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Wednesday, October 31st - Disease Models

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Basso, V., Marchesan, E., Peggion, C., Chakraborty, J., von Stockum, S., Giacomello, M., Ottolini, D., Debattisti, V., Caicci, F., Tasca, E., Pegoraro, V., Angelini, C., Antonini, A., Bertoli, A., Brini, M. and Ziviani, E. (2018). Regulation of ER-mitochondria contacts by Parkin via Mfn2. Pharmacol Res. PubMed ID: 30219582
Summary:
Parkin, an E3 ubiquitin ligase and a Parkinson's disease (PD) related gene, translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy. Mitochondrial pro-fusion protein Mitofusins (Mfn1 and Mfn2) were found to be a target for Parkin mediated ubiquitination. Mfns are transmembrane GTPase embedded in the outer membrane of mitochondria, which are required on adjacent mitochondria to mediate fusion. In mammals, Mfn2 also forms complexes that are capable of tethering mitochondria to endoplasmic reticulum (ER), a structural feature essential for mitochondrial energy metabolism, calcium (Ca(2+)) transfer between the organelles and Ca(2+) dependent cell death. Despite its fundamental physiological role, the molecular mechanisms that control ER-mitochondria cross talk are obscure. Ubiquitination has recently emerged as a powerful tool to modulate protein function, via regulation of protein subcellular localization and protein ability to interact with other proteins. Ubiquitination is also a reversible mechanism, which can be actively controlled by opposing ubiquitination-deubiquitination events. This work found that in Parkin deficient cells and parkin mutant human fibroblasts, the tether between ER and mitochondria is decreased. The site of Parkin dependent ubiquitination was identified, and it was shown that the non-ubiquitinatable Mfn2 mutant fails to restore ER-mitochondria physical and functional interaction. Finally, advantage was taken of an established in vivo model of PD to demonstrate that manipulation of ER-mitochondria tethering by expressing an ER-mitochondria synthetic linker is sufficient to rescue the locomotor deficit associated to an in vivo Drosophila model of PD.
Li, Y. X., Sibon, O. C. M. and Dijkers, P. F. (2018). Inhibition of NF-kappaB in astrocytes is sufficient to delay neurodegeneration induced by proteotoxicity in neurons. J Neuroinflammation 15(1): 261. PubMed ID: 30205834
Summary:
This study examined responses in astrocytes induced by expression of disease-associated, aggregation-prone proteins in other cells. A role was examined for intracellular astrocytic responses in a Drosophila model for Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease), a disease caused by expansion of the polyglutamine (polyQ) stretch in the ATXN3 gene. In this Drosophila SCA3 model, eye-specific expression of a biologically relevant portion of the ATXN3 gene, containing expanded polyQ repeats (SCA3(polyQ78)) was expressed. Eye-specific expression of SCA3(polyQ78) resulted in the presence of astrocytes in the eye, suggesting putative involvement of astrocytes in SCA3. In a candidate RNAi screen, genes in astrocytes were identified that can enhance or suppress SCA3(polyQ78)-induced eye degeneration. Relish, a conserved NF-kappaB transcription factor, was identified as an enhancer of degeneration. Activity of Relish was upregulated in the SCA3 model. Relish can exert its effect via Relish-specific AMPs, since downregulation of these AMPs attenuated degeneration. Relish signaling was examined in astrocytes on neurodegeneration. Selective inhibition of Relish expression specifically in astrocytes extended lifespan of flies that expressed SCA3(polyQ78) exclusively in neurons. Inhibition of Relish signaling in astrocytes also extended lifespan in a Drosophila model for Alzheimer's disease. These data demonstrate that astrocytes respond to proteotoxic stress in neurons, and that these astrocytic responses are important contributors to neurodegeneration. The data provide direct evidence for cell-non-autonomous contributions of astrocytes to neurodegeneration, with possible implications for therapeutic interventions in multiple neurodegenerative diseases.
Furotani, K., Kamimura, K., Yajima, T., Nakayama, M., Enomoto, R., Tamura, T., Okazawa, H. and Sone, M. (2018). Suppression of the synaptic localization of a subset of proteins including APP partially ameliorates phenotypes of the Drosophila Alzheimer's disease model. PLoS One 13(9): e0204048. PubMed ID: 30226901
Summary:
APP (amyloid precursor protein), the causative molecule of Alzheimer's disease, is synthesized in neuronal cell bodies and subsequently transported to synapses. It has been show that the yata gene is required for the synaptic transport of the APP orthologue in Drosophila melanogaster. This study examined the effect of a reduction in yata expression in the Drosophila Alzheimer's disease model, in which expression of human mutant APP was induced. The synaptic localization of APP and other synaptic proteins was differentially inhibited by yata knockdown and null mutation. Expression of APP resulted in abnormal synaptic morphology and the premature death of animals. These phenotypes were partially but significantly rescued by yata knockdown, whereas yata knockdown itself caused no abnormality. Moreover, synaptic transmission accuracy was impaired in this model, and this phenotype was improved by yata knockdown. Thus, these data suggested that the phenotypes caused by APP can be partially prevented by inhibition of the synaptic localization of a subset of synaptic proteins including APP.
Kim, E. Y., Kang, K. H. and Koh, H. (2018). Cyclophilin 1 (Cyp1) mutation ameliorates oxidative stress-induced defects in a Drosophila DJ-1 null mutant. Biochem Biophys Res Commun. PubMed ID: 30297105
Summary:
Drosophila cyclophilin 1 (Cyp1) is a structural and functional homolog of mammalian cyclophilin D (CypD), a unique mitochondrial cyclophilin (Cyp) that regulates the inner mitochondrial membrane permeability transition and cell survival under cellular stresses such as oxidative damage. This study generated and characterized a Drosophila Cyp1 mutant. Cyp1 mutant flies successfully developed into adults and showed no significant defects in mitochondrial morphology, function, and content. However, oxidative damage significantly decreased in Cyp1 mutant flies, and inhibition of Cyp1 expression substantially increased the survival under various oxidative stress paradigms. Moreover, Cyp1 mutation successfully ameliorated survival rates, locomotor activity, and dopaminergic neuron quantity in a Drosophila DJ-1 mutant under oxidative stress, further confirming the protective role of Cyp1 mutation against oxidative stress. In conclusion, these results suggest Cyp1 and its human homolog CypD as putative molecular targets for the treatment of DJ-1 deficiency-associated diseases, including Parkinson's disease.
Kunduri, G., Turner-Evans, D., Konya, Y., Izumi, Y., Nagashima, K., Lockett, S., Holthuis, J., Bamba, T., Acharya, U. and Acharya, J. K. (2018). Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants. Proc Natl Acad Sci U S A 115(38): E8919-e8928. PubMed ID: 30185559
Summary:
Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. This study shows an animal model of PSE, in Drosophila, owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase (cpes)-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glial membranes. Further, this study shows the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype.
Mallik, M., Catinozzi, M., Hug, C. B., Zhang, L., Wagner, M., Bussmann, J., Bittern, J., Mersmann, S., Klambt, C., Drexler, H. C. A., Huynen, M. A., Vaquerizas, J. M. and Storkebaum, E. (2018). Xrp1 genetically interacts with the ALS-associated FUS orthologue caz and mediates its toxicity. J Cell Biol. PubMed ID: 30209068
Summary:
Cabeza (caz) is the single Drosophila melanogaster orthologue of the human FET proteins FUS, TAF15, and EWSR1, which have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. This study identified Xrp1, a nuclear chromatin-binding protein, as a key modifier of caz mutant phenotypes. Xrp1 expression was strongly up-regulated in caz mutants, and Xrp1 heterozygosity rescued their motor defects and life span. Interestingly, selective neuronal Xrp1 knockdown was sufficient to rescue, and neuronal Xrp1 overexpression phenocopied caz mutant phenotypes. The caz/Xrp1 genetic interaction depended on the functionality of the AT-hook DNA-binding domain in Xrp1, and the majority of Xrp1-interacting proteins are involved in gene expression regulation. Consistently, caz mutants displayed gene expression dysregulation, which was mitigated by Xrp1 heterozygosity. Finally, Xrp1 knockdown substantially rescued the motor deficits and life span of flies expressing ALS mutant FUS in motor neurons, implicating gene expression dysregulation in ALS-FUS pathogenesis.

Tuesday, October 30th - Chromatin

Armstrong, R. L., Penke, T. J. R., Strahl, B. D., Matera, A. G., McKay, D. J., MacAlpine, D. M. and Duronio, R. J. (2018). Chromatin conformation and transcriptional activity are permissive regulators of DNA replication initiation in Drosophila. Genome Res. PubMed ID: 30279224
Summary:
Chromatin structure has emerged as a key contributor to spatial and temporal control over the initiation of DNA replication. Nevertheless, a causal relationship between chromatin structure and replication initiation remains elusive. This study combined histone gene engineering and whole-genome sequencing in Drosophila to determine how perturbing chromatin structure affects replication initiation. Most pericentric heterochromatin was found to remain late replicating in H3K9R mutants, even though H3K9R pericentric heterochromatin is depleted of HP1a, more accessible, and transcriptionally active. These data indicate that HP1a loss, increased chromatin accessibility, and elevated transcription do not result in early replication of heterochromatin. Nevertheless, a small amount of pericentric heterochromatin with increased accessibility replicates earlier in H3K9R mutants. Transcription is de-repressed in these regions of advanced replication, but not in those regions of the H3K9R mutant genome that replicate later, suggesting that transcriptional repression may contribute to late replication. This study also explored relationships among chromatin, transcription, and replication in euchromatin by analyzing H4K16R mutants. In Drosophila, the X Chromosome is upregulated 2-fold and replicates earlier in XY males than it does in XX females. This study found that H4K16R mutation prevents normal male development and abrogates hyper-expression and earlier replication of the male X, consistent with previously established genome-wide correlations between transcription and early replication. By contrast, H4K16R females are viable and fertile, indicating that H4K16 modification is dispensable for genome replication and gene expression.
Hanlon, S. L., Miller, D. E., Eche, S. and Hawley, R. S. (2018). Origin, composition, and structure of the supernumerary B chromosome of Drosophila melanogaster. Genetics. PubMed ID: 30249684
Summary:
The number of chromosomes carried by an individual species is one of its defining characteristics. Some species, however, can also carry supernumerary chromosomes referred to as B chromosomes. B chromosomes were recently identified in a laboratory stock of Drosophila melanogaster enabling extensive molecular analysis. The B chromosomes were isolated by pulsed-field gel electrophoresis and determined their composition through next-generation sequencing. Although these B chromosomes carry no known euchromatic sequence, they are rich in transposable elements and long arrays of short nucleotide repeats, the most abundant being the uncharacterized AAGAT satellite repeat. Fluorescent in-situ hybridization on metaphase chromosome spreads revealed this repeat is located on Chromosome 4, strongly suggesting the origin of the B chromosomes is Chromosome 4. Cytological and quantitative comparisons of signal intensity between Chromosome 4 and the B chromosomes supports the hypothesis that the structure of the B chromosome is an isochromosome. Also, the identification is reported of a new B chromosome variant in a related laboratory stock. This B chromosome has a similar repeat signature as the original but is smaller and much less prevalent. Additional stocks with similar genotypes were examined and B chromosomes were found, but these stocks lacked the AAGAT satellite repeat. This molecular characterization of D. melanogaster B chromosomes is the first step towards understanding how supernumerary chromosomes arise from essential chromosomes and what may be necessary for their stable inheritance.
Cheutin, T. and Cavalli, G. (2018). Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis. Nat Commun 9(1): 3898. PubMed ID: 30254245
Summary:
Polycomb-group proteins are conserved chromatin factors that maintain the silencing of key developmental genes, notably the Hox gene clusters, outside of their expression domains. Depletion of Polycomb repressive complex 1 (PRC1) proteins typically results in chromatin unfolding, as well as ectopic transcription. To disentangle these two phenomena, this study analyzed the temporal function of two PRC1 proteins, Polyhomeotic (Ph) and Polycomb (Pc), on Hox gene clusters during Drosophila embryogenesis. The absence of Ph or Pc affects the higher-order chromatin folding of Hox clusters prior to ectopic Hox gene transcription, demonstrating that PRC1 primary function during early embryogenesis is to compact its target chromatin. Moreover, the differential effects of Ph and Pc on Hox cluster folding match the differences in ectopic Hox gene expression observed in these two mutants. These data suggest that PRC1 maintains gene silencing by folding chromatin domains and impose architectural layer to gene regulation.
Foglizzo, M., Middleton, A. J., Burgess, A. E., Crowther, J. M., Dobson, R. C. J., Murphy, J. M., Day, C. L. and Mace, P. D. (2018). A bidentate Polycomb Repressive-Deubiquitinase complex is required for efficient activity on nucleosomes. Nat Commun 9(1): 3932. PubMed ID: 30258054
Summary:
Attachment of ubiquitin to lysine 119 of Histone 2A (H2AK119Ub) is an epigenetic mark characteristic of repressed developmental genes, which is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. This study reports the crystal structure of the Drosophila PR-DUB, revealing that the deubiquitinase Calypso and its activating partner ASX form a 2:2 complex. The bidentate Calypso-ASX complex is generated by dimerisation of two activated Calypso proteins through their coiled-coil regions. Disrupting the Calypso dimer interface does not affect inherent catalytic activity, but inhibits removal of H2AK119Ub as a consequence of impaired recruitment to nucleosomes. Mutating the equivalent surface on the human counterpart, BAP1, also compromises activity on nucleosomes. Together, this suggests that high local concentrations drive assembly of bidentate PR-DUB complexes on chromatin-providing a mechanistic basis for enhanced PR-DUB activity at specific genomic foci, and the impact of distinct classes of PR-DUB mutations in tumorigenesis.
Yao, B., Li, Y., Wang, Z., Chen, L., Poidevin, M., Zhang, C., Lin, L., Wang, F., Bao, H., Jiao, B., Lim, J., Cheng, Y., Huang, L., Phillips, B. L., Xu, T., Duan, R., Moberg, K. H., Wu, H. and Jin, P. (2018). Active N(6)-methyladenine demethylation by DMAD regulates gene expression by coordinating with Polycomb Protein in neurons. Mol Cell 71(5): 848-857.e846. PubMed ID: 30078725
Summary:
A ten-eleven translocation (TET) ortholog exists as a DNA N(6)-methyladenine (6mA) demethylase (DMAD) in Drosophila. However, the molecular roles of 6mA and DMAD remain unexplored. Through genome-wide 6mA and transcriptome profiling in Drosophila brains and neuronal cells, this study found that 6mA may epigenetically regulate a group of genes involved in neurodevelopment and neuronal functions. Mechanistically, DMAD interacts with the Trithorax-related complex protein Wds to maintain active transcription by dynamically demethylating intragenic 6mA. Accumulation of 6mA by depleting DMAD coordinates with Polycomb proteins and contributes to transcriptional repression of these genes. These findings suggest that active 6mA demethylation by DMAD plays essential roles in fly CNS by orchestrating through added epigenetic mechanisms.
Luzhin, A. V., Flyamer, I. M., Khrameeva, E. E., Ulianov, S. V., Razin, S. V. and Gavrilov, A. A. (2018). Quantitative differences in TAD border strength underly the TAD hierarchy in Drosophila chromosomes. J Cell Biochem. PubMed ID: 30260021
Summary:
Chromosomes in many organisms, including Drosophila and mammals, are folded into topologically associating domains (TADs). Increasing evidence suggests that TAD folding is hierarchical, wherein subdomains combine to form larger superdomains, instead of a sequence of nonoverlapping domains. This study examined the hierarchical structure of TADs in Drosophila. The boundaries of TADs of different hierarchical levels are characterized by the presence of different portions of active chromatin, but do not vary in the binding of architectural proteins, such as CCCTC binding factor or cohesin. The apparent hierarchy of TADs in Drosophila chromosomes is not likely to have functional importance but rather reflects various options of long-range chromatin folding directed by the distribution of active and inactive chromatin segments and may represent population average.

Monday, October 29th - Adult Neural Function

Koon, A. C., Chen, Z. S., Peng, S., Fung, J. M. S., Zhang, X., Lembke, K. M., Chow, H. K., Frank, C. A., Jiang, L., Lau, K. F. and Chan, H. Y. E. (2018). Drosophila Exo70 is essential for neurite extension and survival under thermal stress. J Neurosci 38(37): 8071-8086. PubMed ID: 30209205
Summary:
The octomeric exocyst complex governs the final step of exocytosis in both plants and animals. Its roles, however, extend beyond exocytosis and include organelle biogenesis, ciliogenesis, cell migration, and cell growth. Exo70 is a conserved component of the exocyst whose function in Drosophila is unclear. This study characterized two mutant alleles of Drosophila exo70. exo70 mutants exhibit reduced synaptic growth, locomotor activity, glutamate receptor density, and mEPSP amplitude. Presynaptic Exo70 is necessary for normal synaptic growth at the neuromuscular junction (NMJ). At the neuromuscular junction, exo70 genetically interacts with the small GTPase ralA to regulate synaptic growth. Loss of Exo70 leads to the blockage of JNK signaling-, activity-, and temperature-induced synaptic outgrowths. This phenotype is associated with an impairment of integral membrane protein transport to the cell surface at synaptic terminals. In octopaminergic motor neurons, Exo70 is detected in synaptic varicosities, as well as the regions of membrane extensions in response to activity stimulation. Strikingly, mild thermal stress causes severe neurite outgrowth defects and pharate adult lethality in exo70 mutants. exo70 mutants also display defective locomotor activity in response to starvation stress. These results demonstrated that Exo70 is an important regulator of induced synaptic growth and is crucial for an organism's adaptation to environmental changes.
Fujiwara, Y., Hermann-Luibl, C., Katsura, M., Sekiguchi, M., Ida, T., Helfrich-Forster, C. and Yoshii, T. (2018). The CCHamide1 neuropeptide expressed in the anterior dorsal neuron 1 conveys a circadian signal to the ventral lateral neurons in Drosophila melanogaster. Front Physiol 9: 1276. PubMed ID: 30246807
Summary:
Drosophila possesses ~150 brain clock neurons that control circadian behavioral rhythms. This study investigated the role of CCHamide1 (CCHa1), a neuropeptide expressed in the anterior dorsal neuron 1 (DN1a), in intercellular communication of the clock neurons. CCHa1 connects the DN1a clock neurons to the ventral lateral clock neurons (LNv) via the CCHa1 receptor, which is a homolog of the gastrin-releasing peptide receptor playing a role in circadian intercellular communications in mammals. CCHa1 knockout or knockdown flies have a generally low activity level with a special reduction of morning activity. In addition, they exhibit advanced morning activity under light-dark cycles and delayed activity under constant dark conditions, which correlates with an advance/delay of PAR domain Protein 1 (PDP1) oscillations in the small-LNv (s-LNv) neurons that control morning activity. The terminals of the s-LNv neurons show rather high levels of Pigment-dispersing factor (PDF) in the evening, when PDF is low in control flies, suggesting that the knockdown of CCHa1 leads to increased PDF release; PDF signals the other clock neurons and evidently increases the amplitude of their PDP1 cycling. A previous study showed that high-amplitude PDP1 cycling increases the siesta of the flies, and indeed, CCHa1 knockout or knockdown flies exhibit a longer siesta than control flies. The DN1a neurons are known to be receptive to PDF signaling from the s-LNv neurons; thus, these results suggest that the DN1a and s-LNv clock neurons are reciprocally coupled via the neuropeptides CCHa1 and PDF, and this interaction fine-tunes the timing of activity and sleep.
Hans, V. R., Wendt, T. I., Patel, A. M., Patel, M. M., Perez, L., Talbot, D. E. and Jemc, J. C. (2018). Raw regulates glial population of the eye imaginal disc. Genesis. PubMed ID: 30288928
Summary:
Glia are critical for proper development, support, and function of the nervous system. The Drosophila eye has proven an excellent model for gaining significant insight into the molecular mechanisms regulating glial development and function. Recent studies have demonstrated that Raw is required in glia of the central and peripheral nervous systems; however, the function of Raw in glia of the developing eye has not been explored. These studies demonstrate that raw knockdown results in a reduction in the number of glia in the third instar eye imaginal disc and reduced glial spreading across the field of differentiating photoreceptor neurons. Expression of a raw enhancer trap reveals that raw is expressed in eye disc glia. Exploration of the mechanism by which raw knockdown results in glial reduction reveals that Raw is required for glial proliferation and migration into the eye disc. In addition, Raw negatively regulates Jun N-terminal kinase (JNK) signaling in glia of the developing eye and increased JNK signaling results in a reduction in the number of glia populating the eye disc, similar to that observed upon raw knockdown. Thus, Raw functions as a critical regulator of glial population of the eye imaginal disc by regulating glial proliferation and migration and inhibiting JNK signaling.
Lamaze, A., Kratschmer, P., Chen, K. F., Lowe, S. and Jepson, J. E. C. (2018). A wake-promoting circadian output circuit in Drosophila. Curr Biol 28(19): 3098-3105.e3093. PubMed ID: 30270186
Summary:
Circadian clocks play conserved roles in gating sleep and wake states throughout the day-night cycle. In the fruit fly Drosophila melanogaster, DN1p clock neurons have been reported to play both wake- and sleep-promoting roles, suggesting a complex coupling of DN1p neurons to downstream sleep and arousal centers. However, the circuit logic by which DN1p neurons modulate sleep remains poorly understood. This study shows that DN1p neurons can be divided into two morphologically distinct subsets. Projections from one subset surround the pars intercerebralis, a previously defined circadian output region. In contrast, the second subset also sends presynaptic termini to a visual processing center, the anterior optic tubercle (AOTU). Within the AOTU, DN1p neurons inhibit a class of tubercular-bulbar (TuBu) neurons that act to promote consolidated sleep. These TuBu neurons in turn form synaptic connections with R neurons of the ellipsoid body, a region linked to visual feature detection, locomotion, spatial memory, and sleep homeostasis. These results define a second output arm from DN1p neurons and suggest a role for TuBu neurons as regulators of sleep drive.
Guo, F., Holla, M., Diaz, M. M. and Rosbash, M. (2018). A circadian output circuit controls sleep-wake arousal in Drosophila. Neuron. PubMed ID: 30269992
Summary:
The Drosophila core circadian circuit contains distinct groups of interacting neurons that give rise to diurnal sleep-wake patterns. Previous work showed that a subset of dorsal neurons 1 (DN1s) are sleep-promoting through their inhibition of activity-promoting circadian pacemakers. This study shows that these anterior-projecting DNs (APDNs) also "exit" the circadian circuitry and communicate with the homeostatic sleep center in higher brain regions to regulate sleep and sleep-wake arousal. These APDNs connect to a small, discrete subset of tubercular-bulbar neurons, which are connected in turn to specific sleep-centric ellipsoid body (EB)-ring neurons of the central complex. Remarkably, activation of the APDNs produces sleep-like oscillations in the EB and affects arousal. The data indicate that this APDN-TuBusup-EB circuit temporally regulates sleep-wake arousal in addition to the previously defined role of the TuBu-EB circuit in vision, navigation, and attention.
Li, M. T., Cao, L. H., Xiao, N., Tang, M., Deng, B., Yang, T., Yoshii, T. and Luo, D. G. (2018). Hub-organized parallel circuits of central circadian pacemaker neurons for visual photoentrainment in Drosophila. Nat Commun 9(1): 4247. PubMed ID: 30315165
Summary:
Circadian rhythms are orchestrated by a master clock that emerges from a network of circadian pacemaker neurons. The master clock is synchronized to external light/dark cycles through photoentrainment, but the circuit mechanisms underlying visual photoentrainment remain largely unknown. This study reports that Drosophila has eye-mediated photoentrainment via a parallel pacemaker neuron organization. Patch-clamp recordings of central circadian pacemaker neurons reveal that light excites most of them independently of one another. Light-responding pacemaker neurons were shown to send their dendrites to a neuropil called accessary medulla (aMe), where they make monosynaptic connections with Hofbauer-Buchner eyelet photoreceptors and interneurons that transmit compound-eye signals. Laser ablation of aMe and eye removal both abolish light responses of circadian pacemaker neurons, revealing aMe as a hub to channel eye inputs to central circadian clock. Taken together, this study demonstrates that the central clock receives eye inputs via hub-organized parallel circuits in Drosophila.

Friday, October 26th - RNA

Mach, J., Atkins, M., Gajewski, K. M., Mottier-Pavie, V., Sansores-Garcia, L., Xie, J., Mills, R. A., Kowalczyk, W., Van Huffel, L., Mills, G. B. and Halder, G. (2018). Modulation of the Hippo pathway and organ growth by RNA processing proteins. Proc Natl Acad Sci U S A. PubMed ID: 30257938
Summary:
The Hippo tumor-suppressor pathway regulates organ growth, cell proliferation, and stem cell biology. Defects in Hippo signaling and hyperactivation of its downstream effectors-Yorkie (Yki) in Drosophila and YAP/TAZ in mammals-result in progenitor cell expansion and overgrowth of multiple organs and contribute to cancer development. Deciphering the mechanisms that regulate the activity of the Hippo pathway is key to understanding its function and for therapeutic targeting. However, although the Hippo kinase cascade and several other upstream inputs have been identified, the mechanisms that regulate Yki/YAP/TAZ activity are still incompletely understood. To identify new regulators of Yki activity, Drosophila was screened for suppressors of tissue overgrowth and Yki activation caused by overexpression of atypical protein kinase C (aPKC), a member of the apical cell polarity complex. This screen identified mutations in the heterogeneous nuclear ribonucleoprotein Hrb27C that strongly suppressed the tissue defects induced by ectopic expression of aPKC. Hrb27C was required for aPKC-induced tissue growth and Yki target gene expression but did not affect general gene expression. Genetic and biochemical experiments showed that Hrb27C affects Yki phosphorylation. Other RNA-binding proteins known to interact with Hrb27C for mRNA transport in oocytes were also required for normal Yki activity, although they suppressed Yki output. Based on the known functions of Hrb27C, it is concluded that Hrb27C-mediated control of mRNA splicing, localization, or translation is essential for coordinated activity of the Hippo pathway.
Kelleher, E. S., Azevedo, R. B. R. and Zheng, Y. (2018). The evolution of small-RNA-mediated silencing of an invading transposable element. Genome Biol Evol. PubMed ID: 30252073
Summary:
Transposable elements (TEs) are genomic parasites that impose fitness costs on their hosts by producing deleterious mutations and disrupting gametogenesis. Host genomes avoid these costs by regulating TE activity, particularly in germline cells where new insertions are heritable and TEs are exceptionally active. However, the capacity of different TE-associated fitness costs to select for repression in the host, and the role of selection in the evolution of TE regulation more generally, remain controversial. This study used forward, individual-based simulations to examine the evolution of small-RNA-mediated TE regulation, a conserved mechanism for TE repression that is employed by both prokaryotes and eukaryotes. To design and parameterize a biologically realistic model, this study drew on an extensive survey of empirical studies of the transposition and regulation of P-element DNA transposons in Drosophila melanogaster. Even under conservative assumptions, where small-RNA-mediated regulation reduces transposition only, repression evolves rapidly and adaptively after the genome is invaded by a new TE in simulated populations. It was further shown that the spread of repressor alleles through simulated populations is greatly enhanced by two additional TE-imposed fitness costs: dysgenic sterility and ectopic recombination. Finally, it was demonstrated that the adaptive mutation rate to repression is a critical parameter that influences both the evolutionary trajectory of host repression and the associated proliferation of TEs after invasion in simulated populations. These findings suggest that adaptive evolution of TE regulation may be stronger and more prevalent than previously appreciated, and provide a framework for interpreting empirical data.
Barckmann, B., El-Barouk, M., Pelisson, A., Mugat, B., Li, B., Franckhauser, C., Fiston Lavier, A. S., Mirouze, M., Fablet, M. and Chambeyron, S. (2018). The somatic piRNA pathway controls germline transposition over generations. Nucleic Acids Res 46(18): 9524-9536. PubMed ID: 30312469
Summary:
Transposable elements (TEs) are parasitic DNA sequences that threaten genome integrity by replicative transposition in host gonads. The Piwi-interacting RNAs (piRNAs) pathway is assumed to maintain Drosophila genome homeostasis by downregulating transcriptional and post-transcriptional TE expression in the ovary. However, the bursts of transposition that are expected to follow transposome derepression after piRNA pathway impairment have not yet been reported. This study shows, at a genome-wide level, that piRNA loss in the ovarian somatic cells boosts several families of the endogenous retroviral subclass of TEs, at various steps of their replication cycle, from somatic transcription to germinal genome invasion. For some of these TEs, the derepression caused by the loss of piRNAs is backed up by another small RNA pathway (siRNAs) operating in somatic tissues at the post transcriptional level. Derepressed transposition during 70 successive generations of piRNA loss exponentially increases the genomic copy number by up to 10-fold.
Kennerdell, J. R., Liu, N. and Bonini, N. M. (2018). MiR-34 inhibits polycomb repressive complex 2 to modulate chaperone expression and promote healthy brain aging. Nat Commun 9(1): 4188. PubMed ID: 30305625
Summary:
Aging is a prominent risk factor for neurodegenerative disease. Defining gene expression mechanisms affecting healthy brain aging should lead to insight into genes that modulate susceptibility to disease. To define such mechanisms, analysis of miR-34 mutants have been pursued in Drosophila. The miR-34 mutant brain displays a gene expression profile of accelerated aging, and miR-34 upregulation is a potent suppressor of polyglutamine-induced neurodegeneration. Pcl and Su(z)12, two components of polycomb repressive complex 2, (PRC2), are targets of miR-34, with implications for age-associated processes. Because PRC2 confers the repressive H3K27me3 mark, it is hypothesized that miR-34 modulates PRC2 activity to relieve silencing of genes promoting healthful aging. Gene expression profiling of the brains of hypomorphic mutants in Enhancer of zeste (E(z)), the enzymatic methyltransferase component of PRC2, revealed a younger brain transcriptome profile and identified the small heat shock proteins as key genes reduced in expression with age.
Kuznetsova, K. G., Kliuchnikova, A. A., Ilina, II, Chernobrovkin, A. L., Novikova, S., Farafonova, T. E., Karpov, D. S., Ivanov, M. V., Goncharov, A. O., Ilgisonis, E. V., Voronko, O. E., Nasaev, S. S., Zgoda, V. G., Zubarev, R. A., Gorshkov, M. V. and Moshkovskii, S. A. (2018). Proteogenomics of adenosine-to-inosine RNA editing in the fruit fly. J Proteome Res. PubMed ID: 30298734
Summary:
Adenosine-to-inosine RNA editing (see Drosophila Adar) is one of the most common types of RNA editing, a posttranscriptional modification made by special enzymes. A proteomic study on this phenomenon is presented for Drosophila melanogaster. Three proteome data sets were used in the study: two taken from public repository and the third one obtained in this study. A customized protein sequence database was generated using results of genome-wide adenosine-to-inosine RNA studies and applied for identifying the edited proteins. The total number of 68 edited peptides belonging to 59 proteins was identified in all data sets. Eight of them being shared between the whole insect, head and brain proteomes. Seven edited sites belonging to synaptic vesicle and membrane trafficking proteins were selected for validation by orthogonal analysis by Multiple Reaction Monitoring. Five editing events in cpx, Syx1A, Cadps, CG4587 and EndoA were validated in fruit fly brain tissue at the proteome level using isotopically labeled standards. Ratios of unedited-to-edited proteoforms varied from 35:1 (Syx1A) to 1:2 (EndoA). Lys-137 to Glu editing of endophilin A may have functional consequences for its interaction to membrane. The work demonstrates the feasibility to identify the RNA editing event at the proteome level using shotgun proteomics and customized edited protein database.
Nayak, A., Kim, D. Y., Trnka, M. J., Kerr, C. H., Lidsky, P. V., Stanley, D. J., Rivera, B. M., Li, K. H., Burlingame, A. L., Jan, E., Frydman, J., Gross, J. D. and Andino, R. (2018). A viral protein restricts Drosophila RNAi immunity by regulating Argonaute activity and stability. Cell Host Microbe 24(4): 542-557.e549. PubMed ID: 30308158
Summary:
The dicistrovirus, Cricket paralysis virus (CrPV) encodes an RNA interference (RNAi) suppressor, 1A, which modulates viral virulence. Using the Drosophila model, structural, biochemical, and virological approaches were combined to elucidate the strategies by which CrPV-1A restricts RNAi immunity. The atomic resolution structure of CrPV-1A uncovered a flexible loop that interacts with Argonaute 2 (Ago-2), thereby inhibiting Ago-2 endonuclease-dependent immunity. Mutations disrupting Ago-2 binding attenuates viral pathogenesis in wild-type but not Ago-2-deficient flies. CrPV-1A also contains a BC-box motif that enables the virus to hijack a host Cul2-Rbx1-EloBC ubiquitin ligase complex, which promotes Ago-2 degradation and virus replication. This study uncovers a viral-based dual regulatory program that restricts antiviral immunity by direct interaction with and modulation of host proteins. While the direct inhibition of Ago-2 activity provides an efficient mechanism to establish infection, the recruitment of a ubiquitin ligase complex enables CrPV-1A to amplify Ago-2 inactivation to restrict further antiviral RNAi immunity.

Thursday, October 25th - Signaling

Kakad, P. P., Penserga, T., Davis, B. P., Henry, B., Boerner, J., Riso, A., Pielage, J. and Godenschwege, T. A. (2018). An ankyrin-binding motif regulates nuclear levels of L1-type neuroglian and expression of the oncogene Myc in Drosophila neurons. J Biol Chem. PubMed ID: 30257867
Summary:
L1 cell adhesion molecule (L1CAM) is well known for its importance in nervous system development and cancer progression. In addition to its role as a plasma membrane protein in cytoskeletal organization, recent in vitro studies have revealed that both transmembrane and cytosolic fragments of proteolytically cleaved vertebrate L1CAM translocate to the nucleus. In vitro studies indicate that nuclear L1CAM affects genes with functions in DNA post-replication repair, cell cycle control, and cell migration and differentiation, but its in vivo role and how its nuclear levels are regulated is less well understood. This study reports that mutations in the conserved ankyrin-binding domain affect nuclear levels of the sole Drosophila homolog neuroglian (Nrg) and that it also has a noncanonical role in regulating transcript levels of the oncogene Myc in the adult nervous system. Altered nuclear levels of Nrg correlate with altered transcript levels of Myc in neurons similar to what has been reported for human glioblastoma stem cells. However, whereas previous in vitro studies suggest that increased nuclear levels of L1CAM promote tumor cell survival, this study found that elevated levels of nuclear Nrg in neurons are associated with increased sensitivity to oxidative stress and reduced lifespan of adult animals. It is therefore concluded that these findings are of potential relevance to the management of neurodegenerative diseases associated with oxidative stress and cancer.
Karney-Grobe, S., Russo, A., Frey, E., Milbrandt, J. and DiAntonio, A. (2018). HSP90 is a chaperone for DLK and is required for axon injury signaling. Proc Natl Acad Sci U S A. PubMed ID: 30275300
Summary:
Peripheral nerve injury induces a robust proregenerative program that drives axon regeneration. A loss-of-function pharmacological screen was carried out in cultured adult mouse sensory neurons for proteins required to activate this program. Well-characterized inhibitors were present as injury signaling was induced but were removed before axon outgrowth to identify molecules that block induction of the program. Of 480 compounds, 35 prevented injury-induced neurite regrowth. The top hits were inhibitors to heat shock protein 90 (HSP90), a chaperone with no known role in axon injury. HSP90 inhibition blocks injury-induced activation of the proregenerative transcription factor cJun and several regeneration-associated genes. These phenotypes mimic loss of the proregenerative kinase, dual leucine zipper kinase (DLK), a critical neuronal stress sensor that drives axon degeneration, axon regeneration, and cell death. HSP90 is an atypical chaperone that promotes the stability of signaling molecules. HSP90 and DLK show two hallmarks of HSP90-client relationships: (i) HSP90 binds DLK, and (ii) HSP90 inhibition leads to rapid degradation of existing DLK protein. Moreover, HSP90 is required for DLK stability in vivo, where HSP90 inhibitor reduces DLK protein in the sciatic nerve. This phenomenon is evolutionarily conserved in Drosophila. Genetic knockdown of Drosophila HSP90, Hsp83, decreases levels of Drosophila DLK, Wallenda, and blocks Wallenda-dependent synaptic terminal overgrowth and injury signaling. These findings support the hypothesis that HSP90 chaperones DLK and is required for DLK functions, including proregenerative axon injury signaling.
Herman, J. A., Willits, A. B. and Bellemer, A. (2018). Galphaq and Phospholipase Cbeta signaling regulate nociceptor sensitivity in Drosophila melanogaster larvae. PeerJ 6: e5632. PubMed ID: 30258723
Summary:
Drosophila melanogaster larvae detect noxious thermal and mechanical stimuli in their environment using polymodal nociceptor neurons whose dendrites tile the larval body wall. Activation of these nociceptors by potentially tissue-damaging stimuli elicits a stereotyped escape locomotion response. The cellular and molecular mechanisms that regulate nociceptor function are increasingly well understood, but gaps remain in knowledge of the broad mechanisms that control nociceptor sensitivity. This study used cell-specific knockdown and overexpression to show that nociceptor sensitivity to noxious thermal and mechanical stimuli is correlated with levels of Galphaq and phospholipase Cbeta signaling. Genetic manipulation of these signaling mechanisms does not result in changes in nociceptor morphology, suggesting that changes in nociceptor function do not arise from changes in nociceptor development, but instead from changes in nociceptor activity. These results demonstrate roles for Galphaq and phospholipase Cbeta signaling in facilitating the basal sensitivity of the larval nociceptors to noxious thermal and mechanical stimuli and suggest future studies to investigate how these signaling mechanisms may participate in neuromodulation of sensory function.
Kastl, P., Manikowski, D., Steffes, G., Schurmann, S., Bandari, S., Klambt, C. and Grobe, K. (2018). Disrupting Hedgehog Cardin-Weintraub sequence and positioning changes cellular differentiation and compartmentalization in vivo. Development 145(18). PubMed ID: 30242104
Summary:
Metazoan Hedgehog (Hh) morphogens are essential regulators of growth and patterning at significant distances from their source, despite being produced as N-terminally palmitoylated and C-terminally cholesteroylated proteins, which firmly tethers them to the outer plasma membrane leaflet of producing cells and limits their spread. One mechanism to overcome this limitation is proteolytic processing of both lipidated terminal peptides, called shedding, but molecular target site requirements for effective Hh shedding remained undefined. This work used Drosophila melanogaster as a model to show that mutagenesis of the N-terminal Cardin-Weintraub (CW) motif inactivates recombinant Hh proteins to variable degrees and, if overexpressed in the same compartment, converts them into suppressors of endogenous Hh function. In vivo, additional removal of N-palmitate membrane anchors largely restored endogenous Hh function, supporting the hypothesis that proteolytic CW processing controls Hh solubilization. Importantly, it was also observed that CW repositioning impairs anterior/posterior compartmental boundary maintenance in the third instar wing disc. This demonstrates that Hh shedding not only controls the differentiation of anterior cells, but also maintains the sharp physical segregation between these receiving cells and posterior Hh-producing cells.
Jayakumar, S., Richhariya, S., Deb, B. K. and Hasan, G. (2018). A multi-component neuronal response encodes the larval decision to pupariate upon amino acid starvation. J Neurosci. PubMed ID: 30301757
Summary:
Organisms need to co-ordinate growth with development, particularly in the context of nutrient availability. Thus, multiple ways have evolved to survive extrinsic nutrient deprivation during development. In Drosophila, growth occurs during larval development. Larvae are thus critically dependant on nutritional inputs but post critical weight they pupariate even when starved. How nutrient availability is coupled to the internal metabolic state for the decision to pupariate needs better understanding. Glutamatergic interneurons have been identified in the ventral ganglion that regulate pupariation on a protein-deficient diet. This study report that Drosophila third instar larvae (either sex) sense arginine to evaluate their nutrient environment using an amino-acid transporter Slimfast. The glutamatergic interneurons integrate external protein availability with internal metabolic state through neuropeptide signals. IP3 mediated calcium release and store-operated calcium entry are essential in these glutamatergic neurons for such integration and alter neuronal function by reducing the expression of multiple ion channels.
Huang, J. and Wang, H. (2018). Hsp83/Hsp90 physically associates with Insulin receptor to promote neural stem cell reactivation. Stem Cell Reports 11(4): 883-896. PubMed ID: 30245208
Summary:
Neural stem cells (NSCs) have the ability to exit quiescence and reactivate in response to physiological stimuli. In the Drosophila brain, insulin receptor (InR)/phosphatidylinositol 3-kinase (PI3K)/Akt pathway triggers NSC reactivation. However, intrinsic mechanisms that control the InR/PI3K/Akt pathway during reactivation remain unknown. This study identified heat shock protein 83 (Hsp83/Hsp90), a molecular chaperone, as an intrinsic regulator of NSC reactivation. Hsp83 is both necessary and sufficient for NSC reactivation by promoting the activation of InR pathway in larval brains in the presence of dietary amino acids. Both Hsp83 and its co-chaperone Cdc37 physically associate with InR. Finally, reactivation defects observed in brains depleted of hsp83 were rescued by over-activation of the InR/PI3K/Akt pathway, suggesting that Hsp83 functions upstream of the InR/PI3K/Akt pathway during NSC reactivation. Given the conservation of Hsp83 and the InR pathway, this finding may provide insights into the molecular mechanisms underlying mammalian NSC reactivation.

Wednesday, October 24th - Apoptosis

Williamson, A. P. and Vale, R. D. (2018). Spatial control of Draper receptor signaling initiates apoptotic cell engulfment. J Cell Biol. PubMed ID: 30139739
Summary:
The engulfment of apoptotic cells is essential for tissue homeostasis and recovering from damage. Engulfment is mediated by receptors that recognize ligands exposed on apoptotic cells such as phosphatidylserine (PS). This study converted Drosophila melanogaster S2 cells into proficient phagocytes by transfecting the Draper engulfment receptor and replacing apoptotic cells with PS-coated beads. Similar to the T cell receptor (TCR), PS-ligated Draper forms dynamic microclusters that recruit cytosolic effector proteins and exclude a bulky transmembrane phosphatase, consistent with a kinetic segregation-based triggering mechanism. However, in contrast with the TCR, localized signaling at Draper microclusters results in time-dependent depletion of actin filaments, which facilitates engulfment. The Draper-PS extracellular module can be replaced with FRB and FKBP, respectively, resulting in a rapamycin-inducible engulfment system that can be programmed toward defined targets. Collectively, these results reveal mechanistic similarities and differences between the receptors involved in apoptotic corpse clearance and mammalian immunity and demonstrate that engulfment can be reprogrammed toward nonnative targets.
Cara, F. D., Bulow, M., Simmonds, A. J. and Rachubinski, R. A. (2018). Dysfunctional peroxisomes compromise gut structure and host defense by increased cell death and Tor-dependent autophagy. Mol Biol Cell: mbcE18070434. PubMed ID: 30188767
Summary:
The gut has a central role in digestion and nutrient absorption, but it also serves in defending against pathogens, engages in mutually beneficial interactions with commensals, and is a major source of endocrine signals. Gut homeostasis is necessary for organismal health, and changes to the gut are associated with conditions like obesity and diabetes and inflammatory illnesses like Crohn's disease. This study reports that peroxisomes, organelles involved in lipid metabolism and redox balance, are required to maintain gut epithelium homeostasis and renewal in Drosophila and for survival and development of the organism. Dysfunctional peroxisomes in gut epithelial cells activate Tor kinase-dependent autophagy that increases cell death and epithelial instability, which ultimately alter the composition of the intestinal microbiota, compromise immune pathways in the gut in response to infection, and affect organismal survival. Peroxisomes in the gut effectively function as hubs that coordinate responses from stress, metabolic and immune signaling pathways to maintain enteric health and the functionality of the gut-microbe interface.
Wang, X., Huang, X., Wu, C. and Xue, L. (2018). Pontin/Tip49 negatively regulates JNK-mediated cell death in Drosophila. Cell Death Discov 5: 8. PubMed ID: 30062057
Summary:
Pontin (Pont), also known as Tip49, encodes a member of the AAA+ (ATPases Associated with Diverse Cellular Activities) superfamily and plays pivotal roles in cell proliferation and growth, yet its function in cell death has remained poorly understood. This study performed a genetic screen for dominant modifiers of Eiger-induced JNK-dependent cell death in Drosophila, and identified Pont as a negative regulator of JNK-mediated cell death. In addition, loss of function of Pont is sufficient to induce cell death and activate the transcription of JNK target gene puc. Furthermore, the epistasis analysis indicates that Pont acts downstream of Hep. Finally, this study found that Pont is also required for JNK-mediated thorax development and acts as a negative regulator of JNK phosphorylation. Together, these data suggest that pont encodes a negative component of Egr/JNK signaling pathway in Drosophila through negatively regulating JNK phosphorylation, which provides a novel role of ATPase in Egr-JNK signaling.
Ayala, C. I., Kim, J. and Neufeld, T. P. (2018). Rab6 promotes insulin receptor and cathepsin trafficking to regulate autophagy induction and activity in Drosophila. J Cell Sci. PubMed ID: 30111579
Summary:
The self-degradative process of autophagy is important for energy homeostasis and cytoplasmic renewal. This lysosome-mediated pathway is negatively regulated by the target of rapamycin kinase (TOR) under basal conditions, and requires the vesicle trafficking machinery regulated by Rab GTPases. However, the interactions between autophagy, TOR and Rab proteins remain incompletely understood in vivo. This study identified Rab6 as a critical regulator of the balance between TOR signaling and autolysosome function. Loss of Rab6 causes an accumulation of enlarged autophagic vesicles resulting in part from a failure to deliver lysosomal hydrolases, rendering autolysosomes with a reduced degradative capacity and impaired turnover. Additionally, Rab6 deficient cells are reduced in size and display defective insulin/TOR signaling as a result of mis-sorting and internalization of the insulin receptor. These findings suggest that Rab6 acts to maintain the reciprocal regulation between autophagy and TOR activity during distinct nutrient states, thereby balancing autophagosome production and turnover to avoid autophagic stress.

Tuesday October 23rd - Stem cells

Hakes, A. E., Otsuki, L. and Brand, A. H. (2018). A newly discovered neural stem cell population is generated by the optic lobe neuroepithelium during embryogenesis in Drosophila melanogaster. Development 145(18). PubMed ID: 30254066
Summary:
Neural stem cells must balance symmetric and asymmetric cell divisions to generate a functioning brain of the correct size. In both the developing Drosophila visual system and mammalian cerebral cortex, symmetrically dividing neuroepithelial cells transform gradually into asymmetrically dividing progenitors that generate neurons and glia. As a result, it has been widely accepted that stem cells in these tissues switch from a symmetric, expansive phase of cell divisions to a later neurogenic phase of cell divisions. In the Drosophila optic lobe, this switch is thought to occur during larval development. However, this study found that neuroepithelial cells start to produce neuroblasts during embryonic development, demonstrating a much earlier role for neuroblasts in the developing visual system. These neuroblasts undergo neurogenic divisions, enter quiescence and are retained post-embryonically, together with neuroepithelial cells. Later in development, neuroepithelial cells undergo further cell divisions before transforming into larval neuroblasts. These results demonstrate that the optic lobe neuroepithelium gives rise to neurons and glia over 60 h earlier than was thought previously.
Deng, H., Takashima, S., Paul, M., Guo, M. and Hartenstein, V. (2018). Mitochondrial dynamics regulates Drosophila intestinal stem cell differentiation. Cell Death Discov 5: 17. PubMed ID: 30062062
Summary:
Differentiation of stem/progenitor cells is associated with a substantial increase in mitochondrial mass and complexity. Mitochondrial dynamics, including the processes of fusion and fission, plays an important role for somatic cell reprogramming and pluripotency maintenance in induced pluripotent cells (iPSCs). However, the role of mitochondrial dynamics during stem/progenitor cell differentiation in vivo remains elusive. This study found differentiation of Drosophila intestinal stem cell is accompanied with continuous mitochondrial fusion. Mitochondrial fusion defective (opa1RNAi) ISCs contain less mitochondrial membrane potential, reduced ATP, and increased ROS level. Surprisingly, suppressing fusion also resulted in the failure of progenitor cells to differentiate. Cells did not switch on the expression of differentiation markers, and instead continued to show characteristics of progenitor cells. Meanwhile, proliferation or apoptosis was unaffected. The differentiation defect could be rescued by concomitant inhibition of Drp1, a mitochondrial fission molecule. Moreover, ROS scavenger also partially rescues opa1RNAi-associated differentiation defects via down-regulating JNK activity. It is proposed that mitochondrial fusion plays a pivotal role in controlling the developmental switch of stem cell fate.
Barton, L. J., Duan, T., Ke, W., Luttinger, A., Lovander, K. E., Soshnev, A. A. and Geyer, P. K. (2018). Nuclear lamina dysfunction triggers a germline stem cell checkpoint. Nat Commun 9(1): 3960. PubMed ID: 30262885
Summary:
LEM domain (LEM-D) proteins are conserved components of the nuclear lamina (NL) that contribute to stem cell maintenance through poorly understood mechanisms. The Drosophila emerin homolog Otefin (Ote) is required for maintenance of germline stem cells (GSCs) and gametogenesis. This study shows that ote mutants carry germ cell-specific changes in nuclear architecture that are linked to GSC loss. Strikingly, both GSC death and gametogenesis are rescued by inactivation of the DNA damage response (DDR) kinases, ATR and Chk2. Whereas the germline checkpoint draws from components of the DDR pathway, genetic and cytological features of the GSC checkpoint differ from the canonical pathway. Instead, structural deformation of the NL correlates with checkpoint activation. Despite remarkably normal oogenesis, rescued oocytes do not support embryogenesis. Taken together, these data suggest that NL dysfunction caused by Otefin loss triggers a GSC-specific checkpoint that contributes to maintenance of gamete quality.
Tseng, C. Y., Su, Y. H., Yang, S. M., Lin, K. Y., Lai, C. M., Rastegari, E., Amartuvshin, O., Cho, Y., Cai, Y. and Hsu, H. J. (2018). Smad-independent BMP signaling in somatic cells limits the size of the germline stem cell pool. Stem Cell Reports. PubMed ID: 30122445
Summary:
In developing organisms, proper tuning of the number of stem cells within a niche is critical for the maintenance of adult tissues; however, the involved mechanisms remain largely unclear. This study demonstrated that Thickveins (Tkv), a type I bone morphogenetic protein (BMP) receptor, acts in the Drosophila developing ovarian soma through a Smad-independent pathway to shape the distribution of BMP signal within the niche, impacting germline stem cell (GSC) recruitment and maintenance. Somatic Tkv promotes Egfr signaling to silence transcription of Dally, which localizes BMP signals on the cell surface. In parallel, Tkv promotes Hh signaling, which promotes escort cell cellular protrusions and upregulates expression of the Drosophila BMP homolog, Dpp, forming a positive feedback loop that enhances Tkv signaling and strengthens the niche boundary. These results reveal a role for non-canonical BMP signaling in the soma during GSC establishment and generally illustrate how complex, cell-specific BMP signaling mediates niche-stem cell interactions.
Wu, X., Chen, Z., Gao, Y., Wang, L., Sun, X., Jin, Y. and Liu, W. (2018). The kruppel like factor Dar1 restricts the proliferation of Drosophila intestinal stem cells. FEBS J. PubMed ID: 30188612
Summary:
The kruppel like factors (KLFs) are a family of transcription factor proteins that regulate a wide range of biological processes. In an RNAi-based screening experiment, this study identified Dar1, which is a KLF member in Drosophila, inhibited the proliferation of intestinal stem cells (ISCs). Suppression of Dar1 activated ISC proliferation; as a consequence, the ISCs and the young differentiated cells were increased. On the other hand, overexpression of Dar1 inhibited ISC division and blocked the formation of ISC lineages. In order to explore the molecular mechanism of the Dar1 functions, the gene expression profiles of the Dar1 knockdown and Dar1 overexpression midguts were compared, using the deep RNA sequencing (RNA-Seq) technique. This experiment revealed that Dar1 negatively regulated the expression of several critical cell cycle genes. Evidence is provided that Dar1 has a function upstream of the JAK/STAT signaling pathway, suggesting Dar1 can regulate ISC proliferation through different mechanisms. Consistent with these findings, this study discovered Dar1 was down-regulated in the wounded midguts, allowing increased ISCs proliferation to promote intestinal repair. These data suggest that Dar1 is a functional homolog of the mammalian KLF4 (Wu, 2018).
Ameku, T., Yoshinari, Y., Texada, M. J., Kondo, S., Amezawa, K., Yoshizaki, G., Shimada-Niwa, Y. and Niwa, R. (2018). Midgut-derived neuropeptide F controls germline stem cell proliferation in a mating-dependent manner. PLoS Biol 16(9): e2005004. PubMed ID: 30248087
Summary:
Stem cell maintenance is established by neighboring niche cells that promote stem cell self-renewal. However, it is poorly understood how stem cell activity is regulated by systemic, tissue-extrinsic signals in response to environmental cues and changes in physiological status. This study shows that neuropeptide F (NPF) signaling plays an important role in the pathway regulating mating-induced germline stem cell (GSC) proliferation in the fruit fly Drosophila melanogaster. NPF expressed in enteroendocrine cells (EECs) of the midgut is released in response to the seminal-fluid protein sex peptide (SP) upon mating. This midgut-derived NPF controls mating-induced GSC proliferation via ovarian NPF receptor (NPFR) activity, which modulates bone morphogenetic protein (BMP) signaling levels in GSCs. This study provides a molecular mechanism that describes how a gut-derived systemic factor couples stem cell behavior to physiological status, such as mating, through interorgan communication.

Monday, October 22nd - Signaling

Zhang, N., Parr, C., Birch, A. M., Goldfinger, M. H. and Sastre, M. (2018). The Amyloid Precursor Protein binds to beta-catenin and modulates its cellular distribution. Neurosci Lett. PubMed ID: 30176342
Summary:
Accumulating evidence has shown that the processing of the amyloid precursor protein (APP) and the formation of amyloid-beta are associated with the canonical Wnt/ beta-catenin signalling pathway. It was recently published that the Drosophila homologue of APP (Appl) is a conserved modulator of Wnt PCP signalling, suggesting a potential regulation of this pathway by APP. The aim of this study was to investigate the potential interaction of APP with the canonical Wnt pathway. APP overexpression in N2a cells led to alterations in the subcellular distribution of beta-catenin by physically binding to it, preventing its translocation to the nucleus and precluding the transcription of Wnt target genes. In addition, studies in APP transgenic mice and human Alzheimer's disease (AD) brain tissue showed the cellular co-localization of APP and beta-catenin and binding of both proteins, suggesting the formation physical complexes of APP and beta-catenin, yet not present in healthy controls. Furthermore, a reduction in the levels of nuclear beta-catenin was detected in AD brains compared to controls as well as a decrease in the expression of the inactive phosphorylated Glycogen synthase kinase 3 (GSK3) isoform. Therefore, these findings indicate a reciprocal regulation of Wnt/beta-catenin signalling pathway and APP processing involving a physical interaction between APP and beta-catenin.
Bayer, F. E., Zimmermann, M., Preiss, A. and Nagel, A. C. (2018). Overexpression of the Drosophila ATR homologous checkpoint kinase Mei-41 induces a G2/M checkpoint in Drosophila imaginal tissue. Hereditas 155: 27. PubMed ID: 30202398
Summary:
DNA damage generally results in the activation of ATM/ATR kinases and the downstream checkpoint kinases Chk1/Chk2. In Drosophila melanogaster, the ATR homologue meiotic 41 (mei-41) is pivotal to DNA damage repair and cell cycle checkpoint signalling. Although various mei-41 mutant alleles have been analyzed in the past, no gain-of-function allele is yet available. To fill this gap, transgenic flies were generated allowing temporal and tissue-specific induction of mei-41. Overexpression of mei-41 in wing and eye anlagen affects proliferation and a G2/M checkpoint even in the absence of genomic stress. Similar consequences were observed following the overexpression of the downstream kinase Grapes (Grp) but not of Loki (Lok), encoding the respective Drosophila Chk1 and Chk2 homologues, in agreement with their previously reported activities. Moreover, this study showed that irradiation induced cell cycle arrest was prolonged in the presence of ectopic mei-41 expression. Similar to irradiation stress, mei-41 triggered the occurrence of a slower migrating form of Grp, implying specific phosphorylation of Grp in response to either signal. Using a p53R-GFP biosensor, it was further shown that overexpression of mei-41 was sufficient to elicit a robust p53 activation in vivo. It is concluded that overexpression of the Drosophila ATR homologue mei-41 elicits an effectual DNA damage response irrespective of irradiation.
Benhra, N., Barrio, L., Muzzopappa, M. and Milan, M. (2018). Chromosomal instability induces cellular invasion in epithelial tissues. Dev Cell. PubMed ID: 30245154
Summary:
Most sporadic carcinomas with high metastatic activity show an increased rate of changes in chromosome structure and number, known as chromosomal instability (CIN). However, the role of CIN in driving invasiveness remains unclear. Using an epithelial model in Drosophila, evidence is presented that CIN promotes a rapid and general invasive behavior. Cells with an abnormal number of chromosomes delaminate from the epithelium, extend actin-based cellular protrusions, form membrane blebs, and invade neighboring tissues. This behavior is governed by the activation of non-muscle Myosin II by Rho kinase and by the expression of the secreted EGF/Spitz ligand. This study has unraveled fundamental roles of the mitogen-activated protein kinase pathways mediated by the Fos proto-oncogene and the Capicua tumor suppressor gene in the invasive behavior of CIN-induced aneuploid cells. These results support the proposal that the simple production of unbalanced karyotypes contributes to CIN-induced metastatic progression.
Boese, C. J., Nye, J., Buster, D. W., McLamarrah, T. A., Byrnes, A. E., Slep, K. C., Rusan, N. M. and Rogers, G. C. (2018). Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state. Mol Biol Cell: mbcE18070445. PubMed ID: 30256714
Summary:
Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole 'targeting-factor'. In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. To address how Asl-A phosphorylation specifically affects Plk4 regulation, Asl-A fragment phospho-mutants were generated and expressed in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4's kinase domain. Thus, Asl-A's phosphorylation state determines which of Asl-A's two opposing effects are exerted on Plk4. Initially, non-phosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.
Buhler, K., Clements, J., Winant, M., Bolckmans, L., Vulsteke, V. and Callaerts, P. (2018). Growth control through regulation of insulin-signaling by nutrition-activated steroid hormone in Drosophila. Development. PubMed ID: 30266830
Summary:
Growth and maturation are coordinated processes in all animals. Integration of internal cues, such as signalling pathways, with external cues such as nutritional status is paramount for an orderly progression of development in function of growth. In Drosophila, this involves insulin and steroid signalling, but the underlying mechanisms and their coordination are incompletely understood. This study shows that bioactive 20-hydroxyecdysone production by the enzyme Shade in the fat body is a nutrient-dependent process. Under fed conditions, Shade plays a role in growth control. The trachea and the insulin-producing cells in the brain are identified as direct targets through which 20-hydroxyecdysone regulates insulin-signaling. The identification of the trachea-dependent regulation of insulin-signaling exposes an important variable that may have been overlooked in other studies focusing on insulin-signaling in Drosophila. These findings provide a potentially conserved, novel mechanism by which nutrition can modulate steroid hormone bioactivation, reveal an important caveat of a commonly used transgenic tool to study IPC function and yield further insights as to how steroid and insulin signalling are coordinated during development to regulate growth and developmental timing.
Gordon, O., Henry, C. M., Srinivasan, N., Ahrens, S., Franz, A., Deddouche, S., Chakravarty, P., Phillips, D., George, R., Kjaer, S., Frith, D., Snijders, A. P., Valente, R. S., Simoes da Silva, C. J., Teixeira, L., Thompson, B., Dionne, M. S., Wood, W. and Reis, E. S. C. (2018). alpha-actinin accounts for the bioactivity of actin preparations in inducing STAT target genes in Drosophila melanogaster. Elife 7. PubMed ID: 30260317
Summary:
Damage-associated molecular patterns (DAMPs) are molecules exposed or released by dead cells that trigger or modulate immunity and tissue repair. In vertebrates, the cytoskeletal component F-actin is a DAMP specifically recognised by DNGR-1, an innate immune receptor. Previously work suggested that actin is also a DAMP in Drosophila melanogaster by inducing STAT-dependent genes. This study revise that conclusion and report that alpha-actinin is far more potent than actin at inducing the same STAT response and can be found in trace amounts in actin preparations. Recombinant expression of actin or alpha-actinin in bacteria demonstrated that only alpha-actinin could drive the expression of STAT target genes in Drosophila. The response to injected alpha-actinin required the same signalling cascade that was identified in previous work using actin preparations. Taken together, these data indicate that alpha-actinin rather than actin drives STAT activation when injected into Drosophila.

Friday, October 19th - Adult Physiology

Eichenlaub, T., Villadsen, R., Freitas, F. C. P., Andrejeva, D., Aldana, B. I., Nguyen, H. T., Petersen, O. W., Gorodkin, J., Herranz, H. and Cohen, S. M. (2018). Warburg effect metabolism drives neoplasia in a Drosophila genetic model of epithelial cancer. Curr Biol. PubMed ID: 30293715
Summary:
Cancers develop in a complex mutational landscape. Genetic models of tumor formation have been used to explore how combinations of mutations cooperate to promote tumor formation in vivo. This study identified lactate dehydrogenase (LDH), a key enzyme in Warburg effect metabolism, as a cooperating factor that is both necessary and sufficient for epidermal growth factor receptor (EGFR)-driven epithelial neoplasia and metastasis in a Drosophila model. LDH is upregulated during the transition from hyperplasia to neoplasia, and neoplasia is prevented by LDH depletion. Elevated LDH is sufficient to drive this transition. Notably, genetic alterations that increase glucose flux, or a high-sugar diet, are also sufficient to promote EGFR-driven neoplasia, and this depends on LDH activity. This study provides evidence that increased LDHA expression promotes a transformed phenotype in a human primary breast cell culture model. Furthermore, analysis of publically available cancer data showed evidence of synergy between elevated EGFR and LDHA activity linked to poor clinical outcome in a number of human cancers. Altered metabolism has generally been assumed to be an enabling feature that accelerates cancer cell proliferation. These findings provide evidence that sugar metabolism may have a more profound role in driving neoplasia than previously appreciated.
MacMillan, H. A., Nazal, B., Wali, S., Yerushalmi, G. Y., Misyura, L., Donini, A. and Paluzzi, J. P. (2018). Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance. J Exp Biol. PubMed ID: 30104306
Summary:
For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization, and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of Capability (CAPA) (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been previously demonstrated to cause anti-diuresis in insects, including dipterans. This study provides evidence that low (fM) and high (microM) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K(+) clearance rates and reduce chill tolerance, high doses facilitate K(+) clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, the maximum achievable hormonal titres of CAPA was estimated, and evidence was further found that CAPA may function as an anti-diuretic hormone in Drosophila melanogaster. Evidence is provided of a neuropeptide that can negatively affect cold tolerance in an insect, and further evidence of CAPA functioning as an anti-diuretic peptide in this ubiquitous insect model.
Grangeteau, C., Yahou, F., Everaerts, C., Dupont, S., Farine, J. P., Beney, L. and Ferveur, J. F. (2018). Yeast quality in juvenile diet affects Drosophila melanogaster adult life traits. Sci Rep 8(1): 13070. PubMed ID: 30166573
Summary:
.Diet quality is critical for animal development and survival. Fungi can provide nutrients that are essential to organisms that are unable to synthetize them, such as ergosterol in Drosophila melanogaster. Drosophila studies examining the influence of yeast quality in the diet have generally either provided the diet over the whole life span (larva to adult) or during the adult stage and have rarely focussed on the juvenile diet. This study tested the effect of yeast quality in the larval diet on pre-adult development and adult weight, survival, reproduction and food preference. The yeast Saccharomyces cerevisiae was added in three forms in three treatments-live, heated or dried-to food used as the juvenile diet or was not added (empty treatment). Adults resulting from the larvae raised on these four juvenile diets were all maintained on a similar standard laboratory food diet. The data indicate that yeast quality in the juvenile diet affects larva-to-pupa-but not pupa-to-adult-development. Importantly, adult survival, food preference, mating behaviour and cuticular pheromones strongly varied with the juvenile diet. Therefore, the variation of yeast quality in the pre-adult Drosophila diet affects key adult life traits involved in food search, reproduction and survival.
Fiorino, A., Thompson, D., Yadlapalli, S., Jiang, C., Shafer, O. T., Reddy, P. and Meyhofer, E. (2018). Parallelized, real-time, metabolic-rate measurements from individual Drosophila. Sci Rep 8(1): 14452. PubMed ID: 30262912
Summary:
Significant recent evidence suggests that metabolism is intricately linked to the regulation and dysfunction of complex cellular and physiological responses ranging from altered metabolic programs in cancers and aging to circadian rhythms and molecular clocks. While the metabolic pathways and their fundamental control mechanisms are well established, the precise cellular mechanisms underpinning, for example, enzymatic pathway control, substrate preferences or metabolic rates, remain far less certain. Comprehensive, continuous metabolic studies on model organisms, such as the fruit fly Drosophila melanogaster, may provide a critical tool for deciphering these complex physiological responses. This study describes the development of a high-resolution calorimeter, which combines sensitive thermometry with optical imaging to concurrently perform measurements of the metabolic rate of ten individual flies, in real-time, with ~100 nW resolution. Using this calorimeter the mass-specific metabolic rates of flies of different genotypes, ages, and flies fed with different diets, were measured. This powerful new approach enables systematic studies of the metabolic regulation related to cellular and physiological function and disease mechanisms.
Mochanova, M., Tomcala, A., Svobodova, Z. and Kodrik, D. (2018). Role of adipokinetic hormone during starvation in Drosophila. Comp Biochem Physiol B Biochem Mol Biol 226: 26-35. PubMed ID: 30110658
Summary:
The role of adipokinetic hormone (Drome-AKH) in maintaining the levels of basic nutrients, under starvation conditions, was studied using Drosophila melanogaster mutants with AKH deficiency (Akh(1)) and AKH abundance (EE-Akh). The results showed lipids as the main energy reserve in Drosophila, and their physiological level and metabolism were shown to be under the control of AKH. AKH abundance in the body resulted in lower levels of triacylglycerols and diacylglycerols than in the controls, probably due to a more intensive metabolism; interestingly, there was a disproportional representation of fatty acids in triacylglycerols and diacylglycerols in Drosophila. Lower level of glycogen and its partial control by AKH suggest its lesser role as the storage substance. However, maintenance of free carbohydrate level in Drosophila seemed to be critical; when glycogen stores are exhausted, carbohydrates are synthesized from other sources. Protein levels and their alterations, under starvation, did not seem controlled by AKH. AKH-deficient flies were more resistant while AKH-abundant flies were more sensitive to starvation; females were found to be more resistant than males, regardless of the AKH level, probably due to higher body mass and higher amount of nutrients. However, in accordance with the level of all nutrients, that of AKH also gradually decreased with prolonged starvation.
Keebaugh, E. S., Yamada, R., Obadia, B., Ludington, W. B. and Ja, W. W. (2018). Microbial quantity impacts Drosophila nutrition, Development, and Lifespan. iScience 4: 247-259. PubMed ID: 30240744
Summary:
In Drosophila, microbial association can promote development or extend life. This study tested the impact of microbial association during malnutrition and shows that microbial quantity is a predictor of fly longevity. Although all tested microbes, when abundantly provided, can rescue lifespan on low-protein diet, the effect of a single inoculation seems linked to the ability of that microbial strain to thrive under experimental conditions. Microbes, dead or alive, phenocopy dietary protein, and the calculated dependence on microbial protein content is similar to the protein requirements determined from fly feeding studies, suggesting that microbes enhance host protein nutrition by serving as protein-rich food. Microbes that enhance larval growth are also associated with the ability to better thrive on fly culture medium. These results suggest an unanticipated range of microbial species that promote fly development and longevity and highlight microbial quantity as an important determinant of effects on physiology and lifespan during undernutrition.

Thursday, October 18th - Behavior

Allen, A. M., Anreiter, I., Vesterberg, A., Douglas, S. J. and Sokolowski, M. B. (2018). Pleiotropy of the Drosophila melanogaster foraging gene on larval feeding-related traits. J Neurogenet: 1-11. PubMed ID: 30303018
Summary:
Little is known about the molecular underpinning of behavioral pleiotropy. The Drosophila melanogaster foraging gene is highly pleiotropic, affecting many independent larval and adult phenotypes. Included in foraging's multiple phenotypes are larval foraging path length, triglyceride levels, and food intake. foraging has a complex structure with four promoters and 21 transcripts that encode nine protein isoforms of a cGMP dependent protein kinase (PKG). This study examined if foraging's complex molecular structure underlies the behavioral pleiotropy associated with this gene. Using a promotor analysis strategy, DNA fragments upstream of each of foraging's transcription start sites was cloned and four separate forpr-Gal4s were generated. Supporting the hypothesis of modular function, they had discrete, restricted expression patterns throughout the larva. Promoter specific expression was found in the larval fat body, salivary glands, and body muscle. The modularity of foraging's molecular structure was also apparent in the phenotypic rescues. Path length, triglyceride levels (bordered on significance), and food intake were rescued of forpr0 null larvae using different forpr-Gal4s to drive UAS-for(cDNA). The results refine the spatial expression responsible for foraging's associated phenotypes, as well as the sub-regions of the locus responsible for their expression. foraging's pleiotropy arises at least in part from the individual contributions of its four promoters.
Beauchamp, M., Bertolini, E., Deppisch, P., Steubing, J., Menegazzi, P. and Helfrich-Forster, C. (2018). Closely related fruit fly species living at different latitudes diverge in their circadian clock anatomy and rhythmic behavior. J Biol Rhythms: 748730418798096. PubMed ID: 30203704
Summary:
Differences have been reported in the expression pattern of the blue light-sensitive flavoprotein Cryptochrome (CRY) and the neuropeptide Pigment-dispersing factor (PDF) in the neuronal clock network of high-latitude Drosophila species, belonging to the Drosophila subgenus (virilis-repleta radiation), compared with cosmopolitan D. melanogaster flies, belonging to the Sophophora subgenus. Alterations in rhythmic patterns of activity due to these differences might have adaptive significance for colonizing high-latitude habitats and, hence, adjusting to long photoperiods. This study shows that these differing CRY/PDF expression patterns are only present in those species of the virilis-repleta radiation that colonized high latitudes. The cosmopolitan species D. mercatorum and D. hydei have a D. melanogaster-like clock network and behavior despite belonging to the virilis-repleta radiation. Similarly, two species of the holotropical Zaprionus genus, more closely related to the Drosophila subgenus than to the Sophophora subgenus, retain a D. melanogaster-like clock network and rhythmic behavior. It is therefore suggested that the D. melanogaster-like clock network is the "ancestral fly clock phenotype" and that alterations in the CRY/PDF clock neurochemistry have allowed some species of the virilis-repleta radiation to colonize high-latitude environments.
Crowley-Gall, A., Shaw, M. and Rollmann, S. M. (2018). Host Preference and Olfaction in Drosophila mojavensis. J Hered. PubMed ID: 30299456
Summary:
Many organisms live in complex environments that vary geographically in resource availability. This environmental heterogeneity can lead to changes within species in their phenotypic traits. For example, in many herbivorous insects, variation in host plant availability has been shown to influence insect host preference behavior. This behavior can be mediated in part through the insect olfactory system and the odor-evoked responses of olfactory receptor neurons (ORNs), which are in turn mediated by their corresponding odorant receptor genes. The desert dwelling fly Drosophila mojavensis is a model species for understanding the mechanisms underlying host preference in a heterogeneous environment. Depending on geographic region, one to multiple host plant species are available. Electrophysiological studies were conducted and variation was found in responses of ORNs to host plant volatiles both within and between 2 populations-particularly to the odorant 4-methylphenol. Flies from select localities within each population were found to lack a response to 4-methylphenol. Experiments then assessed the extent to which these electrophysiological differences were associated with difference in several odor-mediated behavioral responses. No association between the presence/absence of these odor-evoked responses and short range olfactory behavior or oviposition behavior was observed. However, differences in odor-induced feeding behavior in response to 4-methylphenol were found. Localities that exhibit an odor-evoked response to the odorant had increased feeding behavior in the presence of the odorant. This study sets the stage for future work examining the functional genetics underlying variation in odor perception.
Feng, G., Zhang, J., Li, M., Shao, L., Yang, L., Song, Q. and Ping, Y. (2018). Control of sleep onset by Shal/Kv4 channels in Drosophila circadian neurons. J Neurosci. PubMed ID: 30185460
Summary:
Sleep is highly conserved across animal species. Both wake- and sleep-promoting neurons are implicated in the regulation of wake-sleep transition at dusk in Drosophila However, little is known about how they cooperate and whether they act via different mechanisms. This study demonstrated that in female Drosophila, sleep onset was specifically delayed by blocking the Shaker cognate L channels (Shal, also known as voltage-gated K(+) channel 4, Kv4) in wake-promoting cells, including large ventral lateral neurons (l-LNvs) and pars intercerebralis (PI), but not in sleep-promoting dorsal neurons (DN1s). Delayed sleep onset was also observed in males by blocking Kv4 activity in wake-promoting neurons. Electrophysiological recordings show that Kv4 channels contribute A-type currents (IA) in LNvs and PI cells, but are much less conspicuous in DN1s. Interestingly, blocking Kv4 in wake-promoting neurons preferentially increased firing rates at dusk around ZT13, when the resting membrane potentials (RMPs) and firing rates were at lower levels. Furthermore, pigment-dispersing factor (PDF) is essential for the regulation of sleep onset by Kv4 in l-LNvs, and downregulation of PDF receptor (PDFR) in PI neurons advanced sleep onset, indicating Kv4 controls sleep onset via regulating PDF/PDFR signaling in wake-promoting neurons. It is proposed that Kv4 acts as a sleep onset controller by suppressing membrane excitability in a clock-dependent manner to balance the wake-sleep transition at dusk. These results have important implications for the understanding and treatment of sleep disorders such as insomnia.
Everaerts, C., Cazale-Debat, L., Louis, A., Pereira, E., Farine, J. P., Cobb, M. and Ferveur, J. F. (2018). Pre-imaginal conditioning alters adult sex pheromone response in Drosophila. PeerJ 6: e5585. PubMed ID: 30280017
Summary:
Pheromones are chemical signals that induce innate responses in individuals of the same species that may vary with physiological and developmental state. In Drosophila melanogaster, the most intensively studied pheromone is 11-cis-vaccenyl acetate (cVA), which is synthezised in the male ejaculatory bulb and is transferred to the female during copulation. Among other effects, cVA inhibits male courtship of mated females. This study found that male courtship inhibition depends on the amount of cVA and this effect is reduced in male flies derived from eggs covered with low to zero levels of cVA. This effect is not observed if the eggs are washed, or if the eggs are laid several days after copulation. This suggests that courtship suppression involves a form of pre-imaginal conditioning, which is shown to occur during the early larval stage. The conditioning effect could not be rescued by synthetic cVA, indicating that it largely depends on conditioning by cVA and other maternally-transmitted factor(s). These experiments suggest that one of the primary behavioral effects of cVA is more plastic and less stereotypical than had hitherto been realised.
Brankatschk, M., Gutmann, T., Knittelfelder, O., Palladini, A., Prince, E., Grzybek, M., Brankatschk, B., Shevchenko, A., Coskun, U. and Eaton, S. (2018). A temperature-dependent switch in feeding preference improves Drosophila development and survival in the cold. Dev Cell 46(6): 781-793.e784. PubMed ID: 30253170
Summary:
How cold-blooded animals acclimate to temperature and what determines the limits of their viable temperature range are not understood. This study shows that Drosophila alter their dietary preference from yeast to plants when temperatures drop below 15 degrees C and that the different lipids present in plants improve survival at low temperatures. Drosophila require dietary unsaturated fatty acids present in plants to adjust membrane fluidity and maintain motor coordination. Feeding on plants extends lifespan and survival for many months at temperatures consistent with overwintering in temperate climates. Thus, physiological alterations caused by a temperature-dependent dietary shift could help Drosophila survive seasonal temperature changes.

Wednesday, October 17th - Adult CNS development and function

Zong, W., Wang, Y., Tang, Q., Zhang, H. and Yu, F. (2018). Prd1 associates with the clathrin adaptor alpha-Adaptin and the kinesin-3 Imac/Unc-104 to govern dendrite pruning in Drosophila. PLoS Biol 16(8): e2004506. PubMed ID: 30142146
Summary:
Refinement of the nervous system depends on selective removal of excessive axons/dendrites, a process known as pruning. Drosophila ddaC sensory neurons prune their larval dendrites via endo-lysosomal degradation of the L1-type cell adhesion molecule (L1-CAM), Neuroglian (Nrg). This study identified a novel gene, pruning defect 1 (prd1), which governs dendrite pruning of ddaC neurons. Prd1 colocalizes with the clathrin adaptor protein alpha-Adaptin (alpha-Ada) and the kinesin-3 immaculate connections (Imac)/Uncoordinated-104 (Unc-104) in dendrites. Moreover, Prd1 physically associates with alpha-Ada and Imac, which are both critical for dendrite pruning. Prd1, alpha-Ada, and Imac promote dendrite pruning via the regulation of endo-lysosomal degradation of Nrg. Importantly, genetic interactions among prd1, alpha-adaptin, and imac indicate that they act in the same pathway to promote dendrite pruning. These findings indicate that Prd1, alpha-Ada, and Imac act together to regulate discrete distribution of alpha-Ada/clathrin puncta, facilitate endo-lysosomal degradation, and thereby promote dendrite pruning in sensory neurons.
Alyagor, I., Berkun, V., Keren-Shaul, H., Marmor-Kollet, N., David, E., Mayseless, O., Issman-Zecharya, N., Amit, I. and Schuldiner, O. (2018). Combining developmental and perturbation-seq uncovers transcriptional modules orchestrating neuronal remodeling. Dev Cell 47(1): 38-52.e36. PubMed ID: 30300589
Summary:
Developmental neuronal remodeling is an evolutionarily conserved mechanism required for precise wiring of nervous systems. Despite its fundamental role in neurodevelopment and proposed contribution to various neuropsychiatric disorders, the underlying mechanisms are largely unknown. This study uncovered the fine temporal transcriptional landscape of Drosophila mushroom body gamma neurons undergoing stereotypical remodeling (see Hierarchical TF Networks Regulate Axon Pruning). The data reveal rapid and dramatic changes in the transcriptional landscape during development. Focusing on DNA binding proteins, eleven were identified that are required for remodeling. Furthermore, developing gamma neurons perturbed for three key transcription factors required for pruning were sequenced. A hierarchical network is described featuring positive and negative feedback loops. Superimposing the perturbation-seq on the developmental expression atlas highlights a framework of transcriptional modules that together drive remodeling. Overall, this study provides a broad and detailed molecular insight into the complex regulatory dynamics of developmental remodeling and thus offers a pipeline to dissect developmental processes via RNA profiling.
Barnhart, E. L., Wang, I. E., Wei, H., Desplan, C. and Clandinin, T. R. (2018). Sequential nonlinear filtering of local motion cues by global motion circuits. Neuron 100(1): 229-243.e223. PubMed ID: 30220510
Summary:
Many animals guide their movements using optic flow, the displacement of stationary objects across the retina caused by self-motion. How do animals selectively synthesize a global motion pattern from its local motion components? To what extent does this feature selectivity rely on circuit mechanisms versus dendritic processing? This study used in vivo calcium imaging to identify pre- and postsynaptic mechanisms for processing local motion signals in global motion detection circuits in Drosophila. Lobula plate tangential cells (LPTCs) detect global motion by pooling input from local motion detectors, T4/T5 neurons. T4/T5 neurons were shown to suppress responses to adjacent local motion signals whereas LPTC dendrites selectively amplify spatiotemporal sequences of local motion signals consistent with preferred global patterns. It is proposed that sequential nonlinear suppression and amplification operations allow optic flow circuitry to simultaneously prevent saturating responses to local signals while creating selectivity for global motion patterns critical to behavior.
Egea-Weiss, A., Renner, A., Kleineidam, C. J. and Szyszka, P. (2018). High precision of spike timing across olfactory receptor neurons allows rapid odor coding in Drosophila. iScience 4: 76-83. PubMed ID: 30240755
Summary:
In recent years, it has become evident that olfaction is a fast sense, and millisecond short differences in stimulus onsets are used by animals to analyze their olfactory environment. In contrast, olfactory receptor neurons are thought to be relatively slow and temporally imprecise. These observations have led to a conundrum: how, then, can an animal resolve fast stimulus dynamics and smell with high temporal acuity? Using parallel recordings from olfactory receptor neurons in Drosophila, this study found hitherto unknown fast and temporally precise odorant-evoked spike responses, with first spike latencies (relative to odorant arrival) down to 3 ms and with a SD below 1 ms. These data provide new upper bounds for the speed of olfactory processing and suggest that the insect olfactory system could use the precise spike timing for olfactory coding and computation, which can explain insects' rapid processing of temporal stimuli when encountering turbulent odor plumes.
Du, J., Zhang, Y., Xue, Y., Zhao, X., Zhao, X., Wei, Y., Li, Z., Zhang, Y. and Zhao, Z. (2018). Diurnal protein oscillation profiles in Drosophila head. FEBS Lett. PubMed ID: 30311939
Summary:
Circadian clocks control daily rhythms in physiology, metabolism, and behavior in most organisms. Proteome-wide analysis of protein oscillations is still lacking in Drosophila. In this study, the total protein and phosphorylated protein in Drosophila heads in a 24-hour daily time-course were assayed by using the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) method, and 10 and 7 oscillating proteins as well as 19 and 22 oscillating phosphoproteins in the w(1118) wild type and Clk(Jrk) mutant strains were separately identified. Lastly, a mini screen was performed to investigate the functions of some oscillating proteins in circadian locomotion rhythms. This study provides the first proteomic profiling of diurnally oscillating proteins in fly heads, thereby providing a basis for further mechanistic studies of these proteins in circadian rhythm.
Barajas-Azpeleta, R., Wu, J., Gill, J., Welte, R., Seidel, C., McKinney, S., Dissel, S. and Si, K. (2018). Antimicrobial peptides modulate long-term memory. PLoS Genet 14(10): e1007440. PubMed ID: 30312294
Summary:
Antimicrobial peptides act as a host defense mechanism and regulate the commensal microbiome. To obtain a comprehensive view of genes contributing to long-term memory. mRNA sequencing from single Drosophila heads was performed following behavioral training that produces long-lasting memory. Surprisingly, it was found that Diptericin B, an immune peptide with antimicrobial activity, is upregulated following behavioral training. Deletion and knock down experiments revealed that Diptericin B and another immune peptide, Gram-Negative Bacteria Binding Protein like 3, regulate long-term but not short-term memory or instinctive behavior in Drosophila. Interestingly, removal of DptB in the head fat body and GNBP-like3 in neurons results in memory deficit. That putative antimicrobial peptides influence memory provides an example of how some immune peptides may have been repurposed to influence the function of nervous system.
Dolan, M. J., Belliart-Guerin, G., Bates, A. S., Frechter, S., Lampin-Saint-Amaux, A., Aso, Y., Roberts, R. J. V., Schlegel, P., Wong, A., Hammad, A., Bock, D., Rubin, G. M., Preat, T., Placais, P. Y. and Jefferis, G. (2018). Communication from learned to innate olfactory processing centers is required for memory retrieval in Drosophila. Neuron. PubMed ID: 30244885
Summary:
The behavioral response to a sensory stimulus may depend on both learned and innate neuronal representations. How these circuits interact to produce appropriate behavior is unknown. In Drosophila, the lateral horn (LH) and mushroom body (MB) are thought to mediate innate and learned olfactory behavior, respectively, although LH function has not been tested directly. This study identified two LH cell types (PD2a1 and PD2b1) that receive input from an MB output neuron required for recall of aversive olfactory memories. These neurons are required for aversive memory retrieval and modulated by training. Connectomics data demonstrate that PD2a1 and PD2b1 neurons also receive direct input from food odor-encoding neurons. Consistent with this, PD2a1 and PD2b1 are also necessary for unlearned attraction to some odors, indicating that these neurons have a dual behavioral role. This provides a circuit mechanism by which learned and innate olfactory information can interact in identified neurons to produce appropriate behavior (Dolan, 2018).
Felsenberg, J., Jacob, P. F., Walker, T., Barnstedt, O., Edmondson-Stait, A. J., Pleijzier, M. W., Otto, N., Schlegel, P., Sharifi, N., Perisse, E., Smith, C. S., Lauritzen, J. S., Costa, M., Jefferis, G., Bock, D. D. and Waddell, S. (2018). Integration of parallel opposing memories underlies memory extinction. Cell. PubMed ID: 30245010
Summary:
Accurately predicting an outcome requires that animals learn supporting and conflicting evidence from sequential experience. In mammals and invertebrates, learned fear responses can be suppressed by experiencing predictive cues without punishment, a process called memory extinction. This study shows that extinction of aversive memories in Drosophila requires specific dopaminergic neurons, which indicate that omission of punishment is remembered as a positive experience. Functional imaging revealed co-existence of intracellular calcium traces in different places in the mushroom body output neuron network for both the original aversive memory and a new appetitive extinction memory. Light and ultrastructural anatomy are consistent with parallel competing memories being combined within mushroom body output neurons that direct avoidance. Indeed, extinction-evoked plasticity in a pair of these neurons neutralizes the potentiated odor response imposed in the network by aversive learning. Therefore, flies track the accuracy of learned expectations by accumulating and integrating memories of conflicting events.

Tuesday, October 16th - Disease Models

Chandran, S., Suggs, J. A., Wang, B. J., Han, A., Bhide, S., Cryderman, D. E., Moore, S. A., Bernstein, S. I., Wallrath, L. L. and Melkani, G. C. (2018). Suppression of myopathic lamin mutations by muscle-specific activation of AMPK and modulation of downstream signaling. Hum Mol Genet. PubMed ID: 30239736
Summary:
Laminopathies are diseases caused by dominant mutations in the human LMNA gene encoding A-type lamins. Lamins are intermediate filaments that line the inner nuclear membrane, provide structural support for the nucleus, and regulate gene expression. Human disease-causing LMNA mutations were modeled in Drosophila Lamin C (LamC) and expressed in indirect flight muscle (IFM). IFM-specific expression of mutant, but not wild-type LamC, caused held-up wings indicative of myofibrillar defects. Analyses of the muscles revealed cytoplasmic aggregates of nuclear envelope (NE) proteins, nuclear and mitochondrial dysmorphology, myofibrillar disorganization, and up-regulation of the autophagy cargo receptor p62. It was hypothesized that the cytoplasmic aggregates of NE proteins trigger signaling pathways that alter cellular homeostasis, causing muscle dysfunction. In support of this hypothesis, transcriptomics data from human muscle biopsy tissue revealed misregulation of the AMPK/4E-BP1/autophagy/proteostatic pathways. S6K mRNA levels were increased and AMPKalpha and mRNAs encoding downstream targets were decreased in muscles expressing mutant LMNA relative controls. The Drosophila laminopathy models were used to determine if altering the levels of these factors modulated muscle pathology. Muscle-specific over-expression of AMPKalpha and down-stream targets 4E-BP, Foxo and PGC1alpha, as well as inhibition of S6K, suppressed the held-up wing phenotype, myofibrillar defects, and LamC aggregation. These findings provide novel insights on mutant LMNA-based disease mechanisms and identify potential targets for drug therapy.
Chakraborty, J., von Stockum, S., Marchesan, E., Caicci, F., Ferrari, V., Rakovic, A., Klein, C., Antonini, A., Bubacco, L. and Ziviani, E. (2018). USP14 inhibition corrects an in vivo model of impaired mitophagy. EMBO Mol Med. PubMed ID: 30249595
Summary:
Mitochondrial autophagy or mitophagy is a key process that allows selective sequestration and degradation of dysfunctional mitochondria to prevent excessive reactive oxygen species, and activation of cell death. Recent studies revealed that ubiquitin-proteasome complex activity and mitochondrial membrane rupture are key steps preceding mitophagy, in combination with the ubiquitination of specific outer mitochondrial membrane (OMM) proteins. The deubiquitinating enzyme ubiquitin-specific peptidase 14 (USP14) has been shown to modulate both proteasome activity and autophagy. This study reports that genetic and pharmacological inhibition of USP14 promotes mitophagy, which occurs in the absence of the well-characterised mediators of mitophagy, PINK1 and Parkin. Critical to USP14-induced mitophagy is the exposure of the LC3 receptor Prohibitin 2 by mitochondrial fragmentation and mitochondrial membrane rupture. Genetic or pharmacological inhibition of USP14 in vivo corrected mitochondrial dysfunction and locomotion behaviour of PINK1/Parkin mutant Drosophila model of Parkinson's disease, an age-related progressive neurodegenerative disorder that is correlated with diminished mitochondrial quality control. This study identifies a novel therapeutic target that ameliorates mitochondrial dysfunction and in vivo PD-related symptoms.
Wang, X., Guo, X., Ma, Y., Wu, C., Li, W. and Xue, L. (2018). APLP2 Modulates JNK-Dependent Cell Migration in Drosophila. Biomed Res Int 2018: 7469714. PubMed ID: 30155482
Summary:
Amyloid precursor-like protein 2 (APLP2) belongs to the APP family and is widely expressed in human cells. Though previous studies have suggested a role of APLP2 in cancer progression, the exact role of APLP2 in cell migration remains elusive. This report shows that ectopic expression of APLP2 in Drosophila induces cell migration which is mediated by JNK signaling, as loss of JNK suppresses while gain of JNK enhances such phenotype. APLP2 is able to activate JNK signaling by phosphorylation of JNK, which triggers the expression of matrix metalloproteinase MMP1 required for basement membranes degradation to promote cell migration. The data presented in this study unraveled an in vivo role of APLP2 in JNK-mediated cell migration.
Aron, R., Pellegrini, P., Green, E. W., Maddison, D. C., Opoku-Nsiah, K., Wong, J. S., Daub, A. C., Giorgini, F. and Finkbeiner, S. (2018). Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease. Nat Commun 9(1): 3191. PubMed ID: 30266909
Summary:
Huntington's disease is a progressive neurodegenerative disorder caused by polyglutamine-expanded mutant huntingtin (mHTT). This study shows that the deubiquitinase Usp12 rescues mHTT-mediated neurodegeneration in Huntington's disease rodent and patient-derived human neurons, and in Drosophila. The neuroprotective role of Usp12 may be specific amongst related deubiquitinases, as the closely related homolog Usp46 does not suppress mHTT-mediated toxicity. Mechanistically, this study identifies Usp12 as a potent inducer of neuronal autophagy. Usp12 overexpression accelerates autophagic flux and induces an approximately sixfold increase in autophagic structures as determined by ultrastructural analyses, while suppression of endogenous Usp12 slows autophagy. Surprisingly, the catalytic activity of Usp12 is not required to protect against neurodegeneration or induce autophagy. These findings identify the deubiquitinase Usp12 as a regulator of neuronal proteostasis and mHTT-mediated neurodegeneration.
Stapper, Z. A. and Jahn, T. R. (2018). Changes in glutathione redox potential are linked to Abeta42-induced neurotoxicity. Cell Rep 24(7): 1696-1703. PubMed ID: 30110626
Summary:
Glutathione is the major low-molecular weight thiol of eukaryotic cells. It is central to one of the two major NADPH-dependent reducing systems and is likely to play a role in combating oxidative stress, a process suggested to play a key role in Alzheimer's disease (AD). However, the nature and relevance of redox changes in the onset and progression of AD are still uncertain. This study combined genetically encoded redox sensors with a Drosophila models of amyloid-beta (Abeta) aggregation. Changes in glutathione redox potential (EGSH) closely correlate with disease onset and progression. This redox imbalance was found to be specifically in neurons, but not in glia cells. EGSH changes and Abeta42 deposition are also accompanied by increased JNK stress signaling. Furthermore, pharmacologic and genetic manipulation of glutathione synthesis modulates Abeta42-mediated neurotoxicity, suggesting a causal relationship between disturbed glutathione redox homeostasis and early AD pathology.
Ast, A., et al. (2018). mHTT seeding activity: A marker of disease progression and neurotoxicity in models of Huntington's disease. Mol Cell 71(5): 675-688.e676. PubMed ID: 30193095
Summary:
Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). This paper reports the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, the neurotoxicity of mHTT seeds was assessed in an inducible Drosophila model transgenic for HTTex1. A strong correlation was found between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.

Monday, October 15th - Transcriptional Regulation

Lim, B., Fukaya, T., Heist, T. and Levine, M. (2018). Temporal dynamics of pair-rule stripes in living Drosophila embryos. Proc Natl Acad Sci U S A 115(33): 8376-8381. PubMed ID: 30061421
Summary:
Traditional studies of gene regulation in the Drosophila embryo centered primarily on the analysis of fixed tissues. These methods provided considerable insight into the spatial control of gene activity, such as the borders of eve stripe 2, but yielded only limited information about temporal dynamics. The advent of quantitative live-imaging and genome-editing methods permits the detailed examination of the temporal control of endogenous gene activity. This study presents evidence that the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) undergo dynamic shifts in gene expression. Sequential anterior shifting of the stripes along the anterior to posterior axis was observed, with stripe 1 exhibiting movement before stripe 2 and the more posterior stripes. Conversely, posterior stripes shift over greater distances (two or three nuclei) than anterior stripes (one or two nuclei). Shifting of the ftz and eve stripes are slightly offset, with ftz moving faster than eve. This observation is consistent with previous genetic studies, suggesting that eve is epistatic to ftz. The precision of pair-rule temporal dynamics might depend on enhancer-enhancer interactions within the eve locus, since removal of the endogenous eve stripe 1 enhancer via CRISPR/Cas9 genome editing led to precocious and expanded expression of eve stripe 2. These observations raise the possibility of an added layer of complexity in the positional information encoded by the segmentation gene regulatory network.
Du, L., Zhou, A., Sohr, A. and Roy, S. (2018). An efficient strategy for generating tissue-specific binary transcription systems in Drosophila by genome editing. J Vis Exp(139). PubMed ID: 30295654
Summary:
Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. This study presents a method to generate highly tissue-specific targeted expression system by employing a CRISPR/Cas-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented in this study for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.
Newcomb, S., Voutev, R., Jory, A., Delker, R. K., Slattery, M. and Mann, R. S. (2018). cis-regulatory architecture of a short-range EGFR organizing center in the Drosophila melanogaster leg. PLoS Genet 14(8): e1007568. PubMed ID: 30142157
Summary:
This study characterized the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. Initial EGFR activation occurs in the center of leg discs by expression of the EGFR ligand Vn and the EGFR ligand-processing protease Rho, each through single enhancers, vnE and rhoE, that integrate inputs from Wg, Dpp, Dll and Sp1. Deletion of vnE and rhoE eliminates vn and rho expression in the center of the leg imaginal discs, respectively. Animals with deletions of both vnE and rhoE (but not individually) show distal but not medial leg truncations, suggesting that the distal source of EGFR ligands acts at short-range to only specify distal-most fates, and that multiple additional 'ring' enhancers are responsible for medial fates. Further, based on the cis-regulatory logic of vnE and rhoE many additional leg enhancers were identified, suggesting that this logic is broadly used by many genes during Drosophila limb development.
Vincent, B. J., Staller, M. V., Lopez-Rivera, F., Bragdon, M. D. J., Pym, E. C. G., Biette, K. M., Wunderlich, Z., Harden, T. T., Estrada, J. and DePace, A. H. (2018). Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos. PLoS Genet 14(9): e1007644. PubMed ID: 30192762
Summary:
Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. This study investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. Evidence was also found that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. These results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. How counter-repression may influence eve stripe 2 regulation and evolution is discussed.
Wang, L. H. and Baker, N. E. (2018). Spatial regulation of expanded transcription in the Drosophila wing imaginal disc. PLoS One 13(7): e0201317. PubMed ID: 30063727
Summary:
Growth and patterning are coordinated during development to define organ size and shape. The growth, proliferation and differentiation of Drosophila wings are regulated by several conserved signaling pathways. This study shows that the Salvador-Warts-Hippo (SWH) and Notch pathways converge on an enhancer in the expanded (ex) gene, which also responds to levels of the bHLH transcription factor Daughterless (Da). Separate cis-regulatory elements respond to Salvador-Warts-Hippo (SWH) and Notch pathways, to bHLH proteins, and to unidentified factors that repress ex transcription in the wing pouch and in the proneural region at the anterior wing margin. Senseless, a zinc-finger transcription factor acting in proneural regions, had a negative impact on ex transcription in the proneural region, but the transcriptional repressor Hairy had no effect. This study suggests that a complex pattern of ex transcription results from integration of a uniform SWH signal with multiple other inputs, rather than from a pattern of SWH signaling.
Wester, J., Lima, C. A. C., Machado, M. C. R., Zampar, P. V., Tavares, S. S. and Monesi, N. (2018). Characterization of a novel Drosophila melanogaster cis-regulatory module that drives gene expression to the larval tracheal system and adult thoracic musculature. Genesis: e23222. PubMed ID: 30096221
Summary:
This study characterized a Drosophila melanogaster cis-regulatory module (CRM) termed TT-CRM. The TT-CRM is 646 bp long and is located in one of the introns of CG32239 (RhoGEF64C) and resides about 3,500 bp upstream of CG13711 and about 620 bp upstream of CG12493. Analysis of 646 bp-lacZ lines revealed that TT-CRM drives gene expression not only to the larval, prepupal, and pupal tracheal system but also to the adult dorsal longitudinal muscles. The patterns of mRNA expression of the transgene and of the CGs that lie in the vicinity of TT-CRM were investigated both in dissected trachea and in adult thoraces. Through RT-qPCR it was observed that in the tracheal system the pattern of expression of 646 bp-lacZ is similar to the pattern of expression of CG32239 and CG13711, whereas in the thoracic muscles 646 bp-lacZ expression accompanies the expression of CG12493. Together, these results suggest new functions for two previously characterized D. melanogaster genes and also contribute to the initial characterization of a novel CRM that drives a dynamic pattern of expression throughout development.

Friday, October 12 - Behavior

Waters, A. J., Capriotti, P., Gaboriau, D. C. A., Papathanos, P. A. and Windbichler, N. (2018). Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster. Sci Rep 8(1): 13125. PubMed ID: 30177778
Summary:
The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. This study examined the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. 13 single guide RNAs (sgRNAs) were directed to the promoters of 7 evolutionary conserved genes, and 11 drivers were used to conduct a misactivation screen. Dominant-lethal activators of the eve locus were identified, and it was found that they disrupt development by strongly activating eve outside its native spatio-temporal context. The same set of sgRNAs were used to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined it was found that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, these results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy presented in this study allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.
Thimgan, M. S., Kress, N., Lisse, J., Fiebelman, C. and Hilderbrand, T. (2018). The acyl-CoA synthetase, pudgy, promotes sleep and is required for the homeostatic response to sleep deprivation. Front Endocrinol (Lausanne) 9: 464. PubMed ID: 30186232
Summary:
The regulation of sleep and the response to sleep deprivation rely on multiple biochemical pathways. A critical connection is the link between sleep and metabolism. Metabolic changes can disrupt sleep, and conversely decreased sleep can alter the metabolic environment. There is building evidence that lipid metabolism, in particular, is a critical part of mounting the homeostatic response to sleep deprivation. This study evaluated an acyl-CoA synthetase, pudgy (pdgy), for its role in sleep and response to sleep deprivation. When pdgy transcript levels are decreased through transposable element disruption of the gene, mutant flies showed lower total sleep times and increased sleep fragmentation at night compared to genetic controls. Consistent with disrupted sleep, mutant flies had a decreased lifespan compared to controls. pdgy disrupted fatty acid handling as pdgy mutants showed increased sensitivity to starvation and exhibited lower fat stores. Moreover, the response to sleep deprivation is reduced when compared to a control flies. When the transcript levels for pdgy were decreased using RNAi, the response to sleep deprivation was decreased compared to background controls. In addition, when pdgy transcription is rescued throughout the fly, the response to sleep deprivation is restored. These data demonstrate that the regulation and function of acyl-CoA synthetase plays a critical role in regulating sleep and the response to sleep deprivation. Endocrine and metabolic signals that alter transcript levels of pdgy impact sleep regulation or interfere with the homeostatic response to sleep deprivation.
Troup, M., Yap, M. H., Rohrscheib, C., Grabowska, M. J., Ertekin, D., Randeniya, R., Kottler, B., Larkin, A., Munro, K., Shaw, P. J. and van Swinderen, B. (2018). Acute control of the sleep switch in Drosophila reveals a role for gap junctions in regulating behavioral responsiveness. Elife 7. PubMed ID: 30109983
Summary:
Sleep is a dynamic process in most animals, involving distinct stages that probably perform multiple functions for the brain. Before sleep functions can be initiated, it is likely that behavioral responsiveness to the outside world needs to be reduced, even while the animal is still awake. Recent work in Drosophila has uncovered a sleep switch in the dorsal fan-shaped body (dFB) of the fly's central brain, but it is not known whether these sleep-promoting neurons also govern the acute need to ignore salient stimuli in the environment during sleep transitions. This study found that optogenetic activation of the sleep switch suppressed behavioral responsiveness to mechanical stimuli, even in awake flies, indicating a broader role for these neurons in regulating arousal. The dFB-mediated suppression mechanism and its associated neural correlates requires innexin6 expression, suggesting that the acute need to reduce sensory perception when flies fall asleep is mediated in part by electrical synapses.
Zhang, W., Guo, C., Chen, D., Peng, Q. and Pan, Y. (2018). Hierarchical control of Drosophila sleep, courtship, and feeding behaviors by male-specific P1 neurons. Neurosci Bull. PubMed ID: 30182322
Summary:
Animals choose among sleep, courtship, and feeding behaviors based on the integration of both external sensory cues and internal states; such choices are essential for survival and reproduction. These competing behaviors are closely related and controlled by distinct neural circuits, but whether they are also regulated by shared neural nodes is unclear. This study investigated how a set of male-specific P1 neurons controls sleep, courtship, and feeding behaviors in Drosophila males. Mild activation of P1 neurons was sufficient to affect sleep, but not courtship or feeding, while stronger activation of P1 neurons labeled by four out of five independent drivers induced courtship, but only the driver that targeted the largest number of P1 neurons affected feeding. These results reveal a common neural node that affects sleep, courtship, and feeding in a threshold-dependent manner, and provide insights into how competing behaviors can be regulated by a shared neural node.
Wienecke, C. F. R., Leong, J. C. S. and Clandinin, T. R. (2018). Linear summation underlies direction selectivity in Drosophila. Neuron 99(4): 680-688.e684. PubMed ID: 30057202
Summary:
While linear mechanisms lay the foundations of feature selectivity in many brain areas, direction selectivity in the elementary motion detector (EMD) of the fly has become a paradigm of nonlinear neuronal computation. This study has bridged this divide by demonstrating that linear spatial summation can generate direction selectivity in the fruit fly Drosophila. Using linear systems analysis and two-photon imaging of a genetically encoded voltage indicator, the emergence was measured of direction-selective (DS) voltage signals in the Drosophila OFF pathway. This study is a direct, quantitative investigation of the algorithm underlying directional signals, with the striking finding that linear spatial summation is sufficient for the emergence of direction selectivity. A linear stage of the fly EMD strongly resembles similar computations in vertebrate visual cortex, demands a reappraisal of the role of upstream nonlinearities, and implicates the voltage-to-calcium transformation in the refinement of feature selectivity in this system.
Jaeger, A. H., Stanley, M., Weiss, Z. F., Musso, P. Y., Chan, R. C., Zhang, H., Feldman-Kiss, D. and Gordon, M. D. (2018). A complex peripheral code for salt taste in Drosophila. Elife 7. PubMed ID: 30307393
Summary:
Each taste modality is generally encoded by a single, molecularly defined, population of sensory cells. However, salt stimulates multiple taste pathways in mammals and insects, suggesting a more complex code for salt taste. This study examined salt coding in Drosophila. After creating a comprehensive molecular map comprised of five discrete sensory neuron classes across the fly labellum, four were found to be sactivated by salt: two exhibiting characteristics of 'low salt' cells, and two 'high salt' classes. Behaviorally, low salt attraction depends primarily on 'sweet' neurons, with additional input from neurons expressing the ionotropic receptor IR94e. High salt avoidance is mediated by 'bitter' neurons and a population of glutamatergic neurons expressing Ppk23. Interestingly, the impact of these glutamatergic neurons depends on prior salt consumption. These results support a complex model for salt coding in flies that combinatorially integrates inputs from across cell types to afford robust and flexible salt behaviors.

Thursday, October 11th - RNA

Zeng, J., Kamiyama, T., Niwa, R. and King-Jones, K. (2018). The Drosophila CCR4-NOT complex is required for cholesterol homeostasis and steroid hormone synthesis. Dev Biol. PubMed ID: 30149007
Summary:
CCR4-NOT is a highly conserved protein complex that regulates gene expression at multiple levels. In yeast, CCR4-NOT functions in transcriptional initiation, heterochromatin formation, mRNA deadenylation and other processes. The range of functions for Drosophila CCR4-NOT is less clear, except for a well-established role as a deadenylase for maternal mRNAs during early embryogenesis. This study report here that CCR4-NOT has an essential function in the Drosophila prothoracic gland (PG), a tissue that predominantly produces the steroid hormone ecdysone. Interfering with the expression of the CCR4-NOT components twin, Pop2, Not1, and Not3 in a PG-specific manner resulted in larval arrest and a failure to initiate metamorphosis. Transcriptome analysis of PG-specific Pop2-RNAi samples revealed that Pop2 is required for the normal expression of ecdysone biosynthetic gene spookier (spok) as well as cholesterol homeostasis genes of the NPC2 family. Interestingly, dietary supplementation with ecdysone and its various sterol precursors showed that 7-dehydrocholesterol and cholesterol completely rescued the larval arrest phenotype, allowing Pop2-RNAi animals to reach pupal stage, and, to a low degree, even survival to adulthood, while the biologically active hormone, 20-Hydroxyecdysone (20E), was significantly less effective. Also, genetic evidence is presented that CCR4-NOT has a nuclear function where CCR4-NOT-depleted cells exhibit aberrant chromatin and nucleoli structures. In summary, these findings indicate that the Drosophila CCR4-NOT complex has essential roles in the PG, where it is required for Drosophila steroid hormone production and cholesterol homeostasis, and likely has functions beyond a mere mRNA deadenylase in Drosophila.
Kordyukova, M., Morgunova, V., Olovnikov, I., Komarov, P. A., Mironova, A., Olenkina, O. M. and Kalmykova, A. (2018). Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis. PLoS One 13(8): e0201787. PubMed ID: 30157274
Summary:
The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. The biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development was studied with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, this study showed that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, it was demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, it is suggested that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants.
Kaschula, R., Pinho, S. and Alonso, C. R. (2018). microRNA-dependent regulation of Hox gene expression sculpts fine-grain morphological patterns in a Drosophila appendage. Development. PubMed ID: 30143542
Summary:
Disruptions of normal Hox gene expression can lead to severe morphological defects revealing a link between the regulation of Hox expression and pattern formation. This study explored these links focusing on the impact of microRNA regulation on the expression of the Drosophila Hox gene Ultrabithorax (Ubx) during haltere development. Through the combination of bioinformatic and transcriptomic analyses the miR-310/313 cluster (miR-310C) as a candidate regulator of Ubx. Several experiments confirm this. First, miR-310C and Ubx protein show complementary expression patterns in haltere imaginal discs; second, artificial activation of miR-310C expression in haltere discs leads to Ubx-like phenotypes. Third, expression of a fluorescent reporter bearing Ubx 3'UTR sequences is reduced when co-expressed with miR-310C. Fourth, deletion of miR-310C leads to Ubx upregulation and changes the array of mechanosensory sensilla at the base of the haltere. Fifth, artificial increase of Ubx levels within the miR-310C expression domain phenocopies the mechanosensory defects observed in miR-310C mutants. It is proposed that miR-310C-mediated repression delimits Ubx fine-grain expression contributing to the sculpting of complex morphologies in the Drosophila haltere. This work reveals a novel role of microRNA regulation in the control of Hox gene expression with impact on morphology.
Theron, E., Maupetit-Mehouas, S., Pouchin, P., Baudet, L., Brasset, E. and Vaury, C. (2018). The interplay between the Argonaute proteins Piwi and Aub within Drosophila germarium is critical for oogenesis, piRNA biogenesis and TE silencing. Nucleic Acids Res. PubMed ID: 30113668
Summary:
Transposable elements (TEs) have invaded most genomes and constitute up to 50% of the human genome. Machinery based on small non-coding piRNAs has evolved to inhibit their expression at the transcriptional and post-transcriptional levels. Surprisingly, this machinery is weakened during specific windows of time in mice, flies or plants, allowing the expression of TEs in germline cells. The function of this de-repression remains unknown. In Drosophila, it has been shown that this developmental window is characterized by a reduction of Piwi expression in dividing germ cells. This study shows that the unique knock-down of Aub in these cells leads to female sterility. It correlates with defects in piRNA amplification, an increased Piwi expression and an increased silencing of transcriptionally silenced TEs. These defects are similar to those observed when Aub is depleted in the whole germline which underlies the crucial role of this developmental window for both oogenesis and TE silencing. This study further showed that, with age, some fertility is recovered which is concomitant to a decrease of Piwi and TE silencing. These data pinpoint the Pilp (Piwi-less pocket - a region of the oocyte lacking Piei) as a tremendously important step for female fertility and genome stability. They further show that such a restricted developmental niche of germ cells may sense environmental changes, such as aging, to protect the germline all along the life.
Tikhonov, M., Utkina, M., Maksimenko, O. and Georgiev, P. (2018). Conserved sequences in the Drosophila mod(mdg4) intron promote poly(A)-independent transcription termination and trans-splicing. Nucleic Acids Res. PubMed ID: 30102331
Summary:
Alternative splicing (AS) is a regulatory mechanism of gene expression that greatly expands the coding capacities of genomes by allowing the generation of multiple mRNAs from a single gene. In Drosophila, the mod(mdg4) locus is an extreme example of AS that produces more than 30 different mRNAs via trans-splicing that joins together the common exons and the 3' variable exons generated from alternative promoters. To map the regions required for trans-splicing, this study has developed an assay for measuring trans-splicing events and identified a 73-bp region in the last common intron that is critical for trans-splicing of three pre-mRNAs synthesized from different DNA strands. It was have also found that conserved sequences in the distal part of the last common intron induce polyadenylation-independent transcription termination and are enriched by paused RNA polymerase II (RNAP II). These results suggest that all mod(mdg4) mRNAs are formed by joining in trans the 5' splice site in the last common exon with the 3' splice site in one of the alternative exons.
Lin, C. J., Hu, F., Dubruille, R., Vedanayagam, J., Wen, J., Smibert, P., Loppin, B. and Lai, E. C. (2018). The hpRNA/RNAi pathway is essential to resolve intragenomic conflict in the Drosophila male germline. Dev Cell 46(3): 316-326.e315. PubMed ID: 30086302
Summary:
Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. This study reveals that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, D. simulans null mutants of Dcr-2 and AGO2 were generated. Strikingly, these core RNAi knockouts massively derepress Dox and MDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, these data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species.

Wednesday, October 10th - Adult Neural Development and Function

Mishra, D., Thorne, N., Miyamoto, C., Jagge, C. and Amrein, H. (2018). The taste of ribonucleosides: Novel macronutrients essential for larval growth are sensed by Drosophila gustatory receptor proteins. PLoS Biol 16(8): e2005570. PubMed ID: 30086130
Summary:
Animals employ various types of taste receptors to identify and discriminate between different nutritious food chemicals. These macronutrients are thought to fall into 3 major groups: carbohydrates/sugars, proteins/amino acids, and fats. This study reports that Drosophila larvae exhibit a novel appetitive feeding behavior towards ribose, ribonucleosides, and RNA. Members of the gustatory receptor (Gr) subfamily 28 (Gr28), expressed in both external and internal chemosensory neurons were identified as molecular receptors necessary for cellular and appetitive behavioral responses to ribonucleosides and RNA. Specifically, behavioral preference assays show that larvae are strongly attracted to ribose- or RNA-containing agarose in a Gr28-dependent manner. Moreover, Ca2+ imaging experiments reveal that Gr28a-expressing taste neurons are activated by ribose, RNA and some ribonucleosides and that these responses can be conveyed to Gr43aGAL4 fructose-sensing neurons by expressing single members of the Gr28 gene family. Lastly, a critical role in behavioral fitness for the Gr28 genes was established by showing that Gr28 mutant larvae exhibit low survival rates when challenged to find ribonucleosides in food. Together, this work identifies a novel taste modality dedicated to the detection of RNA and ribonucleosides, nutrients that are essential for survival during the accelerated growth phase of Drosophila larvae.
Pu, Y., Zhang, Y., Zhang, Y. and Shen, P. (2018). Two Drosophila Neuropeptide Y-like neurons define a reward module for transforming appetitive odor representations to motivation. Sci Rep 8(1): 11658. PubMed ID: 30076343
Summary:
Neuropeptides, many of which are conserved among vertebrate and invertebrate animals, are implicated in the regulation of motivational states that selectively facilitate goal-directed behaviors. After a brief presentation of appetitive odors, Drosophila larvae display an impulsive-like feeding activity in readily accessible palatable food. This innate appetitive response may require coordinated signaling activities of dopamine (DA) and neuropeptide F (NPF; a fly homolog of neuropeptide Y). This study provides anatomical and functional evidence, at single-cell resolution, that two NPF neurons define a reward module in the highest-order brain region for cognitive processing of food-related olfactory representations. First, laser lesioning of these NPF neurons abolished odor induction of appetitive arousal, while their genetic activation mimicked the behavioral effect of appetitive odors. Further, a circuit analysis shows that each of the two NPF neurons relays its signals to a subset of target neurons in the larval hindbrain-like region. Finally, the NPF neurons discriminatively responded to appetitive odor stimuli, and their odor responses were blocked by targeted lesioning of a pair of dopaminergic third-order olfactory neurons that appear to be presynaptic to the NPF neurons. Therefore, the two NPF neurons contribute to appetitive odor induction of impulsive-like feeding by selectively decoding DA-encoded ascending olfactory inputs and relaying NPF-encoded descending motivational outputs for behavioral execution.
Otto, N., Marelja, Z., Schoofs, A., Kranenburg, H., Bittern, J., Yildirim, K., Berh, D., Bethke, M., Thomas, S., Rode, S., Risse, B., Jiang, X., Pankratz, M., Leimkuhler, S. and Klambt, C. (2018). The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia. Nat Commun 9(1): 3514. PubMed ID: 30158546
Summary:
Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, a screen was carried out for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper (Sulfite oxidase) encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, the current findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity.
Wolff, T. and Rubin, G. M. (2018). Neuroarchitecture of the Drosophila central complex: A catalog of nodulus and asymmetrical body neurons and a revision of the protocerebral bridge catalog. J Comp Neurol. PubMed ID: 30084503
Summary:
The central complex, a set of neuropils in the center of the insect brain, plays a crucial role in spatial aspects of sensory integration and motor control. Stereotyped neurons interconnect these neuropils with one another and with accessory structures. Over 5000 Drosophila melanogaster GAL4 lines were screened for expression in two neuropils, the noduli (NO) of the central complex and the asymmetrical body (AB), and multicolor stochastic labelling was used to analyze the morphology, polarity and organization of individual cells in a subset of the GAL4 lines that showed expression in these neuropils. Nine NO and three AB cell types were identified and are described in this study. The morphology of the NO neurons suggests that they receive input primarily in the lateral accessory lobe and send output to each of the six paired noduli. The AB is demonstrated to be a bilateral structure which exhibits asymmetry in size between the left and right bodies. The AB neurons are shown to directly connect the AB to the central complex and accessory neuropils, that they target both the left and right ABs, and that one cell type preferentially innervates the right AB. It is proposed that the AB be considered a central complex neuropil in Drosophila. Finally, highly restricted GAL4 lines are presented for most identified protocerebral bridge, NO and AB cell types. These lines, generated using the split-GAL4 method, will facilitate anatomical studies, behavioral assays, and physiological experiments.
Sheng, C., Javed, U., Gibbs, M., Long, C., Yin, J., Qin, B. and Yuan, Q. (2018). Experience-dependent structural plasticity targets dynamic filopodia in regulating dendrite maturation and synaptogenesis. Nat Commun 9(1): 3362. PubMed ID: 30135566
Summary:
Highly motile dendritic protrusions are hallmarks of developing neurons. These exploratory filopodia sample the environment and initiate contacts with potential synaptic partners. To understand the role for dynamic filopodia in dendrite morphogenesis and experience-dependent structural plasticity, this study analyzed dendrite dynamics, synapse formation, and dendrite volume expansion in developing ventral lateral neurons (LNvs) of the Drosophila larval visual circuit. The findings reveal the temporal coordination between heightened dendrite dynamics with synaptogenesis in LNvs and illustrate the strong influence imposed by sensory experience on the prevalence of dendritic filopodia, which regulate the formation of synapses and the expansion of dendritic arbors. Using genetic analyses, Amphiphysin (Amph), a BAR (Bin/Amphiphysin/Rvs) domain-containing protein, was identified as a required component for tuning the dynamic state of LNv dendrites and promoting dendrite maturation. Taken together, this study establishes dynamic filopodia as the key cellular target for experience-dependent regulation of dendrite development.
Ravi, P., Trivedi, D. and Hasan, G. (2018). FMRFa receptor stimulated Ca2+ signals alter the activity of flight modulating central dopaminergic neurons in Drosophila melanogaster. PLoS Genet 14(8): e1007459. PubMed ID: 30110323
Summary:
Neuropeptide signaling influences animal behavior by modulating neuronal activity and thus altering circuit dynamics. Insect flight is a key innate behavior that very likely requires robust neuromodulation. Cellular and molecular components that help modulate flight behavior are therefore of interest and require investigation. In a genetic RNAi screen for G-protein coupled receptors that regulate flight bout durations, several receptors have been identified, including the receptor for the neuropeptide FMRFa (FMRFaR). To further investigate modulation of insect flight by FMRFa CRISPR-Cas9 mutants were generated in the gene encoding the Drosophila FMRFaR. The mutants exhibit significant flight deficits with a focus in dopaminergic cells. Expression of a receptor specific RNAi in adult central dopaminergic neurons resulted in progressive loss of sustained flight. Further, genetic and cellular assays demonstrated that FMRFaR stimulates intracellular calcium signaling through the IP3R and helps maintain neuronal excitability in a subset of dopaminergic neurons for positive modulation of flight bout durations.

Tuesday, September 9th - Signaling

Rizzo, M. J., Evans, J. P., Burt, M., Saunders, C. J. and Johnson, E. C. (2018). Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking. Biochem Biophys Res Commun 503(3): 1919-1926. PubMed ID: 30064912
Summary:
G protein-coupled receptors are the largest superfamily of cell surface receptors in the Metazoa and play critical roles in transducing extracellular signals into intracellular responses. This action is mediated through conformational changes in the receptor following ligand binding. A number of conserved motifs have critical roles in GPCR function, and this study focused on a highly conserved motif (WxFG) in extracellular loop one (EL1). A phylogenetic analysis documents the presence of the WxFG motif in approximately 90% of Class A GPCRs and the motif is represented in 17 of the 19 Class A GPCR subfamilies. Using site-directed mutagenesis, the conserved tryptophan residue was mutagenized in eight receptors which are members of disparate class A GPCR subfamilies from different taxa. The modification of the Drosophila leucokinin receptor shows that substitution of any non-aromatic amino acid for the tryptophan leads to a loss of receptor function. Additionally, leucine substitutions at this position caused similar signaling defects in the follicle-stimulating hormone receptor (FSHR), Galanin receptor (GALR1), AKH receptor (AKHR), corazonin receptor (CRZR), and muscarinic acetylcholine receptor (mACHR1). Visualization of modified receptors through the incorporation of a fluorescent tag revealed a severe reduction in plasma membrane expression, indicating aberrant trafficking of these modified receptors. Taken together, these results suggest a novel role for the WxFG motif in GPCR trafficking and receptor function.
Patrnogic, J., Heryanto, C. and Eleftherianos, I. (2018). Transcriptional up-regulation of the TGF-beta intracellular signaling transducer Mad of Drosophila larvae in response to parasitic nematode infection. Innate Immun: 1753425918790663. PubMed ID: 30049242
Summary:
The common fruit fly Drosophila melanogaster is an exceptional model for dissecting innate immunity. However, knowledge on responses to parasitic nematode infections still lags behind. Recent studies have demonstrated that the well-conserved TGF-beta signaling pathway participates in immune processes of the fly, including the anti-nematode response. To elucidate the molecular basis of TGF-beta anti-nematode activity, a transcript level analysis of different TGF-beta signaling components was performed following infection of D. melanogaster larvae with the nematode parasite Heterorhabditis gerrardi. No significant changes were found in the transcript level of most extracellular ligands in both bone morphogenic protein (BMP) and activin branches of the TGF-beta signaling pathway between nematode-infected larvae and uninfected controls. However, extracellular ligand, Scw, and Type I receptor, Sax, in the BMP pathway as well as the Type I receptor, Babo, in the activin pathway were substantially up-regulated following H. gerrardi infection. The results suggest that receptor up-regulation leads to transcriptional up-regulation of the intracellular component Mad in response to H. gerrardi following changes in gene expression of intracellular receptors of both TGF-beta signaling branches. These findings identify the involvement of certain TGF-beta signaling pathway components in the immune signal transduction of D. melanogaster larvae against parasitic nematodes.
Casal, J., Ibanez-Jimenez, B. and Lawrence, P. A. (2018). Planar cell polarity: the prickle gene acts independently on both the Ds/Ft and the Stan/Fz systems. Development. PubMed ID: 30154173
Summary:
Epithelial cells are polarised within the plane of the epithelium, forming oriented structures whose coordinated and consistent polarity (planar cell polarity, PCP) relates to the principal axes of the body or organ. In Drosophila at least two separate molecular systems generate and interpret intercellular polarity signals: Dachsous/Fat, and the "core" or Stan/Fz system. This study examined the prickle gene and its protein products Prickle and Spiny leg. Much research on PCP has focused on the asymmetric localisation of core proteins in the cell and as a result prickle was placed in the heart of the Stan/Fz system. This study asked if this view is correct and how the prickle gene relates to the two systems. This study found that prickle can affect, separately, both systems - however, neither Pk nor Sple are essential components of the Ds/Ft or the Stan/Fz system, nor do they act as a functional link between the two systems.
Rode, S., Ohm, H., Anhauser, L., Wagner, M., Rosing, M., Deng, X., Sin, O., Leidel, S. A., Storkebaum, E., Rentmeister, A., Zhu, S. and Rumpf, S. (2018). Differential requirement for translation initiation factor pathways during ecdysone-dependent neuronal remodeling in Drosophila. Cell Rep 24(9): 2287-2299. PubMed ID: 30157424
Summary:
Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, the requirements for translation initiation factors during dendrite pruning were assessed. The canonical cap-binding complex eIF4F was found to be dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. It is proposed that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes.
Sapar, M. L., Ji, H., Wang, B., Poe, A. R., Dubey, K., Ren, X., Ni, J. Q. and Han, C. (2018). Phosphatidylserine externalization results from and causes neurite degeneration in Drosophila. Cell Rep 24(9): 2273-2286. PubMed ID: 30157423
Summary:
Phagocytic clearance of degenerating dendrites or axons is critical for maintaining tissue homeostasis and preventing neuroinflammation. Externalized phosphatidylserine (PS) has been postulated to be an "eat-me" signal allowing recognition of degenerating neurites by phagocytes. This study shows that in Drosophila, PS is dynamically exposed on degenerating dendrites during developmental pruning and after physical injury, but PS exposure is suppressed when dendrite degeneration is genetically blocked. Ectopic PS exposure via phospholipid flippase knockout and scramblase overexpression induced PS exposure preferentially at distal dendrites and caused distinct modes of neurite loss that differ in larval sensory dendrites and in adult olfactory axons. Surprisingly, extracellular lactadherin that lacks the integrin-interaction domain induced phagocyte-dependent degeneration of PS-exposing dendrites, revealing an unidentified bridging function that potentiates phagocytes. These findings establish a direct causal relationship between PS exposure and neurite degeneration in vivo.
Sawant, K., Chen, Y., Kotian, N., Preuss, K. M. and McDonald, J. A. (2018). Rap1 GTPase promotes coordinated collective cell migration in vivo. Mol Biol Cell: mbcE17120752. PubMed ID: 30156466
Summary:
During development and in cancer, cells often move together in small to large collectives. In order to move as a unit, cells within collectives need to stay coupled together and coordinate their motility. How cell collectives remain interconnected and migratory, especially when moving through in vivo environments, is not well understood. The genetically tractable border cell group undergoes a highly polarized and cohesive cluster-type migration in the Drosophila ovary. This study reports that the small GTPase Rap1, through activation by PDZ-GEF, regulates border cell collective migration. Rap1 maintains cell contacts within the cluster, at least in part by promoting the organized distribution of E-cadherin at specific cell-cell junctions. Rap1 also restricts migratory protrusions to the front of the border cell cluster and promotes the extension of protrusions with normal dynamics. Further, Rap1 is required in the outer migratory border cells but not in the central non-migratory polar cells. Such cell specificity correlates well with the spatial distribution of the inhibitory Rapgap1 protein, which is higher in polar cells than in border cells. It is proposed that precisely regulated Rap1 activity reinforces connections between cells and polarizes the cluster, thus facilitating the coordinated collective migration of border cells.

Monday, October 8th - Behavior

Yang, Y. and Edery, I. (2018). Parallel clinal variation in the mid-day siesta of Drosophila melanogaster implicates continent-specific targets of natural selection. PLoS Genet 14(9): e1007612. PubMed ID: 30180162
Summary:
Similar to many diurnal animals, Drosophila melanogaster exhibits a mid-day siesta that is more robust as ambient temperature rises, an adaptive response aimed at minimizing exposure to heat. Mid-day siesta levels are partly regulated by the thermosensitive splicing of a small intron (termed dmpi8) found in the 3' untranslated region (UTR) of the circadian clock gene period (per). Using the well-studied D. melanogaster latitudinal cline along the eastern coast of Australia, this study showed that flies from temperate populations sleep less during the day compared to those from tropical regions. Combinations of four single nucleotide polymorphisms (SNPs) were identified in the 3' UTR of per that yield several different haplotypes. The two most abundant of these haplotypes exhibit a reciprocal tropical-temperate distribution in relative frequency. Intriguingly, transgenic flies with the major tropical isoform manifest increased daytime sleep and reduced dmpi8 splicing compared to those carrying the temperate variant. These results strongly suggest that for a major portion of D. melanogaster in Australia, thermal adaptation of daily sleep behavior included spatially varying selection on ancestrally derived polymorphisms in the per 3' UTR that differentially control dmpi8 splicing efficiency. Prior work showed that African flies from high altitudes manifest reduced mid-day siesta levels, indicative of parallel latitudinal and altitudinal adaptation across continents. However, geographical variation in per 3' UTR haplotypes was not observed for African flies, providing a compelling case for inter-continental variation in factors targeted by natural selection in attaining a parallel adaptation. It is proposed that the ability to calibrate mid-day siesta levels to better match local temperature ranges is a key adaptation contributing to the successful colonization of D. melanogaster beyond its ancestral range in the lowlands of Sub-Saharan Africa.
Mercier, D., Tsuchimoto, Y., Ohta, K. and Kazama, H. (2018). Olfactory landmark-based communication in interacting Drosophila. Curr Biol 28(16): 2624-2631.e2625. PubMed ID: 30078566
Summary:
To communicate with conspecifics, animals deploy various strategies to release pheromones, chemical signals modulating social and sexual behaviors. Importantly, a single pheromone induces different behaviors depending on the context and exposure dynamics. Therefore, to comprehend the ethological role of pheromones, it is essential to characterize how neurons in the recipients respond to temporally and spatially fluctuating chemical signals emitted by donors during natural interactions. In Drosophila melanogaster, the male pheromone 11-cis-vaccenyl acetate (cVA) activates specific olfactory receptor neurons (ORNs) to regulate diverse social and sexual behaviors in recipients. Physicochemical analyses have identified this chemical on an animal's body and in its local environment. However, because these methods are imprecise in capturing spatiotemporal dynamics, it is poorly understood how individual pheromone cues are released, detected, and interpreted by recipients. This study developed a system based on bioluminescence to monitor neural activity in freely interacting Drosophila, and investigated the active detection and perception of the naturally emitted cVA. Unexpectedly, neurons specifically tuned to cVA did not exhibit significant activity during physical interactions between males, and instead responded strongly to olfactory landmarks deposited by males. These landmarks mediated attraction through Or67d receptors and allured both sexes to the marked region. Importantly, the landmarks remained attractive even when a pair of flies was engaged in courtship behavior. In contrast, female deposits did not affect the exploration pattern of either sex. Thus, Drosophila use pheromone marking to remotely signal their sexual identity and to enhance social interactions.
Morley, E. L., Jonsson, T. and Robert, D. (2018). Auditory sensitivity, spatial dynamics, and amplitude of courtship song in Drosophila melanogaster. J Acoust Soc Am 144(2): 734. PubMed ID: 30180716
Summary:
Acoustic communication is an important component of courtship in Drosophila melanogaster. It takes the form of courtship song produced by males through the unilateral extension and vibration of a wing. Following the paradigm of sender-receiver matching, song content is assumed to match tuning in the auditory system, however, D. melanogaster audition is nonlinear and tuning dependent upon signal amplitude. At low stimulus amplitudes or in the absence of sound the antenna is tuned into song frequency, but as amplitude increases the antenna's resonance is shifted up by hundreds of Hertz. Accurate measurements of song amplitude have been elusive because of the strong dependency of amplitude upon the spatial geometry between sender and receiver. In this study, D. melanogaster auditory directional sensitivity and the geometric position between the courting flies are quantified. It is shown that singing occurs primarily from positions resulting in direct stimulation of the female antenna. Using this information, it is established that the majority of song is louder than theoretically predicted and at these sound levels the female antenna should not amplify or be tuned into song. The study implies that Drosophila hearing, and, in particular, its active mechanisms, could function in a broader context than previously surmised.
Park, A., Tran, T. and Atkinson, N. S. (2018). Monitoring food preference in Drosophila by oligonucleotide tagging. Proc Natl Acad Sci U S A 115(36): 9020-9025. PubMed ID: 30127010
Summary:
Drosophila melanogaster is a powerful model organism for dissecting the neurogenetic basis of appetitive and aversive behaviors. However, some methods used to assay food preference require or cause starvation. This can be problematic for fly ethanol research because it can be difficult to dissociate caloric preference for ethanol from pharmacological preference for the drug. BARCODE, a starvation-independent assay that uses trace levels of oligonucleotide tags was designed to differentially mark food types. In BARCODE, flies feed ad libitum, and relative food preference is monitored by qPCR of the oligonucleotides. Persistence of the ingested oligomers within the fly records the feeding history of the fly over several days. Using BARCODE, this study identified a sexually dimorphic preference for ethanol. Females are attracted to ethanol-laden foods, whereas males avoid consuming it. Furthermore, genetically feminizing male mushroom body lobes induces preference for ethanol. In addition, it was demonstrated that BARCODE can be used for multiplex diet measurements when animals are presented with more than two food choices.
Liu, G., Nath, T., Linneweber, G. A., Claeys, A., Guo, Z., Li, J., Bengochea, M., De Backer, S., Weyn, B., Sneyders, M., Nicasy, H., Yu, P., Scheunders, P. and Hassan, B. A. (2018). A simple computer vision pipeline reveals the effects of isolation on social interaction dynamics in Drosophila. PLoS Comput Biol 14(8): e1006410. PubMed ID: 30161262
Summary:
Isolation profoundly influences social behavior in all animals. Longer-term analysis of large groups of flies is hampered by the lack of effective and reliable tools. In this study a new imaging arena was built and the existing tracking algorithm was improved to reliably follow a large number of flies simultaneously. Next, based on the automatic classification of touch and graph-based social network analysis, an algorithm was designed to quantify changes in the social network in response to prior social isolation. It was observed that isolation significantly and swiftly enhanced individual and local social network parameters depicting near-neighbor relationships. The genome-wide molecular correlates of these behavioral changes were explored, and it was found that whereas behavior changed throughout the six days of isolation, gene expression alterations occurred largely on day one. These changes occurred mostly in metabolic genes, and the metabolic changes were varified by showing an increase of lipid content in isolated flies. In summary, this study describes a highly reliable tracking and analysis pipeline for large groups of flies that were use to unravel the behavioral, molecular and physiological impact of isolation on social network dynamics in Drosophila.
Luo, W., Guo, F., McMahon, A., Couvertier, S., Jin, H., Diaz, M., Fieldsend, A., Weerapana, E. and Rosbash, M. (2018). NonA and CPX link the circadian clockwork to locomotor activity in Drosophila. Neuron 99(4): 768-780.e763. PubMed ID: 30057203
Summary:
Drosophila NonA and its mammalian ortholog NONO are members of the Drosophila behavior and human splicing (DBHS) family. NONO also has a strong circadian connection: it associates with the circadian repressor protein Period (Per) and contributes to circadian timekeeping. This study investigated NonA, which is required for proper levels of evening locomotor activity as well as a normal free-running period in Drosophila. NonA is associated with the positive transcription factor Clock/Cycle (Clk/Cyc), interacts directly with complexin (cpx) pre-mRNA, and upregulates gene expression, including the gene cpx. Downregulation of cpx expression in circadian neurons phenocopies NonA downregulation, whereas cpx overexpression rescues the nonA RNAi phenotypes, indicating that cpx is an important NonA target gene. As the cpx protein contributes to proper neurotransmitter and neuropeptide release in response to calcium, these results and others indicate that this control is important for the normal circadian regulation of locomotor activity.
Li, C., Dong, H. and Zhao, K. (2018). A balance between aerodynamic and olfactory performance during flight in Drosophila. Nat Commun 9(1): 3215. PubMed ID: 30097572
Summary:
The ability to track odor plumes to their source (food, mate, etc.) is key to the survival of many insects. During this odor-guided navigation, flapping wings could actively draw odorants to the antennae to enhance olfactory sensitivity, but it is unclear if improving olfactory function comes at a cost to aerodynamic performance. This study computationally quantified the odor plume features around a fruit fly in forward flight and confirmed that the antenna is well positioned to receive a significant increase of odor mass flux (peak 1.8 times), induced by wing flapping, vertically from below the body but not horizontally. This anisotropic odor spatial sampling may have important implications for behavior and the algorithm during plume tracking. Further analysis also suggests that, because both aerodynamic and olfactory functions are indispensable during odor-guided navigation, the wing shape and size may be a balance between the two functions.
Mayseless, O., Berns, D. S., Yu, X. M., Riemensperger, T., Fiala, A. and Schuldiner, O. (2018). Developmental coordination during olfactory circuit remodeling in Drosophila. Neuron. PubMed ID: 30146303
Summary:
Developmental neuronal remodeling is crucial for proper wiring of the adult nervous system. While remodeling of individual neuronal populations has been studied, how neuronal circuits remodel-and whether remodeling of synaptic partners is coordinated-is unknown. This study found that the Drosophila anterior paired lateral (APL) neuron undergoes stereotypic remodeling during metamorphosis in a similar time frame as the mushroom body (MB) -neurons, with whom it forms a functional circuit. By simultaneously manipulating both neuronal populations, this study found that cell-autonomous inhibition of -neuron pruning resulted in the inhibition of APL pruning in a process that is mediated, at least in part, by Ca(2+)-Calmodulin and neuronal activity dependent interaction. Finally, ectopic unpruned MB axons display ectopic connections with the APL, as well as with other neurons, at the adult, suggesting that inhibiting remodeling of one neuronal type can affect the functional wiring of the entire micro-circuit.

Friday, October 5th - Signaling

Mazzotta, G. M., Bellanda, M., Minervini, G., Damulewicz, M., Cusumano, P., Aufiero, S., Stefani, M., Zambelli, B., Mammi, S., Costa, R. and Tosatto, S. C. E. (2018). Calmodulin enhances Cryptochrome binding to INAD in Drosophila photoreceptors. Front Mol Neurosci 11: 280. PubMed ID: 30177872
Summary:
Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila Cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. Previous work has shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. This study predicts and demonstrates a novel Ca(2+)-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses this study to identified and characterized a CaM binding motif in the dCRY C-terminus. Similarly, the CaM binding site of the scaffold protein INAD was also detailed, and it was demonstrated that CaM bridges dCRY and INAD to form a ternary complex in vivo. These results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM.
Li, B., Wong, C., Gao, S. M., Zhang, R., Sun, R., Li, Y. and Song, Y. (2018). The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating notch receptor trafficking. Elife 7. PubMed ID: 30176986
Summary:
The correct establishment and maintenance of unidirectional Notch signaling are critical for the homeostasis of various stem cell lineages. However, the molecular mechanisms that prevent cell-autonomous ectopic Notch signaling activation and deleterious cell fate decisions remain unclear. This study shows that the retromer complex directly and specifically regulates Notch receptor retrograde trafficking in Drosophila neuroblast lineages to ensure the unidirectional Notch signaling from neural progenitors to neuroblasts. Notch polyubiquitination mediated by E3 ubiquitin ligase Itch/Su(dx) is inherently inefficient within neural progenitors, relying on retromer-mediated trafficking to avoid aberrant endosomal accumulation of Notch and cell-autonomous signaling activation. Upon retromer dysfunction, hypo-ubiquitinated Notch accumulates in Rab7(+) enlarged endosomes, where it is ectopically processed and activated in a ligand-dependent manner, causing progenitor-originated tumorigenesis. These results therefore unveil a safeguard mechanism whereby retromer retrieves potentially harmful Notch receptors in a timely manner to prevent aberrant Notch activation-induced neural progenitor dedifferentiation and brain tumor formation.
Ji, S., Samara, N. L., Revoredo, L., Zhang, L., Tran, D. T., Muirhead, K., Tabak, L. A. and Ten Hagen, K. G. (2018). A molecular switch orchestrates enzyme specificity and secretory granule morphology. Nat Commun 9(1): 3508. PubMed ID: 30158631
Summary:
Regulated secretion is an essential process where molecules destined for export are directed to membranous secretory granules, where they undergo packaging and maturation. This study identified a gene (pgant9) that influences the structure and shape of secretory granules within the Drosophila salivary gland. Loss of pgant9, which encodes an O-glycosyltransferase, results in secretory granules with an irregular, shard-like morphology, and altered glycosylation of cargo. Interestingly, pgant9 undergoes a splicing event that acts as a molecular switch to alter the charge of a loop controlling access to the active site of the enzyme. The splice variant with the negatively charged loop glycosylates the positively charged secretory cargo and rescues secretory granule morphology. This study highlights a mechanism for dictating substrate specificity within the O-glycosyltransferase enzyme family. Moreover, these in vitro and in vivo studies suggest that the glycosylation status of secretory cargo influences the morphology of maturing secretory granules.
Jiang, Y., Seimiya, M., Schlumpf, T. B. and Paro, R. (2018). An intrinsic tumour eviction mechanism in Drosophila mediated by steroid hormone signalling. Nat Commun 9(1): 3293. PubMed ID: 30120247
Summary:
Polycomb group proteins are epigenetic regulators maintaining transcriptional memory during cellular proliferation. In Drosophila larvae, malfunction of Polyhomeotic (Ph), a member of the PRC1 silencing complex, results in neoplastic growth. This study reports an intrinsic tumour suppression mechanism mediated by the steroid hormone ecdysone during metamorphosis. Ecdysone alters neoplastic growth into a nontumorigenic state of the mutant ph cells which then become eliminated during adult stage. This study demonstrates that ecdysone exerts this function by inducing a heterochronic network encompassing the activation of the microRNA lethal-7, which suppresses its target gene chronologically inappropriate morphogenesis. This pathway can also promote remission of brain tumours formed in brain tumour mutants, revealing a restraining of neoplastic growth in different tumour types. Given the conserved role of let-7, the identification and molecular characterization of this innate tumour eviction mechanism in flies might provide important clues towards the exploitation of related pathways for human tumour therapy.
Lee, C. H., Kiparaki, M., Blanco, J., Folgado, V., Ji, Z., Kumar, A., Rimesso, G. and Baker, N. E. (2018). A regulatory response to ribosomal protein mutations controls translation, growth, and cell competition. Dev Cell 46(4): 456-469. PubMed ID: 30078730
Summary:
Ribosomes perform protein synthesis but are also involved in signaling processes, the full extent of which are still being uncovered. This study reports that phenotypes of mutating ribosomal proteins (Rps) are largely due to signaling. Using Drosophila, this study discovered that a bZip-domain protein, Xrp1, becomes elevated in Rp mutant cells. Xrp1 reduces translation and growth, delays development, is responsible for gene expression changes, and causes the cell competition of Rp heterozygous cells from genetic mosaics. Without Xrp1, even cells homozygously deleted for Rp genes persist and grow. Xrp1 induction in Rp mutant cells depends on a particular Rp with regulatory effects, RpS12, and precedes overall changes in translation. Thus, effects of Rp mutations, even the reductions in translation and growth, depend on signaling through the Xrp1 pathway and are not simply consequences of reduced ribosome production limiting protein synthesis. One benefit of this system may be to eliminate Rp-mutant cells by cell competition.
Potdar, S. and Sheeba, V. (2018). Wakefulness is promoted during day time by PDFR signalling to dopaminergic neurons in Drosophila melanogaster. eNeuro 5(4). PubMed ID: 30131970
Summary:
Circadian clocks modulate timing of sleep/wake cycles in animals; however, the underlying mechanisms remain poorly understood. In Drosophila melanogaster, large ventral lateral neurons (l-LNv) are known to promote wakefulness through the action of the neuropeptide pigment dispersing factor (PDF), but the downstream targets of PDF signalling remain elusive. In a screen using downregulation or overexpression (OEX) of the gene encoding PDF receptor (pdfr), this study found that a subset of dopaminergic neurons responds to PDF to promote wakefulness during the day. Moreover, this study found that small LNv (s-LNv) and dopaminergic neurons form synaptic contacts, and PDFR signalling inhibited dopaminergic neurons specifically during day time. It is proposed that these dopaminergic neurons that respond to PDFR signalling are sleep-promoting and that during the day when PDF levels are high, they are inhibited, thereby promoting wakefulness. Thus, this study has identified a novel circadian clock pathway that mediates wake promotion specifically during day time.

Thursday, October 4th - Adult Neural Development and Function

Feng, G., Zhang, J., Li, M., Shao, L., Yang, L., Song, Q. and Ping, Y. (2018). Control of sleep onset by Shal/Kv4 channels in Drosophila circadian neurons. J Neurosci. PubMed ID: 30185460
Summary:
Sleep is highly conserved across animal species. Both wake- and sleep-promoting neurons are implicated in the regulation of wake-sleep transition at dusk in Drosophila However, little is known about how they cooperate and whether they act via different mechanisms. This study demonstrated that in female Drosophila, sleep onset was specifically delayed by blocking the Shaker cognate L channels (Shal, also known as voltage-gated K(+) channel 4, Kv4) in wake-promoting cells, including large ventral lateral neurons (l-LNvs) and pars intercerebralis (PI), but not in sleep-promoting dorsal neurons (DN1s). Delayed sleep onset was also observed in males by blocking Kv4 activity in wake-promoting neurons. Electrophysiological recordings show that Kv4 channels contribute A-type currents (IA) in LNvs and PI cells, but are much less conspicuous in DN1s. Interestingly, blocking Kv4 in wake-promoting neurons preferentially increased firing rates at dusk around ZT13, when the resting membrane potentials (RMPs) and firing rates were at lower levels. Furthermore, pigment-dispersing factor (PDF) is essential for the regulation of sleep onset by Kv4 in l-LNvs, and downregulation of PDF receptor (PDFR) in PI neurons advanced sleep onset, indicating Kv4 controls sleep onset via regulating PDF/PDFR signaling in wake-promoting neurons. It is proposed that Kv4 acts as a sleep onset controller by suppressing membrane excitability in a clock-dependent manner to balance the wake-sleep transition at dusk. These results have important implications for the understanding and treatment of sleep disorders such as insomnia.
Jones, S. G., Nixon, K. C. J., Chubak, M. C. and Kramer, J. M. (2018). Mushroom body specific transcriptome analysis reveals dynamic regulation of learning and memory genes after acquisition of long-term courtship memory in Drosophila. G3 (Bethesda). PubMed ID: 30158319
Summary:
The formation and recall of long-term memory (LTM) requires neuron activity-induced gene expression. The complex spatial and temporal dynamics of memory formation creates significant challenges in defining memory-relevant gene expression changes. The Drosophila mushroom body (MB) is a signaling hub in the insect brain that integrates sensory information to form memories across several different experimental memory paradigms. This study performed transcriptome analysis in the MB at two time points after the acquisition of LTM: 1 hour and 24 hours. The MB transcriptome was compared to biologically paired whole head (WH) transcriptomes. In both, more transcript level changes were identified at 1 hour after memory acquisition (WH = 322, MB = 302) than at 24 hours (WH = 23, MB = 20). WH samples showed downregulation of developmental genes and upregulation of sensory response genes. In contrast, MB samples showed vastly different changes in transcripts involved in biological processes that are specifically related to LTM. MB-downregulated genes were highly enriched for metabolic function. MB-upregulated genes were highly enriched for known learning and memory processes, including calcium-mediated neurotransmitter release and cAMP signalling. The neuron activity inducible genes Hr38 and sr were also specifically induced in the MB. These results highlight the importance of sampling time and cell type in capturing biologically relevant transcript level changes involved in learning and memory. The data suggests that MB cells transiently upregulate known memory-related pathways after memory acquisition.
Franconville, R., Beron, C. and Jayaraman, V. (2018). Building a functional connectome of the Drosophila central complex. Elife 7. PubMed ID: 30124430
Summary:
The central complex is a highly conserved insect brain region composed of morphologically stereotyped neurons that arborize in distinctively shaped substructures. The region is implicated in a wide range of behaviors and several modeling studies have explored its circuit computations. Most studies have relied on assumptions about connectivity between neurons based on their overlap in light microscopy images. This study presents an extensive functional connectome of Drosophila melanogaster's central complex at cell-type resolution. Using simultaneous optogenetic stimulation, calcium imaging and pharmacology, the connectivity was tested between 70 presynaptic-to-postsynaptic cell-type pairs. Numerous inputs to the central complex were identified, but only a small number of output channels. Additionally, the connectivity of this highly recurrent circuit appears to be sparser than anticipated from light microscopy images. Finally, the connectivity matrix highlights the potentially critical role of a class of bottleneck interneurons. All data is provided for interactive exploration on a website.
Kinold, J. C., Pfarr, C. and Aberle, H. (2018). Sidestep-induced neuromuscular miswiring causes severe locomotion defects in Drosophila larvae. Development 145(17). PubMed ID: 30166331
Summary:
Mutations in motor axon guidance molecules cause aberrant projection patterns of motor nerves. As most studies in Drosophila have analysed these molecules in fixed embryos, the consequences for larval locomotion are entirely unexplored. This study took advantage of sidestep (side)-mutant larvae that display severe locomotion defects because of irreparable innervation errors. Mutations in side affected all motor nerve branches and all body wall regions. Innervation defects were non-stereotypical, showing unique innervation patterns in each hemisegment. Premature activation of Side in muscle precursors abrogated dorsal migration of motor nerves, resulting in larvae with a complete loss of neuromuscular junctions on dorsal-most muscles. High-speed videography showed that these larvae failed to maintain substrate contact and inappropriately raised both head and tail segments above the substrate, resulting in unique 'arching' and 'lifting' phenotypes. These results show that guidance errors in side mutants are maintained throughout larval life and are asymmetrical with respect to the bilateral body axis. Together with similar findings in mice, this study also suggests that miswiring could be an underlying cause of inherited movement disorders.
Li, J., Guajardo, R., Xu, C., Wu, B., Li, H., Li, T., Luginbuhl, D. J., Xie, X. and Luo, L. (2018). Stepwise wiring of the Drosophila olfactory map requires specific Plexin B levels. Elife 7. PubMed ID: 30136927
Summary:
The precise assembly of a neural circuit involves many consecutive steps. The conflict between a limited number of wiring molecules and the complexity of the neural network impels each molecule to execute multiple functions at different steps. This study examined the cell-type specific distribution of endogenous levels of axon guidance receptor Plexin B (PlexB) in the developing antennal lobe, the first olfactory processing center in Drosophila. Different classes of olfactory receptor neurons (ORNs) express PlexB at different levels in two wiring steps - axonal trajectory choice and subsequent target selection. In line with its temporally distinct patterns, the proper levels of PlexB control both steps in succession. Genetic interactions further revealed that the effect of high-level PlexB is antagonized by its canonical partner Sema2b. Thus, PlexB plays a multifaceted role in instructing the assembly of the Drosophila olfactory circuit through temporally-regulated expression patterns and expression level-dependent effects.
Loewen, C., Boekhoff-Falk, G., Ganetzky, B. and Chtarbanova, S. (2018). A novel mutation in brain tumor causes both neural over-proliferation and neurodegeneration in adult Drosophila. G3 (Bethesda). PubMed ID: 30126833
Summary:
A screen for neuroprotective genes in Drosophila melanogaster led to the identification of a mutation that causes extreme, progressive loss of adult brain neuropil in conjunction with massive brain overgrowth. The mutation was mapped to the brain tumor (brat) locus, which encodes a tripartite motif-NCL-1, HT2A, and LIN-41 (TRIM-NHL) RNA-binding protein with established roles limiting stem cell proliferation in developing brain and ovary. However, a neuroprotective role for brat in the adult Drosophila brain has not been described previously. The new allele, bratcheesehead(bratchs), carries a mutation in the coiled-coil domain of the TRIM motif, and is temperature-sensitive. mRNA and protein levels of neural stem cell genes are increased in heads of adult bratchs mutants; the over-proliferation phenotype initiates prior to adult eclosion. It is also reported that disruption of an uncharacterized gene coding for a presumptive prolyl-4-hydroxylase strongly enhances the over-proliferation and neurodegeneration phenotypes. Together, these results reveal an unexpected role for brat that could be relevant to human cancer and neurodegenerative diseases.

Wednesday, October 3rd - Chromatin

Hundertmark, T., Gartner, S. M. K., Rathke, C. and Renkawitz-Pohl, R. (2018). Nejire/dCBP-mediated histone H3 acetylation during spermatogenesis is essential for male fertility in Drosophila melanogaster. PLoS One 13(9): e0203622. PubMed ID: 30192860
Summary:
Spermatogenesis in many species including Drosophila melanogaster is accompanied by major reorganisation of chromatin in post-meiotic stages, involving a nearly genome-wide displacement of histones by protamines, Mst77F and Protamine-like 99C. A proposed prerequisite for the histone-to-protamine transition is massive histone H4 hyper-acetylation prior to the switch. This study investigated the pattern of histone H3 lysine acetylation and general lysine crotonylation in D. melanogaster spermiogenesis to elucidate a possible role of these marks in chromatin reorganisation. Lysine crotonylation was strongest prior to remodelling and the deposition of this mark depended on the acetylation status of the spermatid chromatin. In contrast to H4 acetylation, individual H3 acetylation marks displayed surprisingly distinct patterns during the histone-to-protamine transition. Nejire, a histone acetyl transferase, is expressed during the time of histone-to-protamine transition. Nejire knock down led to strongly reduced fertility, which correlated with misshaped spermatid nuclei and a lack of mature sperm. protA and prtl99C transcript levels were reduced after knocking down Nejire. ProtB-eGFP, Mst77F-eGFP and Prtl99C-eGFP were synthesized at the late canoe stage, while histones were often not detectable. However, in some cysts histones persist in parallel to protamines. Therefore, it was hypothesized that complete histone removal requires multiple histone modifications besides H3K18ac and H3K27ac. In summary, H3K18 and H3K27 acetylation during Drosophila spermatogenesis is dependent on Nejire or a yet uncharacterized acetyl transferase. Nejire is required for male fertility since Nejire contributes to efficient transcription of protA and prtl99C, but not Mst77F, in spermatocytes, and to maturation of sperm.
Deutschman, E., Ward, J. R., Ho, A. L. K. T., Alban, T. J., Zhang, D., Willard, B., Lemieux, M. E., Lathia, J. D. and Longworth, M. S. (2018). Comparing and contrasting the effects of Drosophila Condensin II subunit dCAP-D3 overexpression and depletion in vivo. Genetics. PubMed ID: 30068527
Summary:
The Condensin II complex plays important, conserved roles in genome organization throughout the cell cycle and in the regulation of gene expression. Previous studies have linked decreased Condensin II subunit expression with a variety of diseases. This study shows that elevated levels of Condensin II subunits are detected in somatic cancers. To evaluate potential biological effects of elevated Condensin II levels, the Condensin II subunit, dCAP-D3, was overexpressed in Drosophila melanogaster larval tissues and the effects were examined on the mitotic and interphase specific functions of Condensin II. Interestingly, while ubiquitous overexpression resulted in pupal lethality, tissue specific overexpression of dCAP-D3 caused formation of nucleoplasmic protein aggregates which slowed mitotic prophase progression, mimicking results observed when dCAP-D3 levels are depleted. Surprisingly, dCAP-D3 aggregate formation resulted in faster transitions from metaphase to anaphase. Overexpressed dCAP-D3 protein failed to precipitate other Condensin II subunits in non-dividing tissues, but did cause changes to gene expression which occurred in a manner opposite of what was observed when dCAP-D3 levels were depleted in both dividing and non-dividing tissues. These findings show that altering dCAP-D3 levels in either direction has detrimental effects on mitotic timing, the regulation of gene expression, and organism development. Taken together, these data suggest that the different roles for Condensin II throughout the cell cycle may be independent of each other and/or that dCAP-D3 may possess functions that are separate from those involving its association with the Condensin II complex. If conserved, these findings could have implications for tumors harboring elevated CAP-D3 levels.
Hua, B. L., Bell, G. W., Kashevsky, H., Von Stetina, J. R. and Orr-Weaver, T. L. (2018). Dynamic changes in ORC localization and replication fork progression during tissue differentiation. BMC Genomics 19(1): 623. PubMed ID: 30134926
Summary:
Genomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role. This study exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, sites of ORC binding were found to show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited. These studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation.
Fedotova, A., Aoki, T., Rossier, M., Mishra, R., Clendine, C., Kyrchanova, O., Wolle, D., Bonchuk, A., Maeda, R., Mutero, A., Cleard, F., Mogila, V., Karch, F., Georgiev, P. and Schedl, P. (2018). The BEN domain protein insensitive binds to the Fab-7 chromatin boundary to establish proper segmental identity in Drosophila. Genetics. PubMed ID: 30082280
Summary:
Boundaries (insulators) in the Drosophila bithorax complex (BX-C) delimit autonomous regulatory domains that orchestrate the parasegment (PS)-specific expression of the BX-C homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains, which control Abd-B expression in PS11 and PS12, respectively. This boundary is composed of multiple functionally redundant elements and has two key functions: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass. Tnis study shows that two BEN domain protein complexes, Insensitive and Elba, bind to multiple sequences located in the Fab-7 nuclease hypersensitive regions. Two of these sequences are recognized by both Insv and Elba and correspond to a CCAATTGG palindrome. Elba also binds to a related CCAATAAG sequence, while Insv does not. However, the third Insv recognition sequences is ~100 bp in length and contains the CCAATAAG sequence at one end. Both Insv and Elba are assembled into large complexes (~420 kD and ~265-290 kD, respectively) in nuclear extracts. Using a sensitized genetic background this study showed that the Insv protein is required for Fab-7 boundary function, and that PS11 identity is not properly established in insv mutants. This is the first demonstration that a BEN domain protein is important for the functioning of an endogenous fly boundary.
Mahajan, S., Wei, K. H., Nalley, M. J., Gibilisco, L. and Bachtrog, D. (2018). De novo assembly of a young Drosophila Y chromosome using single-molecule sequencing and chromatin conformation capture. PLoS Biol 16(7): e2006348. PubMed ID: 30059545
Summary:
While short-read sequencing technology has resulted in a sharp increase in the number of species with genome assemblies, these assemblies are typically highly fragmented. Repeats pose the largest challenge for reference genome assembly, and pericentromeric regions and the repeat-rich Y chromosome are typically ignored from sequencing projects. This study assemble the genome of Drosophila miranda using long reads for contig formation, chromatin interaction maps for scaffolding and short reads, and optical mapping and bacterial artificial chromosome (BAC) clone sequencing for consensus validation. The assembly recovers entire chromosomes and contains large fractions of repetitive DNA, including about 41.5 Mb of pericentromeric and telomeric regions, and >100 Mb of the recently formed highly repetitive neo-Y chromosome. While Y chromosome evolution is typically characterized by global sequence loss and shrinkage, the neo-Y increased in size by almost 3-fold because of the accumulation of repetitive sequences. This high-quality assembly allows reconstruction og the chromosomal events that have led to the unusual sex chromosome karyotype in D. miranda, including the independent de novo formation of a pair of sex chromosomes at two distinct time points, or the reversion of a former Y chromosome to an autosome.
Guio, L., Vieira, C. and Gonzalez, J. (2018). Stress affects the epigenetic marks added by natural transposable element insertions in Drosophila melanogaster. Sci Rep 8(1): 12197. PubMed ID: 30111890
Summary:
Transposable elements are emerging as an important source of cis-acting regulatory sequences and epigenetic marks that could influence gene expression. However, few studies have dissected the role of specific transposable element insertions on epigenetic gene regulation. Bari-Jheh is a natural transposon that mediates resistance to oxidative stress by adding cis-regulatory sequences that affect expression of nearby genes. This work, integrated publicly available ChIP-seq and piRNA data with chromatin immunoprecipitation experiments to get a more comprehensive picture of Bari-Jheh molecular effects. Bari-Jheh was shown to be enriched for H3K9me3 in nonstress conditions, and for H3K9me3, H3K4me3 and H3K27me3 in oxidative stress conditions, which is consistent with expression changes in adjacent genes. It was further shown that under oxidative stress conditions, H3K4me3 and H3K9me3 spread to the promoter region of Jheh1 gene. Finally, another insertion of the Bari1 family was associated with increased H3K27me3 in oxidative stress conditions suggesting that Bari1 histone marks are copy-specific. It is concluded that besides adding cis-regulatory sequences, Bari-Jheh influences gene expression by affecting the local chromatin state.

Tuesday, October 2nd - Signaling

Alpar, L., Bergantinos, C. and Johnston, L. A. (2018). Spatially restricted regulation of Spatzle/Toll signaling during cell competition. Dev Cell. PubMed ID: 30146479
Summary:
Cell competition employs comparisons of fitness to selectively eliminate cells sensed as less healthy. In Drosophila, apoptotic elimination of the weaker "loser" cells from growing wing discs is induced by a signaling module consisting of the Toll ligand Spatzle (Spz), several Toll-related receptors, and NF-kappaB factors. How this module is activated and restricted to competing disc cells is unknown. This study used Myc-induced cell competition to demonstrate that loser cell elimination requires local wing disc synthesis of Spz. Spz processing enzyme (SPE) and modular serine protease (ModSP) are identified as activators of Spz-regulated competitive signaling, and "winner" cells are shown to trigger elimination of nearby WT cells by boosting SPE production. Moreover, Spz requires both Toll and Toll-8 to induce apoptosis of wing disc cells. Thus, during cell competition, Spz-mediated signaling is strictly confined to the imaginal disc, allowing errors in tissue fitness to be corrected without compromising organismal physiology.
Cohen, E., Allen, S. R., Sawyer, J. K. and Fox, D. T. (2018). Fizzy-related dictates a cell cycle switch during organ repair and tissue growth responses in the Drosophila hindgut. Elife 7. PubMed ID: 30117808
Summary:
Ploidy-increasing cell cycles drive tissue growth in many developing organs. Such cycles, including endocycles, are increasingly appreciated to drive tissue growth following injury or activated growth signaling in mature organs. In these organs, the regulation and distinct roles of different cell cycles remains unclear. This study uncovered a programmed switch between cell cycles in the Drosophila hindgut pylorus. Using an acute injury model, mitosis was identified as the response in larval pyloric cells, whereas endocycles occur in adult pyloric cells. By developing a novel genetic method, DEMISE (Dual-Expression-Method-for-Induced-Site-specific-Eradication), it was shown that the cell cycle regulator Fizzy-related dictates the decision between mitosis and endocycles. After injury, both cycles accurately restore tissue mass and genome content. However, in response to sustained growth signaling, only endocycles preserve epithelial architecture. These data reveal distinct cell cycle programming in response to similar stimuli in mature vs. developmental states and reveal a tissue-protective role of endocycles.
Hou, X., Burstein, S. R. and Long, S. B. (2018).. Structures reveal opening of the store-operated calcium channel Orai. Elife 7. PubMed ID: 30160233
Summary:
The store-operated calcium (Ca(2+)) channel Orai governs Ca(2+) influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca(2+) stored in the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Previous work revealed the structure of Orai with a closed pore. In this study, using a gain-of-function mutation that constitutively activates the channel, an X-ray structure is presented of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca(2+) selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, a sequence is proposed for store-operated activation.
Peters, K. A., Detmar, E., Sepulveda, L., Del Valle, C., Valsquier, R., Ritz, A., Rogers, S. L. and Applewhite, D. A. (2018). A cell-based assay to investigate non-muscle Myosin II contractility via the Folded-gastrulation signaling pathway in Drosophila S2R+ cells. J Vis Exp(138). PubMed ID: 30176023
Summary:
A cell-based assay has been developed using Drosophila cells that recapitulates apical constriction initiated by Folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a Galpha12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

Monday, October 1st - RNA

Hirashima, T., Tanaka, R., Yamaguchi, M. and Yoshida, H. (2018). The ABD on the nascent polypeptide and PH domain are required for the precise Anillin localization in Drosophila syncytial blastoderm. Sci Rep 8(1): 12910. PubMed ID: 30150713
Summary:
Targeting proteins to regions where they are required is essential for proper development of organisms. For achievement of this, subcellular mRNA localization is one of the critical mechanisms. Subcellular mRNA localization is an evolutionarily conserved phenomenon from E. coli to human and contributes to limiting the regions at which its products function and efficiently supplies substrates for protein translation. During early Drosophila embryogenesis, while 71% of the 3370 mRNAs analyzed have shown prominent subcellular localization, the underlying molecular mechanisms have not been elucidated. This study revealed that anillin mRNA, one of the localized mRNAs in early Drosophila embryo, localizes to the tip of the pseudo-cleavage furrow in the Drosophila syncytial blastoderm using in situ hybridization combined with immunohistochemistry. Localization analyses with transgenic fly lines carrying a series of deletion mRNAs indicate that this localization is dependent on its own nascent polypeptides including the actin binding domain (ABD). In addition to the mRNA localization, it is revealed that the pleckstrin homology (PH) domain of Anillin protein is also required for its proper localization. Thus, this study indicates that the precise localization of Anillin protein is tightly regulated by the ABD on the nascent polypeptide and PH domain in the Drosophila syncytial blastoderm.
Monedero Cobeta, I., Stadler, C. B., Li, J., Yu, P., Thor, S. and Benito-Sipos, J. (2018). Specification of Drosophila neuropeptidergic neurons by the splicing component brr2. PLoS Genet 14(8): e1007496. PubMed ID: 30133436
Summary:
During embryonic development, a number of genetic cues act to generate neuronal diversity. While intrinsic transcriptional cascades are well-known to control neuronal sub-type cell fate, the target cells can also provide critical input to specific neuronal cell fates. Such signals, denoted retrograde signals, are known to provide critical survival cues for neurons, but have also been found to trigger terminal differentiation of neurons. One salient example of such target-derived instructive signals pertains to the specification of the Drosophila FMRFamide neuropeptide neurons, the Tv4 neurons of the ventral nerve cord. Tv4 neurons receive a BMP signal from their target cells, which acts as the final trigger to activate the FMRFa gene. A recent FMRFa-eGFP genetic screen identified several genes involved in Tv4 specification, two of which encode components of the U5 subunit of the spliceosome: brr2 (l(3)72Ab) and Prp8. This study focused on the role of RNA processing during target-derived signaling. brr2 and Prp8 were found to play crucial roles in controlling the expression of the FMRFa neuropeptide specifically in six neurons of the VNC (Tv4 neurons). Detailed analysis of brr2 revealed that this control is executed by two independent mechanisms, both of which are required for the activation of the BMP retrograde signaling pathway in Tv4 neurons: (1) Proper axonal pathfinding to the target tissue in order to receive the BMP ligand. (2) Proper RNA splicing of two genes in the BMP pathway: the thickveins (tkv) gene, encoding a BMP receptor subunit, and the Medea gene, encoding a co-Smad. These results reveal involvement of specific RNA processing in diversifying neuronal identity within the central nervous system.
Burow, D. A., Martin, S., Quail, J. F., Alhusaini, N., Coller, J. and Cleary, M. D. (2018). Attenuated codon optimality contributes to neural-specific mRNA decay in Drosophila. Cell Rep 24(7): 1704-1712. PubMed ID: 30110627
Summary:
Tissue-specific mRNA stability is important for cell fate and physiology, but the mechanisms involved are not fully understood. This study found that zygotic mRNA stability in Drosophila correlates with codon content: optimal codons are enriched in stable transcripts associated with metabolic functions like translation, while non-optimal codons are enriched in unstable transcripts, including those associated with neural development. Bioinformatic analyses and reporter assays revealed that similar codons stabilize or destabilize mRNAs in the nervous system and other tissues, but the link between codon content and stability is attenuated in the nervous system. This study confirmed that optimal codons are decoded by abundant tRNAs while non-optimal codons are decoded by less abundant tRNAs in embryos and in the nervous system. It is concluded that codon optimality is a general determinant of zygotic mRNA stability, and attenuation of codon optimality allows trans-acting factors to exert greater influence over mRNA decay in the nervous system.
Dong, H., Zhu, M., Meng, L., Ding, Y., Yang, D., Zhang, S., Qiang, W., Fisher, D. W. and Xu, E. Y. (2018). Pumilio2 regulates synaptic plasticity via translational repression of synaptic receptors in mice. Oncotarget 9(63): 32134-32148. PubMed ID: 30181804
Summary:
PUMILIO 2 (PUM2) is a member of Pumilio and FBF (PUF) family, an RNA binding protein family with phylogenetically conserved roles in germ cell development. The Drosophila Pumilio homolog is also required for dendrite morphogenesis and synaptic function via translational control of synaptic proteins, such as glutamate receptors. Now PUM2 influences synaptic function in vivo and, subsequently, seizures is not known. This study found that PUM2 is highly expressed in the brain, especially in the temporal lobe, and knockout of Pum2 (Pum2(-/-) ) resulted in significantly increased pyramidal cell dendrite spine and synapse density. In addition, multiple proteins associated with excitatory synaptic function, including glutamate receptor 2 (GLUR2), are up-regulated in Pum2(-/-) mice. The expression of GLUR2 protein but not mRNA is increased in the Pum2(-/-) mutant hippocampus, Glur2 transcripts are increased in mutant polysome fractions, and overexpression of PUM2 led to repression of reporter expression containing the 3'Untranslated Region (3'UTR) of Glur2, suggesting translation of GLUR2 was increased in the absence of Pum2. Overall, these studies provide a molecular mechanism for the increased temporal lobe excitability observed with PUM2 loss and suggest PUM2 might contribute to intractable temporal lobe epilepsy.
Volin, M., Zohar-Fux, M., Gonen, O., Porat-Kuperstein, L. and Toledano, H. (2018). microRNAs selectively protect hub cells of the germline stem cell niche from apoptosis. J Cell Biol. PubMed ID: 30093492
Summary:
Genotoxic stress such as irradiation causes a temporary halt in tissue regeneration. The ability to regain regeneration depends on the type of cells that survived the assault. Previous studies showed that this propensity is usually held by the tissue-specific stem cells. However, stem cells cannot maintain their unique properties without the support of their surrounding niche cells. This study shows that exposure of Drosophila melanogaster to extremely high levels of irradiation temporarily arrests spermatogenesis and kills half of the stem cells. In marked contrast, the hub cells that constitute a major component of the niche remain completely intact. It was further shown that this atypical resistance to cell death relies on the expression of certain antiapoptotic microRNAs (miRNAs) that are selectively expressed in the hub and keep the cells inert to apoptotic stress signals. It is proposed that at the tissue level, protection of a specific group of niche cells from apoptosis underlies ongoing stem cell turnover and tissue regeneration.
Pai, A. A., Paggi, J. M., Yan, P., Adelman, K. and Burge, C. B. (2018). Numerous recursive sites contribute to accuracy of splicing in long introns in flies. PLoS Genet 14(8): e1007588. PubMed ID: 30148878
Summary:
Recursive splicing, a process by which a single intron is removed from pre-mRNA transcripts in multiple distinct segments, has been observed in a small subset of Drosophila melanogaster introns. However, detection of recursive splicing requires observation of splicing intermediates that are inherently unstable, making it difficult to study. This study developed new computational approaches to identify recursively spliced introns and applied them, in combination with existing methods, to nascent RNA sequencing data from Drosophila S2 cells. These approaches identified hundreds of novel sites of recursive splicing, expanding the catalog of recursively spliced fly introns by 4-fold. A subset of recursive sites were validated by RT-PCR and sequencing. Recursive sites occur in most very long (> 40 kb) fly introns, including many genes involved in morphogenesis and development, and tend to occur near the midpoints of introns. Suggesting a possible function for recursive splicing, it was observed that fly introns with recursive sites are spliced more accurately than comparably sized non-recursive introns.
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