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


Monday, October 31st, 2016

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Gunnar, E., Bivik, C., Starkenberg, A. and Thor, S. (2016). sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system. Development [Epub ahead of print]. PubMed ID: 27578794
Neural progenitors typically divide asymmetrically to renew themselves, while producing daughters with more limited potential. In the Drosophila embryonic ventral nerve cord, neuroblasts initially produce daughters that divide once to generate two neurons/glia (type I proliferation mode). Subsequently, many neuroblasts switch to generating daughters that differentiate directly (type 0). This programmed type I>0 switch is controlled by Notch signaling, triggered at a distinct point of lineage progression in each neuroblast. However, how Notch signaling onset is gated was unclear. Sequoia (Seq), a C2H2 zinc finger transcription factor with homology to Drosophila Tramtrack and the positive regulatory domain (PRDM) family, has been identified as important for lineage progression. This study found that seq mutants fail to execute the type I>0 daughter proliferation switch, and also display increased neuroblast proliferation. Genetic interaction studies reveal that seq interacts with the Notch pathway, and seq furthermore affects expression of a Notch pathway reporter. These findings suggest that seq may act as a context-dependent regulator of Notch signaling, and underscore the growing connection between Seq, Ttk, the PRDM family and Notch signaling.
Liu, Y., Du, S., Lv, L., Lei, B., Shi, W., Tang, Y., Wang, L. and Zhong, Y. (2016). Hippocampal activation of Rac1 regulates the forgetting of object recognition memory. Curr Biol 26: 2351-2357. PubMed ID: 27593377
Evolutionary Homolog Study:
Forgetting is a universal feature for most types of memories. The best-defined and extensively characterized behaviors that depict forgetting are natural memory decay and interference-based forgetting. In Drosophila, training-induced activation of the small G protein Rac1 mediates natural memory decay and interference-based forgetting of aversive conditioning memory. In mice, the activation of photoactivable-Rac1 in recently potentiated spines in a motor learning task erases the motor memory. These lines of evidence prompted an investigation of the role for Rac1 in time-based natural memory decay and interference-based forgetting in mice. The inhibition of Rac1 activity in hippocampal neurons through targeted expression of a dominant-negative Rac1 form extended object recognition memory from less than 72 hr to over 72 hr, whereas Rac1 activation accelerated memory decay within 24 hr. Interference-induced forgetting of this memory was correlated with Rac1 activation and was completely blocked by inhibition of Rac1 activity. Electrophysiological recordings of long-term potentiation provided independent evidence that further supported a role for Rac1 activation in forgetting. Thus, Rac1-dependent forgetting is evolutionarily conserved from invertebrates to vertebrates.
Newquist, G., Novenschi, A., Kohler, D. and Mathew, D. (2016). Differential contributions of olfactory receptor neurons in a Drosophila olfactory circuit. eNeuro 3. PubMed ID: 27570823
The ability of an animal to detect, discriminate, and respond to odors depends on the functions of its olfactory receptor neurons (ORNs). The extent to which each ORN, upon activation, contributes to chemotaxis is not well understood. It was hypothesized that strong activation of each ORN elicits a different behavioral response in the Drosophila melanogaster larva by differentially affecting the composition of its navigational behavior. To test this hypothesis, Drosophila larvae were exposed to specific odorants to analyze the effect of individual ORN activity on chemotaxis. Two different behavioral paradigms were used to analyze the chemotaxis response of larvae to odorants. When tested with five different odorants that elicit strong physiological responses from single ORNs, larval behavioral responses toward each odorant differed in the strength of attraction as well as in the composition of discrete navigational elements, such as runs and turns. Further, behavioral responses to odorants did not correlate with either the strength of odor gradients tested or the sensitivity of each ORN to its cognate odorant. Finally, evidence is provided that wild-type larvae with all ORNs intact exhibit higher behavioral variance than mutant larvae that have only a single pair of functional ORNs. It is concluded that individual ORNs contribute differently to the olfactory circuit that instructs chemotactic responses. The results, along with recent studies from other groups, suggest that ORNs are functionally nonequivalent units. These results have implications for understanding peripheral odor coding.
Rezaval, C., Pattnaik, S., Pavlou, H. J., Nojima, T., Bruggemeier, B., D'Souza, L. A., Dweck, H. K. and Goodwin, S. F. (2016). Activation of latent courtship circuitry in the brain of Drosophila females induces male-like behaviors. Curr Biol 26: 2508-2515. PubMed ID: 27568592
Courtship in Drosophila melanogaster offers a powerful experimental paradigm for the study of innate sexually dimorphic behaviors. Fruit fly males exhibit an elaborate courtship display toward a potential mate. Females never actively court males, but their response to the male's display determines whether mating will actually occur. Sex-specific behaviors are hardwired into the nervous system via the actions of the sex determination genes doublesex (dsx) and fruitless (fru). Activation of male-specific dsx/fru+ P1 neurons in the brain initiates the male's courtship display, suggesting that neurons unique to males trigger this sex-specific behavior. In females, dsx+ neurons play a pivotal role in sexual receptivity and post-mating behaviors. This study manipulated the function of dsx+ neurons in the female brain to investigate higher-order neurons that drive female behaviors. Surprisingly, it was found that activation of female dsx+ neurons in the brain induces females to behave like males by promoting male-typical courtship behaviors. Activated females display courtship toward conspecific males or females, as well other Drosophila species. Specific dsx+ neurons critical for driving male courtship were uncovered and pheromones were identified that trigger such behaviors in activated females. While male courtship behavior was thought to arise from male-specific central neurons, this study shows that the female brain is equipped with latent courtship circuitry capable of inducing this male-specific behavioral program.

Sunday, October 30th

Katzenberger, R. J., Ganetzky, B. and Wassarman, D. A. (2016). Age and diet affect genetically separable secondary injuries that cause acute mortality following traumatic brain injury in Drosophila. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27754853
Outcomes of traumatic brain injury (TBI) vary because of differences in primary and secondary injuries. Primary injuries occur at the time of a traumatic event, whereas secondary injuries occur later as a result of cellular and molecular events activated in the brain and other tissues by primary injuries. This study used a Drosophila melanogaster TBI model to investigate secondary injuries that cause acute mortality. By analyzing percent mortality within 24 hours of primary injuries, it was previously found that age at the time of primary injuries and diet afterward affect the severity of secondary injuries. This study shows that secondary injuries peaked in activity 1-8 hours after primary injuries. Additionally, it was demonstrated that age and diet activated distinct secondary injuries in a genotype-specific manner and that concurrent activation of age- and diet-regulated secondary injuries synergistically increased mortality. To identify genes involved in secondary injuries that cause mortality, genome-wide mRNA expression profiles were compared of uninjured and injured flies under age and diet conditions that had different mortalities. During the peak period of secondary injuries, innate immune response genes were the predominant class of genes that changed expression. Furthermore, age and diet affected the magnitude of the change in expression of some innate immune response genes, suggesting roles for these genes in inhibiting secondary injuries that cause mortality. These results indicate that the complexity of TBI outcomes is due in part to distinct, genetically controlled, age- and diet-regulated mechanisms that promote secondary injuries and that involve a subset of innate immune response genes.
Nystrand, M., Cassidy, E. J. and Dowling, D. K. (2016). Transgenerational plasticity following a dual pathogen and stress challenge in fruit flies. BMC Evol Biol 16: 171. PubMed ID: 27567640
Phenotypic plasticity operates across generations, when the parental environment affects phenotypic expression in the offspring. Understanding whether this plasticity is adaptive requires a factorial design in which both mothers and their offspring are subjected to either the pathogen challenge or a control, in experimentally matched and mismatched combinations. Most prior studies exploring the capacity for pathogen-mediated transgenerational plasticity have, however, failed to adopt such a design. Furthermore, it is currently poorly understood whether the magnitude or direction of pathogen-mediated transgenerational responses will be sensitive to environmental heterogeneity. This study explored the transgenerational consequences of a dual pathogen and stress challenge administered in the maternal generation in the fruit fly, Drosophila melanogaster. Prospective mothers were assigned to a non-infectious pathogen treatment consisting of an injection with heat-killed bacteria or a procedural control, and a stress treatment consisting of sleep deprivation or control. Their daughters and sons were similarly assigned to the same pathogen treatment, prior to measurement of their reproductive success. Transgenerational interactions involving pathogen treatments of mothers and their offspring were observed, on the reproductive success of daughters but not sons. These interactions were unaffected by sleep deprivation. It is concluded the the direction of the transgenerational effects was not consistent with that predicted under a scenario of adaptive transgenerational plasticity. Instead, they were indicative of expectations based on terminal investment, in which an individual perceiving an imminent threat to survival should reallocate their resources to current reproduction at the expense of somatic maintenance and survival
Neyen, C., Runchel, C., Schupfer, F., Meier, P. and Lemaitre, B. (2016). The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors. Nat Immunol [Epub ahead of print]. PubMed ID: 27548432
The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. This study describes rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. It is proposed that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat.
Purice, M. D., Speese, S. D. and Logan, M. A. (2016). Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity. Nat Commun 7: 12871. PubMed ID: 27647497
Advanced age is the greatest risk factor for neurodegenerative disorders, but the mechanisms that render the senescent brain vulnerable to disease are unclear. Glial immune responses provide neuroprotection in a variety of contexts. Thus, this study explored how glial responses to neurodegeneration are altered with age. Glia-axon phagocytic interactions were shown to change dramatically in the aged Drosophila brain. Aged glia clear degenerating axons slowly due to low phosphoinositide-3-kinase (PI3K) signalling and, subsequently, reduced expression of the conserved phagocytic receptor Draper/MEGF10. Importantly, boosting PI3K/Draper activity in aged glia significantly reverses slow phagocytic responses. Moreover, several hours post axotomy, early hallmarks of Wallerian degeneration (WD) are delayed in aged flies. It is proposed that slow clearance of degenerating axons is mechanistically twofold, resulting from deferred initiation of axonal WD and reduced PI3K/Draper-dependent glial phagocytic function. Interventions that boost glial engulfment activity, however, can substantially reverse delayed clearance of damaged neuronal debris.

Morris, O., Liu, X., Domingues, C., Runchel, C., Chai, A., Basith, S., Tenev, T., Chen, H., Choi, S., Pennetta, G., Buchon, N. and Meier, P. (2016). Signal integration by the IkappaB protein Pickle shapes Drosophila innate host defense. Cell Host Microbe 20: 283-295. PubMed ID: 27631699
Pattern recognition receptors are activated following infection and trigger transcriptional programs important for host defense. Tight regulation of NF-κB activation is critical to avoid detrimental and misbalanced responses. This study describes Pickle (CG5118), a Drosophila nuclear IκB that integrates signaling inputs from both the Imd and Toll pathways by skewing the transcriptional output of the NF-κB dimer repertoire. Pickle interacts with the NF-kappaB protein Relish and the histone deacetylase dHDAC1, selectively repressing Relish homodimers while leaving other NF-κB dimer combinations unscathed. Pickle's ability to selectively inhibit Relish homodimer activity contributes to proper host immunity and organismal health. Although loss of pickle results in hyper-induction of Relish target genes and improved host resistance to pathogenic bacteria in the short term, chronic inactivation of pickle causes loss of immune tolerance and shortened lifespan. Pickle therefore allows balanced immune responses that protect from pathogenic microbes while permitting the establishment of beneficial commensal host-microbe relationships.
Sekihara, S., Shibata, T., Hyakkendani, M. and Kawabata, S. I. (2016). RNA interference directed against the Transglutaminase gene triggers dysbiosis of gut microbiota in Drosophila. J Biol Chem [Epub ahead of print]. PubMed ID: 27760824
Transglutaminase (TG) suppresses immune deficiency pathway-controlled antimicrobial peptides (IMD-AMPs), thereby conferring immune tolerance to gut microbes, and RNAi of the TG gene in flies has been shown to decrease the lifespan compared with non-TG-RNAi flies. In this study, analysis of the bacterial composition of the Drosophila gut by next-generation sequencing revealed that gut microbiota comprising one dominant genus of Acetobacter in non-TG-RNAi flies was shifted to that comprising two dominant genera of Acetobacter and Providencia in TG-RNAi flies. Four bacterial strains, including Acetobacter persici SK1 and Acetobacter indonesiensis SK2, Lactobacillus pentosus SK3, and Providencia rettgeri SK4, were isolated from the midgut of TG-RNAi flies. SK1 exhibited the highest resistance to the IMD-AMPs Cecropin A1 and Diptericin among the isolated bacteria. In contrast, SK4 exhibited considerably lower resistance against Cecropin A1, whereas SK4 exhibited high resistance to hypochlorous acid. The resistance of strains SK1-4 against IMD-AMPs in in vitro assays could not explain the shift of the microbiota in the gut of TG-RNAi flies. The lifespan was reduced in gnotobiotic flies that ingested both SK4 and SK1, concomitant with the production of reactive oxygen species and apoptosis in the midgut, whereas survival rate was not altered in gnotobiotic flies that mono-ingested either SK4 or SK1.

Saturday, October 29th

Tauscher, P. M., Gui, J. and Shimmi, O. (2016). Adaptive protein divergence of BMP ligands takes place under developmental and evolutionary constraints. Development [Epub ahead of print]. PubMed ID: 27578781
The bone morphogenetic protein (BMP) signaling network, comprising evolutionary conserved BMP2/4/Decapentaplegic (Dpp) and Chordin/Short gastrulation (Sog), is widely utilized for dorsal-ventral (DV) patterning during animal development. A similar network is required for posterior crossvein (PCV) formation in the Drosophila pupal wing. Although both transcriptional and post-transcriptional regulation of co-factors in the network appears to give rise to tissue-specific and species-specific properties, their mechanisms are incompletely understood. In Drosophila, BMP5-8 type ligands, Screw (Scw) and /aGlass bottom boat (Gbb), form heterodimers with Dpp for DV patterning and PCV development, respectively. Sequence analysis indicates that the Scw ligand contains two N-glycosylation motifs; one being highly conserved between BMP2/4 and BMP5-8 type ligands, and the other being Scw ligand-specific. The data reveal that N-glycosylation of the Scw ligand boosts BMP signaling both in cell culture and in the embryo. In contrast, N-glycosylation modifications of Gbb or Scw ligands reduce the consistency of PCV development. These results suggest that tolerance for structural changes of BMP5-8 type ligands is dependent on developmental constraints. Furthermore, gain and loss of N-glycosylation motifs in conserved signaling molecules under evolutionary constraints appear to constitute flexible modules to adapt to developmental processes.
Porcelli, D., Westram, A. M., Pascual, M., Gaston, K. J., Butlin, R. K. and Snook, R. R. (2016). Gene expression clines reveal local adaptation and associated trade-offs at a continental scale. Sci Rep 6: 32975. PubMed ID: 27599812
Local adaptation, where fitness in one environment comes at a cost in another, should lead to spatial variation in trade-offs between life history traits and may be critical for population persistence. Recent studies have sought genomic signals of local adaptation, but often have been limited to laboratory populations representing two environmentally different locations of a species' distribution. This study measured gene expression, as a proxy for fitness, in males of Drosophila subobscura, occupying a 20 degrees latitudinal and 11 ° C thermal range. Uniquely, six populations were sampled, and both common garden and semi-natural responses to identify signals of local adaptation were identified. Contrasting patterns of investment were found: transcripts with expression positively correlated to latitude were enriched for metabolic processes, expressed across all tissues whereas negatively correlated transcripts were enriched for reproductive processes, expressed primarily in testes. When using only the end populations, to compare the results to previous studies, it was found that locally adaptive patterns were obscured. While phenotypic trade-offs between metabolic and reproductive functions across widespread species are well-known, the results identify underlying genetic and tissue responses at a continental scale that may be responsible for this. This may contribute to understanding population persistence under environmental change.
Niwa, H., Nakamura, A., Urata, M., Shirae-Kurabayashi, M., Kuraku, S., Russell, S. and Ohtsuka, S. (2016). The evolutionally-conserved function of group B1 Sox family members confers the unique role of Sox2 in mouse ES cells. BMC Evol Biol 16: 173. PubMed ID: 27582319
Evolutionary Homolog Study:
In mouse ES cells, the function of Sox2 is essential for the maintenance of pluripotency. Since the Sox-family of transcription factors are well conserved in the animal kingdom, addressing the evolutionary origin of Sox2 function in pluripotent stem cells is intriguing from the perspective of understanding the origin of pluripotency. This question was approached using a functional complementation assay in inducible Sox2-null ES cells. Assaying mouse Sox proteins from different Groups, it was found that only Group B1 and Group G proteins were able to support pluripotency. Interestingly, invertebrate homologs of mammalian Group B1 Sox proteins were able to replace the pluripotency-associated function of mouse Sox2. Moreover, the mouse ES cells rescued by the Drosophila SoxNeuro protein are able to contribute to chimeric embryos. These data indicate that the function of mouse Sox2 supporting pluripotency is based on an evolutionally conserved activity of the Group B1 Sox family. Since pluripotent stem cell population in developmental process could be regarded as the evolutional novelty in vertebrates, it could be regarded as a co-optional use of their evolutionally conserved function.
Shindey, R., Varma, V., Nikhil, K. L. and Sharma, V. K. (2016). Evolution of robust circadian clocks in Drosophila melanogaster populations reared in constant dark for over 330 generations. Naturwissenschaften 103: 74. PubMed ID: 27585442
Robustness, the ability to maintain stable biological phenotypes across environments, is thought to be of adaptive value. Previous studies have reported higher intrinsic activity levels and power of rhythm in Drosophila populations (stocks) reared in constant darkness (DD stocks) as compared to those reared in constant light (LL stocks) and 12:12-h light-dark cycles (LD stocks) for over 19 years (approximately 330 generations). The current study intended to examine whether the enhanced levels of activity observed in DD stocks persist under various environments such as photoperiods, ambient temperatures, non-24-h light-dark (LD) cycles, and semi-natural conditions (SN). DD stocks largely retain their phenotype of enhanced activity levels across most of the above-mentioned environments suggesting the evolution of robust circadian clocks in DD stocks. Furthermore, the change in peak activity levels upon entrainment was not significantly different across the three stocks for any of the examined environmental conditions. This suggests that the enhancement of activity levels in DD stocks is not due to differential sensitivity to environment. Thus, these results suggest that rearing in constant darkness (DD) leads to evolution of robust circadian clocks suggesting a possible adaptive value of possessing such rhythms under constant dark environments.

Okada, H., Ebhardt, H. A., Vonesch, S. C., Aebersold, R. and Hafen, E. (2016). Proteome-wide association studies identify biochemical modules associated with a wing-size phenotype in Drosophila melanogaster. Nat Commun 7: 12649. PubMed ID: 27582081
The manner by which genetic diversity within a population generates individual phenotypes is a fundamental question of biology. To advance the understanding of the genotype-phenotype relationships towards the level of biochemical processes, a proteome-wide association study (PWAS) was performed of a complex quantitative phenotype. The variation of wing imaginal disc proteomes was quantified in Drosophila genetic reference panel (DGRP) lines using SWATH mass spectrometry. In spite of the very large genetic variation (1/36 bp) between the lines, proteome variability is surprisingly small, indicating strong molecular resilience of protein expression patterns. Proteins associated with adult wing size form tight co-variation clusters that are enriched in fundamental biochemical processes. Wing size correlates with some basic metabolic functions, positively with glucose metabolism but negatively with mitochondrial respiration and not with ribosome biogenesis. This study highlights the power of PWAS to filter functional variants from the large genetic variability in natural populations.
Crowley-Gall, A., Date, P., Han, C., Rhodes, N., Andolfatto, P., Layne, J. E. and Rollmann, S. M. (2016). Population differences in olfaction accompany host shift in Drosophila mojavensis. Proc Biol Sci 283. PubMed ID: 27581882
Evolutionary shifts in plant-herbivore interactions provide a model for understanding the link among the evolution of behaviour, ecological specialization and incipient speciation. Drosophila mojavensis uses different host cacti across its range, and volatile chemicals emitted by the host are the primary cue for host plant identification. This study shows that changes in host plant use between distinct D. mojavensis populations are accompanied by changes in the olfactory system. Specifically, differences were observed in olfactory receptor neuron specificity and sensitivity, as well as changes in sensillar subtype abundance, between populations. Additionally, RNA-seq analyses reveal differential gene expression between populations for members of the odorant receptor gene family. Hence, alterations in host preference are associated with changes in development, regulation and function at the olfactory periphery.

Friday October 28th

Fried, P., Sanchez-Aragon, M., Aguilar-Hidalgo, D., Lehtinen, B., Casares, F. and Iber, D. (2016). A model of the spatio-temporal dynamics of Drosophila eye disc development. PLoS Comput Biol 12: e1005052. PubMed ID: 27626238
Patterning and growth are linked during early development and have to be tightly controlled to result in a functional tissue or organ. During the development of the Drosophila eye, this linkage is particularly clear: the growth of the eye primordium mainly results from proliferating cells ahead of the morphogenetic furrow (MF), a moving signaling wave that sweeps across the tissue from the posterior to the anterior side, that induces proliferating cells anterior to it to differentiate and become cell cycle quiescent in its wake. Therefore, final eye disc size depends on the proliferation rate of undifferentiated cells and on the speed with which the MF sweeps across the eye disc. A spatio-temporal model of the growing eye disc was developed in this study based on the regulatory interactions controlled by the signals Decapentaplegic (Dpp), Hedgehog (Hh) and the transcription factor Homothorax (Hth) and how the signaling patterns affect the movement of the MF and impact on eye disc growth was explored. Published and new quantitative data were used to parameterize the model. In particular, two crucial parameter values, the degradation rate of Hth and the diffusion coefficient of Hh, were measured. The model is able to reproduce the linear movement of the MF and the termination of growth of the primordium. It was further shown that the model can explain several mutant phenotypes, but fails to reproduce the previously observed scaling of the Dpp gradient in the anterior compartment (Fried, 2016).
Vega-Macaya, F., Manieu, C., Valdivia, M., Mlodzik, M. and Olguin, P. (2016). Establishment of the muscle-tendon junction during thorax morphogenesis in Drosophila requires the Rho-kinase. Genetics [Epub ahead of print]. PubMed ID: 27585845
The assembly of the musculoskeletal system in Drosophila relies on the integration of chemical and mechanical signaling between the developing muscles with ectodermal cells specialized as "tendon cells". Mechanical tension generated at the junction of flight muscles and tendon cells of the notum epithelium is required for muscle morphogenesis and is balanced by the epithelium in order to not deform. Drosophila Rho kinase (Drok) is necessary in tendon cells to assemble stable myotendinous junctions (MTJ), which are required for muscle morphogenesis and survival. In addition, Drok is required in tendon cells to maintain epithelial shape and cell orientation in the notum, independently of chascon (chas). Loss of Drok function in tendon cells results in miss-orientation of tendon cell extensions and abnormal accumulation of Thrombospondin and βPS-integrin, which may cause abnormal myotendinous junction formation and muscle morphogenesis. This role does not depend exclusively on non-muscular Myosin-II activation (Myo-II), indicating that other DRok targets are key in this process. It is proposed that DRok function in tendon cells is key to promote the establishment of MTJ attachment and to balance mechanical tension generated at the MTJ by muscle compaction.
Green, N., Odell, N., Zych, M., Clark, C., Wang, Z. H., Biersmith, B., Bajzek, C., Cook, K. R., Dushay, M. S. and Geisbrecht, E. (2016). A common suite of coagulation proteins function in Drosophila muscle attachment. Genetics [Epub ahead of print]. PubMed ID: 27585844
The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. The surprising discovery was made that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster. One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes thrombospondin (Tsp) and tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. It was further demonstrated that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, it is proposed that shared extracellular protein-protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment.
Hayashi, S., Manabe, I., Suzuki, Y., Relaix, F. and Oishi, Y. (2016). Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice. Elife [Epub ahead of print]. PubMed ID: 27743478
Evolutionary Homolog Study:
Kruppel-like factor 5 (Klf5) (see Drosophila dar1) is a zinc-finger transcription factor that controls various biological processes, including cell proliferation and differentiation. This study shows that Klf5 is also an essential mediator of skeletal muscle regeneration and myogenic differentiation (see myogenesis in Drosophila). During muscle regeneration after injury (cardiotoxin injection), Klf5 is induced in the nuclei of differentiating myoblasts and newly formed myofibers expressing myogenin in vivo. Satellite cell-specific Klf5 deletion severely impairs muscle regeneration, and myotube formation is suppressed in Klf5-deleted cultured C2C12 myoblasts and satellite cells. Klf5 knockdown suppresses induction of muscle differentiation-related genes, including myogenin. Klf5 ChIP-seq revealed that Klf5 binding overlaps that of MyoD (see Drosophila nau) and Mef2 (see Drosophila Mef2), and Klf5 physically associates with both MyoD and Mef2. In addition, MyoD recruitment is greatly reduced in the absence of Klf5. These results indicate that Klf5 is an essential regulator of skeletal muscle differentiation, acting in concert with myogenic transcription factors such as MyoD and Mef2.

Thursday, October 27th

Guo, Y., Flegel, K., Kumar, J., McKay, D.J. and Buttitta, L.A. (2016). Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells. Biol Open [Epub ahead of print]. PubMed ID: 27737823
During development cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. This study examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. A cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa. In the wing, ecdysone signaling at the larva to puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva to puparium pulse during early metamorphosis, Broad expression plummets allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing.

Bourouh, M., Dhaliwal, R., Rana, K., Sinha, S., Guo, Z. and Swan, A. (2016). Distinct and overlapping requirements for Cyclins A, B and B3 in Drosophila female meiosis. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27652889
Meiosis, like mitosis depends on the activity of the mitotic Cyclin dependent kinase, Cdk1 and its cyclin partners. This study examined the specific requirements for the three mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3 in meiosis of Drosophila melanogaster. All three cyclins were found to contribute redundantly to nuclear envelope breakdown, though Cyclin A appears to make the most important individual contribution. Cyclin A is also required for bi-orientation of homologues in meiosis I. Cyclin B3, as previously reported, is required for anaphase progression in meiosis I and in meiosis II. Cyclin B3 also plays a redundant role, with Cyclin A, in preventing DNA replication during meiosis. Cyclin B is required for maintenance of the metaphase I arrest in mature oocytes, for spindle organization and for timely progression through the 2nd meiotic division. It is also essential for polar body formation at the completion of meiosis. With the exception of its redundant role in meiotic maturation, Cyclin B appears to function independently of Cyclins A and B3 through most of meiosis. The study concludes that the 3 mitotic Cyclin-Cdk complexes have distinct and overlapping functions in Drosophila female meiosis.

Ye, A. A., Torabi, J. and Maresca, T. J. (2016). Aurora A kinase amplifies a midzone phosphorylation gradient to promote high-fidelity cytokinesis. Biol Bull 231: 61-72. PubMed ID: 27638695
During cytokinesis, aurora B kinase (ABK) relocalizes from centromeres to the spindle midzone, where it is thought to provide a spatial cue for cytokinesis. While global ABK inhibition in Drosophila S2 cells results in macro- and multi-nucleated large cells, mislocalization of midzone ABK (mABK) by depletion of Subito (Drosophila MKLP2) does not cause notable cytokinesis defects. Subito depletion was, therefore, used to investigate the contribution of other molecules to cytokinesis in the absence of mABK. Inhibiting potential polar relaxation pathways via removal of centrosomes (CNN RNAi) or a kinetochore-based phosphatase gradient (Sds22 RNAi) did not result in cytokinesis defects on their own or in combination with loss of mABK. Disruption of aurora A kinase (AAK) activity resulted in midzone assembly defects, but did not significantly affect contractile ring positioning or cytokinesis. Live-cell imaging of an aurora kinase phosphorylation sensor revealed that midzone substrates were less phosphorylated in AAK-inhibited cells, despite the fact that midzone levels of active phosphorylated ABK (pABK) were normal. The data suggest that equatorial stimulation rather than polar relaxation mechanisms is the major determinant of contractile ring positioning and high-fidelity cytokinesis in Drosophila S2 cells. Furthermore, it is proposed that equatorial stimulation is mediated primarily by the delivery of factors to the cortex by noncentrosomal microtubules (MTs), as well as a midzone-derived phosphorylation gradient that is amplified by the concerted activities of mABK and a soluble pool of AAK.
Stubenvoll, M. D., Medley, J. C., Irwin, M. and Song, M. H. (2016). ATX-2, the C. elegans ortholog of human Ataxin-2, regulates centrosome size and microtubule dynamics. PLoS Genet 12: e1006370. PubMed ID: 27689799
Evolutionary Homolog Study
Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT) to form mitotic bipolar spindles that ensure proper chromosome segregation. This study reports a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2 (see Drosophila Ataxin-2), in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, γ-Tubulin), increasing MT nucleating activity but impeding MT growth. ATX-2 influences MT behavior through γ-Tubulin (see Drosophila γ-Tubulin) at the centrosome. These data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics.

Wednesday, October 26th

Lin, C. C., Riabinina, O. and Potter, C. J. (2016). Olfactory behaviors assayed by computer tracking of Drosophila in a four-quadrant olfactometer. J Vis Exp [Epub ahead of print]. PubMed ID: 27585032
A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory). With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. This paper presents a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.
Branco, A.T., Schilling, L., Silkaitis, K., Dowling, D.K. and Lemos, B. (2016). Reproductive activity triggers accelerated male mortality and decreases lifespan: genetic and gene expression determinants in Drosophila. Heredity (Edinb) [Epub ahead of print]. PubMed ID: 27731328
Reproduction and aging evolved to be intimately associated. Experimental selection for early-life reproduction drives the evolution of decreased longevity in Drosophila whereas experimental selection for increased longevity leads to changes in reproduction. Although life history theory offers hypotheses to explain these relationships, the genetic architecture and molecular mechanisms underlying reproduction-longevity associations remain a matter of debate. This study shows that mating triggers accelerated mortality in males and identifies hundreds of genes that are modulated upon mating in the fruit fly Drosophila melanogaster. Interrogation of genome-wide gene expression in virgin and recently mated males revealed coherent responses, with biological processes that are upregulated (testis-specific gene expression) or downregulated (metabolism and mitochondria-related functions) upon mating. Furthermore, using a panel of genotypes from the Drosophila Synthetic Population Resource (DSPR) as a source of naturally occurring genetic perturbation, abundant variation in longevity and reproduction-induced mortality among genotypes was uncovered. Genotypes display more than fourfold variation in longevity and reproduction-induced mortality that can be traced to variation in specific segments of the genome. The data reveal individual variation in sensitivity to reproduction and physiological processes that are enhanced and suppressed upon mating. These results raise the prospect that variation in longevity and age-related traits could be traced to processes that coordinate germline and somatic function.

Schlichting, M., Menegazzi, P., Lelito, K. R., Yao, Z., Buhl, E., Dalla Benetta, E., Bahle, A., Denike, J., Hodge, J. J., Helfrich-Forster, C. and Shafer, O. T. (2016). A neural network underlying circadian entrainment and photoperiodic adjustment of sleep and activity in Drosophila. J Neurosci 36: 9084-9096. PubMed ID: 27581451
A sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. This study shows that the Hofbauer-Buchner eyelets, located between the retina and the medulla in the fly optic lobes, differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, it was shown that the large LNvs interact with subsets of "evening cells" to adjust the timing of the evening peak of activity in a day length-dependent manner. This work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms.
Caballero, J., Mazo, C., Rodriguez-Pinto, I. and Theobald, J. C. (2015). A visual horizon affects steering responses during flight in fruit flies. J Exp Biol 218: 2942-2950. PubMed ID: 26232414
To navigate well through three-dimensional environments, animals must in some way gauge the distances to objects and features around them. Humans use a variety of visual cues to do this, but insects, with their small size and rigid eyes, are constrained to a more limited range of possible depth cues. For example, insects attend to relative image motion when they move, but cannot change the optical power of their eyes to estimate distance. On clear days, the horizon is one of the most salient visual features in nature, offering clues about orientation, altitude and, for humans, distance to objects. This study set out to determine whether flying fruit flies treat moving features as farther off when they are near the horizon. Tethered flies respond strongly to moving images they perceive as close. The strength of steering responses was measured while independently varying the elevation of moving stimuli and the elevation of a virtual horizon. Responses to vertical bars were found to be increased by negative elevations of their bases relative to the horizon, closely correlated with the inverse of apparent distance. In other words, a bar that dips far below the horizon elicits a strong response, consistent with using the horizon as a depth cue. Wide-field motion also had an enhanced effect below the horizon, but this was only prevalent when flies were additionally motivated with hunger. These responses may help flies tune behaviors to nearby objects and features when they are too far off for motion parallax.

Koenig, S., Wolf, R. and Heisenberg, M. (2016). Visual attention in flies-dopamine in the mushroom bodies mediates the after-effect of cueing. PLoS One 11: e0161412. PubMed ID: 27571359
Visual environments may simultaneously comprise stimuli of different significance. Often such stimuli require incompatible responses. Selective visual attention allows an animal to respond exclusively to the stimuli at a certain location in the visual field. In the process of establishing its focus of attention the animal can be influenced by external cues. This study characterized the behavioral properties and neural mechanism of cueing in the fly Drosophila melanogaster. A cue can be attractive, repulsive or ineffective depending upon (e.g.) its visual properties and location in the visual field. Dopamine signaling in the brain is required to maintain the effect of cueing once the cue has disappeared. Raising or lowering dopamine at the synapse abolishes this after-effect. Specifically, dopamine is necessary and sufficient in the αβ-lobes of the mushroom bodies. Evidence is provided for an involvement of the αβposterior Kenyon cells.
Getahun, M. N., Thoma, M., Lavista-Llanos, S., Keesey, I., Fandino, R. A., Knaden, M., Wicher, D., Olsson, S. B. and Hansson, B. S. (2016). Intracellular regulation of the insect chemoreceptor complex impacts odor localization in flying insects. J Exp Biol [Epub ahead of print]. PubMed ID: 27591307
Flying insects are well-known for airborne odor tracking, and evolved diverse chemoreceptors. While ionotropic receptors (IRs) are found across Protostomes, insect odorant receptors (ORs) have only been identified in winged insects. It was therefore hypothesized that the unique signal transduction of ORs offers an advantage for odor localization in flight. Using Drosophila, expression and increased activity of the intracellular signaling protein, PKC, in was found antennal sensilla following odor stimulation. Odor stimulation also enhances phosphorylation of the OR coreceptor, Orco, in vitro, while site directed mutation of Orco or mutations in PKC subtypes reduces sensitivity and dynamic ranges of OR-expressing neurons in vivo, but not IRs. It was ultimately shown that these mutations reduce competence for odor localization of flies in flight. We conclude that intracellular regulation of OR sensitivity is necessary for efficient odor localization, which suggests a mechanistic advantage for the evolution of the OR complex in flying insects.

Tuesday, October 25th

Zhou, Q., DeSantis, D. F., Friedrich, M. and Pignoni, F. (2016). Shared and distinct mechanisms of atonal regulation in Drosophila ocelli and compound eyes. Dev Biol 418: 10-16. PubMed ID: 27565023
The fruit fly Drosophila melanogaster has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli. Here we show that a 2kb genomic DNA fragment contains distinct and common regulatory elements necessary for ato induction in compound eyes and ocelli. The three binding sites that mediate direct regulation by the RD factors Sine oculis and Eyeless in the compound eye are also required in the ocelli. However, in the latter, these sites mediate control by Sine oculis and the other Pax6 factor of Drosophila, Twin of eyeless, which can bind the Pax6 sites in vitro. Moreover, the three sites are differentially utilized in the ocelli: all three are similarly essential for atonal induction in the posterior ocelli, but show considerable redundancy in the anterior ocellus. Strikingly, this difference parallels the distinct control of ato transcription in the posterior and anterior progenitors of the developing compound eyes. From a comparative perspective, these findings suggest that the ocelli of arthropods may have originated through spatial partitioning from the dorsal edge of an ancestral compound eye.
Merenciano, M., Ullastres, A., de Cara, M. A., Barron, M. G. and Gonzalez, J. (2016). Multiple independent retroelement insertions in the promoter of a stress response gene have variable molecular and functional effects in Drosophila. PLoS Genet 12: e1006249. PubMed ID: 27517860
Transposable elements significantly contribute to promote diversity. This work analyzed in detail one of the transposable element insertions, named FBti0019985, that has been co-opted to drive expression of CG18446, a candidate stress response gene. Strains from different natural populations were analyzed and it was found that besides FBti0019985, there are another eight independent transposable elements inserted in the proximal promoter region of CG18446. All nine insertions are solo-LTRs that belong to the roo family. The sequence of the nine roo insertions was examined, and whether the different insertions were functionally equivalent was examined by performing 5'-RACE, gene expression, and cold-stress survival experiments. Different insertions were found to have different molecular and functional consequences. The exact position where the transposable elements are inserted matters, as they all showed highly conserved sequences but only two of the analyzed insertions provided alternative transcription start sites, and only the FBti0019985 insertion consistently affects CG18446 expression. The phenotypic consequences of the different insertions also vary: only FBti0019985 was associated with cold-stress tolerance. Interestingly, the only previous report of transposable elements inserting repeatedly and independently in a promoter region in D. melanogaster, were also located upstream of a stress response gene. These results suggest that functional validation of individual structural variants is needed to resolve the complexity of insertion clusters.
Li, H., Qi, Y. and Jasper, H. (2016). Ubx dynamically regulates Dpp signaling by repressing Dad expression during copper cell regeneration in the adult Drosophila midgut. Dev Biol. PubMed ID: 27570230
The gastrointestinal (GI) tract of metazoans is lined by a series of regionally distinct epithelia. To maintain structure and function of the GI tract, regionally diversified differentiation of somatic stem cell (SC) lineages is critical. The adult Drosophila midgut provides an accessible model to study SC regulation and specification in a regionally defined manner. SCs of the posterior midgut (PM) have been studied extensively, but the control of SCs in the middle midgut (MM) is less well understood. The MM contains a stomach-like copper cell region (CCR) that is regenerated by gastric stem cells (GSSCs) and contains acid-secreting copper cells (CCs). Bmp-like Decapentaplegic (Dpp) signaling determines the identity of GSSCs, and is required for CC regeneration, yet the precise control of Dpp signaling activity in this lineage remains to be fully established. This study shows that Dad, a negative feedback regulator of Dpp signaling, is dynamically regulated in the GSSC lineage to allow CC differentiation. Dad is highly expressed in GSSCs and their first daughter cells, the gastroblasts (GBs), but has to be repressed in differentiating CCs to allow Dpp-mediated differentiation into CCs. WThe Hox gene Ultrabithorax (Ubx) is required for this regulation. Loss of Ubx prevents Dad repression in the CCR, resulting in defective CC regeneration. This study highlights the need for dynamic control of Dpp signaling activity in the differentiation of the GSSC lineage and identifies Ubx as a critical regulator of this process.
Miki, T.S., Carl, S.H., Stadler, M.B. and Großhans, H. (2016). XRN2 autoregulation and control of polycistronic gene expression in Caenorhabditis elegans. PLoS Genet 12: e1006313. PubMed ID: 27631780
Evolutionary Homolog Study
XRN2 (see Drosophila Rat1) is a conserved 5'→3' exoribonuclease that complexes with proteins that contain XRN2-binding domains (XTBDs). In Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 (see Drosophila CG31301) stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) (see Drosophila CG7789)  through hydrolysis of an endogenous XRN inhibitor 3'-phosphoadenosine-5'-phosphate (PAP). Through unbiased screening, this study found that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. De-repression appears to be triggered such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Indeed, two distinct XRN2 repression mechanisms are alleviated at different thresholds of XRN2 inactivation. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and additional operons under its control were identified, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but a part of the mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons. 

Monday, October 24th

Rao, K., Stone, M. C., Weiner, A. T., Gheres, K. W., Zhou, C., Deitcher, D. L., Levitan, E. S. and Rolls, M. M. (2016). Spastin, atlastin and ER relocalization are involved in axon, but not dendrite, regeneration. Mol Biol Cell. PubMed ID: 27605706
Mutations in over 50 genes including spastin and atlastin lead to Hereditary Spastic Paraplegia (HSP). It was previously demonstrated that reduction of spastin leads to a deficit in axon regeneration in a Drosophila model. Axon regeneration was similarly impaired in neurons when HSP proteins atlastin, seipin and spichthyin were reduced. Impaired regeneration was dependent on genetic background, and was observed when partial reduction of HSP proteins was combined with expression of dominant-negative microtubule regulators, suggesting HSP proteins work with microtubules to promote regeneration. Microtubule rearrangements triggered by axon injury were, however, normal in all genotypes. Other markers were examined to identify additional changes associated with regeneration. While mitochondria, endosomes and ribosomes did not exhibit dramatic repatterning during regeneration, the endoplasmic reticulum (ER) was frequently concentrated near the tip of the growing axon. In atlastin RNAi and spastin mutant animals, ER accumulation near single growing axon tips was impaired. ER tip concentration was only observed during axon regeneration, and not during dendrite regeneration. In addition, dendrite regeneration was unaffected by reduction of spastin or atlastin. It is proposed that the HSP proteins Spastin and Atlastin promote axon regeneration by coordinating concentration of the ER and microtubules at the growing axon tip.
Martin, F. and Alcorta, E. (2016). Measuring activity in olfactory receptor neurons in Drosophila: Focus on spike amplitude. J Insect Physiol 95: 23-41. PubMed ID: 27614176
Olfactory responses at the receptor level have been thoroughly described in Drosophila melanogaster by electrophysiological methods. Single sensilla recordings (SSRs) measure neuronal activity in intact individuals in response to odors. For sensilla that contain more than one olfactory receptor neuron (ORN), their different spontaneous spike amplitudes can distinguish each signal under resting conditions. However, activity is mainly described by spike frequency. Some reports on ORN response dynamics studied two components in the olfactory responses of ORNs: a fast component that is reflected by the spike frequency and a slow component that is observed in the LFP (local field potential, the single sensillum counterpart of the electroantennogram, EAG). However, no apparent correlation was found between the two elements. This report shows that odorant stimulation produces two different effects in the fast component, affecting spike frequency and spike amplitude. Spike amplitude clearly diminishes at the beginning of a response, but it recovers more slowly than spike frequency after stimulus cessation, suggesting that ORNs return to resting conditions long after they recover a normal spontaneous spike frequency. Moreover, spike amplitude recovery follows the same kinetics as the slow voltage component measured by the LFP, suggesting that both measures are connected. These results were obtained in ab2 and ab3 sensilla in response to two odors at different concentrations. Both spike amplitude and LFP kinetics depend on odorant, concentration and neuron, suggesting that like the EAG they may reflect olfactory information.
Pitts, S., Pelser, E., Meeks, J. and Smith, D. (2016). Odorant responses and courtship behaviors influenced by at4 neurons in Drosophila. PLoS One 11: e0162761. PubMed ID: 27617442
In insects, pheromones function as triggers to elicit complex behavior programs, such as courtship and mating behavior. In most species, the neurons tuned to pheromones are localized in a specific subset of olfactory sensilla located on the antenna called trichoid sensilla. In Drosophila there are two classes of trichoid sensilla, at1 sensilla that contain the dendrites of a single neuron that is specifically tuned to the male-specific pheromone 11-cis vaccenyl acetate (cVA), and at4 sensilla that contain three neurons with relatively poorly defined chemical specificity and function. Using a combination of odorant receptor mutant analysis, single sensillum electrophysiology and optogenetics, this study examined the chemical tuning and behavioral consequences of the three at4 olfactory neuron classes. The results indicate that one class, Or65abc neurons, are unresponsive to cVA pheromone, and function to inhibit courtship behaviors in response to an unknown ligand, while the other two neuron classes, Or88a and Or47b neurons, are sensitive to a diverse array of fly and non-fly odors, and activation of these neurons has little direct impact on courtship behaviors.
Li, Z., Zhang, T., Lin, Z., Hou, C., Zhang, J., Men, Y., Li, H. and Gao, J. (2016). Lgl1 is required for olfaction and development of olfactory bulb in mice. PLoS One 11: e0162126. PubMed ID: 27603780
Evolutionary Homolog Study:
Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and functioned as a key regulator of epithelial polarity and asymmetric cell division. This study generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in olfactory bulb (OB). Next, the effects were examined of Lgl1 loss in the OB. First, the expression patterns of Lgl1 in the neurogenic regions of the embryonic dorsal region of the LGE (dLGE) and postnatal OB were determined. Furthermore, the Lgl1 conditional mutants exhibited abnormal morphological characteristics of the OB. Behavioral analysis exhibited greatly impaired olfaction in Lgl1 mutant mice. To elucidate the possible mechanisms of impaired olfaction in Lgl1 mutant mice, the development of the OB was investigated. Interestingly, reduced thickness of the mitral cell layer and decreased density of mitral cells (MCs) were observed in Lgl1 mutant mice. Additionally, a dramatic loss in SP8+ interneurons (e.g. calretinin and GABAergic/non-dopaminergic interneurons) was observed in the glomerular layer of the olfactory bulb. The results demonstrate that Lgl1 is required for the development of the olfactory bulb and the deletion of Lgl1 results in impaired olfaction in mice.

Sunday, October 23rd

Michel, M., Aliee, M., Rudolf, K., Bialas, L., Julicher, F. and Dahmann, C. (2016). The selector gene apterous and Notch are required to locally increase mechanical cell bond tension at the Drosophila dorsoventral compartment boundary. PLoS One 11: e0161668. PubMed ID: 27552097
In Drosophila, the wing imaginal disc is subdivided into a dorsal and a ventral compartment. Cells of the dorsal, but not ventral, compartment express the selector gene apterous. Apterous expression sets in motion a gene regulatory cascade that leads to the activation of Notch signaling in a few cell rows on either side of the dorsoventral compartment boundary. Both Notch and apterous mutant clones disturb the separation of dorsal and ventral cells. Maintenance of the straight shape of the dorsoventral boundary involves a local increase in mechanical tension at cell bonds along the boundary. The mechanisms by which cell bond tension is locally increased however remain unknown. This study used a combination of laser ablation of cell bonds, quantitative image analysis, and genetic mutants to show that Notch and Apterous are required to increase cell bond tension along the dorsoventral compartment boundary. Moreover, clonal expression of the Apterous target gene capricious results in cell separation and increased cell bond tension at the clone borders. Finally, using a vertex model to simulate tissue growth, an increase in cell bond tension at the borders of cell clones, but not throughout the cell clone, was found to lead to cell separation. It is concluded that Apterous and Notch maintain the characteristic straight shape of the dorsoventral compartment boundary by locally increasing cell bond tension.
Satoh, T., Nakamura, Y. and Satoh, A. K. (2016). The roles of Syx5 in Golgi morphology and rhodopsin transport in Drosophila photoreceptors. Biol Open [Epub ahead of print]. PubMed ID: 27591190
SNAREs (SNAP receptors) are the key components of protein complexes that drive membrane fusion. This study reports the function of a SNARE, Syntaxin 5 (Syx5), in the development of photoreceptors in Drosophila In wild type photoreceptors, Syx5 localizes to cis-Golgi, along with cis-Golgi markers, Rab1, and GM130. It was observed that Syx5-deficient photoreceptors shows notable accumulation of these cis-Golgi markers accompanying drastic accumulation of vesicles between ER and Golgi cisternae. Extensive analysis of Rh1 (rhodopsin 1) trafficking revealed that in Syx5-deficient photoreceptors, Rh1 is exported from the ER with normal kinetics, retained in cis-Golgi region along with GM130 for a prolonged period, then subsequently degraded presumable by endoplasmic-reticulum-associated protein degradation (ERAD) after retrieved to the ER. Unlike a previous report of Rab6-deficient photoreceptors-where two apical transport pathways are specifically inhibited-vesicle transport pathways to all of plasma membrane domains are inhibited in Syx5-deficient photoreceptors, implying that Rab6 and Syx5 are acting in different steps of intra-Golgi transport. These results indicate that Syx5 is crucial for membrane protein transport, presumably during ER-derived vesicle fusion to form cis-Golgi cisternae.
Cordoba, S., Requena, D., Jory, A., Saiz, A. and Estella, C. (2016). The evolutionary conserved transcription factor Sp1 controls appendage growth through Notch signaling. Development 143(19):3623-3631. PubMed ID: 27578786
The appendages of arthropods and vertebrates are not homologous structures, although the underlying genetic mechanisms that pattern them are highly conserved. Members of the Sp family of transcription factors are expressed in the developing limbs and their function is required for limb growth in both insects and chordates. Despite the fundamental and conserved role that these transcription factors play during appendage development, their target genes and the mechanisms in which they participate to control limb growth are mostly unknown. This study analyzed the individual contributions of two Drosophila Sp members, buttonhead (btd) and Sp1, during leg development. Sp1 plays a more prominent role controlling leg growth than btd. A regulatory function of Sp1 in Notch signaling was identified, and a genome wide transcriptome analysis was performed to identify other potential Sp1 target genes contributing to leg growth. The data suggest a mechanism by which the Sp factors control appendage growth through the Notch signaling.
Bi, P., Yue, F., Sato, Y., Wirbisky, S., Liu, W., Shan, T., Wen, Y., Zhou, D., Freeman, J. and Kuang, S. (2016). Stage-specific effects of Notch activation during skeletal myogenesis. Elife 5. PubMed ID: 27644105
Evolutionary Homolog Study
Skeletal myogenesis involves sequential activation, proliferation, self-renewal/differentiation and fusion of myogenic stem cells (satellite cells). Notch signaling is known to be essential for the maintenance of satellite cells, but its function in late-stage myogenesis, i.e. post-differentiation myocytes and post-fusion myotubes, is unknown. Using stage-specific Cre alleles, distinct roles were found of Notch1 in mononucleated myocytes and multinucleated myotubes. Specifically, constitutive Notch1 activation dedifferentiates myocytes into Pax7+ quiescent satellite cells, leading to severe defects in muscle growth and regeneration, and postnatal lethality. By contrast, myotube-specific Notch1 activation improves the regeneration and exercise performance of aged and dystrophic muscles. Mechanistically, Notch1 activation in myotubes upregulates the expression of Notch ligands, which modulate Notch signaling in the adjacent satellite cells to enhance their regenerative capacity. These results highlight context-dependent effects of Notch activation during myogenesis, and demonstrate that Notch1 activity improves myotube's function as a stem cell niche.

Saturday, October 22nd

Maartens, A.P., Wellmann, J., Wictome, E., Klapholz, B., Green, H. and Brown, N.H. (2016). Drosophila vinculin is more harmful when hyperactive than absent, and can circumvent integrin to form adhesion complexes. J Cell Sci [Epub ahead of print]. PubMed ID: 27737911
Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. This study examines how altering vinculin activity perturbs integrin function within the context of Drosophila development. Whereas loss of vinculin produces relatively minor phenotypes, gain of vinculin activity, via a loss of head-tail autoinhibition, causes lethality. The minimal domain capable of inducing lethality is the talin-binding D1 domain, and this appears to require talin-binding activity, as lethality is suppressed by competition with single vinculin binding sites from talin. Activated Drosophila vinculin triggers the formation of cytoplasmic adhesion complexes via the rod of talin, but independently of integrin. These complexes contain a subset of adhesion proteins but no longer link the membrane to actin. The negative effects of hyperactive vinculin are segregated into morphogenetic defects caused by the whole head domain, and lethality caused by the D1 domain. These findings demonstrate the critical importance of the tight control of vinculin's activity.

Chuang, M., Hsiao, T.I., Tong, A., Xu, S. and Chisholm, A.D. (2016). DAPK interacts with Patronin and the microtubule cytoskeleton in epidermal development and wound repair. Elife [Epub ahead of print]. PubMed ID: 27661253
Evolutionary Homology Study:
Epidermal barrier epithelia form a first line of defense against the environment, protecting animals against infection and repairing physical damage. In C. elegans, death-associated protein kinase (DAPK-1) (see Drosophila Drak, which promotes phosphorylation of Spaghetti squash at sites known to stimulate actomyosin contractility) regulates epidermal morphogenesis, innate immunity and wound repair. This study found that DAPK-1 maintains epidermal tissue integrity through regulation of the microtubule (MT) cytoskeleton. dapk-1 epidermal phenotypes are suppressed by treatment with microtubule-destabilizing drugs and mimicked or enhanced by microtubule-stabilizing drugs. Loss of function in ptrn-1 (see Drosophila Patronin), the C. elegans member of the Patronin/Nezha/CAMSAP family of MT minus-end binding proteins, suppresses dapk-1 epidermal and innate immunity phenotypes. Over-expression of the MT-binding CKK domain of PTRN-1 triggers epidermal and immunity defects resembling those of dapk-1 mutants, and PTRN-1 localization is regulated by DAPK-1. DAPK-1 and PTRN-1 physically interact in co-immunoprecipitation experiments, and DAPK-1 itself undergoes MT-dependent transport. These data uncover an unexpected interdependence of DAPK-1 and the microtubule cytoskeleton in maintenance of epidermal integrity.

Huelsmann, S., Rintanen, N., Sethi, R., Brown, N. H. and Ylanne, J. (2016). Evidence for the mechanosensor function of filamin in tissue development. Sci Rep 6: 32798. PubMed ID: 27597179
Cells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. This study tested how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, these results show that an appropriate level of mechanical sensitivity is required for filamin's function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates filamin binding to cellular components.
Shwartz, A., Dhanyasi, N., Schejter, E.D. and Shilo, B.Z. (2016). The Drosophila formin Fhos is a primary mediator of sarcomeric thin-filament array assembly. Elife 5. PubMed ID: 27731794
Actin-based thin filament arrays constitute a fundamental core component of muscle sarcomeres. This study used formation of the Drosophila indirect flight musculature for studying the assembly and maturation of thin-filament arrays in a skeletal muscle model system. Employing GFP-tagged actin monomer incorporation, several distinct phases in the dynamic construction of thin-filament arrays were identified. This sequence includes assembly of nascent arrays after an initial period of intensive microfilament synthesis, followed by array elongation, primarily from filament pointed-ends, radial growth of the arrays via recruitment of peripheral filaments and continuous barbed-end turnover. Using genetic approaches, the single Drosophila homolog of the FHOD sub-family of formins, Fhos, was identified as a primary and versatile mediator of IFM thin-filament organization. Localization of Fhos to the barbed-ends of the arrays, achieved via a novel N-terminal domain, appears to be a critical aspect of its sarcomeric roles.

Friday, October 22

Carvajal-Gonzalez, J.M., Mulero-Navarro, S., Smith, M. and Mlodzik, M. (2016). A novel Frizzled-based screening tool identifies genetic modifiers of planar cell polarity in Drosophila wings. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27729438
Most mutant alleles in the Fz-PCP pathway genes have been discovered in classic Drosophila screens looking for recessive loss-of-function mutations. Nonetheless, although Fz-PCP signaling is sensitive to increased doses of PCP gene products, not many screens have been performed in the wing under genetically engineered Fz over-expression conditions, mostly because the Fz phenotypes are strong and/or not easy to score and quantify. This study presents a screen based on an unexpected mild Frizzled gain-of-function phenotype. The leakiness of a chimeric Frizzled protein designed to be accumulated in the endoplasmic reticulum generates a reproducible Frizzled gain-of-function phenotype in Drosophila wings. Using this genotype, a genome-wide collection of large deficiencies was screened and 16 strongly interacting genomic regions were found. 7 of these regions were narrowed down to finally 116 candidate genes. Using this approach, 8 new loci, with a potential function in the PCP context, were identified. Further, krasavietz and its interactor short-stop were identified and confirmed as new genes acting during planar cell polarity establishment with a function related to actin and microtubules dynamics.

Redhai, S., et al. (2016). Regulation of dense-core granule replenishment by autocrine BMP signalling in Drosophila secondary cells. PLoS Genet 12: e1006366. PubMed ID: 27727275
Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although how many secretagogues stimulate DCG release is understood, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. This study demonstrates that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, this study shows that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. This analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.
Ding, L., Lei, Y., Han, Y., Li, Y., Ji, X. and Liu, L. (2016). Vimar is a novel regulator of mitochondrial fission through Miro. PLoS Genet 12: e1006359. PubMed ID: 27716788
As fundamental processes in mitochondrial dynamics, mitochondrial fusion, fission and transport are regulated by several core components, including Miro. As an atypical Rho-like small GTPase with high molecular mass, the exchange of GDP/GTP in Miro may require assistance from a guanine nucleotide exchange factor (GEF). However, the GEF for Miro has not been identified. While studying mitochondrial morphology in Drosophila, it was incidentally observed that the loss of vimar, a gene encoding an atypical GEF, enhanced mitochondrial fission under normal physiological conditions. Because Vimar could co-immunoprecipitate with Miro in vitro, it was speculated that Vimar might be the GEF of Miro. In support of this hypothesis, a loss-of-function (LOF) vimar mutant rescued mitochondrial enlargement induced by a gain-of-function (GOF) Miro transgene; whereas a GOF vimar transgene enhanced Miro function. In addition, vimar lost its effect under the expression of a constitutively GTP-bound or GDP-bound Miro mutant background. These results indicate a genetic dependence of vimar on Miro. Moreover, mitochondrial fission was found to play a functional role in high-calcium induced necrosis, and a LOF vimar mutant rescued the mitochondrial fission defect and cell death. This result can also be explained by vimar's function through Miro, because Miro's effect on mitochondrial morphology is altered upon binding with calcium. In addition, a PINK1 mutant, which induced mitochondrial enlargement and had been considered as a Drosophila model of Parkinson's disease (PD), caused fly muscle defects, and the loss of vimar could rescue these defects. Furthermore, it was found that the mammalian homolog of Vimar, RAP1GDS1, played a similar role in regulating mitochondrial morphology, suggesting a functional conservation of this GEF member. The Miro/Vimar complex may be a promising drug target for diseases in which mitochondrial fission and fusion are dysfunctional.
Portegijs, V., et al. (2016). Multisite phosphorylation of NuMA-related LIN-5 controls mitotic spindle positioning in C. elegans. PLoS Genet 12: e1006291. PubMed ID: 27711157
Evolutionary Homolog Study
During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein (see Drosophila Dynein) by a conserved ternary complex of Gα (see Drosophila G-iα65A), GPR-1/2 (see Drosophila Pins), and LIN-5 (see Drosophila Mushroom body defect) proteins in C. elegans (Galpha-LGN-NuMA in mammals). Previous studies showed that the polarity kinase PKC-3 (see Drosophila aPKC) phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. This study investigated whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which was identified by reverse two-hybrid screening. Evidence is provided for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5-GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B (see Drosophila CyclinB) in vitro. This study applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and evidence is provided for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. These data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation.

Thursday October 20th

Ma, X., Han, Y., Song, X., Do, T., Yang, Z., Ni, J and Xie, T. (2016). DNA damage-induced CHK2 activation compromises germline stem cell self-renewal and lineage differentiation. Development [Epub ahead of print]. PubMed ID: 27729408
This study used germline stem cells (GSCs) in the Drosophila ovary to show that DNA damage retards stem cell self-renewal and lineage differentiation in a CHK2 kinase-dependent manner. Both heatshock-inducible endonuclease I-CreI expression and X-ray irradiation can efficiently introduce double-strand breaks in GSCs and their progeny, resulting in a rapid GSC loss and an accumulation of ill-differentiated GSC progeny. Elimination of CHK2 or its kinase activity can almost fully rescue the GSC loss and the progeny differentiation defect caused by DNA damage induced by I-CreI or X-ray. Surprisingly, checkpoint kinases ATM and ATR have distinct functions from CHK2 in GSCs in response to DNA damage. The reduction in BMP signaling and E-cadherin only makes limited contribution to DNA damage-induced GSC loss. Finally, DNA damage also decreases the expression of the master differentiation factor Bam in a CHK2-dependent manner, which helps explain the GSC progeny differentiation defect. Therefore, this study demonstrates, for the first time in vivo, that CHK2 kinase activation is required for the DNA damage-mediated disruption of adult stem cell self-renewal and lineage differentiation, and might also offer novel insight into how DNA damage causes tissue aging and cancer formation. It also demonstrates that inducible I-CreI is a convenient genetic system for studying DNA damage responses in stem cells.

McClenahan, F.K., Sharma, H., Shan, X., Eyermann, C. and Colognato, H. (2016). Dystroglycan suppresses Notch to regulate stem cell niche structure and function in the developing postnatal subventricular zone. Dev Cell [Epub ahead of print]. PubMed ID: 27569418Evolutionary Homolog Study
While the extracellular matrix (ECM) is known to regulate neural stem cell quiescence (see Drosophila neuroblast) in the adult subventricular zone (SVZ), the function of ECM in the developing SVZ remains unknown. This study reports that the ECM receptor dystroglycan (see Drosophila Dystroglycan) regulates a unique developmental restructuring of ECM in the early postnatal SVZ. Dystroglycan is furthermore required for ependymal cell differentiation and assembly of niche pinwheel structures, at least in part by suppressing Notch (see Drosophila Notch) activation in radial glial cells, which leads to the increased expression of MCI (see Drosophila geminin), Myb (see Drosophila Myb), and FoxJ1 (see Drosophila CG32006), transcriptional regulators necessary for acquisition of the multiciliated phenotype. Dystroglycan also regulates perinatal radial glial cell proliferation and transition into intermediate gliogenic progenitors, such that either acute or constitutive loss of function in dystroglycan results in increased oligodendrogenesis. These findings reveal a role for dystroglycan in orchestrating both the assembly and function of the SVZ neural stem cell niche.

Amoyel, M., Hillion, K. H., Margolis, S. R. and Bach, E. A. (2016). Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues. Development [Epub ahead of print]. PubMed ID: 27633989
Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on losing access to these signals. This study demonstrates that differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot properly differentiate. An insulin peptide produced by somatic cells immediately outside of the stem cell niche was found to act locally to promote somatic differentiation through Insulin receptor (InR) activation. These results indicate that there is a local 'differentiation' niche which upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, it was demonstrated that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, this study shows that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that local production of positive and negative InR signals regulate the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation is actively induced by signaling.

Singh, S.R., Zeng, X., Zhao, J., Liu, Y., Hou, G., Liu, H. and Hou, S.X. (2016). The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila. Nature [Epub ahead of print]. PubMed ID: 27680705
Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. This study investigated stem-cell death in the digestive system of adult Drosophila melanogaster. It was found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively kills normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spares differentiated cells. The dying stem cells are engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduce CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers.

Wednesday, October 19th

Wakabayashi, S., Sawamura, N., Voelzmann, A., Broemer, M., Asahi, T. and Hoch, M. (2016). Ohgata, the single Drosophila ortholog of human Cereblon, regulates insulin signaling-dependent organismic growth. J Biol Chem [Epub ahead of print]. PubMed ID: 27702999
Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that is highly conserved in animals and plants. CRBN proteins have been implicated in various biological processes such as development, metabolism, learning and memory formation and their impairment has been linked to autosomal recessive non-syndromic intellectual disability and cancer. Furthermore, human CRBN has been identified as the primary target of thalidomide teratogenicity. Data on functional analysis of CRBN family members in vivo is, however, still scarce. This study identified Ohgata (OHGT), the Drosophila ortholog of CRBN as regulator of insulin signaling-mediated growth. Using ohgt mutants generated by targeted mutagenesis, it was shown that its loss results in increased body weight and organ size without changes of the body proportions. Ohgt knockdown in the fat body, an organ analogous to mammalian liver and adipose tissue, phenocopies the growth phenotypes. Overgrowth is due to an elevation of insulin signaling in ohgt mutants and to the downregulation of inhibitory cofactors of circulating Drosophila Insulin-like Peptides (Dilps), named Acid Labile Subunit (ALS) and Imaginal morphogenesis protein-Late 2 (Imp-L2). The two inhibitory proteins have been previously shown to be components of a heterotrimeric complex with growth promoting Dilp2 and Dilp5. This study reveals OHGT as a novel regulator of insulin-dependent organismic growth in Drosophila.

Hong, C.J. and Hamilton, B.A. (2016). Zfp423 regulates Sonic Hedgehog signaling via primary cilium function. PLoS Genet 12: e1006357. PubMed ID: 27727273
Evolutionary Homolog Study:
Zfp423 (see Drosophila Oaz) encodes a 30-zinc finger transcription factor that intersects several canonical signaling pathways. Zfp423 mutations result in ciliopathy-related phenotypes, including agenesis of the cerebellar vermis in mice and Joubert syndrome (JBTS19) and nephronophthisis (NPHP14) in humans. Unlike most ciliopathy genes, Zfp423 encodes a nuclear protein and its developmental expression is complex, leading to alternative proposals for cellular mechanisms. This study shows that Zfp423 is expressed by cerebellar granule cell precursors, that loss of Zfp423 in these precursors leads to cell-intrinsic reduction in proliferation, loss of response to Shh (see Drosophila hh), and primary cilia abnormalities that include diminished frequency of both Smoothened (see Drosophila smo) and IFT88 (see Drosophila nompB) localization. Loss of Zfp423 alters expression of several genes encoding key cilium components, including increased expression of Tulp3 (see Drosophila ktub). Tulp3 is a direct binding target of Zfp423 and reducing the overexpression of Tulp3 in Zfp423-deficient cells suppresses Smoothened translocation defects. These results define Zfp423 deficiency as a bona fide ciliopathy, acting upstream of Shh signaling, and indicate a mechanism intrinsic to granule cell precursors for the resulting cerebellar hypoplasia.

Huang, H. and Kornberg, T. B. (2016). Cells must express components of the planar cell polarity system and extracellular matrix to support cytonemes. Elife 5 [Epub ahead of print]. PubMed ID: 27591355
Development of Drosophila dorsal air sac, a tracheal tube that grows toward Branchless FGF-expressing cells in the wing imaginal disc, depends on Decapentaplegic (Dpp) and Fibroblast growth factor (FGF) proteins produced by the wing imaginal disc and transported by cytonemes to the air sac primordium (ASP). Dpp and FGF signaling in the ASP was dependent on components of the planar cell polarity (PCP) system in the disc, and neither Dpp- nor FGF-receiving cytonemes extended over mutant disc cells that lacked them. ASP cytonemes normally navigate through extracellular matrix (ECM) composed of collagen, laminin, Dally and Dally-like (Dlp) proteins that are stratified in layers over the disc cells. However, ECM over PCP mutant cells had reduced levels of laminin, Dally and Dlp, and whereas Dpp-receiving ASP cytonemes navigated in the Dally layer and required Dally (but not Dlp), FGF-receiving ASP cytonemes navigated in the Dlp layer, requiring Dlp (but not Dally). These findings suggest that cytonemes interact directly and specifically with proteins in the stratified ECM.
Zhou, W., He, Q., Zhang, C., He, X., Cui, Z., Liu, F. and Li, W. (2016). BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development. Elife [Epub ahead of print]. PubMed ID: 27719760
Evolutionary Homolog Study:
Notch signaling plays a crucial role in the control of proliferation and differentiation of stem and progenitor cells during embryogenesis or organogenesis (see embryogenesis and organogenesis in Drosophila), but its regulation is incompletely understood. BLOS2, encoded by the Bloc1s2 gene (see Drosophila Blos2), is a shared subunit of two lysosomal trafficking complexes, biogenesis of lysosome-related organelles complex-1 (BLOC-1) and BLOC-1 related complex. Bloc1s2-/- mice are embryonic lethal and exhibit defects in cortical development and hematopoiesis. Loss of BLOS2 results in elevated Notch signaling, which consequently increases the proliferation of neural progenitor cells and inhibits neuronal differentiation in cortices. Likewise, ablation of bloc1s2 in zebrafish or mice leads to increased hematopoietic stem and progenitor cell production in the aorta-gonad-mesonephros region. BLOS2 physically interacts with Notch1 in endo-lysosomal trafficking of Notch1. These findings suggest that BLOS2 is a novel negative player in regulating Notch signaling through lysosomal trafficking by controlling multiple stem and progenitor cell homeostasis in vertebrates.

Kakanj, P., Moussian, B., Grönke, S., Bustos, V., Eming, S.A., Partridge, L. and Leptin, M. (2016). Insulin and TOR signal in parallel through FOXO and S6K to promote epithelial wound healing. Nat Commun 7: 12972. PubMed ID: 27713427
The TOR and Insulin/IGF signalling (IIS) network controls growth, metabolism and ageing. Although reducing TOR or insulin signalling can be beneficial for ageing, it can be detrimental for wound healing, but the reasons for this difference are unknown. This study shows that IIS is activated in the cells surrounding an epidermal wound in Drosophila melanogaster larvae, resulting in PI3K activation and redistribution of the transcription factor FOXO. Insulin and TOR signalling are independently necessary for normal wound healing, with FOXO and S6K as their respective effectors. IIS is specifically required in cells surrounding the wound, and the effect is independent of glycogen metabolism. Insulin signalling is needed for the efficient assembly of an actomyosin cable around the wound, and constitutively active myosin II regulatory light chain suppresses the effects of reduced IIS. These findings may have implications for the role of insulin signalling and FOXO activation in diabetic wound healing.

Delanoue, R., Meschi, E., Agrawal, N., Mauri, A., Tsatskis, Y., McNeill, H. and Láopold, P. (2016). Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor. Science 353: 1553-1556. PubMed ID: 27708106
Animals adapt their growth rate and body size to available nutrients by a general modulation of insulin-insulin-like growth factor signaling. In Drosophila, dietary amino acids promote the release in the hemolymph of brain insulin-like peptides (Dilps), which in turn activate systemic organ growth. Dilp secretion by insulin-producing cells involves a relay through unknown cytokines produced by fat cells. This study identifies Methuselah (Mth) as a secretin-incretin receptor subfamily member required in the insulin-producing cells for proper nutrient coupling. Using genetic and ex vivo organ culture experiments, it was shown that the Mth ligand Stunted (Sun) is a circulating insulinotropic peptide produced by fat cells. Therefore, Sun and Mth define a new cross-organ circuitry that modulates physiological insulin levels in response to nutrients.

Tuesday, October 18th

Jovanic, T., Schneider-Mizell, C.M., Shao, M., Masson, J.B., Denisov, G., Fetter, R.D., Mensh, B.D., Truman, J.W., Cardona, A. and Zlatic, M. (2016). Competitive disinhibition mediates behavioral choice and sequences in Drosophila. Cell [Epub ahead of print]. PubMed ID: 27720450
Even a simple sensory stimulus can elicit distinct innate behaviors and sequences. During sensorimotor decisions, competitive interactions among neurons that promote distinct behaviors must ensure the selection and maintenance of one behavior, while suppressing others. The circuit implementation of these competitive interactions is still an open question. By combining comprehensive electron microscopy reconstruction of inhibitory interneuron networks, modeling, electrophysiology, and behavioral studies, this study determined the circuit mechanisms that contribute to the Drosophila larval sensorimotor decision to startle, explore, or perform a sequence of the two in response to a mechanosensory stimulus. Together, these studies reveal that, early in sensory processing, (1) reciprocally connected feedforward inhibitory interneurons implement behavioral choice, (2) local feedback disinhibition provides positive feedback that consolidates and maintains the chosen behavior, and (3) lateral disinhibition promotes sequence transitions. The combination of these interconnected circuit motifs can implement both behavior selection and the serial organization of behaviors into a sequence.
Degen, J., et al. (2016). Honeybees learn landscape features during exploratory orientation flights. Curr Biol. PubMed ID: 27693138
Exploration is an elementary and fundamental form of learning about the structure of the world. Navigating animals explore the environment for safe return to an important place (e.g., a nest site) and to travel between places. Flying central-place foragers like honeybees (Apis mellifera) extend their exploration into distances from which the features of the nest cannot be directly perceived. Bees perform short-range and long-range orientations flights. Short-range flights are performed in the immediate surroundings of the hive and occur more frequently under unfavorable weather conditions, whereas long-range flights lead the bees into different sectors of the surrounding environment. Applying harmonic radar technology for flight tracking, this study addressed the question of whether bees learn landscape features during their first short-range or long-range orientation flight. The homing flights of single bees were compared after they were displaced to areas explored or not explored during the orientation flight. Bees learn the landscape features during the first orientation flight since they returned faster and along straighter flights from explored areas as compared to unexplored areas. The study excluded a range of possible factors that might have guided bees back to the hive based on egocentric navigation strategies (path integration, beacon orientation, and pattern matching of the skyline). It is concluded that bees localize themselves according to learned ground structures and their spatial relations to the hive.
LeDue, E.E., Mann, K., Koch, E., Chu, B., Dakin, R. and Gordon, M.D. (2016). Starvation-induced depotentiation of bitter taste in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 27720624
Nutrient deprivation can lead to dramatic changes in feeding behavior, including acceptance of foods that are normally rejected. In flies, this behavioral shift depends in part on reciprocal sensitization and desensitization of sweet and bitter taste, respectively. However, the mechanisms for bitter taste modulation remain unclear. This study identified a set of brain ventrolateral octopaminergic/tyraminergic neurons, named OA-VLs, that directly modulate bitter sensory neuron output in response to starvation. OA-VLs are in close proximity to bitter sensory neuron axon terminals and show reduced tonic firing following starvation. It was found that octopamine and tyramine potentiate bitter sensory neuron responses, suggesting that starvation-induced reduction in OA-VL activity depotentiates bitter taste. Consistent with this model, artificial silencing of OA-VL activity induces a starvation-like reduction in bitter sensory neuron output. These results demonstrate that OA-VLs mediate a critical step in starvation-dependent bitter taste modulation, allowing flies to dynamically balance the risks associated with bitter food consumption against the threat of severe starvation.

Sokabe, T., Chen, H.C., Luo, J. and Montell, C. (2016). A switch in thermal preference in Drosophila larvae depends on multiple rhodopsins. Cell Rep 17: 336-344. PubMed ID: 27705783
Drosophila third-instar larvae exhibit changes in their behavioral responses to gravity and food as they transition from feeding to wandering stages. Using a thermal gradient encompassing the comfortable range (18°C to 28°C), this study found that third-instar larvae exhibit a dramatic shift in thermal preference. Early third-instar larvae prefer 24°C, which switches to increasingly stronger biases for 18°C-19°C in mid- and late-third-instar larvae. Mutations eliminating either of two rhodopsins, Rh5 and Rh6, wipe out these age-dependent changes in thermal preference. In larvae, Rh5 and Rh6 are thought to function exclusively in the light-sensing Bolwig organ. However, the Bolwig organ was found to be dispensable for the thermal preference. Rather, Rh5 and Rh6 are required in trpA1-expressing neurons in the brain, ventral nerve cord, and body wall. Because Rh1 contributes to thermal selection in the comfortable range during the early to mid-third-instar stage, fine thermal discrimination depends on multiple rhodopsins.

Monday, October 17th

Lee, S.H., Kim, Y.J. and Choi, S.Y. (2016). BMP signaling modulates the probability of neurotransmitter release and readily releasable pools in Drosophila neuromuscular junction synapses. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 27671198
The structure and function of synapses is modulated by the interaction of presynaptic and postsynaptic neurons via cell adhesion molecules or secreted signal molecules. Bone morphogenic protein (BMP) is a secreted molecule mediating retrograde signaling that is involved in the formation and maintenance of synaptic structure throughout many animal species. However, how BMP signaling modulates presynaptic neurotransmitter release is not yet clear. This study analyzed the function of BMP signaling factors in neurotransmitter release in Drosophila neuromuscular synapses using loss-of-function mutants in genes for BMP modulators, Wit, Mad, and Dad. Larvae with mutations in wit and mad commonly show a decreased synaptic bouton number in neuromuscular synapses. Larvae with dad mutations show an increased bouton number. The amplitudes of miniature EJC (mEJC) are normal for these mutants. Wit and mad mutants show decreased evoked EJC (eEJC) amplitude and increased paired pulse facilitation, implying impaired presynaptic neurotransmitter release. A reduction in readily releasable neurotransmitters pool sizes in wit and mad mutants was found. However, dad mutants show a normal probability of neurotransmitter release and readily releasable pool sizes and normal eEJC amplitude even with clear abnormalities in synaptic structure. These results suggest that BMP signaling is critical for each step of presynaptic neurotransmission. Also, that BMP signaling modulates both synaptic structure and function independently and specifically.

Sealover, N. R., Felts, B., Kuntz, C. P., Jarrard, R. E., Hockerman, G. H., Barker, E. L. and Henry, L. K. (2016). The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for MDMA translocation and the induction of substrate efflux. Biochem Pharmacol. PubMed ID: 27638414
The substituted amphetamine MDMA is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic. Previous, studies has identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. This study performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. The findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser.
Rossano, A. J., Kato, A., Minard, K. I., Romero, M. F. and Macleod, G. T. (2016). Na+ /H+ -exchange via the Drosophila vesicular glutamate transporter (DVGLUT) mediates activity-induced acid efflux from presynaptic terminals. J Physiol [Epub ahead of print]. PubMed ID: 27641622
Neuronal activity can result in transient acidification of presynaptic terminals and such shifts in cytosolic pH (pHcyto) likely influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals it was hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation this study investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically-encoded pH-indicator (GEpHI), named 'pHerry', in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity-induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H+ /Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid extruding protein when deposited on the plasma membrane.
Rodrigues, F. F., Shao, W. and Harris, T. J. (2016). The Arf GAP Asap promotes Arf1 function at the Golgi for cleavage furrow biosynthesis in Drosophila. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27535433
Biosynthetic traffic from the Golgi drives plasma membrane growth. For Drosophila embryo cleavage, this growth is rapid, but regulated, for cycles of furrow ingression and regression. The highly conserved small G protein Arf1 organizes Golgi trafficking. Arf1 is activated by guanine nucleotide exchange factors, but essential roles for Arf1 GTPase activating proteins (GAPs) are less clear. This study reports that the conserved Arf GAP Asap is required for cleavage furrow ingression in the early embryo. Since Asap can affect multiple sub-cellular processes, genetic approaches were used to dissect the primary effect of Asap. The data argue against cytoskeletal or endocytic involvement, and reveal a common role for Asap and Arf1 in Golgi organization. Although Asap lacked Golgi enrichment, it was necessary and sufficient for Arf1 accumulation at the Golgi, and a conserved Arf1-Asap binding site was required for Golgi organization and output. Notably, Asap re-localized to the nuclear region at metaphase, a shift that coincided with subtle Golgi re-organization preceding cleavage furrow regression. It is concluded that Asap is essential for Arf1 to function at the Golgi for cleavage furrow biosynthesis. Asap may recycle Arf1 to the Golgi from post-Golgi membranes, providing optimal Golgi output for specific stages of the cell cycle (Ridriguex, 2016).

Loh, K. H., Stawski, P. S., Draycott, A. S., Udeshi, N. D., Lehrman, E. K., Wilton, D. K., Svinkina, T., Deerinck, T. J., Ellisman, M. H., Stevens, B., Carr, S. A. and Ting, A. Y. (2016). Proteomic analysis of unbounded cellular compartments: Synaptic clefts. Cell 166: 1295-1307 e1221. PubMed ID: 27565350
Evolutionary Homolog Study
Cellular compartments that cannot be biochemically isolated are challenging to characterize. This study demonstrates the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, the proteomes of two of the most common excitatory and inhibitory synaptic clefts were mapped in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed identification of Mdga2 as a potential specificity factor influencing Neuroligin-2 (see Drosophila Nlg2) recruitment of presynaptic neurotransmitters at inhibitory synapses.
Zeng, M., Shang, Y., Araki, Y., Guo, T., Huganir, R. L. and Zhang, M. (2016). Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Plasticity. Cell 166: 1163-1175 e1112. PubMed ID: 27565345
Evolutionary Homolog Study
Postsynaptic densities (PSDs) are membrane semi-enclosed, submicron protein-enriched cellular compartments beneath postsynaptic membranes, which constantly exchange their components with bulk aqueous cytoplasm in synaptic spines. Formation and activity-dependent modulation of PSDs is considered as one of the most basic molecular events governing synaptic plasticity in the nervous system. This study discovered that SynGAP, one of the most abundant PSD proteins and a Ras/Rap GTPase activator, forms a homo-trimer and binds to multiple copies of PSD-95 (see Drosophila Dlg1). Binding of SynGAP to PSD-95 induces phase separation of the complex, forming highly concentrated liquid-like droplets reminiscent of the PSD. The multivalent nature of the SynGAP/PSD-95 complex is critical for the phase separation to occur and for proper activity-dependent SynGAP dispersions from the PSD. In addition to revealing a dynamic anchoring mechanism of SynGAP at the PSD, these results also suggest a model for phase-transition-mediated formation of PSD.

Sunday, October 16th

Hackett, J. L., Wang, X., Smith, B. R. and Macdonald, S. J. (2016). Mapping QTL contributing to variation in posterior lobe morphology between strains of Drosophila melanogaster. PLoS One 11: e0162573. PubMed ID: 27606594
Closely-related, and otherwise morphologically similar insect species frequently show striking divergence in the shape and/or size of male genital structures, a phenomenon thought to be driven by sexual selection. Comparative interspecific studies can help elucidate the evolutionary forces acting on genital structures to drive this rapid differentiation. However, genetic dissection of sexual trait divergence between species is frequently hampered by the difficulty generating interspecific recombinants. Intraspecific variation can be leveraged to investigate the genetics of rapidly-evolving sexual traits; this study carried out out a genetic analysis of variation in the posterior lobe within D. melanogaster. The lobe is a male-specific process emerging from the genital arch of D. melanogaster and three closely-related species, is essential for copulation, and shows radical divergence in form across species. There is also abundant variation within species in the shape and size of the lobe, and while this variation is considerably more subtle than that seen among species, it nonetheless provides the raw material for QTL mapping. An advanced intercross population was created from a pair of phenotypically-different inbred strains, and after phenotyping and genotyping-by-sequencing the recombinants, several QTL contributing to various measures of lobe morphology were mapped. The additional generations of crossing over in the mapping population led to QTL intervals that are smaller than is typical for an F2 mapping design. The intervals that were mapped overlap with a pair of lobe QTL that was previously identified in an independent mapping cross, potentially suggesting a level of shared genetic control of trait variation. The QTL additionally implicate a suite of genes that have been shown to contribute to the development of the posterior lobe. These loci are strong candidates to harbor naturally-segregating sites contributing to phenotypic variation within D. melanogaster, and may also be those contributing to divergence in lobe morphology between species.
Piontkivska, H., Matos, L. F., Paul, S., Scharfenberg, B., Farmerie, W. G., Miyamoto, M. M. and Wayne, M. L. (2016). Role of host-driven mutagenesis in determining genome evolution of sigma virus (DMelSV; Rhabdoviridae) in Drosophila melanogaster. Genome Biol Evol. PubMed ID: 27614234
Sigma virus (DMelSV) is ubiquitous in natural populations of Drosophila melanogaster Host-mediated, selective RNA editing of adenosines to inosines (ADAR) may contribute to control of viral infection by preventing transcripts from being transported into the cytoplasm or being translated accurately; or by increasing the viral genomic mutation rate. Previous PCR-based studies showed that ADAR mutations occur in DMelSV at low frequency. This study used SOLiDTM deep sequencing of flies from a single host population from Athens, GA, USA to comprehensively evaluate patterns of sequence variation in DMelSV with respect to ADAR. GA dinucleotides, which are weak targets of ADAR, are strongly overrepresented in the positive strand of the virus, consistent with selection to generate ADAR resistance on this complement of the transient, double-stranded, RNA intermediate in replication and transcription. Potential ADAR sites in a worldwide sample of viruses are more likely to be "resistant" if the sites do not vary among samples. Either variable sites are less constrained and hence are subject to weaker selection than conserved sites, or the variation is driven by ADAR. Evidence was also found of mutations segregating within hosts, hereafter referred to as hypervariable sites. Some of these sites were variable only in one or two flies (i.e. rare); others were shared by four or even all five of the flies (i.e. common). Rare and common hypervariable sites were indistinguishable with respect to susceptibility to ADAR; however, polymorphism in rare sites were more likely to be consistent with the action of ADAR than in common ones, again suggesting that ADAR is deleterious to the virus. Thus, in DMelSV, host mutagenesis is constraining viral evolution both within and between hosts.
Faria, V.G., Martins, N.E., Magalhães, S., Paulo, T.F., Nolte, V., Schlötterer, C., Sucena, É and Teixeira, L. (2016). Drosophila adaptation to viral infection through defensive symbiont evolution. PLoS Genet 12: e1006297. PubMed ID: 27684942
Microbial symbionts can modulate host interactions with biotic and abiotic factors. Such interactions may affect the evolutionary trajectories of both host and symbiont. Wolbachia protects Drosophila melanogaster against several viral infections and the strength of the protection varies between variants of this endosymbiont. Since Wolbachia is maternally transmitted, its fitness depends on the fitness of its host. Therefore, Wolbachia populations may be under selection when Drosophila is subjected to viral infection. This study shows that in D. melanogaster populations selected for increased survival upon infection with Drosophila C virus there is a strong selection coefficient for specific Wolbachia variants, leading to their fixation. Flies carrying these selected Wolbachia variants have higher survival and fertility upon viral infection when compared to flies with the other variants. These findings demonstrate how the interaction of a host with pathogens shapes the genetic composition of symbiont populations. Furthermore, host adaptation can result from the evolution of its symbionts, with host and symbiont functioning as a single evolutionary unit.

Bastide, H., Lange, J. D., Lack, J. B., Yassin, A. and Pool, J. E. (2016). A variable genetic architecture of melanic evolution in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 27638419
Unraveling the genetic architecture of adaptive phenotypic divergence is a fundamental quest in evolutionary biology. In Drosophila melanogaster, high-altitude melanism has evolved in separate mountain ranges in sub-Saharan Africa, potentially as an adaptation to UV intensity. The genetic basis of this melanism was investigated in three populations using a new bulk segregant analysis mapping method. Nineteen distinct QTL regions were identified from 9 mapping crosses, with several QTL peaks overlapping between two or all populations, and yet different crosses involving the same melanic population commonly yielded distinct QTLs. The strongest QTLs often overlapped well-known pigmentation genes, but wide signals of genetic differentiation (FST) was typically not found between lightly and darkly pigmented populations at these genes. Instead, small numbers of highly differentiated SNPs were found at the probable causative genes. A simulation analysis showed that these patterns of polymorphism were consistent with selection on standing genetic variation. Overall, these results suggest that even for potentially simpler traits like pigmentation, the complexity of adaptive trait evolution poses important challenges for QTL mapping and population genetic analysis.
Shahrestani, P., Wilson, J. B., Mueller, L. D. and Rose, M. R. (2016). Patterns of physiological decline due to age and selection in Drosophila melanogaster. Evolution [Epub ahead of print]. PubMed ID: 27624548
In outbred sexually reproducing populations, age-specific mortality rates reach a plateau in late life following the exponential increase in mortality rates that marks aging. Little is known about what happens to physiology when cohorts transition from aging to late life. Age-specific values were measured for starvation resistance, desiccation resistance, time-in-motion and geotaxis in ten Drosophila melanogaster populations: five populations selected for rapid development and five control populations. Adulthood was divided into two stages, the aging phase and the late-life phase according to demographic data. Consistent with previous studies, populations selected for rapid development entered the late-life phase at an earlier age than the controls. Age-specific rates of change for all physiological phenotypes showed differences between the aging phase and the late-life phase. This result suggests that late life is physiologically distinct from aging. The ages of transitions in physiological characteristics from aging to late life statistically match the age at which the demographic transition from aging to late life occurs, in all cases but one. These experimental results support evolutionary theories of late life that depend on patterns of decline and stabilization in the forces of natural selection.
Jungreis, I., Chan, C. S., Waterhouse, R. M., Fields, G., Lin, M. F. and Kellis, M. (2016). Evolutionary dynamics of abundant stop codon readthrough. Mol Biol Evol [Epub ahead of print]. PubMed ID: 27604222
This study leveraged comparative genomic evidence across 21 Anopheles mosquitoes to systematically annotate translational stop codon readthrough genes in the malaria vector Anopheles gambiae, and to provide the first study of abundant readthrough evolution, by comparison with 20 Drosophila species. Evolutionary signatures were identified of conserved, functional readthrough of 353 stop codons in the malaria vector, Anopheles gambiae, and of 51 additional Drosophila melanogaster stop codons. Most differences between the readthrough repertoires of the two species arose from readthrough gain or loss in existing genes, rather than birth of new genes or gene death; that readthrough-associated RNA structures are sometimes gained or lost while readthrough persists; that readthrough is more likely to be lost at TAA and TAG stop codons; and that readthrough is under continued purifying evolutionary selection in mosquito, based on population genetic evidence. Readthrough-associated gene properties were determined that predate readthrough, and differences were identified in the characteristic properties of readthrough genes between clades. More than 600 functional readthrough stop codons were identified in mosquito and 900 in fruit fly, provide evidence of readthrough control of peroxisomal targeting, and the phylogenetic extent of abundant readthrough as following divergence from centipede was refined.

Saturday, October 15th

Horvath, M., Mihajlovic, Z., Slaninova, V., Perez Gomez, R., Moshkin, Y. and Krejci, A. (2016). The silent information regulator 1 (Sirt1) is a positive regulator of the Notch pathway in Drosophila. Biochem J [Epub ahead of print]. PubMed ID: 27623778
The silent information regulator 1 (Sirt1) has previously been shown to have negative effects on the Notch pathway in several contexts. This study brings evidence that Sirt1 has a positive effect on Notch activation in Drosophila, in the context of sensory organ precursor specification and during wing development. The phenotype of Sirt1 mutant resembles weak Notch loss of function phenotypes and genetic interactions of Sirt1 with the components of the Notch pathway also suggest a positive role of Sirt1 in Notch signalling. Sirt1 is necessary for the efficient activation of E(spl) genes by Notch in S2N cells. Additionally, the Notch dependent response of several E(spl) genes is sensitive to metabolic stress caused by 2-deoxyglucose treatment, in a Sirt1 dependent manner. Sirt1 associates with several proteins involved in Notch repression as well as activation, including the cofactor exchange factor Ebi (TBL1), the RLAF/LAF histone chaperon complex and the Tip60 acetylation complex. Moreover, Sirt1 participates in the deacetylation of the CSL transcription factor Su(H). The role of Sirt1 in Notch signalling is therefore more complex than previously recognised and its diverse effects may be explained by a plethora of Sirt1 substrates involved in the regulation of Notch signalling.
Soriano, E. V., Ivanova, M. E., Fletcher, G., Riou, P., Knowles, P. P., Barnouin, K., Purkiss, A., Kostelecky, B., Saiu, P., Linch, M., Elbediwy, A., Kjaer, S., O'Reilly, N., Snijders, A. P., Parker, P. J., Thompson, B. J. and McDonald, N. Q. (2016). aPKC Inhibition by Par3 CR3 Flanking Regions Controls Substrate Access and Underpins Apical-Junctional Polarization. Dev Cell 38: 384-398. PubMed ID: 27554858
Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). This study shows that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/alphaB/alphaC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. These results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.
Nguyen, M.B., Vuong, L.T. and Choi, K.W. (2016). Ebi modulates wing growth by ubiquitin-dependent downregulation of Crumbs in Drosophila. Development 143: 3506-3513. PubMed ID: 27702784
Notch signaling at the dorsoventral (DV) boundary is essential for patterning and growth of wings in Drosophila. The WD40 domain protein Ebi has been implicated in the regulation of Notch signaling at the DV boundary. This study shows that Ebi regulates wing growth by antagonizing the function of the transmembrane protein Crumbs (Crb). Ebi physically binds to the extracellular domain of Crb (Crbext), and this interaction is specifically mediated by WD40 repeats 7-8 of Ebi and a laminin G domain of Crbext. Wing notching resulting from reduced levels of Ebi is suppressed by decreasing the Crb function. Consistent with this antagonistic genetic relationship, Ebi knockdown in the DV boundary elevates the Crb protein level. Furthermore, Ebi is required for downregulation of Crb by ubiquitylation. Taken together, the study proposes that the interplay of Crb expression in the DV boundary and ubiquitin-dependent Crb downregulation by Ebi provides a mechanism for the maintenance of Notch signaling during wing development.

Zhang, Y., Wang, X., Matakatsu, H., Fehon, R. and Blair, S.S. (2016). The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dachs. Elife 5. PubMed ID: 27692068
Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, this study performed a structure-function analysis of Dachs, and used this to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein that was named Dlish. It was found that Dlish is regulated by Fat and Dachs, that Dlish also binds Fat and the Dachs regulator Approximated, and that Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. Evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs.

Friday, October 14th

Timmons, A.K., Mondragon, A.A., Meehan, T.L. and McCall, K. (2016). Control of non-apoptotic nurse cell death by engulfment genes in Drosophila. Fly (Austin) [Epub ahead of print]. PubMed ID: 27686122
Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, fifteen germline-derived nurse cells transfer their cytoplasmic contents into the oocyte and die. Disruption of apoptosis or autophagy only partially inhibits the death of the nurse cells, indicating that other mechanisms significantly contribute to nurse cell death. It has been demonstrated that the surrounding stretch follicle cells non-autonomously promote nurse cell death during late oogenesis and that phagocytosis genes including draper, ced-12, and the JNK pathway are crucial for this process. This study shows that when phagocytosis genes are inhibited in the follicle cells, events specifically associated with death of the nurse cells are impaired. Death of the nurse cells is not completely blocked in draper mutants, suggesting that other engulfment receptors are involved. Indeed, it was found that the integrin subunit, αPS3, is enriched on stretch follicle cells during late oogenesis and is required for elimination of the nurse cells. Moreover, double mutant analysis revealed that integrins act in parallel to draper. Death of nurse cells in the Drosophila ovary is a unique example of programmed cell death that is both non-apoptotic and non-cell autonomously controlled.

Koerver, L., Melzer, J., Roca, E. A., Teichert, D., Glatter, T., Arama, E. and Broemer, M. (2016). The de-ubiquitylating enzyme DUBA is essential for spermatogenesis in Drosophila. Cell Death Differ[Epub ahead of print]. PubMed ID: 27518434
De-ubiquitylating enzymes (DUBs) reverse protein ubiquitylation and thereby control essential cellular functions. A screen for a DUB that counteracts caspase ubiquitylation to regulate cell survival identified the Drosophila ovarian tumour-type DUB DUBA (CG6091). DUBA physically interact with the initiator caspase Death regulator Nedd2-like caspase (Dronc) and de-ubiquitylates it, thereby contributing to efficient inhibitor of apoptosis-antagonist-induced apoptosis in the fly eye. Searching also for non-apoptotic functions of DUBA, Duba-null mutants were found to be male sterile and display defects in spermatid individualisation, a process that depends on non-apoptotic caspase activity. Spermatids of DUBA-deficient flies showed reduced caspase activity and lack critical structures of the individualisation process. Biochemical characterisation revealed an obligate activation step of DUBA by phosphorylation. With genetic rescue experiments it was demonstrated that DUBA phosphorylation and catalytic activity are crucial in vivo for DUBA function in spermatogenesis. These results demonstrate for the first time the importance of de-ubiquitylation for fly spermatogenesis.
Catrina, I. E., Bayer, L. V., Yanez, G., McLaughlin, J. M., Malaczek, K., Bagaeva, E., Marras, S. A. and Bratu, D. P. (2016). The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11. RNA Biol: 1-16. PubMed ID: 27654348
To achieve proper RNA transport and localization, RNA viruses exploit cellular vesicular trafficking pathways. AGFG1, a host protein essential for HIV-1 and Influenza A replication, has been shown to mediate release of intron-containing viral RNAs from the perinuclear region. It is still unknown what its precise role in this release is, or whether AGFG1 also participates in cytoplasmic transport. This study reports for the first time the expression patterns during oogenesis for the Arf GTPase activating protein Drongo, the fruit fly homolog of AGFG1. Temporally controlled Drongo expression is achieved by translational repression of drongo mRNA within P-bodies. This study shows a first link between the recycling endosome pathway and Drongo and finds that proper Drongo localization at the oocyte's cortex during mid-oogenesis requires functional Rab11.
Klein, J. D., Qu, C., Yang, X., Fan, Y., Tang, C. and Peng, J. C. (2016). c-Fos repression by Piwi regulates Drosophila ovarian germline formation and tissue morphogenesis. PLoS Genet 12: e1006281. PubMed ID: 27622269
Drosophila melanogaster Piwi functions within the germline stem cells (GSCs) and the somatic niche to regulate GSC self-renewal and differentiation. How Piwi influences GSCs is largely unknown. This study uncovered a genetic interaction between Piwi and c-Fos in the somatic niche that influences GSCs. c-Fos is a proto-oncogene that influences many cell and developmental processes. In wild-type ovarian cells, c-Fos is post-transcriptionally repressed by Piwi, which destabilized the c-Fos mRNA by promoting the processing of its 3' untranslated region (UTR) into Piwi-interacting RNAs (piRNAs). The c-Fos 3' UTR was sufficient to trigger Piwi-dependent destabilization of a GFP reporter. Piwi represses c-Fos in the somatic niche to regulate GSC maintenance and differentiation and in the somatic follicle cells to affect somatic cell disorganization, tissue dysmorphogenesis, oocyte maturation arrest, and infertility.

Thursday, October 13th

Bahrampour, S. and Thor, S. (2016). Ctr9, a key component of the Paf1 complex affects proliferation and terminal differentiation in the developing Drosophila nervous system. G3 (Bethesda). PubMed ID: 27520958
The Paf1 protein complex (Paf1C) promotes H3K4 and H3K36 trimethylation, H2BK123 ubiquitination, RNA Pol II transcriptional termination, and also RNA-mediated gene silencing. Paf1C contains five canonical protein components including Paf1 and Ctr9, that are critical for overall complex integrity, as well as , Leo1 and Cdc73/Hyrax. This study provide the first detailed phenotypic study of Ctr9 function in Drosophila. Ctr9 mutants die at late embryogenesis or early larval life, but can be partly rescued by nervous system re-expression of Ctr9. A number of phenotypes are observed in Ctr9 mutants, including increased neuroblast numbers, increased nervous system proliferation, as well as down-regulation of many neuropeptide genes. Analysis of cell cycle and regulatory gene expression reveals up-regulation of the E2f1 cell cycle factor, as well as changes in Antennapedia and Grainy head expression. Reduction of H3K4me3 modification was found in the embryonic nervous system. Genome-wide transcriptome analysis points to additional downstream genes that may underlie these Ctr9 phenotypes, revealing gene expression changes in Notch pathway target genes, cell cycle genes and neuropeptide genes. In addition, significant effects were found on the gene expression of metabolic genes. These findings reveal that Ctr9 is an essential gene that is necessary at multiple stages of nervous system development, and provides a starting point for future studies of the Paf1C in Drosophila.
Alavi, M., Song, M., King, G.L., Gillis, T., Propst, R., Lamanuzzi, M., Bousum, A., Miller, A., Allen, R. and Kidd, T. (2016). Dscam1 forms a complex with Robo1 and the N-terminal fragment of Slit to promote the growth of longitudinal axons. PLoS Biol 14: e1002560. PubMed ID: 27654876
The Slit protein is a major midline repellent for central nervous system (CNS) axons. In vivo, Slit is proteolytically cleaved into N- and C-terminal fragments, but the biological significance of this is unknown. Analysis in the Drosophila ventral nerve cord of a slit allele (slit-UC) that cannot be cleaved revealed that midline repulsion is still present but longitudinal axon guidance is disrupted, particularly across segment boundaries. Double mutants for the Slit receptors Dscam1 and robo1 strongly resemble the slit-UC phenotype, suggesting they cooperate in longitudinal axon guidance, and through biochemical approaches, it was found that Dscam1 and Robo1 form a complex dependent on Slit-N. In contrast, Robo1 binding alone shows a preference for full-length Slit, whereas Dscam1 only binds Slit-N. Using a variety of transgenes, it was demonstrated that Dscam1 appears to modify the output of Robo/Slit complexes so that signaling is no longer repulsive. These data suggest that the complex is promoting longitudinal axon growth across the segment boundary. The ability of Dscam1 to modify the output of other receptors in a ligand-dependent fashion may be a general principle for Dscam proteins.

Shin, D. H. and Hong, J. W. (2016). Transcriptional activity of the short gastrulation primary enhancer in the ventral midline requires its early activity in the presumptive neurogenic ectoderm. BMB Rep [Epub ahead of print]. PubMed ID: 27616358
The short gastrulation (sog) shadow enhancer directs early and late sog expression in the neurogenic ectoderm and the ventral midline of the developing Drosophila embryo, respectively. Evidence is presented that the sog primary enhancer also has both activities, with the late enhancer activity dependent on the early activity. Computational analyses showed that the sog primary enhancer contains five Dorsal (Dl)-, four Zelda (Zld)-, three Bicoid (Bcd)-, and no Single-minded (Sim)-binding sites. In contrast to many ventral midline enhancers, the primary enhancer can direct lacZ expression in the ventral midline as well as in the neurogenic ectoderm without a canonical Sim-binding site. Intriguingly, the impaired transcriptional synergy between Dl and either Zld or Bcd led to aberrant and abolished lacZ expression in the neurogenic ectoderm and in the ventral midline, respectively. These findings suggest that the two enhancer activities of the sog primary enhancer are functionally consolidated and geographically inseparable.
Neijts, R., Amin, S., van Rooijen, C., Tan, S., Creyghton, M.P., de Laat, W. and Deschamps, J. (2016). Polarized regulatory landscape and Wnt responsiveness underlie Hox activation in embryos. Genes Dev 30: 1937-1942. PubMed ID: 27633012
Evolutionary Homolog Study:
Sequential 3'-to-5' activation of the Hox gene clusters in early embryos is a most fascinating issue in developmental biology. Neither the trigger nor the regulatory elements involved in the transcriptional initiation of the 3'-most Hox genes have been unraveled in any organism. This study demonstrates that a series of enhancers in the mouse, some of which are Wnt-dependent (see Drosophila Wg), is located within a HoxA (see Drosophila lab) 3' subtopologically associated domain (subTAD). This subTAD forms the structural basis for multiple layers of 3'-polarized features, including DNA accessibility and enhancer activation. Deletion of the cassette of Wnt-dependent enhancers proves its crucial role in initial transcription of HoxA at the 3' side of the cluster.

Wednesday, October 12

Ricolo, D., Deligiannaki, M., Casanova, J. and Araújo, S.J. (2016). Centrosome amplification increases single-cell branching in post-mitotic cells. Curr Biol [Epub ahead of print]. PubMed ID: 27693136
Centrosome amplification is a hallmark of cancer, although how this process affects tumorigenesis is still not understood. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. This study analyzed the effects of centrosome amplification in post-mitotic cells during single-cell branching. It was shown that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. Mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. Rca1 and CycA post-mitotic cells have supernumerary centrosomes and other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. The study proposes that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects.

Urbansky, S., González Avalos, P., Wosch, M. and Lemke, S. (2016). Folded gastrulation and T48 drive the evolution of coordinated mesoderm internalization in flies. Elife 5. PubMed ID: 27685537
Gastrulation constitutes a fundamental yet diverse morphogenetic process of metazoan development. Modes of gastrulation range from stochastic translocation of individual cells to coordinated infolding of an epithelial sheet. How such morphogenetic differences are genetically encoded and whether they have provided specific developmental advantages is unclear. This study identified two genes, folded gastrulation and t48, which in the evolution of fly gastrulation acted as a likely switch from an ingression of individual cells to the invagination of the blastoderm epithelium. Both genes are expressed and required for mesoderm invagination in the fruit fly Drosophila melanogaster but do not appear during mesoderm ingression of the midge Chironomus riparius. Early expression of either or both of these genes in C.riparius is sufficient to invoke mesoderm invagination similar to D.melanogaster. The possible genetic simplicity and a measurable increase in developmental robustness might explain repeated evolution of similar transitions in animal gastrulation.

Raza, Q. and Jacobs, J. R. (2016). Guidance signalling regulates leading edge behaviour during collective cell migration of cardiac cells in Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 27618756
Collective cell migration is the coordinated movement of cells, which organize tissues during morphogenesis, repair and some cancers. The motile cell membrane of the advancing front in collective cell migration is termed the Leading Edge. The embryonic development of the vertebrate and Drosophila hearts are both characterized by the coordinated medial migration of a bilateral cluster of mesodermal cells. In Drosophila, the cardioblasts form cohesive bilateral rows that migrate collectively as a unit towards the dorsal midline to form the dorsal vessel. This study characterized the collective cell migration of cardioblasts as an in vivo quantitative model to study the behaviour of the Leading Edge. Whether guidance signalling through Slit and Netrin pathways plays a role in cell migration during heart development was investigated. Through time-lapse imaging and quantitative assessment of migratory behaviour of the cardioblasts in loss-of-function mutants, both Slit and Netrin mediated signals are autonomously and concomitantly required to maximize migration velocity, filopodial and lamellipodial activities. Additionally, another Slit and Netrin receptor, Dscam1, the role of which during heart development was previously unknown, is required for both normal migration of cardioblasts and luminal expansion. Leading edge behaviour analysis revealed a dosage dependent genetic interaction between Slit and Netrin receptors suggesting that downstream signalling through these receptors converge on a common output that increases leading edge activity of the cardioblasts. Finally, guidance signalling was found to maintain the balance between epithelial and mesenchymal characteristics of the migrating cardioblasts.
Lujan, E., Bornemann, D. J., Rottig, C., Bayless, B. A., Stocker, H., Hafen, E., Arora, K. and Warrior, R. (2016). Analysis of novel alleles of brother of tout-velu, the Drosophila ortholog of human EXTL3 using a newly developed FRT42D ovoD chromosome. Genesis [Epub ahead of print]. PubMed ID: 27636555

The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovoD dominant female-sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini-white (w+mW.hs), or rosy (ry+) and neomycin (neoR) transgenes are in common use. Parallel ovoD lines were developed using the w+mW.hs FRT insertions on the X and chromosomes 2R and 3L, as well as the ry+, neoR FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry+, neoR FRT background, are not amenable to germline clonal analysis without labor-intensive recombination onto chromosome arms containing a w+mW.hs FRT. This study reports the creation of a new ovoD line for the ry+, neoR FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent, the maternal-effect phenotypes of novel brother of tout-velu alleles generated on an FRT42D chromosome were characterized in a somatic mosaic screen. An apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages.

Tuesday, October 11th

Xu, N., Lu, X., Kavi, H., Emelyanov, A.V., Bernardo, T.J., Vershilova, E., Skoultchi, A.I. and Fyodorov, D.V. (2016). BEN domain protein Elba2 can functionally substitute for linker histone H1 in Drosophila in vivo. Sci Rep 6: 34354. PubMed ID: 27687115
Metazoan linker histones are essential for development and play crucial roles in organization of chromatin, modification of epigenetic states and regulation of genetic activity. Vertebrates express multiple linker histone H1 isoforms, which may function redundantly. In contrast, H1 isoforms are not present in Dipterans, including D. melanogaster, except for an embryo-specific, distantly related dBigH1. This study shows that Drosophila BEN domain protein Elba2, which is expressed in early embryos and has been hypothesized to have insulator-specific functions, can compensate for the loss of H1 in vivo. Although the Elba2 gene is not essential, its mutation causes a disruption of normal internucleosomal spacing of chromatin and reduced nuclear compaction in syncytial embryos. Elba2 protein is distributed ubiquitously in polytene chromosomes and strongly colocalizes with H1. In H1-depleted animals, ectopic expression of Elba2 rescues the increased lethality and ameliorates abnormalities of chromosome architecture and heterochromatin functions. Ectopic expression of BigH1 similarly complements the deficiency of H1 protein. Thus, in organisms that do not express redundant H1 isoforms, the structural and biological functions performed by canonical linker histones in later development, may be shared in early embryos by weakly homologous proteins, such as BigH1, or even unrelated, non-homologous proteins, such as Elba2.

Chaturvedi, D., Inaba, M., Scoggin, S. and Buszczak, M. (2016). Drosophila CG2469 encodes a homolog of human CTR9 and is essential for development. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27678520
Conserved from yeast to humans, the Paf1 complex participates in a number of diverse processes including transcriptional initiation and polyadenylation. This complex typically includes 5 proteins: Paf1, Rtf1, Cdc73, Leo1 and Ctr9. Previous efforts have identified clear Drosophila homologs of Paf1, Rtf1 and Cdc73 based on sequence similarity. Further work has showed that these proteins help to regulate gene expression and are required for viability. To date, a Drosophila homolog of Ctr9 has remained uncharacterized. This study shows that the gene CG2469 encodes a functional Drosophila Ctr9 homolog. Both human and Drosophila Ctr9 localize to the nuclei of Drosophila cells and appear enriched in histone locus bodies. RNAi knock-down of Drosophila Ctr9 results in a germline stem cell loss phenotype marked by defects in the morphology of germ cell nuclei. A molecular null mutation of Drosophila Ctr9 results in lethality and a human cDNA Ctr9 transgene rescues this phenotype. Clonal analysis in the ovary using this null allele reveals that loss of Drosophila Ctr9 results in a reduction of global levels of histone H3 trimethylation of lysine 4 (H3K4me3) but does not compromise the maintenance of stem cells in ovaries. Given the differences between the null mutant and RNAi knockdown phenotypes, the germ cell defects caused by RNAi likely result from the combined loss of Drosophila Ctr9 and other unidentified genes. These data provide further evidence that the function of this Paf1 complex component is conserved across species.

Hong, S.T. and Choi, K.W. (2016). Antagonistic roles of Drosophila Tctp and Brahma in chromatin remodelling and stabilizing repeated sequences. Nat Commun 7: 12988. PubMed ID: 27687497
Genome stability is essential for all organisms. Translationally controlled tumour protein (TCTP) is a conserved protein associated with cancers. TCTP is involved in multiple intracellular functions, but its role in transcription and genome stability is poorly understood. This study demonstrates new functions of Drosophila TCTP (Tctp) in transcription and the stability of repeated sequences (rDNA and pericentromeric heterochromatin). Tctp binds Brahma (Brm) chromatin remodeler to negatively modulate its activity. Tctp mutants show abnormally high levels of transcription in a large set of genes and transposons. These defects are ameliorated by brm mutations. Furthermore, Tctp promotes the stability of repeated sequences by opposing the Brm function. Additional regulation of pericentromeric heterochromatin by Tctp is mediated by su(var)3-9 transcriptional regulation. Altogether, Tctp regulates transcription and the stability of repeated sequences by antagonizing excess Brm activity. This study provides insights into broader nuclear TCTP functions for the maintenance of genome stability.

Snyder, M.J., Lau, A.C., Brouhard, E.A., Davis, M.B., Jiang, J., Sifuentes, M.H. and Csankovszki, G. (2016). Anchoring of heterochromatin to the nuclear lamina reinforces dosage compensation-mediated gene repression. PLoS Genet 12: e1006341. PubMed ID: 27690361
Evolutionary Homolog Study:
Compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensationin C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, it was found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. The study proposes a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression.

Monday, October 10th

McCommis, K. S., Hodges, W. T., Bricker, D. K., Wisidagama, D. R., Compan, V., Remedi, M. S., Thummel, C. S. and Finck, B. N. (2016). An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion. Mol Metab 5: 602-614. PubMed ID: 27656398
Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC) is an important and rate-limiting step in its metabolism. In pancreatic β-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion. To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, genetically-engineered Drosophila and mice were used with loss of MPC activity in insulin-producing cells. In both species, MPC deficiency results in elevated blood sugar concentrations and glucose intolerance accompanied by impaired glucose-stimulated insulin secretion. In mouse islets, β-cell MPC-deficiency resulted in decreased respiration with glucose, ATP-sensitive potassium (KATP) channel hyperactivity, and impaired insulin release. Moreover, treatment of pancreas-specific MPC knockout mice with glibenclamide, a sulfonylurea KATP channel inhibitor, improved defects in islet insulin secretion and abnormalities in glucose homeostasis in vivo. Finally, using a recently-developed biosensor for MPC activity, it was shown that the MPC is rapidly stimulated by glucose treatment in INS-1 insulinoma cells suggesting that glucose sensing is coupled to mitochondrial pyruvate carrier activity. Altogether, these studies suggest that the MPC plays an important and ancestral role in insulin-secreting cells in mediating glucose sensing, regulating insulin secretion, and controlling systemic glycemia.
Hakonardottir, G. K., Lopez-Ceballos, P., Herrera-Reyes, A. D., Das, R., Coombs, D. and Tanentzapf, G. (2015). In vivo quantitative analysis of Talin turnover in response to force. Mol Biol Cell 26: 4149-4162. PubMed ID: 26446844
Cell adhesion to the extracellular matrix (ECM) allows cells to form and maintain three-dimensional tissue architecture. Cell-ECM adhesions are stabilized upon exposure to mechanical force. This study used quantitative imaging and mathematical modeling to gain mechanistic insight into how integrin-based adhesions respond to increased and decreased mechanical forces. A critical means of regulating integrin-based adhesion is provided by modulating the turnover of integrin and its adhesion complex (integrin adhesion complex [IAC]). The turnover of the IAC component Talin, a known mechanosensor, was analyzed using fluorescence recovery after photobleaching. Experiments were carried out in live, intact flies in genetic backgrounds that increased or decreased the force applied on sites of adhesion. This analysis showed that when force is elevated, the rate of assembly of new adhesions increases such that cell-ECM adhesion is stabilized. Moreover, under conditions of decreased force, the overall rate of turnover, but not the proportion of adhesion complex components undergoing turnover, increases. Using point mutations, the key functional domains of Talin were identified that mediate its response to force. Finally, by fitting a mathematical model to the data, the mechanisms that mediate the stabilization of ECM-based adhesion during development were uncovered.
McClatchey, S.T., Wang, Z., Linden, L.M., Hastie, E.L., Wang, L., Shen, W., Chen, A., Chi, Q. and Sherwood, D.R. (2016). Boundary cells restrict dystroglycan trafficking to control basement membrane sliding during tissue remodeling. Elife [Epub ahead of print]. PubMed ID: 27661254
Evolutionary Homolog Study:
Epithelial cells and their underlying basement membranes (BMs) slide along each other to renew epithelia, shape organs, and enlarge BM openings. How BM sliding is controlled, however, is poorly understood. Using genetic and live cell imaging approaches during uterine-vulval attachment in C. elegans, this study discovered that the invasive uterine anchor cell activates Notch signaling (see Notch in Drosophila) in neighboring uterine cells at the boundary of the BM gap through which it invades to promote BM sliding. Through an RNAi screen, it was found that Notch activation upregulates expression of ctg-1 (see CG13893 in Drosophila), which encodes a Sec14-GOLD protein and member of the Sec14 phosphatidylinositol-transfer protein superfamily that is implicated in vesicle trafficking. Through photobleaching, targeted knockdown, and cell-specific rescue, these results suggest that CTG-1 restricts BM adhesion receptor DGN-1 (dystroglycan) (see Dg in Drosophila) trafficking to the cell-BM interface, which promotes BM sliding. Together, these studies reveal a new morphogenetic signaling pathway that controls BM sliding to remodel tissues.

Cerikan, B., Shaheen, R., Colo, G.P., Gläßer, C., Hata, S., Knobeloch, K.P., Alkuraya, F.S., Fässler, R. and Schiebel, E. (2016). Cell-intrinsic adaptation arising from chronic ablation of a key rho GTPase regulator. Dev Cell [Epub ahead of print]. PubMed ID: 27693507
Evolutionary Homology Study:
Genome-editing technologies allow systematic inactivation of human genes. Whether knockout phenotypes always reflect gene functions as determined by acute RNAi is an important question. This study shows how the acute knockdown of the Adams-Oliver syndrome (AOS) gene DOCK6 (see Drosophila Zir), coding for a RAC1/CDC42 guanine nucleotide exchange factor, results in strikingly different phenotypes to those generated by genomic DOCK6 disruption. Cell-intrinsic adaptation compensates for loss of DOCK6 function. Prolonged DOCK6 loss impacts upon the MRTF-A/SRF (see Drosophila bs) transcription factor, reducing levels of the ubiquitin-like modifier ISG15 (see Drosophila Nedd8). Reduced ISGylation of the IQGAP1 protein increases levels of active CDC42 and RAC1 to compensate for DOCK6 disruption. Similar downregulation of ISG15 in cells from DOCK6 AOS patients indicates that such adaptation can compensate for genetic defects during development. Thus, phenotypes of gene inactivation are critically dependent on the timescale, as acute knockdown reflects a transient state of adjustment to a new equilibrium that is attained following compensation.

Chu, C. W. and Sokol, S. Y. (2016). Wnt proteins can direct planar cell polarity in vertebrate ectoderm. Elife 5. PubMed ID: 27658614
Evolutionary Homolog Study
The coordinated orientation of cells across the tissue plane, known as planar cell polarity (PCP), is manifested by the segregation of core PCP proteins to different sides of the cell. Secreted Wnt ligands are involved in many PCP-dependent processes, yet whether they act as polarity cues has been controversial. This study shows that in Xenopus early ectoderm, the Prickle3/Vangl2 complex (see Drosophila Prickle and Vang) was polarized to anterior cell edges and this polarity was disrupted by several Wnt antagonists. In midgastrula embryos, Wnt5a, Wnt11, and Wnt11b, but not Wnt3a, acted across many cell diameters to orient Prickle3/Vangl2 complexes away from their sources regardless of their positions relative to the body axis. Planar polarity of endogenous Vangl2 in the neuroectoderm was similarly redirected by an ectopic Wnt source and disrupted after depletion of Wnt11b in the presumptive posterior region of the embryo. These observations provide evidence for the instructive role of Wnt ligands in vertebrate PCP.
Wang, I. E., Lapan, S. W., Scimone, M. L., Clandinin, T. R. and Reddien, P. W. (2016). Hedgehog signaling regulates gene expression in planarian glia. Elife 5. PubMed ID: 27612382
Evolutionary Homolog Study
Hedgehog signaling is critical for vertebrate central nervous system (CNS) development, but its role in CNS biology in other organisms is poorly characterized. In the planarian Schmidtea mediterranea, hedgehog (hh; see Drosophila Hedgehog) is expressed in medial cephalic ganglia neurons, suggesting a possible role in CNS maintenance or regeneration. RNA sequencing of planarian brain tissue was performed following RNAi of hh and patched (ptc; see Drosophila Patched), which encodes the Hh receptor. Two misregulated genes, intermediate filament-1 (if-1) and calamari (cali), were expressed in a previously unidentified non-neural CNS cell type. These cells expressed orthologs of astrocyte-associated genes involved in neurotransmitter uptake and metabolism, and extended processes enveloping regions of high synapse concentration. It is proposed that these cells are planarian glia. Planarian glia were distributed broadly, but only expressed if-1 and cali in the neuropil near hh+ neurons. Planarian glia and their regulation by Hedgehog signaling present a novel tractable system for dissection of glia biology.

Sunday, October 9th

Chen, J., Reiher, W., Hermann-Luibl, C., Sellami, A., Cognigni, P., Kondo, S., Helfrich-Förster, C., Veenstra, J.A. and Wegener, C. (2016). Allatostatin A signalling in Drosophila regulates feeding and sleep and is modulated by PDF. PLoS Genet 12: e1006346. PubMed ID: 27689358
Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. This study shows that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing posterior lateral protocerebrum (PLP) neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. The PLP neurons are identified to be a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, these results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF.

Berendes, V., Zill, S.N., Büschges, A. and Bockemühl, T. (2016). Speed-dependent interplay between local pattern-generating activity and sensory signals during walking in Drosophila. J Exp Biol [Epub ahead of print]. PubMed ID: 27688052
In insects, the coordinated motor output required for walking is based on the interaction between local pattern-generating networks providing basic rhythmicity and leg sensory signals which modulate this output on a cycle-to-cycle basis. How this interplay changes speed-dependently and thereby gives rise to the different coordination patterns observed at different speeds is understood insufficiently. This study used amputation to reduce sensory signals in single legs and decouple them mechanically during walking in Drosophila. This allows for the dissociation between locally-generated motor output in the stump and coordinating influences from intact legs. Leg stumps are still rhythmically active during walking. While the oscillatory frequency in intact legs is dependent on walking speed, stumps show a high and relatively constant oscillation frequency at all walking speeds. At low walking speeds strict cycle-to-cycle coupling between stumps and intact legs is not present. In contrast, at high walking speeds stump oscillations are strongly coupled to the movement of intact legs on a 1-to-1 basis. While during slow walking there is no preferred phase between stumps and intact legs, a preferred time interval between touch-down or lift-off events in intact legs and levation or depression of stumps is present. Based on these findings, the study hypothesizes that, as in other insects, walking speed in Drosophila is predominantly controlled by indirect mechanisms and that direct modulation of basic pattern-generating circuits plays a subsidiary role. Furthermore, inter-leg coordination strength seems to be speed-dependent and greater coordination is evident at higher walking speeds.

Mansourian, S., Corcoran, J., Enjin, A., Lofstedt, C., Dacke, M. and Stensmyr, M. C. (2016). Fecal-derived phenol induces egg-laying aversion in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 27641770
Feces is an abundant, rich source of energy, utilized by a myriad of organisms, not least by members of the order Diptera, i.e., flies. How Drosophila melanogaster reacts to fecal matter remains unclear. This study examined oviposition behavior toward a range of fecal samples from mammals native to the putative Southeast African homeland of the fly. D. melanogaster displays a strong oviposition aversion toward feces from carnivorous mammals but indifference or even attraction toward herbivore dung. A set of four predictor volatiles were identified that can be used to differentiate fecal from non-fecal matter, as well as separate carnivore from herbivore feces. Of these volatiles, phenol-indicative of carnivore feces-confers egg-laying aversion and is detected by a single class of sensory neurons expressing Or46a. The Or46a-expressing neurons are necessary and sufficient for oviposition site aversion. It was further demonstrated that carnivore feces-unlike herbivore dung-contain a high rate of pathogenic bacteria taxa. These harmful bacteria produce phenol from L-tyrosine, an amino acid specifically enriched in high protein diets, such as consumed by carnivores. Finally, it was demonstrated that carnivore feces, as well as phenol, is also avoided by a ball-rolling species of dung beetle, suggesting that phenol is a widespread avoidance signal because of its association with pathogenic bacteria.
Du, E. J., Ahn, T. J., Wen, X., Seo, D. W., Na, D. L., Kwon, J. Y., Choi, M., Kim, H. W., Cho, H. and Kang, K. (2016). Nucleophile sensitivity of Drosophila TRPA1 underlies light-induced feeding deterrence. Elife 5. PubMed ID: 27656903
Solar irradiation including ultraviolet (UV) light causes tissue damage by generating reactive free radicals that can be electrophilic or nucleophilic due to unpaired electrons. Little is known about how free radicals induced by natural sunlight are rapidly detected and avoided by animals. This study found that Drosophila Transient Receptor Potential Ankyrin 1 (TRPA1), previously known only as an electrophile receptor, sensitively detects photochemically active sunlight through nucleophile sensitivity. Rapid light-dependent feeding deterrence in Drosophila was was found to be mediated only by the TRPA1(A) isoform, despite the TRPA1(A) and TRPA1(B) isoforms having similar electrophile sensitivities. Such isoform dependence re-emerges in the detection of structurally varied nucleophilic compounds and nucleophilicity-accompanying hydrogen peroxide (H2O2). Furthermore, these isoform-dependent mechanisms require a common set of TRPA1(A)-specific residues dispensable for electrophile detection. Collectively, TRPA1(A) rapidly responds to natural sunlight intensities through its nucleophile sensitivity as a receptor of photochemically generated radicals, leading to an acute light-induced behavioral shift in Drosophila.

Saturday, October 8th

Cattie, D.J., Richardson, C.E., Reddy, K.C., Ness-Cohn, E.M., Droste, R., Thompson, M.K., Gilbert, W.V. and Kim, D.H. (2016). Mutations in nonessential eIF3k and eIF3l genes confer lifespan extension and enhanced resistance to ER stress in Caenorhabditis elegans. PLoS Genet 12: e1006326. PubMed ID: 27690135
Evolutionary Homolog Study:
The translation initiation factor eIF3 is a multi-subunit protein complex that coordinates the assembly of the 43S pre-initiation complex in eukaryotes. Prior studies have demonstrated that not all subunits of eIF3 are essential for the initiation of translation, suggesting that some subunits may serve regulatory roles. This study shows that loss-of-function mutations in the genes encoding the conserved eIF3k (see Drosophila CG10306) and eIF3l subunits of the translation initiation complex eIF3 result in a 40% extension in lifespan and enhanced resistance to endoplasmic reticulum (ER) stress in Caenorhabditis elegans. In contrast to previously described mutations in genes encoding translation initiation components that confer lifespan extension in C. elegans, loss-of-function mutations in eif-3.K or eif-3.L are viable, and mutants show normal rates of growth and development, and have wild-type levels of bulk protein synthesis. Lifespan extension resulting from EIF-3.K or EIF-3.L deficiency is suppressed by a mutation in the Forkhead family transcription factor DAF-16 (see Drosophila foxo). Mutations in eif-3.K or eif-3.L also confer enhanced resistance to ER stress, independent of IRE-1-XBP-1 (see Drosophila Ire1), ATF-6 (see Drosophila Atf6), and PEK-1 (see Drosophila PEK), and independent of DAF-16. These data suggest a pivotal functional role for conserved eIF3k and eIF3l accessory subunits of eIF3 in the regulation of cellular and organismal responses to ER stress and aging.

Matsukawa, K., Hashimoto, T., Matsumoto, T., Ihara, R., Chihara, T., Miura, M., Wakabayashi, T. and Iwatsubo, T. (2016). Familial ALS-linked mutations in Profilin 1 exacerbate TDP-43-induced degeneration in the retina of Drosophila melanogaster through an increase in the cytoplasmic localization of TDP-43. J Biol Chem [Epub ahead of print]. PubMed ID: 27634045
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive and selective loss of motor neurons. Causative genes for familial ALS (fALS) include mutations within profilin 1 (PFN1; see Drosophila Chickadee) have recently been identified in ALS18. Transgenic Drosophila melanogaster were generated overexpressing human PFN1 in the retinal photoreceptor neurons. Overexpression of wild-type or fALS mutant PFN1 caused no degenerative phenotypes in the retina. Double overexpression of fALS mutant PFN1 and human TDP-43 (see Drosophila TDP-43) markedly exacerbated the TDP-43-induced retinal degeneration, i.e., vacuolation and thinning of the retina, whereas co-expression of wild-type PFN1 did not aggravate the degenerative phenotype. Notably, co-expression of TDP-43 with fALS mutant PFN1 increased the cytoplasmic localization of TDP-43, the latter being remained in nuclei upon co-expression with wild-type PFN1, whereas co-expression of TDP-43 lacking the nuclear localization signal with fALS mutant PFN1 did not aggravate the retinal degeneration. Knockdown of endogenous Drosophila PFN1 did not alter the degenerative phenotypes of the retina in flies overexpressing wild-type TDP-43. These data suggest that ALS-linked PFN1 mutations exacerbate TDP-43-induced neurodegeneration in a gain-of-function manner, possibly by shifting the localization of TDP-43 from nuclei to cytoplasm.
M'Angale, P.G. and Staveley, B.E. (2016). Bcl-2 homologue Debcl enhances α-synuclein-induced phenotypes in Drosophila. PeerJ 4: e2461. PubMed ID: 27672511
The common hallmark for both sporadic and familial forms of Parkinson disease (PD) is mitochondrial dysfunction. Mammals have at least twenty proapoptotic and antiapoptotic Bcl-2 family members, in contrast, only two Bcl-2 family genes have been identified in Drosophila melanogaster, the proapoptotic mitochondrial localized Debcl and the antiapoptotic Buffy. The expression of the human transgene α-synuclein, a gene that is strongly associated with inherited forms of PD, in dopaminergic neurons (DA) of Drosophila, results in loss of neurons and locomotor dysfunction to model PD in flies. The altered expression of Debcl in the DA neurons and neuron-rich eye and along with the expression of α-synuclein offers an opportunity to highlight the role of Debcl in mitochondrial-dependent neuronal degeneration and death. The directed overexpression of Debcl using the Ddc-Gal4 transgene in the DA of Drosophila results in flies with severely decreased survival and a premature age-dependent loss in climbing ability. The inhibition of Debcl results in enhanced survival and improved climbing ability whereas the overexpression of Debcl in the α-synuclein-induced Drosophila model of PD results in more severe phenotypes. In addition, the co-expression of Debcl along with Buffy partially counteracts the Debcl-induced phenotypes, to improve the lifespan and the associated loss of locomotor ability observed. In complementary experiments, the overexpression of Debcl along with the expression of α-synuclein in the eye, enhances the eye ablation that results from the overexpression of Debcl. The co-expression of Buffy along with Debcl overexpression results in the rescue of the moderate developmental eye defects. The co-expression of Buffy along with inhibition of Debcl partially restores the eye to a roughened eye phenotype. Taken all together these results clarify on the role for Debcl in neurodegenerative disorders.

Griffin, R.M., Schielzeth, H. and Friberg, U. (2016). Autosomal and X linked additive genetic variation for lifespan and aging: Comparisons within and between the sexes in Drosophila melanogaster. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27678519
Theory makes several predictions concerning differences in genetic variation between the X chromosome and the autosomes due to male X hemizygosity. The X chromosome should i) typically show relatively less standing genetic variation than the autosomes, ii) exhibit more variation in males compared to females because of dosage compensation, and iii) potentially be enriched with sex-specific genetic variation. This study addressed each of these predictions for lifespan and aging in Drosophila melanogaster. To achieve unbiased estimates of X and autosomal additive genetic variance, 80 chromosome substitution lines were used; 40 for the X chromosome and 40 combining the two major autosomes, which were assayed for sex-specific and cross-sex genetic (co)variation. Significant X and autosomal additive genetic variance for both traits in both sexes (with reservation for X linked variation of aging in females) were found, but no conclusive evidence for depletion of X linked variation (measured through females) was found. Males display more X linked variation for lifespan than females, but it is unclear if this is due to dosage compensation since also autosomal variation is larger in males. Finally, the X chromosome is enriched for sex-specific genetic variation in lifespan. Results are overall less conclusive for aging. Collectively these data suggest the X chromosome has reduced capacity to respond to sexually concordant selection on lifespan from standing genetic variation, while its ability to respond to sexually antagonistic selection may be augmented.

Deshpande, M., Feiger, Z., Shilton, A. K., Luo, C. C., Silverman, E. and Rodal, A. A. (2016). Role of BMP receptor traffic in synaptic growth defects in an ALS model. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27535427
TAR DNA-binding protein 43 (TDP-43) is genetically and functionally linked to Amyotrophic Lateral Sclerosis (ALS), and regulates transcription, splicing, and transport of thousands of RNA targets that function in diverse cellular pathways. In ALS, pathologically altered TDP-43 is thought to lead to disease by toxic gain-of-function effects on RNA metabolism, as well as by sequestering endogenous TDP-43 and causing its loss of function. However, it remains unclear which of the numerous cellular processes disrupted downstream of TDP-43 dysfunction lead to neurodegeneration. This study found that both loss- and gain-of-function of TDP-43 in Drosophila cause a reduction of synaptic-growth-promoting Bone Morphogenic Protein (BMP) signaling at the neuromuscular junction (NMJ). Further, a shift of BMP receptors from early to recycling endosomes was observed along with increased mobility of BMP receptor-containing compartments at the NMJ. Inhibition of the recycling endosome GTPase Rab11 partially rescued TDP-43-induced defects in BMP receptor dynamics and distribution, and suppressed BMP signaling, synaptic growth, and larval crawling defects. These results indicate that defects in receptor traffic lead to neuronal dysfunction downstream of TDP-43 misregulation, and that rerouting receptor traffic may be a viable strategy for rescuing neurological impairment.
Akbar, M. A., Mandraju, R., Tracy, C., Hu, W., Pasare, C. and Kramer, H. (2016). ARC syndrome-linked Vps33B protein is required for inflammatory endosomal maturation and signal termination. Immunity 45: 267-279. PubMed ID: 27496733
Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) sense microbial ligands and initiate signaling to induce inflammatory responses. Although the quality of inflammatory responses is influenced by internalization of TLRs, the role of endosomal maturation in clearing receptors and terminating inflammatory responses is not well understood. This study reports that Drosophila and mammalian Vps33B proteins play critical roles in the maturation of phagosomes and endosomes following microbial recognition. Vps33B was necessary for clearance of endosomes containing internalized PRRs, failure of which resulted in enhanced signaling and expression of inflammatory mediators. Lack of Vps33B had no effect on trafficking of endosomes containing non-microbial cargo. These findings indicate that Vps33B function is critical for determining the fate of signaling endosomes formed following PRR activation. Exaggerated inflammatory responses dictated by persistence of receptors in aberrant endosomal compartments could therefore contribute to symptoms of Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, a disease linked to loss of Vps33B.

Fernandez-Funez, P., Sanchez-Garcia, J., de Mena, L., Zhang, Y., Levites, Y., Khare, S., Golde, T. E. and Rincon-Limas, D. E. (2016). Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila. Proc Natl Acad Sci U S A 113: E5212-5221. PubMed ID: 27531960
Alzheimer's disease (AD) is the most prevalent of a large group of related proteinopathies for which there is currently no cure. This study used Drosophila to explore a strategy to block Aβ42 neurotoxicity through engineering of the Heat shock protein 70 (Hsp70), a chaperone that has demonstrated neuroprotective activity against several intracellular amyloids. To target its protective activity against extracellular Aβ42, a signal peptide was added to Hsp70. This secreted form of Hsp70 (secHsp70) suppresses Aβ42 neurotoxicity in adult eyes, reduces cell death, protects the structural integrity of adult neurons, alleviates locomotor dysfunction, and extends lifespan. SecHsp70 binding to Aβ42 through its holdase domain is neuroprotective, but its ATPase activity is not required in the extracellular space. Thus, the holdase activity of secHsp70 masks Aβ42 neurotoxicity by promoting the accumulation of nontoxic aggregates. Combined with other approaches, this strategy may contribute to reduce the burden of AD and other extracellular proteinopathies.
De Rose, F., Corda, V., Solari, P., Sacchetti, P., Belcari, A., Poddighe, S., Kasture, S., Solla, P., Marrosu, F. and Liscia, A. (2016). Drosophila mutant model of Parkinson's disease revealed an unexpected olfactory performance: Morphofunctional evidences. Parkinsons Dis 2016: 3508073. PubMed ID: 27648340
Parkinson's disease (PD) is one of the most common neurodegenerative diseases characterized by the clinical triad: tremor, akinesia, and rigidity. Several studies have suggested that PD patients show disturbances in olfaction as one of the earliest, nonspecific nonmotor symptoms of disease onset. This study sought to use the fruit fly Drosophila melanogaster as a model organism to explore olfactory function in LRRK loss-of-function mutants, which was previously demonstrated to be a useful model for PD. Surprisingly, the results showed that the LRRK mutant, compared to the wild flies, presents a dramatic increase in the amplitude of the electroantennogram responses and this is coupled with a higher number of olfactory sensilla. In spite of the above reported results, the behavioural response to olfactory stimuli in mutant flies is impaired compared to that obtained in wild type flies. Thus, behaviour modifications and morphofunctional changes in the olfaction of LRRK loss-of-function mutants might be used as an index to explore the progression of parkinsonism in this specific model, also with the aim of studying and developing new treatments.

Friday, October 7th

Yang, D., Lian, T., Tu, J., Gaur, U., Mao, X., Fan, X., Li, D., Li, Y. and Yang, M. (2016). LncRNA mediated regulation of aging pathways in Drosophila melanogaster during dietary restriction. Aging (Albany NY) 8: 2182-2203. PubMed ID: 27687893
Dietary restriction (DR) extends lifespan in many species which is a well-known phenomenon. Long non-coding RNAs (lncRNAs) play an important role in regulation of cell senescence and important age-related signaling pathways. This study profiled the lncRNA and mRNA transcriptome of fruit flies at 7 day and 42 day during DR and fully-fed conditions, respectively. A large number of differentially expressed lncRNAs and their targets enriched in GO and KEGG analysis were found. Some new aging related signaling pathways during DR, such as hippo signaling pathway-fly, phototransduction-fly and protein processing in endoplasmic reticulum etc were also found. Novel lncRNAs XLOC_092363 and XLOC_166557 were found to be located in 10 kb upstream sequences of hairy and ems promoters, respectively. Furthermore, tissue specificity of some novel lncRNAs was analyzed at 7 day of DR in fly head, gut and fat body. Also the silencing of lncRNA XLOC_076307 resulted in altered expression level of its targets including Gadd45 (involved in FoxO signaling pathway). Together, these results implicate many lncRNAs closely associated with dietary restriction, which could provide a resource for lncRNA in aging and age-related disease field.

Sharma, S., Poetz, F., Bruer, M., Ly-Hartig, T. B., Schott, J., Seraphin, B. and Stoecklin, G. (2016). Acetylation-dependent control of global poly(A) RNA degradation by CBP/p300 and HDAC1/2. Mol Cell 63: 927-938. PubMed ID: 27635759
Acetylation of histones and transcription-related factors is known to exert epigenetic and transcriptional control of gene expression. This study reports that histone acetyltransferases (HATs) and histone deacetylases (HDACs) also regulate gene expression at the posttranscriptional level by controlling poly(A) RNA stability. Inhibition of HDAC1 and HDAC2 induces massive and widespread degradation of normally stable poly(A) RNA in mammalian and Drosophila cells. Acetylation-induced RNA decay depends on the HATs p300 and CBP, which acetylate the exoribonuclease CAF1a, a catalytic subunit of the CCR4-CAF1-NOT deadenlyase complex and thereby contribute to accelerating poly(A) RNA degradation. Taking adipocyte differentiation as a model, global stabilization of poly(A) RNA was observed during differentiation, concomitant with loss of CBP/p300 expression. This study uncovers reversible acetylation as a fundamental switch by which HATs and HDACs control the overall turnover of poly(A) RNA.
Ote, M., Ueyama, M. and Yamamoto, D. (2016). Wolbachia protein TomO targets nanos mRNA and restores germ stem cells in Drosophila Sex-lethal mutants. Curr Biol [Epub ahead of print]. PubMed ID: 27498563
Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, this study identified the Wolbachia gene WD1278 encoding a novel protein called toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs.
Catrina, I.E., Bayer, L.V., Yanez, G., McLaughlin, J.M., Malaczek, K., Bagaeva, E., Marras, S.A. and Bratu, D.P. (2016). The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11. RNA Biol [Epub ahead of print]. PubMed ID: 27654348
To achieve proper RNA transport and localization, RNA viruses exploit cellular vesicular trafficking pathways. AGFG1, a host protein essential for HIV-1 and Influenza A replication, has been shown to mediate release of intron-containing viral RNAs from the perinuclear region. It is still unknown what its precise role in this release is, or whether AGFG1 also participates in cytoplasmic transport. This study reports the expression patterns during oogenesis for Drongo, the fruit fly homolog of AGFG1. It was found that temporally controlled Drongo expression is achieved by translational repression of drongo mRNA within P-bodies. A link was found between the recycling endosome pathway and Drongo, and proper Drongo localization at the oocyte's cortex during mid-oogenesis was found to require functional Rab11.

Thursday, October 6th

Sears, J. C. and Broihier, H. T. (2016). FoxO regulates microtubule dynamics and polarity to promote dendrite branching in Drosophila sensory neurons. Dev Biol [Epub ahead of print]. PubMed ID: 27546375
The size and shape of dendrite arbors are defining features of neurons and critical determinants of neuronal function. The molecular mechanisms establishing arborization patterns during development are not well understood, though properly regulated microtubule (MT) dynamics and polarity are essential. It has been found that FoxO regulates axonal MTs, raising the question of whether it also regulates dendritic MTs and morphology. This study demonstrates that FoxO promotes dendrite branching in all classes of Drosophila dendritic arborization (da) neurons. FoxO is required both for initiating growth of new branches and for maintaining existing branches. To elucidate FoxO function, MT organization was characterized in both foxO null and overexpressing neurons. FoxO was found to directs MT organization and dynamics in dendrites. Moreover, it is both necessary and sufficient for anterograde MT polymerization, which is known to promote dendrite branching. Lastly, FoxO promotes proper larval nociception, indicating a functional consequence of impaired da neuron morphology in foxO mutants. Together, these results indicate that FoxO regulates dendrite structure and function and suggest that FoxO-mediated pathways control MT dynamics and polarity.
Teckchandani, A. and Cooper, J. A. (2016). The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells. Elife 5. PubMed ID: 27656905
Evolutionary Homolog Study
Cell migration requires the cyclical assembly and disassembly of focal adhesions. Adhesion induces phosphorylation of focal adhesion proteins, including Cas (Crk-associated substrate/p130Cas/BCAR1; see Drosophila Cas). However, Cas phosphorylation stimulates adhesion turnover. This raises the question of how adhesion assembly occurs against opposition from phospho-Cas. This study shows that suppressor of cytokine signaling 6 (SOCS6; see Drosophila Socs36E) and Cullin 5 (see Drosophila Cullin 5), two components of the CRL5SOCS6 ubiquitin ligase, inhibit Cas-dependent focal adhesion turnover at the front but not rear of migrating epithelial cells. The front focal adhesions contain phospho-Cas which recruits SOCS6. If SOCS6 cannot access focal adhesions, or cullins or the proteasome are inhibited, adhesion disassembly is stimulated. This suggests that the localized targeting of phospho-Cas within adhesions by CRL5SOCS6 and concurrent cullin and proteasome activity provide a negative feedback loop, ensuring that adhesion assembly predominates over disassembly at the leading edge. By this mechanism, ubiquitination provides a new level of spatio-temporal control over cell migration.
Vieillard, J., Paschaki, M., Duteyrat, J. L., Augiere, C., Cortier, E., Lapart, J. A., Thomas, J. and Durand, B. (2016). Transition zone assembly and its contribution to axoneme formation in Drosophila male germ cells. J Cell Biol 214: 875-889. PubMed ID: 27646273
The ciliary transition zone (TZ) is a complex structure found at the cilia base. Defects in TZ assembly are associated with human ciliopathies. In most eukaryotes, three protein complexes (CEP290, NPHP, and MKS) cooperate to build the TZ. This study shows that in Drosophila melanogaster, mild TZ defects are observed in the absence of MKS components. In contrast, Cby and Azi1 cooperate to build the TZ by acting upstream of Cep290 and MKS components. Without Cby and Azi1, centrioles fail to form the TZ, precluding sensory cilia assembly, and no ciliary membrane cap associated with sperm ciliogenesis is made. This ciliary cap is critical to recruit the tubulin-depolymerizing kinesin Klp59D, required for regulation of axonemal growth. These results show that Drosophila TZ assembly in sensory neurons and male germ cells involves cooperative actions of Cby and Dila. They further reveal that temporal control of membrane cap assembly by TZ components and microtubule elongation by kinesin-13 is required for axoneme formation in male germ cells.
Aigouy, B. and Le Bivic, A. (2016). The PCP pathway regulates Baz planar distribution in epithelial cells. Sci Rep 6: 33420. PubMed ID: 27624969
The localisation of apico-basal polarity proteins along the Z-axis of epithelial cells is well understood while their distribution in the plane of the epithelium is poorly characterised. This study provides a systematic description of the planar localisation of apico-basal polarity proteins in the Drosophila ommatidial epithelium. The adherens junction proteins Shotgun and Armadillo, as well as the baso-lateral complexes, are bilateral, i.e. present on both sides of cell interfaces. In contrast, it is reported that other key adherens junction proteins, Bazooka and the myosin regulatory light chain (Spaghetti squash) are unilateral, i.e. present on one side of cell interfaces. Furthermore, planar cell polarity (PCP) and not the apical determinants Crumbs and Par-6 control Bazooka unilaterality in cone cells. Altogether, this work unravels an unexpected organisation and combination of apico-basal, cytoskeletal and planar polarity proteins that is different on either side of cell-cell interfaces and unique for the different contacts of the same cell.

Wednesday, October 5th

Busto, G. U., Guven-Ozkan, T., Chakraborty, M. and Davis, R. L. (2016). Developmental inhibition of miR-iab8-3p disrupts mushroom body neuron structure and adult learning ability. Dev Biol [Epub ahead of print]. PubMed ID: 27634569
MicroRNAs are small non-coding RNAs that inhibit protein expression post-transcriptionally. They have been implicated in many different physiological processes, but little is known about their individual involvement in learning and memory. Several miRNAs have been identified that either increased or decreased intermediate-term memory when inhibited in the central nervous system, including miR-iab8-3p. This paper reports a new developmental role for this miRNA. Blocking the expression of miR-iab8-3p during the development of the organism leads to hypertrophy of individual mushroom body neuron soma, a reduction in the field size occupied by axonal projections, and adult intellectual disability. Four potential mRNA targets of miR-iab8-3p were identified whose inhibition modulates intermediate-term memory including ceramide phosphoethanolamine synthase, which may account for the behavioral effects produced by miR-iab8-3p inhibition. These results offer important new information on a microRNA required for normal neurodevelopment and the capacity to learn and remember normally.
Zwarts, L., Goossens, T., Clements, J., Kang, Y. Y. and Callaerts, P. (2016). Axon branch-specific Semaphorin-1a signaling in Drosophila mushroom body development. Front Cell Neurosci 10: 210. PubMed ID: 27656129
Correct wiring of the mushroom body (MB) neuropil in the Drosophila brain involves appropriate positioning of different axonal lobes, as well as the sister branches that develop from individual axons. This positioning requires the integration of various guidance cues provided by different cell types, which help the axons find their final positions within the neuropil. Semaphorins are well-known for their conserved roles in neuronal development and axon guidance. This study investigated the role of Sema-1a in MB development more closely. Sema-1a is expressed in the MBs as well as surrounding structures, including the glial transient interhemispheric fibrous ring, throughout development. By loss- and gain-of-function experiments, it was shown that the MB axons display lobe and sister branch-specific Sema-1a signaling, which controls different aspects of axon outgrowth and guidance. Furthermore, these effects are modulated by the integration of MB intrinsic and extrinsic Sema-1a signaling pathways involving PlexA and PlexB. Finally, a role is also shown for neuronal- glial interaction in Sema-1a dependent β-lobe outgrowth.
Purice, M. D., Speese, S. D. and Logan, M. A. (2016). Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity. Nat Commun 7: 12871. PubMed ID: 27647497
Advanced age is the greatest risk factor for neurodegenerative disorders, but the mechanisms that render the senescent brain vulnerable to disease are unclear. Glial immune responses provide neuroprotection in a variety of contexts. Thus, this study explored how glial responses to neurodegeneration are altered with age. Glia-axon phagocytic interactions were shown to change dramatically in the aged Drosophila brain. Aged glia clear degenerating axons slowly due to low phosphoinositide-3-kinase (PI3K) signalling and, subsequently, reduced expression of the conserved phagocytic receptor Draper/MEGF10. Importantly, boosting PI3K/Draper activity in aged glia significantly reverses slow phagocytic responses. Moreover, several hours post axotomy, early hallmarks of Wallerian degeneration (WD) are delayed in aged flies. It is proposed that slow clearance of degenerating axons is mechanistically twofold, resulting from deferred initiation of axonal WD and reduced PI3K/Draper-dependent glial phagocytic function. Interventions that boost glial engulfment activity, however, can substantially reverse delayed clearance of damaged neuronal debris.
Jeong, Y. T., Oh, S. M., Shim, J., Seo, J. T., Kwon, J. Y. and Moon, S. J. (2016). Mechanosensory neurons control sweet sensing in Drosophila. Nat Commun 7: 12872. PubMed ID: 27641708
Animals discriminate nutritious food from toxic substances using their sense of taste. Since taste perception requires taste receptor cells to come into contact with water-soluble chemicals, it is a form of contact chemosensation. Concurrent with that contact, mechanosensitive cells detect the texture of food and also contribute to the regulation of feeding. Little is known, however, about the extent to which chemosensitive and mechanosensitive circuits interact. This study shows Drosophila prefers soft food at the expense of sweetness and that this preference requires labellar mechanosensory neurons (MNs) and the mechanosensory channel Nanchung. Activation of these labellar MNs causes GABAergic inhibition of sweet-sensing gustatory receptor neurons, reducing the perceived intensity of a sweet stimulus. These findings expand understanding of the ways different sensory modalities cooperate to shape animal behaviour.

Tuesday, October 4th

Wood, J.G., Jones, B.C., Jiang, N., Chang, C., Hosier, S., Wickremesinghe, P., Garcia, M., Hartnett, D.A., Burhenn, L., Neretti, N. and Helfand, S.L. (2016). Chromatin-modifying genetic interventions suppress age-associated transposable element activation and extend life span in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27621458
Transposable elements (TEs) are mobile genetic elements, highly enriched in heterochromatin, that constitute a large percentage of the DNA content of eukaryotic genomes. Aging in Drosophila melanogaster is characterized by loss of repressive heterochromatin structure and loss of silencing of reporter genes in constitutive heterochromatin regions. Using next-generation sequencing, this study found that transcripts of many genes native to heterochromatic regions and TEs increased with age in fly heads and fat bodies. A dietary restriction regimen, known to extend life span, represses the age-related increased expression of genes located in heterochromatin, as well as TEs. A corresponding age-associated increase in TE transposition in fly fat body cells was also observed that was delayed by dietary restriction. Furthermore, manipulating genes known to affect heterochromatin structure, including overexpression of Sir2, Su(var)3-9, and Dicer-2, as well as decreased expression of Adar, mitigate age-related increases in expression of TEs. Increasing expression of either Su(var)3-9 or Dicer-2 also leads to an increase in life span. Mutation of Dicer-2 leads to an increase in DNA double-strand breaks. Treatment with the reverse transcriptase inhibitor 3TC results in decreased TE transposition as well as increased life span in TE-sensitized Dicer-2 mutants. Together, these data support the retrotransposon theory of aging, which hypothesizes that epigenetically silenced TEs become deleteriously activated as cellular defense and surveillance mechanisms break down with age. Furthermore, interventions that maintain repressive heterochromatin and preserve TE silencing may prove key to preventing damage caused by TE activation and extending healthy life span.

Swain, A., Misulovin, Z., Pherson, M., Gause, M., Mihindukulasuriya, K., Rickels, R. A., Shilatifard, A. and Dorsett, D. (2016). Drosophila TDP-43 RNA-binding protein facilitates association of sister chromatid cohesion proteins with genes, enhancers and polycomb response elements. PLoS Genet 12: e1006331. PubMed ID: 27662615
The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. This study shows that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs). RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers.
Loubiere, V., Delest, A., Thomas, A., Bonev, B., Schuettengruber, B., Sati, S., Martinez, A. M. and Cavalli, G. (2016). Coordinate redeployment of PRC1 proteins suppresses tumor formation during Drosophila development. Nat Genet [Epub ahead of print]. PubMed ID: 27643538
Polycomb group proteins form two main complexes, PRC2 and PRC1, which generally coregulate their target genes. This study shows that PRC1 components act as neoplastic tumor suppressors independently of PRC2 function. By mapping the distribution of PRC1 components and trimethylation of histone H3 at Lys27 (H3K27me3) across the genome, a large set of genes were identified that acquire PRC1 in the absence of H3K27me3 in Drosophila larval tissues. These genes massively outnumber canonical targets and are mainly involved in the regulation of cell proliferation, signaling and polarity. Alterations in PRC1 components specifically deregulate this set of genes, whereas canonical targets are derepressed in both PRC1 and PRC2 mutants. In human embryonic stem cells, PRC1 components colocalize with H3K27me3 as in Drosophila embryos, whereas in differentiated cell types they are selectively recruited to a large set of proliferation and signaling-associated genes that lack H3K27me3, suggesting that the redeployment of PRC1 components during development is evolutionarily conserved.
Ye, Y., Gu, L., Chen, X., Shi, J., Zhang, X. and Jiang, C. (2016). Chromatin remodeling during the in vivo glial differentiation in early Drosophila embryos. Sci Rep 6: 33422. PubMed ID: 27634414
Chromatin remodeling plays a critical role in gene regulation and impacts many biological processes. However, little is known about the relationship between chromatin remodeling dynamics and in vivo cell lineage commitment. This study revealed the patterns of histone modification change and nucleosome positioning dynamics and their epigenetic regulatory roles during the in vivo glial differentiation in early Drosophila embryos. The genome-wide average H3K9ac signals in promoter regions are decreased in the glial cells compared to the neural progenitor cells. However, H3K9ac signals are increased in a group of genes that are up-regulated in glial cells and involved in gliogenesis. There occurs extensive nucleosome remodeling including shift, loss, and gain. Nucleosome depletion regions (NDRs) form in both promoters and enhancers. As a result, the associated genes are up-regulated. Intriguingly, NDRs form in two fashions: nucleosome shift and eviction. Moreover, the mode of NDR formation is independent of the original chromatin state of enhancers in the neural progenitor cells.

Monday, October 3rd

Li, H., Hu, C., Bai, L., Li, H., Li, M., Zhao, X., Czajkowsky, D. M. and Shao, Z. (2016). Ultra-deep sequencing of ribosome-associated poly-adenylated RNA in early Drosophila embryos reveals hundreds of conserved translated sORFs. DNA Res [Epub ahead of print]. PubMed ID: 27559081
There is growing recognition that small open reading frames (sORFs) encoding peptides shorter than 100 amino acids are an important class of functional elements in the eukaryotic genome, with several already identified to play critical roles in growth, development, and disease. However, understanding of their biological importance has been hindered owing to the significant technical challenges limiting their annotation. This study combined ultra-deep sequencing of ribosome-associated poly-adenylated RNAs with rigorous conservation analysis to identify a comprehensive population of translated sORFs during early Drosophila embryogenesis. In total, 399 sORFs were identified, including those previously annotated but without evidence of translational capacity, those found within transcripts previously classified as non-coding, and those not previously known to be transcribed. Further, evidence was found for translation of many sORFs with different isoforms, suggesting their regulation is as complex as longer ORFs. Furthermore, many sORFs are found not associated with ribosomes in late-stage Drosophila S2 cells, suggesting that many of the translated sORFs may have stage-specific functions during embryogenesis. These results thus provide the first comprehensive annotation of the sORFs present during early Drosophila embryogenesis, a necessary basis for a detailed delineation of their function in embryogenesis and other biological processes.
Myat, M. M. and Patel, U. (2016). Receptor-type Guanylyl cyclase at 76C (Gyc76C) regulates de novo lumen formation during Drosophila tracheal development. PLoS One 11: e0161865. PubMed ID: 27642749
Lumen formation and maintenance are important for the development and function of essential organs such as the lung, kidney and vasculature. In the Drosophila embryonic trachea, lumena form de novo to connect the different tracheal branches into an interconnected network of tubes. This study identified a novel role for the receptor type guanylyl cyclase at 76C (Gyc76C) in de novo lumen formation in the Drosophila trachea. In embryos mutant for gyc76C or its downsteam effector protein kinase G (PKG) 1, tracheal lumena are disconnected. Dorsal trunk (DT) cells of gyc76C mutant embryos migrate to contact each other and complete the initial steps of lumen formation, such as the accumulation of E-cadherin (E-cad) and formation of an actin track at the site of lumen formation. However, the actin track and E-cad contact site of gyc76C mutant embryos did not mature to become a new lumen and DT lumena did not fuse. Also failure was observed of the luminal protein Vermiform to be secreted into the site of new lumen formation in gyc76C mutant trachea. These DT lumen formation defects were accompanied by altered localization of the Arf-like 3 GTPase (Arl3), a known regulator of vesicle-vesicle and vesicle-membrane fusion. In addition to the DT lumen defect, lumena of gyc76C mutant terminal cells were shorter compared to wild-type cells. These studies show that Gyc76C and downstream PKG-dependent signaling regulate de novo lumen formation in the tracheal DT and terminal cells, most likely by affecting Arl3-mediated luminal secretion.
Harumoto, T., Anbutsu, H., Lemaitre, B. and Fukatsu, T. (2016). Male-killing symbiont damages host's dosage-compensated sex chromosome to induce embryonic apoptosis. Nat Commun 7: 12781. PubMed ID: 27650264
Some symbiotic bacteria are capable of interfering with host reproduction in selfish ways. How such bacteria can manipulate host's sex-related mechanisms is of fundamental interest encompassing cell, developmental and evolutionary biology. This study uncovered the molecular and cellular mechanisms underlying Spiroplasma-induced embryonic male lethality in Drosophila melanogaster. Transcriptomic analysis reveals that many genes related to DNA damage and apoptosis are up-regulated specifically in infected male embryos. Detailed genetic and cytological analyses demonstrate that male-killing Spiroplasma causes DNA damage on the male X chromosome interacting with the male-specific lethal (MSL) complex. The damaged male X chromosome exhibits a chromatin bridge during mitosis, and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways. Notably, the MSL complex is not only necessary but also sufficient for this cytotoxic process. These results highlight symbiont's sophisticated strategy to target host's sex chromosome and recruit host's molecular cascades toward massive apoptosis in a sex-specific manner.
Ordan, E. and Volk, T. (2016). Amontillado is required for Drosophila Slit processing and for tendon-mediated muscle patterning. Biol Open [Epub ahead of print]. PubMed ID: 27628033
Slit cleavage into an N-terminal and C-terminal polypeptides is essential for restricting the range of Slit activity. Although the Slit cleavage site has been characterized previously and is evolutionally conserved, the identity of the protease that cleaves Slit remains elusive. Previous analysis has indicated that Slit cleavage is essential to immobilize the active Slit-N at the tendon cell surfaces, mediating the arrest of muscle elongation. In an attempt to identify the protease required for Slit cleavage, this study performed an RNAi-based assay in the ectoderm and followed the process of elongation of the lateral transverse muscles toward tendon cells. The Drosophila homolog of Pheromone Convertase 2 (PC2) Amontillado (Amon) was identified as an essential protease for Slit cleavage. Further analysis indicated that Slit mobility on SDS polyacrylamide gel electrophoresis is slightly up-shifted in amon mutants, and its conventional cleavage into the Slit-N and Slit-C polypeptides is attenuated. Consistent with the requirement for Amon to promote Slit cleavage and membrane immobilization of Slit-N, the muscle phenotype of amon mutant embryos is rescued by co-expressing a membrane-bound form of full-length Slit lacking the cleavage site and knocked into the slit locus. The identification of a novel protease component essential for Slit processing may represent an additional regulatory step in the Slit signaling pathway.

Sunday, October 2nd

Marco Antonio, D. S. and Hartfelder, K. (2016).. Toward an understanding of divergent compound eye development in drones and workers of the honeybee (Apis mellifera L.): A correlative analysis of morphology and gene expression. J Exp Zool B Mol Dev Evol [Epub ahead of print]. PubMed ID: 27658924
Eye development in insects is best understood in Drosophila melanogaster, but little is known for other holometabolous insects. Combining a morphological with a gene expression analysis, this study investigated eye development in the honeybee, putting emphasis on the sex-specific differences in eye size. Optic lobe development starts from an optic lobe anlage in the larval brain, which sequentially gives rise to the lobula, medulla, and lamina. The lamina differentiates in the last larval instar, when it receives optic nerve projections from the developing retina. The expression analysis focused on seven genes important for Drosophila eye development: eyes absent, sine oculis, embryonic lethal abnormal vision, minibrain, small optic lobes, epidermal growth factor receptor, and roughest. All except small optic lobes were more highly expressed in third-instar drone larvae, but then, in the fourth and fifth instar, their expression was sex-specifically modulated, showing shifts in temporal dynamics. The clearest differences were seen for small optic lobes, which is highly expressed in the developing eye of workers, and minibrain and roughest, which showed a strong expression peak coinciding with retina differentiation. A microarray analysis for optic lobe/retina complexes revealed the differential expression of several metabolism-related genes, as well as of two micro-RNAs. While major morphological differences were not seen in the developing eye structures before the pupal stage, the expression differences observed for the seven candidate genes and in the transcriptional microarray profiles indicate that molecular signatures underlying sex-specific optic lobe and retina development become established throughout the larval stages.
Hickner, P. V., Rivaldi, C. L., Johnson, C. M., Siddappaji, M., Raster, G. J. and Syed, Z. (2016). The making of a pest: Insights from the evolution of chemosensory receptor families in a pestiferous and invasive fly, Drosophila suzukii. BMC Genomics 17: 648. PubMed ID: 27530109
Drosophila suzukii differs from other melanogaster group members in their proclivity for laying eggs in fresh fruit rather than in fermenting fruits. Earlier work has revealed how the olfactory landscape of D. suzukii is dominated by volatiles derived from its unique niche. This study annotated the Olfactory receptors and Gustatory Receptors in D. suzukii and two close relatives, D. biarmipes and D. takahashii, to identify candidate chemoreceptors associated with D. suzukii's unusual niche utilization. A total of 71 Or genes were annotated in D. suzukii, with nine of those being pseudogenes (12.7 %). Alternative splicing of two genes brings the total to 62 genes encoding 66 Ors. Duplications of Or23a and Or67a expanded D. suzukii's Or repertoire, while pseudogenization of Or74a, Or85a, and Or98b reduced the number of functional Ors to roughly the same as other annotated species in the melanogaster group. Seventy-one intact Gr genes and three pseudogenes were annotated in D. suzukii. Alternative splicing in three genes brings the total number of Grs to 81. Signatures of positive selection were identified in two Ors and three Grs at nodes leading to D. suzukii, while three copies in the largest expanded Or lineage, Or67a, also showed signs of positive selection at the external nodes. This analysis of D. suzukii's chemoreceptor repertoires in the context of nine melanogaster group drosophilids, including two of its closest relatives (D. biarmipes and D. takahashii), revealed several candidate receptors associated with the adaptation of D. suzukii to its unique ecological niche.
Huang, Y. and Agrawal, A. F. (2016). Experimental evolution of gene expression and plasticity in alternative selective regimes. PLoS Genet 12: e1006336. PubMed ID: 27661078
Little is known of how gene expression and its plasticity evolves as populations adapt to different environmental regimes. Expression is expected to evolve adaptively in all populations but only those populations experiencing environmental heterogeneity are expected to show adaptive evolution of plasticity. This study measured the transcriptome in a cadmium-enriched diet and a salt-enriched diet for experimental populations of Drosophila melanogaster that evolved for ~130 generations in one of four selective regimes: two constant regimes maintained in either cadmium or salt diets and two heterogeneous regimes that varied either temporally or spatially between the two diets. For populations evolving in constant regimes, a strong signature of counter-gradient evolution was found; the evolved expression differences between populations adapted to alternative diets is opposite to the plastic response of the ancestral population that is naive to both diets. Based on expression patterns in the ancestral populations, a set of genes was identified for which selection in heterogeneous regimes was predicted to result in increases in plasticity, and the expected pattern was found. In contrast, a set of genes where reduced plasticity was predicted did not follow expectation. Nonetheless, both gene sets showed a pattern consistent with adaptive expression evolution in heterogeneous regimes, highlighting the difference between observing 'optimal' plasticity and improvements in environment-specific expression. Looking across all genes, there is evidence in all regimes of differences in biased allele expression across environments ('allelic plasticity') and this is more common among genes with plasticity in total expression.
Tejeda, M. T., Arredondo, J., Liedo, P., Perez-Staples, D., Ramos-Morales, P. and Diaz-Fleischer, F. (2016). Reasons for success: rapid evolution for desiccation resistance and life-history changes in the polyphagous fly Anastrepha ludens. Evolution [Epub ahead of print]. PubMed ID: 27641541
Evolutionary Homolog Study
Species that exhibit broad ranges of distribution may successfully navigate environmental changes by modifying some of their life history traits. Environmental humidity imposes a critical stress that organisms may overcome by increasing their resistance to desiccation. This study used experimental evolution to investigate adaptation to desiccation in the tephritid Anastrepha ludens, a species with high fecundity, late maturation and long lifespan. This study measured morphological, physiological, developmental as well as demographic changes involved in the adaptation to desiccation. Notwithstanding a low heritability (h2 = 0.237), desiccation resistance evolved extremely rapidly and few negative trade-offs were detected. Selected flies exhibited correlated increases in longevity, body size, the amount of body lipids and bulk water content, and in the duration of the pupal stage. Females further delayed sexual maturation, decreased daily fecundity but retained high lifetime reproductive potential. No differences in male mating competitiveness were found. Selected and control lines differed in longevity but not in total female fecundity, demonstrating that A. ludens flies have the capability for fast adaptation to desiccation without loosing their reproductive capability. Thus, it seems that a rapid evolutionary response to desiccation in this polyphagous insect works as a buffer for environmental variation and reduces the strength of selection on reproductive traits.

Cheng, C. and Kirkpatrick, M. (2016). Sex-specific selection and sex-biased gene expression in humans and flies. PLoS Genet 12: e1006170. PubMed ID: 27658217
Sexual dimorphism results from sex-biased gene expression, which evolves when selection acts differently on males and females. While there is an intimate connection between sex-biased gene expression and sex-specific selection, few empirical studies have studied this relationship directly. This study compare the two on a genome-wide scale in humans and flies. A distinctive "Twin Peaks" pattern in humans that relates the strength of sex-specific selection, quantified by genetic divergence was yound between male and female adults at autosomal loci, to the degree of sex-biased expression. Genes with intermediate degrees of sex-biased expression show evidence of ongoing sex-specific selection, while genes with either little or completely sex-biased expression do not. This pattern apparently results from differential viability selection in males and females acting in the current generation. The Twin Peaks pattern is also found in Drosophila using a different measure of sex-specific selection acting on fertility. A simple model was developed that successfully recapitulates the Twin Peaks. These results suggest that many genes with intermediate sex-biased expression experience ongoing sex-specific selection in humans and flies.
Sulak, M., Fong, L., Mika, K., Chigurupati, S., Yon, L., Mongan, N. P., Emes, R. D. and Lynch, V. J. (2016). TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants. Elife 5. PubMed ID: 27642012
Evolutionary Homolog Study
A major constraint on the evolution of large body sizes in animals is an increased risk of developing cancer. There is no correlation, however, between body size and cancer risk. This lack of correlation is often referred to as 'Peto's Paradox'. This study showed that the elephant genome encodes 20 copies of the tumor suppressor gene TP53 (see Drosophila p53) and that the increase in TP53 copy number occurred coincident with the evolution of large body sizes, the evolution of extreme sensitivity to genotoxic stress, and a hyperactive TP53 signaling pathway in the elephant (Proboscidean) lineage. Furthermore several of the TP53 retrogenes (TP53RTGs) were shown to be transcribed and likely translated. While TP53RTGs do not appear to directly function as transcription factors, they do contribute to the enhanced sensitivity of elephant cells to DNA damage and the induction of apoptosis by regulating activity of the TP53 signaling pathway. These results suggest that an increase in the copy number of TP53 may have played a direct role in the evolution of very large body sizes and the resolution of Peto's paradox in Proboscideans.

Saturday, October 1st

Williams, M. J., Perland, E., Eriksson, M. M., Carlsson, J., Erlandsson, D., Laan, L., Mahebali, T., Potter, E., Frediksson, R., Benedict, C. and Schioth, H. B. (2016). Recurrent sleep fragmentation induces insulin and neuroprotective mechanisms in middle-aged flies. Front Aging Neurosci 8: 180. PubMed ID: 27531979
Lack of quality sleep increases central nervous system oxidative stress and impairs removal of neurotoxic soluble metabolites from brain parenchyma. During aging poor sleep quality, caused by sleep fragmentation, increases central nervous system cellular stress. Currently, it is not known how organisms offset age-related cytotoxic metabolite increases in order to safeguard neuronal survival. Furthermore, it is not understood how age and sleep fragmentation interact to affect oxidative stress protection pathways. This study demonstrates that sleep fragmentation increases systems that protect against oxidative damage and neuroprotective endoplasmic reticulum molecular chaperones, as well as neuronal insulin and dopaminergic expression in middle-aged Drosophila males. Interestingly, even after sleep recovery the expression of these genes was still upregulated in middle-aged flies. Finally, sleep fragmentation generates higher levels of reactive oxygen species (ROS) in middle-aged flies and after sleep recovery these levels remain significantly higher than in young flies. The fact that neuroprotective pathways remain upregulated in middle-aged flies beyond sleep fragmentation suggests it might represent a strong stressor for the brain during later life.
Laranjeira, A., Schulz, J. and Dotti, C. G. (2016). Genes related to fatty acid beta-oxidation play a role in the functional decline of the Drosophila brain with age. PLoS One 11: e0161143. PubMed ID: 27518101
In living organisms, ageing is widely considered to be the result of a multifaceted process consisting of the progressive accumulation of damage over time, having implications both in terms of function and survival. The study of ageing presents several challenges, from the different mechanisms implicated to the great diversity of systems affected over time. The current study set out to identify genes involved in the functional decline of the brain with age and study its relevance in a tissue dependent manner using Drosophila melanogaster as a model system. The age-dependent upregulation is reported of genes involved in the metabolic process of fatty acid beta-oxidation in the nervous tissue of female wild-type flies. Downregulation of CG10814, dHNF4 and lipid mobilizing genes bmm and dAkh rescues the functional decline of the brain with age, both at the cellular and behaviour level, while over-expression worsens performance. The data proposes the occurrence of a metabolic alteration in the fly brain with age, whereby the process of beta-oxidation of fatty acids experiences a genetic gain-of-function. This event proved to be one of the main causes contributing to the functional decline of the brain with age.
Klepsatel, P., Galikova, M., Xu, Y. and Kuhnlein, R. P. (2016). Thermal stress depletes energy reserves in Drosophila. Sci Rep 6: 33667. PubMed ID: 27641694
Understanding how environmental temperature affects metabolic and physiological functions is of crucial importance to assess the impacts of climate change on organisms. This study used different laboratory strains and a wild-caught population of the fruit fly Drosophila melanogaster to examine the effect of temperature on the body energy reserves of an ectothermic organism. Permanent ambient temperature elevation or transient thermal stress was shown to cause significant depletion of body fat stores. Surprisingly, transient thermal stress induces a lasting "memory effect" on body fat storage, which also reduces survivorship of the flies upon food deprivation later after stress exposure. Functional analyses revealed that an intact heat-shock response is essential to protect flies from temperature-dependent body fat decline. Moreover, it was found that the temperature-dependent body fat reduction is caused at least in part by apoptosis of fat body cells, which might irreversibly compromise the fat storage capacity of the flies. Altogether, these results provide evidence that thermal stress has a significant negative impact on organismal energy reserves, which in turn might affect individual fitness.
Ahmadi, M. and Roy, R. (2016). AMPK acts as a molecular trigger to coordinate glutamatergic signals and adaptive behaviours during acute starvation. Elife 5. PubMed ID: 27642785
Evolutionary Homolog Study
The stress associated with starvation is accompanied by compensatory behaviours that enhance foraging efficiency and increase the probability of encountering food. However, the molecular details of how hunger triggers changes in the activity of neural circuits to elicit these adaptive behavioural outcomes remains to be resolved. This study shows that AMP-activated protein kinase (AMPK; see Drosophila AMPKα) regulates neuronal activity to elicit appropriate behavioural outcomes in response to acute starvation, and this effect is mediated by the coordinated modulation of glutamatergic inputs. AMPK targets both the AMPA-type glutamate receptor GLR-1 (see Drosophila Glu-RIIA) and the metabotropic glutamate receptor MGL-1 (see Drosophila mGluR) in one of the primary circuits that governs behavioural response to food availability in C. elegans. Overall, this study suggests that AMPK acts as a molecular trigger in the specific starvation-sensitive neurons to modulate glutamatergic inputs and to elicit adaptive behavioural outputs in response to acute starvation.

Vaccaro, A., Birman, S. and Klarsfeld, A. (2016). Chronic jet lag impairs startle-induced locomotion in Drosophila. Exp Gerontol 85: 24-27. PubMed ID: 27639775
Endogenous circadian clocks with ~24-h periodicity are found in most organisms from cyanobacteria to humans. Daylight synchronizes these clocks to solar time. In humans, shift-work and jet lag perturb clock synchronization, and such perturbations, when repeated or chronic, are strongly suspected to be detrimental to healthspan. This study investigated locomotor aging and longevity in Drosophila melanogaster with genetically or environmentally disrupted clocks. Two mutations in period (per, a gene essential for circadian rhythmicity in Drosophila) were compared, after introducing them in a common reference genetic background: the arrhythmic per01, and perT which displays robust short 16-h rhythms. Compared to the wild type, both per mutants showed reduced longevity and decreased startle-induced locomotion in aging flies, while spontaneous locomotor activity was not impaired. The per01 phenotypes were generally less severe than those of perT, suggesting that chronic jet lag is more detrimental to aging than arrhythmicity in Drosophila. Interestingly, the adjustment of environmental light-dark cycles to the endogenous rhythms of the perT mutant fully suppressed the acceleration in the age-related decline of startle-induced locomotion, while it accelerated this decline in wild-type flies. Overall, these results show that chronic jet lag accelerates a specific form of locomotor aging in Drosophila, and that this effect can be alleviated by environmental changes that ameliorate circadian rhythm synchronization.
Yerushalmi, G. Y., Misyura, L., Donini, A. and MacMillan, H. A. (2016). Chronic dietary salt stress mitigates hyperkalemia and facilitates chill coma recovery in Drosophila melanogaster. J Insect Physiol 95: 89-97. PubMed ID: 27642001
Chill susceptible insects like Drosophila lose the ability to regulate water and ion homeostasis at low temperatures. This loss of hemolymph ion and water balance drives a hyperkalemic state that depolarizes cells, causing cellular injury and death. The ability to maintain ion homeostasis at low temperatures and/or recover ion homeostasis upon rewarming is closely related to insect cold tolerance. It was thus hypothesized that changes to organismal ion balance, which can be achieved in Drosophila through dietary salt loading, could alter whole animal cold tolerance phenotypes. Drosophila melanogaster was put in the presence of diets highly enriched in NaCl, KCl, xylitol (an osmotic control) or sucrose (a dietary supplement known to impact cold tolerance) for 24h, and it was confirmed that they consumed the novel food. Independently of their osmotic effects, NaCl, KCl, and sucrose supplementation all improved the ability of flies to maintain K+ balance in the cold, which allowed for faster recovery from chill coma after 6h at 0° C. These supplements, however, also slightly increased the critical thermal minimum and had little impact on survival rates following chronic cold stress (24h at 0° C), suggesting that the effect of diet on cold tolerance depends on the measure of cold tolerance assessed. In contrast to prolonged salt stress, brief feeding (1.5h) on diets high in salt slowed coma recovery, suggesting that the long-term effects of NaCl and KCl on chilling tolerance result from phenotypic plasticity, induced in response to a salty diet, rather than simply the presence of the diet in the gut lumen.

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