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Monday, April 30th - Drosophila Disease Models

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Rossi, F., Stephan-Otto Attolini, C., Mosquera, J. L. and Gonzalez, C. (2018). Drosophila larval brain neoplasms present tumour-type dependent genome instability. G3 (Bethesda). PubMed ID: 29467187
Summary:
Single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) are found at different rates in human cancer. To determine if these genetic lesions appear in Drosophila tumours this study sequenced the genomes of 17 malignant neoplasms caused by mutations in l(3)mbt, brat, aurA, or lgl. CNVs and SNPs were found in all the tumours. Tumour-linked CNVs range between 11 and 80 per sample, affecting between 92 and 1546 coding sequences. CNVs are in average less frequent in l(3)mbt than in brat lines. Nearly half of the CNVs fall within the 10 to 100Kb range, all tumour samples contain CNVs larger that 100 Kb and some have CNVs larger than 1Mb. The rates of tumour-linked SNPs change more than 20-fold depending on the tumour type: at late time points brat, l(3)mbt, and aurA and lgl lines present median values of SNPs/Mb of exome of 0.16, 0.48, and 3.6, respectively. Higher SNP rates are mostly accounted for by C>A transversions, which likely reflect enhanced oxidative stress conditions in the affected tumours. Both CNVs and SNPs turn over rapidly. No evidence was found for selection of a gene signature affected by CNVs or SNPs in the cohort. Altogether, these results show that the rates of CNVs and SNPs, as well as the distribution of CNV sizes in this cohort of Drosophila tumours are well within the range of those reported for human cancer. Genome instability is therefore inherent to Drosophila malignant neoplastic growth at a variable extent that is tumour type dependent.
Talmat-Amar, Y., Arribat, Y. and Parmentier, M. L. (2018). Vesicular Axonal Transport is Modified In Vivo by Tau Deletion or Overexpression in Drosophila. Int J Mol Sci 19(3). PubMed ID: 29509687
Summary:
Structural microtubule associated protein Tau is found in high amount in axons and is involved in several neurodegenerative diseases. Although many studies have highlighted the toxicity of an excess of Tau in neurons, the in vivo understanding of the endogenous role of Tau in axon morphology and physiology is poor. Indeed, knock-out mice display no strong cytoskeleton or axonal transport phenotype, probably because of some important functional redundancy with other microtubule-associated proteins (MAPs). This study took advantage of the model organism Drosophila, which genome contains only one homologue of the Tau/MAP2/MAP4 family to decipher (endogenous) Tau functions. Tau depletion was found to lead to a decrease in microtubule number and microtubule density within axons, while Tau excess leads to the opposite phenotypes. Analysis of vesicular transport in tau mutants showed altered mobility of vesicles, but no change in the total amount of putatively mobile vesicles, whereas both aspects were affected when Tau was overexpressed. In conclusion, this study shows that loss of Tau in tau mutants not only leads to a decrease in axonal microtubule density, but also impairs axonal vesicular transport, albeit to a lesser extent compared to the effects of an excess of Tau.
Spurrier, J., Shukla, A. K., McLinden, K., Johnson, K. and Giniger, E. (2018). Altered expression of the Cdk5 activator-like protein, Cdk5alpha, causes neurodegeneration in part by accelerating the rate of aging. Dis Model Mech. PubMed ID: 29469033
Summary:
Aging is the greatest risk factor for neurodegeneration, but the connection between the two processes remains opaque. This is in part for want of a rigorous way to define physiological age, as opposed to chronological age. This study developed a comprehensive metric for physiological age in Drosophila, based on genome-wide expression profiling. This metric was applied to a model of adult-onset neurodegeneration, increased or decreased expression of the activating subunit of the Cdk5 protein kinase, encoded by the gene Cdk5alpha, the ortholog of mammalian p35. Cdk5alpha-mediated degeneration was associated with a 27-150% acceleration of the intrinsic rate of aging, depending on the tissue and genetic manipulation. Gene ontology analysis and direct experimental tests revealed that affected, age-associated processes included numerous core phenotypes of neurodegeneration, including enhanced oxidative stress and impaired proteostasis. Taken together, these results suggest that Cdk5alpha-mediated neurodegeneration results from accelerated aging, in combination with cell-autonomous neuronal insults. These data fundamentally recast the picture of the relationship between neurodegeneration and its most prominent risk factor, natural aging.
Walls, S. M., Cammarato, A., Chatfield, D. A., Ocorr, K., Harris, G. L. and Bodmer, R. (2018). Ceramide-protein interactions modulate ceramide-associated lipotoxic cardiomyopathy. Cell Rep 22(10): 2702-2715. PubMed ID: 29514098
Summary:
Lipotoxic cardiomyopathy (LCM) is characterized by abnormal myocardial accumulation of lipids, including ceramide; however, the contribution of ceramide to the etiology of LCM is unclear. This study investigated the association of ceramide metabolism and ceramide-interacting proteins (CIPs) in LCM in the Drosophila heart model. Ceramide feeding or ceramide-elevating genetic manipulations are strongly associated with cardiac dilation and defects in contractility. High ceramide-associated LCM is prevented by inhibiting ceramide synthesis, establishing a robust model of direct ceramide-associated LCM, corroborating previous indirect evidence in mammals. Several CIPs were identified from mouse heart and Drosophila extracts, including caspase activator Annexin-X, myosin chaperone Unc-45, and lipogenic enzyme FASN1, and remarkably, their cardiac-specific manipulation can prevent LCM. Collectively, these data suggest that high ceramide-associated lipotoxicity is mediated, in part, through altering caspase activation, sarcomeric maintenance, and lipogenesis, thus providing evidence for conserved mechanisms in LCM pathogenesis in mammals.
Moraru, A., Wiederstein, J., Pfaff, D., Fleming, T., Miller, A. K., Nawroth, P. and Teleman, A. A. (2018). Elevated levels of the reactive metabolite methylglyoxal recapitulate progression of type 2 diabetes. Cell Metab. PubMed ID: 29551588
Summary:
The molecular causes of type 2 diabetes (T2D) are not well understood. Both type 1 diabetes (T1D) and T2D are characterized by impaired insulin signaling and hyperglycemia. From analogy to T1D, insulin resistance and hyperglycemia are thought to also play causal roles in T2D. Recent clinical studies, however, found that T2D patients treated to maintain glycemia below the diabetes definition threshold (HbA1c < 6.5%) still develop diabetic complications. This suggests additional insulin- and glucose-independent mechanisms could be involved in T2D progression and/or initiation. T2D patients have elevated levels of the metabolite methylglyoxal (MG). This study shows, using Drosophila glyoxalase 1 knockouts, that animals with elevated methylglyoxal recapitulate several core aspects of T2D: insulin resistance, obesity, and hyperglycemia. Thus elevated MG could constitute one root cause of T2D, suggesting that the molecular causes of elevated MG warrant further study.
Tsai, P. I., et al. (2018). PINK1 phosphorylates MIC60/Mitofilin to control structural plasticity of mitochondrial crista junctions. Mol Cell 69(5): 744-756.e746. PubMed ID: 29456190
Summary:
Mitochondrial crista structure partitions vital cellular reactions and is precisely regulated by diverse cellular signals. This study shows that, in Drosophila, mitochondrial cristae undergo dynamic remodeling among distinct subcellular regions and the Parkinson's disease (PD)-linked Ser/Thr kinase PINK1 participates in their regulation. Mitochondria increase crista junctions and numbers in selective subcellular areas, and this remodeling requires PINK1 to phosphorylate the inner mitochondrial membrane protein MIC60/mitofilin, which stabilizes MIC60 oligomerization. Expression of MIC60 restores crista structure and ATP levels of PINK1-null flies and remarkably rescues their behavioral defects and dopaminergic neurodegeneration. In an extension to human relevance, the PINK1-MIC60 pathway was found to be conserved in human neurons, and expression of several MIC60 coding variants in the mitochondrial targeting sequence found in PD patients in Drosophila impairs crista junction formation and causes locomotion deficits. These findings highlight the importance of maintenance and plasticity of crista junctions to cellular homeostasis in vivo.

Friday, April 27th - Adult Neural Development and Function

Umezaki, Y., Hayley, S. E., Chu, M. L., Seo, H. W., Shah, P. and Hamada, F. N. (2018). Feeding-state-dependent modulation of temperature preference requires insulin signaling in Drosophila warm-sensing neurons. Curr Biol 28(5): 779-787.e773. PubMed ID: 29478858
Summary:
Starvation is life-threatening and therefore strongly modulates many aspects of animal behavior and physiology. In mammals, hunger causes a reduction in body temperature and metabolism, resulting in conservation of energy for survival. However, the molecular basis of the modulation of thermoregulation by starvation remains largely unclear. Whereas mammals control their body temperature internally, small ectotherms, such as Drosophila, set their body temperature by selecting an ideal environmental temperature through temperature preference behaviors. This study demonstrates in Drosophila that starvation results in a lower preferred temperature, which parallels the reduction in body temperature in mammals. The insulin/insulin-like growth factor (IGF) signaling (IIS) pathway is involved in starvation-induced behaviors and physiology and is well conserved in vertebrates and invertebrates. Insulin-like peptide 6 (Ilp6) in the fat body (fly liver and adipose tissues) is responsible for the starvation-induced reduction in preferred temperature (Tp). Temperature preference behavior is controlled by the anterior cells (ACs), which respond to warm temperatures via transient receptor potential A1 (TrpA1). This study demonstrated that starvation decreases the responding temperature of ACs via insulin signaling, resulting in a lower Tp than in nutrient-rich conditions. Thus, this study shows that hunger information is conveyed from fat tissues via Ilp6 and influences the sensitivity of warm-sensing neurons in the brain, resulting in a lower temperature set point. Because starvation commonly results in a lower body temperature in both flies and mammals, it is proposed that insulin signaling is an ancient mediator of starvation-induced thermoregulation.
Shaikh, M. N. and Tejedor, F. J. (2018). Mnb/Dyrk1A orchestrates a transcriptional network at the transition from self-renewing neurogenic progenitors to postmitotic neuronal precursors. J Neurogenet 32(1): 37-50. PubMed ID: 29495936
Summary:
The Down syndrome and microcephaly related gene Mnb/Dyrk1A encodes an evolutionary conserved protein kinase subfamily that plays important roles in neurodevelopment. minibrain (mnb) mutants of Drosophila melanogaster (Dm) exhibit reduced adult brains due to neuronal deficits generated during larval development. These deficits are the consequence of the apoptotic cell death of numerous neuronal precursors that fail to properly exit the cell cycle and differentiate. Recent studies have found that in both the Dm larval brain and the embryonic vertebrate central nervous system (CNS), a transient expression of Mnb/Dyrk1A promotes the cell cycle exit of newborn neuronal precursors by upregulating the expression of the cyclin-dependent kinase inhibitor p27kip1 (called Dacapo in Dm). In the larval brain, Mnb performs this action by regulating the expression of three transcription factors, Asense (Ase), Deadpan (Dpn) and Prospero (Pros), which are key regulators of the self-renewal, proliferation, and terminal differentiation of neural progenitor cells. The cellular/temporal expression pattern of Ase, Dpn, Pros and Mnb was studied in detail, and possible regulatory effects were analyzed among them at the transitions from neurogenic progenitors to postmitotic neuronal precursors in the Dm larval brain. The emerging picture of this analysis reveals an intricate regulatory network in which Mnb appears to play a pivotal role helping to delineate the dynamics of the expression patterns of Ase, Dpn and Pros, as well as their specific functions in the aforementioned transitions. The results also show that Ase, Dpn and Pros perform several cross-regulatory actions and contribute to shape the precise cellular/temporal expression pattern of Mnb. It is proposed that Mnb/Dyrk1A plays a central role in CNS neurogenesis by integrating molecular mechanisms that regulate progenitor self-renewal, cell cycle progression and neuronal differentiation.
Schubert, F. K., Hagedorn, N., Yoshii, T., Helfrich-Forster, C. and Rieger, D. (2018). Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their functional role in the circadian system. J Comp Neurol 526(7): 1209-1231. PubMed ID: 29424420
Summary:
Drosophila melanogaster is a long-standing model organism in the circadian clock research. A major advantage is the relative small number of about 150 neurons, which built the circadian clock in Drosophila. A recent work focused on the neuroanatomical properties of the lateral neurons of the clock network. By applying the multicolor-labeling technique Flybow it was possible to identify the anatomical similarity of the previously described E2 subunit of the evening oscillator of the clock, which is built by the 5th small ventrolateral neuron (5th s-LNv ) and one ITP positive dorsolateral neuron (LNd ). These two clock neurons share the same spatial and functional properties. Both neurons were found innervating the same brain areas with similar pre- and postsynaptic sites in the brain. The anatomical findings support their shared function as a main evening oscillator in the clock network like also found in previous studies. A second quite surprising finding addresses the large lateral ventral PDF-neurons (l-LNv s). It was shown that the four hardly distinguishable l-LNv s consist of two subgroups with different innervation patterns. While three of the neurons reflect the well-known branching pattern reproduced by PDF immunohistochemistry, one neuron per brain hemisphere has a distinguished innervation profile and is restricted only to the proximal part of the medulla-surface. This neuron was named "extra" l-LNv (l-LNv x). The anatomical findings reflect different functional properties of the two l-LNv subgroups.
Selcho, M., Muhlbauer, B., Hensgen, R., Shiga, S., Wegener, C. and Yasuyama, K. (2018). Anatomical characterization of PDF-tri neurons and peptidergic neurons associated with eclosion behavior in Drosophila. J Comp Neurol. PubMed ID: 29427506
Summary:
The peptidergic Pigment-dispersing factor (PDF)-Tri neurons are a group of non-clock neurons that appear transiently around the time of adult ecdysis (=eclosion) in the fruit fly Drosophila melanogaster. This specific developmental pattern points to a function of these neurons in eclosion or other processes that are active around pupal-adult transition. As a first step to understand the role of these neurons, the anatomy of the PDF-Tri neurons were characterized. In addition, a further set of peptidergic neurons is described that have been associated with eclosion behavior, eclosion hormone (EH), and crustacean cardioactive peptide (CCAP) neurons, to single cell level in the pharate adult brain. PDF-Tri neurons as well as CCAP neurons co-express a classical transmitter indicated by the occurrence of small clear vesicles in addition to dense-core vesicles containing the peptides. In the tritocerebrum, gnathal ganglion and the superior protocerebrum PDF-Tri neurites contain peptidergic varicosities and both pre- and postsynaptic sites, suggesting that the PDF-Tri neurons represent modulatory rather than pure interneurons that connect the subesophageal zone with the superior protocerebrum. The extensive overlap of PDF-Tri arborizations with neurites of CCAP- and EH-expressing neurons in distinct brain regions provides anatomical evidence for a possible function of the PDF-Tri neurons in eclosion behavior.
Schatton, A., Mendoza, E., Grube, K. and Scharff, C. (2018). FoxP in bees: A comparative study on the developmental and adult expression pattern in three bee species considering isoforms and circuitry. J Comp Neurol. PubMed ID: 29536541
Summary:
Mutations in the transcription factors FOXP1, FOXP2 and FOXP4 affect human cognition, including language. The FoxP gene locus is evolutionarily ancient and highly conserved in its DNA-binding domain. In Drosophila melanogaster FoxP has been implicated in courtship behavior, decision making and specific types of motor-learning. Because honeybees (Apis mellifera, Am) excel at navigation and symbolic dance communication, they are a particularly suitable insect species to investigate a potential link between neural FoxP expression and cognition. Two AmFoxP isoforms were characterized and their expression was mapped in the brain during development and in adult foragers. Using a custom-made antiserum and in situ hybridization, 11 AmFoxP expressing neuron populations are described. FoxP was expressed in equivalent patterns in two other representatives of Apidae; a closely related dwarf bee and a bumblebee species. Neural tracing revealed that the largest FoxP expressing neuron cluster in honeybees projects into a posterior tract that connects the optic lobe to the posterior lateral protocerebrum, predicting a function in visual processing. These data provide an entry point for future experiments assessing the function of FoxP in eusocial Hymenoptera.
Tsao, C. H., Chen, C. C., Lin, C. H., Yang, H. Y. and Lin, S. (2018). Drosophila mushroom bodies integrate hunger and satiety signals to control innate food-seeking behavior. Elife 7. PubMed ID: 29547121
Summary:
The fruit fly can evaluate its energy state and decide whether to pursue food-related cues. This study reveals that the mushroom body (MB) integrates hunger and satiety signals to control food-seeking behavior. Five pathways in the MB were found to be essential for hungry flies to locate and approach food. Blocking the MB-intrinsic Kenyon cells (KCs) and the MB output neurons (MBONs) in these pathways impairs food-seeking behavior. Starvation bi-directionally modulates MBON responses to a food odor, suggesting that hunger and satiety controls occur at the KC-to-MBON synapses. These controls are mediated by six types of dopaminergic neurons (DANs). By manipulating these DANs, it was possible to inhibit food-seeking behavior in hungry flies or promote food seeking in fed flies. Finally, this study showed that the DANs potentially receive multiple inputs of hunger and satiety signals. This work demonstrates an information-rich central circuit in the fly brain that controls hunger-driven food-seeking behavior.

Thursday, April 26th - Immune Response

Lindberg, B. G., Tang, X., Dantoft, W., Gohel, P., Seyedoleslami Esfahani, S., Lindvall, J. M. and Engstrom, Y. (2018). Nubbin isoform antagonism governs Drosophila intestinal immune homeostasis. PLoS Pathog 14(3): e1006936. PubMed ID: 29499056
Summary:
Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. This study shows that the POU/Oct gene nubbin (nub) encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-kappaB), JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic transcription factor isoforms.
Troha, K., Im, J. H., Revah, J., Lazzaro, B. P. and Buchon, N. (2018). Comparative transcriptomics reveals CrebA as a novel regulator of infection tolerance in D. melanogaster. PLoS Pathog 14(2): e1006847. PubMed ID: 29394281
Summary:
Host responses to infection encompass many processes in addition to activation of the immune system, including metabolic adaptations, stress responses, tissue repair, and other reactions. The response to bacterial infection in Drosophila melanogaster has been classically described in studies that focused on the immune response elicited by a small set of largely avirulent microbes. Thus, there is surprisingly limited knowledge of responses to infection that are outside the canonical immune response, of how the response to pathogenic infection differs from that to avirulent bacteria, or even of how generic the response to various microbes is and what regulates that core response. This study addresses these questions by profiling the D. melanogaster transcriptomic response to 10 bacteria that span the spectrum of virulence. Each bacterium triggers a unique transcriptional response, with distinct genes making up to one third of the response elicited by highly virulent bacteria. A core set of 252 genes was identified that are differentially expressed in response to the majority of bacteria tested. Among these, the transcription factor CrebA is a novel regulator of infection tolerance. Knock-down of CrebA significantly increased mortality from microbial infection without any concomitant change in bacterial number. Upon infection, CrebA is upregulated by both the Toll and Imd pathways in the fat body, where it is required to induce the expression of secretory pathway genes. Loss of CrebA during infection triggered endoplasmic reticulum (ER) stress and activated the unfolded protein response (UPR), which contributed to infection-induced mortality. Altogether, this study reveals essential features of the response to bacterial infection and elucidates the function of a novel regulator of infection tolerance.
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Lee, K. A., Cho, K. C., Kim, B., Jang, I. H., Nam, K., Kwon, Y. E., Kim, M., Hyeon, D. Y., Hwang, D., Seol, J. H. and Lee, W. J. (2018). Inflammation-modulated metabolic reprogramming is required for DUOX-dependent gut immunity in Drosophila. Cell Host Microbe 23(3): 338-352.e335. PubMed ID: 29503179
Summary:
DUOX, a member of the NADPH xidase family, acts as the first line of defense against enteric pathogens by producing microbicidal reactive oxygen species. DUOX is activated upon enteric infection, but the mechanisms regulating DUOX activity remain incompletely understood. Using Drosophila genetic tools, this study shows that enteric infection results in "pro-catabolic" signaling that initiates metabolic reprogramming of enterocytes toward lipid catabolism, which ultimately governs DUOX homeostasis. Infection induces signaling cascades involving TRAF3 and kinases AMPK and WTS, which regulate TOR kinase to control the balance of lipogenesis versus lipolysis. Enhancing lipogenesis blocks DUOX activity, whereas stimulating lipolysis via ATG1-dependent lipophagy is required for DUOX activation. Drosophila with altered activity in TRAF3-AMPK/WTS-ATG1 pathway components exhibit abolished infection-induced lipolysis, reduced DUOX activation, and enhanced susceptibility to enteric infection. Thus, this work uncovers signaling cascades governing inflammation-induced metabolic reprogramming and provides insight into the pathophysiology of immune-metabolic interactions in the microbe-laden gut epithelia.
Turelli, M., et al. (2018). Rapid Global Spread of wRi-like Wolbachia across Multiple Drosophila. Curr Biol 28(6): 963-971.e968. PubMed ID: 29526588
Summary:
Maternally transmitted Wolbachia, Spiroplasma, and Cardinium bacteria are common in insects, but their interspecific spread is poorly understood. Because Wolbachia cannot survive outside host cells, spread between distantly related host species requires horizontal transfers that are presumably rare. This study documents spread of wRi-like Wolbachia among eight highly diverged Drosophila hosts (10-50 million years) over only about 14,000 years (5,000-27,000). Comparing 110 wRi-like genomes, it was found </=0.02% divergence from the wRi variant that spread rapidly through California populations of D. simulans. The hosts include both globally invasive species (D. simulans, D. suzukii, and D. ananassae) and narrowly distributed Australian endemics (D. anomalata and D. pandora). Phylogenetic analyses that include mtDNA genomes indicate introgressive transfer of wRi-like Wolbachia between closely related species D. ananassae, D. anomalata, and D. pandora but no horizontal transmission within species. These analyses suggest D. ananassae as the Wolbachia source for the recent wRi invasion of D. simulans and D. suzukii as the source of Wolbachia in its sister species D. subpulchrella. Although six of these wRi-like variants cause strong cytoplasmic incompatibility, two cause no detectable reproductive effects, indicating that pervasive mutualistic effects complement the reproductive manipulations for which Wolbachia are best known. "Super spreader" variants like wRi may be particularly useful for controlling insect pests and vector-borne diseases with Wolbachia transinfections.
Franz, A., Wood, W. and Martin, P. (2018). Fat body cells are motile and actively migrate to wounds to drive repair and prevent infection. Dev Cell 44(4): 460-470.e463. PubMed ID: 29486196
Summary:
Adipocytes have many functions in various tissues beyond energy storage, including regulating metabolism, growth, and immunity. However, little is known about their role in wound healing. This study used live imaging of fat body cells, the equivalent of vertebrate adipocytes in Drosophila, to investigate their potential behaviors and functions following skin wounding. Pupal fat body cells are not immotile, as previously presumed, but actively migrate to wounds using an unusual adhesion-independent, actomyosin-driven, peristaltic mode of motility. Once at the wound, fat body cells collaborate with hemocytes, Drosophila macrophages, to clear the wound of cell debris; they also tightly seal the epithelial wound gap and locally release antimicrobial peptides to fight wound infection. Thus, fat body cells are motile cells, enabling them to migrate to wounds to undertake several local functions needed to drive wound repair and prevent infections.
Issa, N., Guillaumot, N., Lauret, E., Matt, N., Schaeffer-Reiss, C., Van Dorsselaer, A., Reichhart, J. M. and Veillard, F. (2018). The circulating protease Persephone is an immune sensor for microbial proteolytic activities upstream of the Drosophila toll pathway. Mol Cell 69(4): 539-550.e536. PubMed ID: 29452635
Summary:
Microbial or endogenous molecular patterns as well as pathogen functional features can activate innate immune systems. Whereas detection of infection by pattern recognition receptors has been investigated in details, sensing of virulence factors activities remains less characterized. In Drosophila, genetic evidences indicate that the serine protease Persephone belongs to a danger pathway activated by abnormal proteolytic activities to induce Toll signaling. However, neither the activation mechanism of this pathway nor its specificity has been determined. This study identified a unique region in the pro-domain of Persephone that functions as bait for exogenous proteases independently of their origin, type, or specificity. Cleavage in this bait region constitutes the first step of a sequential activation and licenses the subsequent maturation of Persephone to the endogenous cysteine cathepsin 26-29-p. These results establish Persephone itself as an immune receptor able to sense a broad range of microbes through virulence factor activities rather than molecular patterns.

Wednesday, April 25 - Cytoskeleton

Das, A., Cesario, J., Hinman, A. M., Jang, J. K. and McKim, K. S. (2018). Kinesin 6 regulation in Drosophila female meiosis by the non-conserved N- and C-terminal domains. G3 (Bethesda). PubMed ID: 29514846
Summary:
Bipolar spindle assembly occurs in the absence of centrosomes in the oocytes of most organisms. In the absence of centrosomes in Drosophila oocytes, it has been proposed proposed that the kinesin 6 Subito, a MKLP-2 homolog, is required for establishing spindle bipolarity and chromosome biorientation by assembling a robust central spindle during prometaphase I. Although the functions of the conserved motor domains of kinesins is well studied, less is known about the contribution of the poorly conserved N- and C- terminal domains to motor function. This study has investigated the contribution of these domains to kinesin 6 functions in meiosis and early embryonic development. The N-terminal domain has antagonistic elements that regulate localization of the motor to microtubules. Other parts of the N- and C- terminal domains are not required for microtubule localization but are required for motor function. Some of these elements of Subito are more important for either mitosis or meiosis, as revealed by separation-of-function mutants. One of the functions for both the N- and C-terminals domains is to restrict the CPC to the central spindle in a ring around the chromosomes. Evidence is provided that CDK1 phosphorylation of Subito regulates its activity associated with homolog bi-orientation. These results suggest the N- and C-terminal domains of Subito, while not required for localization to the central spindle microtubules, have important roles regulating Subito, by interacting with other spindle proteins and promoting activities such as bipolar spindle formation and homologous chromosome bi-orientation during meiosis.
Glasheen, B. M., Ramanath, S., Patel, M., Sheppard, D., Puthawala, J. T., Riley, L. A. and Swank, D. M. (2018). Five Alternative Myosin converter domains influence muscle power, stretch activation, and kinetics. Biophys J 114(5): 1142-1152. PubMed ID: 29539400
Summary:
Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. The influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, was studied using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a-e). Five transgenic fly lines were generated, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b-e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics.
Resnik-Docampo, M., Sauer, V., Schinaman, J. M., Clark, R. I., Walker, D. W. and Jones, D. L. (2018). Keeping it tight: The relationship between bacterial dysbiosis, septate junctions, and the intestinal barrier in Drosophila. Fly (Austin): 1-7. PubMed ID: 29455581
Summary:
Maladaptive changes in the intestinal flora, typically referred to as bacterial dysbiosis, have been linked to intestinal aging phenotypes, including an increase in intestinal stem cell (ISC) proliferation, activation of inflammatory pathways, and increased intestinal permeability. However, the causal relationships between these phenotypes are only beginning to be unravelled. Age-related changes have been characterized that occur to septate junctions (SJ) between adjacent, absorptive enterocytes (EC) in the fly intestine. Changes could be observed in the overall level of SJ proteins, as well as the localization of a subset of SJ proteins. Such age-related changes were particularly noticeable at tricellular junctions (TCJ). Acute loss of the Drosophila TCJ protein Gliotactin (Gli) in ECs led to rapid activation of stress signalling in stem cells and an increase in ISC proliferation, even under axenic conditions; a gradual disruption of the intestinal barrier was also observed. The uncoupling of changes in bacteria from alterations in ISC behaviour and loss of barrier integrity has allowed exploration of the interrelationship of these intestinal aging phenotypes in more detail and has shed light on the importance of the proteins that contribute to maintenance of the intestinal barrier.
Roque, H., Saurya, S., Pratt, M. B., Johnson, E. and Raff, J. W. (2018). Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation. PLoS Genet 14(2): e1007198. PubMed ID: 29425198
Summary:
Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. This study examined Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane-although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. PLP helps assemble "pericentriolar clouds" of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. It is proposed that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects observed in Plp mutant cells.
Del Signore, S. J., Cilla, R. and Hatini, V. (2018). The WAVE regulatory complex and branched F-actin counterbalance contractile force to control cell shape and packing in the Drosophila eye. Dev Cell 44(4): 471-483.e474. PubMed ID: 29396116
Summary:
Contractile forces eliminate cell contacts in many morphogenetic processes. However, mechanisms that balance contractile forces to promote subtler remodeling remain unknown. To address this gap, this study investigated remodeling of Drosophila eya lattice cells (LCs), which preserve cell contacts as they narrow to form the edges of a multicellular hexagonal lattice. It was found that during narrowing, LC-LC contacts dynamically constrict and expand. Similar to other systems, actomyosin-based contractile forces promote pulses of constriction. Conversely, it was found that WAVE-dependent branched F-actin accumulates at LC-LC contacts during expansion and functions to expand the cell apical area, promote shape changes, and prevent elimination of LC-LC contacts. Finally, it was found that small Rho GTPases regulate the balance of contractile and protrusive dynamics. These data suggest a mechanism by which WAVE regulatory complex-based F-actin dynamics antagonize contractile forces to regulate cell shape and tissue topology during remodeling and thus contribute to the robustness and precision of the process.
Streichan, S. J., Lefebvre, M. F., Noll, N., Wieschaus, E. F. and Shraiman, B. I. (2018). Global morphogenetic flow is accurately predicted by the spatial distribution of myosin motors. Elife 7. PubMed ID: 29424685
Summary:
During embryogenesis tissue layers undergo morphogenetic flow rearranging and folding into specific shapes. While developmental biology has identified key genes and local cellular processes, global coordination of tissue remodeling at the organ scale remains unclear. This study combined in toto light-sheet microscopy of the Drosophila embryo with quantitative analysis and physical modeling to relate cellular flow with the patterns of force generation during the gastrulation process. The complex spatio-temporal flow pattern can be predicted from the measured meso-scale myosin density and anisotropy using a simple, effective viscous model of the tissue, achieving close to 90% accuracy with one time dependent and two constant parameters. This analysis uncovers the importance of a) spatial modulation of myosin distribution on the scale of the embryo and b) the non-locality of its effect due to mechanical interaction of cells, demonstrating the need for the global perspective in the study of morphogenetic flow.

Tuesday, April 24th - RNAs

Luo, S., He, F., Luo, J., Dou, S., Wang, Y., Guo, A. and Lu, J. (2018). Drosophila tsRNAs preferentially suppress general translation machinery via antisense pairing and participate in cellular starvation response. Nucleic Acids Res. PubMed ID: 29548011
Summary:
Transfer RNA-derived small RNAs (tsRNAs) are an emerging class of small RNAs, yet their regulatory roles have not been well understood. The molecular mechanisms and consequences of tsRNA-mediated regulation in Drosophila was studied. By analyzing 495 public small RNA libraries, it was demonstrated that most tsRNAs are conserved, prevalent and abundant in Drosophila. By carrying out mRNA sequencing and ribosome profiling of S2 cells transfected with single-stranded tsRNA mimics and mocks, this study showed that tsRNAs recognize target mRNAs through conserved complementary sequence matching and suppress target genes by translational inhibition. The target prediction suggests that tsRNAs preferentially suppress translation of the key components of the general translation machinery, which explains how tsRNAs inhibit the global mRNA translation. Serum starvation experiments confirm tsRNAs participate in cellular starvation responses by preferential targeting the ribosomal proteins and translational initiation or elongation factors. Knock-down of AGO2 in S2 cells under normal and starved conditions reveals a dependence of the tsRNA-mediated regulation on AGO2. The repressive effects of representative tsRNAs on cellular global translation and specific targets was evaluated with luciferase reporter assays. This study suggests the tsRNA-mediated regulation might be crucial for the energy homeostasis and the metabolic adaptation in the cellular systems.
Meers, M. P., Adelman, K., Duronio, R. J., Strahl, B. D., McKay, D. J. and Matera, A. G. (2018). Transcription start site profiling uncovers divergent transcription and enhancer-associated RNAs in Drosophila melanogaster. BMC Genomics 19(1): 157. PubMed ID: 29466941
Summary:
High-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors. However, TSS profiling has not been investigated in an orthogonal in vivo setting. This study presents a comprehensive dataset that links TSS dynamics with nucleosome occupancy and gene expression in the wandering third instar larva, a developmental stage characterized by large-scale shifts in transcriptional programs in preparation for metamorphosis. The data recapitulate major regulatory classes of TSSs, based on peak width, promoter-proximal polymerase pausing, and cis-regulatory element density. The paucity of divergent transcription units was consfirmed in D. melanogaster, but also notable exceptions were identified. Furthermore, thousands of novel initiation events occurring at unannotated TSSs that can be classified into functional categories by their local density of histone modifications. Interestingly, a sub-class of these unannotated TSSs overlaps with functionally validated enhancer elements, consistent with a regulatory role for "enhancer RNAs" (eRNAs) in defining developmental transcription programs. It is concluded that High-depth TSS mapping is a powerful strategy for identifying and characterizing low-abundance and/or low-stability RNAs. Global analysis of transcription initiation patterns in a developing organism reveals a vast number of novel initiation events that identify potential eRNAs as well as other non-coding transcripts critical for animal development.
Nishida, K. M., Sakakibara, K., Iwasaki, Y. W., Yamada, H., Murakami, R., Murota, Y., Kawamura, T., Kodama, T., Siomi, H. and Siomi, M. C. (2018). Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis. Nature 555(7695): 260-264. PubMed ID: 29489748
Evolutionary Homolog Study
PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that bind to PIWI proteins to control transposons and maintain genome integrity in animal germ lines. piRNA 3' end formation in the silkworm Bombyx mori has been shown to be mediated by the 3'-to-5' exonuclease Trimmer (Trim; known as PNLDC1 in mammals), and piRNA intermediates are bound with PIWI anchored onto mitochondrial Tudor domain protein Papi. However, it remains unclear whether the Zucchini (Zuc) endonuclease and Nibbler (Nbr) 3'-to-5' exonuclease, both of which have pivotal roles in piRNA biogenesis in Drosophila, are required for piRNA processing in other species. This study showed that the loss of Zuc in Bombyx had no effect on the levels of Trim and Nbr, but resulted in the aberrant accumulation of piRNA intermediates within the Papi complex, and that these were processed to form mature piRNAs by recombinant Zuc. Papi exerted its RNA-binding activity only when bound with PIWI and phosphorylated, suggesting that complex assembly involves a hierarchical process. Both the 5' and 3' ends of piRNA intermediates within the Papi complex showed hallmarks of PIWI 'slicer' activity, yet no phasing pattern was observed in mature piRNAs. The loss of Zuc did not affect the 5'- and 3'-end formation of the intermediates, strongly supporting the idea that the 5' end of Bombyx piRNA is formed by PIWI slicer activity, but independently of Zuc, whereas the 3' end is formed by the Zuc endonuclease. The Bombyx piRNA biogenesis machinery is simpler than that of Drosophila, because Bombyx has no transcriptional silencing machinery that relies on phased piRNAs.

Skrajna, A., Yang, X. C., Dadlez, M., Marzluff, W. F. and Dominski, Z. (2018). Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker. Nucleic Acids Res. PubMed ID: 29529248
Summary:
3' end cleavage of metazoan replication-dependent histone pre-mRNAs requires the multi-subunit holo-U7 snRNP and the stem-loop binding protein (SLBP). The exact composition of the U7 snRNP and details of SLBP function in processing remain unclear. To identify components of the U7 snRNP in an unbiased manner, a novel approach was developed for purifying processing complexes from Drosophila and mouse nuclear extracts. In this method, catalytically active processing complexes are assembled in vitro on a cleavage-resistant histone pre-mRNA containing biotin and a photo-sensitive linker, and eluted from streptavidin beads by UV irradiation for direct analysis by mass spectrometry. In the purified processing complexes, Drosophila and mouse U7 snRNP have a remarkably similar composition, always being associated with CPSF73, CPSF100, symplekin and CstF64. Many other proteins previously implicated in the U7-dependent processing are not present. Drosophila U7 snRNP bound to histone pre-mRNA in the absence of SLBP contains the same subset of polyadenylation factors but is catalytically inactive and addition of recombinant SLBP is sufficient to trigger cleavage. This result suggests that Drosophila SLBP promotes a structural rearrangement of the processing complex, resulting in juxtaposition of the CPSF73 endonuclease with the cleavage site in the pre-mRNA substrate.

Knuckles, P., Lence, T., Haussmann, I. U., Jacob, D., Kreim, N., Carl, S. H., Masiello, I., Hares, T., Villasenor, R., Hess, D., Andrade-Navarro, M. A., Biggiogera, M., Helm, M., Soller, M., Buhler, M. and Roignant, J. Y. (2018). Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m(6)A machinery component Wtap/Fl(2)d. Genes Dev. PubMed ID: 29535189
Summary:
N(6)-methyladenosine (m(6)A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m(6)A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. This study identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m(6)A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m(6)A levels and is involved in sex determination in Drosophila. Flacc promotes m(6)A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, this work advances the molecular understanding of conservation and regulation of the m(6)A machinery.
Lu, K. L., Nelson, J. O., Watase, G. J., Warsinger-Pepe, N. and Yamashita, Y. M. (2018). Transgenerational dynamics of rDNA copy number in Drosophila male germline stem cells. Elife 7. PubMed ID: 29436367
Summary:
rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. This study investigated the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. This study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in young animals that inherited reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, it is proposed that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline.

Monday, April 23rd - Signaling Pathways

Paglia, S., Sollazzo, M., Di Giacomo, S., de Biase, D., Pession, A. and Grifoni, D. (2017). Failure of the PTEN/aPKC/Lgl axis primes formation of adult brain tumours in Drosophila. Biomed Res Int 2017: 2690187. PubMed ID: 29445734
Summary:
Different regions in the mammalian adult brain contain immature precursors, reinforcing the concept that brain cancers, such as glioblastoma multiforme (GBM), may originate from cells endowed with stem-like properties. Alterations of the tumour suppressor gene PTEN are very common in primary GBMs. Very recently, PTEN loss was shown to undermine a specific molecular axis, whose failure is associated with the maintenance of the GBM stem cells in mammals. This axis is composed of PTEN, aPKC, and the polarity determinant Lethal giant larvae (Lgl): PTEN loss promotes aPKC activation through the PI3K pathway, which in turn leads to Lgl inhibition, ultimately preventing stem cell differentiation. To find the neural precursors responding to perturbations of this molecular axis, this study targeted different neurogenic regions of the Drosophila brain. PTEN mutation was shown to impact aPKC and Lgl protein levels also in Drosophila. Moreover, it was demonstrated that PI3K activation is not sufficient to trigger tumourigenesis, while aPKC promotes hyperplastic growth of the neuroepithelium and a noticeable expansion of the type II neuroblasts. Finally, this study showed that these neuroblasts form invasive tumours that persist and keep growing in the adult, leading the affected animals to untimely death, thus displaying frankly malignant behaviours.
Preiss, A., Nagel, A. C., Praxenthaler, H. and Maier, D. (2018). Complex genetic interactions of novel Suppressor of Hairless alleles deficient in co-repressor binding. PLoS One 13(3): e0193956. PubMed ID: 29509808
Summary:
In Drosophila, repression of Notch target genes involves the CSL homologue Suppressor of Hairless (Su(H)) and the Notch (N) antagonist Hairless (H) that together form a repressor complex. Guided by crystal structure, three mutations Su(H)LL, Su(H)LLF and Su(H)LLL were generated that specifically affect interactions with the repressor H, and were introduced into the endogenous Su(H) locus by gene engineering. In contrast to the wild type isoform, these Su(H) mutants are incapable of repressor complex formation. Accordingly, Notch signalling activity is dramatically elevated in the homozygotes, resembling complete absence of H activity. It was noted, however, that heterozygotes do not display a dominant H loss of function phenotype. This work addressed genetic interactions the three H-binding deficient Su(H) mutants display in combination with H and N null alleles. A null mutant was included of Delta (Dl), encoding the ligand of the Notch receptor, as well as of Su(H) itself in the genetic analyses. H, N or Dl mutations cause dominant wing phenotypes that are sensitive to gene dose of the others. Moreover, H heterozygotes lack bristle organs and develop bristle sockets instead of shafts. The latter phenotype is suppressed by Su(H) null alleles but not by H-binding deficient Su(H) alleles which was attributed to the socket cell specific activity of Su(H). Modification of the dominant wing phenotypes of either H, N or Dl, however, suggested some lack of repressor activity in the Su(H) null allele and likewise in the H-binding deficient Su(H) alleles. Overall, Su(H) mutants are recessive perhaps reflecting self-adjusting availability of Su(H) protein.
Shan, Z., Tu, Y., Yang, Y., Liu, Z., Zeng, M., Xu, H., Long, J., Zhang, M., Cai, Y. and Wen, W. (2018). Basal condensation of Numb and Pon complex via phase transition during Drosophila neuroblast asymmetric division. PLoS One 13(3): e0193956. Nat Commun 9(1): 737. PubMed ID: 29467404
Summary:
Uneven distribution and local concentration of protein complexes on distinct membrane cortices is a fundamental property in numerous biological processes, including Drosophila neuroblast (NB) asymmetric cell divisions and cell polarity in general. In NBs, the cell fate determinant Numb forms a basal crescent together with Pon and is segregated into the basal daughter cell to initiate its differentiation. This study discovered that Numb PTB domain, using two distinct binding surfaces, recognizes repeating motifs within Pon in a previously unrecognized mode. The multivalent Numb-Pon interaction leads to high binding specificity and liquid-liquid phase separation of the complex. Perturbations of the Numb/Pon complex phase transition impair the basal localization of Numb and its subsequent suppression of Notch signaling during NB asymmetric divisions. Such phase-transition-mediated protein condensations on distinct membrane cortices may be a general mechanism for various cell polarity regulatory complexes.
Roth, S. W., Bitterman, M. D., Birnbaum, M. J. and Bland, M. L. (2018). Innate immune signaling in Drosophila blocks insulin Signaling by uncoupling PI(3,4,5)P3 production and Akt activation. Cell Rep 22(10): 2550-2556. PubMed ID: 29514084
Summary:
In obese adipose tissue, Toll-like receptor signaling in macrophages leads to insulin resistance in adipocytes. Similarly, Toll signaling in the Drosophila larval fat body blocks insulin-dependent growth and nutrient storage. This study finds that Toll acts cell autonomously to block growth but not PI(3,4,5)P3 production in fat body cells expressing constitutively active PI3K. Fat body Toll signaling blocks whole-animal growth in rictor mutants lacking TORC2 activity, but not in larvae lacking Pdk1. Phosphorylation of Akt on the Pdk1 site, Thr342, is significantly reduced by Toll signaling, and expression of mutant Akt(T342D) rescues cell and animal growth, nutrient storage, and viability in animals with active Toll signaling. Altogether, these data show that innate immune signaling blocks insulin signaling at a more distal level than previously appreciated, and they suggest that manipulations affecting the Pdk1 arm of the pathway may have profound effects on insulin sensitivity in inflamed tissues.
Saito-Diaz, K., Benchabane, H., Tiwari, A., Tian, A., Li, B., Thompson, J. J., Hyde, A. S., Sawyer, L. M., Jodoin, J. N., Santos, E., Lee, L. A., Coffey, R. J., Beauchamp, R. D., Williams, C. S., Kenworthy, A. K., Robbins, D. J., Ahmed, Y. and Lee, E. (2018). APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway. Dev Cell 44(5): 566-581. PubMed ID: 29533772
Summary:
Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting beta-catenin degradation downstream of Wnt receptors. Unexpectedly, this study found that blocking Wnt receptor activity in APC-deficient cells inhibits Wnt signaling independently of Wnt ligand. Inducible loss of APC is rapidly followed by Wnt receptor activation and increased beta-catenin levels. In contrast, APC2 loss does not promote receptor activation. This study shows that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, conservation of this mechanism in was demonstrated in Drosophila intestinal stem cells. A model is proposed in which APC and APC2 function to promote beta-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.
Musselman, L. P., Fink, J. L., Maier, E. J., Gatto, J. A., Brent, M. R. and Baranski, T. J. (2018). Seven-up is a novel regulator of insulin signaling. Genetics [Epub ahead of print]. PubMed ID: 29487137
Summary:
Insulin resistance is associated with obesity, cardiovascular disease, non-alcoholic fatty liver disease, and type 2 diabetes. These complications are exacerbated by a high-calorie diet, which this study uses to model type 2 diabetes in Drosophila melanogaster. These studies focused on the fat body, an adipose- and liver-like tissue that stores fat and maintains circulating glucose. A gene regulatory network was constructed to predict potential regulators of insulin signaling in this tissue. Genomic characterization of fat bodies suggested a central role for the transcription factor Seven-up (Svp). This study describes a new role for Svp as a positive regulator of insulin signaling. Tissue-specific loss-of-function showed that Svp is required in the fat body to promote glucose clearance, lipid turnover, and insulin signaling. Svp appears to promote insulin signaling, at least in part, by inhibiting ecdysone signaling. Svp also impairs the immune response possibly via inhibition of antimicrobial peptide expression in the fat body. Taken together, these studies show that gene regulatory networks can help identify positive regulators of insulin signaling and metabolic homeostasis using the Drosophila fat body.

Friday April 20th - Neural Function Adult

Nagy, D., Andreatta, G., Bastianello, S., Martin Anduaga, A., Mazzotta, G., Kyriacou, C. P. and Costa, R. (2018). A semi-natural approach for sTudying seasonal diapause in Drosophila melanogaster reveals robust photoperiodicity. J Biol Rhythms: 748730417754116. PubMed ID: 29415605
Summary:
The fruit fly Drosophila melanogaster survives thermally stressful conditions in a state of reproductive dormancy (diapause), manifested by reduced metabolic activity and arrested ovarian development in females. Unlike insects that rely primarily on photoperiodic stimuli to initiate the diapause program, in this species dormancy is regulated by low temperature and enhanced by shorter photoperiods. Overwintering phenotypes are usually studied under simple laboratory conditions, where animals are exposed to rectangular light-dark (LD) cycles at a constant temperature. This study sought to adopt more realistic diapause protocols by generating LD profiles that better mimic outdoor conditions. Experimental flies were subjected to semi-natural late autumn and summer days, while control females received the same amounts of light but in rectangular LD cycles (LD 8:16 and LD 15:9, respectively). Semi-natural autumnal days were found to induce a higher proportion of females to enter dormancy, while females in semi-natural summer days showed reduced diapause compared with their corresponding rectangular controls, generating an impressive photoperiodic response. In contrast, under rectangular light regimes, the diapause of Drosophila field lines exhibited minimal photoperiodicity. This semi-natural method reveals that D. melanogaster diapause is considerably more photoperiodic than previously believed and suggests that this seasonal response is best studied under simulated natural lighting conditions.
Rossi, A. M. and Fernandes, V. M. (2018). Wrapping glial morphogenesis and signaling control the timing and pattern of neuronal differentiation in the Drosophila lamina. J Exp Neurosci 12: 1179069518759294. PubMed ID: 29531474
Summary:
Various regions of the developing brain coordinate their construction so that the correct types and numbers of cells are generated to build a functional network. Previous work has discovered that wrapping glia in the Drosophila visual system are essential for coordinating retinal and lamina development. Wrapping glia, which ensheath photoreceptor axons, respond to an epidermal growth factor cue from photoreceptors by secreting insulins. Wrapping glial insulins activate the mitogen-activated protein kinase (MAPK) pathway downstream of insulin receptor in lamina precursors to induce neuronal differentiation. The signaling relay via wrapping glia introduces a delay that allows the lamina to assemble the correct stoichiometry and physical alignment of precursors before differentiating and imposes a stereotyped spatiotemporal pattern that is relevant for specifying the individual lamina neuron fates. This study further describes how wrapping glia morphogenesis correlates with the timing of lamina neuron differentiation by 2-photon live imaging. Although MAPK activity in lamina precursors drives neuronal differentiation, the upstream receptor driving MAPK activation in lamina precursors and the ligand secreted by wrapping glia to trigger it differentially affect lamina neuron differentiation. These results highlight differences in MAPK signaling properties and confirm that communication between photoreceptors, wrapping glia, and lamina precursors must be precisely controlled to build a complex neural network.
Ohhara, Y., Kobayashi, S., Yamakawa-Kobayashi, K. and Yamanaka, N. (2018). Adult-specific insulin-producing neurons in Drosophila melanogaster. J Comp Neurol. PubMed ID: 29424424
Summary:
Holometabolous insects undergo metamorphosis to reorganize their behavioral and morphological features into adult-specific ones. In the central nervous system (CNS), some larval neurons undergo programmed cell death, whereas others go through remodeling of axonal and dendritic arbors to support functions of re-established adult organs. Although there are multiple neuropeptides that have stage-specific roles in holometabolous insects, the reorganization pattern of the entire neuropeptidergic system through metamorphosis still remains largely unclear. This study conducted a mapping and lineage tracing of peptidergic neurons in the larval and adult CNS by using Drosophila genetic tools. Diuretic hormone 44-producing median neurosecretory cells start expressing Insulin-like peptide 2 in the pharate adult stage. This neuronal cluster projects to the corpora cardiaca and dorsal vessel in both larval and adult stages, and also innervates an adult-specific structure in the digestive tract, the crop. It is proposed that the adult-specific insulin-producing cells may regulate functions of the digestive system in a stage-specific manner. This study provides a neuroanatomical basis for understanding remodeling of the neuropeptidergic system during insect development and evolution.
Molnar, C., Estrada, B. and de Celis, J. F. (2018). Tay bridge and ERK activity are required for motoneuron function in the Drosophila neural system. Genes Brain Behav. PubMed ID: 29524312
Summary:
Extracellular regulated kinase (Erk) activity is required during neural development for the specification of cell fates in neuroblasts and neuronal lineages, and also regulates several aspects of the activity and survival of mature neurons. The activation of Erk is regulated at multiple levels by kinases and phosphatases that alter its phosphorylation state and by other proteins that regulate its subcellular localization. This study finds that tay bridge (tay), a negative regulator of Erk in Drosophila imaginal discs, is required in the motoneurons to regulate the number and size of neuromuscular synapses in these cells. The expression of Tay is maximal in motoneurons with low levels of activated ERK, suggesting that Tay modulates the activity of Erk in these cells. Loss of tay expression and increased Erk activity specifically in the motoneurons cause a reversible decrease in walking speed. Impaired motoneurons activity may be caused by alterations in the functionality and number of synaptic boutons developing at the neuromuscular junction in tay mutants.
Peng, J., Santiago, I. J., Ahn, C., Gur, B., Tsui, C. K., Su, Z., Xu, C., Karakhanyan, A., Silies, M. and Pecot, M. Y. (2018). Drosophila Fezf coordinates laminar-specific connectivity through cell-intrinsic and cell-extrinsic mechanisms. Elife 7. PubMed ID: 29513217
Summary:
Laminar arrangement of neural connections is a fundamental feature of neural circuit organization. Identifying mechanisms that coordinate neural connections within correct layers is thus vital for understanding how neural circuits are assembled. In the medulla of the Drosophila visual system neurons form connections within ten parallel layers. The M3 layer receives input from two neuron types that sequentially innervate M3 during development. This study shows that M3-specific innervation by both neurons is coordinated by Drosophila Fezf (dFezf; Earmuff), a conserved transcription factor that is selectively expressed by the earlier targeting input neuron. In this cell, dFezf instructs layer specificity and activates the expression of a secreted molecule (Netrin) that regulates the layer specificity of the other input neuron. It is proposed that employment of transcriptional modules that cell-intrinsically target neurons to specific layers, and cell-extrinsically recruit other neurons is a general mechanism for building layered networks of neural connections.
Saumweber, T., Rohwedder, A., Schleyer, M., Eichler, K., Chen, Y. C., Aso, Y., Cardona, A., Eschbach, C., Kobler, O., Voigt, A., Durairaja, A., Mancini, N., Zlatic, M., Truman, J. W., Thum, A. S. and Gerber, B. (2018). Functional architecture of reward learning in mushroom body extrinsic neurons of larval Drosophila. Nat Commun 9(1): 1104. PubMed ID: 29549237
Summary:
The brain adaptively integrates present sensory input, past experience, and options for future action. The insect mushroom body exemplifies how a central brain structure brings about such integration. This study used a combination of systematic single-cell labeling, connectomics, transgenic silencing, and activation experiments to study the mushroom body at single-cell resolution, focusing on the behavioral architecture of its input and output neurons (MBINs and MBONs), and of the mushroom body intrinsic APL neuron. The results reveal the identity and morphology of almost all of these 44 neurons in stage 3 Drosophila larvae. Upon an initial screen, functional analyses focusing on the mushroom body medial lobe uncover sparse and specific functions of its dopaminergic MBINs, its MBONs, and of the GABAergic APL neuron across three behavioral tasks, namely odor preference, taste preference, and associative learning between odor and taste. These results thus provide a cellular-resolution study case of how brains organize behavior.

Thursday, April 19th- Transcriptional Regulation

Cusanovich, D. A., Reddington, J. P., Garfield, D. A., Daza, R. M., Aghamirzaie, D., Marco-Ferreres, R., Pliner, H. A., Christiansen, L., Qiu, X., Steemers, F. J., Trapnell, C., Shendure, J. and Furlong, E. E. M. (2018). The cis-regulatory dynamics of embryonic development at single-cell resolution. Nature. PubMed ID: 29539636
Summary:
This study investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), chromatin accessibility was profiled in over 20,000 single nuclei from fixed Drosophila embryos spanning three embryonic stages: stage 5 blastoderm nuclei; 6-8 h after egg laying, to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12 h after egg laying, when cells are undergoing terminal differentiation. The results show that there is spatial heterogeneity in the accessibility of the regulatory genome before gastrulation, a feature that aligns with future cell fate, and that nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility while maintaining a signature of their germ layer of origin. Analysis of the data reveals overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program that is reminiscent of the mesendoderm lineage in other species. 30,075 distal regulatory elements were identified that exhibit tissue-specific accessibility. The germ-layer specificity of a subset of these predicted enhancers was validated in transgenic embryos, achieving an accuracy of 90%. Overall, these results demonstrate the power of shotgun single-cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types.
Papagianni, A., Fores, M., Shao, W., He, S., Koenecke, N., Andreu, M. J., Samper, N., Paroush, Z., Gonzalez-Crespo, S., Zeitlinger, J. and Jimenez, G. (2018). Capicua controls Toll/IL-1 signaling targets independently of RTK regulation. Proc Natl Acad Sci U S A 115(8): 1807-1812. PubMed ID: 29432195
Summary:
The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: In the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling down-regulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. This study characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-kappaB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a mode of Cic regulation unrelated to the well-established RTK/Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, these results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.
Mikhaylichenko, O., Bondarenko, V., Harnett, D., Schor, I. E., Males, M., Viales, R. R. and Furlong, E. E. M. (2018). The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription. Genes Dev [Epub ahead of print]. PubMed ID: 29378788
Summary:
Gene expression is regulated by promoters, which initiate transcription, and enhancers, which control their temporal and spatial activity. However, the discovery that mammalian enhancers also initiate transcription questions the inherent differences between enhancers and promoters. This study investigate the transcriptional properties of enhancers during Drosophila embryogenesis using characterized developmental enhancers. While the timing of enhancer transcription is generally correlated with enhancer activity, the levels and directionality of transcription are highly varied among active enhancers. To assess how this impacts function, a dual transgenic assay was developed to simultaneously measure enhancer and promoter activities from a single element in the same embryo. Extensive transgenic analysis revealed a relationship between the direction of endogenous transcription and the ability to function as an enhancer or promoter in vivo, although enhancer RNA (eRNA) production and activity are not always strictly coupled. Some enhancers (mainly bidirectional) can act as weak promoters, producing overlapping spatio-temporal expression. Conversely, bidirectional promoters often act as strong enhancers, while unidirectional promoters generally cannot. The balance between enhancer and promoter activity is generally reflected in the levels and directionality of eRNA transcription and is likely an inherent sequence property of the elements themselves.
Boisclair Lachance, J. F., Webber, J. L., Hong, L., Dinner, A. and Rebay, I. (2018). Cooperative recruitment of Yan via a high-affinity ETS supersite organizes repression to confer specificity and robustness to cardiac cell fate specification. Genes Dev. PubMed ID: 29535190
Summary:
Cis-regulatory modules (CRMs) are defined by unique combinations of transcription factor-binding sites. Emerging evidence suggests that the number, affinity, and organization of sites play important roles in regulating enhancer output and, ultimately, gene expression. This study investigated how the cis-regulatory logic of a tissue-specific CRM responsible for even-skipped (eve) induction during cardiogenesis organizes the competing inputs of two E-twenty-six (ETS) members: the activator Pointed (Pnt) and the repressor Yan. Using a combination of reporter gene assays and CRISPR-Cas9 gene editing, it is suggested that Yan and Pnt have distinct syntax preferences. Not only does Yan prefer high-affinity sites, but an overlapping pair of such sites is necessary and sufficient for Yan to tune Eve expression levels in newly specified cardioblasts and block ectopic Eve induction and cell fate specification in surrounding progenitors. Mechanistically, the efficient Yan recruitment promoted by this high-affinity ETS supersite not only biases Yan-Pnt competition at the specific CRM but also organizes Yan-repressive complexes in three dimensions across the eve locus. Taken together, these results uncover a novel mechanism by which differential interpretation of CRM syntax by a competing repressor-activator pair can confer both specificity and robustness to developmental transitions.
Roeske, M. J., Camino, E. M., Grover, S., Rebeiz, M. and Williams, T. M. (2018). Cis-regulatory evolution integrated the Bric-a-brac transcription factors into a novel fruit fly gene regulatory network. Elife 7. PubMed ID: 29297463
Summary:
Gene expression evolution through gene regulatory network (GRN) changes has gained appreciation as a driver of morphological evolution. However, understanding how GRNs evolve is hampered by finding relevant cis-regulatory element (CRE) mutations, and interpreting the protein-DNA interactions they alter. This study investigated evolutionary changes in the duplicated Bric-a-brac (Bab) transcription factors and a key Bab target gene in a GRN underlying the novel dimorphic pigmentation of D. melanogaster and its relatives. It has remained uncertain how Bab was integrated within the pigmentation GRN. This study shows that the ancestral transcription factor activity of Bab gained a role in sculpting sex-specific pigmentation through the evolution of binding sites in a CRE of the pigment-promoting yellow gene. This work demonstrates how a new trait can evolve by incorporating existing transcription factors into a GRN through CRE evolution, an evolutionary path likely to predominate newly evolved functions of transcription factors.
Preger-Ben Noon, E., Sabaris, G., Ortiz, D. M., Sager, J., Liebowitz, A., Stern, D. L. and Frankel, N. (2018). Comprehensive analysis of a cis-regulatory region reveals pleiotropy in enhancer function. Cell Rep 22(11): 3021-3031. PubMed ID: 29539428
Summary:
Developmental genes can have complex cis-regulatory regions with multiple enhancers. Early work revealed remarkable modularity of enhancers, whereby distinct DNA regions drive gene expression in defined spatiotemporal domains. Nevertheless, a few reports have shown that enhancers function in multiple developmental stages, implying that enhancers can be pleiotropic. This paper has studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. All seven shavenbaby enhancers drive expression in multiple tissues and developmental stages. This study explores how enhancer pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, revealing site pleiotropy, whereas for a second enhancer, these roles are encoded by distinct sites. Enhancer pleiotropy may be a common feature of cis-regulatory regions of developmental genes, and site pleiotropy may constrain enhancer evolution in some cases.

Wednesday, March 18th - Chromatin

Melnikova, L., Kostyuchenko, M., Parshikov, A., Georgiev, P. and Golovnin, A. (2018). Role of Su(Hw) zinc finger 10 and interaction with CP190 and Mod(mdg4) proteins in recruiting the Su(Hw) complex to chromatin sites in Drosophila. PLoS One 13(2): e0193497. PubMed ID: 29474480
Summary:
Su(Hw) belongs to the class of proteins that organize chromosome architecture and boundaries/insulators between regulatory domains. This protein contains a cluster of 12 zinc finger domains most of which are responsible for binding to three different modules in the consensus site. Su(Hw) forms a complex with CP190 and Mod(mdg4)-67.2 proteins that binds to well-known Drosophila insulators. To understand how Su(Hw) performs its activities and binds to specific sites in chromatin, this study examined the previously described su(Hw)f mutation that disrupts the 10th zinc finger (ZF10) responsible for Su(Hw) binding to the upstream module. The results have shown that Su(Hw)f loses the ability to interact with CP190 in the absence of DNA. In contrast, complete deletion of ZF10 does not prevent the interaction between Su(Hw)Delta10 and CP190. Having studied insulator complex formation in different mutant backgrounds, it is concluded that both association with CP190 and Mod(mdg4)-67.2 partners and proper organization of DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators.
Nagalingam, K., Lorenc, M. T., Manoli, S., Cameron, S. L., Clarke, A. R. and Dudley, K. J. (2018). Chromatin immunoprecipitation (ChIP) method for non-model fruit flies (Diptera: Tephritidae) and evidence of histone modifications. PLoS One 13(3): e0194420. PubMed ID: 29543899
Summary:
Combining ChIP with genomic screening technologies and Next Generation Sequencing (e.g. ChIP-seq), it has become possible to profile DNA-protein interactions (including covalent histone modifications) across entire genomes. However, the applicability of ChIP-chip and ChIP-seq has rarely been extended to non-model species because of a number of technical challenges. This study reports a method that can be used to identify genome wide covalent histone modifications in a group of non-model fruit fly species (Diptera: Tephritidae). The method was developed by testing and refining protocols that have been used in model organisms, including Drosophila melanogaster. This method was found to be suitable for a group of economically important pest fruit fly species, viz., Bactrocera dorsalis, Ceratitis capitata, Zeugodacus cucurbitae and Bactrocera tryoni. An example ChIP-seq dataset for B. tryoni is reported, providing evidence for histone modifications in the genome of a tephritid fruit fly for the first time. Since tephritids are major agricultural pests globally, this methodology will be a valuable resource to study taxa-specific evolutionary questions and to assist with pest management. It also provides a basis for researchers working with other non-model species to undertake genome wide DNA-protein interaction studies.
Nazer, E., Dale, R. K., Chinen, M., Radmanesh, B. and Lei, E. P. (2018). Argonaute2 and LaminB modulate gene expression by controlling chromatin topology. PLoS Genet 14(3): e1007276. PubMed ID: 29529026
Summary:
Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. This study identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, this study identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, these findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression.
Nishioka, K., Wang, X. F., Miyazaki, H., Soejima, H. and Hirose, S. (2018). Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from degradation by Pacman. Development 145(5). PubMed ID: 29523653
Summary:
Under stress conditions, the coactivator Multiprotein bridging factor 1 (Mbf1) translocates from the cytoplasm into the nucleus to induce stress-response genes. However, its role in the cytoplasm, where it is mainly located, has remained elusive. This study shows that Drosophila Mbf1 associates with E(z) mRNA and protects it from degradation by the exoribonuclease Pacman (Pcm), thereby ensuring Polycomb silencing. In genetic studies, loss of mbf1 function enhanced a Polycomb phenotype in Polycomb group mutants, and was accompanied by a significant reduction in E(z) mRNA expression. Furthermore, a pcm mutation suppressed the Polycomb phenotype and restored the expression level of E(z) mRNA, while pcm overexpression exhibited the Polycomb phenotype in the mbf1 mutant but not in the wild-type background. In vitro, Mbf1 protected E(z) RNA from Pcm activity. These results suggest that Mbf1 buffers fluctuations in Pcm activity to maintain an E(z) mRNA expression level sufficient for Polycomb silencing.
Becker, L., Nogueira, M. S., Klima, C., de Angelis, M. H. and Peleg, S. (2018). Rapid and transient oxygen consumption increase following acute HDAC/KDAC inhibition in Drosophila tissue. Sci Rep 8(1): 4199. PubMed ID: 29520020
Summary:
Epigenetic deregulation, such as the reduction of histone acetylation levels, is thought to be causally linked to various maladies associated with aging. Consequently, histone deacetylase inhibitors are suggested to serve as epigenetic therapy by increasing histone acetylation. However, previous work suggests that many non-histone proteins, including metabolic enzymes, are also acetylated and that post transitional modifications may impact their activity. Furthermore, deacetylase inhibitors were recently shown to impact the acetylation of a variety of proteins. By utilizing a novel technique to measure oxygen consumption rate from whole living tissue, this study demonstrated that treatment of whole living fly heads by the HDAC/KDAC inhibitors sodium butyrate and Trichostatin A, induces a rapid and transient increase of oxygen consumption rate. In addition, this study indicates that the rate increase is markedly attenuated in midlife fly head tissue. Overall, this data suggest that HDAC/KDAC inhibitors may induce enhanced mitochondrial activity in a rapid manner. This observed metabolic boost provides further, but novel evidence, that treating various maladies with deacetylase inhibitors may be beneficial.
Harrer, N., Schindler, C. E. M., Bruetzel, L. K., Forne, I., Ludwigsen, J., Imhof, A., Zacharias, M., Lipfert, J. and Mueller-Planitz, F. (2018). Structural architecture of the nucleosome remodeler ISWI determined from cross-Linking, mass spectrometry, SAXS, and modeling. Structure 26(2): 282-294.e286. PubMed ID: 29395785
Summary:
Chromatin remodeling factors assume critical roles by regulating access to nucleosomal DNA. To determine the architecture of the Drosophila ISWI remodeling enzyme, this study developed an integrative structural approach that combines protein cross-linking, mass spectrometry, small-angle X-ray scattering, and computational modeling. The resulting structural model shows the ATPase module in a resting state with both ATPase lobes twisted against each other, providing support for a conformation that was recently trapped by crystallography. The autoinhibiting NegC region does not protrude from the ATPase module as suggested previously. The regulatory NTR domain is located near both ATPase lobes. The full-length enzyme is flexible and can adopt a compact structure in solution with the C-terminal HSS domain packing against the ATPase module. These data imply a series of conformational changes upon activation of the enzyme and illustrate how the NTR, NegC, and HSS domains contribute to regulation of the ATPase module.

Tuesday, April 17 - Adult development

Torres-Oliva, M., Schneider, J., Wiegleb, G., Kaufholz, F. and Posnien, N. (2018). Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity. PLoS Genet 14(1): e1007180. PubMed ID: 29360820
Summary:
Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. This study obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. The expression profiles of all genes expressed during eye-antennal disc development was revealed, and temporally co-expressed genes was revealed by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, the transcriptome dataset was combined with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, this study shows that the transcription factor Hunchback (Hb) regulates a significant number of genes that are expressed during late differentiation stages. It was confirmed that hb is expressed in two polyploid subperineurial glia cells (carpet cells) and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, functional data is provided indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, the expression data was combined with a de novo Hb motif search to reveal stage specific putative target genes of which a significant number was found to be expressed in carpet cells.
Palliyil, S., Zhu, J., Baker, L. R., Neuman, S. D., Bashirullah, A. and Kumar, J. P. (2018). Allocation of distinct organ fates from a precursor field requires a shift in expression and function of gene regulatory networks. PLoS Genet 14(1): e1007185. PubMed ID: 29351292
Summary:
A common occurrence in metazoan development is the rise of multiple tissues/organs from a single uniform precursor field. One example is the anterior forebrain of vertebrates, which produces the eyes, hypothalamus, diencephalon, and telencephalon. Another instance is the Drosophila wing disc, which generates the adult wing blade, the hinge, and the thorax. Gene regulatory networks (GRNs) that are comprised of signaling pathways and batteries of transcription factors parcel the undifferentiated field into discrete territories. This simple model is challenged by two observations. First, many GRN members that are thought to control the fate of one organ are actually expressed throughout the entire precursor field at earlier points in development. Second, each GRN can simultaneously promote one of the possible fates choices while repressing the other alternatives. It is therefore unclear how GRNs function to allocate tissue fates if their members are uniformly expressed and competing with each other within the same populations of cells. This paradigm was addressed by studying fate specification in the Drosophila eye-antennal disc. The disc, which begins its development as a homogeneous precursor field, produces a number of adult structures including the compound eyes, the ocelli, the antennae, the maxillary palps, and the surrounding head epidermis. Several selector genes that control the fates of the eye and antenna, respectively, are first expressed throughout the entire eye-antennal disc. This study shows that during early stages, these genes are tasked with promoting the growth of the entire field. Upon segregation to distinct territories within the disc, each GRN continues to promote growth while taking on the additional roles of promoting distinct primary fates and repressing alternate fates. The timing of both expression pattern restriction and expansion of functional duties is an elemental requirement for allocating fates within a single field.
Nagai, T., Honda, H. and Takemura, M. (2018). Simulation of cell patterning triggered by cell death and differential adhesion in Drosophila wing. Biophys J 114(4): 958-967. PubMed ID: 29490255
Summary:
The Drosophila wing exhibits a well-ordered cell pattern, especially along the posterior margin, where hair cells are arranged in a zigzag pattern in the lateral view. Based on an experimental result observed during metamorphosis of Drosophila, it was considered that a pattern of initial cells autonomously develops to the zigzag pattern through cell differentiation, intercellular communication, and cell death (apoptosis), and computer simulations were performed of a cell-based model of vertex dynamics for tissues. The model describes the epithelial tissue as a monolayer cell sheet of polyhedral cells. Their vertices move according to equations of motion, minimizing the sum total of the interfacial and elastic energies of cells. The interfacial energy densities between cells are introduced consistently with an ideal zigzag cell pattern, extracted from the experimental result. The apoptosis of cells is modeled by gradually reducing their equilibrium volume to zero and by assuming that the hair cells prohibit neighboring cells from undergoing apoptosis. Based on experimental observations, wing elongation along the proximal-distal axis was also assumed. Starting with an initial cell pattern similar to the micrograph experimentally obtained just before apoptosis, the simulations were carried out according to the model mentioned above, and the ideal zigzag cell pattern was successfully reproduced. This elucidates a physical mechanism of patterning triggered by cell apoptosis theoretically and exemplifies a new framework to study apoptosis-induced patterning. It is concluded that the zigzag cell pattern is formed by an autonomous communicative process among the participant cells.
Hara, Y., Sudo, T., Togane, Y., Akagawa, H. and Tsujimura, H. (2018). Cell death in neural precursor cells and neurons before neurite formation prevents the emergence of abnormal neural structures in the Drosophila optic lobe. Dev Biol. PubMed ID: 29447906
Summary:
Programmed cell death is a conserved strategy for neural development both in vertebrates and invertebrates and is recognized at various developmental stages in the brain from neurogenesis to adulthood. This study investigated the effect of inhibition of cell death on optic lobe development. Cell death was shown to occur in neural precursor cells and neurons before neurite formation and functions to prevent various developmental abnormalities. When neuronal cell death was inhibited by an effector caspase inhibitor, p35, multiple abnormal neuropil structures arose during optic lobe development-e.g., enlarged or fused neuropils, misrouted neurons and abnormal neurite lumps. Inhibition of cell death also induced morphogenetic defects in the lamina and medulla development. These defects were reproduced in the mutant of an initiator caspase, dronc. If cell death was a mechanism for removing the abnormal neuropil structures, it would also be expected to be observed in mutants defective for corpse clearance. However, they were not observed in these mutants. When dead cell-membranes were visualized with Apoliner, they were observed only in cortices and not in neuropils. These results suggest that the cell death occurs before mature neurite formation. Moreover, it was found that inhibition of cell death induced ectopic neuroepithelial cells, neuroblasts and ganglion mother cells in late pupal stages, at sites where the outer and inner proliferation centers were located at earlier developmental stages. Caspase-3 activation was observed in the neuroepithelial cells and neuroblasts in the proliferation centers. These results indicate that cell death is required for elimination of the precursor cells composing the proliferation centers. This study substantiates an essential role of early neural cell death for ensuring normal development of the central nervous system.
Morrison, C. A., Chen, H., Cook, T., Brown, S. and Treisman, J. E. (2018). Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye. PLoS Genet 14(1): e1007173. PubMed ID: 29324767
Summary:
Transcriptional regulators can specify different cell types from a pool of equivalent progenitors by activating distinct developmental programs. The Glass transcription factor is expressed in all progenitors in the developing Drosophila eye, and is maintained in both neuronal and non-neuronal cell types. Glass is required for neuronal progenitors to differentiate as photoreceptors, but its role in non-neuronal cone and pigment cells is unknown. To determine whether Glass activity is limited to neuronal lineages, the effects were compared of misexpressing it in neuroblasts of the larval brain and in epithelial cells of the wing disc. Glass activated overlapping but distinct sets of genes in these neuronal and non-neuronal contexts, including markers of photoreceptors, cone cells and pigment cells. Coexpression of other transcription factors such as Pax2, Eyes absent, Lozenge and Escargot enabled Glass to induce additional genes characteristic of the non-neuronal cell types. Cell type-specific glass mutations generated in cone or pigment cells using somatic CRISPR revealed autonomous developmental defects, and expressing Glass specifically in these cells partially rescued glass mutant phenotypes. These results indicate that Glass is a determinant of organ identity that acts in both neuronal and non-neuronal cells to promote their differentiation into functional components of the eye.
Petrovsky, R., Krohne, G. and Grosshans, J. (2018). Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes. Eur J Cell Biol 97(2): 102-113. PubMed ID: 29395481
Summary:
The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. This study analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, distinct morphological features specific for the respective protein were detected. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope.

Monday, April 16th - Signaling

Koch, M., Nicolas, M., Zschaetzsch, M., de Geest, N., Claeys, A., Yan, J., Morgan, M. J., Erfurth, M. L., Holt, M., Schmucker, D. and Hassan, B. A. (2017). A Fat-facets-Dscam1-JNK Pathway enhances axonal growth in development and after injury. Front Cell Neurosci 11: 416. PubMed ID: 29472843
Summary:
Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. A gain-of-function genetic screen was constructed in Drosophila to identify strong inducers of axonal growth after injury. Focus was placed on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3'-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism.
Lin, W. S., Yeh, S. R., Fan, S. Z., Chen, L. Y., Yen, J. H., Fu, T. F., Wu, M. S. and Wang, P. Y. (2018). Insulin signaling in female Drosophila links diet and sexual attractiveness. Faseb j: fj201800067R. PubMed ID: 29475396
Summary:
Appropriate sexual selection or individual sexual attractiveness is closely associated with the reproductive success of a species. This study reports that young male flies exhibit innate courtship preference for female flies that are raised on higher-yeast diets and that have greater body weight and fecundity, but reduced locomotor activity and shortened lifespan. Male flies discriminate among females that have been fed diets that contain 3 different yeast concentrations-1, 5, and 20% yeast- via gustatory, but not visual or olfactory, perception. Female flies that are raised on higher-yeast diets exhibit elevated expression levels of Drosophila insulin-like peptides (dilps), and it was demonstrated that hypomorphic mutations of dilp2, dilp3, dilp5 or foxo, as well as oenocyte-specific gene disruption of the insulin receptor, all abolish this male courtship preference for high yeast-fed females. Moreover, these data demonstrate that disrupted dilp signaling can alter the expression profile of some cuticular hydrocarbons (CHCs) in female flies, and that genetic inhibition of an enzyme involved in the biosynthesis of CHCs in oenocytes, elongase F, also eliminates the male courtship preference. Together, these findings provide mechanistic insights that link female reproductive potential to sexual attractiveness, thereby encouraging adaptive mating and optimal reproductive success.
Lee, J. J., Sanchez-Martinez, A., Zarate, A. M., Beninca, C., Mayor, U., Clague, M. J. and Whitworth, A. J. (2018). Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. J Cell Biol [Epub ahead of print]. PubMed ID: 29500189
Summary:
The Parkinson's disease factors PINK1 and parkin are strongly implicated in stress-induced mitophagy in vitro, but little is known about their impact on basal mitophagy in vivo. Transgenic Drosophila melanogaster were generated expressing fluorescent mitophagy reporters to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. Mitophagy was readily detectable and abundant in many tissues, including Parkinson's disease-relevant dopaminergic neurons. However, mitolysosomes were not detected in flight muscle. Surprisingly, in Pink1 or parkin null flies, no substantial impact on basal mitophagy was detected. Because these flies exhibit locomotor defects and dopaminergic neuron loss, these findings raise questions about current assumptions of the pathogenic mechanism associated with the PINK1/parkin pathway. The findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure.
Li, S., Li, S., Wang, B. and Jiang, J. (2018). Hedgehog reciprocally controls trafficking of Smo and Ptc through the Smurf family of E3 ubiquitin ligases. Sci Signal 11(516). PubMed ID: 29438012
Summary:
Hedgehog (Hh) induces signaling by promoting the reciprocal trafficking of its receptor Patched (Ptc) and the signal transducer Smoothened (Smo), which is inhibited by Ptc, at the cell surface. Smurf family E3 ubiquitin ligases were identified as essential for Smo ubiquitylation and cell surface clearance, and Smurf family members were found to mediate the reciprocal trafficking of Ptc and Smo in Drosophila melanogaster G protein-coupled receptor kinase 2 (Gprk2)-mediated phosphorylation of Smurf promoted Smo ubiquitylation by increasing the recruitment of Smurf to Smo, whereas protein kinase A (PKA)-mediated phosphorylation of Smo caused Smurf to dissociate from Smo, thereby inhibiting Smo ubiquitylation. Smo and Ptc competed for the same pool of Smurf family E3 ubiquitin ligases, and Hh promoted Ptc ubiquitylation and degradation by disrupting the association of Smurf family E3 ubiquitin ligases with Smo and stimulating their binding to Ptc. This study identifies the E3 ubiquitin ligases that target Smo and provides insight into how Hh regulates the reciprocal trafficking of its receptor and signal transducer.
Kang, D., Wang, D., Xu, J., Quan, C., Guo, X., Wang, H., Luo, J., Yang, Z., Chen, S. and Chen, J. (2018). The InR/Akt/TORC1 Growth-Promoting Signaling Negatively Regulates JAK/STAT Activity and Migratory Cell Fate during Morphogenesis. Dev Cell 44(4): 524-531.e525. PubMed ID: 29456138
Summary:
Cell growth and cell differentiation are two distinct yet coupled developmental processes, but how they are coordinated is not well understood. During Drosophila oogenesis, this study found that the growth-promoting InR/Akt/TOR pathway was involved in suppressing the fate determination of the migratory border cells. The InR/Akt/TOR TOR and Raptor, components of TORC1, to downregulate the JAK/STAT pathway, which is necessary and sufficient for border cell fate determination. TORC1 promotes the protein stability of SOCS36E, the conserved negative regulator of JAK/STAT signaling, through physical interaction, suggesting that TORC1 acts as a key regulator coordinating both cell growth and cell differentiation.
Ma, X., Guo, X., Richardson, H. E., Xu, T. and Xue, L. (2018). POSH regulates Hippo signaling through ubiquitin-mediated expanded degradation. Proc Natl Acad Sci U S A 115(9): 2150-2155. PubMed ID: 29440430
Summary:
The Hippo signaling pathway is a master regulator of organ growth, tissue homeostasis, and tumorigenesis. The activity of the Hippo pathway is controlled by various upstream components, including Expanded (Ex), but the precise molecular mechanism of how Ex is regulated remains poorly understood. This study identified Plenty of SH3s (POSH), an E3 ubiquitin ligase, as a key component of Hippo signaling in Drosophila. POSH overexpression synergizes with loss of Kibra to induce overgrowth and up-regulation of Hippo pathway target genes. Furthermore, knockdown of POSH impedes dextran sulfate sodium-induced Yorkie-dependent intestinal stem cell renewal, suggesting a physiological role of POSH in modulating Hippo signaling. Mechanistically, POSH binds to the C-terminal of Ex and is essential for the Crumbs-induced ubiquitination and degradation of Ex. These findings establish POSH as a crucial regulator that integrates the signal from the cell surface to negatively regulate Ex-mediated Hippo activation in Drosophila.

Friday, April 13th - Disease models

Joos, J. P., Saadatmand, A. R., Schnabel, C., Viktorinova, I., Brand, T., Kramer, M., Nattel, S., Dobrev, D., Tomancak, P., Backs, J., Kleinbongard, P., Heusch, G., Lorenz, K., Koch, E., Weber, S. and El-Armouche, A. (2018). Ectopic expression of S28A-mutated Histone H3 modulates longevity, stress resistance and cardiac function in Drosophila. Sci Rep 8(1): 2940. PubMed ID: 29440697
Summary:
Histone H3 serine 28 (H3S28) phosphorylation and de-repression of polycomb repressive complex (PRC)-mediated gene regulation is linked to stress conditions in mitotic and post-mitotic cells. To better understand the role of H3S28 phosphorylation in vivo, a Drosophila strain was studied with ectopic expression of constitutively-activated H3S28A, which prevents PRC2 binding at H3S28, thus mimicking H3S28 phosphorylation. H3S28A mutants showed prolonged life span and improved resistance against starvation and paraquat-induced oxidative stress. Morphological and functional analysis of heart tubes revealed smaller luminal areas and thicker walls accompanied by moderately improved cardiac function after acute stress induction. Whole-exome deep gene-sequencing from isolated heart tubes revealed phenotype-corresponding changes in longevity-promoting and myotropic genes. Changes were also found in genes controlling mitochondrial biogenesis and respiration. Analysis of mitochondrial respiration from whole flies revealed improved efficacy of ATP production with reduced electron transport-chain activity. Finally, posttranslational modification of H3S28 was examined in an experimental heart failure model, and increased H3S28 phosphorylation levels were observed in HF hearts. These data establish a critical role of H3S28 phosphorylation in vivo for life span, stress resistance, cardiac and mitochondrial function in Drosophila. These findings may pave the way for H3S28 phosphorylation as a putative target to treat stress-related disorders such as heart failure.
Chen, A. Y. and Tully, T. (2018). A stress-enhanced model for discovery of disease-modifying gene: Ece1-suppresses the toxicity of alpha-synuclein A30P. Neurobiol Dis 114: 153-163. PubMed ID: 29524599
Summary:
Parkinson's disease (PD) is a progressive motor neurodegenerative disorder, characterized by a selective loss of dopaminergic neurons in the substantia nigra. The complexity of disease etiology includes both genetic and environmental factors. Human alpha-Synuclein A30P (A30P) is a mutant gene identified in early onset PD and has been shown to result selective dopamine neuron loss in transgenic A30P flies and mice. Paraquat (PQ) is an herbicide and an oxidative stress generator, linked to sporadic PD. To identify disease modifier genes, two independently-duplicated experiments were performed of microarray, capturing genome-wide transcriptional changes in A30P flies, chronically fed with PQ-contaminated food. It was hypothesized that the best time point of identifying a disease modifier gene is at time when flies showed maximal combined toxicity of A30P transgene and PQ treatment during an early stage of disease and that effective disease modifiers gene are those showing transcriptional changes to oxidative stress in A30P expressing and not in wild type animals. Fly Neprilysin3 (Nep3) is one identified gene that is highly conserved. Its mouse and human homolog is endothelin-converting enzyme-1 (Ece1). To investigate the neuroprotective effect of Ece1, NS1 cells and mouse midbrain neurons expressing A30P, treated with or without PQ were used. ECE1 expression protected against A30P toxicity on cell viability, on neurite outgrowth and ameliorated A30P accumulation in vitro. Expression of ECE1 in vivo suppressed dopamine neuron loss and alleviated the corresponding motor deficits in mice with A30P-expression. This study leverages a new approach to identify disease modifier genes using a stress-enhanced PD animal model.
Choi, T. Y., Lee, S. H., Kim, Y. J., Bae, J. R., Lee, K. M., Jo, Y., Kim, S. J., Lee, A. R., Choi, S., Choi, L. M., Bang, S., Song, M. R., Chung, J., Lee, K. J., Kim, S. H., Park, C. S. and Choi, S. Y. (2018). Cereblon maintains synaptic and cognitive function by regulating BK channel. J Neurosci. PubMed ID: 29530986
Summary:
Mutations in the cereblon (CRBN) gene cause human intellectual disability, one of the most common cognitive disorders. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. This study investigated the role of CRBN in synaptic function and animal behavior using male mouse and Drosophila models (ohgata). Crbn knockout (KO) mice showed normal brain and spine morphology as well as intact synaptic plasticity; however, they also exhibited decreases in synaptic transmission and presynaptic release probability exclusively in excitatory synapses. Notably, presynaptic function was impaired not only by loss of CRBN expression, but also by expression of pathogenic CRBN mutants (human R419X mutant and Drosophila G552X mutant). BK channel (Slowpoke in Drosophila) blockers paxilline and iberiotoxin reversed this decrease in presynaptic release probability in Crbn KO mice. In addition, paxilline treatment also restored normal cognitive behavior in Crbn KO mice. These results strongly suggest that increased BK channel activity is the pathological mechanism of intellectual disability in CRBN mutations.
Tran, H. H., Dang, S. N. A., Nguyen, T. T., Huynh, A. M., Dao, L. M., Kamei, K., Yamaguchi, M. and Dang, T. T. P. (2018). Drosophila Ubiquitin C-Terminal Hydrolase Knockdown Model of Parkinson's Disease. Sci Rep 8(1): 4468. PubMed ID: 29535397
Summary:
Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Many factors have been shown to contribute to its pathogenesis including genetic and environmental factors. Ubiquitin C-terminal hydrolase L1 (UCHL1) is also known to be involved in the pathogenesis of PD. The study of UCHL1 mas modeled in Drosophila melanogaster and investigated its functions in PD. The specific knockdown of the Drosophila ortholog of UCHL1 (dUCH) in dopaminergic neurons (DA neurons) led to the underdevelopment and/or degeneration of these neurons, specifically in DL1 DA neuron cluster in the larval brain lobe and PPM2, PPM3, PPL2ab, and VUM DA neuron clusters in the adult brain. These defects were followed by a shortage of dopamine in the brain, which subsequently resulted in locomotor dysfunction. The degeneration of DA neurons in dUCH knockdown adult brain, which occurred progressively and severely during the course of aging, mimics the epidemiology of PD. DA neuron and locomotor defects were rescued when dUCH knockdown flies were treated with vitamin C, a well-known antioxidant. These results suggest that dUCH knockdown fly is a promising model for studying the pathogenesis and epidemiology of PD as well as the screening of potential antioxidants for PD therapeutics (Tran, 2018).
Huang, H. W., Brown, B., Chung, J., Domingos, P. M. and Ryoo, H. D. (2018). highroad is a carboxypetidase induced by retinoids to clear mutant Rhodopsin-1 in Drosophila retinitis pigmentosa models. Cell Rep 22(6): 1384-1391. PubMed ID: 29425495
Summary:
Rhodopsins require retinoid chromophores for their function. In vertebrates, retinoids also serve as signaling molecules, but whether these molecules similarly regulate gene expression in Drosophila remains unclear. This study reports the identification of a retinoid-inducible gene in Drosophila, highroad, which is required for photoreceptors to clear folding-defective mutant Rhodopsin-1 proteins. Specifically, knockdown or genetic deletion of highroad blocks the degradation of folding-defective Rhodopsin-1 mutant, ninaEG69D. Moreover, loss of highroad accelerates the age-related retinal degeneration phenotype of ninaEG69D mutants. Elevated highroad transcript levels are detected in ninaEG69D flies, and interestingly, deprivation of retinoids in the fly diet blocks this effect. Consistently, mutations in the retinoid transporter, santa maria, impairs the induction of highroad in ninaEG69D) flies. In cultured S2 cells, highroad expression is induced by retinoic acid treatment. These results indicate that cellular quality-control mechanisms against misfolded Rhodopsin-1 involve regulation of gene expression by retinoids.
Matsumoto, T., Matsukawa, K., Watanabe, N., Kishino, Y., Kunugi, H., Ihara, R., Wakabayashi, T., Hashimoto, T. and Iwatsubo, T. (2018). Self-assembly of FUS through its low-complexity domain contributes to neurodegeneration. Hum Mol Genet. PubMed ID: 29425337
Summary:
Aggregation of fused in sarcoma (FUS; see Drosophila Cabeza) protein, and mutations in FUS gene, are causative to a range of neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. To gain insights into the molecular mechanism whereby FUS causes neurodegeneration, transgenic Drosophila melanogaster were generated overexpressing human FUS in the photoreceptor neurons, which exhibited mild retinal degeneration. Expression of familial ALS-mutant FUS aggravated the degeneration, which was associated with an increase in cytoplasmic localization of FUS. A carboxy-terminally truncated R495X mutant FUS also was localized in cytoplasm, whereas the degenerative phenotype was diminished. Double expression of R495X and wild-type FUS dramatically exacerbated degeneration, sequestrating wild-type FUS into cytoplasmic aggregates. Notably, replacement of all tyrosine residues within the low-complexity domain, which abolished self-assembly of FUS, completely eliminated the degenerative phenotypes. Taken together, it is proposed that self-assembly of FUS through its low-complexity domain contributes to FUS-induced neurodegeneration.

Thursday, April 12 - Stem cells

He, L., Si, G., Huang, J., Samuel, A. D. T. and Perrimon, N. (2018). Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel. Nature 555(7694): 103-106. PubMed ID: 29414942
Summary:
Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. This study shows that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. Piezo was found to be specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Ca(2+) levels, and increases in cytosolic Ca(2+) resemble the Piezo overexpression phenotype, suggesting that Piezo functions through Ca(2+) signalling. Further studies suggest that Ca(2+) signalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Ca(2+) in response to direct mechanical stimulus in a gut compression assay. Thus, this study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.
Kullmann, L. and Krahn, M. P. (2018). Redundant regulation of localization and protein stability of DmPar3. Cell Mol Life Sci. PubMed ID: 29523893
Summary:
Apical-basal polarity is an important characteristic of epithelia and Drosophila neural stem cells. The conserved Par complex, which consists of the atypical protein kinase C and the scaffold proteins Baz and Par6, is a key player in the establishment of apical-basal cell polarity. Membrane recruitment of Baz has been reported to be accomplished by several mechanisms, which might function in redundancy, to ensure the correct localization of the complex. However, none of the described interactions was sufficient to displace the protein from the apical junctions. This study dissected the role of the oligomerization domain and the lipid-binding motif of Baz in vivo in the Drosophila embryo. These domains were found to function in redundancy to ensure the apical junctional localization of Baz: inactivation of only one domain is not sufficient to disrupt the function of Baz during apical-basal polarization of epithelial cells and neural stem cells. In contrast, mutation of both domains results in a strongly impaired protein stability and a phenotype characterized by embryonic lethality and an impaired apical-basal polarity in the embryonic epithelium and neural stem cells, resembling a baz-loss of function allele. Strikingly, the binding of Baz to the transmembrane proteins E-Cadherin, Echinoid, and Starry Night was not affected in this mutant protein. These findings reveal a redundant function of the oligomerization and the lipid-binding domain, which is required for protein stability, correct subcellular localization, and apical-basal cell polarization.
Yin, C. and Xi, R. (2018). A phyllopod-mediated feedback loop promotes intestinal stem cell enteroendocrine commitment in Drosophila. Stem Cell Reports 10(1): 43-57. PubMed ID: 29276156
Summary:
The intestinal epithelium in the Drosophila midgut is maintained by intestinal stem cells (ISCs), which are capable of generating both enterocytes and enteroendocrine cells (EEs) via alternative cell fate specification. Activation of Delta-Notch signaling directs ISCs for enterocyte generation, but how EEs are generated from ISCs remains poorly understood. This study identified Phyllopod (Phyl) as a key regulator that drives EE generation from ISCs. Phyl, which is normally suppressed by Notch, functions as an adaptor protein that bridges Tramtrack 69 (Ttk69) and E3 ubiquitin ligase Sina for degradation. Degradation of Ttk69 allows the activation of the Achaete-Scute Complex (AS-C)-Pros regulatory axis, which promotes EE specification. Interestingly, expression of AS-C genes in turn further induces Phyl expression, thereby establishing a positive feedback loop for continuous EE fate specification and commitment. This positive feedback circuit-driven regulatory mechanism could represent a common strategy for reliable and irreversible cell fate determination from progenitor cells.
Garcia Del Arco, A., Edgar, B. A. and Erhardt, S. (2018). In vivo analysis of centromeric proteins reveals a stem cell-specific asymmetry and an essential role in differentiated, non-proliferating cells. Cell Rep 22(8): 1982-1993. PubMed ID: 29466727
Summary:
Stem cells of the Drosophila midgut (ISCs) are the only mitotically dividing cells of the epithelium and, therefore, presumably the only epithelial cells that require functional kinetochores for microtubule spindle attachment during mitosis. The histone variant CENP-A marks centromeric chromatin as the site of kinetochore formation and spindle attachment during mitotic chromosome segregation. This study shows that centromeric proteins distribute asymmetrically during ISC division. Whereas newly synthesized CENP-A is enriched in differentiating progeny, CENP-C is undetectable in these cells. Remarkably, CENP-A persists in ISCs for weeks without being replaced, consistent with it being an epigenetic mark responsible for maintaining stem cell properties. Furthermore, CENP-A and its loading factor CAL1 were found to be essential for post-mitotic, differentiating cells; removal of any of these factors interferes with endoreduplication. Taken together, it is proposed two additional roles of CENP-A: to maintain stem cell-unique properties and to regulate post-mitotic cells.
Koe, C. T., Tan, Y. S., Lonnfors, M., Hur, S. K., Low, C. S. L., Zhang, Y., Kanchanawong, P., Bankaitis, V. A. and Wang, H. (2018). Vibrator and PI4KIIIalpha govern neuroblast polarity by anchoring non-muscle myosin II. Elife 7. PubMed ID: 29482721
Summary:
A central feature of most stem cells is the ability to self-renew and undergo differentiation via asymmetric division. However, during asymmetric division the role of phosphatidylinositol (PI) lipids and their regulators is not well established. This study shows that the sole type I PI transfer protein, Vibrator, controls asymmetric division of Drosophila neural stem cells (NSCs) by physically anchoring myosin II regulatory light chain, Sqh, to the NSC cortex. Depletion of vib or disruption of its lipid binding and transfer activities disrupts NSC polarity. It is proposed that Vib stimulates PI4KIIIalpha to promote synthesis of a plasma membrane pool of phosphatidylinositol 4-phosphate [PI(4)P] that, in turn, binds and anchors myosin to the NSC cortex. Remarkably, Sqh also binds to PI(4)P in vitro and both Vib and Sqh mediate plasma membrane localization of PI(4)P in NSCs. Thus, reciprocal regulation between Myosin and PI(4)P likely governs asymmetric division of NSCs.
Mohr, J., et al. (2018). The cell fate determinant Scribble is required for maintenance of hematopoietic stem cell function. Leukemia [Epub ahead of print]. PubMed ID: 29467485
Evolutionary Homolog Study
Cell fate determinants influence self-renewal potential of hematopoietic stem cells, Scribble and Llgl1 belong to the Scribble polarity complex and reveal tumor-suppressor function in Drosophila. In hematopoietic cells, genetic inactivation of Llgl1 leads to expansion of the stem cell pool and increases self-renewal capacity without conferring malignant transformation. This study shows that genetic inactivation of its putative complex partner Scribble results in functional impairment of hematopoietic stem cells (HSC) over serial transplantation and during stress. Although loss of Scribble deregulates transcriptional downstream effectors involved in stem cell proliferation, cell signaling, and cell motility, these effectors do not overlap with transcriptional targets of Llgl1. Binding partner analysis of Scribble in hematopoietic cells using affinity purification followed by mass spectometry confirms its role in cell signaling and motility but not for binding to polarity modules described in drosophila. Finally, requirement of Scribble for self-renewal capacity also affects leukemia stem cell function. Thus, Scribble is a regulator of adult HSCs, essential for maintenance of HSCs during phases of cell stress.

Wednesday, April 11th

Rieche, F., Carmine-Simmen, K., Poeck, B., Kretzschmar, D. and Strauss, R. (2018). Drosophila full-length Amyloid precursor protein is required for visual working memory and prevents age-related memory impairment. Curr Biol 28(5): 817-823.e813. PubMed ID: 29478851
Summary:
The β-amyloid precursor protein (APP) plays a central role in the etiology of Alzheimer's disease (AD). APP is cleaved by various secretases whereby sequential processing by the β- and γ-secretases produces the β-amyloid peptide that is accumulating in plaques that typify AD. In addition, this produces secreted N-terminal sAPPβ fragments and the APP intracellular domain (AICD). Alternative cleavage by α-secretase results in slightly longer secreted sAPPalpha fragments and the identical AICD. Whereas the AICD has been connected with transcriptional regulation, sAPPalpha fragments have been suggested to have a neurotrophic and neuroprotective role.Loss of the Drosophila APP-like (APPL) protein impairs associative olfactory memory formation and middle-term memory that can be rescued with a secreted APPL fragment. This study show that APPL is also essential for visual working memory. Interestingly, this short-term memory declines rapidly with age, and this is accompanied by enhanced processing of APPL in aged flies. Furthermore, reducing secretase-mediated proteolytic processing of APPL can prevent the age-related memory loss, whereas overexpression of the secretases aggravates the aging effect. Rescue experiments confirmed that this memory requires signaling of full-length APPL and that APPL negatively regulates the neuronal-adhesion molecule Fasciclin 2. Overexpression of APPL or one of its secreted N termini results in a dominant-negative interaction with the FASII receptor. Therefore, these results show that specific memory processes require distinct APPL products.
Jin, E. J., Kiral, F. R., Ozel, M. N., Burchardt, L. S., Osterland, M., Epstein, D., Wolfenberg, H., Prohaska, S. and Hiesinger, P. R. (2018). Live observation of two parallel membrane degradation pathways at axon terminals. Curr Biol. PubMed ID: 29551411
Summary:
Neurons are highly polarized cells that require continuous turnover of membrane proteins at axon terminals to develop, function, and survive. Yet, it is still unclear whether membrane protein degradation requires transport back to the cell body or whether degradation also occurs locally at the axon terminal, where live observation of sorting and degradation has remained a challenge. This study reports direct observation of two cargo-specific membrane protein degradation mechanisms at axon terminals based on a live-imaging approach in intact Drosophila brains. Different acidification-sensing cargo probes are sorted into distinct classes of degradative 'hub' compartments for synaptic vesicle proteins and plasma membrane proteins at axon terminals. Sorting and degradation of the two cargoes in the separate hubs are molecularly distinct. Local sorting of synaptic vesicle proteins for degradation at the axon terminal is, surprisingly, Rab7 independent, whereas sorting of plasma membrane proteins is Rab7 dependent. The cathepsin-like protease CP1 is specific to synaptic vesicle hubs, and its delivery requires the vesicle SNARE neuronal synaptobrevin. Cargo separation only occurs at the axon terminal, whereas degradative compartments at the cell body are mixed. These data show that at least two local, molecularly distinct pathways sort membrane cargo for degradation specifically at the axon terminal, whereas degradation can occur both at the terminal and en route to the cell body.
Kanai, M. I., Kim, M. J., Akiyama, T., Takemura, M., Wharton, K., O'Connor, M. B. and Nakato, H. (2018). Regulation of neuroblast proliferation by surface glia in the Drosophila larval brain. Sci Rep 8(1): 3730. PubMed ID: 29487331
Summary:
Despite the importance of precisely regulating stem cell division, the molecular basis for this control is still elusive. This study shows that surface glia in the developing Drosophila brain play essential roles in regulating the proliferation of neural stem cells, neuroblasts (NBs). Two classes of extracellular factors, Dally-like (Dlp), a heparan sulfate proteoglycan, and Glass bottom boat (Gbb), a BMP homologue, are required for proper NB proliferation. Interestingly, Dlp expressed in perineural glia (PG), the most outer layer of the surface glia, is responsible for NB proliferation. Consistent with this finding, functional ablation of PG using a dominant-negative form of dynamin showed that PG has an instructive role in regulating NB proliferation. Gbb acts not only as an autocrine proliferation factor in NBs but also as a paracrine survival signal in the PG. It is proposed that bidirectional communication between NBs and glia through TGF-β signaling influences mutual development of these two cell types. The possibility is discussed that PG and NBs communicate via direct membrane contact or transcytotic transport of membrane components. Thus, this study shows that the surface glia acts not only as a simple structural insulator but also a dynamic regulator of brain development.
Lee, P. T., Lin, G., Lin, W. W., Diao, F., White, B. H. and Bellen, H. J. (2018). A kinase-dependent feedforward loop affects CREBB stability and long term memory formation. Elife 7. PubMed ID: 29473541
Summary:
In Drosophila, long-term memory (LTM) requires the cAMP-dependent transcription factor CREBB, expressed in the mushroom bodies (MB) and phosphorylated by PKA. To identify other kinases required for memory formation, Trojan exons encoding T2A-GAL4 were integrated into genes encoding putative kinases and genes expressed in MB were selected for. These lines were screened for learning/memory deficits using UAS-RNAi knockdown based on an olfactory aversive conditioning assay. A novel, conserved kinase, Meng-Po (MP, CG11221, SBK1 in human) was identified, the loss of which severely affects 3 hr memory and 24 hr LTM, but not learning. Remarkably, memory is lost upon removal of the MP protein in adult MB but restored upon its reintroduction. Overexpression of MP in MB significantly increases LTM in wild-type flies showing that MP is a limiting factor for LTM. PKA phosphorylates MP and both proteins synergize in a feedforward loop to control CREBB levels and LTM.
MacWilliam, D., Kowalewski, J., Kumar, A., Pontrello, C. and Ray, A. (2018). Signaling mode of the broad-spectrum conserved CO2 Receptor is one of the important determinants of odor valence in Drosophila. Neuron 97(5): 1153-1167.e1154. PubMed ID: 29429938
Summary:
Odor detection involves hundreds of olfactory receptors from diverse families, making modeling of hedonic valence of an odorant difficult, even in Drosophila melanogaster where most receptors have been deorphanised. This study demonstrates that a broadly tuned heteromeric receptor that detects CO2 (Gr21a, Gr63a) and other odorants is a key determinant of valence along with a few members of the Odorant receptor family in a T-maze, but not in a trap assay. Gr21a and Gr63a have atypically high amino acid conservation in Dipteran insects, and they use both inhibition and activation to convey positive or negative valence for numerous odorants. Inhibitors elicit a robust Gr63a-dependent attraction, while activators, strong aversion. The attractiveness of inhibitory odorants increases with increasing background CO2 levels, providing a mechanism for behavior modulation in odor blends. In mosquitoes, valence is switched and activation of the orthologous receptor conveys attraction. Reverse chemical ecology enables the identification of inhibitory odorants to reduce attraction of mosquitoes to skin.
Kim, H., Kim, H., Kwon, J. Y., Seo, J. T., Shin, D. M. and Moon, S. J. (2018). Drosophila Gr64e mediates fatty acid sensing via the phospholipase C pathway. PLoS Genet 14(2): e1007229. PubMed ID: 29420533
Summary:
Animals use taste to sample and ingest essential nutrients for survival. Free fatty acids (FAs) are energy-rich nutrients that contribute to various cellular functions. Recent evidence suggests FAs are detected through the gustatory system to promote feeding. In Drosophila, phospholipase C (PLC) signaling in sweet-sensing cells is required for FA detection but other signaling molecules are unknown. This study shows that Gr64e is required for the behavioral and electrophysiological responses to FAs. GR64e and TRPA1 are interchangeable when they act downstream of PLC: TRPA1 can substitute for GR64e in FA but not glycerol sensing, and GR64e can substitute for TRPA1 in aristolochic acid but not N-methylmaleimide sensing. In contrast to its role in FA sensing, GR64e functions as a ligand-gated ion channel for glycerol detection. These results identify a novel FA transduction molecule and reveal that Drosophila Grs can act via distinct molecular mechanisms depending on context.

Tuesday, April 10th

Yerushalmi, G. Y., Misyura, L., MacMillan, H. A. and Donini, A. (2018). Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster. J Exp Biol. PubMed ID: 29367271
Summary:
At low temperatures, Drosophila, like most insects, lose the ability to regulate ion and water balance across the gut epithelia, which can lead to a lethal increase of [K(+)] in the hemolymph (hyperkalemia). Cold-acclimation, the physiological response to a prior low temperature exposure, can mitigate or entirely prevent these ion imbalances, but the physiological mechanisms that facilitate this process are not well understood. This study tested whether plasticity in the ionoregulatory physiology of the gut and Malpighian tubules of Drosophila may aid in preserving ion homeostasis in the cold. Upon adult emergence, D. melanogaster females were subjected to seven days at warm (25 ° C) or cold (10 ° C) acclimation conditions. The cold acclimated flies had a lower critical thermal minimum (CTmin), recovered from chill coma more quickly, and better maintained hemolymph K(+) balance in the cold. The improvements in chill tolerance coincided with increased Malpighian tubule fluid secretion and better maintenance of K(+) secretion rates in the cold, as well as reduced rectal K(+) reabsorption in cold-acclimated flies. To test whether modulation of ion-motive ATPases, the main drivers of epithelial transport in the alimentary canal, mediate these changes, the activities were measured of Na(+)-K(+)-ATPase and V-type H(+)-ATPase at the Malpighian tubules, midgut, and hindgut. Na(+)/K(+)-ATPase and V-type H(+)-ATPase activities were lower in the midgut and the Malpighian tubules of cold-acclimated flies, but unchanged in the hindgut of cold acclimated flies, and were not predictive of the observed alterations in K(+) transport. These results suggest that modification of Malpighian tubule and gut ion and water transport likely prevents cold-induced hyperkalemia in cold-acclimated flies and that this process is not directly related to the activities of the main drivers of ion transport in these organs, Na(+)/K(+)- and V-type H(+)-ATPases.
Marelja, Z., Leimkuhler, S. and Missirlis, F. (2018). Iron sulfur and molybdenum cofactor enzymes regulate the Drosophila life cycle by controlling cell metabolism. Front Physiol 9: 50. PubMed ID: 29491838
Summary:
Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, a hypothesis is formulated that (1) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (2) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (3) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (4) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.
Goda, T., Doi, M., Umezaki, Y., Murai, I., Shimatani, H., Chu, M. L., Nguyen, V. H., Okamura, H. and Hamada, F. N. (2018). Calcitonin receptors are ancient modulators for rhythms of preferential temperature in insects and body temperature in mammals. Genes Dev 32(2): 140-155. PubMed ID: 29440246
Summary:
Daily body temperature rhythm (BTR) is essential for maintaining homeostasis. BTR is regulated separately from locomotor activity rhythms, but its molecular basis is largely unknown. While mammals internally regulate BTR, ectotherms, including Drosophila, exhibit temperature preference rhythm (TPR) behavior to regulate BTR. This study demonstrates that the Diuretic hormone 31 receptor (DH31R) mediates TPR during the active phase in Drosophila. DH31R is expressed in clock cells, and its ligand, DH31, acts on clock cells to regulate TPR during the active phase. Surprisingly, the mouse homolog of DH31R, calcitonin receptor (Calcr), is expressed in the suprachiasmatic nucleus (SCN) and mediates body temperature fluctuations during the active phase in mice. Importantly, DH31R and Calcr are not required for coordinating locomotor activity rhythms. These results represent the first molecular evidence that BTR is regulated distinctly from locomotor activity rhythms and show that DH31R/Calcr is an ancient specific mediator of BTR during the active phase in organisms ranging from ectotherms to endotherms.
Damschroder, D., Reynolds, C. and Wessells, R. (2018). Drosophila tafazzin mutants have impaired exercise capacity. Physiol Rep 6(3). PubMed ID: 29405656
Summary:
Cardiolipin (CL; see CLS) is a mitochondrial phospholipid that helps maintain normal structure of the inner mitochondrial membrane and stabilize the protein complexes of the electron transport chain to promote efficient ATP synthesis. Tafazzin, an acyl-transferase, is required for synthesis of the mature form of CL. Mutations in the tafazzin (TAZ) gene are associated with a human disorder known as Barth syndrome. Symptoms of Barth syndrome often include muscle weakness and exercise intolerance. Previous work demonstrates that Drosophila Taz mutants exhibit motor weakness, as measured by reduced flying and climbing abilities. However, Drosophila TAZ mutants' baseline endurance or response to endurance exercise training has not been assessed. This study finds that TAZ mutants have reduced endurance and do not improve following a stereotypical exercise training paradigm, indicating that loss of TAZ function leads to exercise intolerance in Drosophila. Although cardiac phenotypes are observed in human Barth syndrome patients, TAZ mutants had normal resistance to cardiac pacing. In the future, endurance may be a useful screening tool to identify additional genetic modifiers of tafazzin.
Rhoades, S. D., Nayak, K., Zhang, S., Sehgal, A. and Weljie, A. M. (2018). Circadian- and light-driven metabolic rhythms in Drosophila melanogaster. J Biol Rhythms: 748730417753003. PubMed ID: 29355066
Summary:
Complex interactions of environmental cues and transcriptional clocks drive rhythmicity in organismal physiology. Light directly affects the circadian clock; however, little is known about its relative role in controlling metabolic variations in vivo. This study used high time-resolution sampling in Drosophila at every 2 h to measure metabolite outputs using a liquid-chromatography tandem mass spectrometry (LC-MS/MS) approach. Over 14% of detected metabolites oscillated with circadian periodicity under light-dark (LD) cycles. Many metabolites peaked shortly after lights-on, suggesting responsiveness to feeding and/or activity rather than the preactivity anticipation, as observed in previous transcriptomics analyses. Roughly 9% of measured metabolites uniquely oscillated under constant darkness (DD), suggesting that metabolite rhythms are associated with the transcriptional clock machinery. Strikingly, metabolome differences between LD and constant darkness were observed only during the light phase, highlighting the importance of photic input. Clock mutant flies exhibited strong 12-h ultradian rhythms, including 4 carbohydrate species with circadian periods in wild-type flies, but lacked 24-h circadian metabolic oscillations. A meta-analysis of these results with previous circadian metabolomics experiments uncovered the possibility of conserved rhythms in amino acids, keto-acids, and sugars across flies, mice, and humans and provides a basis for exploring the chrono-metabolic connection with powerful genetic tools in Drosophila.
Ilic, M., Meglic, A., Kreft, M. and Belusic, G. (2018). The fly sensitizing pigment enhances UV spectral sensitivity while preventing polarization-induced artifacts. Front Cell Neurosci 12: 34. PubMed ID: 29467626
Summary:
Microvillar photoreceptors are intrinsically capable of detecting the orientation of e-vector of linearly polarized light. They provide most invertebrates with an additional sensory channel to detect important features of their visual environment. However, polarization sensitivity (PS) of photoreceptors may lead to the detection of polarization-induced false colors and intensity contrasts. Most insect photoreceptors are thus adapted to have minimal PS. Flies have twisted rhabdomeres with microvilli rotated along the length of the ommatidia to reduce PS. The additional UV-absorbing sensitizing pigment on their opsin minimizes PS in the ultraviolet. This study recorded voltage from Drosophila photoreceptors R1-6 to measure the spectral dependence of PS and found that PS in the UV is invariably negligible but can be substantial above 400 nm. Using modeling, it was demonstrated that in R1-6 without the sensitizing pigment, PS in the UV (PS UV) would exceed PS in the visible part of the spectrum (PS VIS) by a factor PS UV /PS VIS = 1.2-1.8, as lower absorption of Rh1 rhodopsin reduces self-screening. Polarimetric imaging of objects relevant to fly polarization vision was used to show that their degree of polarization outdoors is highest in the short-wavelength part of the spectrum. Thus, under natural illumination, the sensitizing pigment in R1-6 renders even those cells with high PS in the visible part unsuitable for proper polarization vision. It is assumed that fly ventral polarization vision can be mediated by R7 alone, with R1-6 serving as an unpolarized reference channel.

Monday, April 9th

Golenkina, S., Chaturvedi, V., Saint, R. and Murray, M. J. (2018). Frazzled can act through distinct molecular pathways in epithelial cells to regulate motility, apical constriction, and localisation of E-Cadherin. PLoS One 13(3): e0194003. PubMed ID: 29518139
Summary:
Netrin receptors of the DCC/NEO/UNC-40/Frazzled family have well established roles in cell migration and axon guidance but can also regulate epithelial features such as adhesion, polarity and adherens junction (AJ) stability. Previous work has shown that overexpression of Drosophila Frazzled (Fra) in the peripodial epithelium (PE) inhibits wing disc eversion and also generates cellular protrusions typical of motile cells. This study tested whether the molecular pathways by which Fra inhibits eversion are distinct from those driving motility. In disc proper (DP) it was shown that in epithelial cells Fra, in addition to inducing F-Actin rich protrusions, can affect localization of AJ components and columnar cell shape. It was then shown that these phenotypes have different requirements for the three conserved Fra cytoplasmic P-motifs and for downstream genes. The formation of protrusions required the P3 motif of Fra, as well as integrins (mys and mew), the Rac pathway (Rac1, wave and, arpc3) and myosin regulatory light chain (Sqh). In contrast, apico-basal cell shape change, which was accompanied by increased myosin phosphorylation, was critically dependent upon the P1 motif and was promoted by RhoGef2 but inhibited by Rac1. Fra also caused a loss of AJ proteins (DE-Cad and Arm) from basolateral regions of epithelial cells. This phenotype required all 3 P-motifs, and was dependent upon the polarity factor par6. par6 was not required for protrusions or cell shape change, but was required to block eversion suggesting that control of AJ components may underlie the ability of Fra to promote epithelial stability. The results imply that multiple molecular pathways act downstream of Fra in epithelial cells.
Daffern, N., Chen, Z., Zhang, Y., Pick, L. and Radhakrishnan, I. (2018). Solution NMR studies of the ligand-binding domain of an orphan nuclear receptor reveals a dynamic helix in the ligand-binding pocket. Biochemistry. PubMed ID: 29547262
Summary:
The ligand-binding domains (LBD) of the NR5A subfamily of nuclear receptors activate transcription via ligand-dependent and ligand-independent mechanisms. The Drosophila Ftz-F1 receptor (NR5A3) belongs to the latter category and its ligand-independence is attributed to a short helical segment (6) within the protein that resides in the canonical ligand-binding pocket (LBP) in the crystalline state. This study shows that the 6 helix is dynamic in solution when Ftz-F1 is bound to the LxxLL motif of its cofactor Ftz, undergoing motions on the fast (picosecond-nanosecond) as well as slow (microsecond-millisecond) timescales. Motions on the slow timescale (ca. 10-3 s) appear to pervade through the domain, most prominently in the LBP and residues at or near the cofactor binding site. The fast timescale motions are ascribed to a solvent-accessible conformation for the 6 helix akin to those described for its orthologs in higher organisms. This conformation is assigned where the LBP is 'open' to a lowly-populated species while the major conformer bears the properties of the crystal structure where the LBP is 'closed'. It is proposed that these conformational transitions signal binding to small molecule ligands and/or play a role in cofactor dissociation from the binding site. Indeed, Ftz-F1 LBD can bind phospholipids, not unlike its orthologs. These studies provide the first detailed insights into intrinsic motions occurring on a variety of timescales in a nuclear receptor LBD and reveal that potentially functionally significant motions could pervade the domain in solution, despite evidence to the contrary implied by the crystal structure.
Fletcher, G. C., Diaz-de-la-Loza, M. D., Borreguero-Munoz, N., Holder, M., Aguilar-Aragon, M. and Thompson, B. J. (2018). Mechanical strain regulates the Hippo pathway in Drosophila. Development 145(5). PubMed ID: 29440303
Summary:
Animal cells are thought to sense mechanical forces via the transcriptional co-activators YAP (or YAP1) and TAZ (or WWTR1), the sole Drosophila homolog of which is named Yorkie (Yki). In mammalian cells in culture, artificial mechanical forces induce nuclear translocation of YAP and TAZ. This study shows that physiological mechanical strain can also drive nuclear localisation of Yki and activation of Yki target genes in the Drosophila follicular epithelium. Mechanical strain activates Yki by stretching the apical domain, reducing the concentration of apical Crumbs, Expanded, Kibra and Merlin, and reducing apical Hippo kinase dimerisation. Overexpressing Hippo kinase to induce ectopic activation in the cytoplasm is sufficient to prevent Yki nuclear localisation even in flattened follicle cells. Conversely, blocking Hippo signalling in warts clones causes Yki nuclear localisation even in columnar follicle cells. No evidence was found for involvement of other pathways, such as Src42A kinase, in regulation of Yki. Finally, the results in follicle cells appear generally applicable to other tissues, as nuclear translocation of Yki is also readily detectable in other flattened epithelial cells such as the peripodial epithelium of the wing imaginal disc, where it promotes cell flattening.
Donohoe, C. D., Csordas, G., Correia, A., Jindra, M., Klein, C., Habermann, B. and Uhlirova, M. (2018). Atf3 links loss of epithelial polarity to defects in cell differentiation and cytoarchitecture. PLoS Genet 14(3): e1007241. PubMed ID: 29494583
Summary:
Interplay between apicobasal cell polarity modules and the cytoskeleton is critical for differentiation and integrity of epithelia. However, this coordination is poorly understood at the level of gene regulation by transcription factors. This study establish the Drosophila activating transcription factor 3 (atf3) as a cell polarity response gene acting downstream of the membrane-associated Scribble polarity complex. Loss of the tumor suppressors Scribble or Dlg1 induces atf3 expression via aPKC but independent of Jun-N-terminal kinase (JNK) signaling. Strikingly, removal of Atf3 from Dlg1 deficient cells restores polarized cytoarchitecture, levels and distribution of endosomal trafficking machinery, and differentiation. Conversely, excess Atf3 alters microtubule network, vesicular trafficking and the partition of polarity proteins along the apicobasal axis. Genomic and genetic approaches implicate Atf3 as a regulator of cytoskeleton organization and function, and identify Lamin C as one of its bona fide target genes. By affecting structural features and cell morphology, Atf3 functions in a manner distinct from other transcription factors operating downstream of disrupted cell polarity.
Henstridge, M. A., Aulsebrook, L., Koyama, T., Johnson, T. K., Whisstock, J. C., Tiganis, T., Mirth, C. K. and Warr, C. G. (2018). Torso-like is a component of the hemolymph and regulates the insulin signalling pathway in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 29440191
Summary:
In Drosophila key developmental transitions are governed by the steroid hormone ecdysone. A number of neuropeptide-activated signalling pathways control ecdysone production in response to environmental signals, including the insulin signalling pathway, which regulates ecdysone production in response to nutrition. This study found that the Membrane Attack Complex/Perforin-like protein Torso-like, best characterised for its role in activating the Torso receptor tyrosine kinase in early embryo patterning, also regulates the insulin signalling pathway in Drosophila. It has been previously reported that the small body size and developmental delay phenotypes of torso-like null mutants resemble those observed when insulin signalling is reduced. This study reports that, in addition to growth defects, torso-like mutants also display metabolic and nutritional plasticity phenotypes characteristic of mutants with impaired insulin signalling. It was further found that in the absence of torso-like the expression of insulin-like peptides is increased, as is their accumulation in the insulin-producing cells. Finally, Torso-like was shown to be a component of the hemolymph and that it is required in the prothoracic gland to control developmental timing and body size. Taken together, these data suggest that the secretion of Torso-like from the prothoracic gland influences the activity of insulin signalling throughout the body in Drosophila.
Kim, J., Bilder, D. and Neufeld, T. P. (2018). Mechanical stress regulates insulin sensitivity through integrin-dependent control of insulin receptor localization. Genes Dev 32(2): 156-164. PubMed ID: 29440263
Summary:
Insulin resistance, the failure to activate insulin signaling in the presence of ligand, leads to metabolic diseases, including type 2 diabetes. Physical activity and mechanical stress have been shown to protect against insulin resistance, but the molecular mechanisms remain unclear. This study addresses this relationship in the Drosophila larval fat body, an insulin-sensitive organ analogous to vertebrate adipose tissue and livers. Insulin signaling in Drosophila fat body cells was abolished in the absence of physical activity and mechanical stress even when excess insulin is present. Physical movement is required for insulin sensitivity in both intact larvae and fat bodies cultured ex vivo. Interestingly, the insulin receptor and other downstream components are recruited to the plasma membrane in response to mechanical stress, and this membrane localization is rapidly lost upon disruption of larval or tissue movement. Sensing of mechanical stimuli is mediated in part by integrins, whose activation is necessary and sufficient for mechanical stress-dependent insulin signaling. Insulin resistance develops naturally during the transition from the active larval stage to the immotile pupal stage, suggesting that regulation of insulin sensitivity by mechanical stress may help coordinate developmental programming with metabolism.

Friday, April 6th

Tas, D., Stickley, L., Miozzo, F., Koch, R., Loncle, N., Sabado, V., Gnagi, B. and Nagoshi, E. (2018). Parallel roles of transcription factors dFOXO and FER2 in the development and maintenance of dopaminergic neurons. PLoS Genet 14(3): e1007271. PubMed ID: 29529025
Summary:
Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson's disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. This study investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD.
Iliadi, K. G., Gluscencova, O. B., Iliadi, N. and Boulianne, G. L. (2018). Mutations in the Drosophila homolog of human PLA2G6 give rise to age-dependent loss of psychomotor activity and neurodegeneration. Sci Rep 8(1): 2939. PubMed ID: 29440694
Summary:
Infantile neuroaxonal dystrophy (INAD) is a fatal neurodegenerative disorder that typically begins within the first few years of life and leads to progressive impairment of movement and cognition. Several years ago, it was shown that >80% of patients with INAD have mutations in the phospholipase gene, PLA2G6. Interestingly, mutations in PLA2G6 are also causative in two other related neurodegenerative diseases, atypical neuroaxonal dystrophy and Dystonia-parkinsonism. While all three disorders give rise to similar defects in movement and cognition, some defects are unique to a specific disorder. At present, the cellular mechanisms underlying PLA2G6-associated neuropathology are poorly understood and there is no cure or treatment that can delay disease progression. This study shows that loss of iPLA2-VIA, the Drosophila homolog of PLA2G6, gives rise to age-dependent defects in climbing and spontaneous locomotion. Moreover, using a newly developed assay, this study shows that iPLA2-VIA mutants also display impairments in fine-tune motor movements, motor coordination and psychomotor learning, which are distinct features of PLA2G6-associated disease in humans. Finally, iPLA2-VIA mutants were shown to exhibit increased sensitivity to oxidative stress, progressive neurodegeneration and a severely reduced lifespan. Altogether, these data demonstrate that Drosophila iPLA2-VIA mutants provide a useful model to study human PLA2G6-associated neurodegeneration.
Jeong, Y., Kim, T., Kim, S., Hong, Y. K., Cho, K. S. and Lee, I. S. (2018). Overexpression of histone methyltransferase NSD in Drosophila induces apoptotic cell death via the Jun-N-terminal kinase pathway. Biochem Biophys Res Commun 496(4): 1134-1140. PubMed ID: 29410178
Summary:
The nuclear receptor-binding SET domain protein gene (NSD) family encodes a group of highly conserved SET domain-containing histone lysine methyltransferases that are important in multiple aspects of development in various organisms. The association of NSD1 duplications has been reported with growth retardation diseases in humans. To gain insight into the molecular mechanisms by which the overexpression of NSD1 influences the disease progression, this study examined the gain-of-function mutant phenotypes of the Drosophila NSD using the GAL4/UAS system. Ubiquitous overexpression of NSD in the fly caused developmental delay and reduced body size at the larval stage, resulting in pupal lethality. Moreover, targeted overexpression in various developing tissues led to significant phenotype alterations, and the gain-of-function phenotypes were rescued by NSD RNAi knockdown. NSD overexpression not only enhanced the transcription of pro-apoptotic genes but also activated caspase. The atrophied phenotype of NSD-overexpressing wing was strongly suppressed by a loss-of-function mutation in hemipterous, which encodes a Drosophila Jun N-terminal kinase. Taken together, these findings suggest that NSD induces apoptosis via the activation of JNK, and thus contributes to the understanding of the molecular mechanisms involved in NSD1-related diseases in humans.
Stephano, F., Nolte, S., Hoffmann, J., El-Kholy, S., von Frieling, J., Bruchhaus, I., Fink, C. and Roeder, T. (2018). Impaired Wnt signaling in dopamine containing neurons is associated with pathogenesis in a rotenone triggered Drosophila Parkinson's disease model. Sci Rep 8(1): 2372. PubMed ID: 29403026
Summary:
Parkinson's disease, which is the one of the most common neurodegenerative movement disorder, is characterized by a progressive loss of dopamine containing neurons. The mechanisms underlying disease initiation and development are not well understood and causative therapies are currently not available. A Drosophila model was used to elucidate the molecular processes during early stages of Parkinson's disease. To induce Parkinson's disease-like phenotypes, flies were treated with the pesticide rotenone, and dopamine producing neurons of animals were isolated that were at an early disease stage. Transcriptomic analyses revealed that gene ontologies associated with regulation of cell death and neuronal functions were significantly enriched. Moreover, the activities of the MAPK/EGFR- and TGF-β signaling pathways were enhanced, while the Wnt pathway was dampened. In order to evaluate the role of Wnt signaling for survival of dopaminergic neurons in the disease model, the reduced Wnt signaling activity was rescued by ectopic overexpression of armadillo/β-catenin. This intervention rescued the rotenone induced movement impairments in the Drosophila model. Taken together, this initial study showed a highly relevant role of Wnt signaling for dopamine producing neurons during pathogenesis in Parkinson's disease and it implies that interfering with this pathway might by a suitable therapeutic option for the future.
Loewen, C. A. and Ganetzky, B. (2018). Mito-nuclear interactions affecting lifespan and neurodegeneration in a Drosophila model of Leigh Syndrome. Genetics 208(4):1535-1552. PubMed ID: 29496745
Summary:
Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. This study describes the characterization of a mutation in Drosophila ND23, a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh Syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23. A key feature of Leigh Syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. It was discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23, including a variant affecting a mitochondrially-encoded component of complex I. Thus, this work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease.
Sowa, A. S., Martin, E., Martins, I. M., Schmidt, J., Depping, R., Weber, J. J., Rother, F., Hartmann, E., Bader, M., Riess, O., Tricoire, H. and Schmidt, T. (2018). Karyopherin alpha-3 is a key protein in the pathogenesis of spinocerebellar ataxia type 3 controlling the nuclear localization of ataxin-3. Proc Natl Acad Sci U S A 115(11): E2624-e2633. PubMed ID: 29476013
Summary:
Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene leading to a polyglutamine expansion in the ataxin-3 protein. The nuclear presence and aggregation of expanded ataxin-3 are critical steps in disease pathogenesis. To identify novel therapeutic targets, this study investigated the nucleocytoplasmic transport system by screening a collection of importins and exportins that potentially modulate this nuclear localization. Using cell, Drosophila, and mouse models, focus was placed on three transport proteins, namely, CRM1, IPO13, KPNA3, and their respective Drosophila orthologs Emb, Cdm, and Kap-alpha3. While overexpression of CRM1/Emb demonstrated positive effects in Drosophila, KPNA3/Kap-alpha3 emerged as the most promising target, as knockdown via multiple RNAi lines demonstrated its ability to shuttle both truncated and full-length expanded ataxin-3, rescue neurodegeneration, restore photoreceptor formation, and reduce aggregation. Furthermore, KPNA3 knockout in SCA3 mice resulted in an amelioration of molecular and behavioral disturbances such as total activity, anxiety, and gait. Since KPNA3 is known to function as an import protein and recognize nuclear localization signals (NLSs), this work unites ataxin-3 structure to the nuclear pore machinery and provides a link between karyopherins, NLS signals, and polyglutamine disease, as well as demonstrates that KPNA3 is a key player in the pathogenesis of SCA3.

Thursday, April 5th

Gowda, S. B. M., Paranjpe, P. D., Reddy, O. V., Thiagarajan, D., Palliyil, S., Reichert, H. and VijayRaghavan, K. (2018). GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila. Proc Natl Acad Sci U S A 115(9): E2115-e2124. PubMed ID: 29440493
Summary:
Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. This study took advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, an algorithm was developed for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, genetic reagents were used for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila. The findings indicate that targeted down-regulation of the GABAA receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila.
Grice, S. J., Sleigh, J. N. and Zameel Cader, M. (2018). Plexin-semaphorin signaling modifies neuromuscular defects in a Drosophila model of peripheral neuropathy. Front Mol Neurosci 11: 55. PubMed ID: 29520219
Summary:
Dominant mutations in GARS, encoding the ubiquitous enzyme glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and Charcot-Marie-Tooth disease type 2D (CMT2D). This genetic disorder exemplifies a recurring paradigm in neurodegeneration, in which mutations in essential genes cause selective degeneration of the nervous system. Recent evidence suggests that the mechanism underlying CMT2D involves extracellular neomorphic binding of mutant GlyRS to neuronally-expressed proteins. Consistent with this, previous studies indicate a non-cell autonomous mechanism, whereby mutant GlyRS is secreted and interacts with the neuromuscular junction (NMJ). In this Drosophila model for CMT2D, it was previously shown that mutant gars expression decreases viability and larval motor function, and causes a concurrent build-up of mutant GlyRS at the larval neuromuscular presynapse. This study reports additional phenotypes that closely mimic the axonal branching defects of Drosophila plexin transmembrane receptor mutants, implying interference of plexin signaling in gars mutants. Individual dosage reduction of two Drosophila Plexins, plexin A (plexA) and B (plexB) enhances and represses the viability and larval motor defects caused by mutant GlyRS, respectively. However, plexB levels, but not plexA levels, modify mutant GlyRS association with the presynaptic membrane. Furthermore, increasing availability of the plexB ligand, Semaphorin-2a (Sema2a), alleviates the pathology and the build-up of mutant GlyRS, suggesting competition for PlexB binding may be occurring between these two ligands. This toxic gain-of-function and subversion of neurodevelopmental processes indicate that signaling pathways governing axonal guidance could be integral to neuropathology and may underlie the non-cell autonomous CMT2D mechanism.
Cassar, M., Sunderhaus, E., Wentzell, J. S., Kuntz, S., Strauss, R. and Kretzschmar, D. (2018). The PKA-C3 catalytic subunit is required in two pairs of interneurons for successful mating of Drosophila. Sci Rep 8(1): 2458. PubMed ID: 29410515
Summary:
Protein kinase A (PKA) has been shown to play a role in a plethora of cellular processes ranging from development to memory formation. Its activity is mediated by the catalytic subunits whereby many species express several paralogs. Drosophila encodes three catalytic subunits (PKA-C1-3) and whereas PKA-C1 has been well studied, the functions of the other two subunits were unknown. PKA-C3 is the orthologue of mammalian PRKX/Pkare and they are structurally more closely related to each other than to other catalytic subunits within their species. PRKX is expressed in the nervous system in mice but its function is also unknown. This study now shows that the loss of PKA-C3 in Drosophila causes copulation defects, though the flies are active and show no defects in other courtship behaviours. This phenotype is specifically due to the loss of PKA-C3 because PKA-C1 cannot replace PKA-C3. PKA-C3 is expressed in two pairs of interneurons that send projections to the ventro-lateral protocerebrum and the mushroom bodies and that synapse onto motor neurons in the ventral nerve cord. Rescue experiments show that expression of PKA-C3 in these interneurons is sufficient for copulation, suggesting a role in relaying information from the sensory system to motor neurons to initiate copulation.
Cohen, D., van Swinderen, B. and Tsuchiya, N. (2018). Isoflurane impairs low-frequency feedback but leaves high-frequency feedforward connectivity intact in the fly brain. eNeuro 5(1). PubMed ID: 29541686
Summary:
Hierarchically organized brains communicate through feedforward (FF) and feedback (FB) pathways. In mammals, FF and FB are mediated by higher and lower frequencies during wakefulness. FB is preferentially impaired by general anesthetics in multiple mammalian species. This suggests FB serves critical functions in waking brains. The brain of Drosophila melanogaster is also hierarchically organized, but the presence of FB in these brains is not established. This study examined FB in the fly brain, by simultaneously recording local field potentials (LFPs) from low-order peripheral structures and higher-order central structures. The data was analyzed using Granger causality (GC), the first application of this analysis technique to recordings from the insect brain. The analysis revealed that low frequencies (0.1-5 Hz) mediated FB from the center to the periphery, while higher frequencies (10-45 Hz) mediated FF in the opposite direction. Further, isoflurane anesthesia preferentially reduced FB. The results imply that the spectral characteristics of FF and FB may be a signature of hierarchically organized brains that is conserved from insects to mammals. It is speculated that general anesthetics may induce unresponsiveness across species by targeting the mechanisms that support FB.
Enriquez, J., Rio, L. Q., Blazeski, R., Bellemin, S., Godement, P., Mason, C. and Mann, R. S. (2018). Differing strategies despite shared lineages of motor neurons and glia to achieve robust development of an adult neuropil in Drosophila. Neuron 97(3): 538-554.e535. PubMed ID: 29395908
Summary:
In vertebrates and invertebrates, neurons and glia are generated in a stereotyped manner from neural stem cells, but the purpose of invariant lineages is not understood. This study shows that two stem cells that produce leg motor neurons in Drosophila also generate neuropil glia, which wrap and send processes into the neuropil where motor neuron dendrites arborize. The development of the neuropil glia and leg motor neurons is highly coordinated. However, although motor neurons have a stereotyped birth order and transcription factor code, the number and individual morphologies of the glia born from these lineages are highly plastic, yet the final structure they contribute to is highly stereotyped. It is suggested that the shared lineages of these two cell types facilitate the assembly of complex neural circuits and that the two birth order strategies-hardwired for motor neurons and flexible for glia-are important for robust nervous system development, homeostasis, and evolution.
Jang, W., Baek, M., Han, Y. S. and Kim, C. (2018). Duox mediates ultraviolet injury-induced nociceptive sensitization in Drosophila larvae. Mol Brain 11(1): 16. PubMed ID: 29540218
Summary:
Nociceptive sensitization is an increase in pain perception in response to stimulus. Following brief irradiation of Drosophila larvae with UV, nociceptive sensitization occurs in class IV multiple dendritic (mdIV) neurons, which are polymodal sensory nociceptors. Diverse signaling pathways have been identified that mediate nociceptive sensitization in mdIV neurons, including TNF, Hedgehog, BMP, and Tachykinin, yet the underlying mechanisms are not completely understood. This study reports that duox heterozygous mutant larvae, which have normal basal nociception, exhibit an attenuated hypersensitivity response to heat and mechanical force following UV irradiation. Employing the ppk-Gal4 line, which is exclusively expressed in mdIV neurons, this study further shows that silencing duox in mdIV neurons attenuates UV-induced sensitization. These findings reveal a novel role for duox in nociceptive sensitization of Drosophila larvae, and will enhance understanding of the mechanisms underlying this process in Drosophila sensory neurons.

Wednesday, April 4th

Spinner, M. A., Walla, D. A. and Herman, T. G. (2018). Drosophila Syd-1 has RhoGAP activity that is required for presynaptic clustering of Bruchpilot/ELKS but not Neurexin-1. Genetics 208(2): 705-716. PubMed ID: 29217522
Summary:
Syd-1 proteins are required for presynaptic development in worm, fly, and mouse. Syd-1 proteins in all three species contain a Rho GTPase activating protein (GAP)-like domain of unclear significance: invertebrate Syd-1s are thought to lack GAP activity, and mouse mSYD1A has GAP activity that is thought to be dispensable for its function. This study shows that Drosophila melanogaster Syd-1 can interact with all six fly Rhos and has GAP activity toward Rac1 and Cdc42. During development, fly Syd-1 clusters multiple presynaptic proteins at the neuromuscular junction (NMJ), including the cell adhesion molecule Neurexin (Nrx-1) and the active zone (AZ) component Bruchpilot (Brp), both of which Syd-1 binds directly. A mutant form of Syd-1 that specifically lacks GAP activity localizes normally to presynaptic sites and is sufficient to recruit Nrx-1 but fails to cluster Brp normally. Evidence is provided that Syd-1 participates with Rac1 in two separate functions: (1) together with the Rac guanine exchange factor (RacGEF) Trio, GAP-active Syd-1 is required to regulate the nucleotide-bound state of Rac1, thereby promoting Brp clustering; and (2) Syd-1, independent of its GAP activity, is required for the recruitment of Nrx-1 to boutons, including the recruitment of Nrx-1 that is promoted by GTP-bound Rac1. It is concluded that, contrary to current models, the GAP domain of fly Syd-1 is active and required for presynaptic development; it is suggested that the same may be true of vertebrate Syd-1 proteins. In addition, the data provide new molecular insight into the ability of Rac1 to promote presynaptic development.
Itoh, K., Akimoto, Y., Kondo, S., Ichimiya, T., Aoki, K., Tiemeyer, M. and Nishihara, S. (2018). Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles. Dev Biol [Epub ahead of print]. PubMed ID: 29499182
Summary:
T antigen (Galβ1-3GalNAcalpha1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcalpha1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. This study has unraveled biological roles of glucuronylated T antigen. The data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). dGlcAT-P null mutants were created, and it was found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. It was also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, a genetic interaction was shown between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles.
Dung, V. M., Suong, D. N. A., Okamaoto, Y., Hiramatsu, Y., Thao, D. T. P., Yoshida, H., Takashima, H. and Yamaguchi, M. (2018). Neuron-specific knockdown of Drosophila PDHB induces reduction of lifespan, deficient locomotive ability, abnormal morphology of motor neuron terminals and photoreceptor axon targeting. Exp Cell Res. PubMed ID: 29501567
Summary:
Pyruvate dehydrogenase complex deficiency (PDCD) is a common primary cause of defects in mitochondrial function and also can lead to peripheral neuropathy. Pyruvate dehydrogenase E1 component subunit β (PDHB) is a subunit of pyruvate dehydrogenase E1, which is a well-known component of PDC. In Drosophila melanogaster, the CG11876 (dPDHB) gene is a homolog of human PDHB. This study established a Drosophila model with neuron-specific knockdown of dPDHB to investigate its role in neuropathy pathogenesis. Knockdown of dPDHB in pan-neurons induced locomotor defects in both larval and adult stages, which was consistent with abnormal morphology of the motor neuron terminals at neuromuscular junctions and mitochondrial fragmentation in brains. Moreover, neuron-specific knockdown of dPDHB also shortened the lifespan of adult flies. In addition, flies with knockdown of dPDHB manifested a rough eye phenotype and aberrant photoreceptor axon targeting. These results with the Drosophila model suggest the involvement of PDHB in peripheral neuropathy.
Azpurua, J., Mahoney, R. E. and Eaton, B. A. (2018). Transcriptomics of aged Drosophila motor neurons reveals a matrix metalloproteinase that impairs motor function. Aging Cell 17(2). PubMed ID: 29411505
Summary:
The neuromuscular junction (NMJ) is responsible for transforming nervous system signals into motor behavior and locomotion. In the fruit fly Drosophila melanogaster, an age-dependent decline in motor function occurs, analogous to the decline experienced in mice, humans, and other mammals. By specifically profiling the transcriptome of Drosophila motor neurons across age using custom microarrays, this study found that the expression of the matrix metalloproteinase 1 (dMMP1) gene reproducibly increased in motor neurons in an age-dependent manner. Modulation of physiological aging also altered the rate of dMMP1 expression, validating dMMP1 expression as a bona fide aging biomarker for motor neurons. Temporally controlled overexpression of dMMP1 specifically in motor neurons was sufficient to induce deficits in climbing behavior and cause a decrease in neurotransmitter release at neuromuscular synapses. These deficits were reversible if the dMMP1 expression was shut off again immediately after the onset of motor dysfunction. Additionally, repression of dMMP1 enzymatic activity via overexpression of a tissue inhibitor of metalloproteinases delayed the onset of age-dependent motor dysfunction. MMPs are required for proper tissue architecture during development. These results support the idea that matrix metalloproteinase 1 is acting as a downstream effector of antagonistic pleiotropy in motor neurons and is necessary for proper development, but deleterious when reactivated at an advanced age.
Constance, W. D., Mukherjee, A., Fisher, Y. E., Pop, S., Blanc, E., Toyama, Y. and Williams, D. W. (2018). Neurexin and Neuroligin-based adhesion complexes drive axonal arborisation growth independent of synaptic activity. Elife 7. PubMed ID: 29504935
Summary:
Building arborisations of the right size and shape is fundamental for neural network function. Live imaging in vertebrate brains strongly suggests that nascent synapses are critical for branch growth during development. The molecular mechanisms underlying this are largely unknown. This study presents a novel system in Drosophila for studying the development of complex arborisations live, in vivo during metamorphosis. In growing arborisations branch dynamics and localisations of presynaptic proteins are seen, very similar to the 'synaptotropic growth' described in fish/frogs. These accumulations of presynaptic proteins do not appear to be presynaptic release sites and are not paired with neurotransmitter receptors. Knockdowns of either evoked or spontaneous neurotransmission do not impact arbor growth. Instead, axonal branch growth was found to be regulated by dynamic, focal localisations of Neurexin and Neuroligin. These adhesion complexes provide stability for filopodia by a 'stick-and-grow' based mechanism wholly independent of synaptic activity.
Migh, E., Gotz, T., Foldi, I., Szikora, S., Gombos, R., Darula, Z., Medzihradszky, K. F., Maleth, J., Hegyi, P., Sigrist, S. and Mihaly, J. (2018). Microtubule organization in presynaptic boutons relies on the formin DAAM. Development 145(6). PubMed ID: 29487108
Summary:
Regulation of the cytoskeleton is fundamental to the development and function of synaptic terminals, such as neuromuscular junctions. Despite the identification of numerous proteins that regulate synaptic actin and microtubule dynamics, the mechanisms of cytoskeletal control during terminal arbor formation have remained largely elusive. This study shows that DAAM, a member of the formin family of cytoskeleton organizing factors, is an important presynaptic regulator of neuromuscular junction development in Drosophila. The actin filament assembly activity of DAAM plays a negligible role in terminal formation; rather, DAAM is necessary for synaptic microtubule organization. Genetic interaction studies consistently link DAAM with the Wg/Ank2/Futsch module of microtubule regulation and bouton formation. Finally, evidence is provided that DAAM is tightly associated with the synaptic active zone scaffold, and electrophysiological data point to a role in the modulation of synaptic vesicle release. Based on these results, it is proposed that DAAM is an important cytoskeletal effector element of the Wg/Ank2 pathway involved in the determination of basic synaptic structures, and, additionally, that DAAM may couple the active zone scaffold to the presynaptic cytoskeleton.

Tuesday, April 3rd

Guo, J., Tang, H. W., Li, J., Perrimon, N. and Yan, D. (2018). Xio is a component of the Drosophila sex determination pathway and RNA N(6)-methyladenosine methyltransferase complex. Proc Natl Acad Sci U S A. PubMed ID: 29555755
Summary:
N(6)-methyladenosine (m(6)A), the most abundant chemical modification in eukaryotic mRNA, has been implicated in Drosophila sex determination by modifying Sex-lethal (Sxl) pre-mRNA and facilitating its alternative splicing. This study identified a sex determination gene, CG7358, and renamed it xio according to its loss-of-function female-to-male transformation phenotype. xio encodes a conserved ubiquitous nuclear protein of unknown function. Xio was shown to colocalize and interacts with all previously known m(6)A writer complex subunits (METTL3, METTL14, Fl(2)d/WTAP, Vir/KIAA1429, and Nito/Rbm15) and that loss of xio is associated with phenotypes that resemble other m(6)A factors, such as sexual transformations, Sxl splicing defect, held-out wings, flightless flies, and reduction of m(6)A levels. Thus, Xio encodes a member of the m(6)A methyltransferase complex involved in mRNA modification. Since its ortholog ZC3H13 (or KIAA0853) also associates with several m(6)A writer factors, the function of Xio in the m(6)A pathway is likely evolutionarily conserved.
Duan, H., de Navas, L. F., Hu, F., Sun, K., Mavromatakis, Y. E., Viets, K., Zhou, C., Kavaler, J., Johnston, R. J., Jr., Tomlinson, A. and Lai, E. C. (2018). The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye. Development. PubMed ID: 29540498
Summary:
Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling, mediated by the Epidermal Growth Factor receptor (EGFR) and Sevenless receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that normal Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of mir-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. mir-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently de-represses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev, which otherwise completely lack this cell fate. Altogether, this study reveals a rare setting of developmental cell specification that substantially requires miRNA control.
Zhu, L., Kandasamy, S. K. and Fukunaga, R. (2018). Dicer partner protein tunes the length of miRNAs using base-mismatch in the pre-miRNA stem. Nucleic Acids Res. PubMed ID: 29373753
Summary:
Dicer partner proteins Drosophila Loquacious-PB (Loqs-PB) and human TRBP tune the length of miRNAs produced by Dicer from a subset of pre-miRNAs and thereby alter their target repertoire, by an unknown mechanism. This study developed a novel high-throughput method that was named Dram-seq (Dice randomized pre-miRNA pool and seq) to study length distributions of miRNAs produced from thousands of different pre-miRNA variants. Using Dram-seq, it was found that a base-mismatch in the pre-miRNA stem can alter the length of miRNAs compared with a base-pair at the same position in both Drosophila and human, and is important for the miRNA length tuning by Loqs-PB. Loqs-PB directly bound base-mismatched nucleotides in the pre-miRNA stem. It is speculated that Loqs-PB tunes miRNA length by changing the conformation of base-mismatched nucleotides in the pre-miRNA stem to that of base-paired ones and thereby altering the distance of the pre-miRNA stem.
Hara, M., Lourido, S., Petrova, B., Lou, H. J., Von Stetina, J. R., Kashevsky, H., Turk, B. E. and Orr-Weaver, T. L. (2018). Identification of PNG kinase substrates uncovers interactions with the translational repressor TRAL in the oocyte-to-embryo transition. Elife 7. PubMed ID: 29480805
Summary:
The Drosophila Pan Gu (PNG) kinase complex regulates hundreds of maternal mRNAs that become translationally repressed or activated as the oocyte transitions to an embryo. A previous paper (Hara, 2017), demonstrated PNG activity is under tight developmental control and restricted to this transition. This study's examination of PNG specificity showed it to be a Thr-kinase yet lacking a clear phosphorylation site consensus sequence. An unbiased biochemical screen for PNG substrates identified the conserved translational repressor Trailer Hitch (TRAL). Phosphomimetic mutation of the PNG phospho-sites in TRAL reduced its ability to inhibit translation in vitro. In vivo, mutation of tral dominantly suppressed png mutants and restored Cyclin B protein levels. The repressor Pumilio (PUM) has the same relationship with PNG, and PUM was shown to be a PNG substrate. Furthermore, PNG can phosphorylate BICC and ME31B, repressors that bind TRAL in cytoplasmic RNPs. Therefore, PNG likely promotes translation at the oocyte-to-embryo transition by phosphorylating and inactivating translational repressors.
Funakoshi, M., Tsuda, M., Muramatsu, K., Hatsuda, H., Morishita, S. and Aigaki, T. (2018). Overexpression of Larp4B downregulates dMyc and reduces cell and organ sizes in Drosophila. Biochem Biophys Res Commun 497(2): 762-768. PubMed ID: 29462618
Summary:
Regulation of cell and organ sizes is fundamental for all organisms, but its molecular basis is not fully understood. A gain-of-function screen was performed, and larp4B was identified whose overexpression reduces cell and organ sizes in Drosophila melanogaster. Larp4B is a member of La-related proteins (LARPs) containing an LA motif and an adjacent RNA recognition motif (RRM), and play diverse roles in RNA metabolism. However, the function of Larp4B has remained poorly characterized. Transgenic flies were generated overexpressing wild-type mammalian Larp4B or a deletion variant lacking the LA and RRM domains, and it was demonstrated that the RNA-binding domains are essential for Larp4B to reduce cell and organ sizes. The larp4B-induced phenotype was suppressed by dMyc overexpression, which promotes cell growth and survival. Furthermore, overexpression of larp4B decreased dMyc protein levels, whereas its loss-of-function mutation had an opposite effect. These results suggest that Larp4B is a negative regulator of dMyc.
Fukunaga, R. (2018). Loquacious-PD removes phosphate inhibition of Dicer-2 processing of hairpin RNAs into siRNAs. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 29550490
Summary:
Drosophila Dicer-2 processes RNA substrates into short interfering RNAs (siRNAs). Loquacious-PD (Loqs-PD), a dsRNA-binding protein that associates with Dicer-2, is required for processing of a subset of RNA substrates including hairpin RNAs into siRNAs. Inorganic phosphate-a small molecule present in all cell types-inhibits Dicer-2 from processing precursor of microRNAs (pre-miRNAs), which are processed by Dicer-1. Whether or how Loqs-PD modulates the inhibitory effect of inorganic phosphate on Dicer-2 processing of RNA substrates is unknown. To address this question, an in vitro hairpin RNA processing assay was performed with Dicer-2 in the presence or absence of Loqs-PD and/or inorganic phosphate. Inorganic phosphate was found to inhibits Dicer-2 alone, but not Dicer-2 + Loqs-PD, from processing blunt-end hairpin RNAs into siRNAs. Thus, Loqs-PD removes the inhibitory effect of inorganic phosphate on Dicer-2 processing of blunt-end hairpin RNAs, allowing siRNA production in the presence of inorganic phosphate.

Monday, April 2nd

Fropf, R., Zhou, H. and Yin, J. C. P. (2018). The clock gene period differentially regulates sleep and memory in Drosophila. Neurobiol Learn Mem [Epub ahead of print]. PubMed ID: 29474956
Summary:
Circadian regulation is a conserved phenomenon across the animal kingdom, and its disruption can have severe behavioral and physiological consequences. Core circadian clock proteins are likewise well conserved from Drosophila to humans. While the molecular clock interactions that regulate circadian rhythms have been extensively described, additional roles for clock genes during complex behaviors are less understood. This study showed that mutations in the clock gene period (per) result in differential time-of-day effects on acquisition and long-term memory of aversive olfactory conditioning. Sleep is also altered in period mutants: while its overall levels don't correlate with memory, sleep plasticity in different genotypes correlates with immediate performance after training. This study further describes distinct anatomical bases for Period function by manipulating Period activity in restricted brain cells and testing the effects on specific aspects of memory and sleep. In the null mutant background, different features of sleep and memory are affected when a form of the period gene is reintroduce in glia, lateral neurons, and the fan-shaped body. The results indicate that the role of the clock gene period may be separable in specific aspects of sleep or memory; further studies into the molecular mechanisms of these processes suggest independent neural circuits and molecular cascades that mediate connections between the distinct phenomena.
Filosevic, A., Al-Samarai, S. and Andretic Waldowski, R. (2018). High throughput measurement of locomotor sensitization to volatilized cocaine in Drosophila melanogaster. Front Mol Neurosci 11: 25. PubMed ID: 29459820
Summary:
This study developed a new behavioral test, FlyBong, which combines delivery of volatilized cocaine (vCOC) to individually housed flies with objective quantification of their locomotor activity. There are two main advantages of FlyBong: it is high-throughput and it allows for comparisons of locomotor activity of individual flies before and after single or multiple exposures. At the population level, exposure to vCOC leads to transient and concentration-dependent increase in locomotor activity, representing sensitivity to an acute dose. A second exposure leads to further increase in locomotion, representing locomotor sensitization. FlyBong was validated by showing that locomotor sensitization at either the population or individual level is absent in the mutants for circadian genes period (per), Clock (Clk), and cycle (cyc). The locomotor sensitization that is present in timeless (tim) and pigment dispersing factor (pdf) mutant flies is in large part not cocaine specific, but derived from increased sensitivity to warm air. Circadian genes are not only integral part of the neural mechanism that is required for development of locomotor sensitization, but in addition, they modulate the intensity of locomotor sensitization as a function of the time of day. Motor-activating effects of cocaine are sexually dimorphic and require a functional dopaminergic transporter. FlyBong is a new and improved method for inducing and measuring locomotor sensitization to cocaine in individual Drosophila. Because of its high-throughput nature, FlyBong can be used in genetic screens or in selection experiments aimed at the unbiased identification of functional genes involved in acute or chronic effects of volatilized psychoactive substances.
Deakin, A., Mendl, M., Browne, W. J., Paul, E. S. and Hodge, J. J. L. (2018). State-dependent judgement bias in Drosophila: evidence for evolutionarily primitive affective processes. Biol Lett 14(2). PubMed ID: 29491031
Summary:
Affective states influence decision-making under ambiguity in humans and other animals. Individuals in a negative state tend to interpret ambiguous cues more negatively than individuals in a positive state. This study demonstrates that the fruit fly, Drosophila melanogaster, also exhibits state-dependent changes in cue interpretation. Drosophila were trained on a Go/Go task to approach a positive (P) odour associated with a sugar reward and actively avoid a negative (N) odour associated with shock. Trained flies were then either shaken to induce a purported negative state or left undisturbed (control), and given a choice between: air or P; air or N; air or ambiguous odour (1 : 1 blend of P : N). Shaken flies were significantly less likely to approach the ambiguous odour than control flies. This 'judgement bias' may be mediated by changes in neural activity that reflect evolutionarily primitive affective states. It cannot be said whether such states are consciously experienced, but use of this model organism's versatile experimental tool kit may facilitate elucidation of their neural and genetic basis.
Burgos, A., Honjo, K., Ohyama, T., Qian, C. S., Shin, G. J., Gohl, D. M., Silies, M., Tracey, W. D., Zlatic, M., Cardona, A. and Grueber, W. B. (2018). Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila. Elife 7. PubMed ID: 29528286
Summary:
Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. In Drosophila larvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. A population of interneurons in the nerve cord of Drosophila, termed Down-and-Back (DnB) neurons, was identified that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior.
Potdar, S., Daniel, D. K., Thomas, F. A., Lall, S. and Sheeba, V. (2018). Sleep deprivation negatively impacts reproductive output in Drosophila melanogaster. J Exp Biol. PubMed ID: 29361608
Summary:
Most animals sleep or exhibit a sleep-like state, yet the adaptive significance of this phenomenon remains unclear. Although reproductive deficits are associated with lifestyle induced sleep deficiencies, how sleep loss affects reproductive physiology is poorly understood, even in model organisms. This study aimed to bridge this mechanistic gap by impairing sleep in female fruit flies and testing its effect on egg output. Sleep deprivation by feeding caffeine or by mechanical perturbation was shown to result in decreased egg output. Transient activation of wake-promoting dopaminergic neurons decreases egg output in addition to sleep levels, thus demonstrating a direct negative impact of sleep deficit on reproductive output. Similarly, loss-of-function mutation in dopamine transporter fumin (fmn) leads to both significant sleep loss and lowered fecundity. This demonstration of a direct relationship between sleep and reproductive fitness indicates a strong driving force for the evolution of sleep.
Brenman-Suttner, D. B., Long, S. Q., Kamesan, V., de Belle, J. N., Yost, R. T., Kanippayoor, R. L. and Simon, A. F. (2018). Progeny of old parents have increased social space in Drosophila melanogaster. Sci Rep 8(1): 3673. PubMed ID: 29487349
Summary:
This study report the effects of aging and parental age in Drosophila melanogaster on two types of responses to social cues: the choice of preferred social spacing in an undisturbed group and the response to the Drosophila stress odorant (dSO) emitted by stressed flies. The patterns of changes during aging were notably different for these two social responses. Flies were initially closer in space and then became further apart. However, the pattern of change in response to dSO followed a more typical decline in performance, similarly to changes in locomotion. Interestingly, the increased social space of old parents, as well as their reduced performance in avoiding dSO, was passed on to their progeny, such that young adults adopted the behavioural characteristic of their old parents. While the response to social cues was inherited, the changes in locomotion were not. It was possible to scale the changes in the social space of parents and their progeny by accelerating or decelerating the physiological process of aging by increasing temperatures and exposure to oxidative stress, or via caloric restriction, respectively. Finally, when only one parent was aged, only the male progeny of old fathers and the progeny of very old mothers were more distant.
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