What's hot today:
Current papers in developmental biology and gene function


Wednesday February 28th, 2018

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Bademosi, A. T., Lauwers, E., Amor, R., Verstreken, P., van Swinderen, B. and Meunier, F. A. (2018). In vivo single-molecule tracking at the Drosophila presynaptic motor nerve terminal. J Vis Exp(131). PubMed ID: 29364242
An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. This study describes a technique to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. This study describes how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.
Romano, G., Holodkov, N., Klima, R., Grilli, F., Guarnaccia, C., Nizzardo, M., Rizzo, F., Garcia, R. and Feiguin, F. (2018). Downregulation of glutamic acid decarboxylase in Drosophila TDP-43-null brains provokes paralysis by affecting the organization of the neuromuscular synapses. Sci Rep 8(1): 1809. PubMed ID: 29379112
Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects the motor system, comprised of motoneurons and associated glia. Accordingly, neuronal or glial defects in TDP-43 function provoke paralysis due to the degeneration of the neuromuscular synapses in Drosophila. To identify the responsible molecules and mechanisms, a genome wide proteomic analysis was performed to determine differences in protein expression between wild-type and TDP-43-minus fly heads. The data established that mutant insects presented reduced levels of the enzyme glutamic acid decarboxylase (Gad1) and increased concentrations of extracellular glutamate. Genetic rescue of Gad1 activity in neurons or glia was sufficient to recuperate flies locomotion, synaptic organization and glutamate levels. Analogous recovery was obtained by treating TDP-43-null flies with glutamate receptor antagonists demonstrating that Gad1 promotes synapses formation and prevents excitotoxicity. Similar suppression of TDP-43 provoked the downregulation of GAD67, the Gad1 homolog protein in human neuroblastoma cell lines and analogous modifications were observed in iPSC-derived motoneurons from patients carrying mutations in TDP-43, uncovering conserved pathological mechanisms behind the disease.
Heo, K., Nahm, M., Lee, M. J., Kim, Y. E., Ki, C. S., Kim, S. H. and Lee, S. (2017). The Rap activator Gef26 regulates synaptic growth and neuronal survival via inhibition of BMP signaling. Mol Brain 10(1): 62. PubMed ID: 29282074
In Drosophila, precise regulation of BMP signaling is essential for normal synaptic growth at the larval neuromuscular junction (NMJ) and neuronal survival in the adult brain. However, the molecular mechanisms underlying fine-tuning of BMP signaling in neurons remain poorly understood. This study shows that loss of the Drosophila PDZ guanine nucleotide exchange factor Gef26 significantly increases synaptic growth at the NMJ and enhances BMP signaling in motor neurons. It was further shown that Gef26 functions upstream of Rap1 in motor neurons to restrain synaptic growth. Synaptic overgrowth in gef26 or rap1 mutants requires BMP signaling, indicating that Gef26 and Rap1 regulate synaptic growth via inhibition of BMP signaling. Gef26 is involved in the endocytic downregulation of surface expression of the BMP receptors thickveins (Tkv) and wishful thinking (Wit). Loss of Gef26 also induces progressive brain neurodegeneration through Rap1- and BMP signaling-dependent mechanisms. Taken together, these results suggest that the Gef26-Rap1 signaling pathway regulates both synaptic growth and neuronal survival by controlling BMP signaling.
Ashley, J., Cordy, B., Lucia, D., Fradkin, L. G., Budnik, V. and Thomson, T. (2018). Retrovirus-like Gag protein Arc1 binds RNA and traffics across synaptic boutons. Cell 172(1-2): 262-274.e211. PubMed ID: 29328915
Arc/Arg3.1 is required for synaptic plasticity and cognition, and mutations in this gene are linked to autism and schizophrenia. Arc bears a domain resembling retroviral/retrotransposon Gag-like proteins, which multimerize into a capsid that packages viral RNA. The significance of such a domain in a plasticity molecule is uncertain. This study reports that the Drosophila Arc1 protein forms capsid-like structures that bind darc1 mRNA in neurons and is loaded into extracellular vesicles that are transferred from motorneurons to muscles. This loading and transfer depends on the darc1-mRNA 3' untranslated region, which contains retrotransposon-like sequences. Disrupting transfer blocks synaptic plasticity, suggesting that transfer of dArc1 complexed with its mRNA is required for this function. Notably, cultured cells also release extracellular vesicles containing the Gag region of the Copia retrotransposon complexed with its own mRNA. Taken together, these results point to a trans-synaptic mRNA transport mechanism involving retrovirus-like capsids and extracellular vesicles.
Kerwin, S. K., Li, J. S. S., Noakes, P. G., Shin, G. J. and Millard, S. S. (2018). Regulated alternative splicing of Drosophila Dscam2 is necessary for attaining the appropriate number of photoreceptor synapses. Genetics 208(2): 717-728. PubMed ID: 29208630
How the brain makes trillions of synaptic connections using a genome of only 20,000 genes is a major question in modern neuroscience. Alternative splicing is one mechanism that can increase the number of proteins produced by each gene, but its role in regulating synapse formation is poorly understood. In Drosophila, photoreceptors form a synapse with multiple postsynaptic elements including lamina neurons L1 and L2. L1 and L2 express distinct isoforms of the homophilic repulsive protein Dscam2, and since these isoforms cannot bind to each other, cell-specific expression has been proposed to be necessary for preventing repulsive interactions that could disrupt the synapse. This study shows that the number of synapses are reduced in flies that express only one isoform, and L1 and L2 dendritic morphology is perturbed. It is proposed that these defects result from inappropriate interactions between L1 and L2 dendrites. It is concluded that regulated Dscam2 alternative splicing is necessary for the proper assembly of photoreceptor synapses.
Bademosi, A. T., Steeves, J., Karunanithi, S., Zalucki, O. H., Gormal, R. S., Liu, S., Lauwers, E., Verstreken, P., Anggono, V., Meunier, F. A. and van Swinderen, B. (2018). Trapping of Syntaxin1a in presynaptic nanoclusters by a clinically relevant general anesthetic. Cell Rep 22(2): 427-440. PubMed ID: 29320738
Propofol is the most commonly used general anesthetic in humans. Understanding of its mechanism of action has focused on its capacity to potentiate inhibitory systems in the brain. However, it is unknown whether other neural mechanisms are involved in general anesthesia. This study demonstrates that the synaptic release machinery is also a target. Using single-particle tracking photoactivation localization microscopy, it was shown that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility. Removing the interaction with the t-SNARE partner SNAP-25 abolishes propofol-induced syntaxin1A confinement, indicating that syntaxin1A and SNAP-25 together form an emergent drug target. Impaired syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated syntaxin1A construct that interacts with SNAP-25. These results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional syntaxin1A nanoclusters.

Tuesday, February 27th

Long, D. M. and Giebultowicz, J. M. (2017). Age-related changes in the expression of the circadian clock protein PERIOD in Drosophila glial cells. Front Physiol 8: 1131. PubMed ID: 29375400
Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Immunocytochemistry experiments have been conducted to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. The data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain.
Prahlad, A., Spalthoff, C., Kong, D., Grosshans, J., Gopfert, M. C. and Schmidt, C. F. (2017). Mechanical properties of a Drosophila larval chordotonal organ. Biophys J 113(12): 2796-2804. PubMed ID: 29262372
Proprioception is an integral part of the feedback circuit that is essential for locomotion control in all animals. Chordotonal organs perform proprioceptive and other mechanosensory functions in insects and crustaceans. The mechanical properties of these organs are believed to be adapted to the sensory functions, but had not been probed directly. This study measured mechanical properties of a particular chordotonal organ-the lateral pentascolopidial (lch5) organ of Drosophila larvae-which plays a key role in proprioceptive locomotion control. Tension was applied to the whole organ in situ by transverse deflection. Upon release of force, the organ displayed overdamped relaxation with two widely separated time constants, tens of milliseconds and seconds, respectively. When the muscles covering the lch5 organ were excised, the slow relaxation was absent, and the fast relaxation became faster. Interestingly, most of the strain in the stretched organ is localized in the cap cells, which account for two-thirds of the length of the entire organ, and could be stretched by approximately 10% without apparent damage. In laser ablation experiments it was found that cap cells retracted by approximately 100 mμm after being severed from the neurons, indicating considerable steady-state stress and strain in these cells. Given the fact that actin as well as myosin motors are abundant in cap cells, the results point to a mechanical regulatory role of the cap cells in the lch5 organ.
Leonhardt, A., Meier, M., Serbe, E., Eichner, H. and Borst, A. (2017). Neural mechanisms underlying sensitivity to reverse-phi motion in the fly. PLoS One 12(12): e0189019. PubMed ID: 29261684
Optical illusions provide powerful tools for mapping the algorithms and circuits that underlie visual processing, revealing structure through atypical function. Of particular note in the study of motion detection has been the reverse-phi illusion. When contrast reversals accompany discrete movement, detected direction tends to invert. This occurs across a wide range of organisms, spanning humans and invertebrates. This study mapped an algorithmic account of the phenomenon onto neural circuitry in the fruit fly Drosophila melanogaster. Through targeted silencing experiments in tethered walking flies as well as electrophysiology and calcium imaging, it was demonstrated that ON- or OFF-selective local motion detector cells T4 and T5 are sensitive to certain interactions between ON and OFF. A biologically plausible detector model accounts for subtle features of this particular form of illusory motion reversal, like the re-inversion of turning responses occurring at extreme stimulus velocities. In light of comparable circuit architecture in the mammalian retina, it is suggested that similar mechanisms may apply even to human psychophysics.
Sitaraman, D., Kramer, E. F., Kahsai, L., Ostrowski, D. and Zars, T. (2017). Discrete serotonin systems mediate memory enhancement and escape latencies after unpredicted aversive experience in Drosophila place memory. Front Syst Neurosci 11: 92. PubMed ID: 29321732
Feedback mechanisms in operant learning are critical for animals to increase reward or reduce punishment. However, not all conditions have a behavior that can readily resolve an event. Animals must then try out different behaviors to better their situation through outcome learning. Learned helplessness, as a type of outcome learning, manifests in part as increases in escape latency in the face of repeated unpredicted shocks. Little is known about the mechanisms of outcome learning. When fruit flies are exposed to unpredicted high temperatures in a place learning paradigm, flies both increase escape latencies and have a higher memory when given control of a place/temperature contingency. This study describes discrete serotonin neuronal circuits that mediate aversive reinforcement, escape latencies, and memory levels after place learning in the presence and absence of unexpected aversive events. The results show that two features of learned helplessness depend on the same modulatory system as aversive reinforcement. Moreover, changes in aversive reinforcement and escape latency depend on local neural circuit modulation, while memory enhancement requires larger modulation of multiple behavioral control circuits.
Suzuki-Sawano, E., Ueno, K., Naganos, S., Sawano, Y., Horiuchi, J. and Saitoe, M. (2017). A Drosophila ex vivo model of olfactory appetitive learning. Sci Rep 7(1): 17725. PubMed ID: 29255174
During olfactory appetitive learning, animals associate an odor, or conditioned stimulus (CS), with an unconditioned stimulus (US), often a sugar reward. This association induces feeding behavior, a conditioned response (CR), upon subsequent exposure to the CS. In this study, a model of this behavior was developed in isolated Drosophila brains. Artificial activation of neurons expressing the Gr5a sugar-responsive gustatory receptor (Gr5a GRNs) induces feeding behavior in starved flies. Consistent with this, it was found that in dissected brains, activation of Gr5a GRNs induces Ca(2+) transients in motor neurons, MN11 + 12, required for ingestion. Significantly, activation of Gr5a GRNs can substitute for presentation of sugar rewards during olfactory appetitive learning. Similarly, in dissected brains, coincident stimulation of Gr5a GRNs and the antennal lobe (AL), which processes olfactory information, results in increased Ca(2+) influx into MN11 + 12 cells upon subsequent AL stimulation. Importantly, olfactory appetitive associations are not formed in satiated flies. Likewise, AL-evoked Ca(2+) transients in MN11 + 12 are not produced in ex vivo brains from satiated flies. These results suggest that a starved/satiated state is maintained in dissected brains, and that this ex vivo system will be useful for identification of neural networks involved in olfactory appetitive learning.
Astigarraga, S., Douthit, J., Tarnogorska, D., Creamer, M. S., Mano, O., Clark, D. A., Meinertzhagen, I. A. and Treisman, J. E. (2018). Drosophila Sidekick is required in developing photoreceptors to enable visual motion detection. Development [Epub ahead of print]. PubMed ID: 29361567
The assembly of functional neuronal circuits requires growth cones to extend in defined directions and recognize the correct synaptic partners. Homophilic adhesion between vertebrate Sidekick proteins promotes synapse formation between retinal neurons involved in visual motion detection.This study shows that that Drosophila Sidekick accumulates in specific synaptic layers of the developing motion detection circuit and is necessary for normal optomotor behavior. Sidekick is required in photoreceptors, but not their target lamina neurons, to promote the alignment of lamina neurons into columns and subsequent sorting of photoreceptor axons into synaptic modules based on their precise spatial orientation. Sidekick is also localized to the dendrites of the direction-selective T4 and T5 cells, and is expressed in some of their presynaptic partners. In contrast to its vertebrate homologues, Sidekick is not essential for T4 and T5 to direct their dendrites to the appropriate layers or to receive synaptic contacts. These results illustrate a conserved requirement for Sidekick proteins to establish visual motion detection circuits that is achieved through distinct cellular mechanisms in Drosophila and vertebrates.

Monday, February 25th

Daniel, S. G., Russ, A. D., Guthridge, K. M., Raina, A. I., Estes, P. S., Parsons, L. M., Richardson, H. E., Schroeder, J. A. and Zarnescu, D. C. (2018). miR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila. Biol Open 7(1). PubMed ID: 29361610
Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, it was hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, it was found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling, a core set of ten miRNAs were identified that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which was found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, it was overexpressed in the context of lgl knock-down by RNAi, and it was found to be able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. This work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.
Akulenko, N., Ryazansky, S., Morgunova, V., Komarov, P. A., Olovnikov, I., Vaury, C., Jensen, S. and Kalmykova, A. (2018). Transcriptional and chromatin changes accompanying de novo formation of transgenic piRNA clusters. RNA [Epub ahead of print]. PubMed ID: 29358235
Expression of transposable elements in the germline is controlled by Piwi-interacting (pi) RNAs produced by genomic loci termed piRNA clusters and associated with Rhino, a Heterochromatin Protein 1 (HP1) homolog. Previously, it was shown that transgenes containing a fragment of the I retrotransposon form de novo piRNA clusters in the Drosophila germline providing suppression of I-element activity. It was noted that identical transgenes located in different genomic sites vary considerably in piRNA production, and they were classified as "strong" and "weak" piRNA clusters. This study investigated what chromatin and transcriptional changes occur at the transgene insertion sites after their conversion into piRNA clusters. The formation of a transgenic piRNA cluster was found to be accompanied by activation of transcription from both genomic strands that likely initiates at multiple random sites. The chromatin of all transgene-associated piRNA clusters contain high levels of trimethylated lysine 9 of histone H3 (H3K9me3) and HP1a, whereas Rhino binding is considerably higher at the strong clusters. None of these chromatin marks was revealed at the "empty" sites before transgene insertion. Finally, this study showed that in the nucleus of polyploid nurse cells, the formation of a piRNA cluster at a given transgenic genomic copy works according to an "all- or- nothing" model: either there is high Rhino enrichment or there is no association with Rhino at all. As a result, genomic copies of a weak piRNA transgenic cluster show a mosaic association with Rhino foci, while the majority of strong transgene copies associate with Rhino and are hence involved in piRNA production.
Sgromo, A., Raisch, T., Backhaus, C., Keskeny, C., Alva, V., Weichenrieder, O. and Izaurralde, E. (2017). Drosophila Bag-of-marbles directly interacts with the CAF40 subunit of the CCR4-NOT complex to elicit repression of mRNA targets. RNA [Epub ahead of print]. PubMed ID: 29255063
Drosophila melanogaster Bag-of-marbles (Bam) promotes germline stem cell (GSC) differentiation by repressing the expression of mRNAs encoding stem cell maintenance factors. Bam interacts with Benign gonial cell neoplasm (Bgcn) and the CCR4 deadenylase, a catalytic subunit of the CCR4-NOT complex. Bam has been proposed to bind CCR4 and displace it from the CCR4-NOT complex. This study investigated the interaction of Bam with the CCR4-NOT complex by using purified recombinant proteins. Unexpectedly, it was found that Bam does not interact with CCR4 directly but instead binds to the CAF40 subunit of the complex in a manner mediated by a conserved N-terminal CAF40-binding motif (CBM). The crystal structure of the Bam CBM bound to CAF40 reveals that the CBM peptide adopts an alpha-helical conformation after binding to the concave surface of the crescent-shaped CAF40 protein. It was further shown that Bam-mediated mRNA decay and translational repression depend entirely on Bam's interaction with CAF40. Thus, Bam regulates the expression of its mRNA targets by recruiting the CCR4-NOT complex through interaction with CAF40.
Yan, S., Acharya, S., Groning, S. and Grosshans, J. (2017). Slam protein dictates subcellular localization and translation of its own mRNA. PLoS Biol 15(12): e2003315. PubMed ID: 29206227
Many mRNAs specifically localize within the cytoplasm and are present in RNA-protein complexes. It is generally assumed that localization and complex formation of these RNAs are controlled by trans-acting proteins encoded by genes different than the RNAs themselves. This study analyzed slow as molasses (slam) mRNA that prominently colocalizes with its encoded protein at the basal cortical compartment during cellularization. The functional implications of this striking colocalization have been unknown. This study showed that slam mRNA translation is spatiotemporally controlled. Translation was found to be largely restricted to the onset of cellularization when Slam protein levels at the basal domain sharply increase. slam mRNA was translated locally, at least partially, as not yet translated mRNA transiently accumulated at the basal region. Slam RNA accumulated at the basal domain only if Slam protein was present. Furthermore, a slam RNA with impaired localization but full coding capacity was only weakly translated. A biochemical interaction of slam mRNA and protein was detected as demonstrated by specific co-immunoprecipitation from embryonic lysate. The intimate relationship of slam mRNA and protein may constitute a positive feedback loop that facilitates and controls timely and rapid accumulation of Slam protein at the prospective basal region.
Wang, M., Branco, A. T. and Lemos, B. (2017). The Y chromosome modulates splicing and sex-biased intron retention rates in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 29263027
The Drosophila Y chromosome is a 40MB segment of mostly repetitive DNA; it harbors a handful of protein coding genes and a disproportionate amount of satellite repeats, transposable elements, and multicopy DNA arrays. Intron retention (IR) is a type of alternative splicing (AS) event by which one or more introns remain within the mature transcript. IR recently emerged as a deliberate cellular mechanism to modulate gene expression levels and has been implicated in multiple biological processes. However, the extent of sex differences in IR and the contribution of the Y chromosome to the modulation of alternative splicing and intron retention rates has not been addressed. This study showed pervasive intron retention (IR) in the fruit fly Drosophila melanogaster with thousands of novel IR events, hundreds of which displayed extensive sex-bias. The data also revealed an unsuspected role for the Y chromosome in the modulation of alternative splicing and intron retention. The majority of sex-biased IR events introduced premature termination codons and the magnitude of sex-bias was associated with gene expression differences between the sexes. Surprisingly, an extra Y chromosome in males (X^YY genotype) or the presence of a Y chromosome in females (X^XY genotype) significantly modulated IR and recapitulated natural differences in IR between the sexes. These results highlight the significance of sex-biased IR in tuning sex differences and the role of the Y chromosome as a source of variable IR rates between the sexes. Modulation of splicing and intron retention rates across the genome represent new and unexpected outcomes of the Drosophila Y chromosome.
Shu, Z., Huang, Y. C., Palmer, W. H., Tamori, Y., Xie, G., Wang, H., Liu, N. and Deng, W. M. (2017). Systematic analysis reveals tumor-enhancing and -suppressing microRNAs in Drosophila epithelial tumors. Oncotarget 8(65): 108825-108839. PubMed ID: 29312571
Despite their emergence as an important class of noncoding RNAs involved in cancer cell transformation, invasion, and migration, the precise role of microRNAs (miRNAs) in tumorigenesis remains elusive. To gain insights into how miRNAs contribute to primary tumor formation, an RNA sequencing (RNA-Seq) analysis was conducted of Drosophila wing disc epithelial tumors induced by knockdown of a neoplastic tumor-suppressor gene (nTSG) lethal giant larvae (lgl), combined with overexpression of an active form of oncogene Ras (Ras(V12)), and 51 mature miRNAs were identified that changed significantly in tumorous discs. Followed by in vivo tumor enhancer and suppressor screens in sensitized genetic backgrounds, ten tumor-enhancing (TE) miRNAs and eleven tumor-suppressing (TS) miRNAs were identified that contributed to the nTSG defect-induced tumorigenesis. Among these, four TE and three TS miRNAs have human homologs. From this study, 29 miRNAs were identified that individually had no obvious role in enhancing or alleviating tumorigenesis despite their changed expression levels in nTSG tumors. This systematic analysis, which includes both RNA-Seq and in vivo functional studies, helps to categorize miRNAs into different groups based on their expression profile and functional relevance in epithelial tumorigenesis, whereas the evolutionarily conserved TE and TS miRNAs provide potential therapeutic targets for epithelial tumor treatment.
Malik, S., Jang, W. and Kim, C. (2017). Protein interaction mapping of translational regulators affecting expression of the critical stem cell factor nos. Dev Reprod 21(4): 449-456. PubMed ID: 29354790
The germline stem cells of the Drosophila ovary continuously produce eggs throughout the life- span. Intricate regulation of stemness and differentiation is critical to this continuous production. The translational regulator Nos is an intrinsic factor that is required for maintenance of stemness in germline stem cells. Nos expression is reduced in differentiating cells at the post-transcriptional level by diverse translational regulators. However, molecular mechanisms underlying Nos repression are not completely understood. Through three distinct protein-protein interaction experiments, this study identified specific molecular interactions between translational regulators involved in Nos repression. The findings suggest a model in which protein complexes assemble on the 3' untranslated region of Nos mRNA in order to regulate Nos expression at the post-transcriptional level.
Pai, A. A., Henriques, T., McCue, K., Burkholder, A., Adelman, K. and Burge, C. B. (2017). The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture. Elife 6. PubMed ID: 29280736
Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning ('intron definition') or exon-spanning ('exon definition') pairs. To understand how exon and intron length and splice site recognition mode impact splicing, splicing rates were measured genome-wide in Drosophila, using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. The modal intron length range of 60-70 nt was found to represent a local maximum of splicing rates, but much longer exon-defined introns are spliced even faster and more accurately.Unexpectedly low variation was observed in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and multiple gene level variables associated with splicing rate were identified. Together these data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance the rate or accuracy of splicing.

Friday, February 23rd

Lee, H., Cho, D. Y., Wojtowicz, D., Harbison, S. T., Russell, S., Oliver, B. and Przytycka, T. (2017). Dosage-dependent expression variation suppressed on the Drosophila male X chromosome. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 29242386
DNA copy number variation is associated with many high phenotypic heterogeneity disorders. This study systematically examined the impact of Drosophila deletions on gene expression profiles to ask if increased expression variability due to reduced gene dose might underlie this phenotypic heterogeneity. Indeed, one dose genes were found to have higher gene expression variability relative to two dose genes. Interestingly, expression variability was related to the magnitude of expression compensation, suggesting that gene dose reduction induced regulation is noisy. In a remarkable exception to this rule the single X chromosome of males showed reduced expression variability, even compared to two dose genes. Analysis of sex transformed flies indicates that X expression variability is independent of the male differentiation program. Instead, a correlation was uncovered between occupancy of the chromatin modifying protein encoded by males absent on first (mof) and expression variability, linking noise suppression to the specialized X chromosome dosage compensation system. MOF occupancy on autosomes in both sexes lowered transcriptional noise as well. The results demonstrate that gene deletions can lead to heterogeneous responses, which are often noisy. This has implications for understanding gene network regulatory interactions and phenotypic heterogeneity. Additionally, chromatin modification appears to play a role in dampening transcriptional noise.
Batut, P. J. and Gingeras, T. R. (2017). Conserved noncoding transcription and core promoter regulatory code in early Drosophila development. Elife 6 [Epub ahead of print]. PubMed ID: 29260710
Multicellular development is driven by regulatory programs that orchestrate the transcription of protein-coding and noncoding genes. To decipher this genomic regulatory code, and to investigate the developmental relevance of noncoding transcription, genome-wide promoter activity throughout embryogenesis was compared in 5 Drosophila species. Core promoters, generally not thought to play a significant regulatory role, in fact impart restrictions on the developmental timing of gene expression on a global scale. A hierarchical regulatory model is proposed in which core promoters define broad windows of opportunity for expression, by defining a range of transcription factors from which they can receive regulatory inputs. This two-tiered mechanism globally orchestrates developmental gene expression, including extremely widespread noncoding transcription. Noncoding transcription was found to be more widespread than anticipated in Drosophila, with 3973 promoters driving the expression of lncRNAs during embryogenesis. This study functionally characterize the FBgn0264479 (long non-coding RNA:CR4388) locus, which expresses a lncRNA in a highly conserved spatiotemporal pattern suggestive of a role in early dorsoventral patterning. The sequence and expression specificity of noncoding RNA promoters were found to be evolutionarily conserved, implying biological relevance. Overall, this work introduces a hierarchical model for developmental gene regulation, and reveals a major role for noncoding transcription in animal development.
Kudron, M. M., et al. (2017). The modERN Resource: Genome-Wide Binding Profiles for Hundreds of Drosophila and Caenorhabditis elegans Transcription Factors. Genetics [Epub ahead of print]. PubMed ID: 29284660
In order to develop a catalog of regulatory sites in two major model organisms, Drosophilia melanogaster and Caenorhabditis elegans, the modERN consortium has systematically assayed the binding sites of transcription factors (TFs). Combined with data produced by modENCODE, data is now available for 262 TFs identifying 1.23M sites in the fly genome and 217 TFs identifying 0.67M sites in the worm genome. Because sites from different TFs are often overlapping and tightly clustered, they fall into 91,011 and 59,150 regions in the fly and worm, respectively, and these binding sites span as little as 8.7 Mb and 5.8 Mb in the two organisms. Clusters with large numbers of sites (so-called HOT regions) predominantly associate with broadly expressed genes, whereas clusters containing sites from just a few factors are associated with genes expressed in tissue specific patterns. All of the strains expressing GFP-tagged TFs are available at the stock centers and the ChIP-seq data are available through the ENCODE DCC, and also through a simple interface (http://epic.gs.washington.edu/modERN/) that facilitates rapid accessibility of processed datasets. These data will facilitate a vast number of scientific inquiries into the fuction of individual TFs in key developmental, metabolic, defense and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks and globally across the lifespans of these two key model organisms.
Orengo, D. J., Aguade, M. and Juan, E. (2017). Characterization of dFOXO binding sites upstream of the Insulin Receptor P2 promoter across the Drosophila phylogeny. PLoS One 12(12): e0188357. PubMed ID: 29200426
The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. This study has focused on the Insulin Receptor (InR) expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO). Transcription factor binding sites were finely characterized in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, the effect of mutations in the characterized dFOXO sites of D. melanogaster was characterized in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription.
Glaser-Schmitt, A. and Parsch, J. (2018). Functional characterization of adaptive variation within a cis-regulatory element influencing Drosophila melanogaster growth. PLoS Biol 16(1): e2004538. PubMed ID: 29324742
Gene expression variation is a major contributor to phenotypic diversity within species and is thought to play an important role in adaptation. However, examples of adaptive regulatory polymorphism are rare, especially those that have been characterized at both the molecular genetic level and the organismal level. This study consisted of a functional analysis of the Drosophila melanogaster CG9509 enhancer, a cis-regulatory element that shows evidence of adaptive evolution in populations outside the species' ancestral range in sub-Saharan Africa. Three single nucleotide polymorphisms were found to be responsible for the difference in CG9509 expression observed between sub-Saharan African and cosmopolitan populations. Interestingly, while two of these variants appear to have been the targets of a selective sweep outside of sub-Saharan Africa, the variant with the largest effect on expression remains polymorphic in cosmopolitan populations, suggesting it may be subject to a different mode of selection. To elucidate the function of CG9509, a series of functional and tolerance assays was performed on flies in which CG9509 expression was disrupted. CG9509 was found to play a role in larval growth and influences adult body and wing size, as well as wing loading. Furthermore, variation in several of these traits was associated with variation within the CG9509 enhancer. The effect on growth appears to result from a modulation of active ecdysone levels and expression of growth factors. Taken together, these findings suggest that selection acted on 3 sites within the CG9509 enhancer to increase CG9509 expression and, as a result, reduce wing loading as D. melanogaster expanded out of sub-Saharan Africa.
Henriques, T., Scruggs, B. S., Inouye, M. O., Muse, G. W., Williams, L. H., Burkholder, A. B., Lavender, C. A., Fargo, D. C. and Adelman, K. (2018). Widespread transcriptional pausing and elongation control at enhancers. Genes Dev [Epub ahead of print]. PubMed ID: 29378787
Regulation by gene-distal enhancers is critical for cell type-specific and condition-specific patterns of gene expression. Thus, to understand the basis of gene activity in a given cell type or tissue, the precise locations of enhancers must be determined and their behaviors functionally characterized. This study demonstrates that transcription is a nearly universal feature of enhancers in Drosophila and mammalian cells and that nascent RNA sequencing strategies are optimal for identification of both enhancers and superenhancers. This study dissected the mechanisms governing enhancer transcription and discovered remarkable similarities to transcription at protein-coding genes. RNA polymerase II (RNAPII) undergoes regulated pausing and release at enhancers. However, as compared with mRNA genes, RNAPII at enhancers is less stable and more prone to early termination. Furthermore, it was found that the level of histone H3 Lys4 (H3K4) methylation at enhancers corresponds to transcriptional activity such that highly active enhancers display H3K4 trimethylation rather than the H3K4 monomethylation considered a hallmark of enhancers. Finally, this work provides insights into the unique characteristics of superenhancers, which stimulate high-level gene expression through rapid pause release; interestingly, this property renders associated genes resistant to the loss of factors that stabilize paused RNAPII.
Anderson, C., Reiss, I., Zhou, C., Cho, A., Siddiqi, H., Mormann, B., Avelis, C. M., Deford, P., Bergland, A., Roberts, E., Taylor, J., Vasiliauskas, D. and Johnston, R. J. (2017). Natural variation in stochastic photoreceptor specification and color preference in Drosophila. Elife 6 [Epub ahead of print]. PubMed ID: 29251595
Each individual perceives the world in a unique way, but little is known about the genetic basis of variation in sensory perception. In the fly eye, the random mosaic of color-detecting R7 photoreceptor subtypes is determined by stochastic on/off expression of the transcription factor Spineless (Ss). In a genome-wide association study, a naturally occurring insertion in a regulatory DNA element in ss was identified that lowers the ratio of Ss(ON) to Ss(OFF) cells. This change in photoreceptor fates shifts the innate color preference of flies from green to blue. The genetic variant increases the binding affinity for Klumpfuss (Klu), a zinc finger transcriptional repressor that regulates ss expression. Klu is expressed at intermediate levels to determine the normal ratio of Ss(ON) to Ss(OFF) cells. Thus, binding site affinity and transcription factor levels are finely tuned to regulate stochastic expression, setting the ratio of alternative fates and ultimately determining color preference.
Xu, X. S., Gantz, V. M., Siomava, N. and Bier, E. (2017). CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity. Elife 6. PubMed ID: 29274230
The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. This study employed diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these 'in locus' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.

Thursday, February 22nd

Prince, L. M. and Rand, M. D. (2017). Notch target gene E(spl)mdelta is a mediator of methylmercury-induced myotoxicity in Drosophila. Front Genet 8: 233. PubMed ID: 29379520
Methylmercury (MeHg) is a ubiquitous environmental contaminant and neurotoxicant that has long been known to cause a variety of motor deficits. These motor deficits have primarily been attributed to MeHg targeting of developing neurons and induction of oxidative stress and calcium dysregulation. Studies in Drosophila have revealed that MeHg perturbs embryonic muscle formation and upregulates Notch target genes, reflected predominantly by expression of the downstream transcriptional repressor Enhancer of Split mdelta [E(spl)mdelta]. An E(spl)mdelta reporter gene shows expression primarily in the myogenic domain, and both MeHg exposure and genetic upregulation of E(spl)mdelta can disrupt embryonic muscle development. This study tested the hypothesis that developing muscle is targeted by MeHg via upregulation of E(spl)mdelta. Developmental MeHg exposure causes a decreased rate of eclosion that parallels gross disruption of indirect flight muscle (IFM) development. An increase in E(spl) expression across the pupal stages, with preferential E(spl)mdelta upregulation occurring at early (p5) stages, is also observed. E(spl)mdelta overexpression in myogenic lineages under the Mef2 promoter was seen to phenocopy eclosion and IFM effects of developmental MeHg exposure; whereas reduced expression of E(spl)mdelta shows rescue of eclosion and IFM morphology effects of MeHg exposure. No effects were seen on eclosion with E(spl)mdelta overexpression in neural and gut tissues. These data indicate that muscle development is a target for MeHg and that E(spl)mdelta is a muscle-specific mediator of this myotoxicity.
Yang, D., Thomas, J. M., Li, T., Lee, Y., Liu, Z. and Smith, W. (2017). Drosophila hep pathway mediates Lrrk2-induced neurodegeneration. Biochem Cell Biol [Epub ahead of print]. PubMed ID: 29268033
Although the pathogenesis of Parkinson's disease (PD) remains unclear, mutations in leucine-rich repeat kinase 2 (Lrrk2) are among the major causes of familial PD. Most of these mutations disrupt Lrrk2 kinase and/or GTPase domain function, resulting in neuronal degeneration. However, the signal pathways underlying Lrrk2-induced neuronal degeneration are not fully understood. There is an expanding body of evidence that suggests a link between Lrrk2 function and MAP kinase (MAPK) cascades. To further investigate this link in vivo, genetic RNAi screens of the MAPK pathways were performed in a Drosophila model to identify genetic modifier(s) that can suppress G2019S-Lrrk2-induced PD-like phenotypes. The results revealed that the knockdown of hemipterous (hep, or JNKK) increased fly survival time, improved locomotor function and reduced loss of dopaminergic neurons in G2019S-Lrrk2 transgenic flies. Expression of the dominant-negative allele of JNK (JNK-DN), a kinase that is downstream of hep in G2019S-Lrrk2 transgenic flies, elicited a similar effect. Moreover, treatment with the JNK inhibitor SP600125 partially reversed the G2019S-Lrrk2-induced loss of dopaminergic neurons. These results indicate that the hep pathway plays an important role in Lrrk2-linked Parkinsonism in flies. These studies provide new insights into the molecular mechanisms underlying Lrrk2-linked PD pathogenesis and aid in identifying potential therapeutic targets.
Uechi, H., Kuranaga, E., Iriki, T., Takano, K., Hirayama, S., Miura, M., Hamazaki, J. and Murata, S. (2017). Ubiquitin-binding protein CG5445 suppresses aggregation and cytotoxicity of Amyotrophic Lateral Sclerosis-linked TDP-43 in Drosophila. Mol Cell Biol [Epub ahead of print]. PubMed ID: 29109084
Ubiquitin-mediated protein degradation plays essential roles in proteostasis and is involved in the pathogenesis of neurodegenerative diseases in which ubiquitin-positive aberrant proteins accumulate. However, how such aberrant proteins are processed inside cells has not been fully explored. This study show that the product of CG5445, a previously uncharacterized Drosophila gene, prevents accumulation of aggregate-prone ubiquitinated proteins. Ubiquitin conjugates were associated with CG5445, knockdown of which caused accumulation of detergent-insoluble ubiquitinated proteins. Furthermore, CG5445 rescued eye degeneration caused by the amyotrophic lateral sclerosis (ALS)-linked mutant TAR DNA-binding protein 43-kDa (TDP-43), which often forms ubiquitin-positive aggregates in cells through the capacity of CG5445 to bind to ubiquitin chains. Biochemically, CG5445 inhibited accumulation of insoluble forms and promoted their clearance. These results demonstrate a new possible mechanism by which cells maintain ubiquitinated aggregation-prone proteins in a soluble form to decrease their cytotoxicity until they are degraded.
Yang, C. N., Wu, M. F., Liu, C. C., Jung, W. H., Chang, Y. C., Lee, W. P., Shiao, Y. J., Wu, C. L., Liou, H. H., Lin, S. K. and Chan, C. C. (2017). Differential protective effects of connective tissue growth factor against Abeta neurotoxicity on neurons and glia. Hum Mol Genet 26(20): 3909-3921. PubMed ID: 29016849
Impaired clearance of amyloid-beta peptide (Abeta; see Drosophila Appl) leads to abnormal extracellular accumulation of this neurotoxic protein that drives neurodegeneration in sporadic Alzheimer's disease (AD). Connective tissue growth factor (CTGF/CCN2) expression is elevated in plaque-surrounding astrocytes in AD patients. However, the role of CTGF in AD pathogenesis remains unclear. This study characterized the neuroprotective activity of CTGF. CTGF facilitates Abeta uptake and subsequent degradation within primary glia and neuroblastoma cells. CTGF enhanced extracellular Abeta degradation via membrane-bound matrix metalloproteinase-14 (MMP14) in glia and extracellular MMP13 in neurons. In the brain of a Drosophila AD model, glial-expression of CTGF reduced Abeta deposits, improved locomotor function, and rescued memory deficits. Neuroprotective potential of CTGF against Abeta42-induced photoreceptor degeneration was disrupted through silencing MMPs. Therefore, CTGF may represent a node for potential AD therapeutics as it intervenes in glia-neuron communication via specific MMPs to alleviate Abeta neurotoxicity in the central nervous system.
Min, S., Yoon, W., Cho, H. and Chung, J. (2017). Misato underlies visceral myopathy in Drosophila. Sci Rep 7(1): 17700. PubMed ID: 29255146
Genetic mechanisms for the pathogenesis of visceral myopathy (VM) have been rarely demonstrated. This study reports the visceral role of in Drosophila and its implications for the pathogenesis of VM. Depletion of mst using three independent RNAi lines expressed by a pan-muscular driver elicited characteristic symptoms of VM, such as abnormal dilation of intestinal tracts, reduced gut motility, feeding defects, and decreased life span. By contrast, exaggerated expression of mst reduced intestine diameters, but increased intestinal motilities along with thickened muscle fibers, demonstrating a critical role of mst in the visceral muscle. Mst expression was detected in the adult intestine with its prominent localization to actin filaments and was required for maintenance of intestinal tubulin and actomyosin structures. Consistent with the subcellular localization of Mst, the intestinal defects induced by mst depletion were dramatically rescued by exogenous expression of an actin member. Upon ageing the intestinal defects were deteriorative with marked increase of apoptotic responses in the visceral muscle. Taken together, it is proposed that the impairment of actomyosin structures induced by mst depletion in the visceral muscle as a pathogenic mechanism for VM.
Ring, J., et al. (2017). Mitochondrial energy metabolism is required for lifespan extension by the spastic paraplegia-associated protein spartin. Microb Cell 4(12): 411-422. PubMed ID: 29234670
Hereditary spastic paraplegias, a group of neurodegenerative disorders, can be caused by loss-of-function mutations in the protein spartin. However, the physiological role of spartin remains largely elusive. This study shows that heterologous expression of human or Drosophila spartin extends chronological lifespan of yeast, reducing age-associated ROS production, apoptosis, and necrosis. Spartin localizes to the proximity of mitochondria and physically interacts with proteins related to mitochondrial and respiratory metabolism. Interestingly, Nde1, the mitochondrial external NADH dehydrogenase, and Pda1, the core enzyme of the pyruvate dehydrogenase complex, are required for spartin-mediated cytoprotection. Furthermore, spartin interacts with the glycolysis enhancer phospo-fructo-kinase-2,6 (Pfk26) and is sufficient to complement for PFK26-deficiency at least in early aging. It is concluded that mitochondria-related energy metabolism is crucial for spartin's vital function during aging; this study uncovers a network of specific interactors required for this function.
Vivien Chiu, W. Y., Koon, A. C., Ki Ngo, J. C., Edwin Chan, H. Y. and Lau, K. F. (2017). GULP1/CED-6 ameliorates amyloid-beta toxicity in a Drosophila model of Alzheimer's disease. Oncotarget 8(59): 99274-99283. PubMed ID: 29245900
Amyloidogenic processing of APP (see Drosophila Appl) by beta- and gamma-secretases leads to the generation of amyloid-beta peptide (Abeta), and the accumulation of Abeta in senile plaques is a hallmark of Alzheimer's disease (AD). Understanding the mechanisms of APP processing is therefore paramount. Increasing evidence suggests that APP intracellular domain (AICD) interacting proteins influence APP processing. This study characterized the overexpression of AICD interactor GULP1 in a Drosophila AD model expressing human BACE and APP695. Transgenic GULP1 significantly lowered the levels of both Abeta1-40 and Abeta1-42 without decreasing the BACE and APP695 levels. Overexpression of GULP1 also reduced APP/BACE-mediated retinal degeneration, rescued motor dysfunction and extended longevity of the flies. These results indicate that GULP1 regulate APP processing and reduce neurotoxicity in a Drosophila AD model.
Dragh, M. A., Xu, Z., Al-Allak, Z. S. and Hong, L. (2017). Vitamin K2 Prevents Lymphoma in Drosophila. Sci Rep 7(1): 17047. PubMed ID: 29213118
Previous studies have established the anticancer effect of vitamin K2 (VK2). However, its effect on lymphoma induced by UBIAD1/heix mutation in Drosophila remains unknown. Therefore, this study aimed to develop an in vivo model of lymphoma for the precise characterization of lymphoma phenotypes. This study also aimed to improve the understanding of the mechanisms that underlie the preventative effects of VK2 on lymphoma. The results demonstrated that VK2 prevents lymphoma by acting as an electron carrier and by correcting the function and structure of mitochondria by inhibiting mitochondrial reactive oxygen species production mtROS. This work identifies mitochondria as a key player in cancer therapy strategies.

Wednesday, February 21st

Myers, L., Perera, H., Alvarado, M. G. and Kidd, T. (2017). The Drosophila Ret gene functions in the stomatogastric nervous system with the Maverick TGFbeta ligand and the Gfrl co-receptor. Development [Epub ahead of print]. PubMed ID: 29361562
The RET receptor tyrosine kinase is critical for the development of the enteric nervous system (ENS), acting as a receptor for Glial Cell Line-Derived Neurotrophic Factor (GDNF) via GFR co-receptors. Drosophila has a well-conserved RET homologue (Ret) that has been proposed to function independently of the Gfr-like co-receptor (Gfrl). This study found that Ret is required for development of the stomatogastric (enteric) nervous system (SNS) in both embryos and larvae, and its loss results in feeding defects. Live imaging analysis suggests that peristaltic waves are initiated but not propagated in mutant midguts. Examination of axons innervating the midgut reveals increased branching but the area covered by the branches is decreased. This phenotype can be rescued by Ret expression. Additionally, Gfrl shares the same ENS and feeding defects, suggesting that Ret and Gfrl might function together to respond to a ligand. This study identified the TGFbeta family member Maverick (Mav) as a ligand for Gfrl and a Mav chromosomal deficiency displayed similar embryonic ENS defects. These results suggest that the Ret and Gfrl families co-evolved before the separation of invertebrate and vertebrate lineages.
Romani, P., Duchi, S., Gargiulo, G. and Cavaliere, V. (2017). Evidence for a novel function of Awd in maintenance of genomic stability. Sci Rep 7(1): 16820. PubMed ID: 29203880
The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/NME2 metastasis suppressor genes. Awd acts in multiple tissues where its function is critical in establishing and maintaining epithelial integrity. This study analysed awd gene function in Drosophila epithelial cells using transgene-mediated RNA interference and genetic mosaic analysis. awd knockdown in larval wing disc epithelium leads to chromosomal instability (CIN) and induces apoptosis mediated by activation of c-Jun N-terminal kinase. Forced maintenance of Awd depleted cells, by expressing the cell death inhibitor p35, downregulates atypical protein kinase C and DE-Cadherin. Consistent with their loss of cell polarity and enhanced level of matrix metalloproteinase 1, cells delaminate from wing disc epithelium. Furthermore, the DNA content profile of these cells indicates that they are aneuploid. Overall, these data demonstrate a novel function for awd in maintenance of genomic stability. These results are consistent with other studies reporting that NME1 down-regulation induces CIN in human cell lines and suggest that Drosophila model could be successfully used to study in vivo the impact of NME/Awd - induced genomic instability on tumour development and metastasis formation.
Langridge, P. D. and Struhl, G. (2017). Epsin-dependent ligand endocytosis activates Notch by force. Cell 171(6): 1383-1396.e1312. PubMed ID: 29195077
DSL ligands activate Notch by inducing proteolytic cleavage of the receptor ectodomain, an event that requires ligand to be endocytosed in signal-sending cells by the adaptor protein Epsin. Two classes of explanation for this unusual requirement are (1) recycling models, in which the ligand must be endocytosed to be modified or repositioned before it binds Notch and (2) pulling models, in which the ligand must be endocytosed after it binds Notch to exert force that exposes an otherwise buried site for cleavage. This study demonstrates in vivo that ligands that cannot enter the Epsin pathway nevertheless bind Notch but fail to activate the receptor because they cannot exert sufficient force. This argues against recycling models and in favor of pulling models. These results also suggest that once ligand binds receptor, activation depends on a competition between Epsin-mediated ligand endocytosis, which induces cleavage, and transendocytosis of the ligand by the receptor, which aborts the incipient signal.
Richhariya, S. and Hasan, G. (2017). Ral function in muscle is required for flight maintenance in Drosophila. Small GTPases: 1-6. PubMed ID: 29284321
Ral is a small GTPase of the Ras superfamily that is important for a number of cellular functions. Recently work has shown that expression of Ral is regulated by store-operated calcium entry (SOCE) in Drosophila neurons. In this study, through genetic and behavioural experiments, it was shown that Ral function is required in differentiated muscles for flight. Reducing Ral function in muscles, specifically reduced duration of flight bouts but not other motor functions, like climbing. Interestingly, unlike in the nervous system, Ral expression in the muscle is not regulated by SOCE. Moreover, either knockdown or genetic inhibition of SOCE in muscles does not affect flight. These findings demonstrate that a multiplicity of signalling mechanisms very likely regulate Ral expression in different tissues.
Steinmetz, E. L., Dewald, D. N. and Walldorf, U. (2017). Homeodomain-interacting protein kinase phosphorylates the Drosophila Paired box protein 6 (Pax6) homologues Twin of eyeless and Eyeless. Insect Mol Biol [Epub ahead of print]. PubMed ID: 29205612
Homeodomain-interacting protein kinase (Hipk), the Drosophila homologue of mammalian HIPK2, plays several important roles in regulating differentiation, proliferation, apoptosis, and stress responses and acts as a mediator for signals of diverse pathways, such as Notch or Wingless signalling. The Paired box protein 6 (Pax6) has two Drosophila homologues, Twin of eyeless (Toy) and Eyeless (Ey). Both stand atop the retinal determination gene network (RDGN), which is essential for proper eye development in Drosophila. This study set Hipk and the master regulators Toy and Ey in an enzyme-substrate relationship. Furthermore, a physical interaction is proven between Toy and Hipk in vivo using bimolecular fluorescence complementation. Using in vitro kinase assays with different truncated Toy constructs and mutational analyses, four Hipk phosphorylation sites of Toy were mapped, one in the paired domain (Ser(121)) and three in the C-terminal transactivation domain of Toy (Thr(395) , Ser(410) and Thr(452)). The interaction and phosphorylation of the master regulator Toy by Hipk may be important for precise tuning of signalling within the RDGN and therefore for Drosophila eye development.
Riedel, F., Galindo, A., Muschalik, N. and Munro, S. (2017). The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases. J Cell Biol [Epub ahead of print]. PubMed ID: 29273580
Originally identified in yeast, transport protein particle (TRAPP) complexes are Rab GTPase exchange factors that share a core set of subunits. TRAPPs were initially found to act on Ypt1, the yeast orthologue of Rab1, but recent studies have found that yeast TRAPPII can also activate the Rab11 orthologues Ypt31/32. Mammals have two TRAPP complexes, but their role is less clear, and they contain subunits that are not found in the yeast complexes but are essential for cell growth. To investigate TRAPP function in metazoans, this study shows that Drosophila melanogaster have two TRAPP complexes similar to those in mammals and that both activate Rab1, whereas one, TRAPPII, also activates Rab11. TRAPPII is not essential but becomes so in the absence of the gene parcas that encodes the Drosophila orthologue of the SH3BP5 family of Rab11 guanine nucleotide exchange factors (GEFs). Thus, in metazoans, Rab1 activation requires TRAPP subunits not found in yeast, and Rab11 activation is shared by TRAPPII and an unrelated GEF that is metazoan specific.

Tuesday, February 20th

Viktorinova, I., Henry, I. and Tomancak, P. (2017). Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations. PLoS Genet 13(11): e1007107. PubMed ID: 29176774
Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. This study identified a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. Evidence is provided that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. It is proposed that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.
Tseng, C. Y. and Hsu, H. J. (2017). Decreased expression of lethal giant larvae causes ovarian follicle cell outgrowth in the Drosophila Scutoid mutant. PLoS One 12(12): e0188917. PubMed ID: 29261681
Snail, a zinc-finger transcription factor, controls the process of epithelial-mesenchymal transition, and ectopic expression of this protein may produce cells with stem cell properties. Because the effect of Snail expression in ovarian epithelial cells remains unclear, this study generated Drosophila ovarian follicle stem cells (FSCs) with homozygous Scutoid (Sco) mutation. The Sco mutation is a reciprocal transposition that is known to induce ectopic Snail activity. Sco mutant FSCs showed excess proliferation and high competitiveness for niche occupancy, and the descendants of this lineage formed outgrowths that failed to enter the endocycle. Surprisingly, such phenotypes were not rescued by suppressing Snail expression, but were completely restored by supplying Lethal giant larvae (Lgl). The lgl allele is a cell polarity gene that is often mutated in the genome. Importantly, Sco mutants survived in a complementation test with lgl. This result was probably obtained because the Sco-associated lgl allele appears to diminish, but not ablate lgl expression. While these data do not rule out the possibility that the Sco mutation disrupts a regulator of lgl transcription, the results strongly suggest that the phenotypes found in Sco mutants are more closely associated with the lgl allele than ectopic Snail activity.
Deady, L. D., Li, W. and Sun, J. (2017). The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles. Elife 6. PubMed ID: 29256860
Follicle rupture, the final step in ovulation, utilizes conserved molecular mechanisms including matrix metalloproteinases (Mmps), steroid signaling, and adrenergic signaling. It is still unknown how follicles become competent for follicle rupture/ovulation. This study identified a zinc-finger transcription factor Hindsight (Hnt) as the first transcription factor regulating follicle's competency for ovulation in Drosophila. Hnt is not expressed in immature stage-13 follicle cells but is upregulated in mature stage-14 follicle cells, which is essential for follicle rupture/ovulation. Hnt upregulates Mmp2 expression in posterior follicle cells (essential for the breakdown of the follicle wall) and Oamb expression in all follicle cells (the receptor for receiving adrenergic signaling and inducing Mmp2 activation). Hnt's role in regulating Mmp2 and Oamb can be replaced by its human homolog Ras-responsive element-binding protein 1 (RREB-1). These data suggest that Hnt/RREB-1 plays conserved role in regulating follicle maturation and competency for ovulation.
Blom-Dahl, D. and Azpiazu, N. (2017). The Pax protein Eyegone (Eyg) interacts with the pi-RNA component Aubergine (Aub) and controls egg chamber development in Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 29278721
The eyegone (eyg) gene encodes Eyg, a transcription factor of the Pax family with multiple roles during Drosophila development. Eyg has been shown to be nuclear in the cells where it functions. This report describes a new functional cytoplasmic distribution of Eyg during egg chamber development in the female ovarioles. The protein is present from the germarium until stage 10 of cyst development. The majority of egg chambers that develop in the absence of Eyg arrest their development before stage 10, show augmented levels of the telomeric retro-transposon TART-A and low levels of heterochromatin marks in the oocyte nucleus. During the maternal to zygotic transition (MTZ) Eyg seems to play a role in destabilizing germ cell less (gcl) and oo 16 RNA binding protein (orb) mRNAs. Eyg interacts with Aubergine (Aub), a component of the pi-RNA pathway during egg chamber development. This interaction could be essential for Eyg to be retained in the cytoplasm and fulfill its functions there.
Carbonell, A., Perez-Montero, S., Climent-Canto, P., Reina, O. and Azorin, F. (2017). The germline linker histone dBigH1 and the translational regulator Bam form a repressor loop essential for male germ stem cell differentiation. Cell Rep 21(11): 3178-3189. PubMed ID: 29241545
Drosophila spermatogenesis constitutes a paradigmatic system to study maintenance, proliferation, and differentiation of adult stem cell lineages. Each Drosophila testis contains 6-12 germ stem cells (GSCs) that divide asymmetrically to produce gonialblast cells that undergo four transit-amplifying (TA) spermatogonial divisions before entering spermatocyte differentiation. Mechanisms governing these crucial transitions are not fully understood. This study reports the essential role of the germline linker histone dBigH1 during early spermatogenesis. These results suggest that dBigH1 is a general silencing factor that represses Bam, a key regulator of spermatogonia proliferation that is silenced in spermatocytes. Reciprocally, Bam represses dBigH1 during TA divisions. This double-repressor mechanism switches dBigH1/Bam expression from off/on in spermatogonia to on/off in spermatocytes, regulating progression into spermatocyte differentiation. dBigH1 is also required for GSC maintenance and differentiation. These results show the critical importance of germline H1s for male GSC lineage differentiation, unveiling a regulatory interaction that couples transcriptional and translational repression.
Meiselman, M. R., Kingan, T. G. and Adams, M. E. (2018). Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation. BMC Biol 16(1): 18. PubMed ID: 29382341
Environmental stressors induce changes in endocrine state, leading to energy re-allocation from reproduction to survival. Female Drosophila melanogaster respond to thermal and nutrient stressors by arresting egg production through elevation of the steroid hormone ecdysone. However, the mechanisms through which this reproductive arrest occurs are not well understood. This study reports that stress-induced elevation of ecdysone is accompanied by decreased levels of ecdysis triggering hormone (ETH). Depressed levels of circulating ETH lead to attenuated activity of its targets, including juvenile hormone-producing corpus allatum and, as described in this study for the first time, octopaminergic neurons of the oviduct. Elevation of steroid thereby results in arrested oogenesis, reduced octopaminergic input to the reproductive tract, and consequent suppression of ovulation. ETH mitigates heat or nutritional stress-induced attenuation of fecundity, which suggests that its deficiency is critical to reproductive adaptability. These findings indicate that, as a dual regulator of octopamine and juvenile hormone release, ETH provides a link between stress-induced elevation of ecdysone levels and consequent reduction in fecundity.

Monday, February 19th

Urban, J., Kuzu, G., Bowman, S., Scruggs, B., Henriques, T., Kingston, R., Adelman, K., Tolstorukov, M. and Larschan, E. (2017). Enhanced chromatin accessibility of the dosage compensated Drosophila male X-chromosome requires the CLAMP zinc finger protein. PLoS One 12(10): e0186855. PubMed ID: 29077765
The essential process of dosage compensation is required to equalize gene expression of X-chromosome genes between males (XY) and females (XX). In Drosophila, the conserved Male-specific lethal (MSL) histone acetyltransferase complex mediates dosage compensation by increasing transcript levels from genes on the single male X-chromosome approximately two-fold. Consistent with its increased levels of transcription, the male X-chromosome has enhanced chromatin accessibility, distinguishing it from the autosomes. This study demonstrates that the non-sex-specific CLAMP (Chromatin-linked adaptor for MSL proteins) zinc finger protein that recognizes GA-rich sequences genome-wide promotes the specialized chromatin environment on the male X-chromosome and can act over long genomic distances (~14 kb). Although MSL complex is required for increasing transcript levels of X-linked genes, it is not required for enhancing global male X-chromosome chromatin accessibility, and instead works cooperatively with CLAMP to facilitate an accessible chromatin configuration at its sites of highest occupancy. Furthermore, CLAMP regulates chromatin structure at strong MSL complex binding sites through promoting recruitment of the Nucleosome Remodeling Factor (NURF) complex. In contrast to the X-chromosome, CLAMP regulates chromatin and gene expression on autosomes through a distinct mechanism that does not involve NURF recruitment. Overall, these results support a model where synergy between a non-sex-specific transcription factor (CLAMP) and a sex-specific cofactor (MSL) creates a specialized chromatin domain on the male X-chromosome.
Wakisaka, K. T., Ichiyanagi, K., Ohno, S. and Itoh, M. (2017). Diversity of P-element piRNA production among M' and Q strains and its association with P-M hybrid dysgenesis in Drosophila melanogaster. Mob DNA 8: 13. PubMed ID: 29075336
Transposition of P elements in the genome causes P-M hybrid dysgenesis in Drosophila melanogaster. For the P strain, the P-M phenotypes are associated with the ability to express a class of small RNAs, called piwi-interacting small RNAs (piRNAs), that suppress the P elements in female gonads. However, little is known about the extent to which piRNAs are involved in the P-M hybrid dysgenesis in M' and Q strains, which show different abilities to regulate the P elements from P strains. To elucidate the molecular basis of the suppression of paternally inherited P elements, this study analyzed the mRNA and piRNA levels of P elements in the F1 progeny between males of a P strain and nine-line females of M' or Q strains (M' or Q progenies). M' progenies showed the hybrid dysgenesis phenotype, while Q progenies did not. Consistently, the levels of P-element mRNA in both the ovaries and F1 embryos were higher in M' progenies than in Q progenies, indicating that the M' progenies have a weaker ability to suppress P-element expression. The level of P-element mRNA was inversely correlated to the level of piRNAs in F1 embryos. Importantly, the M' progenies were characterized by a lower abundance of P-element piRNAs in both young ovaries and F1 embryonic bodies. The Q progenies showed various levels of piRNAs in both young ovaries and F1 embryonic bodies despite all of the Q progenies suppressing P-element transposition in their gonad. These results are consistent with an idea that the level of P-element piRNAs is a determinant for dividing strain types between M' and Q and that the suppression mechanisms of transposable elements, including piRNAs, are varied between natural populations.
Urban, J. A., Urban, J. M., Kuzu, G. and Larschan, E. N. (2017). The Drosophila CLAMP protein associates with diverse proteins on chromatin. PLoS One 12(12): e0189772. PubMed ID: 29281702
Gaining new insights into gene regulation involves an in-depth understanding of protein-protein interactions on chromatin. A powerful model for studying mechanisms of gene regulation is dosage compensation, a process that targets the X-chromosome to equalize gene expression between XY males and XX females. Previous work has identified a zinc finger protein in Drosophila melanogaster that plays a sex-specific role in targeting the Male-specific lethal (MSL) dosage compensation complex to the male X-chromosome, called the Chromatin-Linked Adapter for MSL Proteins (CLAMP). CLAMP has been found to have non-sex-specific roles as an essential protein that regulates chromatin accessibility at promoters genome-wide. To identify associations between CLAMP and other factors in both male and female cells, two complementary mass spectrometry approaches were used. This study demonstrates that CLAMP associates with the transcriptional regulator complex Negative Elongation Factor (NELF; see Nelf-E) in both sexes and determine that CLAMP reduces NELF recruitment to several target genes. In sum, this study has identified many new CLAMP-associated factors and provide a resource for further study of this little understood essential protein.
Pereira, A. and Paro, R. (2017). Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus. Epigenetics Chromatin 10(1): 57. PubMed ID: 29208012
Numerous target genes of the Polycomb group (PcG) are transiently activated by a stimulus and subsequently repressed. However, mechanisms by which PcG proteins regulate such target genes remain elusive. This study employed the heat shock-responsive hsp70 locus in Drosophila to study the chromatin dynamics of PRC1 and its interplay with known regulators of the locus before, during and after heat shock. We detected mutually exclusive binding patterns for HSF and PRC1 at the hsp70 locus. Pleiohomeotic (Pho), a DNA-binding PcG member, dynamically interacts with Spt5, an elongation factor. The dynamic interaction switch between Pho and Spt5 is triggered by the recruitment of HSF to chromatin. Mutation in the protein-protein interaction domain (REPO domain) of Pho interferes with the dynamics of its interaction with Spt5. The transcriptional kinetics of the heat shock response is negatively affected by a mutation in the REPO domain of Pho. It is proposed that a dynamic interaction switch between PcG proteins and an elongation factor enables stress-inducible genes to efficiently switch between ON/OFF states in the presence/absence of the activating stimulus.
Zolotarev, N., Maksimenko, O., Kyrchanova, O., Sokolinskaya, E., Osadchiy, I., Girardot, C., Bonchuk, A., Ciglar, L., Furlong, E. E. M. and Georgiev, P. (2017). Opbp is a new architectural/insulator protein required for ribosomal gene expression. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 29036346
A special class of poorly characterized architectural proteins is required for chromatin topology and enhancer-promoter interactions. This study identified Opbp as a new Drosophila architectural protein, interacting with CP190 both in vivo and in vitro. Opbp binds to a very restrictive set of genomic regions, through a rare sequence specific motif. These sites are co-bound by CP190 in vivo, and generally located at bidirectional promoters of ribosomal protein genes. This study shows that Opbp is essential for viability, and loss of opbp function, or destruction of its motif, leads to reduced ribosomal protein gene expression, indicating a functional role in promoter activation. As characteristic of architectural/insulator proteins, the Opbp motif is sufficient for distance-dependent reporter gene activation and enhancer-blocking activity, suggesting an Opbp-mediated enhancer-promoter interaction. Rather than having a constitutive role, Opbp represents a new type of architectural protein with a very restricted, yet essential, function in regulation of housekeeping gene expression.
Ramachandran, S., Ahmad, K. and Henikoff, S. (2017). Transcription and remodeling produce asymmetrically unwrapped nucleosomal intermediates. Mol Cell 68(6): 1038-1053.e1034. PubMed ID: 29225036
Nucleosomes are disrupted during transcription and other active processes, but the structural intermediates during nucleosome disruption in vivo are unknown. To identify intermediates, This study mapped subnucleosomal protections in Drosophila cells using Micrococcal Nuclease followed by sequencing. At the first nucleosome position downstream of the transcription start site, unwrapped intermediates were identified, including hexasomes that lack either proximal or distal contacts. Inhibiting topoisomerases or depleting histone chaperones increased unwrapping, whereas inhibiting release of paused RNAPII or reducing RNAPII elongation decreased unwrapping. These results indicate that positive torsion generated by elongating RNAPII causes transient loss of histone-DNA contacts. Using this mapping approach, it was found that nucleosomes flanking human CTCF insulation sites are similarly disrupted. Diagnostic subnucleosomal particle remnants were idenfied in cell-free human DNA data as a relic of transcribed genes from apoptosing cells. Thus identification of subnucleosomal fragments from nuclease protection data represents a general strategy for structural epigenomics.
Verghese, S. and Su, T. T. (2017). STAT, Wingless, and Nurf-38 determine the accuracy of regeneration after radiation damage in Drosophila. PLoS Genet 13(10): e1007055. PubMed ID: 29028797
Regeneration in Drosophila larval wing imaginal discs has been studied after damage by ionizing radiation. Faithful regeneration was detected that restored a wing disc and abnormal regeneration that produced an extra wing disc. A sequence of changes in cell number, location and fate is described that occur to produce an ectopic disc. A group of cells was identified that not only participate in ectopic disc formation but also recruit others to do so. STAT92E (Drosophila STAT3/5) and Nurf-38, which encodes a member of the Nucleosome Remodeling Factor complex, oppose each other in these cells to modulate the frequency of ectopic disc growth. The picture that emerges is one in which activities like STAT increase after radiation damage and fulfill essential roles in rebuilding the tissue. But such activities must be kept in check so that one and only one wing disc is regenerated.
Marshall, O. J. and Brand, A. H. (2017). Chromatin state changes during neural development revealed by in vivo cell-type specific profiling. Nat Commun 8(1): 2271. PubMed ID: 29273756
A key question in developmental biology is how cellular differentiation is controlled during development. While transitions between trithorax-group (TrxG) and polycomb-group (PcG) chromatin states are vital for the differentiation of ES cells to multipotent stem cells, little is known regarding the role of chromatin states during development of the brain. This study shows that large-scale chromatin remodelling occurs during Drosophila neural development. The majority of genes activated during neuronal differentiation are silent in neural stem cells (NSCs) and occupy black chromatin and a TrxG-repressive state. In neurons, almost all key NSC genes are switched off via HP1-mediated repression. PcG-mediated repression does not play a significant role in regulating these genes, but instead regulates lineage-specific transcription factors that control spatial and temporal patterning in the brain. Combined, these data suggest that forms of chromatin other than canonical PcG/TrxG transitions take over key roles during neural development.

Friday, February 16th

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
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 addressed these questions by profiling the D. melanogaster transcriptomic response to 10 bacteria that span the spectrum of virulence. Each bacterium was found to trigger 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, it was determined that 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.
Duneau, D. F., Kondolf, H. C., Im, J. H., Ortiz, G. A., Chow, C., Fox, M. A., Eugenio, A. T., Revah, J., Buchon, N. and Lazzaro, B. P. (2017). The Toll pathway underlies host sexual dimorphism in resistance to both Gram-negative and Gram-positive bacteria in mated Drosophila. BMC Biol 15(1): 124. PubMed ID: 29268741
This study used Drosophila melanogaster to assess and dissect sexual dimorphism in the innate response to systemic bacterial infection. Both virgin and mated females are more susceptible than mated males to most, but not all, infections. The lower resistance of females to infection with Providencia rettgeri, a Gram-negative bacterium that naturally infects D. melanogaster was investigated. Females were found to have a higher number of phagocytes than males, and ablation of hemocytes does not eliminate the dimorphism in resistance to P. rettgeri, so the observed dimorphism does not stem from differences in the cellular response. The Imd pathway is critical for the production of antimicrobial peptides in response to Gram-negative bacteria, but mutants for Imd signaling continued to exhibit dimorphism even though both sexes showed strongly reduced resistance. Instead, it was found that the Toll pathway is responsible for the dimorphism in resistance. The Toll pathway is dimorphic in genome-wide constitutive gene expression and in induced response to infection. Toll signaling is dimorphic in both constitutive signaling and in induced activation in response to P. rettgeri infection. The dimorphism in pathway activation can be specifically attributed to Persephone-mediated immune stimulation, by which the Toll pathway is triggered in response to pathogen-derived virulence factors. It was additionally found that, in absence of Toll signaling, males become more susceptible than females to the Gram-positive Enterococcus faecalis. This reversal in susceptibility between male and female Toll pathway mutants compared to wildtype hosts highlights the key role of the Toll pathway in D. melanogaster sexual dimorphism in resistance to infection. Altogether, these data demonstrate that Toll pathway activity differs between male and female D. melanogaster in response to bacterial infection.
Perez-Zamorano, B., Rosas-Madrigal, S., Lozano, O. A. M., Castillo Mendez, M. and Valverde-Garduno, V. (2017). Identification of cis-regulatory sequences reveals potential participation of lola and Deaf1 transcription factors in Anopheles gambiae innate immune response. PLoS One 12(10): e0186435. PubMed ID: 29028826
The innate immune response of Anopheles gambiae involves the transcriptional upregulation of effector genes. Therefore, the cis-regulatory sequences and their cognate binding factors play essential roles in the mosquito's immune response. However, the genetic control of the mosquito's innate immune response is not yet fully understood. To gain further insight on the elements, the factors and the potential mechanisms involved, an open chromatin profiling was carried out on A. gambiae-derived immune-responsive cells. This study reports the identification of cis-regulatory sites, immunity-related transcription factor binding sites, and cis-regulatory modules. A de novo motif discovery carried out on this set of cis-regulatory sequences identified immunity-related motifs and cis-regulatory modules. These modules contain motifs that are similar to binding sites for REL-, STAT-, lola- and Deaf1-type transcription factors. Sequence motifs similar to the binding sites for GAGA were found within a cis-regulatory module, together with immunity-related transcription factor binding sites. The presence of Deaf1- and lola-type binding sites, along with REL- and STAT-type binding sites, suggests that the immunity function of these two factors could have been conserved both in Drosophila and Anopheles gambiae.
Ragheb, R., Chuyen, A., Torres, M., Defaye, A., Seyres, D., Kremmer, L., Fernandez-Nunez, N., Tricoire, H., Rihet, P., Nguyen, C., Roder, L. and Perrin, L. (2017). Interplay between trauma and Pseudomonas entomophila infection in flies: a central role of the JNK pathway and of CrebA. Sci Rep 7(1): 16222. PubMed ID: 29176735
In mammals, both sterile wounding and infection induce inflammation and activate the innate immune system, and the combination of both challenges may lead to severe health defects, revealing the importance of the balance between the intensity and resolution of the inflammatory response for the organism's fitness. Underlying mechanisms remain however elusive. Using Drosophila, this study showed that, upon infection with the entomopathogenic bacterium Pseudomonas entomophila (Pe), a sterile wounding induces a reduced resistance and increased host mortality. To identify the molecular mechanisms underlying the susceptibility of wounded flies to bacterial infection, the very first steps of the process were analyzed by comparing the transcriptome landscape of infected (simple hit flies, SH), wounded and infected (double hit flies, DH) and wounded (control) flies. Overexpressed genes in DH flies compared to SH ones are significantly enriched in genes related to stress, including members of the JNK pathway. The JNK pathway plays a central role in the DH phenotype by manipulating the Jra/dJun activity. Moreover, the CrebA/Creb3-like transcription factor (TF) and its targets were up-regulated in SH flies, and CrebA was shown to be required for mounting an appropriate immune response. Drosophila thus appears as a relevant model to investigate interactions between trauma and infection and allows unraveling of key pathways involved.
Yanagawa, A., Neyen, C., Lemaitre, B. and Marion-Poll, F. (2017). The gram-negative sensing receptor PGRP-LC contributes to grooming induction in Drosophila. PLoS One 12(11): e0185370. PubMed ID: 29121087
Behavioral resistance protects insects from microbial infection. However, signals inducing insect hygiene behavior are still relatively unexplored. A previous study demonstrated that olfactory signals from microbes enhance insect hygiene behavior, and gustatory signals even induce the behavior. In this paper, cross-talk between behavioral resistance and innate immunity is postulated. To examine this hypothesis, a previously validated behavioral test was employed to examine the function of taste signals in inducing a grooming reflex in decapitated flies. Microbes, which activate different pattern recognition systems upstream of immune pathways, were applied to see if there was any correlation between microbial perception and grooming reflex. To narrow down candidate elicitors, the grooming induction tests were conducted with highly purified bacterial components. Lastly, the role of DAP-type peptidoglycan in grooming induction was confirmed. These results demonstrate that cleaning behavior can be triggered through recognition of diaminopimelic acid-type bacterial peptidoglycan by its receptor PGRP-LC.
Sung, E. J., et al. (2017). Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress. Proc Natl Acad Sci U S A 114(52): 13786-13791. PubMed ID: 29229844
This study used Drosophila to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca(2+) signaling pathway mediates innate immune responses to GBP, this study conducted a dsRNA library screen for genes that modulate Ca(2+) mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. The metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This was established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. It was further reported that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these homeostatic attributes of GBP: Elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan.
Patrnogic, J. and Leclerc, V. (2017). The serine protease homolog spheroide is involved in sensing of pathogenic Gram-positive bacteria. PLoS One 12(12): e0188339. PubMed ID: 29211760
In Drosophila, recognition of pathogens such as Gram-positive bacteria and fungi triggers the activation of proteolytic cascades and the subsequent activation of the Toll pathway. This response can be achieved by either detection of pathogen associated molecular patterns or by sensing microbial proteolytic activities ("danger signals"). Previous data suggested that certain serine protease homologs (serine protease folds that lack an active catalytic triad) could be involved in the pathway. This study generated a null mutant of the serine protease homolog spheroide (sphe). These mutant flies are susceptible to Enterococcus faecalis infection and unable to fully activate the Toll pathway. Sphe is required to activate the Toll pathway after challenge with pathogenic Gram-Positive bacteria. Sphe functions in the danger signal pathway, downstream or at the level of Persephone.
Tusco, R., Jacomin, A. C., Jain, A., Penman, B. S., Larsen, K. B., Johansen, T. and Nezis, I. P. (2017). Kenny mediates selective autophagic degradation of the IKK complex to control innate immune responses. Nat Commun 8(1): 1264. PubMed ID: 29097655
Selective autophagy is a catabolic process with which cellular material is specifically targeted for degradation by lysosomes. The function of selective autophagic degradation of self-components in the regulation of innate immunity is still unclear. This study shows that Drosophila Kenny, the homolog of mammalian IKKgamma, is a selective autophagy receptor that mediates the degradation of the IkappaB kinase complex. Selective autophagic degradation of the IkappaB kinase complex prevents constitutive activation of the immune deficiency pathway in response to commensal microbiota. Autophagy-deficient flies have a systemic innate immune response that promotes a hyperplasia phenotype in the midgut. Remarkably, human IKKgamma does not interact with mammalian Atg8-family proteins. Using a mathematical model, this suggests mechanisms by which pathogen selection might have driven the loss of LIR motif functionality during evolution. These results suggest that there may have been an autophagy-related switch during the evolution of the IKKgamma proteins in metazoans.

Thursday, February 15th

Cabirol-Pol, M. J., Khalil, B., Rival, T., Faivre-Sarrailh, C. and Besson, M. T. (2017). Glial lipid droplets and neurodegeneration in a Drosophila model of complex I deficiency. Glia [Epub ahead of print]. PubMed ID: 29285794
Mitochondrial defects associated with respiratory chain complex I deficiency lead to heterogeneous fatal syndromes. While the role of NDUFS8, an essential subunit of the core assembly of the complex I, is established in mitochondrial diseases, the mechanisms underlying neuropathology are poorly understood. This study developed a Drosophila model of NDUFS8 deficiency by knocking down the expression of its fly homologue in neurons or in glial cells. Downregulating ND23 in neurons resulted in shortened lifespan, and decreased locomotion. Although total brain ATP levels were decreased, histological analysis did not reveal any signs of neurodegeneration except for photoreceptors of the retina. Interestingly, ND23 deficiency-associated phenotypes were rescued by overexpressing the glucose transporter hGluT3 demonstrating that boosting glucose metabolism in neurons was sufficient to bypass altered mitochondrial functions and to confer neuroprotection. The consequences of ND23 knockdown was then studied in glial cells. In contrast to neuronal knockdown, loss of ND23 in glia did not lead to significant behavioral defects nor to reduced lifespan, but induced brain degeneration, as visualized by numerous vacuoles found all over the nervous tissue. This phenotype was accompanied by the massive accumulation of lipid droplets at the cortex-neuropile boundaries, suggesting an alteration of lipid metabolism in glia. These results demonstrate that complex I deficiency triggers metabolic alterations both in neurons and glial cells which may contribute to the neuropathology.
Damrau, C., Toshima, N., Tanimura, T., Brembs, B. and Colomb, J. (2017). Octopamine and tyramine contribute separately to the counter-regulatory response to sugar deficit in Drosophila. Front Syst Neurosci 11: 100. PubMed ID: 29379421
All animals constantly negotiate external with internal demands before and during action selection. Energy homeostasis is a major internal factor biasing action selection. For instance, in addition to physiologically regulating carbohydrate mobilization, starvation-induced sugar shortage also biases action selection toward food-seeking and food consumption behaviors (the counter-regulatory response). Biogenic amines are often involved when such widespread behavioral biases need to be orchestrated. In mammals, norepinephrine (noradrenalin) is involved in the counterregulatory response to starvation-induced drops in glucose levels. The invertebrate homolog of noradrenalin, octopamine (OA) and its precursor tyramine (TA) are neuromodulators operating in many different neuronal and physiological processes. Tyrosine-β-hydroxylase (tβh) mutants are unable to convert TA into OA. It was hypothesized that tβh mutant flies may be aberrant in some or all of the counter-regulatory responses to starvation and that techniques restoring gene function or amine signaling may elucidate potential mechanisms and sites of action. Corroborating this hypothesis, starved mutants show a reduced sugar response and their hemolymph sugar concentration is elevated compared to control flies. When starved, they survive longer. Temporally controlled rescue experiments revealed an action of the OA/TA-system during the sugar response, while spatially controlled rescue experiments suggest actions also outside of the nervous system. Additionally, the analysis of two OA- and four TA-receptor mutants suggests an involvement of both receptor types in the animals' physiological and neuronal response to starvation.
Lau, M. T., Lin, Y. Q., Kisling, S., Cotterell, J., Wilson, Y. A., Wang, Q. P., Khuong, T. M., Bakhshi, N., Cole, T. A., Oyston, L. J., Cole, A. R. and Neely, G. G. (2017). A simple high throughput assay to evaluate water consumption in the fruit fly. Sci Rep 7(1): 16786. PubMed ID: 29196744
Water intake is essential for survival and thus under strong regulation. This study describes a simple high throughput system to monitor water intake over time in Drosophila. The design of the assay involves dehydrating fly food and then adding water back separately so flies either eat or drink. Water consumption is then evaluated by weighing the water vessel and comparing this back to an evaporation control. This system is high throughput, does not require animals to be artificially dehydrated, and is simple both in design and implementation. Initial characterisation of homeostatic water consumption shows high reproducibility between biological replicates in a variety of experimental conditions. Water consumption was dependent on ambient temperature and humidity and was equal between sexes when corrected for mass. By combining this system with the Drosophila genetics tools, it was possible to confirm a role for ppk28 and DopR1 in promoting water consumption, and through functional investigation of RNAseq data from dehydrated animals, it was found that DopR1 expression in the mushroom body was sufficient to drive consumption and enhance water taste sensitivity. Together, this study provides a simple high throughput water consumption assay that can be used to dissect the cellular and molecular machinery regulating water homeostasis in Drosophila.
Mohr, S. E., Rudd, K., Hu, Y., Song, W. R., Gilly, Q., Buckner, M., Housden, B. E., Kelley, C., Zirin, J., Tao, R., Amador, G., Sierzputowska, K., Comjean, A. and Perrimon, N. (2017). Zinc detoxification: A functional genomics and transcriptomics analysis in Drosophila melanogaster cultured cells. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 29223976
This paper presents the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wildtype or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, 47 genes were identified for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and more than 1800 putative zinc-responsive genes were identified as well. Analysis of the 'omics data points to the relevance of ion transporters, glutathione-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). Knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells and rdog is transcriptionally up-regulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the 'omics datasets presented in this study provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.
Monck, H., Toppe, D., Michael, E., Sigrist, S., Richter, V., Hilpert, D., Raccuglia, D., Efetova, M. and Schwarzel, M. (2017). A new method to characterize function of the Drosophila heart by means of optical flow. J Exp Biol 220(Pt 24): 4644-4653. PubMed ID: 29237767
The minuteness of Drosophila poses a challenge to quantify performance of its tubular heart and computer-aided analysis of its beating heart has evolved as a resilient compromise between instrumental costs and data robustness. This paper introduces an optical flow algorithm (OFA) that continuously registers coherent movement within videos of the beating Drosophila heart and uses this information to subscribe the time course of observation with characteristic phases of cardiac contraction or relaxation. The OFA combines high discriminatory power with robustness to characterize the performance of the Drosophila tubular heart using indicators from human cardiology. Proof of this concept is provided using the test bed of established cardiac conditions that include the effects of ageing, knockdown of the slow repolarizing potassium channel subunit KCNQ and ras-mediated hypertrophy of the heart tube. Together, this establishes the analysis of coherent movement as a suitable indicator of qualitative changes of the heart's beating characteristics, which improves the usefulness of Drosophila as a model of cardiac diseases.
Martinelli, E., Ludke, A., Adamo, P., Strauch, M., Di Natale, C. and Galizia, C. G. (2017). Normalizing brain activity across individuals using functional reference mapping. Sci Rep 7(1): 17128. PubMed ID: 29214995
Neural activity can be mapped across individuals using brain atlases, but when spatial relationships are not equal, these techniques collapse. This study mapped activity across individuals using functional registration, based on physiological responses to predetermined reference stimuli. Data from several individuals are integrated into a common multidimensional stimulus space, where dimensionality and axes are defined by these reference stimuli. This technique was used to discriminate volatile compounds with a cohort of Drosophila flies, by recording odor responses in receptor neurons on the flies' antennae. This technique is proposed for the development of reliable biological sensors when activity raw data cannot be calibrated. In particular, this technique will be useful for evaluating physiological measurements in natural chemosensory systems, and therefore will allow to exploit the sensitivity and selectivity of olfactory receptors present in the animal kingdom for analytical purposes.

Wednesday, February 14th

Kavaler, J., Duan, H., Aradhya, R., de Navas, L. F., Joseph, B., Shklyar, B. and Lai, E. C. (2017). miRNA suppression of a Notch repressor directs non-neuronal fate in Drosophila mechanosensory organs. J Cell Biol 217(2):571-583. PubMed ID: 29196461
Although there is abundant evidence that individual microRNA (miRNA) loci repress large cohorts of targets, large-scale knockout studies suggest that most miRNAs are phenotypically dispensable. This study identified a rare case of developmental cell specification that is highly dependent on miRNA control of an individual target. Binary cell fate choice in the Drosophila melanogaster peripheral sensory organ lineage is controlled by the non-neuronally expressed mir-279/996 cluster, with a majority of notum sensory organs exhibiting transformation of sheath cells into ectopic neurons. The mir-279/996 defect phenocopies Notch loss of function during the sheath-neuron cell fate decision, suggesting the miRNAs facilitate Notch signaling. Consistent with this, mir-279/996 knockouts are strongly enhanced by Notch heterozygosity, and activated nuclear Notch is impaired in the miRNA mutant. Although Hairless (H) is the canonical nuclear Notch pathway inhibitor, and H heterozygotes exhibit bristle cell fate phenotypes reflecting gain-of-Notch signaling, H/+ does not rescue mir-279/996 mutants. Instead, Insensible (Insb), another neural nuclear Notch pathway inhibitor, was identified as a critical direct miR-279/996 target. Insb is posttranscriptionally restricted to neurons by these miRNAs, and its heterozygosity strongly suppresses ectopic peripheral nervous system neurons in mir-279/996 mutants. Thus, proper assembly of multicellular mechanosensory organs requires a double-negative circuit involving miRNA-mediated suppression of a Notch repressor to assign non-neuronal cell fate.
Sanfilippo, P., Wen, J. and Lai, E. C. (2017). Landscape and evolution of tissue-specific alternative polyadenylation across Drosophila species. Genome Biol 18(1): 229. PubMed ID: 29191225
Drosophila melanogaster has one of best-described transcriptomes of any multicellular organism. Nevertheless, the paucity of 3'-sequencing data in this species precludes comprehensive assessment of alternative polyadenylation (APA), which is subject to broad tissue-specific control. This study generated deep 3'-sequencing data from 23 developmental stages, tissues, and cell lines of Drosophila, yielding a comprehensive atlas of ~ 62,000 polyadenylated ends. These data broadly extend the annotated transcriptome, identify ~ 40,000 novel 3' termini, and reveal that two-thirds of Drosophila genes are subject to APA. Furthermore, this study dramatically expands the numbers of genes known to be subject to tissue-specific APA, such as 3' untranslated region (UTR) lengthening in head and shortening in testis, and characterizes new tissue and developmental 3' UTR patterns. These thorough 3' UTR annotations permit reassessment of post-transcriptional regulatory networks, via conserved miRNA and RNA binding protein sites. To evaluate the evolutionary conservation and divergence of APA patterns, developmental and tissue-specific 3'-seq libraries from Drosophila yakuba and Drosophila virilis were generated. Broadly analogous tissue-specific APA trends were documented in these species, but also significant alterations were observed in 3' end usage across orthologs. The population of functionally evolving poly(A) sites was exploited to gain clear evidence that evolutionary divergence in core polyadenylation signal (PAS), and downstream sequence element (DSE) motifs drive broad alterations in 3' UTR isoform expression across the Drosophila phylogeny. These data provide a critical resource for the Drosophila community and offer many insights into the complex control of alternative tissue-specific 3' UTR formation and its consequences for post-transcriptional regulatory networks.
Tants, J. N., Fesser, S., Kern, T., Stehle, R., Geerlof, A., Wunderlich, C., Juen, M., Hartlmuller, C., Bottcher, R., Kunzelmann, S., Lange, O., Kreutz, C., Forstemann, K. and Sattler, M. (2017). Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference. Nucleic Acids Res 45(21):12536-12550. PubMed ID: 29040648
RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing it was demonstrated that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC.
Wong, J. T., Akhbar, F., Ng, A. Y. E., Tay, M. L., Loi, G. J. E. and Pek, J. W. (2017). DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1. Nat Commun 8(1): 759. PubMed ID: 28970471
Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. This study reports that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, this study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila. Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. The study shows that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis.
Lou, W. P., et al. (2017). Regulation of adult CNS axonal regeneration by the post-transcriptional regulator Cpeb1. Front Mol Neurosci 10: 445. PubMed ID: 29379413
Adult mammalian central nervous system (CNS) neurons are unable to regenerate following axonal injury, leading to permanent functional impairments. Yet, the reasons underlying this regeneration failure are not fully understood. This study examined the transcriptome and translatome shortly after spinal cord injury. Profiling of the total and ribosome-bound RNA in injured and naive spinal cords identified a substantial post-transcriptional regulation of gene expression. In particular, transcripts associated with nervous system development were down-regulated in the total RNA fraction while remaining stably loaded onto ribosomes. Interestingly, motif association analysis of post-transcriptionally regulated transcripts identified the cytoplasmic polyadenylation element (CPE) as enriched in a subset of these transcripts that was more resistant to injury-induced reduction at the transcriptome level. Modulation of these transcripts by overexpression of the CPE binding protein, Cpeb1, in mouse and Drosophila CNS neurons promoted axonal regeneration following injury. This study uncovered a global evolutionarily conserved post-transcriptional mechanism enhancing regeneration of injured CNS axons.
Luhur, A., Buddika, K., Ariyapala, I. S., Chen, S. and Sokol, N. S. (2017). Opposing post-transcriptional control of InR by FMRP and LIN-28 adjusts stem cell-based tissue growth. Cell Rep 21(10): 2671-2677. PubMed ID: 29212015
Although the intrinsic mechanisms that control whether stem cells divide symmetrically or asymmetrically underlie tissue growth and homeostasis, they remain poorly defined. This study reports that the RNA-binding protein fragile X mental retardation protein (FMRP) limits the symmetric division, and resulting expansion, of the stem cell population during adaptive intestinal growth in Drosophila. The elevated insulin sensitivity that FMRP-deficient progenitor cells display contributes to their accelerated expansion, which is suppressed by the depletion of insulin-signaling components. This FMRP activity is mediated solely via a second conserved RNA-binding protein, LIN-28, known to boost insulin signaling in stem cells. Via LIN-28, FMRP controls progenitor cell behavior by post-transcriptionally repressing the level of Insulin receptor (InR). This study identifies the stem cell-based mechanism by which FMRP controls tissue adaptation, and it raises the possibility that defective adaptive growth underlies the accelerated growth, gastrointestinal, and other symptoms that affect fragile X syndrome patients.

Tuesday, February 13th

Brown, H. E., Reichert, M. C. and Evans, T. A. (2017). In vivo functional analysis of Drosophila Robo1 fibronectin type III repeats. G3 (Bethesda). PubMed ID: 29217730
The repellant ligand Slit and its Roundabout (Robo) family receptors regulate midline crossing of axons during development of the embryonic CNS. Slit proteins are produced at the midline and signal through Robo receptors to repel axons from the midline. Disruption of Slit-Robo signaling causes ectopic midline crossing phenotypes in the CNS of a broad range of animals, including insects and vertebrates. While previous studies have investigated the roles of Drosophila melanogaster Robo1's five Immunoglobulin-like (Ig) domains, little is known about the importance of the three evolutionarily conserved Fibronectin (Fn) type-III repeats. Each of each of Drosophila Robo1's three Fn repeats were individually deleted, and then these Robo1 variants were tested in vitro to determine their ability to bind Slit in cultured Drosophila cells and in vivo to investigate the requirement for each domain in regulating Robo1's embryonic expression pattern, axonal localization, midline repulsive function, and sensitivity to Commissureless (Comm) downregulation. We demonstrate that the Fn repeats are not required for Robo1 to bind Slit or for proper expression of Robo1 in Drosophila embryonic neurons. When expressed in a robo1 mutant background, these variants are able to restore midline repulsion to an extent equivalent to full-length Robo1. A novel requirement is identified for Fn3 in the exclusion of Robo1 from commissures and downregulation of Robo1 by Comm. These results indicate that each of the Drosophila Robo1 Fn repeats are individually dispensable for the protein's role in midline repulsion, despite the evolutionarily conserved "5+3" protein structure.
Fuenzalida-Uribe, N. and Campusano, J. M. (2017). Unveiling the dual role of the dopaminergic system on locomotion and the innate value for an aversive olfactory stimulus in Drosophila. Neuroscience 371: 433-444. PubMed ID: 29292079
The communication between sensory systems and the specific brain centers that process this information is crucial to develop adequate behavioral responses. Modulatory systems, including dopaminergic circuits, regulate this communication to finely tune the behavioral response associated to any given stimulus. For instance, the Mushroom Body (MB), an insect brain integration center that receives and processes several sensory stimuli and organizes the execution of motor programs, communicates with MB output neurons (MBONs) to develop behavioral responses associated to olfactory stimuli. This communication is modulated by dopaminergic neural systems. This study shows that silencing dopaminergic neurons increases the aversive response observed in adult flies exposed to Benzaldehyde (Bz) or octanol. The contribution was studied of two dopaminergic clusters that innervate different zones of MB, Protocerebral anterior medial (PAM) and Protocerebral posterior lateral 1 (PPL1), on the innate value to the aversive stimulus and the associated locomotor behavior. In order to do this, the synaptic transmission of these neural clusters was manipulated through the expression of Tetanus toxin, Kir2.1 and Transient receptor potential cation channel A1 (TrpA1) channels. The results show that neurons in PPL1 and PAM differentially modulate the innate value to Bz in adult flies. On the other hand, blocking neurotransmission or genetic silencing of PAM neurons results in decreased locomotor behavior in flies, an effect not observed when silencing PPL1. These results suggest that as in mammals, specific dopaminergic pathways differentially modulate locomotor behavior and the innate value for an odorant, a limbic-like response in Drosophila.
Chen, Y. D. and Dahanukar, A. (2017). Molecular and cellular organization of taste neurons in adult Drosophila pharynx. Cell Rep 21(10): 2978-2991. PubMed ID: 29212040
The Drosophila pharyngeal taste organs are poorly characterized despite their location at important sites for monitoring food quality. Functional analysis of pharyngeal neurons has been hindered by the paucity of molecular tools to manipulate them, as well as their relative inaccessibility for neurophysiological investigations. This study generated receptor-to-neuron maps of all three pharyngeal taste organs by performing a comprehensive chemoreceptor-GAL4/LexA expression analysis. The organization of pharyngeal neurons reveals similarities and distinctions in receptor repertoires and neuronal groupings compared to external taste neurons. The mapping results were validated by pinpointing a single pharyngeal neuron required for feeding avoidance of L-canavanine. Inducible activation of pharyngeal taste neurons reveals functional differences between external and internal taste neurons and functional subdivision within pharyngeal sweet neurons. These results provide roadmaps of pharyngeal taste organs in an insect model system for probing the role of these understudied neurons in controlling feeding behaviors.
Walsh, K. T. and Doe, C. Q. (2017). Drosophila embryonic type II neuroblasts: origin, temporal patterning, and contribution to the adult central complex. Development 144: 4552-4562. PubMed ID: 29158446
Drosophila neuroblasts are an excellent model for investigating how neuronal diversity is generated. Most brain neuroblasts generate a series of ganglion mother cells (GMCs) that each make two neurons (type I lineage), but sixteen brain neuroblasts generate a series of intermediate neural progenitors (INPs) that each produce 4-6 GMCs and 8-12 neurons (type II lineage). Thus, type II lineages are similar to primate cortical lineages, and may serve as models for understanding cortical expansion. Yet the origin of type II neuroblasts remains mysterious: do they form in the embryo or larva? If they form in the embryo, do their progeny populate the adult central complex, as do the larval type II neuroblast progeny? This study presents molecular and clonal data showing that all type II neuroblasts form in the embryo, produce INPs, and express known temporal transcription factors. Embryonic type II neuroblasts and INPs undergo quiescence, and produce embryonic-born progeny that contribute to the adult central complex. These results provide a foundation for investigating the development of the central complex, and tools for characterizing early-born neurons in central complex function.
Ahn, J. E., Chen, Y. and Amrein, H. (2017). Molecular basis of fatty acid taste in Drosophila. Elife 6. PubMed ID: 29231818
Behavioral studies have established that Drosophila appetitive taste responses towards fatty acids are mediated by sweet sensing Gustatory Receptor Neurons (GRNs). This study shows that sweet GRN activation requires the function of the Ionotropic Receptor genes IR25a, IR76b and IR56d. The former two IR genes are expressed in several neurons per sensillum, while IR56d expression is restricted to sweet GRNs. Importantly, loss of appetitive behavioral responses to fatty acids in IR25a and IR76b mutant flies can be completely rescued by expression of respective transgenes in sweet GRNs. Interestingly, appetitive behavioral responses of wild type flies to hexanoic acid reach a plateau at ~1%, but decrease with higher concentration, a property mediated through IR25a/IR76b independent activation of bitter GRNs. With previous report on sour taste, these studies suggest that IR-based receptors mediate different taste qualities through cell-type specific IR subunits.
Huang, T. H., Niesman, P., Arasu, D., Lee, D., De La Cruz, A. L., Callejas, A., Hong, E. J. and Lois, C. (2017). Tracing neuronal circuits in transgenic animals by transneuronal control of transcription (TRACT). Elife 6. PubMed ID: 29231171
Understanding the computations that take place in brain circuits requires identifying how neurons in those circuits are connected to one another. This study describes a technique called TRACT (TRAnsneuronal Control of Transcription) based on ligand-induced intramembrane proteolysis to reveal monosynaptic connections arising from genetically labeled neurons of interest. In this strategy, neurons expressing an artificial ligand ('donor' neurons) bind to and activate a genetically-engineered artificial receptor on their synaptic partners ('receiver' neurons). Upon ligand-receptor binding at synapses the receptor is cleaved in its transmembrane domain and releases a protein fragment that activates transcription in the synaptic partners. Using TRACT in Drosophila this study has confirmed the connectivity between olfactory receptor neurons and their postsynaptic targets, and have discovered potential new connections between neurons in the circadian circuit. These results demonstrate that the TRACT method can be used to investigate the connectivity of neuronal circuits in the brain.

Monday, February 12th

Starble, R. and Pokrywka, N. J. (2017). The retromer subunit Vps26 mediates Notch signaling during Drosophila oogenesis. Mech Dev. PubMed ID: 29031909
During endocytosis, molecules are internalized by the cell through the invagination of the plasma membrane. Endocytosis is required for proper cell function and for normal development in Drosophila. One component of the endocytic pathway is the retromer complex, which recycles transmembrane proteins to other parts of the cell such as the plasma membrane and the trans-Golgi network. Previous studies have shown that mutations to the retromer complex result in developmental defects in Drosophila. In humans, retromer dysfunction has been implicated in Alzheimer's and Parkinson's disease, but little is known about the role of the retromer complex in Drosophila oogenesis. This project examined the role of the retromer protein Vps26 in oogenesis by characterizing the phenotype of vps26 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins and vesicular trafficking markers in mutant egg chambers. vps26 germline clones exhibit a signaling defect between the germline cells and follicle cells indicated by an increase in LysoTracker staining of the border cells in the mutants. This signaling defect in vps26 mutants may be the result of impaired Notch signaling based on the misexpression of multiple proteins in the Notch signaling pathway in vps26 mutants.
Zhang, L., Ribbeck, K., Turner, B. and Ten Hagen, K. G. (2017). Loss of the mucosal barrier alters the progenitor cell niche via JAK/STAT signaling. J Biol Chem 292(52):21231-21242. PubMed ID: 29127201
The mucous barrier of the digestive tract is the first line of defense against pathogens and damage. Disruptions in this barrier are associated with diseases such as Crohn's disease, colitis and colon cancer, but mechanistic insights into these processes and diseases are limited. Loss of a conserved O-glycosyltransferase (PGANT4) in Drosophila has been shown to result in aberrant secretion of components of the peritrophic/mucous membrane in the larval digestive tract. This study shows that loss of pgant4 disrupts the mucosal barrier, resulting in epithelial expression of the IL-6-like cytokine Upd3, leading to activation of JAK/STAT signaling, differentiation of cells that form the progenitor cell niche and abnormal proliferation of progenitor cells. This niche disruption could be recapitulated by overexpressing upd3 and rescued by deleting upd3, highlighting a crucial role for this cytokine. Moreover, niche integrity and cell proliferation in pgant4-deficient animals could be rescued by overexpression of the conserved cargo receptor Tango1 and partially rescued by supplementation with exogenous mucins or treatment with antibiotics. These findings help elucidate the paracrine signaling events activated by a compromised mucosal barrier and provide a novel in vivo screening platform for mucin mimetics and other strategies to treat diseases of the oral mucosa and digestive tract.
Yadav, S., Thakur, R., Georgiev, P., Deivasigamani, S., K, H., Ratnaparkhi, G. and Raghu, P. (2017). RDGBalpha localization and function at a membrane contact site is regulated by FFAT/VAP interactions. J Cell Sci [Epub ahead of print]. PubMed ID: 29180517
Phosphatidylinositol transfer proteins (PITPs) are essential regulators of PLC signalling. The PI transfer domain (PITPd) of multi-domain PITPs is reported to be sufficient for in vivo function questioning the relevance of other domains in the protein. In Drosophila photoreceptors, loss of RDGBalpha, a multi-domain PITP localized to membrane contact sites (MCS), results in multiple defects during PLC signalling. This study report that the PITPd of RDGBalpha does not localize to MCS and fails to support function during strong PLC stimulation. The MCS localization of RDGBalpha depends on the interaction of its FFAT motif with dVAP-A. Disruption of the FFAT motif (RDGB(FF/AA)) or downregulation of dVAP-A, both result in mislocalization of RDGBalpha and are associated with loss of function. Importantly, the ability of the PITPd in full-length RDGB(FF/AA) to rescue mutant phenotypes was significantly worse than that of the PITPd alone indicating that an intact FFAT motif is necessary for PITPd activity in vivo. Thus the interaction between the FFAT motif and dVAP-A confers not only localization but also intramolecular regulation on lipid transfer by the PITPd of RDGBalpha.
Cheong, H. S. J. and VanBerkum, M. F. A. (2017). Long disordered regions of the C-terminal domain of Abelson tyrosine kinase have specific and additive functions in regulation and axon localization. PLoS One 12(12): e0189338. PubMed ID: 29232713
Abelson tyrosine kinase (Abl) is a key regulator of actin-related morphogenetic processes including axon guidance, where it functions downstream of several guidance receptors. While the long C-terminal domain (CTD) of Abl is required for function, its role is poorly understood. In this study, a battery of mutants of Drosophila Abl was created that systematically deleted large segments of the CTD from Abl or added them back to the N-terminus alone. The functionality of these Abl transgenes was assessed through rescue of axon guidance defects and adult lethality in Abl loss-of-function, as well as through gain-of-function effects in sensitized slit or frazzled backgrounds that perturb midline guidance in the Drosophila embryonic nerve cord. Two regions of the CTD play important and distinct roles, but additive effects for other regions were also detected. The first quarter of the CTD, including a conserved PxxP motif and its surrounding sequence, regulates Abl function while the third quarter localizes Abl to axons. These regions feature long stretches of intrinsically disordered sequence typically found in hub proteins and are associated with diverse protein-protein interactions. Thus, the CTD of Abl appears to use these disordered regions to establish a variety of different signaling complexes required during formation of axon tracts.
Billmann, M., Chaudhary, V., ElMaghraby, M. F., Fischer, B. and Boutros, M. (2017). Widespread rewiring of genetic networks upon Wnt cancer signaling pathway activation. Cell Syst [Epub ahead of print]. PubMed ID: 29199019
Cellular signaling networks coordinate physiological processes in all multicellular organisms. Within networks, modules switch their function to control signaling activity in response to the cellular context. However, systematic approaches to map the interplay of such modules have been lacking. This study generated a context-dependent genetic interaction network of a metazoan's signaling pathway. Using Wnt signaling in Drosophila as a model, >290,000 double perturbations were measured of the pathway in a baseline state, after activation by Wnt ligand, or after loss of the tumor suppressor APC. Genetic interactions within the Wnt network were found to globally rewire after pathway activation. Between-state networks were derived that showed how genes changed their function between state-specific networks. This related pathway inhibitors across states and identified genes required for pathway activation. For instance, it was predicted and confirmed that the ER-resident protein Catsup is required for ligand-mediated Wnt signaling activation. Together, state-dependent and between-state genetic interaction networks identify responsive functional modules that control cellular pathways.
Bayer, F. E., Zimmermann, M., Fischer, P., Gromoll, C., Preiss, A. and Nagel, A. C. (2017). p53 and cyclin G cooperate in mediating genome stability in somatic cells of Drosophila. Sci Rep 7(1): 17890. PubMed ID: 29263364
One of the key players in genome surveillance is the tumour suppressor p53 mediating the adaptive response to a multitude of stress signals. This study identified Cyclin G (CycG) as co-factor of p53-mediated genome stability. CycG has been shown before to be involved in double-strand break repair during meiosis. Moreover, it is also important for mediating DNA damage response in somatic tissue. This study finds it in protein complexes together with p53, and shows that the two proteins interact physically in vitro and in vivo in response to ionizing irradiation. In contrast to mammals, Drosophila Cyclin G is no transcriptional target of p53. Genetic interaction data reveal that p53 activity during DNA damage response requires the presence of CycG. Morphological defects caused by overexpression of p53 are ameliorated in cycG null mutants. Moreover, using a p53 biosensor, it was shown that p53 activity is impeded in cycG mutants. As both p53 and CycG are likewise required for DNA damage repair and longevity it is proposed that CycG plays a positive role in mediating p53 function in genome surveillance of Drosophila.

Friday, February 9th

Vaughan, L., Marley, R., Miellet, S. and Hartley, P. S. (2017). The impact of SPARC on age-related cardiac dysfunction and fibrosis in Drosophila. Exp Gerontol. PubMed ID: 29032244
Tissue fibrosis, an accumulation of extracellular matrix proteins such as collagen, accompanies cardiac ageing in humans and this is linked to an increased risk of cardiac failure. The mechanisms driving age-related tissue fibrosis and cardiac dysfunction are unclear, yet clinically important. Drosophila is amenable to the study of cardiac ageing as well as collagen deposition; however it is unclear whether collagen accumulates in the ageing Drosophila heart. This work examined collagen deposition and cardiac function in ageing Drosophila, in the context of reduced expression of collagen-interacting protein SPARC (Secreted Protein Acidic and Rich in Cysteine) an evolutionarily conserved protein linked with fibrosis. Heart function was measured using high frame rate videomicroscopy. Collagen deposition was monitored using a fluorescently-tagged collagen IV reporter (encoded by the Viking gene) and staining of the cardiac collagen, Pericardin. The Drosophila heart accumulated collagen IV and Pericardin as flies aged. Associated with this was a decline in cardiac function. SPARC heterozygous flies lived longer than controls and showed little to no age-related cardiac dysfunction. As flies of both genotypes aged, cardiac levels of collagen IV (Viking) and Pericardin increased similarly. Over-expression of SPARC caused cardiomyopathy and increased Pericardin deposition. The findings demonstrate that, like humans, the Drosophila heart develops a fibrosis-like phenotype as it ages. Although having no gross impact on collagen accumulation, reduced SPARC expression extended Drosophila lifespan and cardiac health span. It is proposed that cardiac fibrosis in humans may develop due to the activation of conserved mechanisms and that SPARC may mediate cardiac ageing by mechanisms more subtle than gross accumulation of collagen.
Walkowicz, L., Kijak, E., Krzeptowski, W., Gorska-Andrzejak, J., Stratoulias, V., Woznicka, O., Chwastek, E., Heino, T. I. and Pyza, E. M. (2017). Downregulation of DmMANF in glial cells results in neurodegeneration and affects sleep and lifespan in Drosophila melanogaster. Front Neurosci 11: 610. PubMed ID: 29163014
In Drosophila melanogaster, mesencephalic astrocyte-derived neurotrophic factor (DmMANF) is an evolutionarily conserved ortholog of mammalian MANF and cerebral dopamine neurotrophic factor (CDNF), which have been shown to promote the survival of dopaminergic neurons in the brain. Especially high levels of DmMANF were observed in the visual system of Drosophila, particularly in the first optic neuropil (lamina). In the lamina, DmMANF was found in glial cells (surface and epithelial glia), photoreceptors and interneurons. Interestingly, silencing of DmMANF in all neurons or specifically in photoreceptors or L2 interneurons had no impact on the structure of the visual system. However, downregulation of DmMANF in glial cells induced degeneration of the lamina. Remarkably, this degeneration in the form of holes and/or tightly packed membranes was observed only in the lamina epithelial glial cells. Those membranes seem to originate from the endoplasmic reticulum, which forms autophagosome membranes. Moreover, capitate projections, the epithelial glia invaginations into photoreceptor terminals that are involved in recycling of the photoreceptor neurotransmitter histamine, were less numerous after DmMANF silencing either in neurons or glial cells. The distribution of the alpha subunit of Na+/K+-ATPase protein in the lamina cell membranes was also changed. At the behavioral level, silencing of DmMANF either in neurons or glial cells affected the daily activity/sleep pattern, and flies showed less activity during the day but higher activity during the night than did controls. In the case of silencing in glia, the lifespan of flies was also shortened. The obtained results showed that DmMANF regulates many functions in the brain, particularly those dependent on glial cells.
Zandawala, M., Marley, R., Davies, S. A. and Nassel, D. R. (2017). Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila. Cell Mol Life Sci [Epub ahead of print]. PubMed ID: 29043393
Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. This study investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. It was also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. These data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles.
Zarndt, R., Walls, S. M., Ocorr, K. and Bodmer, R. (2017). Reduced cardiac Calcineurin expression mimics long-term hypoxia-induced heart defects in Drosophila. Circ Cardiovasc Genet 10(5). PubMed ID: 28986453
Hypoxia is often associated with cardiopulmonary diseases, which represent some of the leading causes of mortality worldwide. Long-term hypoxia exposures, whether from disease or environmental condition, can cause cardiomyopathy and lead to heart failure. Indeed, hypoxia-induced heart failure is a hallmark feature of chronic mountain sickness in maladapted populations living at high altitude. In a previously established Drosophila heart model for long-term hypoxia exposure, it was found that hypoxia caused heart dysfunction. Calcineurin is known to be critical in cardiac hypertrophy under normoxia, but its role in the heart under hypoxia is poorly understood. The present study explored the function of calcineurin, a gene candidate that was found downregulated in the Drosophila heart after lifetime and multigenerational hypoxia exposure. Roles of 2 homologs of Calcineurin A, CanA14F, and Pp2B, were examined in the Drosophila cardiac response to long-term hypoxia. Knockdown of these calcineurin catalytic subunits caused cardiac restriction under normoxia that are further aggravated under hypoxia. Conversely, cardiac overexpression of Pp2B under hypoxia was lethal, suggesting that a hypertrophic signal in the presence of insufficient oxygen supply is deleterious. These results suggest a key role for calcineurin in cardiac remodeling during long-term hypoxia with implications for diseases of chronic hypoxia, and it likely contributes to mechanisms underlying these disease states.
Weisz, E. D., Towheed, A., Monyak, R. E., Toth, M. S., Wallace, D. C. and Jongens, T. A. (2017). Loss of Drosophila FMRP leads to alterations in energy metabolism and mitochondrial function. Hum Mol Genet 27(1): 95-106. PubMed ID: 29106525
Fragile X Syndrome (FXS), the most prevalent form of inherited intellectual disability and the foremost monogenetic cause of autism, is caused by loss of expression of the FMR1 gene. This study shows that dfmr1 modulates the global metabolome in Drosophila. Despite a previous discovery of increased brain insulin signaling, the results indicate that dfmr1 mutants have reduced carbohydrate and lipid stores and are hypersensitive to starvation stress. The observed metabolic deficits cannot be explained by feeding behavior, as it is reported that dfmr1 mutants are hyperphagic. Rather, the data identify dfmr1 as a regulator of mitochondrial function. Under supersaturating conditions, dfmr1 mutant mitochondria have significantly increased maximum electron transport system (ETS) capacity. Moreover, electron micrographs of indirect flight muscle reveal striking morphological changes in the dfmr1 mutant mitochondria. Taken together, these results illustrate the importance of dfmr1 for proper maintenance of nutrient homeostasis and mitochondrial function.
Branch, A., Zhang, Y. and Shen, P. (2017). Genetic and neurobiological analyses of the noradrenergic-like system in vulnerability to sugar overconsumption using a Drosophila model. Sci Rep 7(1): 17642. PubMed ID: 29247240
Regular overconsumption of sugar is associated with obesity and type-2 diabetes, but how genetic factors contribute to variable sugar preferences and intake levels remains mostly unclear. This study provides evidence for the usefulness of a Drosophila larva model to investigate genetic influence on vulnerability to sugar overconsumption. Using genetic and RNA interference approaches, this study shows that the activity of the Oamb gene, which encodes a receptor for octopamine (OA, the invertebrate homologue of norepinephrine), plays a major role in controlled sugar consumption. Furthermore, Oamb appears to suppress sugar food intake in fed larvae in an acute manner, and neurons expressing this Oamb receptor do not overlap with neurons expressing Octbeta3R, another OA receptor previously implicated in hunger-driven exuberant sugar intake. Together, these results suggest that two separate sub-circuits, defined by Oamb and Octbeta3R respectively, co-regulate sugar consumption according to changes in energy needs. It is proposed that the noradrenergic-like system defines an ancient regulatory mechanism for prevention of sugar overload.

Thursday, February 8th

Wolfstetter, G., Pfeifer, K., van Dijk, J. R., Hugosson, F., Lu, X. and Palmer, R. H. (2017). The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila. Sci Signal 10(502). PubMed ID: 29066538
In Drosophila melanogaster, the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb) are required to specify muscle founder cells in the visceral mesoderm. This study identified a critical role for the scaffolding protein Cnk (Connector enhancer of kinase suppressor of Ras) in this signaling pathway. Embryos that ectopically expressed the minimal Alk interaction region in the carboxyl terminus of Cnk or lacked maternal and zygotic cnk did not generate visceral founder cells or a functional gut musculature, phenotypes that resemble those of jeb and Alk mutants. Deletion of the entire Alk-interacting region in the cnk locus affected the Alk signaling pathway in the visceral mesoderm and not other RTK signaling pathways in other tissues. In addition, the Cnk-interacting protein Aveugle (Ave) was critical for Alk signaling in the developing visceral mesoderm. Alk signaling stimulates the MAPK/ERK pathway, but the scaffolding protein Ksr, which facilitates activation of this pathway, was not required to promote visceral founder cell specification. Thus, Cnk and Ave represent critical molecules downstream of Alk, and their loss genocopies the lack of visceral founder cell specification of Alk and jeb mutants, indicating their essential roles in Alk signaling.
Wen, J. K., Wang, Y. T., Chan, C. C., Hsieh, C. W., Liao, H. M., Hung, C. C. and Chen, G. C. (2017). Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila. Elife 6. PubMed ID: 29144896
Autophagy is essential for maintaining cellular homeostasis and survival under various stress conditions. Autophagy-related gene 9 (Atg9) encodes a multipass transmembrane protein thought to act as a membrane carrier for forming autophagosomes. However, the molecular regulation and physiological importance of Atg9 in animal development remain largely unclear. This study generated Atg9 null mutant flies and found that loss of Atg9 led to shortened lifespan, locomotor defects, and increased susceptibility to stress. Atg9 loss also resulted in aberrant adult midgut morphology with dramatically enlarged enterocytes. Interestingly, inhibiting the TOR signaling pathway rescued the midgut defects of the Atg9 mutants. In addition, Atg9 interacted with PALS1-associated tight junction protein (Patj), which associates with TSC2 to regulate TOR activity. Depletion of Atg9 caused a marked decrease in TSC2 levels. These findings revealed an antagonistic relationship between Atg9 and TOR signaling in the regulation of cell growth and tissue homeostasis.
Xu, T. et al. (2017). RBPJ/CBF1 interacts with L3MBTL3/MBT1 to promote repression of Notch signaling via histone demethylase KDM1A/LSD1. Embo J 36(21): 3232-3249. PubMed ID: 29030483
Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, this study identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ (Suppressor of Hairless in Drosophila) In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.
Vanderploeg, J. and Jacobs, J. R. (2017). Mapping heart development in flies: Src42A acts non-autonomously to promote heart tube formation in Drosophila. Vet Sci 4(2). PubMed ID: 29056682
Congenital heart defects, clinically identified in both small and large animals, are multifactorial and complex. Although heritable factors are known to have a role in cardiovascular disease, the full genetic aetiology remains unclear. Model organism research has proven valuable in providing a deeper understanding of the essential factors in heart development. For example, mouse knock-out studies reveal a role for the Integrin adhesion receptor in cardiac tissue. Recent research in Drosophila melanogaster (the fruit fly), a powerful experimental model, has demonstrated that the link between the extracellular matrix and the cell, mediated by Integrins, is required for multiple aspects of cardiogenesis. This study tested the hypothesis that Integrins signal to the heart cells through Src42A kinase. Using the powerful genetics and cell biology analysis possible in Drosophila, it was demonstrated that Src42A acts in early events of heart tube development. Careful examination of mutant heart tissue and genetic interaction data suggests that Src42A's role is independent of Integrin and the Integrin-related Focal Adhesion Kinase. Rather, Src42A acts non-autonomously by promoting programmed cell death of the amnioserosa, a transient tissue that neighbors the developing heart.
Yan, Y., Wang, H., Hu, M., Jiang, L., Wang, Y., Liu, P., Liang, X., Liu, J., Li, C., Lindstrom-Battle, A., Lam, S. M., Shui, G., Deng, W. M. and Jiao, R. (2017). HDAC6 suppresses age-dependent ectopic fat accumulation by maintaining the proteostasis of PLIN2 in Drosophila. Dev Cell 43(1): 99-111.e115. PubMed ID: 28966044
Age-dependent ectopic fat accumulation (EFA) in animals contributes to the progression of tissue aging and diseases such as obesity, diabetes, and cancer. However, the primary causes of age-dependent EFA remain largely elusive. This study characterized the occurrence of age-dependent EFA in Drosophila and identified HDAC6, a cytosolic histone deacetylase, as a suppressor of EFA. Loss of HDAC6 leads to significant age-dependent EFA, lipid composition imbalance, and reduced animal longevity on a high-fat diet. The EFA and longevity phenotypes are ameliorated by a reduction of the lipid-droplet-resident protein PLIN2. HDAC6 was found to be associated physically with the chaperone protein dHsc4/Hsc70 to maintain the proteostasis of PLIN2. These findings indicate that proteostasis collapse serves as an intrinsic cue to cause age-dependent EFA. This study suggests that manipulation of proteostasis could be an alternative approach to the treatment of age-related metabolic diseases such as obesity and diabetes.
Vasudevan, D., Clark, N. K., Sam, J., Cotham, V. C., Ueberheide, B., Marr, M. T., and Ryoo, H. D. (2017). The GCN2-ATF4 signaling pathway induces 4E-BP to bias translation and boost antimicrobial peptide synthesis in response to bacterial infection. Cell Rep 21(8): 2039-2047. PubMed ID: 29166596
Bacterial infection often leads to suppression of mRNA translation, but hosts are nonetheless able to express immune response genes through as yet unknown mechanisms. This study used a Drosophila model to demonstrate that antimicrobial peptide (AMP) production during infection is paradoxically stimulated by the inhibitor of cap-dependent translation, 4E-BP (eIF4E-binding protein; encoded by the Thor gene). 4E-BP was found to be induced upon infection with pathogenic bacteria by the stress-response transcription factor ATF4 and its upstream kinase, GCN2. Loss of gcn2, atf4, or 4e-bp compromised immunity. While AMP transcription is unaffected in 4e-bp mutants, AMP protein levels are substantially reduced. The 5' UTRs of AMPs score positive in cap-independent translation assays, and this cap-independent activity is enhanced by 4E-BP. These results are corroborated in vivo using transgenic 5' UTR reporters. These observations indicate that ATF4-induced 4e-bp contributes to innate immunity by biasing mRNA translation toward cap-independent mechanisms, thus enhancing AMP synthesis.

Wednesday, February 7th

Pandey, H., Bourahmoune, K., Honda, T., Honjo, K., Kurita, K., Sato, T., Sawa, A. and Furukubo-Tokunaga, K. (2017). Genetic interaction of DISC1 and Neurexin in the development of fruit fly glutamatergic synapses. NPJ Schizophr 3(1): 39. PubMed ID: 29079805
Originally identified at the breakpoint of a (1;11)(q42.1; q14.3) chromosomal translocation in a Scottish family with a wide range of mental disorders, the DISC1 gene has been a focus of intensive investigations as an entry point to study the molecular mechanisms of diverse mental dysfunctions. Perturbations of the DISC1 functions lead to behavioral changes in animal models, which are relevant to psychiatric conditions in patients. This work expressed the human DISC1 gene in Drosophila and performed a genetic screening for the mutations of psychiatric risk genes that cause modifications of DISC1 synaptic phenotypes at the neuromuscular junction. DISC1 was found to interact with dnrx1, the Drosophila homolog of the human Neurexin (NRXN1) gene, in the development of glutamatergic synapses. While overexpression of DISC1 suppressed the total bouton area on the target muscles and stimulated active zone density in wild-type background, a partial reduction of the dnrx1 activity negated the DISC1-mediated synaptic alterations. Likewise, overexpression of DISC1 stimulated the expression of a glutamate receptor component, DGLURIIA, in wild-type background but not in the dnrx1 heterozygous background. In addition, DISC1 caused mislocalization of Discs large, the Drosophila PSD-95 homolog, in the dnrx1 heterozygous background. Analyses with a series of domain deletions have revealed the importance of axonal localization of the DISC1 protein for efficient suppression of DNRX1 in synaptic boutons. These results thus suggest an intriguing converging mechanism controlled by the interaction of DISC1 and Neurexin in the developing glutamatergic synapses.
Wu, J., Tao, N., Tian, Y., Xing, G., Lv, H., Han, J., Lin, C. and Xie, W. (2017). Proteolytic maturation of Drosophila Neuroligin 3 by tumor necrosis factor alpha-converting enzyme in the nervous system. Biochim Biophys Acta 1862(3):440-450. PubMed ID: 29107812
The functions of autism-associated Neuroligins (Nlgs) are modulated by their post-translational modifications, such as proteolytic cleavage. A previous study has shown that there are different endogenous forms of DNlg3 in Drosophila, indicating it may undergo proteolytic processing. However, the molecular mechanism underlying DNlg3 proteolytic processing is unknown. This study reports a novel proteolytic mechanism that is essential for DNlg3 maturation and function in the nervous system. Molecular cloning, cell culture, immunohistochemistry, western blotting and genetic studies were employed to map the DNlg3 cleavage region, identify the protease and characterize the cleavage manner. Behavior analysis, immunohistochemistry and genetic manipulations were employed to study the functions of different DNlg3 forms in nervous system and neuromuscular junction (NMJs). Tumor necrosis factor alpha-converting enzyme (TACE) was shown to cleave DNlg3 exclusively at its extracellular acetylcholinesterase-like domain to generate the N-terminal fragment and the short membrane-anchored fragment (sDNlg3). DNlg3 was constitutively processed in an activity-independent manner. Interestingly, DNlg3 was cleaved intracellularly in the Golgi apparatus before it arrived at the cell surface, a unique cleavage mechanism that is distinct from 'conventional' ectodomain shedding of membrane proteins, including rodent Nlg1. Genetic studies showed that sDNlg3 was essential for maintaining proper locomotor activity in Drosophila. These results revealed a unique cleavage mechanism of DNlg3 and a neuron-specific role for DNlg3 maturation which is important in locomotor activity. This study provides a new insight into a cleavage mechanism of Nlgs maturation in nervous system.
Santana, E. and Casas-Tinto, S. (2017). Orb2 as modulator of Brat and their role at the neuromuscular junction. J Neurogenet 31(4):181-188. PubMed ID: 29105522
How synapses are built and dismantled is a central question in neurobiology. A wide range of proteins and processes from gene transcription to protein degradation are involved. Orb2 regulates mRNA translation depending on its monomeric or oligomeric state to modulate nervous system development and memory. Orb2 is expressed in Drosophila larval brain and neuromuscular junction (NMJ), Orb2 knockdown causes a reduction of synapse number and defects in neuronal morphology. Brain tumor (Brat) is an Orb2 target; it is expressed in larval brain related with cell growth and proliferation. Brat downregulation induces an increase in synapse number and abnormal growth of buttons and branches in neurons. In absence of Orb2, Brat is overexpressed suggesting that Orb2 is negatively regulating Brat mRNA translation. Orb2 or Brat control the expression of specific genes related to neuronal function. Orb2 is required for Liprin and Synaptobrevin transcription meanwhile Brat is required for Synaptobrevin and Synaptotagmin transcription. This study presents evidences of a novel genetic mechanism to regulate synapse fine tuning during development and propose an equilibrium between Orb2 conformational state and nervous system formation.
Wu, S., Gan, G., Zhang, Z., Sun, J., Wang, Q., Gao, Z., Li, M., Jin, S., Huang, J., Thomas, U., Jiang, Y. H., Li, Y., Tian, R. and Zhang, Y. Q. (2017). A presynaptic function of Shank protein in Drosophila. J Neurosci 37(48): 11592-11604. PubMed ID: 29074576
Human genetic studies support that loss-of-function mutations in the SH3 domain and ankyrin repeat containing family proteins (SHANK1-3), the large synaptic scaffolding proteins enriched at the postsynaptic density of excitatory synapses, are causative for autism spectrum disorder and other neuropsychiatric disorders in humans. To better understand the in vivo functions of Shank and facilitate dissection of neuropathology associated with SHANK mutations in human, multiple mutations were generated in the Shank gene, the only member of the SHANK family in Drosophila melanogaster. Both male and female Shank null mutants were fully viable and fertile with no apparent morphological or developmental defects. Expression analysis revealed apparent enrichment of Shank in the neuropils of the CNS. Specifically, Shank coexpressed with another PSD scaffold protein, Homer, in the calyx of mushroom bodies in the brain. Consistent with high expression in mushroom body calyces, Shank mutants show an abnormal calyx structure and reduced olfactory acuity. These morphological and functional phenotypes were fully rescued by pan-neuronal reexpression of Shank, and only partially rescued by presynaptic but no rescue by postsynaptic reexpression of Shank. These findings thus establish a previously unappreciated presynaptic function of Shank.
Gao, H., He, F., Lin, X. and Wu, Y. (2017). Drosophila VAMP7 regulates Wingless intracellular trafficking. PLoS One 12(10): e0186938. PubMed ID: 29065163
Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. This study showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, this study found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. It is hypothesized that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling.
Chang, H. F., et al. (2017). Cytotoxic granule endocytosis depends on the Flower protein. J Cell Biol [Epub ahead of print]. PubMed ID: 29288152
Evolutionary Homolog Study
Cytotoxic T lymphocytes (CTLs) kill target cells by the regulated release of cytotoxic substances from granules at the immunological synapse. To kill multiple target cells, CTLs use endocytosis of membrane components of cytotoxic granules. This study examined the potential calcium dependence of endocytosis in mouse CTLs on Flower, which mediates the calcium dependence of synaptic vesicle endocytosis. In Drosophila melanogaster Flower is predominantly localized on intracellular vesicles that move to the synapse on target cell contact. Endocytosis is entirely blocked at an early stage in Flower-deficient CTLs and is rescued to wild-type level by reintroducing Flower or by raising extracellular calcium. A Flower mutant lacking binding sites for the endocytic adaptor AP-2 proteins fails to rescue endocytosis, indicating that Flower interacts with proteins of the endocytic machinery to mediate granule endocytosis. Thus, these data identify Flower as a key protein mediating granule endocytosis.
Yeates, C. J., Zwiefelhofer, D. J. and Frank, C. A. (2017). The maintenance of synaptic homeostasis at the Drosophila neuromuscular junction is reversible and sensitive to high temperature. eNeuro 4(6). PubMed ID: 29255795
Homeostasis is a vital mode of biological self-regulation. The hallmarks of homeostasis for any biological system are a baseline set point of physiological activity, detection of unacceptable deviations from the set point, and effective corrective measures to counteract deviations. Homeostatic synaptic plasticity (HSP) is a form of neuroplasticity in which neurons and circuits resist environmental perturbations and stabilize levels of activity. One assumption is that if a perturbation triggers homeostatic corrective changes in neuronal properties, those corrective measures should be reversed upon removal of the perturbation. This study tested the reversibility and limits of HSP at the well-studied Drosophila melanogaster neuromuscular junction (NMJ). At the Drosophila NMJ, impairment of glutamate receptors causes a decrease in quantal size, which is offset by a corrective, homeostatic increase in the number of vesicles released per evoked presynaptic stimulus, or quantal content. This process has been termed presynaptic homeostatic potentiation (PHP). Taking advantage of the GAL4/GAL80(TS)/UAS expression system, PHP was triggered by expressing a dominant-negative glutamate receptor subunit GluRIIA at the NMJ. PHP was then reversed by halting expression of the dominant-negative receptor. These data show that PHP is fully reversible over a time course of 48-72 h after the dominant-negative glutamate receptor stops being genetically expressed. As an extension of these experiments, it was found that when glutamate receptors are impaired, neither PHP nor NMJ growth is reliably sustained at high culturing temperatures (30-32 degrees C). These data suggest that a limitation of homeostatic signaling at high temperatures could stem from the synapse facing a combination of challenges simultaneously.
Liao, E. H., Gray, L., Tsurudome, K., El Mounzer, W., Elazzouzi, F., Baim, C., Farzin, S., Calderon, M. R., Kauwe, G. and Haghighi, A. P. (2018). Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction. PLoS Genet 14(1): e1007184. PubMed ID: 29373576
Retrograde signaling is essential for neuronal growth, function and survival; however, little is known about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. This study identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but a significant decrease in the number of presynaptic release sites was found. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Overexpression of the type II TGFβ receptor Wit enhanced presynaptic pMad levels. In Khc-73 mutants, this enhancement was significantly suppressed. Similarly, muscle overexpression of the ligand Gbb enhanced pMAD levels in presynaptic boutons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, loss of Khc-73 was shown to curb the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. These findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons.

Tuesday, February 6th

Jia, Q., Liu, S., Wen, D., Cheng, Y., Bendena, W. G., Wang, J. and Li, S. (2017). Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation. J Biol Chem 292(52):21504-21516. PubMed ID: 29118190
Tissue remodeling is a crucial process in animal development and disease progression. Coordinately controlled by the two main insect hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), tissues are remodeled context-specifically during insect metamorphosis. Previous work has discovered that two matrix metalloproteinases (Mmps) cooperatively induce fat body cell dissociation in Drosophila. However, the molecular events involved in this Mmps-mediated dissociation are unclear. This study reports that JH and 20E coordinately and precisely control the developmental timing of Mmps-induced fat body cell dissociation. During the larval-prepupal transition, the anti-metamorphic factor Kr-h1 was found to transduce JH signaling, which directly inhibited Mmps expression and activated expression of tissue inhibitor of metalloproteinases (timp), and thereby suppressed Mmps-induced fat body cell dissociation. It is also noted that upon a decline in the JH titer, a prepupal peak of 20E suppresses Mmps-induced fat body cell dissociation through the 20E primary-response genes, E75 and <Blimp-1, which inhibited expression of the nuclear receptor and competence factor betaftz-F1. Moreover, upon a decline in the 20E titer, betaftz-F1 expression was induced by the 20E early-late response gene DHR3, and then betaftz-F1 directly activated Mmps expression and inhibited timp expression, causing Mmps-induced fat body cell dissociation during 6-12 hrs after puparium formation. In conclusion, coordinated signaling via JH and 20E finely tunes the developmental timing of Mmps-induced fat body cell dissociation. These findings shed critical light on hormonal regulation of insect metamorphosis.
Sawala, A. and Gould, A. P. (2017). The sex of specific neurons controls female body growth in Drosophila. PLoS Biol 15(10): e2002252. PubMed ID: 28976974
Sexual dimorphisms in body size are widespread throughout the animal kingdom but their underlying mechanisms are not well characterized. Most models for how sex chromosome genes specify size dimorphism have emphasized the importance of gonadal hormones and cell-autonomous influences in mammals versus strictly cell-autonomous mechanisms in Drosophila melanogaster. This study used tissue-specific genetics to investigate how sexual size dimorphism (SSD) is established in Drosophila. The larger body size characteristic of Drosophila females is established very early in larval development via an increase in the growth rate per unit of body mass. The female sex determination gene Sex-lethal (Sxl) was shown to function in central nervous system (CNS) neurons as part of a relay that specifies the early sex-specific growth trajectories of larval but not imaginal tissues. Neuronal Sxl acts additively in 2 neuronal subpopulations, one of which corresponds to 7 median neurosecretory cells: the insulin-producing cells (IPCs). Surprisingly, however, male-female differences in the production of insulin-like peptides (Ilps) from the IPCs do not appear to be involved in establishing SSD in early larvae, although they may play a later role. These findings support a relay model in which Sxl in neurons and Sxl in local tissues act together to specify the female-specific growth of the larval body. They also reveal that, even though the sex determination pathways in Drosophila and mammals are different, they both modulate body growth via a combination of tissue-autonomous and nonautonomous inputs.
Neuert, H., Yuva-Aydemir, Y., Silies, M. and Klambt, C. (2017). Different modes of APC/C activation control growth and neuron-glia interaction in the developing Drosophila eye. Development 144(24):4673-4683. PubMed ID: 29084807
The development of the nervous system requires tight control of cell division, fate specification and migration. The anaphase promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that affects different steps of cell cycle progression, as well as having postmitotic functions in nervous system development. It can therefore link different developmental stages in one tissue. The two adaptor proteins Fizzy/Cdc20 and Fizzy-Related/Cdh1 confer APC/C substrate specificity. This study shows that two distinct modes of APC/C function act during Drosophila eye development. Fizzy/Cdc20 controls the early growth of the eye disc anlage and the concomitant entry of glial cells onto the disc. In contrast, fzr/cdh1 acts during neuronal patterning and photoreceptor axon growth, and subsequently affects neuron-glia interaction. To further address the postmitotic role of Fzr/Cdh1 in controlling neuron-glia interaction, a series of novel APC/C candidate substrates were identified. Four of the candidate genes are required for fzr/cdh1 dependent neuron-glia interaction, including the dynein light chain Dlc90F. Taken together, these data show how different modes of APC/C activation can couple early growth and neuron-glia interaction during eye disc development.
Wang, X. F., Shen, Y., Cheng, Q., Fu, C. L., Zhou, Z. Z., Hirose, S. and Liu, Q. X. (2017). Apontic directly activates hedgehog and cyclin E for proper organ growth and patterning. Sci Rep 7(1): 12470. PubMed ID: 28963499
Hedgehog (Hh) signaling pathway and Cyclin E are key players in cell proliferation and organ development. Hyperactivation of hh and cyclin E has been linked to several types of cancer. However, coordination of the expression of hh and cyclin E was not well understood. This study shows that an evolutionarily conserved transcription factor Apontic (Apt) directly activates hh and cyclin E through its binding site in the promoter regions of hh and cyclin E. This Apt-dependent proper expression of hh and cyclin E is required for cell proliferation and development of the Drosophila wing. Furthermore, Fibrinogen silencer-binding protein (FSBP), a mammalian homolog of Apt, also positively regulates Sonic hh (Shh), Desert hh (Dhh), Cyclin E1 (CCNE1) and Cyclin E2 (CCNE2) in cultured human cells, suggesting evolutionary conservation of the mechanism. Apt-mediated expression of hh and cyclin E can direct proliferation of Hh-expressing cells and simultaneous growth, patterning and differentiation of Hh-recipient cells. The discovery of the simultaneous expression of Hh and principal cell-cycle regulator Cyclin E by Apt implicates insight into the mechanism by which deregulated hh and cyclin E promotes tumor formation.
Uryu, O., Ou, Q., Komura-Kawa, T., Kamiyama, T., Iga, M., Syrzycka, M., Hirota, K., Kataoka, H., Honda, B. M., King-Jones, K. and Niwa, R. (2017). Cooperative control of ecdysone biosynthesis in Drosophila by transcription factors Seance, Ouija board, and Molting Defective. Genetics [Epub ahead of print]. PubMed ID: 29187506
Insect ecdysteroids are steroid hormones that control many aspects of development and physiology. During larval development, ecdysone is synthesized in an endocrine organ called the prothoracic gland (PG) through a series of ecdysteroidogenic enzymes encoded by the Halloween genes. The expression of the Halloween genes is highly restricted and dynamic, indicating that their spatiotemporal regulation is mediated by their tight transcriptional control. This study reports that three ZAD-C2H2 zinc finger transcription factors-Seance (Sean), Ouija board (Ouib), and Molting defective (Mld)-cooperatively control ecdysone biosynthesis in the fruit fly Drosophila melanogaster. Sean and Ouib act in cooperation with Mld to positively regulate the transcription of neverland and spookier, respectively, two Halloween genes. Remarkably, loss-of-function mutations in sean, ouib, or mld can be rescued by the expression of neverland,spookier, or both, respectively. These results suggest that the three transcription factors have distinct roles in coordinating the expression of just two genes in Drosophila. Given that neverland and spookier are located in constitutive heterochromatin, Sean, Ouib, and Mld represent the first example of a transcription factor subset that regulates genes located in constitutive heterochromatin.
Kim, A. R., Choi, E. B., Kim, M. Y. and Choi, K. W. (2017). Angiotensin-converting enzyme Ance is cooperatively regulated by Mad and Pannier in Drosophila imaginal discs. Sci Rep 7(1): 13174. PubMed ID: 29030610
Angiotensin-converting enzyme (ACE) is an evolutionarily conserved peptidyl dipeptidase. Mammalian ACE converts angiotensin I to the active vasoconstrictor angiotensin II, thus playing a critical role for homeostasis of the renin-angiotensin system. In Drosophila, the ACE homolog Ance is expressed in specific regions of developing organs, but its regulatory mechanism has not been identified. This study provides evidence that Ance expression is regulated by a combination of Mad and Pannier (Pnr) in imaginal discs. Ance expression in eye and wing discs depends on Dpp signaling. The Mad binding site of Ance regulatory region is essential for Ance expression. Ance expression in imaginal discs is also regulated by the GATA family transcription factor Pnr. Pnr directly regulates Ance expression by binding to a GATA site of Ance enhancer. In addition, Pnr and Mad physically and genetically interact. Ance null mutants are morphologically normal but show genetic interaction with dpp mutants. Furthermore, human SMAD2 and GATA4 were shown to physically interact, and ACE expression in HEK293 cells is regulated by SMAD2 and GATA4. Taken together, this study reveals a cooperative mechanism of Ance regulation by Mad and Pnr. The data also suggest a conserved transcriptional regulation of human ACE.

Monday, February 5th

Tauber, J. M., Brown, E. B., Li, Y., Yurgel, M. E., Masek, P. and Keene, A. C. (2017). A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste. PLoS Genet 13(11): e1007059. PubMed ID: 29121639
Fat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. The highly palatable taste of free fatty acids (FAs), one of the building blocks of fat, promotes food consumption, activates reward circuitry. A broad population of sugar-sensing taste neurons expressing Gustatory Receptor 64f (Gr64f) is required for reflexive feeding responses to both FAs and sugars. This study reports a genetic silencing screen to identify specific populations of taste neurons that mediate fatty acid (FA) taste. Neurons identified by expression of Ionotropic Receptor 56d (IR56d) were found to be necessary and sufficient for reflexive feeding response to FAs. Functional imaging reveals that IR56d-expressing neurons are responsive to short- and medium-chain FAs. Silencing IR56d neurons selectively abolishes FA taste, and their activation is sufficient to drive feeding responses. Analysis of co-expression with Gr64f identifies two subpopulations of IR56d-expressing neurons. While physiological imaging reveals that both populations are responsive to FAs, IR56d/Gr64f neurons are activated by medium-chain FAs and are sufficient for reflexive feeding response to FAs. Moreover, flies can discriminate between sugar and FAs in an aversive taste memory assay, indicating that FA taste is a unique modality in Drosophila. Taken together, these findings localize FA taste within the Drosophila gustatory center and provide an opportunity to investigate discrimination between different categories of appetitive tastants.
Uchizono, S., Itoh, T. Q., Kim, H., Hamada, N., Kwon, J. Y. and Tanimura, T. (2017). Deciphering the genes for taste receptors for fructose in Drosophila. Mol Cells 40(10): 731-736. PubMed ID: 29047261
Taste sensitivity to sugars plays an essential role in the initiation of feeding behavior. In Drosophila melanogaster, recent studies have identified several gustatory receptor (Gr) genes required for sensing sweet compounds. However, it is as yet undetermined how these GRs function as taste receptors tuned to a wide range of sugars. Among sugars, fructose has been suggested to be detected by a distinct receptor from other sugars. While GR43A has been reported to sense fructose in the brain, it is not expressed in labellar gustatory receptor neurons that show taste response to fructose. In contrast, the Gr64a-Gr64f gene cluster was recently shown to be associated with fructose sensitivity. This study sought to decipher the genes required for fructose response among Gr64a-Gr64f genes. Unexpectedly, the qPCR analyses for these genes show that labellar expression levels of Gr64d and Gr64e are higher in fructose low-sensitivity flies than in high-sensitivity flies. Moreover, gustatory nerve responses to fructose in labellar sensilla are higher in Gr64d and Gr64f mutant lines than in mutant flies of the other Gr64a-Gr64f genes. These data suggest the possibility that deletion of GR64D or GR64F may indirectly induce enhanced fructose sensitivity in the labellum. Finally, it is concluded that response to fructose cannot be explained by a single one of the Gr64a-Gr64f genes.
Talay, M., Richman, E. B., Snell, N. J., Hartmann, G. G., Fisher, J. D., Sorkac, A., Santoyo, J. F., Chou-Freed, C., Nair, N., Johnson, M., Szymanski, J. R. and Barnea, G. (2017). Transsynaptic mapping of second-order taste neurons in flies by trans-tango. Neuron 96(4): 783-795.e784. PubMed ID: 29107518
Mapping neural circuits across defined synapses is essential for understanding brain function. This study describes trans-Tango, a technique for anterograde transsynaptic circuit tracing and manipulation. At the core of trans-Tango is a synthetic signaling pathway that is introduced into all neurons in the animal. This pathway converts receptor activation at the cell surface into reporter expression through site-specific proteolysis. Specific labeling is achieved by presenting a tethered ligand at the synapses of genetically defined neurons, thereby activating the pathway in their postsynaptic partners and providing genetic access to these neurons. Trans-Tango was first validated in the Drosophila olfactory system and then implemented in the gustatory system, where projections beyond the first-order receptor neurons are not fully characterized. Putative second-order neurons were identified within the sweet circuit that include projection neurons targeting known neuromodulation centers in the brain. These experiments establish trans-Tango as a flexible platform for transsynaptic circuit analysis.
Sung, H. Y., Jeong, Y. T., Lim, J. Y., Kim, H., Oh, S. M., Hwang, S. W., Kwon, J. Y. and Moon, S. J. (2017). Heterogeneity in the Drosophila gustatory receptor complexes that detect aversive compounds. Nat Commun 8(1): 1484. PubMed ID: 29133786
Animals must detect aversive compounds to survive. Bitter taste neurons express heterogeneous combinations of bitter receptors that diversify their response profiles, but this remains poorly understood. This study describes groups of taste neurons in Drosophila that detect the same bitter compounds using unique combinations of gustatory receptors (GRs). These distinct complexes also confer responsiveness to non-overlapping sets of additional compounds. While either GR32a/GR59c/GR66a or GR22e/GR32a/GR66a heteromultimers are sufficient for lobeline, berberine, and denatonium detection, only GR22e/GR32a/GR66a responds to strychnine. Thus, despite minimal sequence-similarity, Gr22e and Gr59c show considerable but incomplete functional overlap. Since the gain- or loss-of-function of Gr22e or Gr59c alters bitter taste response profiles, it is concluded that a taste neuron's specific combination of Grs determines its response profile. It is suspected that the heterogeneity of Gr expression in Drosophila taste neurons diversifies bitter compound detection, improving animal fitness under changing environmental conditions that present a variety of aversive compounds.
Tsubouchi, A., Yano, T., Yokoyama, T. K., Murtin, C., Otsuna, H. and Ito, K. (2017). Topological and modality-specific representation of somatosensory information in the fly brain. Science 358(6363): 615-623. PubMed ID: 29097543
Insects and mammals share similarities of neural organization underlying the perception of odors, taste, vision, sound, and gravity. This study observed that insect somatosensation also corresponds to that of mammals. In Drosophila, the projections of all the somatosensory neuron types to the insect's equivalent of the spinal cord segregated into modality-specific layers comparable to those in mammals. Some sensory neurons innervate the ventral brain directly to form modality-specific and topological somatosensory maps. Ascending interneurons with dendrites in matching layers of the nerve cord send axons that converge to respective brain regions. Pathways arising from leg somatosensory neurons encode distinct qualities of leg movement information and play different roles in ground detection. Establishment of the ground pattern and genetic tools for neuronal manipulation should provide the basis for elucidating the mechanisms underlying somatosensation.
Tanaka, R., Higuchi, T., Kohatsu, S., Sato, K. and Yamamoto, D. (2017). Optogenetic activation of the fruitless-labeled circuitry in Drosophila subobscura males induces mating motor acts. J Neurosci 37(48): 11662-11674. PubMed ID: 29109241
It remains an enigma how the nervous system of different animal species produces different behaviors. The neural circuitry for mating behavior was studied in Drosophila subobscura, a species that displays unique courtship actions not shared by other members of the genera including the genetic model D. melanogaster, in which the core courtship circuitry has been identified. The D. subobscura fruitless (fru) gene, a master regulator for the courtship circuitry formation in D. melanogaster, was disrupted resulting in complete loss of mating behavior. frusoChrimV was also developed that expresses the optogenetic activator Chrimson fused with a fluorescent marker under the native fru promoter. The fru-labeled circuitry in D. subobscura visualized by frusoChrimV revealed differences between females and males, optogenetic activation of which in males induced mating behavior including attempted copulation. These findings provide a substrate for neurogenetic dissection and manipulation of behavior in non-model animals, and will help to elucidate the neural basis for behavioral diversification.

Friday, February 2nd

Tsai, A., Muthusamy, A. K., Alves, M. R., Lavis, L. D., Singer, R. H., Stern, D. L. and Crocker, J. (2017). Nuclear microenvironments modulate transcription from low-affinity enhancers. Elife 6. PubMed ID: 29095143
Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. This study reports that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.
Frank, H. O., Sanchez, D. G., de Freitas Oliveira, L., Kobarg, J. and Monesi, N. (2017). The Drosophila melanogaster Eip74EF-PA transcription factor directly binds the sciarid BhC4-1 promoter. Genesis 55(11). PubMed ID: 28971561
The DNA puff BhC4-1 gene of Bradysia hygida (Diptera, Sciaridae) is amplified and expressed in the salivary glands at the end of the last larval instar. Even though there are no BhC4-1 orthologs in Drosophila melanogaster, the mechanisms that regulate BhC4-1 gene expression in B. hygida are for the most part conserved in D. melanogaster. The BhC4-1 promoter contains a 129bp (-186/-58) cis-regulatory module (CRM) that drives developmentally regulated expression in transgenic salivary glands at the onset of metamorphosis. Both in the sciarid and in transgenic D. melanogaster, BhC4-1 gene expression is induced by the increase in ecdysone titers that triggers metamorphosis. Genetic interaction experiments revealed that in the absence of the Eip74EF-PA early gene isoform BhC4-1-lacZ levels of expression in the salivary gland are severely reduced. This study shows that the overexpression of the Eip74EF-PA transcription factor is sufficient to anticipate BhC4-1-lacZ expression in transgenic D. melanogaster. Through yeast one-hybrid assays it was confirmed that the Eip74EF-PA transcription factor directly binds to the 129 bp sciarid CRM. Together, these results contribute to the characterization of an insect CRM and indicate that the ecdysone gene regulatory network that promotes metamorphosis is conserved between D. melanogaster and the sciarid B. hygida.
Baumann, D. G. and Gilmour, D. S. (2017). A sequence-specific core promoter-binding transcription factor recruits TRF2 to coordinately transcribe ribosomal protein genes. Nucleic Acids Res 45(18): 10481-10491. PubMed ID: 28977400
Ribosomal protein (RP) genes must be coordinately expressed for proper assembly of the ribosome yet the mechanisms that control expression of RP genes in metazoans are poorly understood. Recently, TATA-binding protein-related factor 2 (TRF2) rather than the TATA-binding protein (TBP) was found to function in transcription of RP genes in Drosophila. Unlike TBP, TRF2 lacks sequence-specific DNA binding activity, so the mechanism by which TRF2 is recruited to promoters is unclear. This study shows that the transcription factor M1BP, which associates with the core promoter region, activates transcription of RP genes. Moreover, M1BP directly interacts with TRF2 to recruit it to the RP gene promoter. High resolution ChIP-exo was used to analyze in vivo the association of M1BP, TRF2 and TFIID subunit, TAF1. Despite recent work suggesting that TFIID does not associate with RP genes in Drosophila, it was found that TAF1 is present at RP gene promoters and that its interaction might also be directed by M1BP. Although M1BP associates with thousands of genes, its colocalization with TRF2 is largely restricted to RP genes, suggesting that this combination is key to coordinately regulating transcription of the majority of RP genes in Drosophila.
Samee, M. A. H., Lydiard-Martin, T., Biette, K. M., Vincent, B. J., Bragdon, M. D., Eckenrode, K. B., Wunderlich, Z., Estrada, J., Sinha, S. and DePace, A. H. (2017). Quantitative measurement and thermodynamic modeling of fused enhancers support a two-tiered mechanism for interpreting regulatory DNA. Cell Rep 21(1): 236-245. PubMed ID: 28978476
Computational models of enhancer function generally assume that transcription factors (TFs) exert their regulatory effects independently, modeling an enhancer as a "bag of sites." These models fail on endogenous loci that harbor multiple enhancers, and a "two-tier" model appears better suited: in each enhancer TFs work independently, and the total expression is a weighted sum of their expression readouts. These two opposing views on how cis-regulatory information is integrated were tested in this study. Two Drosophila blastoderm enhancers were fused, their readouts were measured, and the above two models were applied to these data. The two-tier mechanism better fits these readouts, suggesting that these fused enhancers comprise multiple independent modules, despite having sequence characteristics typical of single enhancers. Short-range TF-TF interactions are not sufficient to designate such modules, suggesting unknown underlying mechanisms. These results underscore that mechanisms of how modules are defined and how their outputs are combined remain to be elucidated.
Boija, A., Mahat, D. B., Zare, A., Holmqvist, P. H., Philip, P., Meyers, D. J., Cole, P. A., Lis, J. T., Stenberg, P. and Mannervik, M. (2017). CBP regulates recruitment and release of promoter-proximal RNA polymerase II. Mol Cell 68(3): 491-503. PubMed ID: 29056321
Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. This study identifies a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. Drosophila CBP inhibition results in "dribbling" of Pol II from the pause site to positions further downstream but impedes transcription through the +1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes.
Chowdhury, Z. S., Sato, K. and Yamamoto, D. (2017). The core-promoter factor TRF2 mediates a Fruitless action to masculinize neurobehavioral traits in Drosophila. Nat Commun 8(1): 1480. PubMed ID: 29133872
In fruit flies, the male-specific fruitless (fru) gene product FruBM plays a central role in establishing the neural circuitry for male courtship behavior by orchestrating the transcription of genes required for the male-type specification of individual neurons. This study identified the core promoter recognition factor gene Trf2 as a dominant modifier of fru actions. Trf2 knockdown in the sexually dimorphic mAL neurons leads to the loss of a male-specific neurite and a reduction in male courtship vigor. TRF2 forms a repressor complex with FruBM, strongly enhancing the repressor activity of FruBM at the promoter region of the robo1 gene, whose function is required for inhibiting the male-specific neurite formation. In females that lack FruBM, TRF2 stimulates robo1 transcription. These results suggest that TRF2 switches its own role from an activator to a repressor of transcription upon binding to FruBM, thereby enabling the ipsilateral neurite formation only in males.

Thursday, February 1st

Tomer, D., Chippalkatti, R., Mitra, K. and Rikhy, R. (2018). ERK regulates mitochondrial membrane potential in fission deficient Drosophila follicle cells during differentiation. Dev Biol 434(1):48-62. PubMed ID: 29157562
Mitochondrial morphology regulatory proteins interact with signaling pathways involved in differentiation. In Drosophila oogenesis, EGFR signaling regulates mitochondrial fragmentation in posterior follicle cells (PFCs). EGFR driven oocyte patterning and Notch signaling mediated differentiation are abrogated when PFCs are deficient for the mitochondrial fission protein Drp1. It is not known whether fused mitochondrial morphology in drp1 mutant PFCs exerts its effects on these signaling pathways through a change in mitochondrial electron transport chain (ETC) activity. This study shows that aggregated mitochondria in drp1 mutant PFCs have increased mitochondrial membrane potential. Experiments were performed to assess the signaling pathway regulating mitochondrial membrane potential and how this impacts follicle cell differentiation. drp1 mutant PFCs were found to show an increase in phosphorylated ERK (dpERK) formed downstream of EGFR signaling. ERK regulates high mitochondrial membrane potential in drp1 mutant PFCs. PFCs depleted of ERK and drp1 are able to undergo Notch mediated differentiation. Notably mitochondrial membrane potential decrease via ETC inhibition activates Notch signaling at an earlier stage in wild type and suppresses the Notch signaling defect in drp1 mutant PFCs. Thus, this study shows that the EGFR pathway maintains mitochondrial morphology and mitochondrial membrane potential in follicle cells for its functioning and decrease in mitochondrial membrane potential is needed for Notch mediated differentiation.
Tomaru, M., Ohsako, T., Watanabe, M., Juni, N., Matsubayashi, H., Sato, H., Takahashi, A. and Yamamoto, M. T. (2017). Severe fertility effects of sheepish sperm caused by failure to enter female sperm storage organs in Drosophila melanogaster. G3 (Bethesda) 8(1):149-160. PubMed ID: 29158336
In Drosophila, mature sperm are transferred from males to females during copulation, stored in the sperm storage organs of females, and then utilized for fertilization. This study reports a gene named sheepish (shps) of D. melanogaster that is essential for sperm storage in females. shps mutant males, although producing morphologically normal and motile sperm that are effectively transferred to females, produce very few offspring. Direct counts of sperm indicated that the primary defect was correlated to failure of shps sperm to migrate into the female sperm storage organs. Increased sperm motion parameters were seen in the control after transfer to females, whereas sperm from shps males have characteristics of the motion parameters different from the control. The few sperm that occasionally entered the female sperm storage organs showed no obvious defects in fertilization and early embryo development. The female post-mating responses after copulation with shps males appeared normal at least with respect to conformational changes of uterus, mating plug formation and female remating rates. The shps gene encodes a protein with homology to amine oxidases, including as observed in mammals, with a transmembrane region at the C-terminal end. The shps mutation was characterized by a nonsense replacement in the third exon of CG13611 and shps was rescued by transformants of the wild-type copy of CG13611 Thus, shps may define a new class of gene responsible for sperm storage.
Rothenbusch-Fender, S., Fritzen, K., Bischoff, M. C., Buttgereit, D., Oenel, S. F. and Renkawitz-Pohl, R. (2017). Myotube migration to cover and shape the testis of Drosophila depends on Heartless, Cadherin/Catenin, and myosin II. Biol Open 6(12):1876-1888. PubMed ID: 29122742
During Drosophila metamorphosis, nascent testis myotubes migrate from the prospective seminal vesicle of the genital disc onto pupal testes and then further to cover the testes with multinucleated smooth-like muscles. This study shows that DWnt2 is likely required for determination of testis-relevant myoblasts on the genital disc. Knock down of FGFR Heartless by RNAi and a dominant-negative version revealed multiple functions of Heartless, namely regulation of the amount of myoblasts on the genital disc, connection of seminal vesicles and testes, and migration of muscles along the testes. Live imaging indicated that the downstream effector Stumps is required for migration of testis myotubes on the testis towards the apical tip. After myoblast fusion, myosin II is needed for migration of nascent testis myotubes, in which Thisbe-dependent FGF signaling is activated. Cadherin-N is essential for connecting these single myofibers and for creating a firm testis muscle sheath that shapes and stabilizes the testis tubule. Based on these results, a model is proposed for the migration of testis myotubes in which nascent testis myotubes migrate as a collective onto and along the testis, dependent on FGF-regulated expression of myosin II.
Tiwari, B., Kurtz, P., Jones, A. E., Wylie, A., Amatruda, J. F., Boggupalli, D. P., Gonsalvez, G. B. and Abrams, J. M. (2017). Retrotransposons mimic germ plasm determinants to promote transgenerational inheritance. Curr Biol 27(19): 3010-3016.e3013. PubMed ID: 28966088
Retrotransposons are a pervasive class of mobile elements present in the genomes of virtually all forms of life. In metazoans, these are preferentially active in the germline, which, in turn, mounts defenses that restrain their activity. This study report that certain classes of retrotransposons ensure transgenerational inheritance by invading presumptive germ cells before they are formed. Using sensitized Drosophila and zebrafish models, this study found that diverse classes of retrotransposons migrate to the germ plasm, a specialized region of the oocyte that prefigures germ cells and specifies the germline of descendants in the fertilized egg. In Drosophila, evidence was found for a "stowaway" model, whereby Tahre retroelements traffic to the germ plasm by mimicking oskar RNAs and engaging the Staufen-dependent active transport machinery. Consistent with this, germ plasm determinants attracted retroelement RNAs even when these components were ectopically positioned in bipolar oocytes. Likewise, vertebrate retrotransposons similarly migrated to the germ plasm in zebrafish oocytes. Together, these results suggest that germ plasm targeting represents a fitness strategy adopted by some retrotransposons to ensure transgenerational propagation.
Dai, W., Peterson, A., Kenney, T., Burrous, H. and Montell, D. J. (2017). Quantitative microscopy of the Drosophila ovary shows multiple niche signals specify progenitor cell fate. Nat Commun 8(1): 1244. PubMed ID: 29093440
Adult stem cells commonly give rise to transit-amplifying progenitors, whose progeny differentiate into distinct cell types. It is unclear if stem cell niche signals coordinate fate decisions within the progenitor pool. This study used quantitative analysis of Wnt, Hh, and Notch signalling reporters and the cell fate markers Eyes Absent (Eya) and Castor (Cas) to study the effects of hyper-activation and loss of niche signals on progenitor development in the Drosophila ovary. Follicle stem cell (FSC) progeny adopt distinct polar, stalk, and main body cell fates. Wnt signalling transiently inhibits expression of the main body cell fate determinant Eya, and Wnt hyperactivity strongly biases cells towards polar and stalk fates. Hh signalling independently controls the proliferation to differentiation transition. Notch is permissive but not instructive for differentiation of multiple cell types. These findings reveal that multiple niche signals coordinate cell fates and differentiation of progenitor cells.
Chlasta, J., Milani, P., Runel, G., Duteyrat, J. L., Arias, L., Lamire, L. A., Boudaoud, A. and Grammont, M. (2017). Variations in basement membrane mechanics are linked to epithelial morphogenesis. Development 144(23): 4350-4362. PubMed ID: 29038305
The regulation of morphogenesis by the basement membrane (BM) may rely on changes in its mechanical properties. To test this, an atomic force microscopy-based method was developed to measure BM mechanical stiffness during two key processes in Drosophila ovarian follicle development. First, follicle elongation depends on epithelial cells that collectively migrate, secreting BM fibrils perpendicularly to the anteroposterior axis. These data show that BM stiffness increases during this migration and that fibril incorporation enhances BM stiffness. In addition, stiffness heterogeneity, due to oriented fibrils, is important for egg elongation. Second, epithelial cells change their shape from cuboidal to either squamous or columnar. This study proves that BM softens around the squamous cells and that this softening depends on the TGFbeta pathway (the ligands Gbb and Dpp signalling to follicle cells). It was also demonstrated that interactions between BM constituents are necessary for cell flattening. Altogether, these results show that BM mechanical properties are modified during development and that, in turn, such mechanical modifications influence both cell and tissue shapes.
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