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Current papers in developmental biology and gene function





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Monday, August 31st, 2020 - Gonads

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Zhang, J., Luo, J., Chen, J., Dai, J. and Montell, C. (2020). The Role of Y Chromosome Genes in Male Fertility in Drosophila melanogaster. Genetics. PubMed ID: 32404399
Summary:
The Y chromosome of Drosophila melanogaster is pivotal for male fertility. Yet, only 16 protein-coding genes reside on this chromosome. The Y chromosome is comprised primarily of heterochromatic sequences, including DNA repeats and satellite DNA, and most of the Y chromosome is still missing from the genome sequence. Furthermore, the functions of the majority of genes on the Y chromosome remain elusive. Through multiple genetic strategies, six distinct segments on the Y chromosome have been identified as "male fertility factors," and candidate gene sequences corresponding to each of these loci have been ascribed. In one case, kl-3, a specific protein coding sequence for a fertility factor has been confirmed molecularly. This study employed CRISPR/Cas9 to generate mutations, and RNAi, to interrogate the requirements of protein coding sequences on the Y chromosome for male fertility. CRISPR/Cas9-mediated editing of kl-2 and kl-5 causes male sterility, supporting the model that these gene sequences correspond to the cognate fertility factors. Another gene, CCY, also functions in male fertility and may be the ks-2 fertility factor. This study demonstrates that editing of kl-2, kl-3 and kl-5, and RNAi knockdown of CCY, disrupts nuclear elongation, and leads to defects in sperm individualization, including impairments in the individualization complex (IC) and synchronization. However, CRISPR/Cas9 mediated knockout of some genes on the Y chromosome, such as FDY, Ppr-Y and Pp1-Y2 do cause sterility, indicating that not all Y chromosome genes are essential for male fertility.
Chebbo, S., Josway, S., Belote, J. M. and Manier, M. K. (2020). A putative novel role for Eip74EF in male reproduction in promoting sperm elongation at the cost of male fecundity. J Exp Zool B Mol Dev Evol. PubMed ID: 32725718
Summary:
Spermatozoa are the most morphologically variable cell type, yet little is known about genes controlling natural variation in sperm shape. Drosophila fruit flies have the longest sperm known, which are evolving under postcopulatory sexual selection, driven by sperm competition and cryptic female choice. Long sperm outcompete short sperm but primarily when females have a long seminal receptacle (SR), the primary sperm storage organ. Thus, the selection on sperm length is mediated by SR length, and the two traits are coevolving across the Drosophila lineage, driven by a genetic correlation and fitness advantage of long sperm and long SR genotypes in both males and females. Ecdysone-induced protein 74EF (Eip74EF) is expressed during postmeiotic stages of spermatogenesis when spermatid elongation occurs, and this study found that it is rapidly evolving under positive selection in Drosophila. Hypomorphic knockout of the E74A isoform leads to shorter sperm but does not affect SR length, suggesting that E74A may be involved in promoting spermatid elongation but is not a genetic driver of male-female coevolution. It was also found that E74A knockout has opposing effects on fecundity in males and females, with an increase in fecundity for males but a decrease in females, consistent with its documented role in oocyte maturation. These results suggest a novel function of Eip74EF in spermatogenesis and demonstrates that this gene influences both male and female reproductive success. This study speculates on possible roles for E74A in spermatogenesis and male reproductive success.
Fingerhut, J. M. and Yamashita, Y. M. (2020). mRNA localization mediates maturation of cytoplasmic cilia in Drosophila spermatogenesis. J Cell Biol 219(9). PubMed ID: 32706373
Summary:
Cytoplasmic cilia, a specialized type of cilia in which the axoneme resides within the cytoplasm rather than within the ciliary compartment, are proposed to allow for the efficient assembly of very long cilia. Despite being found diversely in male gametes (e.g., Plasmodium falciparum microgametocytes and human and Drosophila melanogaster sperm), very little is known about cytoplasmic cilia assembly. This study shows that a novel RNP granule containing the mRNAs for axonemal dynein motor proteins becomes highly polarized to the distal end of the cilia during cytoplasmic ciliogenesis in Drosophila sperm. This allows for the incorporation of these axonemal dyneins into the axoneme directly from the cytoplasm, possibly by localizing translation. This RNP granule contains the proteins Reptin and Pontin, loss of which perturbs granule formation and prevents incorporation of the axonemal dyneins, leading to sterility. It is proposed that cytoplasmic cilia assembly requires the precise localization of mRNAs encoding key axonemal constituents, allowing these proteins to incorporate efficiently into the axoneme.
Hu, Q., Duncan, F. E., Nowakowski, A. B., Antipova, O. A., Woodruff, T. K., O'Halloran, T. V. and Wolfner, M. F. (2020). Zinc Dynamics during Drosophila Oocyte Maturation and Egg Activation. iScience 23(7): 101275. PubMed ID: 32615472
Summary:
Temporal fluctuations in zinc concentration are essential signals, including during oogenesis and early embryogenesis. In mammals, zinc accumulation and release are required for oocyte maturation and egg activation, respectively. This study demonstrates that zinc flux occurs in Drosophila oocytes and activated eggs, and that zinc is required for female fertility. Synchrotron-based X-ray fluorescence microscopy reveals zinc as the most abundant transition metal in Drosophila oocytes. Its levels increase during oocyte maturation, accompanied by the appearance of zinc-enriched intracellular granules in the oocyte, which depend on transporters. Subsequently, in egg activation, which mediates the transition from oocyte to embryo, oocyte zinc levels decrease significantly, as does the number of zinc-enriched granules. This pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, extending the parallels in female gamete processes between Drosophila and mammals and establishing Drosophila as a model for dissecting reproductive roles of zinc.
Hu, Q. and Wolfner, M. F. (2020). Regulation of Trpm activation and calcium wave initiation during Drosophila egg activation. Mol Reprod Dev. PubMed ID: 32735035
Summary:
The transition from a developmentally arrested mature oocyte to a developing embryo requires a series of highly conserved events, collectively known as egg activation. All of these events are preceded by a ubiquitous rise of intracellular calcium, which results from influx of external calcium and/or calcium release from internal storage. In Drosophila, this calcium rise initiates from the pole(s) of the oocyte by influx of external calcium in response to mechanical triggers. It is thought to trigger calcium responsive kinases and/or phosphatases, which in turn alter the oocyte phospho-proteome to initiate downstream events. Recent studies revealed that external calcium enters the activating Drosophila oocyte through Trpm channels, a feature conserved in mouse. The local entry of calcium raises the question of whether Trpm channels are found locally at the poles of the oocyte or are localized around the oocyte periphery, but activated only at the poles. This study shows that Trpm is distributed all around the oocyte. This requires that it thus be specially regulated at the poles to allow calcium wave initiation. Neither egg shape nor local pressure is sufficient to explain this local activation of Trpm channels.
Miao, G., Godt, D. and Montell, D. J. (2020). Integration of Migratory Cells into a New Site In vivo Requires Channel-Independent Functions of Innexins on Microtubules. Dev Cell. PubMed ID: 32668209
Summary:
During embryonic development and cancer metastasis, migratory cells must establish stable connections with new partners at their destinations. This study established Drosophila border cells as a model for this multistep process. During oogenesis, border cells delaminate from the follicular epithelium and migrate. When they reach their target, the oocyte, they undergo a stereotypical series of steps to adhere to it, then connect with another migrating epithelium. Gap-junction-forming innexin proteins were identified as critical. Surprisingly, the channel function is dispensable. Instead, Innexins 2 and 3 function within the border cells, and Innexin 4 functions within the germline, to regulate microtubules. The microtubule-dependent border cell-oocyte interaction is essential to brace the cells against external morphogenetic forces. Thus, this study established an experimental model and used genetic, thermogenetic, and live-imaging approaches to uncover the contributions of Innexins and microtubules to a cell-biological process important in development and cancer.

Friday, August 28th - Behavior

Meichtry, L. B., Poetini, M. R., Dahleh, M. M. M., Araujo, S. M., Musachio, E. A. S., Bortolotto, V. C., de Freitas Couto, S., Somacal, S., Emanuelli, T., Gayer, M. C., Roehrs, R., Guerra, G. P. and Prigol, M. (2020). Addition of saturated and trans-fatty acids to the diet induces depressive and anxiety-like behaviors in Drosophila melanogaster. Neuroscience. PubMed ID: 32738432
Summary:
This study aimed to evaluate the effects of the addition of saturated fat and hydrogenated vegetable fat (HVF) to the diet on depressive and anxiety-like behaviors in Drosophila melanogaster. Flies were exposed to experimental diets: regular diet (RD), or HVF in the concentrations of the substitute (SHVF), HVF 10% and HVF 20%, or Lard (L) in the concentrations of the substitute (SL), L 10% and L 20%, during seven days. The results showed that flies fed with the HVF diet presented similar behaviors to depression, anxiety, and a higher number of aggressive events. Flies exposed to L showed only depressive-like behavior. Regarding serotonin levels (5HT), there was a significant reduction in the flies exposed to SHVF, HVF 10%, HVF 20%, and L 20%. Regarding the levels of octopamine (OA), there was a significant reduction in the flies exposed to both HVF and L rich diets when compared with the RD group. Also, there was a significant negative correlation between 5HT or OA levels and behaviors of aggressiveness, negative geotaxis, immobility time, light/dark, and grooming in the flies. This study shows that Drosophila melanogaster can serve as a valuable model for understanding psychiatric disorders and that the type of fatty acid (FA) offered in the diet can influence these disorders. This demonstrates the importance of the composition of the FAs in the neural pathways, being able to influence the signaling of neurotransmitters, such as 5HT and OA, and thus, cause behavioral changes.
Mishra, A., Tung, S., Shree Sruti, V. R., Srivathsa, S. V. and Dey, S. (2020). Mate-finding dispersal reduces local mate limitation and sex bias in dispersal. J Anim Ecol. PubMed ID: 32535925
Summary:
Sex-biased dispersal (SBD) often skews the local sex ratio in a population. This can result in a shortage of mates for individuals of the less-dispersive sex. Such mate limitation can lead to Allee effects in populations that are small or undergoing range expansion, consequently affecting their survival, growth, stability and invasion speed. Theory predicts that mate shortage can lead to either an increase or a decrease in the dispersal of the less-dispersive sex. However, neither of these predictions have been empirically validated. To investigate how SBD-induced mate limitation affects dispersal of the less-dispersive sex, Drosophila melanogaster populations with varying dispersal propensities were used. To rule out any mate-independent density effects, the behavioral plasticity was examined of dispersal in presence of mates as well as same-sex individuals with differential dispersal capabilities. In the presence of high-dispersive mates, the dispersal of both male and female individuals was significantly increased. However, the magnitude of this increase was much larger in males than in females, indicating that the former show greater mate-finding dispersal. Moreover, the dispersal of either sex did not change when dispersing alongside high- or low-dispersive individuals of the same sex. This suggested that the observed plasticity in dispersal was indeed due to mate-finding dispersal, and not mate-independent density effects. Strong mate-finding dispersal can diminish the magnitude of sex bias in dispersal. This can modulate the evolutionary processes that shape range expansions and invasions, depending on the population size. In small populations, mate-finding dispersal can ameliorate Allee effects. However, in large populations, it can dilute the effects of spatial sorting.
Melnattur, K., Zhang, B. and Shaw, P. J. (2020). Disrupting flight increases sleep and identifies a novel sleep-promoting pathway in Drosophila. Sci Adv 6(19): eaaz2166. PubMed ID: 32494708
Summary:
Sleep is plastic and is influenced by ecological factors and environmental changes. The mechanisms underlying sleep plasticity are not well understood. This study shows that manipulations that impair flight in Drosophila increase sleep as a form of sleep plasticity. Flight was disrupted by blocking the wing-expansion program, genetically disrupting flight, and by mechanical wing perturbations. A new sleep regulatory circuit was defined starting with specific wing sensory neurons, their target projection neurons in the ventral nerve cord, and the neurons they connect to in the central brain. In addition, a critical neuropeptide (Burs) and its receptor (Rickets) were identified that link wing expansion and sleep. Disrupting flight activates these sleep-promoting projection neurons, as indicated by increased cytosolic calcium levels, and stably increases the number of synapses in their axonal projections. These data reveal an unexpected role for flight in regulating sleep and provide new insight into how sensory processing controls sleep need.
Park, S. J. and Ja, W. W. (2020). Absolute ethanol intake predicts ethanol preference in Drosophila melanogaster. J Exp Biol 223(Pt 11). PubMed ID: 32366685
Summary:
Factors that mediate ethanol preference in Drosophila melanogaster are not well understood. A major confound has been the use of diverse methods to estimate ethanol consumption. This study measured fly consumptive ethanol preference on base diets varying in nutrients, taste and ethanol concentration. Both sexes showed an ethanol preference that was abolished on high nutrient concentration diets. Additionally, manipulating total food intake without altering the nutritive value of the base diet or the ethanol concentration was sufficient to evoke or eliminate ethanol preference. Absolute ethanol intake and food volume consumed were stronger predictors of ethanol preference than caloric intake or the dietary caloric content. These findings suggest that the effect of the base diet on ethanol preference is largely mediated by total consumption associated with the delivery medium, which ultimately determines the level of ethanol intake. It is speculated that a physiologically relevant threshold for ethanol intake is essential for preferential ethanol consumption.
Rooke, R., Rasool, A., Schneider, J. and Levine, J. D. (2020). Drosophila melanogaster behaviour changes in different social environments based on group size and density. Commun Biol 3(1): 304. PubMed ID: 32533063
Summary:
Many organisms, when alone, behave differently from when they are among a crowd. Drosophila similarly display social behaviour and collective behaviour dynamics within groups not seen in individuals. In flies, these emergent behaviours may be in response to the global size of the group or local nearest-neighbour density. This study investigated i) which aspect of social life flies respond to: group size, density, or both and ii) whether behavioural changes within the group are dependent on olfactory support cells. Behavioural assays demonstrate that flies adjust their interactive behaviour to group size but otherwise compensate for density by achieving a standard rate of movement, suggesting that individuals are aware of the number of others within their group. It was shown that olfactory support cells are necessary for flies to behave normally in large groups. These findings shed insight into the subtle and complex life of Drosophila within a social setting.
Moulin, T. C., Ferro, F., Berkins, S., Hoyer, A., Williams, M. J. and Schioth, H. B. (2020). Transient Administration of Dopaminergic Precursor Causes Inheritable Overfeeding Behavior in Young Drosophila melanogaster Adults. Brain Sci 10(8). PubMed ID: 32731370
Summary:
Imbalances in dopaminergic signaling during development have been indicated as part of the underlying neurobiology of several psychiatric illnesses. Yet, how transient manipulation of dopaminergic signaling influences long-lasting behavioral consequences, or if these modifications can induce inheritable traits, it is still not understood. This study used the Drosophila model to test if transient pharmacological activation of the dopaminergic system leads to modulations of feeding and locomotion in adult flies. Transient administration of a dopaminergic precursor, levodopa, at 6 h, 3 days or 5 days post-eclosion, induced overfeeding behavior, while no significant effects on locomotion were found. Moreover, this phenotype was inherited by the offspring of flies treated 6 h or 3 days post-eclosion, but not the offspring of those treated 5 days post-eclosion. These results indicate that transient alterations in dopaminergic signaling can produce behavioral alterations in adults, which can then be carried to descendants. These findings provide novel insights into the conditions in which environmental factors can produce transgenerational eating disorders.

Thursday, August 27th - Signal Transduction

Kang, Y. Y., Wachi, Y., Engdorf, E., Fumagalli, E., Wang, Y., Myers, J., Massey, S., Greiss, A., Xu, S. and Roman, G. (2020). Normal Ethanol Sensitivity and Rapid Tolerance Require the G Protein Receptor Kinase 2 in Ellipsoid Body Neurons in Drosophila. Alcohol Clin Exp Res. PubMed ID: 32573992
Summary:
G protein signaling pathways are key neuromodulatory mechanisms for behaviors and neurological functions that affect the impact of ethanol (EtOH) on locomotion, arousal, and synaptic plasticity. This study reports a novel role for the Drosophila G protein-coupled receptor kinase 2 (GPRK2) as a member of the GRK4/5/6 subfamily in modulating EtOH-induced behaviors. The requirement of Drosophila Gprk2 for naive sensitivity to EtOH sedation and ability of the fly to develop rapid tolerance after a single exposure to EtOH were studied using the loss of righting reflex (LORR) and fly group activity monitor (FlyGrAM) assays. Loss-of-function Gprk2 mutants demonstrate an increase in alcohol-induced hyperactivity, reduced sensitivity to the sedative effects of EtOH, and diminished rapid tolerance after a single intoxicating exposure. The requirement for Gprk2 in EtOH sedation and rapid tolerance maps to ellipsoid body neurons within the Drosophila brain, suggesting that wild-type Gprk2 is required for modulation of locomotion and alertness. However, even though Gprk2 loss of function leads to decreased and fragmented sleep, this change in the sleep state does not depend on Gprk2 expression in the ellipsoid body. This work on GPRK2 has established a role for this GRK4/5/6 subfamily member in EtOH sensitivity and rapid tolerance.
Liu, Y., Mattila, J. and Hietakangas, V. (2020). Systematic Screen for Drosophila Transcriptional Regulators Phosphorylated in Response to Insulin/mTOR Pathway. G3 (Bethesda). PubMed ID: 32554565
Summary:
Insulin/insulin-like growth factor signaling (IIS) is a conserved mechanism to regulate animal physiology in response to nutrition. IIS activity controls gene expression, but only a subset of transcriptional regulators (TRs) targeted by the IIS pathway is currently known. This study reports the results of an unbiased screen for Drosophila TRs phosphorylated in an IIS-dependent manner. To conduct the screen, a library was built of 857 V5/Strep-tagged TRs under the control of Copper-inducible metallothionein promoter (pMt). The insulin-induced phosphorylation changes were detected by using Phos-tag SDS-PAGE and Western blotting. Eight proteins were found to display increased phosphorylation after acute insulin treatment. In each case, the insulin-induced phosphorylation was abrogated by mTORC1 inhibitor rapamycin. The hits included two components of the NURF complex (NURF38 and NURF55), bHLHZip transcription factor Max, as well as the Drosophila ortholog of human proliferation-associated 2G4 (dPA2G4). Subsequent experiments revealed that the expression of the dPA2G4 gene was promoted by the mTOR pathway, likely through transcription factor Myc. Furthermore, NURF38 was found to be necessary for growth in larvae, consistent with the role of IIS/mTOR pathway in growth control.
Lien, W. Y., Chen, Y. T., Li, Y. J., Wu, J. K., Huang, K. L., Lin, J. R., Lin, S. C., Hou, C. C., Wang, H. D., Wu, C. L., Huang, S. Y. and Chan, C. C. (2020). Lifespan regulation in alpha/beta posterior neurons of the fly mushroom bodies by Rab27. Aging Cell: e13179. PubMed ID: 32627932
Summary:
Brain function has been implicated to control the aging process and modulate lifespan. However, continuous efforts remain for the identification of the minimal sufficient brain region and the underlying mechanism for neuronal regulation of longevity. This study shows that the Drosophila lifespan is modulated by rab27 functioning in a small subset of neurons of the mushroom bodies (MB), a brain structure that shares analogous functions with mammalian hippocampus and hypothalamus. Depleting rab27 in the α/βp neurons of the MB is sufficient to extend lifespan, enhance systemic stress responses, and alter energy homeostasis, all without trade-offs in major life functions. Within the α/βp neurons, rab27KO causes the mislocalization of phosphorylated S6K thus attenuates TOR signaling, resulting in decreased protein synthesis and reduced neuronal activity. Consistently, expression of dominant-negative S6K in the α/βp neurons increases lifespan. Furthermore, the expression of phospho-mimetic S6 in α/βp neurons of rab27KO rescued local protein synthesis and reversed lifespan extension. These findings demonstrate that inhibiting TOR-mediated protein synthesis in α/βp neurons is sufficient to promote longevity.
Lin, K. Y., Wang, W. D., Lin, C. H., Rastegari, E., Su, Y. H., Chang, Y. T., Liao, Y. F., Chang, Y. C., Pi, H., Yu, B. Y., Chen, S. H., Lin, C. Y., Lu, M. Y., Su, T. Y., Tzou, F. Y., Chan, C. C. and Hsu, H. J. (2020).. Piwi reduction in the aged niche eliminates germline stem cells via Toll-GSK3 signaling. Nat Commun 11(1): 3147. PubMed ID: 32561720
Summary:
Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. This study reports that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade β-catenin. Disruption of β-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and β-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration.
Dong, W., Lu, J., Zhang, X., Wu, Y., Lettieri, K., Hammond, G. R. and Hong, Y. (2020). A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity. J Cell Biol 219(7). PubMed ID: 32580209
Summary:
Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. This study shows that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. The data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.
Lee, J., Cabrera, A. J. H., Nguyen, C. M. T. and Kwon, Y. V. (2020). Dissemination of Ras(V12)-transformed cells requires the mechanosensitive channel Piezo. Nat Commun 11(1): 3568. PubMed ID: 32678085
Summary:
Dissemination of transformed cells is a key process in metastasis. Despite its importance, how transformed cells disseminate from an intact tissue and enter the circulation is poorly understood. This study used a fully developed tissue, Drosophila midgut and describes the morphologically distinct steps and the cellular events occurring over the course of Ras(V12)-transformed cell dissemination. Notably, Ras(V12)-transformed cells formed the Actin- and Cortactin-rich invasive protrusions that were important for breaching the extracellular matrix (ECM) and visceral muscle. Furthermore, the essential roles were uncovered of the mechanosensory channel Piezo in orchestrating dissemination of Ras(V12)-transformed cells. Collectively, this study establishes an in vivo model for studying how transformed cells migrate out from a complex tissue and provides unique insights into the roles of Piezo in invasive cell behavior.

Wednesday, August 26th - Synapse and Vesicles

Goel, P., Nishimura, S., Chetlapalli, K., Li, X., Chen, C. and Dickman, D. (2020). Distinct Target-Specific Mechanisms Homeostatically Stabilize Transmission at Pre- and Post-synaptic Compartments. Front Cell Neurosci 14: 196. PubMed ID: 32676010
Summary:
Neurons must establish and stabilize connections made with diverse targets, each with distinct demands and functional characteristics. At Drosophila neuromuscular junctions (NMJs), synaptic strength remains stable in a manipulation that simultaneously induces hypo-innervation on one target and hyper-innervation on the other. However, the expression mechanisms that achieve this exquisite target-specific homeostatic control remain enigmatic. This study identified the distinct target-specific homeostatic expression mechanisms. On the hypo-innervated target, an increase in postsynaptic glutamate receptor (GluR) abundance is sufficient to compensate for reduced innervation, without any apparent presynaptic adaptations. In contrast, a target-specific reduction in presynaptic neurotransmitter release probability is reflected by a decrease in active zone components restricted to terminals of hyper-innervated targets. Finally, loss of postsynaptic GluRs on one target induces a compartmentalized, homeostatic enhancement of presynaptic neurotransmitter release called presynaptic homeostatic potentiation (PHP) that can be precisely balanced with the adaptations required for both hypo- and hyper-innervation to maintain stable synaptic strength. Thus, distinct anterograde and retrograde signaling systems operate at pre- and post-synaptic compartments to enable target-specific, homeostatic control of neurotransmission.
Guangming, G., Junhua, G., Chenchen, Z., Yang, M. and Wei, X. (2020). Neurexin and Neuroligins Maintain the Balance of Ghost and Satellite Boutons at the Drosophila Neuromuscular Junction. Front Neuroanat 14: 19. PubMed ID: 32581727
Summary:
Ghost and satellite boutons indicate the poor development and overgrowth of the neuromuscular junction (NMJ) boutons. However, the Drosophila neurexin (DNrx) and Drosophila neuroligins (DNlgs) are mainly observed in type Ib boutons, indicating the ultrastructural and developmental phenotypes of the Drosophila NMJ. This study identified the ultrastructural and developmental features of ghost and satellite boutons by utilizing dneurexin (dnrx) and dneuroligins (dnlgs) fly mutants and other associated fly strains. Ghost boutons contain synaptic vesicles with multiple diameters but very rarely contain T-bar structures and swollen or thin subsynaptic reticulum (SSR) membranes. The muscle cell membrane is invaginated at different sites, stretches to the ghost bouton from different directions, forms several layers that enwrap the ghost bouton, and then branches into the complex SSR. Satellite boutons share a common SSR membrane and present either a typical profile in which a main bouton is encircled by small boutons or two atypical profiles in which the small boutons are grouped together or distributed in beads without a main bouton. Electron and confocal microscopy data showed that dnrx, dnlg1, dnlg2, dnlg3, and dnlg4 mutations led to ghost boutons; the overexpression of dnrx, dnlg1, dnlg2, dnlg3, and dnlg4 led to satellite boutons; and the dnlg2;dnlg3 double mutation also led to satellite boutons. These results suggested that DNrx and DNlgs jointly maintain the development and function of NMJ boutons by regulating the balance of ghost and satellite boutons in Drosophila.
Genc, O., An, J. Y., Fetter, R. D., Kulik, Y., Zunino, G., Sanders, S. J. and Davis, G. W. (2020). Homeostatic plasticity fails at the intersection of autism-gene mutations and a novel class of common genetic modifiers. Elife 9. PubMed ID: 32609087
Summary:
This study identified a set of common phenotypic modifiers that interact with five independent autism gene orthologs [RIMS1 (Rim), CHD8 (Kismet), CHD2 (Chd1), WDFY3 (Blue cheese), ASH1L (ASH1)] causing a common failure of presynaptic homeostatic plasticity (PHP) in Drosophila. Heterozygous null mutations in each autism gene are demonstrated to have normal baseline neurotransmission and PHP. However, PHP is sensitized and rendered prone to failure. A subsequent electrophysiology-based genetic screen identifies the first known heterozygous mutations that commonly genetically interact with multiple ASD gene orthologs, causing PHP to fail. Two phenotypic modifiers identified in the screen, PDPK1 and PPP2R5D, are characterized. Finally, transcriptomic, ultrastructural and electrophysiological analyses define one mechanism by which PHP fails; an unexpected, maladaptive up-regulation of CREG, a conserved, neuronally expressed, stress response gene and a novel repressor of PHP. Thus, this study define a novel genetic landscape by which diverse, unrelated autism risk genes may converge to commonly affect the robustness of synaptic transmission.
Harrigan, J., Brambila, D. F., Meera, P., Krantz, D. E. and Schweizer, F. E. (2020). The environmental toxicant ziram enhances neurotransmitter release and increases neuronal excitability via the EAG family of potassium channels. Neurobiol Dis 143: 104977. PubMed ID: 32553709
Summary:
An environmental toxicant ziram is dithiocarbamate known to have neurotoxic effects and to increase the risk of Parkinson's disease. This study has used Drosophila to identify novel molecular pathways by which ziram may disrupt neuronal function. Consistent with previous results in mammalian cells, ziram was found to increase the probability of synaptic vesicle release by dysregulation of the ubiquitin signaling system. In addition, it was found that ziram increases neuronal excitability. Using a combination of live imaging and electrophysiology, ziram was found to increases excitability in both aminergic and glutamatergic neurons. This increased excitability is phenocopied and occluded by null mutant animals of the ether a-go-go (eag) potassium channel. A pharmacological inhibitor of the temperature sensitive hERG (human ether-a-go-go related gene) phenocopies the excitability effects of ziram but only at elevated temperatures. seizure (sei), a fly ortholog of hERG, is thus another candidate target of ziram. Taken together, the eag family of potassium channels emerges as a candidate for mediating some of the toxic effects of ziram. It is proposed that ziram may contribute to the risk of complex human diseases by blockade of human eag and sei orthologs, such as hERG.
Ihara, M., Furutani, S., Shigetou, S., Shimada, S., Niki, K., Komori, Y., Kamiya, M., Koizumi, W., Magara, L., Hikida, M., Noguchi, A., Okuhara, D., Yoshinari, Y., Kondo, S., Tanimoto, H., Niwa, R., Sattelle, D. B. and Matsuda, K. (2020). Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions. Proc Natl Acad Sci U S A 117(28): 16283-16291. PubMed ID: 32611810
Summary:
The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. This study shows that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.
Du, G., Qiao, Y., Zhuo, Z., Zhou, J., Li, X., Liu, Z., Li, Y. and Chen, H. (2020). Lipoic acid rejuvenates aged intestinal stem cells by preventing age-associated endosome reduction. EMBO Rep: e49583. PubMed ID: 32648369
Summary:
The age-associated decline of adult stem cell function is closely related to the decline in tissue function and age-related diseases. However, the underlying mechanisms that ultimately lead to the observed functional decline of stem cells still remain largely unexplored. This study investigated Drosophila midguts and found a continuous downregulation of lipoic acid synthase, which encodes the key enzyme for the endogenous synthesis of alpha-lipoic acid (ALA), upon aging. Importantly, orally administration of ALA significantly reversed the age-associated hyperproliferation of intestinal stem cells (ISCs) and the observed decline of intestinal function, thus extending the lifespan of Drosophila. This study reports that ALA reverses age-associated ISC dysfunction by promoting the activation of the endocytosis-autophagy network, which decreases in aged ISCs. Moreover, this study suggests that ALA may be used as a safe and effective anti-aging compound for the treatment of ISC-dysfunction-related diseases and for the promotion of healthy aging in humans.

Tuesday, April 26th - Adult Physiology and Behavior

Landis, G. N., Doherty, D. V., Yen, C. A., Wang, L., Fan, Y., Wang, I., Vroegop, J., Wang, T., Wu, J., Patel, P., Lee, S., Abdelmesieh, M., Shen, J., Promislow, D. E. L., Curran, S. P. and Tower, J. (2020). Metabolic Signatures of Life Span Regulated by Mating, Sex Peptide and Mifepristone/RU486 in Female Drosophila melanogaster. J Gerontol A Biol Sci Med Sci. PubMed ID: 32648907
Summary:
Mating and transfer of male Sex Peptide (SP), or transgenic expression of SP, causes inflammation and decreased life span in female Drosophila. Mifepristone rescues these effects, yielding dramatic increases in life span. In this study targeted metabolomics data were integrated with further analysis of extant transcriptomic data. Each of seven genes positively correlated with life span were expressed in the brain or eye, and involved regulation of gene expression and signaling. Genes negatively correlated with life span were preferentially expressed in midgut and involved protein degradation, amino acid metabolism, and immune response. Across all conditions, life span was positively correlated with muscle breakdown product 1/3-methylhistadine and purine breakdown product urate, and negatively correlated with tryptophan breakdown product kynurenic acid, suggesting a SP-induced shift from somatic maintenance/turnover pathways to the costly production of energy and lipids from dietary amino acids. Some limited overlap was observed between genes regulated by mifepristone and genes known to be regulated by ecdysone, however, mifepristone was unable to compete with ecdysone for activation of an ecdysone-responsive transgenic reporter. In contrast, genes regulated by mifepristone were highly enriched for genes regulated by Juvenile Hormone (JH), and mifepristone rescued the negative effect of JH analog methoprene on life span in adult virgin females. The data indicate that mifepristone increases life span and decreases inflammation in mated females by antagonizing JH signaling downstream of male SP. Finally, mifepristone increased life span of mated, but not unmated, C. elegans, in two of three trials, suggesting possible evolutionary conservation of mifepristone mechanisms.
ALecheta, M. C., Awde, D. N., O'Leary, T. S., Unfried, L. N., Jacobs, N. A., Whitlock, M. H., McCabe, E., Powers, B., Bora, K., Waters, J. S., Axen, H. J., Frietze, S., Lockwood, B. L., Teets, N. M. and Cahan, S. H. (2020). Integrating GWAS and Transcriptomics to Identify the Molecular Underpinnings of Thermal Stress Responses in Drosophila melanogaster. Front Genet 11: 658. PubMed ID: 32655626
Summary:
Thermal tolerance depends on both the ability to dynamically adjust to a thermal stress and preparatory developmental processes that enhance thermal resistance. This study used a combination of Genome Wide Association mapping (GWAS) and transcriptomic profiling to characterize whether genes associated with thermal tolerance are primarily involved in dynamic stress responses or preparatory processes that influence physiological condition at the time of thermal stress. To test the hypotheses, the critical thermal minimum (CT(min)) and critical thermal maximum (CT(max)) were measured of 100 lines of the Drosophila Genetic Reference Panel (DGRP), and GWAS was used to identify loci that explain variation in thermal limits. Greater variation was observed in lower thermal limits, with CT(min) ranging from 1.81 to 8.60°C, while CT(max) ranged from 38.74 to 40.64°C. 151 and 99 distinct genes associated with CT(min) and CT(max), respectively, were identified, and there was strong support that these genes are involved in both dynamic responses to thermal stress and preparatory processes that increase thermal resistance. Many of the genes identified by GWAS were involved in the direct transcriptional response to thermal stress, and overall GWAS candidates were more likely to be differentially expressed than other genes. Further, several GWAS candidates were regulatory genes that may participate in the regulation of stress responses, and gene ontologies related to development and morphogenesis were enriched, suggesting many of these genes influence thermal tolerance through effects on development and physiological status. Overall, these results suggest that thermal tolerance alleles can influence both dynamic plastic responses to thermal stress and preparatory processes that improve thermal resistance. These results also have utility for directly comparing GWAS and transcriptomic approaches for identifying candidate genes associated with thermal tolerance.
Krittika, S. and Yadav, P. (2020). Dietary protein restriction deciphers new relationships between lifespan, fecundity and activity levels in fruit flies Drosophila melanogaster. Sci Rep 10(1): 10019. PubMed ID: 32572062
Summary:
Drosophila melanogaster has been used in Diet Restriction (DR) studies for a few decades now, due to easy diet implementation and its short lifespan. Since the concentration of protein determines the trade-offs between lifespan and fecundity, it is important to understand the level of protein and the extent of its influence on lifespan, fecundity and activity of fruit flies. This study intended to assess the effect of a series of protein restricted diets from age 1 day of the adult fly on these traits to understand the possible variations in trade-off across tested diets. Lifespan under different protein concentrations remains unaltered, even though protein restricted diets exerted an age-specific influence on fecundity. Interestingly, there was no difference in lifetime activity of the flies in most of the tested protein restricted (PR) diets, even though a sex-dependent influence of protein concentrations was observed. Additionally, it is reported that not all concentrations of PR diet increase activity, thereby suggesting that the correlation between lifespan and the lifetime activity can be challenged under protein-restricted condition. Therefore, the PR does not need to exert its effect on lifespan and fecundity only but can also influence activity levels of the flies, thereby emphasizing the role of nutrient allotment between lifespan, fecundity and activity.
Liu, W., Wang, J., Zhang, H. Y., Yang, Y. C., Kang, R. X., Bai, P., Fu, H., Chen, L. R., Gao, Y. P. and Tan, E. K. (2020). Symbiotic bacteria attenuate Drosophila oviposition repellence to alkaline through acidification. Insect Sci. PubMed ID: 32725723
Summary:
Metazoans harbor a wealth of symbionts that are ever-changing the environment by taking up resources and/or excreting metabolites. One such common environmental modification is a change in pH. Conventional wisdom holds that symbionts facilitate the survival and production of their hosts in the wild, but this notion lacks empirical evidence. This study reports that symbiotic bacteria in the genus Enterococcus attenuate the oviposition avoidance of alkaline environments in Drosophila. The effects of alkalinity on oviposition preference was studied for the first time, and it was found that flies are robustly disinclined to oviposit on alkali-containing substrates. This innate repulsion to alkaline environments is explained, in part, by the fact that alkalinity compromises the health and lifespan of both offspring and parent Drosophila. Enterococcus dramatically diminished or even completely reversed the ovipositional avoidance of alkalinity in Drosophila. Mechanistically, Enterococcus generate abundant lactate during fermentation, which neutralizes the residual alkali in an egg-laying substrate. In conclusion, Enterococcus protects Drosophila from alkali stress by acidifying the ovipositional substrate, and ultimately improves the fitness of the Drosophila population. These results demonstrate that symbionts are profound factors in the Drosophila ovipositional decision, and extend understanding of the intimate interactions between Drosophila and their symbionts.
Moscato, E. H., Dubowy, C., Walker, J. A. and Kayser, M. S. (2020). Social Behavioral Deficits with Loss of Neurofibromin Emerge from Peripheral Chemosensory Neuron Dysfunction. Cell Rep 32(1): 107856. PubMed ID: 32640222
Summary:
Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder associated with social and communicative disabilities. The cellular and circuit mechanisms by which loss of neurofibromin 1 (Nf1) results in social deficits are unknown. This study identified social behavioral dysregulation with Nf1 loss in Drosophila. These deficits map to primary dysfunction of a group of peripheral sensory neurons. Nf1 regulation of Ras signaling in adult ppk23(+) chemosensory cells is required for normal social behaviors in flies. Loss of Nf1 attenuates ppk23(+) neuronal activity in response to pheromones, and circuit-specific manipulation of Nf1 expression or neuronal activity in ppk23(+) neurons rescues social deficits. This disrupted sensory processing gives rise to persistent changes in behavior beyond the social interaction, indicating a sustained effect of an acute sensory misperception. Together these data identify a specific circuit mechanism through which Nf1 regulates social behaviors and suggest social deficits in NF1 arise from propagation of sensory misinformation.
Li, Y., Bai, P., Wei, L., Kang, R., Chen, L., Zhang, M., Tan, E. K. and Liu, W. (2020). Capsaicin Functions as Drosophila Ovipositional Repellent and Causes Intestinal Dysplasia. Sci Rep 10(1): 9963. PubMed ID: 32561812
Summary:
Plants generate a plethora of secondary compounds (toxins) that potently influence the breadth of the breeding niches of animals, including Drosophila. Capsaicin is an alkaloid irritant from hot chili peppers, and can act as a deterrent to affect animal behaviors, such as egg laying choice. However, the mechanism underlying this ovipositional avoidance remains unknown. This study reports that Drosophila females exhibit a robust ovipositional aversion to capsaicin. First, it was found that females were robustly repelled from laying eggs on capsaicin-containing sites. Second, genetic manipulations show that the ovipositional aversion to capsaicin is mediated by activation of nociceptive neurons expressing the painless gene. Finally, it was found that capsaicin compromised the health and lifespan of flies through intestinal dysplasia and oxidative innate immunity. Overall, this study suggests that egg-laying sensation converts capsaicin into an aversive behavior for female Drosophila, mirroring an adaptation to facilitate the survival and fitness of both parents and offspring.

Monday, August 24th - Cytoskeleton and Junctions

Dhanyasi, N., VijayRaghavan, K., Shilo, B. Z. and Schejter, E. D. (2020). Microtubules provide guidance cues for myofibril and sarcomere assembly and growth. Dev Dyn. PubMed ID: 32725855
Summary:
Muscle myofibrils and sarcomeres present exceptional examples of highly ordered cytoskeletal filament arrays, whose distinct spatial organization is an essential aspect of muscle cell functionality. This study utilized ultra-structural analysis to investigate the assembly of myofibrils and sarcomeres within developing myotubes of the indirect flight musculature of Drosophila. A temporal sequence composed of three major processes was identified: subdivision of the unorganized cytoplasm of nascent, multi-nucleated myotubes into distinct organelle-rich and filament-rich domains; initial organization of the filament-rich domains into myofibrils harboring nascent sarcomeric units; and finally, maturation of the highly-ordered pattern of sarcomeric thick (myosin-based) and thin (microfilament-based) filament arrays in parallel to myofibril radial growth. Significantly, organized microtubule arrays were present throughout these stages and exhibited dynamic changes in their spatial patterns consistent with instructive roles. Genetic manipulations confirm these notions, and imply specific and critical guidance activities of the microtubule-based cytoskeleton, as well as structural interdependence between the myosin- and actin-based filament arrays. These observations highlight a surprisingly significant, behind-the-scenes role for microtubules in establishment of myofibril and sarcomere spatial patterns and size, and provide a detailed account of the interplay between major cytoskeletal elements in generating these essential contractile myogenic units.
Lee, S. R., Hong, S. T. and Choi, K. W. (2020). Regulation of epithelial integrity and organ growth by Tctp and Coracle in Drosophila. PLoS Genet 16(6): e1008885. PubMed ID: 32559217
Summary:
Regulation of cell junctions is crucial for the integrity of epithelial tissues and organs. Cell junctions also play roles in controlling cell proliferation for organ growth. Translationally controlled tumor protein (TCTP) is a conserved protein involved in growth control, but its role in cell junctions is unknown. This study shows that Drosophila Tctp directly interacts with the septate junction protein Coracle (Cora) to regulate epithelial integrity and organ growth. Tctp localizes together with Cora in the epidermis of the embryo. Loss of Cora reduces the level of Tctp in the epidermis but not vice versa. cora/+ or Tctp/+ single heterozygotes develop normally to adulthood. However, double heterozygotes for cora and Tctp mutations show severe disruption of epithelia causing synthetic lethality in the embryo. Double knockdown of cora and Tctp in eye imaginal disc synergistically leads to disruption of the eye disc, resulting in a severe reduction or loss of eye and head. Conversely, double knockdown of cora and Tctp in wing disc causes overgrowth as well as cell death. Inhibition of cell death under this condition causes hyperplastic growth of the wing disc. Tctp also shows direct and functional interaction with Cora-associated factors like Yurt and Na+/K+-ATPase. This study suggests that proper levels of Tctp and Cora are essential for the maintenance of the Cora complex and the integrity of epithelia. These data also provide evidence that both Cora and Tctp are required to suppress overgrowth in developing wing.
Dobbelaere, J., Schmidt Cernohorska, M., Huranova, M., Slade, D. and Dammermann, A. (2020). Cep97 Is Required for Centriole Structural Integrity and Cilia Formation in Drosophila. Curr Biol. PubMed ID: 32589908
Summary:
Centrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues within any given tissue, their size is essentially constant. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel, how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrate. This studied Cep97 function in Drosophila melanogaster. Cep97 is shown to be essential for formation of full-length centrioles in multiple tissues of the fly. The microtubule deacetylase Sirt2 was identified as a Cep97 interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells, and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila, Cep97 appears to be only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, these results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis.
Hertzler, J. I., Simonovitch, S. I., Albertson, R. M., Weiner, A. T., Nye, D. M. R. and Rolls, M. M. (2020). Kinetochore proteins suppress neuronal microtubule dynamics and promote dendrite regeneration. Mol Biol Cell: mbcE20040237T. PubMed ID: 32673176
Summary:
Kinetochores connect centromeric chromatin to spindle microtubules during mitosis. Neurons are post-mitotic, so it was surprising to identify transcripts of structural kinetochore (KT) proteins and regulatory chromosome passenger complex (CPC) and spindle assembly checkpoint (SAC) proteins in Drosophila neurons after dendrite injury. To test whether these proteins function during dendrite regeneration, post-mitotic RNAi was performed and dendrites or axons were removed using laser microsurgery. Reduction of KT, CPC and SAC proteins decreased dendrite regeneration without affecting axon regeneration. To understand whether neuronal functions of these proteins rely on microtubules, microtubule behavior was analyzed in uninjured neurons. The number of growing plus, but not minus, ends increased in dendrites with reduced KT, CPC and SAC proteins, while axonal microtubules were unaffected. Increased dendritic microtubule dynamics was independent of DLK-mediated stress, but was rescued by concurrent reduction of γTubulin, the core microtubule nucleation protein. Reduction of γTubulin also rescued dendrite regeneration in backgrounds containing kinetochore RNAi transgenes. It is concluded that kinetochore proteins function post-mitotically in neurons to suppress dendritic microtubule dynamics by inhibiting nucleation.
Greig, J. and Bulgakova, N. A. (2020). Interplay between actomyosin and E-cadherin dynamics regulates cell shape in the Drosophila embryonic epidermis. J Cell Sci. PubMed ID: 32665321
Summary:
Precise regulation of cell shape is vital for building functional tissues. The mechanisms which lead to the formation of highly elongated anisotropic epithelial cells in the Drosophila epidermis were studied. This cell shape is the result of two counteracting mechanisms at the cell surface which regulate the degree of elongation: actomyosin, which inhibits cell elongation downstream of RhoA signalling, and intercellular adhesion, modulated via clathrin-mediated endocytosis of E-cadherin, which promotes cell elongation downstream of the GTPase Arf1. These two mechanisms do not act independently but are interconnected, with RhoA signalling reducing Arf1 recruitment to the plasma membrane. Additionally, cell adhesion itself regulates both mechanisms: p120-catenin, a regulator of intercellular adhesion, promotes the activity of both Arf1 and RhoA. Altogether, this study has uncover a complex network of interactions between cell-cell adhesion, the endocytic machinery, and the actomyosin cortex, and demonstrates how this network regulates cell shape in an epithelial tissue in vivo.
Wang, H., Guo, X., Wang, X., Wang, X. and Chen, J. (2020). Supracellular Actomyosin Mediates Cell-Cell Communication and Shapes Collective Migratory Morphology. iScience 23(6): 101204. PubMed ID: 32535019
Summary:
During collective cell migration, front cells tend to extend a predominant leading protrusion, which is rarely present in cells at the side or rear positions. Using Drosophila border cells (BCs) as a model system of collective migration, this study revealed the presence of a supracellular actomyosin network at the peripheral surface of BC clusters. The Myosin II-mediated mechanical tension was demonstrated as exerted by this peripheral supracellular network not only mediated cell-cell communication between leading BC and non-leading BCs but also restrained formation of prominent protrusions at non-leading BCs. Further analysis revealed that a cytoplasmic dendritic actin network that depends on the function of Arp2/3 complex interacted with the actomyosin network. Together, these data suggest that the outward pushing or protrusive force as generated from Arp2/3-dependent actin polymerization and the inward restraining force as produced from the supracellular actomyosin network together determine the collective and polarized morphology of migratory BCs.

Friday, August 21st - Disease Models

Fernandez-Cruz, I., Sanchez-Diaz, I., Narvaez-Padilla, V. and Reynaud, E. (2020). Rpt2 proteasome subunit reduction causes Parkinson's disease like symptoms in Drosophila. IBRO Rep 9: 65-77. PubMed ID: 32715147
Summary:
The dysfunction of the proteasome-ubiquitin system is commonly reported in several neurodegenerative diseases. Post mortem samples of brains of patients with Parkinson´s disease present cytoplasmic inclusions that are rich in proteins such as ubiquitin, Tau, and α-synuclein. In Parkinson´s disease, a specific reduction of some of the proteasome subunits has also been reported. However, the specific role of the different proteasome subunits in dopaminergic neuron degeneration has not been thoroughly explored. In this work, the Gal4/UAS system was used to test fourteen Drosophila melanogaster RNAi lines from the Bloomington Drosophila Stock Center. Each of these lines targets a different proteasome subunit. To identify the strains that were able to induce neurodegeneration, the expression of these lines was driven to the eye, and they were catagorized as a function of the extent of neurodegeneration that they induced. The targeted proteasomal subunits are conserved in mammals and therefore may be relevant to study proteasome related diseases. The RNAi line among the regulatory subunits with the most penetrant phenotype targeted the proteasomal subunit Rpt2 and its phenotypes were further characterized. Rpt2 knockdown in the Drosophila central nervous system reduced the activity of the proteasome, augmented the amount of insoluble ubiquitinated protein, and elicited motor and non-motor phenotypes that were similar to the ones found in Drosophila and other models for Parkinson's disease. When Rpt2 is silenced pan-neurally, third instar larvae have locomotion dysfunctions and die during pupation. Larval lethality was avoided using the Gal80-Gal4 system to induce the expression of the Rpt2 RNAi to dopaminergic neurons only after pupation. The reduction of Rpt2 in adult dopaminergic neurons causes reduced survival, hyperactivity, neurodegeneration, and sleep loss; probably recapitulating some of the sleep disorders that Parkinson's disease patients have before the appearance of locomotion disorders.
Fliedner, A., Kirchner, P., Wiesener, A., van de Beek, I., Waisfisz, Q., van Haelst, M., Scott, D. A., Lalani, S. R., Rosenfeld, J. A., Azamian, M. S., Xia, F., Dutra-Clarke, M., Martinez-Agosto, J. A., Lee, H., Noh, G. J., Lippa, N., Alkelai, A., Aggarwal, V., Agre, K. E., Gavrilova, R., Mirzaa, G. M., Straussberg, R., Cohen, R., Horist, B., Krishnamurthy, V., McWalter, K., Juusola, J., Davis-Keppen, L., Ohden, L., van Slegtenhorst, M., de Man, S. A., Ekici, A. B., Gregor, A., van de Laar, I. and Zweier, C. (2020). Variants in SCAF4 Cause a Neurodevelopmental Disorder and Are Associated with Impaired mRNA Processing. Am J Hum Genet. PubMed ID: 32730804
Summary:
RNA polymerase II interacts with various other complexes and factors to ensure correct initiation, elongation, and termination of mRNA transcription. One of these proteins is SR-related CTD-associated factor 4 (SCAF4), which is important for correct usage of polyA sites for mRNA termination. Using exome sequencing and international matchmaking, nine likely pathogenic germline variants were identified in SCAF4 including two splice-site and seven truncating variants, all residing in the N-terminal two thirds of the protein. Eight of these variants occurred de novo, and one was inherited. Affected individuals demonstrated a variable neurodevelopmental disorder characterized by mild intellectual disability, seizures, behavioral abnormalities, and various skeletal and structural anomalies. Paired-end RNA sequencing on blood lymphocytes of SCAF4-deficient individuals revealed a broad deregulation of more than 9,000 genes and significant differential splicing of more than 2,900 genes, indicating an important role of SCAF4 in mRNA processing. Knockdown of the SCAF4 ortholog CG4266 in the model organism Drosophila melanogaster resulted in impaired locomotor function, learning, and short-term memory. Furthermore, an increased number of active zones was observed in larval neuromuscular junctions, representing large glutamatergic synapses. These observations indicate a role of CG4266 in nervous system development and function and support the implication of SCAF4 in neurodevelopmental phenotypes. In summary, these data show that heterozygous, likely gene-disrupting variants in SCAF4 are causative for a variable neurodevelopmental disorder associated with impaired mRNA processing.
Cha, S. J., Han, Y. J., Choi, H. J., Kim, H. J. and Kim, K. (2020). Glutathione S-Transferase Rescues Motor Neuronal Toxicity in Fly Model of Amyotrophic Lateral Sclerosis. Antioxidants (Basel) 9(7). PubMed ID: 32674363
Summary:
Transactive response DNA-binding protein-43 (TDP-43; see Drosophila TDP-43) is involved in the pathology of familial and sporadic amyotrophic lateral sclerosis (ALS). TDP-43-mediated ALS models in mice, Drosophila melanogaster, and zebrafish exhibit dysfunction of locomotor function, defective neuromuscular junctions, and motor neuron defects. There is currently no effective cure for ALS, and the underlying mechanisms of TDP-43 in ALS remain poorly understood. In this study, a genetic screen was performed to identify modifiers of human TDP-43 (hTDP-43) in a Drosophila model, and glutathione S-transferase omega 2 (GstO2) was found to be involved in hTDP-43 neurotoxicity. GstO2 overexpressed on recovered defective phenotypes resulting from hTDP-43, including defective neuromuscular junction (NMJ) boutons, degenerated motor neuronal axons, and reduced larvae and adult fly locomotive activity, without modulating the levels of hTDP-43 protein expression. GstO2 modulated neurotoxicity by regulating reactive oxygen species (ROS) produced by hTDP-43 in the Drosophila model of ALS. These results demonstrated that GstO2 was a key regulator in hTDP-43-related ALS pathogenesis and indicated its potential as a therapeutic target for ALS.
Dubey, T., Gorantla, N. V., Chandrashekara, K. T. and Chinnathambi, S. (2020). Photodynamic exposure of Rose-Bengal inhibits Tau aggregation and modulates cytoskeletal network in neuronal cells. Sci Rep 10(1): 12380. PubMed ID: 32704015
Summary:
The intracellular Tau aggregates are known to be associated with Alzheimer's disease. The inhibition of Tau aggregation is an important strategy for screening of therapeutic molecules in Alzheimer's disease. Several classes of dyes possess a unique property of photo-excitation, which is applied as a therapeutic measure against numerous neurological dysfunctions. Rose Bengal is a Xanthene dye, which has been widely used as a photosensitizer in photodynamic therapy. The aggregation inhibition and disaggregation potency of Rose Bengal and photo-excited Rose Bengal were observed by in-vitro fluorescence, circular dichroism, and electron microscopy. Rose Bengal and photo-excited Rose Bengal induce minimal cytotoxicity in neuronal cells. In these studies, it was observed that Rose Bengal and photo-excited Rose Bengal modulate the cytoskeleton network of actin and tubulin. The immunofluorescence studies showed the increased filopodia structures after photo-excited Rose Bengal treatment. Furthermore, Rose Bengal treatment increases the connections between the cells. Rose Bengal and photo-excited Rose Bengal treatment-induced actin-rich podosome-like structures associated with cell membranes. The in-vivo studies on UAS E-14 Tau mutant Drosophila suggested that exposure to Rose Bengal and photo-excited Rose Bengal efficiency rescues the behavioural and memory deficit in flies. Thus, the overall results suggest that Rose Bengal could have a therapeutic potency against Tau aggregation.
Balakrishnan, M., Yu, S. F., Chin, S. M., Soffar, D. B., Windner, S. E., Goode, B. L. and Baylies, M. K. (2020). Cofilin Loss in Drosophila Muscles Contributes to Muscle Weakness through Defective Sarcomerogenesis during Muscle Growth. Cell Rep 32(3): 107893. PubMed ID: 32697999
Summary:
Sarcomeres, the fundamental contractile units of muscles, are conserved structures composed of actin thin filaments and myosin thick filaments. How sarcomeres are formed and maintained is not well understood. This study shows that knockdown of Drosophila cofilin (DmCFL), an actin depolymerizing factor, disrupts both sarcomere structure and muscle function. The loss of DmCFL also results in the formation of sarcomeric protein aggregates and impairs sarcomere addition during growth. The activation of the proteasome delays muscle deterioration in in this model. Furthermore, this study investigated how a point mutation in CFL2 that causes nemaline myopathy (NM) in humans affects CFL function and leads to the muscle phenotypes observed in vivo. These data provide significant insights to the role of CFLs during sarcomere formation, as well as mechanistic implications for disease progression in NM patients.
Groth, C., Vaid, P., Khatpe, A., Prashali, N., Ahiya, A., Andrejeva, D., Chakladar, M., Nagarkar, S., Paul, R., Eichenlaub, T., Herranz, H., Sridhar, T. S., Cohen, S. and Shashidhara, L. S. (2020). Genome Wide Screen for Context-Dependent Tumor Suppressors Identified Using in Vivo Models for Neoplasia in Drosophila. G3 (Bethesda). PubMed ID: 32737065
Summary:
This study reports on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. An independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified in this study are known to be functional in growth control pathways. A number of novel connections to Yki and EGFR driven tissue growth were identified, mostly unique to one of the two. Thus, resources provided in this study would be useful to all researchers who study negative regulators of growth during development and cancer in the context of activated EGFR and/or Yki and positive regulators of growth in the context of activated EGFR. Resources reported in this study are available freely for anyone to use.

Thursday, August 20th - Adult development

Kamiyama, T., Sun, W., Tani, N., Nakamura, A. and Niwa, R. (2020). Poly(A) Binding Protein Is Required for Nuclear Localization of the Ecdysteroidogenic Transcription Factor Molting Defective in the Prothoracic Gland of Drosophila melanogaster. Front Genet 11: 636. PubMed ID: 32676099
Summary:
In insects, ecdysteroids, like ecdysone and the more biologically-active derivative 20-hydroxyecdysone (20E), promote molting and metamorphosis. Ecdysone is biosynthesized in the prothoracic gland (PG), via several steps catalyzed by ecdysteroidogenic enzymes that are encoded by Halloween genes. The transcriptional regulatory mechanism of Halloween genes is still elusive. A previous study has found that the polyadenylated tail [poly(A)] deadenylation complex, called Carbon catabolite repressor 4-Negative on TATA (CCR4-NOT) regulates the expression of spookier (spok), which encodes one of the ecdysteroidogenic enzymes in the fruit fly Drosophila melanogaster. This study reports that poly(A) binding protein (Pabp) is involved in spok expression by regulating nuclear localization of the transcription factor molting defective (Mld). When pabp was knocked down specifically in the PG by transgenic RNAi, both spok mRNA and Spok protein levels were significantly reduced. In addition, the spok promoter-driven green fluorescence protein (GFP) signal was also reduced in the pabp-RNAi PG, suggesting that Pabp is involved in the transcriptional regulation of spok. Which transcription factors are responsible for Pabp-dependent transcriptional regulation was investigated. Among the transcription factors acting in the PG, focus was placed on the zinc-finger transcription factor Mld, as Mld is essential for spok transcription. Mld was localized in the nucleus of the control PG cells, while Mld abnormally accumulated in the cytoplasm of pabp-RNAi PG cells. From these results, it is proposed that Pabp regulates subcellular localization in the PG, specifically of the transcription factor Mld, in the context of ecdysone biosynthesis.
Kannangara, J. R., Henstridge, M. A., Parsons, L. M., Kondo, S., Mirth, C. K. and Warr, C. G. (2020). A New Role for Neuropeptide F Signaling in Controlling Developmental Timing and Body Size in Drosophila melanogaster. Genetics. PubMed ID: 32675276
Summary:
As juvenile animals grow, their behavior, physiology, and development need to be matched to environmental conditions to ensure they survive to adulthood. However, little is known about how behavior and physiology are integrated with development to achieve this outcome. Neuropeptides are prime candidates for achieving this due to their well-known signalling functions in controlling many aspects of behavior, physiology, and development in response to environmental cues. In the growing Drosophila larva, while several neuropeptides have been shown to regulate feeding behavior, and a handful to regulate growth, it is unclear if any of these play a global role in coordinating feeding behavior with developmental programs. This study demonstrates that Neuropeptide F Receptor (NPFR), best studied as a conserved regulator of feeding behavior from insects to mammals, also regulates development in Drosophila. Knocking down NPFR in the prothoracic gland, which produces the steroid hormone ecdysone, generates developmental delay and an extended feeding period, resulting in increased body size. These effects are due to decreased ecdysone production, as these animals have reduced expression of ecdysone biosynthesis genes and lower ecdysone titers. Moreover, these phenotypes can be rescued by feeding larvae food supplemented with ecdysone. Further, this study shows that NPFR negatively regulates the insulin signalling pathway in the prothoracic gland to achieve these effects. Taken together, these data demonstrate that NPFR signalling plays a key role in regulating animal development and may thus play a global role in integrating feeding behavior and development in Drosophila.
Duan, J., Zhao, Y., Li, H., Habernig, L., Gordon, M. D., Miao, X., Engstrom, Y. and Buttner, S. (2020). Bab2 Functions as an Ecdysone-Responsive Transcriptional Repressor during Drosophila Development. Cell Rep 32(4): 107972. PubMed ID: 32726635
Summary:
Drosophila development is governed by distinct ecdysone steroid pulses that initiate spatially and temporally defined gene expression programs. The translation of these signals into tissue-specific responses is crucial for metamorphosis, but the mechanisms that confer specificity to systemic ecdysone pulses are far from understood. This study identified Bric-á-brac 2 (Bab2) as an ecdysone-responsive transcriptional repressor that controls temporal gene expression during larval to pupal transition. Bab2 is necessary to terminate Salivary gland secretion (Sgs) gene expression, while premature Bab2 expression blocks Sgs genes and causes precocious salivary gland histolysis. The timely expression of bab2 is controlled by the ecdysone-responsive transcription factor Broad, and manipulation of EcR/USP/Broad signaling induces inappropriate Bab2 expression and termination of Sgs gene expression. Bab2 directly binds to Sgs loci in vitro and represses all Sgs genes in vivo. This work characterizes Bab2 as a temporal regulator of somatic gene expression in response to systemic ecdysone signaling.
He, Q., Zhang, Y. and Dong, W. (2020). MicroRNA miR-927 targets the juvenile hormone primary response gene Kruppel homolog1 to control Drosophila developmental growth. Insect Mol Biol. PubMed ID: 32715555
Summary:
Kruppel homolog1 (Kr-h1) is a juvenile hormone (JH) response transcriptional factor that transduces JH signaling to repress insect metamorphosis in both hemimetabolous and holometabolous insects. This study identified two miR-927 binding sites within the 3'UTR region of Kr-h1 in Drosophila melanogaster, and miR-927 was found to downregulate the expression of Kr-h1. The expression profiles of miR-927 and Kr-h1 displayed relatively opposite pattern during most of the larval development stages. Overexpression of miR-927 in the fat body significantly decreased the expression of Kr-h1 and resulted in reduced oviposition, increased mortality, delayed pupation, and reduced pupal size. Notably, the co-overexpression of Kr-h1 rescued the developmental and growth defects associated with miR-927 overexpression, indicating that Kr-h1 is a biologically relevant target of miR-927. Moreover, the expression of miR-927 was found to be repressed by JH and its receptor Met/gce, forming a positive regulatory loop of JH signaling. Overall, these studies support a conserved role for the JH/miRNA/Kr-h1 regulatory axis in growth control during insect development.
Carayon, A., Bataille, L., Lebreton, G., Dubois, L., Pelletier, A., Carrier, Y., Wystrach, A., Vincent, A. and Frendo, J. L. (2020). Intrinsic control of muscle attachment sites matching. Elife 9. PubMed ID: 32706334
Summary:
Myogenesis is an evolutionarily conserved process. Little known, however, is how the morphology of each muscle is determined, such that movements relying upon contraction of many muscles are both precise and coordinated. Each Drosophila larval muscle is a single multinucleated fiber whose morphology reflects expression of distinctive identity Transcription Factors (iTFs). By deleting transcription cis-regulatory modules of one iTF, Collier, this study generated viable muscle identity mutants, allowing live imaging and locomotion assays. Both selection of muscle attachment sites and muscle/muscle matching is intrinsic to muscle identity and requires transcriptional reprogramming of syncytial nuclei. Live-imaging shows that the staggered muscle pattern involves attraction to tendon cells and heterotypic muscle-muscle adhesion. Unbalance leads to formation of branched muscles, and this correlates with locomotor behavior deficit. Thus, engineering Drosophila muscle identity mutants allows investigation, in vivo, of physiological and mechanical properties of abnormal muscles.
Ghosh, S., Ghosh, S. and Mandal, L. (2020). Drosophila metamorphosis involves hemocyte mediated macroendocytosis and efferocytosis. Int J Dev Biol 64(4-5-6): 329-339. PubMed ID: 32658992
Summary:
Drosophila hemocytes are majorly associated with immune responses, but they also undertake several non-immune functions that are crucial during various stages of development. The activity and behaviour of hemocytes are least documented during the metamorphic phase of fly development. This study describes the activity, form and behaviour of the most abundant type of hemocyte in Drosophila melanogaster, the "plasmatocyte," throughout pupal development. This study reveals different forms of plasmatocytes laden with varying degrees of histolyzing debris (muscle and fat) which extend beyond the size of the cell itself, highlighting the phagocytic capacity of these plasmatocytes. Interestingly, the engulfment of apoptotic debris by plasmatocytes is an actin-dependent process, and by the end of metamorphosis, clearance is achieved. The uptake of apoptotic debris consisting of muscles and lipids by the plasmatocytes provides a model that can be employed to dissect out the relevant components of macroendocytosis and lipid-loaded phagocytosis. This understanding, by itself, is crucial for addressing the emerging role of phagocytes in physiology and pathophysiology.

Wednesday August 19th - Embryonic development

Aviles-Pagan, E. E., Kang, A. S. W. and Orr-Weaver, T. L. (2020). Identification of New Regulators of the Oocyte-to-Embryo Transition in Drosophila. G3 (Bethesda). PubMed ID: 32690584
Summary:
At the oocyte-to-embryo transition the highly differentiated oocyte arrested in meiosis becomes a totipotent embryo capable of embryogenesis. Oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest and the trigger for the oocyte-to-embryo transition) serve as prerequisites for this transition, both events being controlled posttranscriptionally. Recently, a comprehensive list of proteins was obtained whose levels are developmentally regulated during these events via a high-throughput quantitative proteomic analysis of Drosophila melanogaster oocyte maturation and egg activation. A targeted screen was conducted for potential novel regulators of the oocyte-to-embryo transition, selecting 53 candidates from these proteins. The function of each candidate gene was reduced using transposable element insertion alleles and RNAi, and defects in oocyte maturation or early embryogenesis were screened. Deletion of the aquaporin gene CG7777 did not affect female fertility. However, CG5003 and nebu (CG10960) were identified as new regulators of the transition from oocyte to embryo. Mutations in CG5003, which encodes an F-box protein associated with SCF-proteasome degradation function, cause a decrease in female fertility and early embryonic arrest. Mutations in nebu, encoding a putative glucose transporter, result in defects during the early embryonic divisions, as well as a developmental delay and arrest. nebu mutants also exhibit a defect in glycogen accumulation during late oogenesis. These findings highlight potential previously unknown roles for the ubiquitin protein degradation pathway and sugar transport across membranes during this time, and paint a broader picture of the underlying requirements of the oocyte-to-embryo transition.
Jeon, H., Gim, S., Na, H. and Choe, C. P. (2020). A pair-rule function of odd-skipped in germband stages of Tribolium development. Dev Biol. PubMed ID: 32687895
Summary:
While pair-rule patterning has been observed in most insects examined, the orthologs of Drosophila pair-rule genes have shown divergent roles in insect segmentation. In the beetle Tribolium castaneum, while odd-skipped (Tc-odd) was expressed as a series of pair-rule stripes, RNAi-mediated knockdown of Tc-odd (Tc-odd(RNAi)) resulted in severely truncated, almost asegmental phenotypes rather than the classical pair-rule phenotypes observed in germbands and larval cuticles. However, considering that most segments arise later in germband stages of Tribolium development, the roles of Tc-odd in segmentation of growing germbands could not be analyzed properly in the truncated Tc-odd(RNAi) germbands. This study investigated the segmentation function of Tc-odd in germband stages of Tribolium development by analyzing Tc-odd(RNAi) embryos that resumed germband extension. In the larval cuticles of Tc-odd(RNAi) embryos, normal mandibular and maxillary and loss of the labial segments were consistent in the head, whereas a broad range of segmentation defects including loss or fusion of thoracic and/or abdominal segments was observed in the trunk. Interestingly, a group of Tc-odd(RNAi) germbands showed pair-rule-like defects in the segmental stripes of the segment-polarity genes, engrailed, hedgehog, or wingless, in the abdominal regions. While the pair-rule genes even-skipped, runt, odd, and paired were misregulated in the growing Tc-odd(RNAi) germbands, paired expression required for odd-numbered segment formation was largely abolished, which might cause the pair-rule-like defects. Taken together, these findings suggest that Tc-odd can function as a pair-rule gene in the germband stages of Tribolium development.
Cao, W. X., Kabelitz, S., Gupta, M., Yeung, E., Lin, S., Rammelt, C., Ihling, C., Pekovic, F., Low, T. C. H., Siddiqui, N. U., Cheng, M. H. K., Angers, S., Smibert, C. A., Wuhr, M., Wahle, E. and Lipshitz, H. D. (2020). Precise Temporal Regulation of Post-transcriptional Repressors Is Required for an Orderly Drosophila Maternal-to-Zygotic Transition. Cell Rep 31(12): 107783. PubMed ID: 32579915
Summary:
In animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. The maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster's genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT.
Krueger, D., Pallares Cartes, C., Makaske, T. and De Renzis, S. (2020). betaH-spectrin is required for ratcheting apical pulsatile constrictions during tissue invagination. EMBO Rep: e49858. PubMed ID: 32588528
Summary:
Actomyosin-mediated apical constriction drives a wide range of morphogenetic processes. Activation of myosin-II initiates pulsatile cycles of apical constrictions followed by either relaxation or stabilization (ratcheting) of the apical surface. While relaxation leads to dissipation of contractile forces, ratcheting is critical for the generation of tissue-level tension and changes in tissue shape. How ratcheting is controlled at the molecular level is unknown. This study shows that the actin crosslinker βH-spectrin is upregulated at the apical surface of invaginating mesodermal cells during Drosophila gastrulation. βH-spectrin forms a network of filaments which co-localize with medio-apical actomyosin fibers, in a process that depends on the mesoderm-transcription factor Twist and activation of Rho signaling. βH-spectrin knockdown results in non-ratcheted apical constrictions and inhibition of mesoderm invagination, recapitulating twist mutant embryos. βH-spectrin is thus a key regulator of apical ratcheting during tissue invagination, suggesting that actin cross-linking plays a critical role in this process.
Chen, P., Visokay, S. and Abrams, J. M. (2020). Drosophila GFAT1 and GFAT2 enzymes encode obligate developmental functions. Fly (Austin): 1-7. PubMed ID: 32615907
Summary:
Glutamine: fructose-6-phosphate amidotransferase (GFAT) enzymes catalyse the first committed step of the hexosamine biosynthesis pathway (HBP) using glutamine and fructose-6-phosphate to form glucosamine-6-phosphate (GlcN6P). Numerous species (e.g. mouse, rat, zebrafish, chicken) including humans and Drosophila encode two broadly expressed copies of this enzyme but whether these perform redundant, partially overlapping or distinct functions is not known. To address this question, single gene null mutations were produced in the fly counterparts of gfat1 and gfat2. Deletions for either enzyme were fully lethal and homozygotes lacking either GFAT1 or GFAT2 died at or prior to the first instar larval stage. Therefore, when genetically eliminated, neither isoform was able to compensate for the other. Importantly, dietary supplementation with D-glucosamine-6-phosphate rescued GFAT2 deficiency and restored viability to gfat2(-/-) mutants. In contrast, glucosamine-6-phosphate did not rescue gfat1(-/-) animals.
Nandy, N. and Roy, J. K. (2020). Rab11 is essential for lgl mediated JNK-Dpp signaling in dorsal closure and epithelial morphogenesis in Drosophila. Dev Biol 464(2): 188-201. PubMed ID: 32562757
Summary:
Dorsal closure during Drosophila embryogenesis provides a robust genetic platform to study the basic cellular mechanisms that govern epithelial wound healing and morphogenesis. JNK-Dpp signaling in the dorsolateral epidermis, plays an instrumental role in guiding their fate during this process. A large array of genes have been reported to be involved in the regulation of this core signaling pathway, yet the mechanisms by which they do so is hitherto unclear, which forms the objective of the present study. This study shows a probable mechanism via which lgl, a conserved tumour suppressor gene, regulates the JNK-Dpp pathway during dorsal closure and epithelial morphogenesis. A conditional/targeted knock-down of lgl in the dorsolateral epithelium of embryos results in failure of dorsal closure. Interestingly, a similar phenotype was also observed in a Rab11 knockdown condition. This experiment suggests Rab11 to be interacting with lgl as they seem to synergize in order to regulate the core JNK-Dpp signaling pathway during dorsal closure and also during adult thorax closure process.

Tuesday August 18th, Adult Neural Development and Function

Herrero, A., Yoshii, T., Ispizua, J. I., Colque, C., Veenstra, J. A., Muraro, N. I. and Ceriani, M. F. (2020). Coupling Neuropeptide Levels to Structural Plasticity in Drosophila Clock Neurons. Curr Biol. PubMed ID: 32619484
Summary:
Previous work has reported that pigment dispersing factor (PDF) neurons, which are essential in the control of rest-activity cycles in Drosophila, undergo circadian remodeling of their axonal projections, a phenomenon called circadian structural plasticity. Axonal arborizations display higher complexity during the day and become simpler at night, and this remodeling involves changes in the degree of connectivity. This phenomenon depends on the clock present within the ventrolateral neurons (LNvs) as well as in glia. This work characterized in detail the contribution of the PDF neuropeptide to structural plasticity at different times across the day. Using diverse genetic strategies to temporally restrict its downregulation, it was demonstrated that even subtle alterations to PDF cycling at the dorsal protocerebrum correlate with impaired remodeling, underscoring its relevance for the characteristic morning spread; PDF released from the small LNvs (sLNvs) and the large LNvs (lLNvs) contribute to the process. Moreover, forced depolarization recruits activity-dependent mechanisms to mediate growth only at night, overcoming the restriction imposed by the clock on membrane excitability. Interestingly, the active process of terminal remodeling requires PDF receptor (PDFR) signaling acting locally through the cyclic-nucleotide-gated channel ion channel subunit A (CNGA). Thus, clock-dependent PDF signaling shapes the connectivity of these essential clock neurons on daily basis.
Hidalgo, S., Fuenzalida-Uribe, N., Molina-Mateo, D., Escobar, A. P., Oliva, C., Espana, R. A., Andres, M. E. and Campusano, J. M. (2020). Study of the release of endogenous amines in Drosophila brain in vivo in response to stimuli linked to aversive olfactory conditioning. J Neurochem. PubMed ID: 32596813
Summary:
A highly challenging question in neuroscience is to understand how aminergic neural circuits contribute to the planning and execution of behaviors, including the generation of olfactory memories. In this regard, electrophysiological techniques like Local Field Potential or imaging methods have been used to answer questions relevant to cell and circuit physiology in different animal models, such as the fly Drosophila melanogaster. However, these techniques do not provide information on the neurochemical identity of the circuits of interest. Different approaches including fast scan cyclic voltammetry, allow researchers to identify and quantify in a timely fashion the release of endogenous neuroactive molecules, but have been only used in in vitro Drosophila brain preparations. This study report a procedure to record for the first time the release of endogenous amines -dopamine, serotonin and octopamine- in adult fly brain in vivo, by fast scan cyclic voltammetry. As a proof of principle, recordings were carried out in the calyx region of the Mushroom Bodies, the brain area mainly associated to the generation of olfactory memories in flies. By using principal component regression in normalized training sets for in vivo recordings, an increase was detected in octopamine and serotonin levels in response to electric shock and olfactory cues respectively. This new approach allows the study of dynamic changes in amine neurotransmission that underlie complex behaviors in Drosophila and sheds new light on the contribution of these amines to olfactory processing in this animal model.
Diaz-de-la-Pena, L., Maestro-Paramio, L., Diaz-Benjumea, F. J. and Herrero, P. (2020). Temporal groups of lineage-related neurons have different neuropeptidergic fates and related functions in the Drosophila melanogaster CNS. Cell Tissue Res. PubMed ID: 32556724
Summary:
The central nervous system (CNS) of Drosophila is comprised of the brain and the ventral nerve cord (VNC), which are the homologous structures of the vertebrate brain and the spinal cord, respectively. Neurons of the CNS arise from neural stem cells called neuroblasts (NBs). Each neuroblast gives rise to a specific repertory of cell types whose fate is unknown in most lineages. A combination of spatial and temporal genetic cues defines the fate of each neuron. The origin and specification was studied of a group of peptidergic neurons present in several abdominal segments of the larval VNC that are characterized by the expression of the neuropeptide GPB5, the GPB5-expressing neurons (GPB5-ENs). The data reveal that the progenitor NB that generates the GPB5-ENs also generates the abdominal leucokinergic neurons (ABLKs) in two different temporal windows. This study also shows that these two set of neurons share the same axonal projections in larvae and in adults and, as previously suggested, may both function in hydrosaline regulation. Tenetic analysis of potential specification determinants reveals that Klumpfuss (klu) and huckebein (hkb) are involved in the specification of the GPB5 cell fate. Additionally, GPB5-ENs have a role in starvation resistance and longevity; however, their role in desiccation and ionic stress resistance is not as clear. It is hypothesize that the neurons arising from the same neuroblast lineage are both architecturally similar and functionally related.
Coates, K. E., Calle-Schuler, S. A., Helmick, L. M., Knotts, V. L., Martik, B. N., Salman, F., Warner, L. T., Valla, S. V., Bock, D. D. and Dacks, A. M. (2020). The wiring logic of an identified serotonergic neuron that spans sensory networks. J Neurosci. PubMed ID: 32641403
Summary:
Serotonergic neurons project widely throughout the brain to modulate diverse physiological and behavioral processes. However, a single cell resolution understanding of the connectivity of serotonergic neurons is currently lacking. Using a whole-brain EM dataset of a female Drosophila, this study comprehensively determine the wiring logic of a broadly projecting serotonergic neuron (the "CSDn") that spans several olfactory regions. Within the antennal lobe, the CSDn differentially innervates each glomerulus, yet surprisingly, this variability reflects a diverse set of presynaptic partners, rather than glomerulus-specific differences in synaptic output, which is predominately to local interneurons. Moreover, the CSDn has distinct connectivity relationships with specific local interneuron subtypes, suggesting that the CSDn influences distinct aspects of local network processing. Across olfactory regions, the CSDn has different patterns of connectivity, even having different connectivity with individual projection neurons that also span these regions. Whereas the CSDn targets inhibitory local neurons in the antennal lobe, the CSDn has more distributed connectivity in the LH, preferentially synapsing with principal neuron types based on transmitter content. Lastly, this study identify individual novel synaptic partners associated with other sensory domains that provide strong, top-down input to the CSDn. Taken together, this study reveals the complex connectivity of serotonergic neurons which combine the integration of local and extrinsic synaptic input in a nuanced, region-specific manner.
Guven-Ozkan, T., Busto, G. U., Jung, J. Y., Drago, I. and Davis, R. L. (2020). miR-92a Suppresses Mushroom Body-Dependent Memory Consolidation in Drosophila. eNeuro. PubMed ID: 32737186
Summary:
MicroRNAs fine tune gene expression to regulate many aspects of nervous system physiology. This study shows that miR-92a suppresses memory consolidation that occurs in the αβ and γ mushroom body neurons of Drosophila, making miR-92a a memory suppressor microRNA. Bioinformatics analyses suggested that mRNAs encoding kinesin heavy chain 73 (Khc73), a protein that belongs to Kinesin-3 family of anterograde motor proteins, may be a functional target of miR-92a. Behavioral studies that employed expression of khc73 with and without its 3'UTR containing miR-92a target sites, luciferase assays in HEK cells with reporters containing wild-type and mutant target sequences in the miR-92a 3' UTR, and immunohistochemistry experiments involving Khc73 expression with and without the wild-type khc73 3'UTR all point to the conclusion that khc73 is a major target of miR-92a in its functional role as a microRNA memory suppressor gene.
Goncalves-Pimentel, C., Mazaud, D., Kottler, B., Proelss, S., Hirth, F. and Fanto, M. (2020). A miRNA screen procedure identifies garz as an essential factor in adult glia functions and validates Drosophila as a beneficial 3Rs model to study glial functions and GBF1 biology. F1000Res 9: 317. PubMed ID: 32595956
Summary:
To study the influence of adult glial cells in ageing flies, a genetic screen was performed in Drosophila using a collection of transgenic lines providing conditional expression of micro-RNAs (miRNAs). This study describes a methodological algorithm to identify and rank genes that are candidate to be targeted by miRNAs that shorten lifespan when expressed in adult glia. Four different databases were used for miRNA target prediction in Drosophila but little agreement was found between them, overall. However, top candidate gene analysis shows potential to identify essential genes involved in adult glial functions. One example from the top candidates' analysis is gartenzwerg (garz). It was established that garz is necessary in many glial cell types, that it affects motor behaviour and, at the sub-cellular level, is responsible for defects in cellular membranes, autophagy and mitochondria quality control. The remarkable conservation of functions between garz and its mammalian orthologue, GBF1, was verified validating the use of Drosophila as an alternative 3Rs-beneficial model to knock-out mice for studying the biology of GBF1, potentially involved in human neurodegenerative diseases.

Monday, August 17th - Evolution

Avalos, A., Fang, M., Pan, H., Ramirez Lluch, A., Lipka, A. E., Zhao, S. D., Giray, T., Robinson, G. E., Zhang, G. and Hudson, M. E. (2020). Genomic regions influencing aggressive behavior in honey bees are defined by colony allele frequencies. Proc Natl Acad Sci U S A 117(29): 17135-17141. PubMed ID: 32631983
Summary:
For social animals, the genotypes of group members affect the social environment, and thus individual behavior, often indirectly. This study used genome-wide association studies (GWAS) to determine the influence of individual vs. group genotypes on aggression in honey bees. Aggression in honey bees arises from the coordinated actions of colony members, primarily nonreproductive "soldier" bees, and thus, experiences evolutionary selection at the colony level. This study shows that individual behavior is influenced by colony environment, which in turn, is shaped by allele frequency within colonies. Using a population with a range of aggression, individual whole genomes were sequenced and for genotype-behavior associations were looked for within colonies in a common environment. There were no significant correlations between individual aggression and specific alleles. By contrast, strong correlations were found between colony aggression and the frequencies of specific alleles within colonies, despite a small number of colonies. Associations at the colony level were highly significant and were very similar among both soldiers and foragers, but they covaried with one another. One strongly significant association peak, containing an ortholog of the Drosophila sensory gene dpr4 on linkage group (chromosome) 7, showed strong signals of both selection and admixture during the evolution of gentleness in a honey bee population. Links were thus found between colony genetics and group behavior and also, molecular evidence was found for group-level selection, acting at the colony level. It is concluded that group genetics dominates individual genetics in determining the fatal decision of honey bees to sting.
Fuller, Z. L., Koury, S. A., Leonard, C. J., Young, R. E., Ikegami, K., Westlake, J., Richards, S., Schaeffer, S. W. and Phadnis, N. (2020). Extensive Recombination Suppression and Epistatic Selection Causes Chromosome-Wide Differentiation of a Selfish Sex Chromosome in Drosophila pseudoobscura. Genetics. PubMed ID: 32732371
Summary:
Sex-Ratio (SR) chromosomes are selfish X-chromosomes that distort Mendelian segregation and are commonly associated with inversions. These chromosomal rearrangements suppress recombination with Standard (ST) X-chromosomes and are hypothesized to maintain multiple alleles important for drive in a single large haplotype. A multifaceted study was conducted of the multiply inverted Drosophila pseudoobscura SR chromosome to understand the evolutionary history, genetic architecture, and present-day dynamics that shape this enigmatic selfish chromosome. The D. pseudoobscura SRchromosome has three non-overlapping inversions: basal, medial, and terminal. 23 of 29 Mb of the D. pseudoobscura XR chromosome arm is highly differentiated between the Standard (ST) and SR arrangements, including a 6.6Mb collinear region between the medial and terminal inversions. Although crossing-over is heavily suppressed on this chromosome arm, it is not completely eliminated, with measured rates indicating recombination suppression alone cannot explain patterns of differentiation or the near-perfect association of the three SR chromosome inversions in nature. The ancient basal and medial inversions of the SR chromosome were demonstrated contain genes sufficient to cause weak drive. In contrast, the younger terminal inversion cannot drive by itself, but contains at least one modifier gene necessary for full manifestation of strong sex chromosome drive. By parameterizing population genetic models for chromosome-wide linkage disequilibrium with the experimental results, it is inferred that strong selection acts to maintain the near-perfect association of SR chromosome inversions in present day populations. It is concluded the combined action of suppressed recombination and strong, ongoing, epistatic selection shape the D. pseudoobscura SR arrangement into a highly differentiated chromosome.
McBroome, J., Liang, D. and Corbett-Detig, R. (2020). Fine-scale position effects shape the distribution of inversion breakpoints in Drosophila melanogaster. Genome Biol Evol. PubMed ID: 32437518
Summary:
Chromosomal inversions are among the primary drivers of genome structure evolution in a wide range of natural populations. While there is an impressive array of theory and empirical analyses that have identified conditions under which inversions can be positively selected, comparatively little data is available on the fitness impacts of these genome structural rearrangements themselves. Because inversion breakpoints can disrupt functional elements and alter chromatin domains, the precise positioning of an inversion's breakpoints can strongly affect its fitness. This study compared the fine-scale distribution of low frequency inversion breakpoints with those of high frequency inversions and inversions that have gone to fixation between Drosophila species. A number of differences were identified among frequency classes that may influence inversion fitness. In particular, breakpoints that are proximal to insulator elements, generate large tandem duplications, and minimize impacts on gene coding spans are more prevalent in high frequency and fixed inversions than in rare inversions. The data suggest that natural selection acts to preserve both genes and larger cis-regulatory networks in the occurrence and spread of rearrangements. These factors may act to limit the availability of high fitness arrangements when suppressed recombination is favorable.
Qu, Z., Yiu, W. C....., Bendena, W. G. and Hui, J. H. L. (2020). MicroRNA clusters integrate evolutionary constraints on expression and target affinities: the miR-6/5/4/286/3/309 cluster in Drosophila. Mol Biol Evol. PubMed ID: 32521021
Summary:
A striking feature of microRNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. This study investigated a microRNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this microRNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of microRNA cluster members were also constructed. Expression of individual microRNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient microRNAs together (miR-5/4/286/3/309) or more recently evolved clustered microRNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed down-regulation of leg patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of microRNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct microRNAs. Considered together, the microRNA targets and the evolutionary ages of each microRNA in the cluster demonstrates the importance of microRNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing microRNAs (Zu, 2020).
Cridland, J. M., Majane, A. C., Sheehy, H. K. and Begun, D. J. (2020). Polymorphism and Divergence of Novel Gene Expression Patterns in Drosophila melanogaster. Genetics. PubMed ID: 32737121
Summary:
Transcriptomes may evolve by multiple mechanisms, including the evolution of novel genes, the evolution of transcript abundance, and the evolution of cell, tissue, or organ expression patterns. This study focused on the last of these mechanisms in an investigation of tissue and organ shifts in gene expression in Drosophila melanogaster. In contrast to most investigations of expression evolution, this study sought to provide a framework for understanding the mechanisms of novel expression patterns on a short population genetic timescale. To do so population samples of D. melanogaster transcriptomes were generated from five tissues: accessory gland, testis, larval salivary gland, female head, and first instar larva. These data were combined with comparable data from two outgroups to characterize gains and losses of expression, both polymorphic and fixed, in D. melanogaster. A large number of gain or loss of expression phenotypes were observed, most of which were polymorphic within D. melanogaster. Several polymorphic, novel expression phenotypes were strongly influenced by segregating cis-acting variants. In support of previous literature on the evolution of novelties functioning in male reproduction, many more novel expression phenotypes were found in the testis and accessory gland than in other tissues. Additionally, genes showing novel expression phenotypes tend to exhibit greater tissue specific expression. Finally, in addition to qualitatively novel expression phenotypes, genes exhibiting major quantitative expression divergence in the D. melanogaster lineage were identified.
Davis, H., Sosulski, N. and Civetta, A. (2020). Reproductive isolation caused by azoospermia in sterile male hybrids of Drosophila. Ecol Evol 10(12): 5922-5931. PubMed ID: 32607201
Summary:
Recently diverged populations in the early stages of speciation offer an opportunity to understand mechanisms of isolation and their relative contributions. Drosophila willistoni is a tropical species with broad distribution from Argentina to the southern United States, including the Caribbean islands. A postzygotic barrier between northern populations (North America, Central America, and the northern Caribbean islands) and southern populations (South American and the southern Caribbean islands) has been recently documented and used to propose the existence of two different subspecies. This study has identified premating isolation between populations regardless of their subspecies status. No evidence of postmating prezygotic isolation was found, and this study proceeded to characterize hybrid male sterility between the subspecies. Sterile male hybrids transfer an ejaculate that is devoid of sperm but causes elongation and expansion of the female uterus. In sterile male hybrids, bulging of the seminal vesicle appears to impede the movement of the sperm toward the sperm pump, where sperm normally mixes with accessory gland products. The results highlight a unique form of hybrid male sterility in Drosophila that is driven by a mechanical impediment to transfer sperm rather than by an abnormality of the sperm itself. Interestingly, this form of sterility is reminiscent of a form of infertility (azoospermia) that is caused by lack of sperm in the semen due to blockages that impede the sperm from reaching the ejaculate.

Friday, August 14th - Behavior

Dore, A. A., Bretman, A. and Chapman, T. (2020). Fitness consequences of redundant cues of competition in male Drosophila melanogaster. Ecol Evol 10(12): 5517-5526. PubMed ID: 32607171
Summary:
Phenotypic plasticity can allow animals to adapt their behavior, such as their mating effort, to their social and sexual environment. However, this relies on the individual receiving accurate and reliable cues of the environmental conditions. This can be achieved via the receipt of multimodal cues, which may provide redundancy and robustness. Male Drosophila melanogaster detect presence of rivals via combinations of any two or more redundant cue components (sound, smell, and touch) and respond by extending their subsequent mating duration, which is associated with higher reproductive success. Although alternative combinations of cues of rival presence have previously been found to elicit equivalent increases in mating duration and offspring production, their redundancy in securing success under sperm competition has not previously been tested. This was explicitly tested by exposing male D. melanogaster to alternative combinations of rival cues, and examine reproductive success in both the presence and absence of sperm competition. The results supported previous findings of redundancy of cues in terms of behavioral responses. However, there was no evidence of reproductive benefits accrued by extending mating duration in response to rivals. The lack of identifiable fitness benefits of longer mating under these conditions, both in the presence and absence of sperm competition, contrasted with some previous results, but could be explained by (a) damage sustained from aggressive interactions with rivals leading to reduced ability to increase ejaculate investment, (b) presence of features of the social environment, such as male and female mating status, that obscured the fitness benefits of longer mating, and (c) decoupling of behavioral investment with fitness benefits.
Mishra, A., Tung, S., Shree Sruti, V. R., Srivathsa, S. V. and Dey, S. (2020). Mate-finding dispersal reduces local mate limitation and sex bias in dispersal. J Anim Ecol. PubMed ID: 32535925
Summary:
Sex-biased dispersal (SBD) often skews the local sex ratio in a population. This can result in a shortage of mates for individuals of the less-dispersive sex. Such mate limitation can lead to Allee effects in populations that are small or undergoing range expansion, consequently affecting their survival, growth, stability and invasion speed. Theory predicts that mate shortage can lead to either an increase or a decrease in the dispersal of the less-dispersive sex. However, neither of these predictions have been empirically validated. To investigate how SBD-induced mate limitation affects dispersal of the less-dispersive sex, Drosophila melanogaster populations with varying dispersal propensities were used. To rule out any mate-independent density effects, the behavioral plasticity of dispersal was examined in presence of mates as well as same-sex individuals with differential dispersal capabilities. In the presence of high-dispersive mates, the dispersal of both male and female individuals was significantly increased. However, the magnitude of this increase was much larger in males than in females, indicating that the former show greater mate-finding dispersal. Moreover, the dispersal of either sex did not change when dispersing alongside high- or low-dispersive individuals of the same sex. This suggested that the observed plasticity in dispersal was indeed due to mate-finding dispersal, and not mate-independent density effects. Strong mate-finding dispersal can diminish the magnitude of sex bias in dispersal. This can modulate the evolutionary processes that shape range expansions and invasions, depending on the population size. In small populations, mate-finding dispersal can ameliorate Allee effects. However, in large populations, it can dilute the effects of spatial sorting.
Park, S. J. and Ja, W. W. (2020). Absolute ethanol intake predicts ethanol preference in Drosophila melanogaster. J Exp Biol 223(Pt 11). PubMed ID: 32366685
Summary:
Factors that mediate ethanol preference in Drosophila melanogaster are not well understood. A major confound has been the use of diverse methods to estimate ethanol consumption. This study measured fly consumptive ethanol preference on base diets varying in nutrients, taste and ethanol concentration. Both sexes showed an ethanol preference that was abolished on high nutrient concentration diets. Additionally, manipulating total food intake without altering the nutritive value of the base diet or the ethanol concentration was sufficient to evoke or eliminate ethanol preference. Absolute ethanol intake and food volume consumed were stronger predictors of ethanol preference than caloric intake or the dietary caloric content. These findings suggest that the effect of the base diet on ethanol preference is largely mediated by total consumption associated with the delivery medium, which ultimately determines the level of ethanol intake. It is speculated that a physiologically relevant threshold for ethanol intake is essential for preferential ethanol consumption.
Dombrovski, M., Kuhar, R., Mitchell, A., Shelton, H. and Condron, B. (2020). Cooperative foraging during larval stage affects fitness in Drosophila. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 206(5): 743-755. PubMed ID: 32623493
Summary:
Cooperative behavior can confer advantages to animals. This is especially true for cooperative foraging which provides fitness benefits through more efficient acquisition and consumption of food. This study has taken advantage of an experimental model system featuring cooperative foraging behavior in Drosophila. Under crowded conditions, fly larvae form coordinated digging groups (clusters), where individuals are linked together by sensory cues and group membership requires prior experience. However, fitness benefits of Drosophila larval clustering remain unknown. This study demonstrates that animals raised in crowded conditions on food partially processed by other larvae experience a developmental delay presumably due to the decreased nutritional value of the substrate. Intriguingly, same conditions promote the formation of cooperative foraging clusters which further extends larval stage compared to non-clustering animals. Remarkably, this developmental retardation also results in a relative increase in wing size, serving an indicator of adult fitness. Thus, this study finds that the clustering-induced developmental delay is accompanied by fitness benefits. Therefore, cooperative foraging, while delaying development, may have evolved to give Drosophila larvae benefits when presented with competition for limited food resources.
Melnattur, K., Zhang, B. and Shaw, P. J. (2020). Disrupting flight increases sleep and identifies a novel sleep-promoting pathway in Drosophila. Sci Adv 6(19): eaaz2166. PubMed ID: 32494708
Summary:
Sleep is plastic and is influenced by ecological factors and environmental changes. The mechanisms underlying sleep plasticity are not well understood. This study shows that manipulations that impair flight in Drosophila increase sleep as a form of sleep plasticity. Flight was disrupted by blocking the wing-expansion program, genetically disrupting flight, and by mechanical wing perturbations. A new sleep regulatory circuit was defined starting with specific wing sensory neurons, their target projection neurons in the ventral nerve cord, and the neurons they connect to in the central brain. In addition, a critical neuropeptide (Burs) and its receptor (Rickets) were found to link wing expansion and sleep. Disrupting flight activates these sleep-promoting projection neurons, as indicated by increased cytosolic calcium levels, and stably increases the number of synapses in their axonal projections. These data reveal an unexpected role for flight in regulating sleep and provide new insight into how sensory processing controls sleep need.
Rooke, R., Rasool, A., Schneider, J. and Levine, J. D. (2020). Drosophila melanogaster behaviour changes in different social environments based on group size and density. Commun Biol 3(1): 304. PubMed ID: 32533063
Summary:
Many organisms, when alone, behave differently from when they are among a crowd. Drosophila similarly display social behaviour and collective behaviour dynamics within groups not seen in individuals. In flies, these emergent behaviours may be in response to the global size of the group or local nearest-neighbour density. This study investigated i) which aspect of social life flies respond to: group size, density, or both and ii) whether behavioural changes within the group are dependent on olfactory support cells. Behavioural assays demonstrate that flies adjust their interactive behaviour to group size but otherwise compensate for density by achieving a standard rate of movement, suggesting that individuals are aware of the number of others within their group. Olfactory support cells are necessary for flies to behave normally in large groups. These findings shed insight into the subtle and complex life of Drosophila within a social setting.

Thursday, August 13th - Signaling

Blount, J. R., Libohova, K., Silva, G. M. and Todi, S. V. (2020). Isoleucine 44 Hydrophobic Patch Controls Toxicity of Unanchored, Linear Ubiquitin Chains through NF-kappaB Signaling. Cells 9(6). PubMed ID: 32580388
Summary:
Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. Previous work has shown that transgenic, unanchored poly-ubiquitin is not problematic in Drosophila melanogaster. In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. To further explore the notion of unanchored chain toxicity, this study examined when free poly-ubiquitin might become problematic. It was found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies,toxicity from untethered, linear chains was shown to be regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. It is concluded that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted.
Hasan, G. (2020). Surviving nutritional deprivation during development: neuronal intracellular calcium signaling is critical. Int J Dev Biol 64(1-2-3): 249-256. PubMed ID: 32659012
Summary:
Developing cells and tissues in a growing animal need to sense food quality and integrate this information with on-going time-bound developmental programs. The integration of metabolism with development requires cellular and systemic coordination. This laboratory has focused on Ca(2+) signaling arising from the release of Ca(2+) stored in the endoplasmic reticulum (ER), which triggers store-operated Ca(2+) entry. A role is described for ER-store Ca(2+) that operates at the cellular level in various classes of neurons, and eventually drives the systemic coordination required to survive and complete development under conditions of nutritional deprivation. In the model system Drosophila melanogaster, a paradigm was developed to induce nutritional stress during the larval stage and used pupariation as a read-out for development. Applying the vast genetic tool kit available in Drosophila to this paradigm, this study has uncovered novel roles for intracellular Ca (2+) signaling in regulating neuronal activity, at the level of transcription in glutamatergic neurons, and translation in neuropeptidergic neurons. Such regulation of cellular processes is critical for integrating information across a neural circuit at multiple levels, starting from the point of sensing systemic and environmental levels of amino acids to finally connecting with neuropeptide secreting neurons, that communicate with the prothoracic gland, an organ that makes the key developmental hormone, ecdysone. This work underscores the importance of ER-store Ca(2+) for neuronal health, with consequences for animal development.
Ahmed, S. M. H., Maldera, J. A., Krunic, D., Paiva-Silva, G. O., Penalva, C., Teleman, A. A. and Edgar, B. A. (2020). Fitness trade-offs incurred by ovary-to-gut steroid signalling in Drosophila. Nature. PubMed ID: 32641829
Summary:
Sexual dimorphism arises from genetic differences between male and female cells, and from systemic hormonal differences. How sex hormones affect non-reproductive organs is poorly understood, yet highly relevant to health given the sex-biased incidence of many diseases. This study reports that steroid signalling in Drosophila from the ovaries to the gut promotes growth of the intestine specifically in mated females, and enhances their reproductive output. The active ovaries of the fly produce the steroid hormone ecdysone, which stimulates the division and expansion of intestinal stem cells in two distinct proliferative phases via the steroid receptors EcR and Usp and their downstream targets Broad, Eip75B and Hr3. Although ecdysone-dependent growth of the female gut augments fecundity, the more active and more numerous intestinal stem cells also increase female susceptibility to age-dependent gut dysplasia and tumorigenesis, thus potentially reducing lifespan. This work highlights the trade-offs in fitness traits that occur when inter-organ signalling alters stem-cell behaviour to optimize organ size.
Canales Coutino, B., Cornhill, Z. E., Couto, A., Mack, N. A., Rusu, A. D., Nagarajan, U., Fan, Y. N., Hadjicharalambous, M. R., Castellanos Uribe, M., Burrows, A., Lourdusamy, A., Rahman, R., May, S. T. and Georgiou, M. (2020). A Genetic Analysis of Tumor Progression in Drosophila Identifies the Cohesin Complex as a Suppressor of Individual and Collective Cell Invasion. iScience 23(6): 101237. PubMed ID: 32629605
Summary:
Metastasis is the leading cause of death for patients with cancer. Consequently it is imperative to improve understanding of the molecular mechanisms that underlie progression of tumor growth toward malignancy. Advances in genome characterization technologies have been very successful in identifying commonly mutated or misregulated genes in a variety of human cancers. However, the difficulty in evaluating whether these candidates drive tumor progression remains a major challenge. Using the genetic amenability of Drosophila melanogaster, this study generated tumors with specific genotypes in the living animal and carried out a detailed systematic loss-of-function analysis to identify conserved genes that enhance or suppress epithelial tumor progression. This enabled the discovery of functional cooperative regulators of invasion and the establishment of a network of conserved invasion suppressors. This includes constituents of the cohesin complex (see Rad21), whose loss of function either promotes individual or collective cell invasion, depending on the severity of effect on cohesin complex function.
Galeone, A., Adams, J. M., Matsuda, S., Presa, M. F., Pandey, A., Han, S. Y., Tachida, Y., Hirayama, H., Vaccari, T., Suzuki, T., Lutz, C. M., Affolter, M., Zuberi, A. and Jafar-Nejad, H. (2020). Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation. Elife 9. PubMed ID: 32720893
Summary:
During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. Previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner. This study established the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and found, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. This study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.
Chang, Y. C., Tu, H., Huang, T. W., Xu, B. W. and Pi, H. (2020). Upregulated TNF/Eiger signaling mediates stem cell recovery and tissue homeostasis during nutrient resupply in Drosophila testis. Sci Rep 10(1): 11674. PubMed ID: 32669615
Summary:
Stem cell activity and cell differentiation is robustly influenced by the nutrient availability in the gonads. The signal that connects nutrient availability to gonadal stem cell activity remains largely unknown. This study shows that tumor necrosis factor Eiger (Egr) is upregulated in testicular smooth muscles as a response to prolonged protein starvation in Drosophila testis. While Egr is not essential for starvation-induced changes in germline and somatic stem cell numbers, Egr and its receptor Grindelwald influence the recovery dynamics of somatic cyst stem cells (CySCs) upon protein refeeding. Moreover, Egr is also involved in the refeeding-induced, ectopic expression of the CySC self-renewal protein and the accumulation of early germ cells. Egr primarily acts through the Jun N-terminal kinase (JNK) signaling in Drosophila. This study shows that inhibition of JNK signaling in cyst cells suppresses the refeeding-induced abnormality in both somatic and germ cells. In conclusion, this study reveals both beneficial and detrimental effects of Egr upregulation in the recovery of stem cells and spermatogenesis from prolonged protein starvation.

Wednesday, August 12th - Synapse and Vesicles

Romano, G., Klima, R. and Feiguin, F. (2020). TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity. BMC Biol 18(1): 82. PubMed ID: 32620127
Summary:
Mutations in the small RNA-binding protein TDP-43 lead to the formation of insoluble cytoplasmic aggregates that have been associated with the onset and progression of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting homeostasis of the motor system which is also characterized by aberrant expression of retrotransposable elements (RTEs). Although the TDP-43 function was shown to be required in the neurons and glia to maintain the organization of neuromuscular synapses and prevent denervation of the skeletal muscles, the molecular mechanisms involved in physiological dysregulation remain elusive. This issue was addressed using a null mutation of the TDP-43 Drosophila homolog, TBPH. Using genome-wide gene expression profiles, a strong upregulation of RTE expression in was detected TBPH-null Drosophila heads, while the genetic rescue of the TDP-43 function reverted these modifications. Furthermore, this study found that TBPH modulates the small interfering RNA (siRNA) silencing machinery responsible for RTE repression. Molecularly, it was observed that TBPH regulates the expression levels of Dicer-2 by direct protein-mRNA interactions in vivo. Accordingly, the genetic or pharmacological recovery of Dicer-2 activity was sufficient to repress retrotransposon activation and promote motoneuron axonal wrapping and synaptic growth in TBPH-null Drosophila. This study has identified an upregulation of RTE expression in TBPH-null Drosophila heads and demonstrates that defects in the siRNA pathway lead to RTE upregulation and motoneuron degeneration. These results describe a novel physiological role of endogenous TDP-43 in the prevention of RTE-induced neurological alterations through the modulation of Dicer-2 activity and the siRNA pathway.
Kennedy, T., Rinker, D. and Broadie, K. (2020). Genetic background mutations drive neural circuit hyperconnectivity in a fragile X syndrome model. BMC Biol 18(1): 94. PubMed ID: 32731855
Summary:
Intellectual disability (ID) and autism spectrum disorder (ASD) are often characterized by synaptic overgrowth, with the maintenance of immature or inappropriate synapses. Such synaptogenic defects can occur through mutation of a single gene, such as fragile X mental retardation protein (FMRP) loss causing the neurodevelopmental disorder fragile X syndrome (FXS). FXS represents the leading heritable cause of ID and ASD, but many other genes that play roles in ID and ASD have yet to be identified. In a Drosophila FXS disease model, one dfmr150M null mutant stock exhibits previously unreported axonal overgrowths at developmental and mature stages in the giant fiber (GF) escape circuit. These excess axon projections contain both chemical and electrical synapse markers, indicating mixed synaptic connections. Extensive analyses show these supernumerary synapses connect known GF circuit neurons, rather than new, inappropriate partners, indicating hyperconnectivity within the circuit. Despite the striking similarities to well-characterized FXS synaptic defects, this new GF circuit hyperconnectivity phenotype is driven by genetic background mutations in this dfmr150M stock. Similar GF circuit synaptic overgrowth is not observed in independent dfmr1 null alleles. Bulked segregant analysis (BSA) was combined with whole genome sequencing (WGS) to identify the quantitative trait loci (QTL) linked to neural circuit hyperconnectivity. The results reveal 8 QTL associated with inappropriate synapse formation and maintenance in the dfmr150M mutant background. In conclusion, this work identifies a number of genetic regions that contain mutations disrupting proper synaptogenesis within a particularly well-mapped neural circuit. These QTL regions contain potential new genes involved in synapse formation and refinement. Given the similarity of the synaptic overgrowth phenotype to known ID and ASD inherited conditions, identifying these genes should increase understanding of these devastating neurodevelopmental disease states.
Selma-Soriano, E., Llamusi, B., Fernandez-Costa, J. M., Ozimski, L. L., Artero, R. and Redon, J. (2020). Rabphilin involvement in filtration and molecular uptake in Drosophila nephrocytes suggests a similar role in human podocytes. Dis Model Mech. PubMed ID: 32680845
Summary:
Drosophila nephrocyte share functional, structural, and molecular similarities with human podocytes. It is known that podocytes express the Rabphilin 3A (RPH3A)-Rab3A complex, and its expression is altered in mouse and human proteinuric disease. Previous work has identified a polymorphism that suggested a role for RPH3A protein in the development of urinary albumin excretion. Since endocytosis and vesicle trafficking are fundamental pathways for nephrocytes, the objective of this study is to assess the role of the RPH3A ortholog in Drosophila, Rabphilin (Rph), in the structure and function of nephrocytes. It was confirmed that Rph is required for the endocytic pathway to function correctly in pericardial Drosophila nephrocytes. Knockdown of Rph reduces the expression of cubilin and stick and stones genes, which encode proteins that are involved in protein uptake and filtration. This study also found that reduced Rph expression results in a disappearance of the labyrinthine channel structure and a reduction in the number of endosomes, which ultimately leads to changes in the number and volume of nephrocytes. Finally, this study described that administration of retinoic acid to IR-Rph nephrocytes rescued some altered aspects as the filtration and molecular uptake as well as the maintenance of cell fate. According to these data, Rph is crucial for the nephrocyte's filtration and reabsorption and it is required for the maintenance of the nephrocyte's ultrastructure, integrity, and differentiation.
Basargekar, A., Yogi, S., Mushtaq, Z., Deivasigamani, S., Kumar, V., Ratnaparkhi, G. S. and Ratnaparkhi, A. (2020). Drosophila Mon1 and Rab7 interact to regulate glutamate receptor levels at the neuromuscular junction. Int J Dev Biol 64(4-5-6): 299-307. PubMed ID: 32658990
Summary:
Regulation of post-synaptic receptors plays an important role in determining synaptic strength and plasticity. The Drosophila larval neuromuscular junction (nmj) has been used extensively as a model to understand some of these processes. In this context, this study explored in the role of Drosophila Monensin sensitivity protein 1 (DMon1) in regulating glutamate receptor (GluRIIA) levels at the nmj. DMon1 is an evolutionarily conserved protein which, in complex with calcium caffeine zinc sensitivity1 (CCZ1), regulates the conversion of early endosomes to late endosomes through recruitment of Rab7. C-terminal deletion mutants of Dmon1 (Dmon1(Δ181)) exhibit lethality. The escapers have a short life span and exhibit severe motor defects. At the nmj, these mutants show defects in synaptic morphology and a strong increase in GluRIIA levels. The mechanism by which Dmon1 regulates GluRIIA is unclear. This study has characterized an EMS mutant referred to as pog(1) and demonstrate it to be an allele of Dmon1. Further, this study has examined the role of rab7 in regulating GluRIIA. Similar to Dmon1, knock-down of rab7 using RNAi in neurons, but not muscles, leads to an increase in GluRIIA. Loss of one copy each of Dmon1 and rab7 leads to a synergistic increase in receptor expression. Further, overexpression of an activated Rab7 can rescue the GluRIIA phenotype observed in Dmon1 (Δ181) mutants. Together, these results highlight a neuronal role for Rab7 in GluRIIA regulation and underscore the importance of the endo-lysosomal pathway in this process.
Yu, Y., Niccoli, T., Ren, Z., Woodling, N. S., Aleyakpo, B., Szabadkai, G. and Partridge, L. (2020). PICALM rescues glutamatergic neurotransmission, behavioural function, and survival in a Drosophila model of Aβ42 toxicity. Hum Mol Genet. PubMed ID: 32592479
Summary:
Alzheimer's disease (AD) is the most common form of dementia and the most prevalent neurodegenerative disease. Genome Wide Association Studies have linked PICALM to AD risk. PICALM has been implicated in Aβ42 production and turn-over, but whether it plays a direct role in modulating Aβ42 toxicity remains unclear. This study found that increased expression of the Drosophila PICALM orthologue like-AP180 (lap) could rescue Aβ42 toxicity in an adult-onset model of AD, without affecting Aβ42 level. Imbalances in the glutamatergic system, leading to excessive, toxic stimulation have been associated with AD. This study found that Aβ42 caused accumulation of pre-synaptic vesicular glutamate transporter (VGlut) and increased spontaneous glutamate release. Increased lap expression reversed these phenotypes back to control levels, suggesting that lap may modulate glutamatergic transmission. This study also found that lap modulated the localisation of Amph, the homologue of another AD risk factor BIN1, and that Amph itself modulated post-synaptic glutamate receptor (GluRII) localisation. A model is proposed where PICALM modulates glutamatergic transmission, together with BIN1, to ameliorate synaptic dysfunction and disease progression.
Han, T. H., Vicidomini, R., Ramos, C. I., Wang, Q., Nguyen, P., Jarnik, M., Lee, C. H., Stawarski, M., Hernandez, R. X., Macleod, G. T. and Serpe, M. (2020). Neto-alpha Controls Synapse Organization and Homeostasis at the Drosophila Neuromuscular Junction. Cell Rep 32(1): 107866. PubMed ID: 32640231
Summary:
Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), this study shows that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, this study demonstrates that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR (KaiR1D)-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. It is proposed that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.

Tuesday, August 11th - Adult Neural Development and Function

Yuan, D., Ji, X., Hao, S., Gestrich, J., Duan, W., Wang, X., Xiang, Y., Yang, J., Hu, P., Xu, M., Liu, L. and Wei, H. (2020). Lamina feedback neurons regulate the bandpass property of the flicker-induced orientation response in Drosophila. J Neurochem. PubMed ID: 32383496
Summary:
Natural scenes contain complex visual cues with specific features, including color, motion, flicker and position. It is critical to understand how different visual features are processed at the early stages of visual perception to elicit appropriate cellular responses, and even behavioral output. The visual orientation response induced by flickering stripes in a novel behavioral paradigm in Drosophila melanogaster. This study found that free walking flies exhibited bandpass orientation response to flickering stripes of different frequencies. The most sensitive frequency spectrum was confined to low frequencies of 2-4 Hz. Through genetic silencing, this study showed that lamina L1 and L2 neurons, which receive visual inputs from R1-R6 neurons, were the main components in mediating flicker-induced orientation behavior. Moreover, specific blocking of different types of lamina feedback neurons Lawf1, Lawf2, C2, C3 and T1 modulated orientation responses to flickering stripes of particular frequencies, suggesting that bandpass orientation response was generated through cooperative modulation of lamina feedback neurons. Furthermore, this study found that lamina feedback neurons Lawf1 were glutamatergic. Thermal activation of Lawf1 neurons could suppress neural activities in L1 and L2 neurons, which could be blocked by the glutamate-gated chloride channel inhibitor picrotoxin (PTX). In summary, lamina monopolar neurons L1 and L2 are the primary components in mediating flicker-induced orientation response. Meanwhile, lamina feedback neurons cooperatively modulate the orientation response in a frequency-dependent way, which might be achieved through modulating neural activities of L1 and L2 neurons.
Thompson, A. J., Verdin, P. S., Burton, M. J., Davies, T. G. E., Williamson, M. S., Field, L. M., Baines, R. A., Mellor, I. R. and Duce, I. R. (2020). The effects of knock-down resistance mutations and alternative splicing on voltage-gated sodium channels in Musca domestica and Drosophila melanogaster. Insect Biochem Mol Biol 122: 103388. PubMed ID: 32376273
Summary:
Voltage-gated sodium channels (VGSCs) in insects are encoded by only one gene. Using whole cell patch clamping of neurons from pyrethroid susceptible (wild-type) and resistant strains (s-kdr) of housefly, Musca domestica, this study has shown that the V(50) for activation and steady state inactivation of sodium currents (I(Na+)) is significantly depolarised in s-kdr neurons compared with wild-type and that 10 nM deltamethrin significantly hyperpolarised both of these parameters in the neurons from susceptible but not s-kdr houseflies. Similarly, tail currents were more sensitive to deltamethrin in wild-type neurons (EC(15) 14.5 nM) than s-kdr (EC(15) 133 nM). This study also found that in both strains, I(Na+) are of two types: a strongly inactivating (to 6.8% of peak) current, and a more persistent (to 17.1% of peak) current. Analysis of tail currents showed that the persistent current in both strains (wild-type EC(15) 5.84 nM) was more sensitive to deltamethrin than was the inactivating type (wild-type EC(15) 35.1 nM). It has been shown previously, that the presence of exon l in the Drosophila melanogaster VGSC gives rise to a more persistent I(Na+) than does the alternative splice variant containing exon k and this study used PCR with housefly head cDNA to confirm the presence of the housefly orthologues of splice variants k and l. Their effect on deltamethrin sensitivity was determined by examining I(Na+) in Xenopus oocytes expressing either the k or l variants of the Drosophila Para VGSC. Analysis of tail currents, in the presence of various concentrations of deltamethrin, showed that the l splice variant was significantly more sensitive (EC(50) 42 nM) than the k splice variant (EC(50) 866 nM). It is concluded that in addition to the presence of point mutations, target site resistance to pyrethroids may involve the differential expression of splice variants.
Bates, A. S., Schlegel, P., Roberts, R. J. V., Drummond, N., Tamimi, I. F. M., Turnbull, R., Zhao, X., Marin, E. C., Popovici, P. D., Dhawan, S., Jamasb, A., Javier, A., Serratosa Capdevila, L., Li, F., Rubin, G. M., Waddell, S., Bock, D. D., Costa, M. and Jefferis, G. (2020). Complete Connectomic Reconstruction of Olfactory Projection Neurons in the Fly Brain. Curr Biol. PubMed ID: 32619485
Summary:
Nervous systems contain sensory neurons, local neurons, projection neurons, and motor neurons. To understand how these building blocks form whole circuits, it is necessary to distill these broad classes into neuronal cell types and describe their network connectivity. Using an electron micrograph dataset for an entire Drosophila melanogaster brain, this study reconstructed the first complete inventory of olfactory projections connecting the antennal lobe, the insect analog of the mammalian olfactory bulb, to higher-order brain regions in an adult animal brain. This inventory was connected to extant data in the literature, providing synaptic-resolution "holotypes" both for heavily investigated and previously unknown cell types. Projection neurons are approximately twice as numerous as reported by light level studies; cell types are stereotyped, but not identical, in cell and synapse numbers between brain hemispheres. The lateral horn, the insect analog of the mammalian cortical amygdala, is the main target for this olfactory information and has been shown to guide innate behavior. This study found new connectivity motifs, including axo-axonic connectivity between projection neurons, feedback, and lateral inhibition of these axons by a large population of neurons, and the convergence of different inputs, including non-olfactory inputs and memory-related feedback onto third-order olfactory neurons. These features are less prominent in the mushroom body calyx, the insect analog of the mammalian piriform cortex and a center for associative memory. This work provides a complete neuroanatomical platform for future studies of the adult Drosophila olfactory system.
Yuan, X., Sipe, C. W., Suzawa, M., Bland, M. L. and Siegrist, S. E. (2020). Dilp-2-mediated PI3-kinase activation coordinates reactivation of quiescent neuroblasts with growth of their glial stem cell niche. PLoS Biol 18(5): e3000721. PubMed ID: 32463838
Summary:
Dietary nutrients provide macromolecules necessary for organism growth and development. In response to animal feeding, evolutionarily conserved growth signaling pathways are activated, leading to increased rates of cell proliferation and tissue growth. It remains unclear how different cell types within developing tissues coordinate growth in response to dietary nutrients and whether coordinated growth of different cell types is necessary for proper tissue function. This study reports that Drosophila neural stem cells, known as neuroblasts, reactivate from developmental quiescence in a dietary-nutrient-dependent manner. Neuroblast reactivation in the brain requires nonautonomous activation of phosphoinositide 3-kinase (PI3-kinase) signaling from cortex glia and tracheal processes, both of which are closely associated with neuroblasts. Furthermore, PI3-kinase activation in neuroblasts is required nonautonomously for glial membrane expansion and robust neuroblast-glial contact. Finally, PI3-kinase is required cell autonomously for nutrient-dependent growth of neuroblasts, glia, and trachea. Of the 7 Drosophila insulin-like peptides (Dilps), Dilp-2 was found is required for PI3-kinase activation and growth coordination between neuroblasts and glia in the brain. Dilp-2 induces brain cortex glia to initiate membrane growth and make first contact with quiescent neuroblasts. After contact, neuroblasts increase in size and reenter S-phase. Once reactivated from quiescence, neuroblasts promote growth of cortex glia, which, in turn, form a selective membrane barrier around neuroblasts and their newborn progeny. These results highlight the importance of bidirectional growth signaling between neural stem cells and surrounding cell types in the brain in response to nutrition and demonstrate how coordinated growth among different cell types drives tissue morphogenesis and function.
Apostolopoulou, A. A. and Lin, A. C. (2020). Mechanisms underlying homeostatic plasticity in the Drosophila mushroom body in vivo. Proc Natl Acad Sci U S A 117(28): 16606-16615. PubMed ID: 32601210
Summary:
Neural network function requires an appropriate balance of excitation and inhibition to be maintained by homeostatic plasticity. However, little is known about homeostatic mechanisms in the intact central brain in vivo. Homeostatic plasticity was studied in the Drosophila mushroom body, where Kenyon cells receive feedforward excitation from olfactory projection neurons and feedback inhibition from the anterior paired lateral neuron (APL). Prolonged (4-d) artificial activation of the inhibitory APL causes increased Kenyon cell odor responses after the artificial inhibition is removed, suggesting that the mushroom body compensates for excess inhibition. In contrast, there is little compensation for lack of inhibition (blockade of APL). The compensation occurs through a combination of increased excitation of Kenyon cells and decreased activation of APL, with differing relative contributions for different Kenyon cell subtypes. This findings establish the fly mushroom body as a model for homeostatic plasticity in vivo.
Kozlov, A., Koch, R. and Nagoshi, E. (2020). Nitric oxide mediates neuro-glial interaction that shapes Drosophila circadian behavior. PLoS Genet 16(6): e1008312. PubMed ID: 32598344
Summary:
Drosophila circadian behavior relies on the network of heterogeneous groups of clock neurons. Short- and long-range signaling within the pacemaker circuit coordinates molecular and neural rhythms of clock neurons to generate coherent behavioral output. The neurochemistry of circadian behavior is complex and remains incompletely understood. This study demonstrates that the gaseous messenger nitric oxide (NO) is a signaling molecule linking circadian pacemaker to rhythmic locomotor activity. mutants lacking nitric oxide synthase (NOS) have behavioral arrhythmia in constant darkness, although molecular clocks in the main pacemaker neurons are unaffected. Behavioral phenotypes of mutants are due in part to the malformation of neurites of the main pacemaker neurons, s-LNvs. Using cell-type selective and stage-specific gain- and loss-of-function of NOS, this study also demonstrated that NO secreted from diverse cellular clusters affect behavioral rhythms. Furthermore, perineurial glia, one of the two glial subtypes that form the blood-brain barrier, as the major source of NO that regulates circadian locomotor output. These results reveal for the first time the critical role of NO signaling in the Drosophila circadian system and highlight the importance of neuro-glial interaction in the neural circuit output.

Monday, August 10th - Adult Physiology

Ceder, M. M., Lekholm, E., Klaesson, A., Tripathi, R., Schweizer, N., Weldai, L., Patil, S. and Fredriksson, R. (2020). Glucose Availability Alters Gene and Protein Expression of Several Newly Classified and Putative Solute Carriers in Mice Cortex Cell Culture and D. melanogaster. Front Cell Dev Biol 8: 579. PubMed ID: 32733888
Summary:
Many newly identified solute carriers (SLCs) and putative transporters have the possibility to be intricately involved in glucose metabolism. This study shows that many transporters of this type display a high degree of regulation at both mRNA and protein level following no or low glucose availability in mouse cortex cultures. This is also the case in Drosophila melanogaster subjected to starvation or diets with different sugar content. Interestingly, re-introduction of glucose to media, or refeeding flies, normalized the gene expression of a number of the targets, indicating a fast and highly dynamic control. These findings demonstrate high conservation of these transporters and how dependent both cell cultures and organisms are on gene and protein regulation during metabolic fluctuations. Several transporter genes were regulated simultaneously maybe to initiate alternative metabolic pathways as a response to low glucose levels, both in the cell cultures and in D. melanogaster. The results display that newly identified SLCs of Major Facilitator Superfamily type, as well as the putative transporters included in this study, are regulated by glucose availability and could be involved in several cellular aspects dependent of glucose and/or its metabolites. Recently, a correlation between dysregulation of glucose in the central nervous system and numerous diseases such as obesity, type 2 diabetes mellitus as well as neurological disease such as Alzheimer's and Parkinson's diseases indicate a complex regulation and fine tuning of glucose levels in the brain. The fact that almost one third of transporters and transporter-related proteins remain orphans with unknown or contradictive substrate profile, location and function, pinpoint the need for further research about them to fully understand their mechanistic role and their impact on cellular metabolism.
Bayliak, M. M., Demianchuk, O. I., Gospodaryov, D. V., Abrat, O. B., Lylyk, M. P., Storey, K. B. and Lushchak, V. I. (2020). Mutations in genes cnc or dKeap1 modulate stress resistance and metabolic processes in Drosophila melanogaster. Comp Biochem Physiol A Mol Integr Physiol 248: 110746. PubMed ID: 32579905
Summary:
The transcription factor Nrf2 and its negative regulator Keap1 play important roles in the maintenance of redox homeostasis in animal cells. Nrf2 activates defenses against oxidative stress and xenobiotics. Homologs of Nrf2 and Keap1 are present in Drosophila melanogaster (CncC and dKeap1, respectively). The aim of this study was to explore effects of CncC deficiency (due to mutation in the cnc gene) or enhanced activity (due to mutation in the dKeap1 gene) on redox status and energy metabolism of young adult flies in relation to behavioral traits and resistance to a number of stressors. Deficiency in either CncC or dKeap1 delayed pupation and increased climbing activity and heat stress resistance in 2-day-old adult flies. Males and females of the Δkeap1 line shared some similarities such as elevated antioxidant defense as well as lower triacylglyceride and higher glucose levels. Males of the Δkeap1 line also had a higher activity of hexokinase, whereas Δkeap1 females showed higher glycogen levels and lower values of respiratory control and ATP production than flies of the control line. Mutation of cnc gene in allele cncEY08884 caused by insertion of P{EPgy2} transposon in cnc promotor did not affect significantly the levels of metabolites and redox parameters, and even activated some components of antioxidant defense. These data suggest that the mutation can be hypomorphic as well as CncC protein can be dispensable for adult fruit flies under physiological conditions. In females, CncC mutation led to lower mitochondrial respiration, higher hexokinase activity and higher fecundity as compared with the control line. Either CncC activation or its deficiency affected stress resistance of flies.
Abrieux, A., Xue, Y., Cai, Y., Lewald, K. M., Nguyen, H. N., Zhang, Y. and Chiu, J. C. (2020). EYES ABSENT and TIMELESS integrate photoperiodic and temperature cues to regulate seasonal physiology in Drosophila. Proc Natl Acad Sci U S A 117(26): 15293-15304. PubMed ID: 32541062
Summary:
Organisms possess photoperiodic timing mechanisms to detect variations in day length and temperature as the seasons progress. The nature of the molecular mechanisms interpreting and signaling these environmental changes to elicit downstream neuroendocrine and physiological responses are just starting to emerge. This study demonstrates that, in Drosophila melanogaster, Eyes absent (Eya) acts as a seasonal sensor by interpreting photoperiodic and temperature changes to trigger appropriate physiological responses. Tissue-specific genetic manipulation of eya expression is sufficient to disrupt the ability of flies to sense seasonal cues, thereby altering the extent of female reproductive dormancy. Specifically, it was observed that Eya proteins, which peak at night in short photoperiod and accumulate at higher levels in the cold, promote reproductive dormancy in female D. melanogaster. Furthermore, evidence is provided indicating that the role of Eya in photoperiodism and temperature sensing is aided by the stabilizing action of the light-sensitive circadian clock protein Timeless (Tim). It is postulated that increased stability and level of Tim at night under short photoperiod together with the production of cold-induced and light-insensitive Tim isoforms facilitate Eya accumulation in winter conditions. This is supported by observations that tim null mutants exhibit reduced incidence of reproductive dormancy in simulated winter conditions, while flies overexpressing tim show an increased incidence of reproductive dormancy even in long photoperiod.
Klepsatel, P., Knoblochova, D., Girish, T. N., Dircksen, H. and Galikova, M. (2020). The influence of developmental diet on reproduction and metabolism in Drosophila. BMC Evol Biol 20(1): 93. PubMed ID: 32727355
Summary:
The adaptive significance of phenotypic changes elicited by environmental conditions experienced early in life has long attracted attention in evolutionary biology. This study used Drosophila melanogaster to test whether the developmental diet produces phenotypes better adapted to cope with similar nutritional conditions later in life. To discriminate among competing hypotheses on the underlying nature of developmental plasticity, a full factorial design was employed with several developmental and adult diets. Specifically, the effects of early- and late-life diets (by varying their yeast and sugar contents) on reproductive fitness and on the amount of energy reserves (fat and glycogen) was examined in two wild-caught populations. Individuals that had developed on either low-yeast or high-sugar diet were found to show decreased reproductive performance regardless of their adult nutritional environment. The lower reproductive fitness might be caused by smaller body size and reduced ovariole number. Overall, these results are consistent with the silver spoon concept, which posits that development in a suboptimal environment negatively affects fitness-associated traits. On the other hand, the higher amount of energy reserves (fat) in individuals that had developed in a suboptimal environment might represent either an adaptive response or a side-effect of compensatory feeding. These findings suggest that the observed differences in the adult physiology induced by early-life diet likely result from inevitable and general effects of nutrition on the development of reproductive and metabolic organs, rather than from adaptive mechanisms.
Bevers, R. P. J., Litovchenko, M., Kapopoulou, A., Braman, V. S., Robinson, M. R., Auwerx, J., Hollis, B. and Deplancke, B. (2019). Mitochondrial haplotypes affect metabolic phenotypes in the Drosophila Genetic Reference Panel. Nat Metab 1(12): 1226-1242. PubMed ID: 32694676
Summary:
The nature and extent of mitochondrial DNA variation in a population and how it affects traits is poorly understood. This study resequenced the mitochondrial genomes of 169 Drosophila Genetic Reference Panel lines, identifying 231 variants that stratify along 12 mitochondrial haplotypes. 1,845 cases of mitonuclear allelic imbalances were identified, thus implying that mitochondrial haplotypes are reflected in the nuclear genome. However, no major fitness effects are associated with mitonuclear imbalance, suggesting that such imbalances reflect population structure at the mitochondrial level rather than genomic incompatibilities. Although mitochondrial haplotypes have no direct impact on mitochondrial respiration, some haplotypes are associated with stress- and metabolism-related phenotypes, including food intake in males. Finally, through reciprocal swapping of mitochondrial genomes, it was demonstrated that a mitochondrial haplotype associated with high food intake can rescue a low food intake phenotype. Together, these findings provide new insight into population structure at the mitochondrial level and point to the importance of incorporating mitochondrial haplotypes in genotype-phenotype relationship studies.
Araujo, S. M., Bortolotto, V. C., Poetini, M. R., Dahleh, M. M. M., Couto, S. F., Pinheiro, F. C., Meichtry, L. B., Musachio, E. A. S., Ramborger, B. P., Roehrs, R., Guerra, G. P. and Prigol, M. (2020). gamma-Oryzanol produces an antidepressant-like effect in a chronic unpredictable mild stress model of depression in Drosophila melanogaster. Stress: 1-12. PubMed ID: 32723199
Summary:
Chronic unpredictable mild stress (CUMS) is a valid model for inducing depression-like symptoms in animal models, causing predictive behavioral, neurochemical, and physiological responses to this condition. This work aims to evaluate the possible antidepressant effect of γ-oryzanol (ORY) in the CUMS-induced depressive model in male Drosophila melanogaster. The CUMS protocol was used to continue a previous study, mimicking a depressive state in these insects. Male flies were subjected to various stressors according to a 10-day randomized schedule and concomitantly treated with ORY or fluoxetine (FLX). After the experimental period, in vivo behavioral tests were performed (open field, forced swimming, aggressiveness test, mating test, male virility, sucrose preference index and light/dark test) and ex vivo analyses measuring serotonin (5HT), dopamine (DA), octopamine (OCT) levels and body weight. ORY-treated flies and concomitant exposure to CUMS did not exhibit obvious behaviors such as prolonged immobility or increased aggressive behavior, reduced male mating and virility behavior, and anxiolytic behavior, in contrast to ORY, not altering sucrose preference and body weight flies exposed to CUMS. ORY effectively prevented 5HT and OCT reduction and partially protected against DA reduction. The data are consistent and provide evidence for the use of ORY as a potential antidepressant compound.

Friday, August 7th - Autophagy and Apoptosis

Barthez, M., Poplineau, M., Elrefaey, M., Caruso, N., Graba, Y. and Saurin, A. J. (2020). Human ZKSCAN3 and Drosophila M1BP are functionally homologous transcription factors in autophagy regulation. Sci Rep 10(1): 9653. PubMed ID: 32541927
Summary:
Autophagy is an essential cellular process that maintains homeostasis by recycling damaged organelles and nutrients during development and cellular stress. ZKSCAN3 is the sole identified master transcriptional repressor of autophagy in human cell lines. How ZKSCAN3 achieves autophagy repression at the mechanistic or organismal level however still remains to be elucidated. Furthermore, Zkscan3 knockout mice display no discernable autophagy-related phenotypes, suggesting that there may be substantial differences in the regulation of autophagy between normal tissues and tumor cell lines. This study demonstrates that vertebrate ZKSCAN3 and Drosophila M1BP are functionally homologous transcription factors in autophagy repression. Expression of ZKSCAN3 in Drosophila prevents premature autophagy onset due to loss of M1BP function and conversely, M1BP expression in human cells can prevent starvation-induced autophagy due to loss of nuclear ZKSCAN3 function. In Drosophila ZKSCAN3 binds genome-wide to sequences targeted by M1BP and transcriptionally regulates the majority of M1BP-controlled genes, demonstrating the evolutionary conservation of the transcriptional repression of autophagy. This study thus  allows the potential for transitioning the mechanisms, gene targets and plethora metabolic processes controlled by M1BP onto ZKSCAN3 and opens up Drosophila as a tool in studying the function of ZKSCAN3 in autophagy and tumourigenesis.
Brooks, D., Naeem, F., Stetsiv, M., Goetting, S. C., Bawa, S., Green, N., Clark, C., Bashirullah, A. and Geisbrecht, E. R. (2020). Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover. PLoS Genet 16(4): e1008700. PubMed ID: 32320396
Summary:
The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. This study shows that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) and CryAB. NUAK biochemically and genetically interacts with Drosophila Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. It was further demonstrated that Fil is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, these results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases.
Li, X., Rommelaere, S., Kondo, S. and Lemaitre, B. (2020). Renal Purge of Hemolymphatic Lipids Prevents the Accumulation of ROS-Induced Inflammatory Oxidized Lipids and Protects Drosophila from Tissue Damage. Immunity 52(2): 374-387. PubMed ID: 32075729
Summary:
Animals require complex metabolic and physiological adaptations to maintain the function of vital organs in response to environmental stresses and infection. This study found that infection or injury in Drosophila induced the excretion of hemolymphatic lipids by Malpighian tubules, the insect kidney. This lipid purge was mediated by a stress-induced lipid-binding protein, Materazzi, which was enriched in Malpighian tubules. Flies lacking materazzi had higher hemolymph concentrations of reactive oxygen species (ROS) and increased lipid peroxidation. These flies also displayed Malpighian tubule dysfunction and were susceptible to infections and environmental stress. Feeding flies with antioxidants rescued the materazzi phenotype, indicating that the main role of Materazzi is to protect the organism from damage caused by stress-induced ROS. These findings suggest that purging hemolymphatic lipids presents a physiological adaptation to protect host tissues from excessive ROS during immune and stress responses, a process that is likely to apply to other organisms.
Kidera, H., Hatabu, T. and Takahashi, K. H. (2020). Apoptosis inhibition mitigates aging effects in Drosophila melanogaster. Genetica. PubMed ID: 32219590
Summary:
Aging is a natural biological process that results in progressive loss of cell, tissue, and organ function. One of the causing factors of the aging process is the decrease in muscle mass, which has not been fully verified in Drosophila. Apoptotic cell death may result in aberrant cell loss and can eventually diminish tissue function and muscle atrophy. If so, inhibition of apoptosis may prolong longevity and reduce motor function and muscle mass decline with age in Drosophila flies. This study used Drosophila melanogaster as study material, and induced the overexpression of Drosophila inhibitor of apoptosis protein 1 gene to inhibit apoptosis, and investigated the effect of apoptosis inhibition on the longevity and age-related declines in flight and climbing ability and muscle mass. As a result, the inhibition of apoptosis tended to mitigate the aging effects and prolonged longevity and reduced climbing ability decline with age. The current study suggests that apoptosis inhibition could mitigate the aging effects in D. melanogaster. Although such effects have already been known in mammals, the current results suggest that the apoptosis may play a similar role in insects as well.
Prieto-Godino, L. L., Silbering, A. F., Khallaf, M. A., Cruchet, S., Bojkowska, K., Pradervand, S., Hansson, B. S., Knaden, M. and Benton, R. (2020). Functional integration of "undead" neurons in the olfactory system. Sci Adv 6(11): eaaz7238. PubMed ID: 32195354
Summary:
Programmed cell death (PCD) is widespread during neurodevelopment, eliminating the surpluses of neuronal production. Using the Drosophila olfactory system, this study examined the potential of cells fated to die to contribute to circuit evolution. Inhibition of PCD is sufficient to generate new cells that express neural markers and exhibit odor-evoked activity. These "undead" neurons express a subset of olfactory receptors that is enriched for relatively recent receptor duplicates and includes some normally found in different chemosensory organs and life stages. Moreover, undead neuron axons integrate into the olfactory circuitry in the brain, forming novel receptor/glomerular couplings. Comparison of homologous olfactory lineages across drosophilids reveals natural examples of fate change from death to a functional neuron. Last, evidence is provided that PCD contributes to evolutionary differences in carbon dioxide-sensing circuit formation in Drosophila and mosquitoes. These results reveal the remarkable potential of alterations in PCD patterning to evolve new neural pathways.
Jacomin, A. C., Petridi, S., Di Monaco, M., Bhujabal, Z., Jain, A., Mulakkal, N. C., Palara, A., Powell, E. L., Chung, B., Zampronio, C., Jones, A., Cameron, A., Johansen, T. and Nezis, I. P. (2020). Regulation of Expression of Autophagy Genes by Atg8a-Interacting Partners Sequoia, YL-1, and Sir2 in Drosophila. Cell Rep 31(8): 107695. PubMed ID: 32460019
Summary:
Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. This study shows that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and it is acetylated in nutrient-rich conditions. Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. These results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2.

Thursday, August 6th - Enhancers, transcription factors and gene regulation

Koreski, K. P., Rieder, L. E., McLain, L. M., Chaubal, A., Marzluff, W. F. and Duronio, R. J. (2020). Drosophila Histone Locus Body assembly and function involves multiple interactions. Mol Biol Cell: mbcE20030176. PubMed ID: 32401666
Summary:
The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and concentrates factors required for RD histone mRNA biosynthesis. The D. melanogaster genome has a single locus comprised of ∼100 copies of a tandemly arrayed 5 kB repeat unit containing one copy of each of the 5 RD histone genes. To determine sequence elements required for D. melanogaster HLB formation and histone gene expression, transgenic gene arrays were used containing 12 copies of the histone repeat unit that functionally complement loss of the ∼200 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were replaced with H2a-H2b promoters (12x(PR)) does not form an HLB or express high levels of RD histone mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is active in HLB assembly and RD histone gene expression in the absence of the endogenous RD histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array contains all known factors present in the wild type HLB including CLAMP, which normally binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein and/or protein-DNA interactions contribute to HLB formation, and that the large number of endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic arrays, thereby preventing HLB formation and gene expression.
Keller, S. H., Jena, S. G., Yamazaki, Y. and Lim, B. (2020). Regulation of spatiotemporal limits of developmental gene expression via enhancer grammar. Proc Natl Acad Sci U S A 117(26): 15096-15103. PubMed ID: 32541043
Summary:
The regulatory specificity of a gene is determined by the structure of its enhancers, which contain multiple transcription factor binding sites. A unique combination of transcription factor binding sites in an enhancer determines the boundary of target gene expression, and their disruption often leads to developmental defects. Despite extensive characterization of binding motifs in an enhancer, it is still unclear how each binding site contributes to overall transcriptional activity. Using live imaging, quantitative analysis, and mathematical modeling, this study measured the contribution of individual binding sites in transcriptional regulation. Binding site arrangement within the Rho-GTPase component t48 enhancer mediates the expression boundary by mainly regulating the timing of transcriptional activation along the dorsoventral axis of Drosophila embryos. By tuning the binding affinity of the Dorsal (Dl) and Zelda (Zld) sites, this study shows that single site modulations are sufficient to induce significant changes in transcription. Yet, no one site seems to have a dominant role; rather, multiple sites synergistically drive increases in transcriptional activity. Interestingly, Dl and Zld demonstrate distinct roles in transcriptional regulation. Dl site modulations change spatial boundaries of t48, mostly by affecting the timing of activation and bursting frequency rather than transcriptional amplitude or bursting duration. However, modulating the binding site for the pioneer factor Zld affects both the timing of activation and amplitude, suggesting that Zld may potentiate higher Dl recruitment to target DNAs. It is proposed that such fine-tuning of dynamic gene control via enhancer structure may play an important role in ensuring normal development.
Brody, T., Yavatkar, A., Kuzin, A. and Odenwald, W. F. (2020). Ultraconserved Non-coding DNA Within Diptera and Hymenoptera. G3 (Bethesda). PubMed ID: 32601058
Summary:
This study has taken advantage of the availability of the assembled genomic sequence of flies, mosquitos, ants and bees to explore the presence of ultraconserved sequence elements in these phylogenetic groups. Non-coding sequences found within and flanking Drosophila developmental genes were compared to homologous sequences in Ceratitis capitata and Musca domestica. Many of the conserved sequence blocks (CSBs) that constitute Drosophila cis-regulatory DNA, recognized by EvoPrinter alignment protocols, are also conserved in Ceratitis and Musca. Also conserved is the position but not necessarily the orientation of many of these ultraconserved CSBs (uCSBs) with respect to flanking genes. Using the mosquito EvoPrint algorithm, uCSBs shared among distantly related mosquito species were identified. Side by side comparison of bee and ant EvoPrints of selected developmental genes identify uCSBs shared between these two Hymenoptera, as well as less conserved CSBs in either one or the other taxon but not in both. Analysis of uCSBs in these dipterans and Hymenoptera will lead to a greater understanding of their evolutionary origin and function of their conserved non-coding sequences and aid in discovery of core elements of enhancers.
Blom-Dahl, D., Cordoba, S., Gabilondo, H., Carr-Baena, P., Diaz-Benjumea, F. J. and Estella, C. (2020). In vivo analysis of the evolutionary conserved BTD-box domain of Sp1 and Btd during Drosophila development. Dev Biol. PubMed ID: 32738261
Summary:
The Sp family of transcription factors plays important functions during development and disease. An evolutionary conserved role for some Sp family members is the control of limb development. The family is characterized by the presence of three C2H2-type zinc fingers and an adjacent 10 aa region with an unknown function called the Buttonhead (BTD) box. The presence of this BTD-box in all Sp family members identified from arthropods to vertebrates, suggests that it plays an essential role during development. However, despite its conservation, the in vivo function of the BTD-box has never been studied. Yhis work has generated specific BTD-box deletion alleles for the Drosophila Sp family members Sp1 and buttonhead (btd) using gene editing tools and analyzed its role during development. Unexpectedly, btd and Sp1 mutant alleles that lack the BTD-box are viable and have almost normal appendages. However, in a sensitized background the requirement of this domain to fully regulate some of Sp1 and Btd target genes is revealed. Furthermore, a novel Sp1 role was identified promoting leg vs antenna identity through the repression of spineless (ss) expression in the leg, a function that also depends on the Sp1 BTD-box.
Bakker, R., Mani, M. and Carthew, R. W. (2020). The Wg and Dpp morphogens regulate gene expression by modulating the frequency of transcriptional bursts. Elife 9. PubMed ID: 32568073
Summary:
Morphogen signaling contributes to the patterned spatiotemporal expression of genes during development. One mode of regulation of signaling-responsive genes is at the level of transcription. Single-cell quantitative studies of transcription have revealed that transcription occurs intermittently, in bursts. Although the effects of many gene regulatory mechanisms on transcriptional bursting have been studied, it remains unclear how morphogen gradients affect this dynamic property of downstream genes. This study adapted single molecule fluorescence in situ hybridization (smFISH) for use in the Drosophila wing imaginal disc in order to measure nascent and mature mRNA of genes downstream of the Wg and Dpp morphogen gradients. The experimental results were compared with predictions from stochastic models of transcription, which indicated that the transcription levels of these genes appear to share a common method of control via burst frequency modulation. These data help further elucidate the link between developmental gene regulatory mechanisms and transcriptional bursting.
Zaytseva, O., Mitchell, N. C., Guo, L., Marshall, O. J., Parsons, L. M., Hannan, R. D., Levens, D. L. and Quinn, L. M. (2020). Transcriptional repression of Myc underlies the tumour suppressor function of AGO1 in Drosophila. Development 147(11). PubMed ID: 32527935
Summary:
This study reports novel tumor suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, these data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.

Wednesday, August 5th - RNA and transposons

Saint-Leandre, B., Capy, P., Hua-Van, A. and Filee, J. (2020). piRNA and transposon dynamics in Drosophila: a female story. Genome Biol Evol. PubMed ID: 32396626
Summary:
The germlines of metazoans contain transposable elements (TEs) causing genetic instability and affecting fitness. To protect the germline from TE activity, gonads of metazoans produce TE-derived piRNAs that silence TE expression. In Drosophila, understanding of piRNA biogenesis is mainly based on studies of the D. melanogaster female germline. However, it is not known whether piRNA functions are also important in the male germline or whether and how piRNAs are affected by the global genomic context. To address these questions, genome sequences, transcriptomes and small RNA libraries extracted from entire testes and ovaries were compared of two sister species: D. melanogaster and D. simulans. Most TE-derived piRNAs were produced in ovaries, and piRNA pathway genes were strongly over-expressed in ovaries compared to testes, indicating that the silencing of TEs by the piRNA pathway mainly took place in the female germline. To study the relationship between host piRNAs and TE landscape, TE genomic features and how they correlate with piRNA production in the two species were analyzed. In D. melanogaster, TE-derived piRNAs were found to target recently active TEs. In contrast, although D. simulans TEs do not display any features of recent activity, the host still intensively produced silencing piRNAs targeting old TE relics. Together, these results show that the piRNA silencing response mainly takes place in Drosophila ovaries and indicate that the host piRNA response is implemented following a burst of TE activity and could persist long after the extinction of active TE families.
Wang, L., Barbash, D. A. and Kelleher, E. S. (2020). Adaptive evolution among cytoplasmic piRNA proteins leads to decreased genomic auto-immunity. PLoS Genet 16(6): e1008861. PubMed ID: 32525870
Summary:
The piRNA pathway acts as a genomic immune system, employing small RNA-mediated silencing to defend host DNA from the harmful effects of transposable elements (TEs). This study used interspecific complementation to test for functional differences between Drosophila melanogaster and D. simulans alleles of three adaptively evolving piRNA pathway proteins: Armitage, Aubergine and Spindle-E. In contrast to piRNA-mediated transcriptional regulators examined in previous studies, these three proteins have cytoplasmic functions in piRNA maturation and post-transcriptional silencing. Across all three proteins interspecific divergence was observed in the regulation of only a handful of TE families, which were more robustly silenced by the heterospecific piRNA pathway protein. This unexpected result suggests that unlike transcriptional regulators, positive selection has not acted on cytoplasmic piRNA effector proteins to enhance their function in TE repression. Rather, TEs may evolve to "escape" silencing by host proteins. It was further discovered that D. simulans alleles of aub and armi exhibit enhanced off-target effects on host transcripts in a D. melanogaster background, as well as modest reductions in the efficiency of piRNA biogenesis, suggesting that promiscuous binding of D. simulans Aub and Armi proteins to host transcripts reduces their participation in piRNA production. Avoidance of genomic auto-immunity may therefore be a critical target of selection. These observations suggest that piRNA effector proteins are subject to an evolutionary trade-off between defending the host genome from the harmful effect of TEs while also minimizing collateral damage to host genes.
Zhang, X., Lyu, J., Jin, X., Yang, W., Lou, Z., Xi, Y., Yang, X. and Ge, W. (2020). A motor neuron protective role of miR-969 mediated by the transcription factor kay. RNA Biol: 1-7. PubMed ID: 32397794
Summary:
Maintenance of motor neuron structure and function is crucial in development and motor behaviour. However, the genetic regulatory mechanism of motor neuron function remains less well understood. This study identified a novel neuroprotective role of the microRNA miR-969 in Drosophila motor neurons. miR-969 is highly expressed in motor neurons. Loss of miR-969 results in early-onset and age-progressive locomotion impairment. Flies lacking miR-969 also exhibit shortened lifespan. Moreover, miR-969 is required in motor neurons. kay was identified as a functionally important target of miR-969. Together, these results indicate that miR-969 can protect motor neuron function by limiting kay activity in Drosophila.
Roy, M., Viginier, B., Saint-Michel, E., Arnaud, F., Ratinier, M. and Fablet, M. (2020). Viral infection impacts transposable element transcript amounts in Drosophila. Proc Natl Acad Sci U S A 117(22): 12249-12257. PubMed ID: 32434916
Summary:
Transposable elements (TEs) are genomic parasites that are found in all genomes, some of which display sequence similarity to certain viruses. In insects, TEs are controlled by the Piwi-interacting small interfering RNA (piRNA) pathway in gonads, while the small interfering RNA (siRNA) pathway is dedicated to TE somatic control and defense against viruses. So far, these two small interfering RNA pathways are considered to involve distinct molecular effectors and are described as independent. Using Sindbis virus (SINV) in Drosophila, this study shows that viral infections affect TE transcript amounts via modulations of the piRNA and siRNA repertoires, with the clearest effects in somatic tissues. These results suggest that viral acute or chronic infections may impact TE activity and, thus, the tempo of genetic diversification. In addition, these results deserve further evolutionary considerations regarding potential benefits to the host, the virus, or the TEs.
Sapiro, A. L., Freund, E. C., Restrepo, L., Qiao, H. H., Bhate, A., Li, Q., Ni, J. Q., Mosca, T. J. and Li, J. B. (2020). Zinc Finger RNA-Binding Protein Zn72D Regulates ADAR-Mediated RNA Editing in Neurons. Cell Rep 31(7): 107654. PubMed ID: 32433963
Summary:
Adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, alters RNA sequences from those encoded by DNA. These editing events are dynamically regulated, but few trans regulators of ADARs are known in vivo. This study screen RNA-binding proteins for roles in editing regulation with knockdown experiments in the Drosophila brain. Zinc-finger protein at 72D (Zn72D) was identified as a regulator of editing levels at a majority of editing sites in the brain. Zn72D both regulates ADAR protein levels and interacts with ADAR in an RNA-dependent fashion, and similar to ADAR, Zn72D is necessary to maintain proper neuromuscular junction architecture and fly mobility. Furthermore, Zn72D's regulatory role in RNA editing is conserved because the mammalian homolog of Zn72D, Zfr, regulates editing in mouse primary neurons. The broad and conserved regulation of ADAR editing by Zn72D in neurons sustains critically important editing events.
Bansal, P., Madlung, J., Schaaf, K., Macek, B. and Bono, F. (2020). An interaction network of RNA-binding proteins involved in Drosophila oogenesis. Mol Cell Proteomics. PubMed ID: 32554711
Summary:
During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, protein-protein associations were examined of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed identification of 26 previously unknown associations, besides recovering several well characterized interactions. Interactions were odemtofoed between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented in this study provides the foundation for future functional and structural studies of mRNA localization across metazoans.

Tuesday, August 4th - Chromatin

Bai, Z., Wei, M., Li, Z. and Xiao, W. (2020). Drosophila Uev1a is dually required for Ben-dependent DNA-damage response and fly mobility. Cell Signal 74: 109719. PubMed ID: 32702441
Summary:
K63-linked polyubiquitination requires the ubiquitin-conjugating enzyme Ubc13 and a Ubc/E2 variant Uev. Lower eukaryotic organisms contain one UEV gene required for DNA-damage tolerance, while vertebrates and higher plants contain multiple UEV genes with distinct functions. In contrast, Drosophila contains only one UEV gene designated dUev1a. This study reports that dUev1a forms a stable heterodimer with Ben, the Drosophila Ubc13 ortholog, that dUev1a-F15E completely abolishes the interaction, and that a conserved dUev1a-F15Y substitution severely reduces its interaction with Ben. dUev1a functionally rescues the corresponding yeast mms2 null mutant from killing by various DNA-damaging agents in a Ben-dependent manner, and the heterozygous dUev1a mutant flies are more sensitive to DNA-damaging agent, indicating that the function of UEV in DNA-damage response is conserved throughout eukaryotes. Meanwhile, dUev1a(+/-) mutant flies displayed reduced mobility characteristic of defects in the central nervous system and reminiscent of the bendless phenotypes, suggesting that dUev1a acts together with Ben in this process. These observations collectively imply that dUev1a is dually required for DNA-damage response and neurological signaling in Drosophila, and that these processes are mediated by the Ben-dUev1a complex that promotes K63-linked polyubiquitination.
Aoki, D., Awazu, A., Fujii, M., Uewaki, J. I., Hashimoto, M., Tochio, N., Umehara, T. and Tate, S. I. (2020). Ultrasensitive Change in Nucleosome Binding by Multiple Phosphorylations to the Intrinsically Disordered Region of the Histone Chaperone FACT. J Mol Biol 432(16): 4637-4657. PubMed ID: 32553729
Summary:
Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. The DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites; the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation ("ultrasensitive response"). This report explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434-624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. It is proposed that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.
Scacchetti, A., Schauer, T., Reim, A., Apostolou, Z., Campos Sparr, A., Krause, S., Heun, P., Wierer, M. and Becker, P. B. (2020). Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms. Elife 9. PubMed ID: 32432549
Summary:
Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In Drosophila, the single Domino chromatin regulator complex is thought to combine both activities via an unknown mechanism. This study shows that alternative isoforms of the Domino nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. This work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. Drosophila generates the same diversity by alternative splicing of a single gene.
Zhao, S., Deanhardt, B., Barlow, G. T., Schleske, P. G., Rossi, A. M. and Volkan, P. C. (2020). Chromatin-based reprogramming of a courtship regulator by concurrent pheromone perception and hormone signaling. Sci Adv 6(21): eaba6913. PubMed ID: 32494751
Summary:
To increase fitness, animals use both internal and external states to coordinate reproductive behaviors. The molecular mechanisms underlying this coordination remain unknown. This study focused on pheromone-sensing Drosophila Or47b neurons, which exhibit age- and social experience-dependent increase in pheromone responses and courtship advantage in males. Fruitless(M) (Fru(M)), a master regulator of male courtship behaviors, drives the effects of social experience and age on Or47b neuron responses and function. Simultaneous exposure to social experience and age-specific juvenile hormone (JH) induces chromatin-based reprogramming of fru(M) expression in Or47b neurons. Group housing and JH signaling increase fru(M) expression in Or47b neurons and active chromatin marks at fru(M) promoter. Conversely, social isolation or loss of JH signaling decreases fru(M) expression and increases repressive marks around fru(M) promoter. These results suggest that fru(M) promoter integrates coincident hormone and pheromone signals driving chromatin-based changes in expression and ultimately neuronal and behavioral plasticity.
Mugat, B., Nicot, S., Varela-Chavez, C., Jourdan, C., Sato, K., Basyuk, E., Juge, F., Siomi, M. C., Pelisson, A. and Chambeyron, S. (2020). The Mi-2 nucleosome remodeler and the Rpd3 histone deacetylase are involved in piRNA-guided heterochromatin formation. Nat Commun 11(1): 2818. PubMed ID: 32499524
Summary:
In eukaryotes, trimethylation of lysine 9 on histone H3 (H3K9) is associated with transcriptional silencing of transposable elements (TEs). In Drosophila ovaries, this heterochromatic repressive mark is thought to be deposited by SetDB1 on TE genomic loci after the initial recognition of nascent transcripts by PIWI-interacting RNAs (piRNAs) loaded on the Piwi protein. This study shows that the nucleosome remodeler Mi-2, in complex with its partner MEP-1, forms a subunit that is transiently associated, in a MEP-1 C-terminus-dependent manner, with known Piwi interactors, including a recently reported SUMO ligase, Su(var)2-10. Together with the histone deacetylase Rpd3, this module is involved in the piRNA-dependent TE silencing, correlated with H3K9 deacetylation and trimethylation. Therefore, Drosophila piRNA-mediated transcriptional silencing involves three epigenetic effectors, a remodeler, Mi-2, an eraser, Rpd3 and a writer, SetDB1, in addition to the Su(var)2-10 SUMO ligase.
Samata, M., Alexiadis, A., Richard, G., Georgiev, P., Nuebler, J., Kulkarni, T., Renschler, G., Basilicata, M. F., Zenk, F. L., Shvedunova, M., Semplicio, G., Mirny, L., Iovino, N. and Akhtar, A. (2020). Intergenerationally Maintained Histone H4 Lysine 16 Acetylation Is Instructive for Future Gene Activation. Cell. PubMed ID: 32502394
Summary:
Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. This study shows that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.

Monday, August 3rd - Synapse and vesicles

Shields, M. C., Bowers, M. R., Kramer, H. L., Fulcer, M. M., Perinet, L. C., Metz, M. J. and Reist, N. E. (2020). The role of the C2A domain of synaptotagmin 1 in asynchronous neurotransmitter release. PLoS One 15(5): e0232991. PubMed ID: 32407359
Summary:
Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments (aspartate to asparagine mutations, sytD-N) had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca2+ binding. Thus, rather than modulating an asynchronous sensor, sytD-N may be mimicking one. To directly test the C2A inhibition hypothesis, an alternate C2A mutation was used that was designed to block Ca2+ binding without mimicking it (an aspartate to glutamate mutation, sytD-E). Analysis of both the original sytD-N mutation and the alternate sytD-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, asynchronous release wass not increased in the sytD-E mutant. Thus, this work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo.
Aponte-Santiago, N. A., Ormerod, K. G., Akbergenova, Y. and Littleton, J. T. (2020). Synaptic plasticity induced by differential manipulation of tonic and phasic motoneurons in Drosophila. J Neurosci. PubMed ID: 32631939
Summary:
Structural and functional plasticity induced by neuronal competition is a common feature of developing nervous systems. However, the rules governing how postsynaptic cells differentiate between presynaptic inputs are unclear. In this study synaptic interactions was characterized following manipulations of tonic Ib or phasic Is glutamatergic motoneurons that co-innervate postsynaptic muscles of male or female Drosophila melanogaster larvae. After identifying drivers for each neuronal subtype, ablation or genetic manipulations was performed to alter neuronal activity and examined the effects on synaptic innervation and function at neuromuscular junctions (NMJs). Ablation of either Ib or Is resulted in decreased muscle response, with some functional compensation occurring in the Ib input when Is was missing. In contrast, the Is terminal failed to show functional or structural changes following loss of the co-innervating Ib input. Decreasing the activity of the Ib or Is neuron with tetanus toxin light chain resulted in structural changes in muscle innervation. Decreased Ib activity resulted in reduced active zone (AZ) number and decreased postsynaptic subsynaptic reticulum (SSR) volume, with the emergence of filopodial-like protrusions from synaptic boutons of the Ib input. Decreased Is activity did not induce structural changes at its own synapses, but the co-innervating Ib motoneuron increased the number of synaptic boutons and AZs it formed. These findings indicate tonic Ib and phasic Is motoneurons respond independently to changes in activity, with either functional or structural alterations in the Ib neuron occurring following ablation or reduced activity of the co-innervating Is input, respectively.
Santana, E., de Los Reyes, T. and Casas-Tinto, S. (2020). Small heat shock proteins determine synapse number and neuronal activity during development. PLoS One 15(5): e0233231. PubMed ID: 32437379
Summary:
Environmental changes cause stress, Reactive Oxygen Species and unfolded protein accumulation which hamper synaptic activity and trigger cell death. Heat shock proteins (HSPs) assist protein refolding to maintain proteostasis and cellular integrity. Mechanisms regulating the activity of HSPs include transcription factors and posttranslational modifications that ensure a rapid response. HSPs preserve synaptic function in the nervous system upon environmental insults or pathological factors and contribute to the coupling between environmental cues and neuron control of development. A biased screening was conducted in Drosophila melanogaster searching for synaptogenic modulators among HSPs during development. The role of two small-HSPs (sHSPs), sHSP23 and sHSP26 was explored in synaptogenesis and neuronal activity. Both sHSPs immunoprecipitate together and the equilibrium between both chaperones is required for neuronal development and activity. The molecular mechanism controlling HSP23 and HSP26 accumulation in neurons relies on a novel gene (CG1561), which was named Pinkman (pkm). It is proposed that sHSPs and Pkm are targets to modulate the impact of stress in neurons and to prevent synapse loss.
Tang, X., Zhang, L., Ma, T., Wang, M., Li, B., Jiang, L., Yan, Y. and Guo, Y. (2020). Molecular mechanisms that regulate export of the planar cell-polarity protein Frizzled-6 out of the endoplasmic reticulum. J Biol Chem. PubMed ID: 32376691
Summary:
Planar cell polarity (PCP) is a process during which cells are polarized along the plane of the epithelium and is regulated by several transmembrane signaling proteins. After their synthesis, these PCP proteins are delivered along the secretory transport pathway to the plasma membrane, where they perform their physiological functions. This study found that the delivery of a PCP protein, Frizzled-6, to the cell surface is regulated by two conserved polybasic motifs: one located in its first intracellular loop and the other in its C-terminal cytosolic domain. The polybasic motif of Frizzled is also important for its surface localization in the Drosophila wing. Frizzled-6 packaging into vesicles at the endoplasmic reticulum (ER) is regulated by a direct interaction between the polybasic motif and the Glu-62 and Glu-63 residues on the secretion-associated Ras-related GTPase 1A (SAR1A) subunit of coat protein complex II (COPII). Moreover, it was found that newly synthesized Frizzled-6 is associated with another PCP protein, cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), in the secretory transport pathway and that this association regulates their surface delivery. These results reveal insight into the molecular machinery that regulates the ER export of Frizzled-6. They also suggest that the association of CELSR1 with Frizzled-6 is important, enabling efficient Frizzled-6 delivery to the cell surface.
Rozbesky, D., Verhagen, M. G., Karia, D., Nagy, G. N., Alvarez, L., Robinson, R. A., Harlos, K., Padilla-Parra, S., Pasterkamp, R. J. and Jones, E. Y. (2020). Structural basis of semaphorin-plexin cis interaction. Embo j: e102926. PubMed ID: 32500924
Summary:
Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell-attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. This study discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. High-resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities.
Petzoldt, A. G., Gotz, T. W. B., Driller, J. H., Lutzkendorf, J., Reddy-Alla, S., Matkovic-Rachid, T., Liu, S., Knoche, E., Mertel, S., Ugorets, V., Lehmann, M., Ramesh, N., Beuschel, C. B., Kuropka, B., Freund, C., Stelzl, U., Loll, B., Liu, F., Wahl, M. C. and Sigrist, S. J. (2020). RIM-binding protein couples synaptic vesicle recruitment to release sites. J Cell Biol 219(7). PubMed ID: 32369542
Summary:
At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. This study shows in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites.
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