhancers that stabilize gene expression patterns (that are called 'static enhancers'). However, more data is needed for a strong support for this or any other alternative models.
Krasnov, A. N., Evdokimova, A. A., Mazina, M. Y., Erokhin, M., Chetverina, D. and Vorobyeva, N. E. (2023). Coregulators Reside within Drosophila Ecdysone-Inducible Loci before and after Ecdysone Treatment. Int J Mol Sci 24(14). PubMed ID: 37511602
Ecdysone signaling in Drosophila remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind ecdysone-response elements with or without the presence of a hormone. For years, EcR enhancers were thought to respond to ecdysone via recruiting coactivator complexes, which replace corepressors and stimulate transcription. However, the exact mechanism of transcription activation by ecdysone remains unclear. This study presents experimental data on 11 various coregulators at ecdysone-responsive loci of Drosophila S2 cells. The regulatory elements are described where coregulators reside within these loci, and changes in their binding levels following 20-hydroxyecdysone treatment were assessed. In the current study, the presence was detected of some coregulators at the TSSs (active and inactive) and boundaries marked with CP190 rather than enhancers of the ecdysone-responsive loci where EcR binds. Minor changes were observed in the coregulators' binding level. Most were present at inducible loci before and after 20-hydroxyecdysone treatment. These findings suggest that: (1) coregulators can activate a particular TSS operating from some distal region (which could be an enhancer, boundary regulatory region, or inactive TSS); (2) coregulators are not recruited after 20-hydroxyecdysone treatment to the responsive loci; rather, their functional activity changes (shown as an increase in H3K27 acetylation marks generated by CBP/p300/Nejire acetyltransferase). Taken together, these findings imply that the 20-hydroxyecdysone signal enhances the functional activity of coregulators rather than promoting their binding to regulatory regions during the ecdysone response.
Khan, S., Pradhan, S. J., Giraud, G., Bleicher, F., Paul, R., Merabet, S. and Shashidhara, L. S. (2023). A Micro-evolutionary Change in Target Binding Sites as a Key Determinant of Ultrabithorax Function in Drosophila. J Mol Evol. PubMed ID: 37341745
Hox genes encode Homeodomain-containing transcription factors, which specify segmental identities along the anterior-posterior axis. Functional changes in Hox genes have been directly implicated in the evolution of body plans across the metazoan lineage. The Hox protein Ultrabithorax (Ubx) is expressed and required in developing third thoracic (T3) segments in holometabolous insects studied so far, particularly, of the order Coleoptera, Lepidoptera and Diptera. Ubx function is key to specify differential development of the second (T2) and T3 thoracic segments in these insects. While Ubx is expressed in the third thoracic segment in developing larvae of Hymenopteran Apis mellifera, the morphological differences between T2 and T3 are subtle. To identify evolutionary changes that are behind the differential function of Ubx in Drosophila and Apis, which are diverged for more than 350 million years, comparative analyses were performed of genome wide Ubx-binding sites between these two insects. These studies reveal that a motif with a TAAAT core is a preferred binding site for Ubx in Drosophila, but not in Apis. Biochemical and transgenic assays suggest that in Drosophila, the TAAAT core sequence in the Ubx binding sites is required for Ubx-mediated regulation of two of its target genes studied here; CG13222, a gene that is normally upregulated by Ubx and vestigial (vg), whose expression is repressed by Ubx in T3. Interestingly, changing the TAAT site to a TAAAT site was sufficient to bring an otherwise unresponsive enhancer of the vg gene from Apis under the control of Ubx in a Drosophila transgenic assay. Taken together, these results suggest an evolutionary mechanism by which critical wing patterning genes might have come under the regulation of Ubx in the Dipteran lineage.
Moudgil, A., Sobti, R. C. and Kaur, T. (2023). In-silico identification and comparison of transcription factor binding sites cluster in anterior-posterior patterning genes in Drosophila melanogaster and Tribolium castaneum. PLoS One 18(8): e0290035. PubMed ID: 37590227
The cis-regulatory data that help in transcriptional regulation is arranged into modular pieces of a few hundred base pairs called CRMs (cis-regulatory modules) and numerous binding sites for multiple transcription factors are prominent characteristics of these cis-regulatory modules. The present study was designed to localize transcription factor binding site (TFBS) clusters on twelve Anterior-posterior (A-P) genes in Tribolium castaneum and compare them to their orthologous gene enhancers in Drosophila melanogaster. Out of the twelve A-P patterning genes, six were gap genes (Kruppel, Knirps, Tailless, Hunchback, Giant, and Caudal) and six were pair rule genes (Hairy, Runt, Even-skipped, Fushi-tarazu, Paired, and Odd-skipped). The genes along with 20 kb upstream and downstream regions were scanned for TFBS clusters using the Motif Cluster Alignment Search Tool (MCAST), a bioinformatics tool that looks for set of nucleotide sequences for statistically significant clusters of non-overlapping occurrence of a given set of motifs. The motifs used in the current study were Hunchback, Caudal, Giant, Kruppel, Knirps, and Even-skipped. The results of the MCAST analysis revealed the maximum number of TFBS for Hunchback, Knirps, Caudal, and Kruppel in both D. melanogaster and T. castaneum, while Bicoid TFBS clusters were found only in D. melanogaster. The size of all the predicted TFBS clusters was less than 1kb in both insect species. These sequences revealed more transversional sites (Tv) than transitional sites (Ti) and the average Ti/Tv ratio was 0.75.
Kyrchanova, O., Ibragimov, A., Postika, N., Georgiev, P. and Schedl, P. (2023). Boundary bypass activity in the abdominal-B region of the Drosophila bithorax complex is position dependent and regulated. Open Biol 13(8): 230035. PubMed ID: 37582404
Expression of Abdominal-B (Abd-B) in abdominal segments A5-A8 is controlled by four regulatory domains, iab-5-iab-8. Each domain has an initiator element (which sets the activity state), elements that maintain this state and tissue-specific enhancers. To ensure their functional autonomy, each domain is bracketed by boundary elements (Mcp, Fab-7, Fab-7 and Fab-8). In addition to blocking crosstalk between adjacent regulatory domains, the Fab boundaries must also have bypass activity so the relevant regulatory domains can 'jump over' intervening boundaries and activate the Abd-B promoter. This study investigated the parameters governing bypass activity. The bypass elements in the Fab-7 and Fab-8 boundaries must be located in the regulatory domain that is responsible for driving Abd-B expression. It is suggested that bypass activity may also be subject to regulation.
Kawasaki, K. and Fukaya, T. (2023). Functional coordination between transcription factor clustering and gene activity. Mol Cell 83(10): 1605-1622. PubMed ID: 37207625
The prevailing view of metazoan gene regulation is that transcription is facilitated through the formation of static activator complexes at distal regulatory regions. This study employed quantitative single-cell live-imaging and computational analysis to provide evidence that the dynamic assembly and disassembly process of transcription factor (TF) clusters at enhancers is a major source of transcriptional bursting in developing Drosophila embryos. It was further shown that the regulatory connectivity between TF clustering and burst induction is highly regulated through intrinsically disordered regions (IDRs). Addition of a poly-glutamine tract to the maternal morphogen Bicoid demonstrated that extended IDR length leads to ectopic TF clustering and burst induction from its endogenous target genes, resulting in defects in body segmentation during embryogenesis. Moreover, this study successfully visualized the presence of 'shared' TF clusters during the co-activation of two distant genes, which provides a concrete molecular explanation for the newly proposed "topological operon" hypothesis in metazoan gene regulation.

Wednesday, November 1st - Disease Models

L. C., Jay, K. L., Andrews, J. C., Walker, M. A., Rodan, L. H., High, F. A., Yamamoto, S., Sweetser, D. A. and Wangler, M. F. (2023). A de novo missense variant in EZH1 associated with developmental delay exhibits functional deficits in Drosophila melanogaster. Genetics 224(4). PubMed ID: 37314226
EZH1, a polycomb repressive complex-2 component, is involved in a myriad of cellular processes. EZH1 represses transcription of downstream target genes through histone 3 lysine27 (H3K27) trimethylation (H3K27me3). Genetic variants in histone modifiers have been associated with developmental disorders, while EZH1 has not yet been linked to any human disease. However, the paralog EZH2 is associated with Weaver syndrome. This study report a previously undiagnosed individual with a novel neurodevelopmental phenotype identified to have a de novo missense variant in EZH1 through exome sequencing. The individual presented in infancy with neurodevelopmental delay and hypotonia and was later noted to have proximal muscle weakness. The variant, p.A678G, is in the SET domain, known for its methyltransferase activity, and an analogous somatic or germline mutation in EZH2 has been reported in patients with B-cell lymphoma or Weaver syndrome, respectively. Human EZH1/2 are homologous to fly Enhancer of zeste (E(z)), an essential gene in Drosophila, and the affected residue (p.A678 in humans, p.A691 in flies) is conserved. To further study this variant, null alleles were obtained, and transgenic flies were generated expressing wildtype [E(z)WT] and the variant [E(z)A691G]. When expressed ubiquitously the variant rescues null-lethality similar to the wildtype. Overexpression of E(z)WT induces homeotic patterning defects but notably the E(z)A691G variant leads to dramatically stronger morphological phenotypes. A dramatic loss is reported of H3K27me2 and a corresponding increase in H3K27me3 in flies expressing E(z)A691G, suggesting this acts as a gain-of-function allele. In conclusion, this study presents a novel EZH1 de novo variant associated with a neurodevelopmental disorder. Furthermore, we found that this variant has a functional impact in Drosophila.
Krupp, S., Tam, O., Gale Hammell, M. and Dubnau, J. (2023). TDP-43 pathology in Drosophila induces glial-cell type specific toxicity that can be ameliorated by knock-down of SF2/SRSF1. bioRxiv. PubMed ID: 37205372
Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer's disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. This study investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, cell-type-specific nuclear RNA sequencing was used to characterize the transcriptional changes were identified that were induced by pathological TDP-43 expression. Numerous glial cell-type specific transcriptional changes. Notably,SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. Further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism.
Herrera, P. and Cauchi, R. J. (2023). Functional characterisation of the ACE2 orthologues in Drosophila provides insights into the neuromuscular complications of COVID-19. Biochim Biophys Acta Mol Basis Dis 1869(8): 166818. PubMed ID: 37495086
SARS-CoV-2, the virus responsible for the coronavirus disease of 2019 (COVID-19), gains cellular entry via interaction with the angiotensin-converting enzyme 2 (ACE2) receptor of host cells. Although SARS-CoV-2 mainly targets the respiratory system, the neuromuscular system also appears to be affected in a large percentage of patients with acute or chronic COVID-19. The cause of the well-described neuromuscular manifestations resulting from SARS-CoV-2 infection remains unresolved. These may result from the neuromuscular-invasive capacity of the virus leading to direct injury. Alternatively, they may be the consequence of ACE2 inactivation either due to viral infection, ACE2 autoantibodies or both. This study made use of the Drosophila model to investigate whether ACE2 downregulation is sufficient to induce neuromuscular phenotypes. Moderate gene silencing of ACE2 orthologues Ance or Ance3 was shown o diminish survival on exposure to thermal stress only upon induction of neuromuscular fatigue driven by increased physical activity. A strong knockdown of Ance or Ance3 directed to muscle reduced or abolished adult viability and caused obvious motoric deficits including reduced locomotion and impaired flight capacity. Selective knockdown of Ance and Ance3 in neurons caused wing defects and an age-dependent decline in motor behaviour, respectively, in adult flies. Interestingly, RNA sequencing led to the discovery of several differentially spliced genes that are required for synaptic function downstream of Ance or Ance3 depletion. These findings are therefore supportive of the notion that loss of a RAS-independent function for ACE2 contributes to the neuromuscular manifestations associated with SARS-CoV-2 infection.
Kervadec, A., Kezos, J., Ni, H., Yu, M., Marchant, J., Spiering, S., Kannan, S., Kwon, C., Andersen, P., Bodmer, R., Grandi, E., Ocorr, K. and Colas, A. R. (2023). Multiplatform modeling of atrial fibrillation identifies phospholamban as a central regulator of cardiac rhythm. Dis Model Mech 16(7). PubMed ID: 37293707
Atrial fibrillation (AF) is a common and genetically inheritable form of cardiac arrhythmia; however, it is currently not known how these genetic predispositions contribute to the initiation and/or maintenance of AF-associated phenotypes. One major barrier to progress is the lack of experimental systems to investigate the effects of gene function on rhythm parameters in models with human atrial and whole-organ relevance. This study assembled a multi-model platform enabling high-throughput characterization of the effects of gene function on action potential duration and rhythm parameters using human induced pluripotent stem cell-derived atrial-like cardiomyocytes and a Drosophila heart model. The findings were validated using computational models of human adult atrial myocytes and tissue. As proof of concept, 20 AF-associated genes were screened and identified phospholamban loss of function as a top conserved hit that shortens action potential duration and increases the incidence of arrhythmia phenotypes upon stress. Mechanistically, this study reveals that phospholamban regulates rhythm homeostasis by functionally interacting with L-type Ca2+ channels and NCX. In summary, this study illustrates how a multi-model system approach paves the way for the discovery and molecular delineation of gene regulatory networks controlling atrial rhythm with application to AF.
Hilsabeck, T. A. U., Narayan, V. P., Wilson, K. A., Carrera, E., Raftery, D., Promislow, D., Brem, R. B., Campisi, J. and Kapahi, P. (2023). Machine Learning identifies conserved traits that influence lifespan and healthspan responses to dietary restriction. bioRxiv. PubMed ID: 37503266
Dietary restriction (DR) is the most robust means to extend lifespan and healthspan across species, but factors such as genetic variation affect how an individual will respond to DR. Additionally, it is unclear how cumulative variations in metabolism and the metabolome influence longevity and health. This study utilized metabolomic, phenotypic, and genome-wide data from Drosophila Genetic Reference Panel strains raised under ad libitum and DR conditions to identify factors which influence longevity and health in response to dietary modulation. Multiple intra-dataset correlations (e.g., metabolites with metabolites) but few inter-dataset correlations (e.g., metabolites with health-related phenotypes) were found. Through random forest modeling across all traits and Mendelian Randomization, key translatable traits were found that influence lifespan or healthspan determination, and the role of multiple metabolites in regulating lifespan was validated. Through these approaches, this study utilized data from flies and humans to elucidate potential therapeutic pathways and metabolomic targets for diet response, lifespan, and healthspan.
Kim, H. S., Parker, D. J., Hardiman, M. M., Munkacsy, E., Jiang, N., Rogers, A. N., Bai, Y., Brent, C., Mobley, J. A., Austad, S. N. and Pickering, A. M. (2023). Early-adulthood spike in protein translation drives aging via juvenile hormone/germline signaling. Nat Commun 14(1): 5021. PubMed ID: 37596266
Protein translation (PT) declines with age in invertebrates, rodents, and humans. It has been assumed that elevated PT at young ages is beneficial to health and PT ends up dropping as a passive byproduct of aging. In Drosophila, this study showed that a transient elevation in PT during early-adulthood exerts long-lasting negative impacts on aging trajectories and proteostasis in later-life. Blocking the early-life PT elevation robustly improves life-/health-span and prevents age-related protein aggregation, whereas transiently inducing an early-life PT surge in long-lived fly strains abolishes their longevity/proteostasis benefits. The early-life PT elevation triggers proteostatic dysfunction, silences stress responses, and drives age-related functional decline via juvenile hormone-lipid transfer protein axis and germline signaling. These findings suggest that PT is adaptively suppressed after early-adulthood, alleviating later-life proteostatic burden, slowing down age-related functional decline, and improving lifespan. This work provides a theoretical framework for understanding how lifetime PT dynamics shape future aging trajectories.
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