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For developmental processes, we know most of the gene networks controlling specific cell responses. We still have to determine how these networks cooperate and how signals become integrated. The JNK pathway is one of the key elements modulating cellular responses during development. Yet, we still know little about how the core components of the pathway interact with additional regulators or how this network modulates cellular responses in the whole organism in homeostasis or during tissue morphogenesis. We have performed a promoter analysis, searching for potential regulatory sequences of puckered (puc) and identified different specific enhancers directing gene expression in different tissues and at different developmental times. Remarkably, some of these domains respond to the JNK activity, but not all. Altogether, these analyses show that puc expression regulation is very complex and that JNK activities participate in non-previously known processes during the development of Drosophila .

Highly specific expression patterns can be caused by the overlapping activities of activator and repressor sequences in enhancers. However, few studies illuminate how these sequences evolve in the origin of new enhancers. Here, we show that expression of the bond gene in the semicircular wall epithelium (swe) of the Drosophila melanogaster male ejaculatory bulb (EB) is controlled by an enhancer consisting of an activator region that requires Abdominal-B driving expression in the entire EB and a repressor region that restricts this expression to the EB swe. Although this expression pattern is independently gained in the distantly related Scaptodrosophila lebanonensis and does not require Abdominal-B, we show that functionally similar repressor sequences are present in Scaptodrosophila and also in species that do not express bond in the EB. We suggest that during enhancer evolution, repressor sequences can precede the evolution of activator sequences and may lead to similar but independently evolved expression patterns., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Associative learning allows animals to use past experience to predict future events. The circuits underlying memory formation support immediate and sustained changes in function, often in response to a single example. Larval Drosophila is a genetic model for memory formation that can be accessed at molecular, synaptic, cellular, and circuit levels, often simultaneously, but existing behavioral assays for larval learning and memory do not address individual animals, and it has been difficult to form long-lasting memories, especially those requiring synaptic reorganization. We demonstrate a new assay for learning and memory capable of tracking the changing preferences of individual larvae. We use this assay to explore how activation of a pair of reward neurons changes the response to the innately aversive gas carbon dioxide (CO 2 ). We confirm that when coupled to CO 2 presentation in appropriate temporal sequence, optogenetic reward reduces avoidance of CO 2 . We find that learning is switch-like: all-or-none and quantized in two states. Memories can be extinguished by repeated unrewarded exposure to CO 2 but are stabilized against extinction by repeated training or overnight consolidation. Finally, we demonstrate long-lasting protein synthesis dependent and independent memory formation., Competing Interests: AL, JT, JW, MG No competing interests declared, (© 2021, Lesar et al.)

Rhythmic rest-activity cycles are controlled by an endogenous clock. In Drosophila , this clock resides in ∼150 neurons organized in clusters whose hierarchy changes in response to environmental conditions. The concerted activity of the circadian network is necessary for the adaptive responses to synchronizing environmental stimuli. Thus far, work was devoted to unravel the logic of the coordination of different clusters focusing on neurotransmitters and neuropeptides. We further explored communication in the adult male brain through ligands belonging to the bone morphogenetic protein (BMP) pathway. Herein we show that the lateral ventral neurons (LNvs) express the small morphogen decapentaplegic (DPP). DPP expression in the large LNvs triggered a period lengthening phenotype, the downregulation of which caused reduced rhythmicity and affected anticipation at dawn and dusk, underscoring DPP per se conveys time-of-day relevant information. Surprisingly, DPP expression in the large LNvs impaired circadian remodeling of the small LNv axonal terminals, likely through local modulation of the guanine nucleotide exchange factor Trio. These findings open the provocative possibility that the BMP pathway is recruited to strengthen/reduce the connectivity among specific clusters along the day and thus modulate the contribution of the clusters to the circadian network. SIGNIFICANCE STATEMENT The circadian clock relies on the communication between groups of so-called clock neurons to coordinate physiology and behavior to the optimal times across the day, predicting and adapting to a changing environment. The circadian network relies on neurotransmitters and neuropeptides to fine-tune connectivity among clock neurons and thus give rise to a coherent output. Herein we show that decapentaplegic, a ligand belonging to the BMP retrograde signaling pathway required for coordinated growth during development, is recruited by a group of circadian neurons in the adult brain to trigger structural remodeling of terminals on a daily basis., (Copyright © 2021 the authors.)

The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. We report a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. We uncover that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The foraging gene in Drosophila melanogaster , which encodes a cGMP-dependent protein kinase, is a highly conserved, complex gene with multiple pleiotropic behavioral and physiological functions in both the larval and adult fly. Adult foraging expression is less well characterized than in the larva. We characterized foraging expression in the brain, gastric system, and reproductive systems using a T2A-Gal4 gene-trap allele. In the brain, foraging expression appears to be restricted to multiple sub-types of glia. This glial-specific cellular localization of foraging was supported by single-cell transcriptomic atlases of the adult brain. foraging is extensively expressed in most cell types in the gastric and reproductive systems. We then mapped multiple cis -regulatory elements responsible for parts of the observed expression patterns by a nested cloned promoter- Gal4 analysis. The mapped cis -regulatory elements were consistently modular when comparing the larval and adult expression patterns. These new data using the T2A-Gal4 gene-trap and cloned foraging promoter fusion GAL4 's are discussed with respect to previous work using an anti-FOR antibody, which we show here to be non-specific. Future studies of foraging 's function will consider roles for glial subtypes and peripheral tissues (gastric and reproductive systems) in foraging 's pleiotropic behavioral and physiological effects.

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of late-onset Parkinson's disease. The pathogenic G2019S mutation enhances LRRK2 kinase activity and induces neurodegeneration in C. elegans, Drosophila and rodent models through unclear mechanisms. Gene expression profiling has the potential to provide detailed insight into the biological pathways modulated by LRRK2 kinase activity. Prior in vivo studies have surveyed the effects of LRRK2 G2019S on genome-wide mRNA expression in complex brain tissues with high cellular heterogeneity, limiting their power to detect more restricted gene expression changes occurring in a cell type-specific manner. Here, we used translating ribosome affinity purification (TRAP) coupled to RNA-seq to profile dopamine neuron-specific gene expression changes caused by LRRK2 G2019S in the Drosophila CNS. A number of genes were differentially expressed in the presence of mutant LRRK2 that represent a broad range of molecular functions including DNA repair (RfC3), mRNA metabolism and translation (Ddx1 and lin-28), calcium homeostasis (MCU), and other categories (Ugt37c1, disp, l(1)G0196, CG6602, CG1126 and CG11068). Further analysis on a subset of these genes revealed that LRRK2 G2019S did not alter their expression across the whole brain, consistent with dopamine neuron-specific effects uncovered by the TRAP approach that may yield insight into the neurodegenerative process. To our knowledge, this is the first study to profile the effects of LRRK2 G2019S specifically on DA neuron gene expression in vivo. Beyond providing a set of differentially expressed gene candidates relevant to LRRK2, we demonstrate the effective use of TRAP to perform high-resolution assessment of dopamine neuron gene expression for the study of PD., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The construction and maturation of the postsynaptic apparatus are crucial for synapse and dendrite development. The fundamental mechanisms underlying these processes are most often studied in glutamatergic central synapses in vertebrates. Whether the same principles apply to excitatory cholinergic synapses, such as those found in the insect central nervous system, is not known. To address this question, we investigated a group of projection neurons in the Drosophila larval visual system, the ventral lateral neurons (LNvs), and identified nAchRα1 ( Dα1 ) and nAchRα6 ( Dα6 ) as the main functional nicotinic acetylcholine receptor (nAchR) subunits in the larval LNvs. Using morphological analyses and calcium imaging studies, we demonstrated critical roles of these two subunits in supporting dendrite morphogenesis and synaptic transmission. Furthermore, our RNA sequencing analyses and endogenous tagging approach identified distinct transcriptional controls over the two subunits in the LNvs, which led to the up-regulation of Dα1 and down-regulation of Dα6 during larval development as well as to an activity-dependent suppression of Dα1 Additional functional analyses of synapse formation and dendrite dynamics further revealed a close association between the temporal regulation of individual nAchR subunits and their sequential requirements during the cholinergic synapse maturation. Together, our findings support transcriptional control of nAchR subunits as a core element of developmental and activity-dependent regulation of central cholinergic synapses., Competing Interests: The authors declare no competing interest.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder characterized pathologically by motor neuron degeneration and associated with aggregation of RNA-binding proteins. TATA-binding protein-associated factor 15 (TAF15) accumulates as cytoplasmic aggregates in neuronal cells, and clearance of these aggregates is considered a potential therapeutic strategy for ALS. However, the exact pathogenic mechanism of TAF15-induced neurotoxicity remains to be elucidated. Glycogen synthase kinase-3 (GSK-3) plays a critical role in the protection of ALS pathology. In the present study, we use a transgenic fly model over-expressing human TAF15 to study the protective effects of Shaggy/GSK3β on TAF15-induced neuronal toxicity in Drosophila brain. Transgenic flies were examined for locomotor activity and lithium treatment. The expression level and solubility of TAF15 were assessed with western blotting, whereas immunohistochemistry was used to assess TAF15 aggregation in Drosophila brain. We have revealed that Shaggy/GSK3β was abnormally activated in neurons of TAF15-expressing flies and its inhibition can suppress the defective phenotypes, thereby preventing retinal degeneration and locomotive activity caused by TAF15. We have also found that Shaggy/GSK3β inhibition in neuronal cells leads to a reduction in TAF15 levels. Indeed, the F-box proteins Slimb and archipelago genetically interact with TAF15 and control TAF15 protein level in Drosophila. Importantly, SCF slimb is a critical regulator for Shaggy/GSK3β-mediated suppression of TAF15-induced toxicity in Drosophila. The present study has provided an in vivo evidence supporting the molecular mechanism of GSK3β inhibition for protection against TAF15-linked proteinopathies., (© 2020 International Society for Neurochemistry.)

Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. Here, we address this question in Drosophila , which encodes for a single germline-specific dBigH1 linker histone. We show that during female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. We find that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, we discuss the situation in testes, where Brat is not expressed, but dBigH1 is translationally silenced too.

Tauopathies belong to a heterogeneous class of neuronal diseases resulting in the metabolic disturbance. A disulfide natural compound of Alpha-Lipoic acid (ALA) has shown numerous pharmacologic, antioxidant, and neuroprotective activities under neuropathological conditions. The aim of this study was to investigate the neuroprotective effects of ALA on the tauopathy-induced oxidative disturbance and behavioral deficits. The transgenic Drosophila model of tauopathy induced by human tau R406W using GAL4/UAS system and effects of ALA (0.001, 0.005, and 0.025 % w/w of diet) on the neuropathology of tau in younger (20 days) and older (30 days) adults were investigated via biochemical, molecular, behavioral and in-situ tissue analyses. Expression of apoptosis-related proteins involving Drosophila Cyt-c-d (trigger of intrinsic apoptosis) and DrICE (effector caspase) were upregulated in both ages (20 and 30 days) and DIAP1 (caspase inhibitor) has reduced only in older model flies compared to the controls. Remarkably, all doses of ALA increased DIAP1 and glutathione (GSH) as well as reducing Cyt-c-d and lipid peroxidation (LPO) in the younger flies compared to the model flies. Moreover, the higher doses of ALA were able to decrease thiol concentrations, to increase total antioxidant capacity, and to improve the behavioral deficits (locomotor function, olfactory memory, and ethanol sensitivity) in the younger flies. On the other hand, only a higher dose of ALA was able to decrease DrICE, Cyt-c-d, LPO, and thiol as well as increasing antioxidant capacity and decreasing ethanol sensitivity (ST50, RT50) in the older flies. TUNEL assay showed that all doses of ALA could potentially increase the DIAP1/DrICE ratio and exert anti-apoptotic effects on younger, but not on the older adults. Furthermore, data obtained from the in-situ ROS assay confirmed that only a higher dose of ALA significantly decreased the ROS level at both ages. Our data showed that an effective neuroprotective dose of ALA and its mechanism of action on this model of tauopathy could potentially be influenced by longevity. Moreover, it was shown that ALA prevents apoptosis and decreases the redox homeostasis, and this partially explains the mechanism by which ALA diminishes behavioral deficits.

The initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc + in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.

The regulation of formation of the Drosophila heart by the Nkx 2.5 homologue Tinman is a key event during embryonic development. In this study, we identify the highly conserved transcription cofactor Akirin as a key factor in the earliest induction of tinman by the Twist transcription cofactor. akirin mutant embryos display a variety of morphological defects in the heart, including abnormal spacing between rows of aortic cells and abnormal patterning of the aortic outflow tract. akirin mutant embryos have a greatly reduced level of tinman transcripts, together with a reduction of Tinman protein in the earliest stages of cardiac patterning. Further, akirin mutants have reduced numbers of Tinman-positive cardiomyoblasts, concomitant with disrupted patterning and organization of the heart. Finally, despite the apparent formation of the heart in akirin mutants, these mutant hearts exhibit fewer coordinated contractions in akirin mutants compared with wild-type hearts. These results indicate that Akirin is crucial for the first induction of tinman by the Twist transcription factor, and that the success of the cardiac patterning program is highly dependent upon establishing the proper level of tinman at the earliest steps of the cardiac developmental pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Our understanding of cancer-specific metabolic changes is currently unclear. In recent years, the fruit fly Drosophila melanogaster with its powerful genetic tools has become an attractive model for studying both tumor autonomous and the systemic processes resulting from the tumor growth. Here we investigated the effect of tumorigenesis on the modulation of lipid droplets (LDs) in the larval fat bodies (mammalian equivalent of adipose tissue). We have overexpressed Notch signaling alone or in combination with the developmental regulator Myocyte enhancer factor 2 (Mef2) using wing-specific and eye-specific drivers, quantified the size of LDs in the fat body of the different tumor bearing larvae, and estimated the expression of genes associated with lipolysis and lipogenesis. We have found that hyperplastic and neoplastic tumor induced by overexpression of Notch and co-expression of Notch and Mef2 respectively triggers impaired lipid metabolism marked by increased size of fat body LDs. The impaired lipid metabolism in tumor carrying larvae is linked to the altered expression of genes that participate in lipolysis and lipogenesis. These findings reveal modulation of LDs as one of the host's specific response upon tumor initiation. This information could potentially uncover mechanisms for designing innovative approaches to modulate cancer growth., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

PTEN-induced putative kinase 1 (PINK1) mutation induces autosomal recessive Parkinson's Disease (PD), mitochondrial dysfunction is the central pathogenic process. However, more and more studies presented the bulk of the damage to neurons with mitochondrial dysfunction stems from the endoplasmic reticulum (ER) stress. In mitochondria damaged PINK1 B9 fly model how protein kinase RNA-like ER kinase (PERK) arm of ER stress functions remains a mystery. Thus, we generated both PERK overexpressed (PEK OE) and down expressed (PEK RNAi) PINK1 B9 flies and monitored their motor activity. We found PEK OE decreased the abnormal wing posture rate and rescued PINK1 B9 flies' motor activity. Furthermore, we observed the increased number of dopaminergic neurons of protocerebral posterior lateral 1 (PPL1) and the tyrosine hydroxylase (TH) protein levels in PINK1 B9 flies. When testing the mitochondrial morphology in flight muscle with TEM, we found that the shape of the mitochondria became normal. The ATP levels of flight muscle tissues were significantly elevated in PEK OE PINK1 B9 flies with the increased function of mitochondrial Electron Transport Chain (ETC) Complex I (CI) but not Complex Ⅱ (CⅡ) which is further confirmed by oxygen consumption experiments, Western Blot, and RT-PCR to examine the corresponding subunits. We suggest that overexpression of PERK can rescue PINK1 B9 PD flies' pathogenic phenotypes and it is linked with the improved mitochondrial function especially CI of ETC but not CⅡ. Our findings may pave a way for the target of the drug for alleviating the suffering of PINK1 mutant autosomal recessive PD patients., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (Na V ) channels are a functional hallmark of the axonal initial segment in vertebrates. We used an intronic Minos -Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole Na V channel in both male and female Drosophila , para Despite being the only Na V channel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS express para , while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of larval neurons may be capable of firing Na V -dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Na v -dependent APs originate in the DAS. Identification of the distal Na V localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates. SIGNIFICANCE STATEMENT The site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (Na V ) channel in the fly, para , and identified that Para is enriched at a distal axonal segment. The distal axonal segment is located distal to where dendrites impinge on axons and is the likely site of AP initiation. Understanding where APs are initiated improves our ability to model neuronal activity and our interpretation of electrophysiological data. Additionally, para is only expressed in 23 ± 1% of third instar larval neurons but is broadly expressed in adults. Single-cell RNA sequencing of the third instar larval brain shows that para expression correlates with the expression of active, differentiated neuronal markers. Therefore, only 23 ± 1% of third instar larval neurons may be able to actively fire Na V -dependent APs., (Copyright © 2020 the authors.)

Fragile X associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by expansion of CGG repeats in the 5' UTR of the fragile X mental retardation 1 (FMR1) gene. Using the well-established FXTAS Drosophila model, we performed a high-throughput chemical screen using 3200 small molecules. NSC363998 was identified to suppress the neurodegeneration caused by riboCGG (rCGG) repeats. Three predicted targets of a NSC363998 derivative are isopeptidases in the neddylation pathway and could modulate the neurotoxicity caused by the rCGG repeats. Decreasing levels of neddylation resulted in enhancing neurodegeneration phenotypes, while up-regulation could rescue the phenotypes. Furthermore, known neddylation substrates, Cul3 and Vhl, and their downstream target, Sima, were found to modulate rCGG 90 -dependent neurotoxicity. Our results suggest that altered neddylation activity can modulate the rCGG repeat-mediated toxicity by regulating Sima protein levels, which could serve as a potential therapeutic target for FXTAS., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the expressions of caspase-3 and survivin in colorectal cancer patients and their possible associations with clinicopathological parameters and the oncological outcome., Methods: Between January 2008 and December 2011, 85 patients with sporadic colorectal cancer were submitted to colectomy with curative intent. Postoperatively, all patients were followed every three months up to the 36th month. Immunohistochemical detection of the apoptosis-related proteins was carried out on 4-μm-thick deparaffinized sections from all primary tumors. Univariate and multivariate analyses were performed by using the R software for Windows, version 3.3.2., Results: Setting the cut-off point for caspase-3 positivity at 5%, 48% of the patients were characterized as caspase-3(+). Caspase-3 positivity was not found related either to any clinicopathological parameter or to the oncological outcome. Choosing simple survivin positivity as the cut-off point for its expression, 78% of the patients were considered as survivin(+). Survivin inexpression predisposed to poorly differentiated tumors of advanced T stage. However, neither a dismal nor a favorable prognostic role for survivin expression or inexpression was disclosed. By dividing all enrolled patients in four different groups, a trend for worse 3-year overall survival rate in the caspase-3(-)/survivin(-) subgroup of patients was noticed (p=0.067)., Conclusion: Caspase-3 expression was unrelated to the oncological outcome in colorectal cancer patients. The proposed favorable prognostic role for survivin inexpression was not confirmed. On the contrary, survivin(-) tumors were mainly of poor differentiation and advanced T stage. An inverse relationship between caspase-3 and survivin expressions was also not confirmed. Future studies focusing on specific survivin isoforms expression or inexpression may give answers on apoptotic-antiapoptotic interactions on cancer cell death.

Background: Differences in core or tissue-specific ribosomal protein (Rp) composition within ribosomes contribute to ribosome heterogeneity and functional variability. Yet, the degree to which ribosome heterogeneity modulates development is unknown. The Drosophila melanogaster eRpL22 family contains structurally diverse paralogues, eRpL22 and eRpL22-like. Unlike ubiquitously expressed eRpL22, eRpL22-like expression is tissue-specific, notably within the male germline and the eye. We investigated expression within the developing eye to uncover tissue/cell types where specific paralogue roles might be defined., Results: Immunohistochemistry analysis confirms ubiquitous eRpL22 expression throughout eye development. In larvae, eRpL22-like is ubiquitously expressed, but highly enriched in the peripodial epithelium (PE). In early pupae, eRpL22-like is broadly distributed in multiple cell types, but later, is primarily enriched in interommatidial hair cells (IoHC). Adult patterns include the ring of accessory cells around ommatidia. Adult retinae IoHC patterning phenotypes (shown by scanning electron microscopy) may be linked to RNAi-mediated eRpL22-like depletion within larval PE. Immunoblots and polysome profile analyses show multiple variants of eRpL22-like across development, with the variant at the expected molecular mass co-sedimenting with active ribosomes., Conclusion: Our data reveal differential patterns of eRpL22-like expression relative to eRpL22 and suggest a specific role for eRpL22-like in developmental patterning of the eye., (© 2020 Wiley Periodicals LLC.)

Octopamine, the invertebrate analog of norepinephrine, is known to modulate a large variety of behaviors in Drosophila including feeding initiation, locomotion, aggression, and courtship, among many others. Significantly less is known about the identity of the neurons that receive octopamine input and how they mediate octopamine-regulated behaviors. Here, we characterize adult neuronal expression of MiMIC-converted Trojan-Gal4 lines for each of the five Drosophila octopamine receptors. Broad neuronal expression was observed for all five octopamine receptors, yet distinct differences among them were also apparent. Use of immunostaining for the octopamine neurotransmitter synthesis enzyme Tdc2, along with a novel genome-edited conditional Tdc2-LexA driver, revealed all five octopamine receptors express in Tdc2/octopamine neurons to varying degrees. This suggests autoreception may be an important circuit mechanism by which octopamine modulates behavior., (© 2020 Wiley Periodicals, Inc.)

Matrix metalloproteinases (MMPs) are evolutionarily conserved extracellular matrix proteinases. Genetic analysis of the Drosophila MMPs, Mmp1 and Mmp2, in vivo reveal that they play vital roles in tissue remodeling. Although the catalytic domain (CD) undertakes most MMP functions, few studies have sought to demonstrate the biochemical properties of the CDs of fly MMPs. Here, we identified the overexpression, purification, and refolding of the CDs of Drosophila Mmp1 and Mmp2 for biochemical studies. Zymography assays and substrate degradation analysis showed that both Mmp1-CD and Mmp2-CD were able to digest casein, gelatin, fibronectin, collagen (types I, IV, and V), while Mmp2-CD showed much higher degradation activity compared with Mmp1-CD. Moreover, human collagen III could be degraded by Mmp1-CD but not Mmp2-CD, and rat collagen I and laminin could be degraded by Mmp2-CD but not Mmp1-CD, suggesting that Drosophila Mmp1 and Mmp2 might have overlapping yet distinct substrate specificity. Using synthetic fluorescent substrates, we further demonstrated that the enzymatic activity of Mmp1-CD and Mmp2-CD could be inhibited by human tissue inhibitors of metalloproteinases (TIMPs). These results reveal the context of the cooperative yet distinct roles of Mmp1 and Mmp2 in tissue remodeling., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In this research, we studied the formation of Drosophila melanogaster FADD (Fas-associated death domain-containing protein) amyloid fiber and its influence on signal transduction in IMD (Immune deficiency) signaling pathway to better understand the regulation mechanism of Drosophila innate immune signaling pathway, which will provide reference for the immune regulation in other species. First, we purified dFADD protein expressed in Escherichia coli and performed Sulfur flavin T binding and transmission electron microscopy to identify the dFADD amyloid fibers formed in vitro. Then we investigated the formation of dFADD polymers in S2 cells using SDD-AGE and confocal microscope. We also constructed dFADD mutants to find out which domain is essential to fiber formation and its effect on IMD signal transduction. Our results revealed that dFADD could be polymerized to form amyloid fiber polymers in vitro and inside the cells. Formation of fibers relies on DED (Death-effector domain) domain of dFADD, since DED domain-deleted mutant existed as a monomer. Dual luciferase reporter assay showed that intact DED domain was required for the induction of downstream antimicrobial peptides, indicating that fiber formation was the key to IMD signal transduction. Our study revealed the role of dFADD in mediating the cascade between IMD and Dredd in the IMD signaling pathway by forming amyloid fibers, suggesting an evolutionarily conserved regulatory mechanism of innate immune signaling pathway.

During prolonged nutrient restriction, developing animals redistribute vital nutrients to favor brain growth at the expense of other organs. In Drosophila , such brain sparing relies on a glia-derived growth factor to sustain proliferation of neural stem cells. However, whether other aspects of neural development are also spared under nutrient restriction is unknown. Here we show that dynamically growing somatosensory neurons in the Drosophila peripheral nervous system exhibit organ sparing at the level of arbor growth: Under nutrient stress, sensory dendrites preferentially grow as compared to neighboring non-neural tissues, resulting in dendrite overgrowth. These neurons express lower levels of the stress sensor FoxO than neighboring epidermal cells, and hence exhibit no marked induction of autophagy and a milder suppression of Tor signaling under nutrient stress. Preferential dendrite growth allows for heightened animal responses to sensory stimuli, indicative of a potential survival advantage under environmental challenges., Competing Interests: AP, YX, CZ, JL, KL, DL, CH No competing interests declared, (© 2020, Poe et al.)

The Decapentaplegic (Dpp) and Wingless (Wg) signaling pathways are essential for animal development. However, how these two signals are integrated in distinct tissues is not fully understood. Here, we describe a novel mode of Dpp-Wg crosstalk during Drosophila wing development. We show that the canonical Dpp signaling is required for Wg target gene activation. In addition, Dpp signaling inhibits the transcription of wg through the schnurri (shn) repressor complex. A Dpp-responsive shn/pMad/Med silencer element (SSE) is identified in the genomic loci of the wg gene. ChIP analysis suggests that shn interacts with this element in vivo. Our findings support a model in which Dpp signaling plays a dual role in transcriptional regulation of both the wg gene and downstream targets., (© 2019 Federation of European Biochemical Societies.)

The main pathological hallmark of Parkinson's disease (PD) is the presence of Lewy bodies, which mainly consist of aggregated α-synuclein. Based on the neurotoxicity of oligomeric α-synuclein and its significance in the aetiology of PD, there has been decades of effort to elucidate an enzyme specifically degrading oligomeric α-synuclein. Here we report an enzyme, Omi, which specifically recognizes and precisely degrades oligomeric α-synuclein but not monomeric α-synuclein. After enzymatic and functional analyses of Omi in in vitro, we developed an in vivo assay system of dual gene interaction in Drosophila to investigate further the etiological role of Omi in PD. Pan-neuronal expression of Omi rescued Parkinsonism in a Drosophila model of PD, while Knockout of Omi exacerbated Parkinsonism. Expression of Omi counteracted the α-synuclein-induced retinal degeneration, providing additional evidence for Omi's protective role oligomeric α-synuclein. This work reports identification of the catabolic pathway of oligomeric α-synuclein as well as showing how Omi functions as the key molecule in the recognition and degradation of toxic oligomeric α-synuclein, a possible cause of neurodegeneration in PD, without affecting monomeric α-synuclein which is a native essential molecule for the normal function of neurons., Competing Interests: Declaration of competing interest None., (Copyright © 2019. Published by Elsevier Inc.)

Guidance cues act during development to guide growth cones to their proper targets in both the central and peripheral nervous systems. Experiments in many species indicate that guidance molecules also play important roles after development, though less is understood about their functions in the adult. The Semaphorin family of guidance cues, signaling through Plexin receptors, influences the development of both axons and dendrites in invertebrates. Semaphorin functions have been extensively explored in Drosophila melanogaster and some other Dipteran species, but little is known about their function in hemimetabolous insects. Here, we characterize sema1a and plexA in the cricket Gryllus bimaculatus. In fact, we found two distinct predicted Sema1a proteins in this species, Sema1a.1 and Sema1a.2, which shared only 48% identity at the amino acid level. We include a phylogenetic analysis that predicted that many other insect species, both holometabolous and hemimetabolous, express two Sema1a proteins as well. Finally, we used in situ hybridization to show that sema1a.1 and sema1a.2 expression patterns were spatially distinct in the embryo, and both roughly overlap with plexA. All three transcripts were also expressed in the adult brain, mainly in the mushroom bodies, though sema1a.2 was expressed most robustly. sema1a.2 was also expressed strongly in the adult thoracic ganglia while sema1a.1 was only weakly expressed and plexA was undetectable., (© 2019 Wiley Periodicals, Inc.)

New cellular functions and developmental processes can evolve by modifying existing genes or creating novel genes. Novel genes can arise not only via duplication or mutation but also by acquiring foreign DNA, also called horizontal gene transfer (HGT). Here we show that HGT likely contributed to the creation of a novel gene indispensable for reproduction in some insects. Long considered a novel gene with unknown origin, oskar has evolved to fulfil a crucial role in insect germ cell formation. Our analysis of over 100 insect Oskar sequences suggests that oskar arose de novo via fusion of eukaryotic and prokaryotic sequences. This work shows that highly unusual gene origin processes can give rise to novel genes that may facilitate evolution of novel developmental mechanisms., Competing Interests: LB, TJ, CE No competing interests declared, (© 2020, Blondel et al.)

Hedgehog proteins, a family of vital cell signaling factors, are expressed in precursor form, which requires specialized autoprocessing, called cholesterolysis, for full biological activity. Cholesterolysis occurs in cis through the action of the precursor's C-terminal enzymatic domain, HhC. In this work, we describe HhC activator compounds (HACs), a novel class of noncovalent modulators that induce autoprocessing infidelity, diminishing native cholesterolysis in favor of precursor autoproteolysis, an otherwise minor and apparently nonphysiological side reaction. HAC-induced autoproteolysis generates hedgehog protein that is cholesterol free and hence signaling deficient. The most effective HAC has an AC 50 of 9 μM, accelerates HhC autoproteolytic activity by 225-fold, and functions in the presence and absence of cholesterol, the native substrate. HACs join a rare class of "antagonists" that suppress native enzymatic activity by subverting mechanistic fidelity.

Long-term memory (LTM) is stored as functional modifications of relevant neural circuits in the brain. A large body of evidence indicates that the initial establishment of such modifications through the process known as memory consolidation requires learning-dependent transcriptional activation and de novo protein synthesis. However, it remains poorly understood how the consolidated memory is maintained for a long period in the brain, despite constant turnover of molecular substrates. Using the Drosophila courtship conditioning assay of adult males as a memory paradigm, here, we show that in Drosophila , environmental light plays a critical role in LTM maintenance. LTM is impaired when flies are kept in constant darkness (DD) during the memory maintenance phase. Because light activates the brain neurons expressing the neuropeptide pigment-dispersing factor (Pdf), we examined the possible involvement of Pdf neurons in LTM maintenance. Temporal activation of Pdf neurons compensated for the DD-dependent LTM impairment, whereas temporal knockdown of Pdf during the memory maintenance phase impaired LTM in light/dark cycles. Furthermore, we demonstrated that the transcription factor cAMP response element-binding protein (CREB) is required in the memory center, namely, the mushroom bodies (MBs), for LTM maintenance, and Pdf signaling regulates light-dependent transcription via CREB. Our results demonstrate for the first time that universally available environmental light plays a critical role in LTM maintenance by activating the evolutionarily conserved memory modulator CREB in MBs via the Pdf signaling pathway. SIGNIFICANCE STATEMENT Temporary memory can be consolidated into long-term memory (LTM) through de novo protein synthesis and functional modifications of neuronal circuits in the brain. Once established, LTM requires continual maintenance so that it is kept for an extended period against molecular turnover and cellular reorganization that may disrupt memory traces. How is LTM maintained mechanistically? Despite the critical importance of LTM maintenance, its molecular and cellular underpinnings remain elusive. This study using Drosophila is significant because it revealed for the first time in any organism that universally available environmental light plays an essential role in LTM maintenance. Interestingly, light does so by activating the evolutionarily conserved transcription factor cAMP response element-binding protein via peptidergic signaling., (Copyright © 2020 the authors.)

Tauopathies such as Alzheimer's disease (AD), Pick's disease (PiD), Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) etc. represent a group of age-related neurodegenerative disorders in which tau protein loses its normal conformation mostly due to hyperphosphorylation and subsequent formation of the aggregates of defined shapes, known as Neurofibrillary Tangles (NFTs). We have demonstrated earlier that reduced dosage of dmyc (Drosophila homolog of human cmyc proto-oncogene) restricts tau WT mediated disease pathogenesis by regulating the phosphorylation status of tau. We demonstrate further that the downregulation of dmyc also alleviates the mutant human-tau (tau V337M ) mediated neurotoxicity in Drosophila by improving disease defects. Moreover, tissue-specific downregulation of dmyc also induces cellular autophagy which facilitates the disposal of misfolded proteins via lysosome-mediated proteostasis. Our findings demonstrate the capability of dmyc in the suppression of different forms of human tauopathies in Drosophila disease models. Interestingly, due to the conserved characteristics of dmyc/cmyc across the animal kingdom, our study strengthens the possibility of utilizing this gene as an effective drug target against tauopathies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Axon degeneration elicits a range of immune responses from local glial cells, including striking changes in glial gene expression, morphology, and phagocytic activity. Here, we describe a detailed set of protocols to assess discrete components of the glial reaction to axotomy in the adult nervous system of Drosophila melanogaster. These methods allow one to visualize and quantify transcriptional, morphological, and functional responses of glia to degenerating axons in a model system that is highly amenable to genetic manipulation.

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1., Competing Interests: EA, MN, YR, AH, CC, CD, LM, MT, GG, GJ, TJ, MZ, KJ No competing interests declared, (© 2019, Auxerre-Plantié et al.)

It has been known for over 20 years that Drosophila melanogaster flies with twelve additional copies of the hsp70 gene encoding the 70 kD heat shock protein lives longer after a non-lethal heat treatment. Since the heat treatment also induces the expression of additional heat shock proteins, the biological effect can be due either to HSP70 acting alone or in combination. This study used the UAS/GAL4 system to determine whether hsp70 is sufficient to affect the longevity and the resistance to thermal, oxidative or desiccation stresses of the whole organism. We observed that HSP70 expression in the nervous system or muscles has no effect on longevity or stress resistance but ubiquitous expression reduces the life span of males. We also observed that the down-regulation of hsp70 using RNAi did not affect longevity., (Copyright © 2019 Xiao et al.)

It is believed that long-term memory (LTM) cannot be formed immediately because it must go through a protein synthesis-dependent consolidation process. However, the current study uses Drosophila aversive olfactory conditioning to show that such processes are dispensable for context-dependent LTM (cLTM). Single-trial conditioning yields cLTM that is formed immediately in a protein-synthesis independent manner and is sustained over 14 days without decay. Unlike retrieval of traditional LTM, which requires only the conditioned odour and is mediated by mushroom-body neurons, cLTM recall requires both the conditioned odour and reinstatement of the training-environmental context. It is mediated through lateral-horn neurons that connect to multiple sensory brain regions. The cLTM cannot be retrieved if synaptic transmission from any one of these centres is blocked, with effects similar to those of altered encoding context during retrieval. The present study provides strong evidence that long-term memory can be formed easily without the need for consolidation.

In complex genetic loci, individual enhancers interact most often with specific basal promoters. Here we investigate the activation of the Bicoid target gene hunchback (hb), which contains two basal promoters (P1 and P2). Early in embryogenesis, P1 is silent, while P2 is strongly activated. In vivo deletion of P2 does not cause activation of P1, suggesting that P2 contains intrinsic sequence motifs required for activation. We show that a two-motif code (a Zelda binding site plus TATA) is required and sufficient for P2 activation. Zelda sites are present in the promoters of many embryonically expressed genes, but the combination of Zelda plus TATA does not seem to be a general code for early activation or Bicoid-specific activation per se. Because Zelda sites are also found in Bicoid-dependent enhancers, we propose that simultaneous binding to both enhancers and promoters independently synchronizes chromatin accessibility and facilitates correct enhancer-promoter interactions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

How circuits assemble starting from stem cells is a fundamental question in developmental neurobiology. We test the hypothesis that, in neuronal stem cells, temporal transcription factors predictably control neuronal terminal features and circuit assembly. Using the Drosophila motor system, we manipulate expression of the classic temporal transcription factor Hunchback (Hb) specifically in the NB7-1 stem cell, which produces U motor neurons (MNs), and then we monitor dendrite morphology and neuromuscular synaptic partnerships. We find that prolonged expression of Hb leads to transient specification of U MN identity, and that embryonic molecular markers do not accurately predict U MN terminal features. Nonetheless, our data show Hb acts as a potent regulator of neuromuscular wiring decisions. These data introduce important refinements to current models, show that molecular information acts early in neurogenesis as a switch to control motor circuit wiring, and provide novel insight into the relationship between stem cell and circuit., Competing Interests: JM, ZM, ML, EH No competing interests declared, (© 2019, Meng et al.)

The interaction of the GAF protein with the promoters of neuron-specific genes during activation and repression of transcription was studied. We showed that, while the Su(Hw) protein remains stably associated with the promoters of these genes at different transcriptional state, the GAF protein level is significantly higher when transcription is activated.

Since Drosophila melanogaster has proven to be a useful model system to study phenotypes of oncogenic mutations and to identify new anti-cancer drugs, we generated human BRAF V600E homologous dRaf mutant (dRaf A572E ) Drosophila melanogaster strains to use these for characterisation of mutant phenotypes and exploit these phenotypes for drug testing. For mutant gene expression, the GAL4/UAS expression system was used. dRaf A572E was expressed tissue-specific in the eye, epidermis, heart, wings, secretory glands and in the whole animal. Expression of dRaf A572E under the control of an eye-specific driver led to semi-lethality and a rough eye phenotype. The vast majority of other tissue-specific and ubiquitous drivers led to a lethal phenotype only. The rough eye phenotype was used to test BRAF inhibitor vemurafenib and MEK1/2 inhibitor cobimetinib. There was no phenotype rescue by this treatment. However, a significant rescue of the lethal phenotype was observed under a gut-specific driver. Here, MEK1/2 inhibitor cobimetinib rescued Drosophila larvae to reach pupal stage in 37% of cases as compared to 1% in control experiments. Taken together, the BRAF V600E homolog dRaf A572E exerts mostly lethal effects in Drosophila. Gut-specific dRaf A572E expression might in future be developed further for drug testing., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

During organogenesis, inductive signals cause cell differentiation and morphogenesis. However, how these phenomena are coordinated to form functional organs is poorly understood. Here, we show that cell differentiation of the Drosophila trachea is sequentially determined in two steps and that the second step is synchronous with the invagination of the epithelial sheet. The master gene trachealess is dispensable for the initiation of invagination, while it is essential for maintaining the invaginated structure, suggesting that tracheal morphogenesis and differentiation are separately induced. trachealess expression starts in bipotential tracheal/epidermal placode cells. After invagination, its expression is maintained in the invaginated cells but is extinguished in the remaining sheet cells. A trachealess cis-regulatory module that shows both tracheal enhancer activity and silencer activity in the surface epidermal sheet was identified. We propose that the coupling of trachealess expression with the invaginated structure ensures that only invaginated cells canalize robustly into the tracheal fate., Competing Interests: TK, SH No competing interests declared, (© 2019, Kondo and Hayashi.)

In the Drosophila model of aggression, males and females fight in same-sex pairings, but a wide disparity exists in the levels of aggression displayed by the 2 sexes. A screen of Drosophila Flylight Gal4 lines by driving expression of the gene coding for the temperature sensitive dTRPA1 channel, yielded a single line ( GMR26E01-Gal4 ) displaying greatly enhanced aggression when thermoactivated. Targeted neurons were widely distributed throughout male and female nervous systems, but the enhanced aggression was seen only in females. No effects were seen on female mating behavior, general arousal, or male aggression. We quantified the enhancement by measuring fight patterns characteristic of female and male aggression and confirmed that the effect was female-specific. To reduce the numbers of neurons involved, we used an intersectional approach with our library of enhancer trap flp-recombinase lines. Several crosses reduced the populations of labeled neurons, but only 1 cross yielded a large reduction while maintaining the phenotype. Of particular interest was a small group (2 to 4 pairs) of neurons in the approximate position of the pC1 cluster important in governing male and female social behavior. Female brains have approximately 20 doublesex (dsx )-expressing neurons within pC1 clusters. Using dsx FLP instead of 357 FLP for the intersectional studies, we found that the same 2 to 4 pairs of neurons likely were identified with both. These neurons were cholinergic and showed no immunostaining for other transmitter compounds. Blocking the activation of these neurons blocked the enhancement of aggression., Competing Interests: The authors declare no conflict of interest.

Rapid or even anticipatory adaptation to environmental conditions can provide a decisive fitness advantage to an organism. The memory of recurring conditions could also benefit future generations; however, neuronally-encoded behavior isn't thought to be inherited across generations. We tested the possibility that environmentally triggered modifications could allow 'memory' of parental experiences to be inherited. In Drosophila melanogaster , exposure to predatory wasps leads to inheritance of a predisposition for ethanol-rich food for five generations. Inhibition of Neuropeptide-F (NPF) activates germline caspases required for transgenerational ethanol preference. Further, inheritance of low NPF expression in specific regions of F 1 brains is required for the transmission of this food preference: a maternally derived NPF locus is necessary for this phenomenon, implicating a maternal epigenetic mechanism of NPF-repression. Given the conserved signaling functions of NPF and its mammalian NPY homolog in drug and alcohol disorders, these observations raise the intriguing possibility of NPY-related transgenerational effects in humans., Competing Interests: JB, BK, GB No competing interests declared, (© 2019, Bozler et al.)

Many metazoan genes express alternative long 3' UTR isoforms in the nervous system, but their functions remain largely unclear. In Drosophila melanogaster, the Dscam1 gene generates short and long (Dscam1-L) 3' UTR isoforms because of alternative polyadenylation (APA). Here, we found that the RNA-binding protein Embryonic Lethal Abnormal Visual System (Elav) impacts Dscam1 biogenesis at two levels, including regulation of long 3' UTR biogenesis and skipping of an upstream exon (exon 19). MinION long-read sequencing confirmed the connectivity of this alternative splicing event to the long 3' UTR. Knockdown or CRISPR deletion of Dscam1-L impaired axon outgrowth in Drosophila. The Dscam1 long 3' UTR was found to be required for correct Elav-mediated skipping of exon 19. Elav thus co-regulates APA and alternative splicing to generate specific Dscam1 transcripts that are essential for neural development. This coupling of APA to alternative splicing might represent a new class of regulated RNA processing., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Development of an organism requires accurate coordination between the growth of a tissue and orientation of cells within the tissue. The large cadherin Fat has been shown to play a role in both of these processes. Fat is involved in the establishment of planar cell polarity and regulates growth through the Hippo pathway, a developmental cascade that controls proliferation and apoptosis. Both Fat and the Hippo pathway are known to regulate transcription of four-jointed, although the nature of this regulation is unknown. In this study, we test whether Fat affects four-jointed transcription via or independently of Hippo pathway. Our analysis of the four-jointed regulatory region reveals a 1.2 kb element that functions as an enhancer for graded expression of Four-jointed in the eye imaginal disc. Within this enhancer element, we identify a 20 bp fragment that is critical for regulation by Fat but not by Hippo. Our findings suggest that Fat and the Hippo pathway control four-jointed expression independently of each other and none of the transcription factors known to function downstream of the Hippo pathway are required to regulate four-jointed expression through the 1.2 kb element., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Mutants which carry mutations in genes encoding mitochondrial ligases MUL1 and PARKIN are convenient Drosophila models of Parkinson's disease (PD). In several studies it has been shown that in Parkinson's disease sleep disturbance occurs, which may be the result of a disturbed circadian clock., Results: We found that the ROS level was higher, while the anti-oxidant enzyme SOD1 level was lower in mul1 A6 and park 1 mutants than in the white mutant used as a control. Moreover, mutations of both ligases affected circadian rhythms and the clock. The expression of clock genes per, tim and clock and the level of PER protein were changed in the mutants. Moreover, expression of ATG5, an autophagy protein also involved in circadian rhythm regulation, was decreased in the brain and in PDF-immunoreactive large ventral lateral clock neurons. The observed changes in the molecular clock resulted in a longer period of locomotor activity rhythm, increased total activity and shorter sleep at night. Finally, the lack of both ligases led to decreased longevity and climbing ability of the flies., Conclusions: All of the changes observed in the brains of these Drosophila models of PD, in which mitochondrial ligases MUL1 and PARKIN do not function, may explain the mechanisms of some neurological and behavioural symptoms of PD.

In many biological systems gene expression at mRNA and protein levels is not identical. Rigorous comparison of such differences on a spatio-temporal scale is still not feasible by high-throughput transcriptomic and proteomic analyses of early embryo development. Here, we characterize differences between mRNA and protein expression of Drosophila segmentation genes at the level of individual gene expression domains. We obtained quantitative imaging data on expression of gap genes gt and hb and pair-rule gene eve for Drosophila wild type embryos, Kr null mutants and Kr+/Kr- heterozygotes. To compare mRNA and protein expression we use several criteria including difference in amplitude and positions of expression domains, pattern shape and positional variability. For a number of gene expression domains we show examples where protein expression does not repeat mRNA expression even after a temporal delay. We calculated time delays between eve pattern formation at the level of mRNA and protein for wild type embryos, Kr mutants and Kr+/Kr- heterozygotes. We detect that in wild type embryos, the amplitudes of eve stripes 3 and 7 do not differ significantly at the level of mRNA, however, stripe 3 is higher than stripe 7 at the protein level. We further show that hb mRNA and protein expression in both anterior and posterior domains significantly differs at specific time points. The formation of hb PS4 stripe at the mRNA level proceeds five times faster than at the level of protein. With regard to spatial expression, we show that the offset between posterior gt mRNA and protein domains is much larger in Kr mutants than in wild type embryos and heterozygotes. Finally, we analyze differences in positional variability of eve stripe 7 expression in Kr mutants and Kr+/Kr- heterozygotes at the level of mRNA and protein. These results enable further perspectives to uncover mechanisms underlying discrepancies between mRNA and protein expression in early embryo., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The in vivo model Drosophila melanogaster was used here to determine the detrimental effects induced by silver nanoparticles (AgNPs) exposure. The main aim was to explore its interaction with the intestinal barrier and the genotoxic effects induced in hemocytes. The observed effects were compared with those obtained by silver nitrate, as an agent acting via the release of silver ions. Larvae were fed in food media containing both forms of silver. Results indicated that silver nitrate was more toxic than AgNPs when the viability "egg-to-adult" was determined. Depigmentation was observed in adults including those exposed to nontoxic concentrations, as indicative of exposure action. Interestingly, AgNPs were able to cross the intestinal barrier affecting hemocytes that show significant increases in the levels of intracellular reactive oxygen species. Additionally, significant levels of genotoxic damage, as determined by the comet assay, were also induced. When the expression of different stress-response genes was determined, for both AgNPs and silver nitrate, significant upregulation of Sod2 and p53 genes was observed. Our results confirm for the first time that in an in vivo model as Drosophila, AgNPs are able to cross the intestinal barriers and produce primary DNA damage (comet assay) via oxidative stress induction. In general, the effects induced by silver nitrate were more pronounced than those induced by AgNPs what would emphasize the role of silver ions in the observed effects. Environ. Mol. Mutagen. 60:277-285, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

In Drosophila, female development is governed by a single RNA-binding protein, Sex-lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Sxl expression in female flies is maintained by an auto-regulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now, it remained unclear how males prevent accidental triggering of the Sxl expression cascade and protect themselves against runaway protein production. Here, we identify the protein Sister-of-Sex-lethal (Ssx) as an inhibitor of Sxl auto-regulatory splicing. Sxl and Ssx have a comparable RNA-binding specificity and compete for binding to RNA regulatory elements present in the Sxl transcript. In cultured Drosophila cells, Sxl-induced changes to alternative splicing can be reverted by the expression of Ssx. Moreover, in adult male flies ablation of the ssx gene results in a low level of productive Sxl mRNA splicing and Sxl protein production in isolated, clonal cell populations. In sum, this demonstrates that Ssx safeguards male animals against Sxl protein production to reinforce a stable, male-specific gene expression pattern., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Drosophila segmentation is regulated by a complex network of transcription factors that include products of the pair-rule genes (PRGs). PRGs are expressed in early embryos in the primorida of alternate segmental units, establishing the repeated, segmental body plan of the fly. Despite detailed analysis of the regulatory logic among segmentation genes, the relationship between these genes and the morphological formation of segments is still poorly understood, since regulation of transcription factor expression is not sufficient to explain how segments actually form and are maintained. Cell surface proteins containing Leucine rich repeats (LRR) play a variety of roles in development, and those expressed in segmental patterns likely impact segment morphogenesis. Here we explore the relationships between the PRG network and segmentally expressed LRR-encoding (sLRR) genes. We examined expression of Toll2, Toll6, Toll7, Toll8 and tartan (trn) in wild type or PRG mutant embryos. Expression of each sLRR-encoding gene is dynamic, but each has a unique register along the anterior-posterior axis. The registers for different sLRRs are off-set from one another resulting in a continually changing set of overlapping expression patterns among the sLRR-encoding genes themselves and between the sLRR-encoding genes and the PRGs. Accordingly, each sLRR-encoding gene is regulated by a unique combination of PRGs. These findings suggest that one role of the PRG network is to promote segmentation by establishing a cell surface code: each row of cells in the two-segment-wide primordia expresses a unique combination of sLRRs, thereby translating regulatory information from the PRGs to direct segment morphogenesis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Gene expression is much altered in aging. We observed age-dependent decline of core clock genes' expression in the whole body of the fruit fly. We hypothesized that inducible overexpression of clock genes (cry, per, tim, cyc and Clk) in the nervous system can improve healthspan of D. melanogaster. We studied the lifespan of transgenic Drosophila and showed life extension for cry, per, cyc and tim genes. It was also the significant positive changes in the stress-resistance of flies overexpressing core clock genes in conditions of hyperthermia, hyperoxia, starvation and persistent lighting. The overexpression of per and cry restore circadian rhythms of locomotor activity. The results presented support the hypotheses that the compensation of circadian oscillator genes expression can improve the healthspan in Drosophila melanogaster., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Gene duplication could promote phenotypic and genetic adaptation to various environments. To understand the effects of gene duplication on transcriptional regulation associated with environmental changes, we focused on the starch hydrolysis pathway, in which amylase enzymes together with maltase enzymes hydrolyze starch into glucose. Drosophila genomes involve ten duplicated Maltase genes. We examined the levels of transcription of the nine of these genes in 36 lines of Drosophila melanogaster collected from a natural population. In the investigated population, the levels of transcription were different between the two dietary carbohydrate sources, glucose and starch. At the transcriptional level, a single Maltase gene, which transcribes the specific Maltase transcripts, worked together with an Amylase gene in the pathway. The three of nine genes responded to carbohydrate changes, and the degree of the response was similar to Amylase gene. Our results suggest that gene duplication could increase capacity of the transcriptional regulation associated with environmental changes., (Copyright © 2018 Elsevier B.V. All rights reserved.)