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


Tuesday, June 30th, 2015

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Menon, K.P., Carrillo, R.A. and Zinn, K. (2015). The translational regulator Cup controls NMJ presynaptic terminal morphology. Mol Cell Neurosci [Epub ahead of print]. PubMed ID: 26102195
During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. It has been previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. This study examines the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. It was shown that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons; these mutants also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and it was shown that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with four genes (EndoA, WASp, Dap160, and Synj) encoding proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. These results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal.

Gokhale, A., et al. (2015). The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity. J Neurosci 35: 7643-7653. PubMed ID: 25972187.
Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis, and in neurons, synaptic vesicle assembly, neurotransmission, and plasticity. Protein networks, or interactomes, downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. This study consisted of a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. Components of the vesicle fusion machinery were identified as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons, among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa, and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function, the role for NSF was studied in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described, this study found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However, neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. These results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity.

Li, X., Wu, Y., Shen, C., Belenkaya, T. Y., Ray, L. and Lin, X. (2015). Drosophila p24 and Sec22 regulate Wingless trafficking in the early secretory pathway. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 26002470
The Wnt signaling pathway is crucial for development and disease. The regulation of Wnt protein trafficking is one of the pivotal issues in the Wnt research field. A genetic screen was performed in Drosophila melanogaster for genes involved in Wingless/Wnt secretion, and the p24 protein family members Baiser, CHOp24, Eclair and a v-SNARE protein Sec22, were identified that are involved in the early secretory pathway of Wingless/Wnt. Genetic evidence is provided demonstrating that loss of p24 proteins or Sec22 impedes Wingless (Wg) secretion in Drosophila wing imaginal discs. Baiser cannot replace other p24 proteins (CHOp24 or Eclair) in escorting Wg, and only Baiser and CHOp24 interact with Wg. Moreover, it was shown that the v-SNARE protein Sec22 and Wg are packaged together with p24 proteins. Taken together, these data provide important insights into the early secretory pathway of Wg/Wnt.

Takats, S., Varga, A., Pircs, K. and Juhasz, G. (2015). Loss of Drosophila Vps16A enhances autophagosome formation through reduced TOR activity. Autophagy [Epub ahead of print] PubMed ID: 26061715
The HOPS tethering complex facilitates autophagosome-lysosome fusion by binding to Syntaxin 17, the autophagosomal SNARE. This study show that loss of the core HOPS complex subunit Vps16A enhances autophagosome formation and slows down Drosophila development. Mechanistically, Tor kinase is less active in Vps16A mutants likely due to impaired endocytic and biosynthetic transport to the lysosome, a site of its activation. Tor reactivation by overexpression of Rheb suppresses autophagosome formation and restores growth and developmental timing in these animals. Thus, Vps16A reduces autophagosome numbers both by indirectly restricting their formation rate and by directly promoting their clearance. In contrast, the loss of Syx17/Syntaxin 17 blocks autophagic flux without affecting the induction step in Drosophila.

Monday, June 29th

Afonso, D.J., Liu, D., Machado, D.R., Pan, H., Jepson, J.E., Rogulja, D. and Koh, K. (2015). TARANIS functions with Cyclin A and Cdk1 in a novel arousal center to control sleep in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 26096977
Sleep is an essential and conserved behavior whose regulation at the molecular and anatomical level remains to be elucidated. This study identifies Taranis (Tara), a Drosophila homolog of the Trip-Br (SERTAD) family of transcriptional coregulators, as a molecule that is required for normal sleep patterns. Through a forward-genetic screen, tara was isolated as a novel sleep gene associated with a marked reduction in sleep amount. Targeted knockdown of tara suggests that it functions in cholinergic neurons to promote sleep. tara encodes a conserved cell-cycle protein that contains a Cyclin A (CycA)-binding homology domain. Tara regulates CycA protein levels and genetically and physically interacts with CycA to promote sleep. Furthermore, decreased levels of Cyclin-dependent kinase 1 (Cdk1), a kinase partner of CycA, rescue the short-sleeping phenotype of tara and CycA mutants, while increased Cdk1 activity mimics the tara and CycA phenotypes, suggesting that Cdk1 mediates the role of Tare and CycA in sleep regulation. Finally, a novel wake-promoting role was described for a cluster of ∼14 CycA-expressing neurons in the pars lateralis (PL), previously proposed to be analogous to the mammalian hypothalamus. The study proposes that Taranis controls sleep amount by regulating CycA protein levels and inhibiting Cdk1 activity in a novel arousal center.

Avila, F. W., et al. (2015). Retention of ejaculate by Drosophila melanogaster females requires the male-derived mating plug protein PEBme. Genetics [Epub ahead of print]. PubMed ID: 26058847.
Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). The Drosophila MP, which is maintained in the mated female for several hours post-mating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in fly reproduction, this study targeted PEBme for RNAi knockdown in males, compromising PMP coagulation in their mates and resulting in a significant reduction in female fertility, adversely affecting post-mating uterine conformation, sperm storage, mating refractoriness, egg-laying and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male and, when he ultimately uncoupled, the ejaculate was pulled out of the female. Thus PEBme and MP coagulation are required for optimal fertility in flies. These results highlight the importance of the MP and the proteins that comprise it in reproduction, and suggest that in Drosophila, the PMP is required to retain the ejaculate within the female reproductive tract to ensure the storage of sperm by mated females.

Akitake, B., Ren, Q., Boiko, N., Ni, J., Sokabe, T., Stockand, J. D., Eaton, B. A. and Montell, C. (2015). Coordination and fine motor control depend on Drosophila TRPgamma. Nat Commun 6: 7288. PubMed ID: 26028119
Motor coordination is broadly divided into gross and fine motor control, both of which depend on proprioceptive organs. However, the channels that function specifically in fine motor control are unknown. This study shows that mutations in trpγ disrupt fine motor control while leaving gross motor proficiency intact. The mutants are unable to coordinate precise leg movements during walking, and are ineffective in traversing large gaps due to an inability in making subtle postural adaptations that are requisite for this task. TRPγ is expressed in proprioceptive organs, and is required in both neurons and glia for gap crossing. TRPγ was expressed in vitro, and its activity was found to be promoted by membrane stretch. A mutation eliminating the Na+/Ca+ exchanger suppresses the gap-crossing phenotype of trpγ flies. These findings indicate that TRPγ contributes to fine motor control through mechanical activation in proprioceptive organs, thereby promoting Ca+ influx, which is required for function.

Sakata, K., Kawasaki, H., Suzuki, T., Ito, K., Negishi, O., Tsuno, T., Tsuno, H., Yamazaki, Y. and Ishida, N. (2015). Inositols affect the mating circadian rhythm of Drosophila melanogaster. Front Pharmacol 6: 111. PubMed ID: 26097456
Accumulating evidence indicates that the molecular circadian clock underlies the mating behavior of Drosophila melanogaster. However, information about which food components affect circadian mating behavior is scant. The ice plant, Mesembryanthemum crystallinum has recently become a popular functional food. This study shows that the close-proximity (CP) rhythm of D. melanogaster courtship behavior is damped under low-nutrient conditions, but is significantly enhanced by feeding the flies with powdered ice plant. Among various components of ice plants, it was found that myo-inositol increase the amplitude and slightly shorten the period of the CP rhythm. Real-time reporter assays show that myo-inositol and D-pinitol shorten the period of the circadian reporter gene Per2-luc in NIH 3T3 cells. These data suggest that the ice plant is a useful functional food and that the ability of inositols to shorten rhythms is a general phenomenon in insects as well as mammals.

Sunday, June 28th

Port, F., Muschalik, N. and Bullock, S. L. (2015). Systematic evaluation of Drosophila CRISPR tools reveals safe and robust alternatives to autonomous gene drives in basic research. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 25999583.
CRISPR/Cas technology allows rapid, site-specific genome modification in a wide variety of organisms. Proof-of-principle studies in Drosophila melanogaster have used various CRISPR/Cas tools and experimental designs, leading to significant uncertainty in the community about how to put this technology into practice. Moreover, it is unclear what proportion of genomic target sites can be modified with high efficiency. This study addressed these issues by systematically evaluating available CRISPR/Cas reagents and methods in Drosophila. The findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. Gene editing was performed at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. This method was contrasted to recently developed autonomous gene drive technology for somatic and germ line genome engineering, and it was concluded that optimized CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk.
Dunst, S., Kazimiers, T., von Zadow, F., Jambor, H., Sagner, A., Brankatschk, B., Mahmoud, A., Spannl, S., Tomancak, P., Eaton, S. and Brankatschk, M. (2015). Endogenously tagged rab proteins: a resource to study membrane trafficking in Drosophila. Dev Cell 33: 351-365. PubMed ID: 25942626
Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. All 27 Drosophila Rabs were tagged in this study with YFP(MYC) at their endogenous chromosomal loci; their expression and subcellular localization was determined in six tissues comprising 23 cell types, and this data was provided in an annotated, searchable image database. The utility of these lines were demonstrated for controlled knockdown, and similar subcellular localization were shown to be able to predict redundant functions. This comprehensive resource was exploited to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments.

Sanders, J., Singh, A., Sterne, G., Ye, B. and Zhou, J. (2015). Learning-guided automatic three dimensional synapse quantification for Drosophila neurons. BMC Bioinformatics 16: 177. PubMed ID: 26017624
The subcellular distribution of synapses is fundamentally important for the assembly, function, and plasticity of the nervous system. Common practices for synapse quantification in neuroscience labs remain largely manual or semi-manual. This is mainly due to computational challenges in automatic quantification of synapses, including large volume, high dimensions and staining artifacts. In the case of confocal imaging, optical limit and xy-z resolution disparity also require special considerations to achieve the necessary robustness. A novel algorithm is presented in the paper for learning-guided automatic recognition and quantification of synaptic markers in 3D confocal images. The method developed a discriminative model based on 3D feature descriptors that detected the centers of synaptic markers. It made use of adaptive thresholding and multi-channel co-localization to improve the robustness. The detected markers then guided the splitting of synapse clumps, which further improved the precision and recall of the detected synapses. Algorithms were tested on lobula plate tangential cells (LPTCs) in the brain of Drosophila melanogaster, for GABAergic synaptic markers on axon terminals as well as dendrites. The presented method was able to overcome the staining artifacts and the fuzzy boundaries of synapse clumps in 3D confocal image, and automatically quantify synaptic markers in a complex neuron such as LPTC. Comparison with some existing tools used in automatic 3D synapse quantification also proved the effectiveness of the proposed method.

Javeed, N., Tardi, N. J., Maher, M., Singari, S. and Edwards, K. A. (2015). Controlled expression of Drosophila homeobox loci using the Hostile takeover system. Dev Dyn 244: 808-825. PubMed ID: 26017699.
Hostile takeover (Hto) is a Drosophila protein trapping system that allows the investigator to both induce a gene and tag its product. The Hto transposon carries a GAL4-regulated promoter expressing an exon encoding a FLAG-mCherry tag. Upon expression, the Hto exon can splice to a downstream genomic exon, generating a fusion transcript and tagged protein. Using rough-eye phenotypic screens, Hto inserts were recovered at eight homeobox or Pax loci: cut, Drgx/CG34340, Pox neuro, araucan, shaven/D-Pax2, Zn finger homeodomain 2, Sex combs reduced (Scr), and the abdominal-A region. The collection yields diverse misexpression phenotypes. Ectopic Drgx was found to alter the cytoskeleton and cell adhesion in ovary follicle cells. Hto expression of cut, araucan, or shaven gives phenotypes similar to those of the corresponding UAS-cDNA constructs. The cut and Pox neuro phenotypes are suppressed by the corresponding RNAi constructs. The Scr and abdominal-A inserts do not make fusion proteins, but may act by chromatin- or RNA-based mechanisms. It is concluded that Hto can effectively express tagged homeodomain proteins from their endogenous loci; the Minos vector allows inserts to be obtained even in transposon cold-spots. Hto screens may recover homeobox genes at high rates because they are particularly sensitive to misexpression.

Saturday, June 27th

Jha, A. R., Miles, C. M., Lippert, N. R., Brown, C. D., White, K. P. and Kreitman, M. (2015). Whole genome resequencing of experimental populations reveals polygenic basis of egg size variation in Drosophila melanogaster. Mol Biol Evol. PubMed ID: 26044351.
Complete genome resequencing of populations holds great promise in deconstructing complex polygenic traits to elucidate molecular and developmental mechanisms of adaptation. Egg size is a classic adaptive trait in insects, birds, and other taxa, but its highly polygenic architecture has prevented high-resolution genetic analysis. This study used replicated experimental evolution in Drosophila melanogaster and whole-genome sequencing to identify consistent signatures of polygenic egg size adaptation. A generalized linear mixed model revealed reproducible allele frequency differences between replicated experimental populations selected for large and small egg volumes at ~4,000 single nucleotide polymorphisms (SNP). Several hundred distinct genomic regions contain clusters of these SNPs and have lower heterozygosity than the genomic background, consistent with selection acting on polymorphisms in these regions. These SNPs are also enriched among genes expressed in Drosophila ovaries and many of these genes have well-defined functions in Drosophila oogenesis. Additional genes regulating egg development, growth, and cell size show evidence of directional selection as genes regulating these biological processes are enriched for highly differentiated SNPs. Genetic crosses performed with a subset of candidate genes demonstrated that these genes influence egg size, at least in the large genetic background. These findings confirm the highly polygenic architecture of this adaptive trait, and suggest the involvement of many novel candidate genes in regulating egg size.
Hoskins, J. L., Ritchie, M. G. and Bailey, N. W. (2015). A test of genetic models for the evolutionary maintenance of same-sex sexual behaviour. Proc Biol Sci 282 [Epub ahead of print]. PubMed ID: 26019160
The evolutionary maintenance of same-sex sexual behaviour (SSB) has received increasing attention because it is perceived to be an evolutionary paradox. The genetic basis of SSB is almost wholly unknown in non-human animals, though this is key to understanding its persistence. Recent theoretical work has yielded broadly applicable predictions centered on two genetic models for SSB: overdominance and sexual antagonism. Using Drosophila melanogaster, natural genetic variation for male SSB was assayed and predictions were empirically tested about the mode of inheritance and fitness consequences of alleles influencing its expression. Fifty inbred lines derived from a wild population were screened for male-male courtship and copulation behaviour, and crosses between the lines were examined for evidence of overdominance and antagonistic fecundity selection. Consistent variation among lines revealed heritable genetic variation for SSB, but the nature of the genetic variation was complex. Phenotypic and fitness variation was consistent with expectations under overdominance, although predictions of the sexual antagonism model were also supported. An unexpected and strong paternal effect on the expression of SSB was found, suggesting possible Y-linkage of the trait. These results inform evolutionary genetic mechanisms that might maintain low but persistently observed levels of male SSB in D. melanogaster, but highlight a need for broader taxonomic representation in studies of its evolutionary causes.

Lee, Y. C. (2015). The role of piRNA-mediated epigenetic silencing in the population dynamics of transposable elements in Drosophila melanogaster. PLoS Genet 11: e1005269. PubMed ID: 26042931.
The piwi-interacting RNAs (piRNA) are small RNAs that target selfish transposable elements (TEs) in many animal genomes. Until now, piRNAs' role in TE population dynamics has only been discussed in the context of their suppression of TE transposition, which alone is not sufficient to account for the skewed frequency spectrum and stable containment of TEs. On the other hand, euchromatic TEs can be epigenetically silenced via piRNA-dependent heterochromatin formation and, similar to the widely known "Position-effect variegation", heterochromatin induced by TEs can "spread" into nearby genes. This study hypothesized that the piRNA-mediated spread of heterochromatin from TEs into adjacent genes has deleterious functional effects and leads to selection against individual TEs. Unlike previously identified deleterious effects of TEs due to the physical disruption of DNA, the functional effect investigated in this study is mediated through the epigenetic influences of TEs. The repressive chromatin mark, H3K9me, was found to be elevated in sequences adjacent to euchromatic TEs at multiple developmental stages in Drosophila melanogaster. Furthermore, the heterochromatic states of genes depend not only on the number of and distance from adjacent TEs, but also on the likelihood that their nearest TEs are targeted by piRNAs. These variations in chromatin status probably have functional consequences, causing genes near TEs to have lower expression. Importantly, stronger selection against TEs were found that lead to higher H3K9me enrichment of adjacent genes, demonstrating the pervasive evolutionary consequences of TE-induced epigenetic silencing. Because of the intrinsic biological mechanism of piRNA amplification, spread of TE heterochromatin could result in the theoretically required synergistic deleterious effects of TE insertions for stable containment of TE copy number. The indirect deleterious impact of piRNA-mediated epigenetic silencing of TEs is a previously unexplored, yet important, element for the evolutionary dynamics of TEs.

Coolon, J. D., Stevenson, K. R., McManus, C. J., Yang, B., Graveley, B. R. and Wittkopp, P. J. (2015). Molecular mechanisms and evolutionary processes contributing to accelerated divergence of gene expression on the Drosophila X chromosome. Mol Biol Evol [Epub ahead of print]. PubMed ID: 26041937
In species with a heterogametic sex, population genetics theory predicts that DNA sequences on the X chromosome can evolve faster than comparable sequences on autosomes. Both neutral and non-neutral evolutionary processes can generate this pattern. Complex traits like gene expression are not predicted to have accelerated evolution by these theories, yet a "faster-X" pattern of gene expression divergence has recently been reported for both Drosophila and mammals. This study tested the hypothesis that accelerated adaptive evolution of cis-regulatory sequences on the X chromosome is responsible for this pattern by comparing the relative contributions of cis- and trans-regulatory changes to patterns of faster-X expression divergence observed between strains and species of Drosophila with a range of divergence times. Support was found for this hypothesis, especially among male-biased genes, when comparing different species. However, evidence was also found that trans-regulatory differences contribute to a faster-X pattern of expression divergence both within and between species. This contribution is surprising because trans-acting regulators of X-linked genes are generally assumed to be randomly distributed throughout the genome. This study found, however, that X-linked transcription factors appear to preferentially regulate expression of X-linked genes, providing a potential mechanistic explanation for this result. The contribution of trans-regulatory variation to faster-X expression divergence was larger within than between species, suggesting it is more likely to result from neutral processes than positive selection. These data show how accelerated evolution of both coding and non-coding sequences on the X chromosome can lead to accelerated expression divergence on the X chromosome relative to autosomes.

Rostant, W. G., Kay, C., Wedell, N. and Hosken, D. J. (2015). Sexual conflict maintains variation at an insecticide resistance locus. BMC Biol 13: 34. PubMed ID: 26032845
The maintenance of genetic variation through sexually antagonistic selection is controversial, partly because specific sexually-antagonistic alleles have not been identified. The Drosophila DDT resistance allele (DDT-R) is an exception. This allele increases female fitness, but simultaneously decreases male fitness, and it has been suggested that this sexual antagonism could explain why polymorphism was maintained at the locus prior to DDT use. This possibility was tested using a genetic model and then used evolving fly populations to test model predictions. Theory confirmed that sexual antagonism is able to maintain genetic variation at this locus, hence explaining why DDT-R did not fix prior to DDT use despite increasing female fitness, and experimentally evolving fly populations verified theoretical predictions. C This demonstrates that sexually antagonistic selection can maintain genetic variation and explains the DDT-R frequencies observed in nature.

Durisko, Z., Kemp, R., Mubasher, R. and Dukas, R. (2014). Dynamics of social behavior in fruit fly larvae. PLoS One 9: e95495. PubMed ID: 24740198
This study quantified the extent and dynamics of social interactions among fruit fly larvae over time. Both a wild-type laboratory population and a recently-caught strain of larvae spontaneously formed social foraging groups. Levels of aggregation initially increased during larval development and then declined with the wandering stage before pupation. Larvae aggregated more on hard than soft food, and more at sites where the surface of the food had been previously broken. Groups of larvae initiated burrowing sooner than solitary individuals, indicating that one potential benefit of larval aggregations is an improved ability to dig and burrow into the food substrate. Two closely related species, D. melanogaster and D. simulans, differ in their tendency to aggregate, which may reflect different evolutionary histories. This protocol for quantifying social behavior in larvae uncovered robust social aggregations in this simple model, which is highly amenable to neurogenetic analyses, and can serve for future research into the mechanisms and evolution of social behavior.

Friday, June 26th

Sen, A., Kalvakuri, S., Bodmer, R. and Cox, R. T. (2015) Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila. Dis Model Mech 8: 577-589. PubMed ID: 26035866
Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease. However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. This study shows that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previous studies have found that clu genetically interacts with park in Drosophila female germ cells. This study shows that clu also genetically interacts with PINK1, and epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.

Holmbeck, M. A., Donner, J. R., Villa-Cuesta, E. and Rand, D. M. (2015) A Drosophila model for mito-nuclear diseases generated by an incompatible tRNA-tRNA synthetase interaction. Dis Model Mech. PubMed ID: 26035388
Communication between the mitochondrial and nuclear genomes is vital for cellular function. The assembly of mitochondrial enzyme complexes that produce the majority of cellular energy requires the coordinated expression and translation of both mitochondrial and nuclear encoded proteins. The joint genetic architecture of this system complicates the basis of mitochondrial diseases, and mutations in both mtDNA- and nuclear-encoded genes have been implicated in mitochondrial dysfunction. Previously, in a set of mitochondrial-nuclear introgression strains, a dual genome epistasis was characterized in which a naturally occurring mutation in the D. simulans simw501 mtDNA-encoded tRNA for tyrosine interacts with a mutation in the nuclear encoded mitochondrial localized tyrosyl-tRNA synthetase from D. melanogaster. This study shows that the incompatible mitochondrial-nuclear combination results in locomotor defects, reduced mitochondrial respiratory capacity, decreased OXPHOS enzyme activity, and severe alterations in mitochondrial morphology. Transgenic rescue strains containing nuclear variants of the tyrosyl-tRNA synthetase are sufficient to rescue many of the deleterious phenotypes identified when paired with the simw501mtDNA. However, the severity of this defective mito-nuclear interaction varies across traits and genetic backgrounds, suggesting that the impact of mitochondrial dysfunction may be tissue specific. Because mutations in mitochondrial tRNATyr are associated with exercise intolerance in humans, this mitochondrial-nuclear introgression model in Drosophila provides a means to dissect the molecular basis of these, and other mitochondrial diseases that are a consequence of the joint genetic architecture of mitochondrial function.

Ott, S., Dziadulewicz, N. and Crowther, D. C. (2015). Iron is a specific cofactor for distinct oxidation- and aggregation-dependent Aβ toxicity mechanisms. Dis Model Mech [Epub ahead of print]. PubMed ID: 26035384
Metals including iron are present at high concentrations in amyloid plaques in patients with Alzheimer's disease where they are also thought to be co-factors in generating oxidative stress and modulating amyloid formation. This study presents data from several Drosophila models of neurodegenerative proteinopathies indicating that the interaction between iron and Aβ (see Drosophila Appl) is specific and is not seen for other aggregation-prone polypeptides. The interaction with iron is likely important in the dimerisation of Aβ and is mediated by three N-terminal histidines. Transgenic fly lines systematically expressing all combinations of His>Ala substitutions in Aβ were generated and were used to study the pathological role of these residues. Developmental eye phenotypes, longevity and histological examinations indicate that the N-terminal histidines have distinct position-dependent and -independent mechanisms. The former mediate the toxic effects of metals and Aβ aggregation under non-oxidising conditions and the latter are relevant under oxidising conditions. Understanding how Aβ mediates neurotoxic effects in vivo will help better targeting of pathological pathways using aggregation-blockers and metal-modifying agents.

Yang, D., Abdallah, A., Li, Z., Lu, Y., Almeida, S. and Gao, F. B. (2015). FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions. Acta Neuropathol [Epub ahead of print]. PubMed ID: 26031661
C9ORF72 repeat expansion is the most common genetic mutation in frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Abnormal dipeptide repeat proteins (DPRs) generated from repeat-associated non-AUG (RAN) translation of repeat-containing RNAs are thought to be pathogenic; however, the mechanisms are unknown. This study reports that (GR)80 and (PR)80 are toxic in neuronal and non-neuronal cells in Drosophila. In contrast to reported shorter poly(GR) forms, (GR)80 is mostly localized throughout the cytosol without detectable accumulation in the nucleolus, accompanied by suppression of Notch signaling and cell loss in the wing. Some Notch target genes are also downregulated in brains and iPSC-derived cortical neurons of C9ORF72 patients. Increased Notch expression largely suppressed (GR)80-induced cell loss in the wing. When co-expressed in Drosophila, HeLa cells, or human neurons, (GA)80 recruited (GR)80 into cytoplasmic inclusions, partially decreasing the toxicity of (GR)80 and restoring Notch signaling in Drosophila. Thus, different DPRs have opposing roles in cell loss and this study has identified the Notch pathway as one of the receptor signaling pathways that might be compromised in C9ORF72 FTD/ALS.

Thursday, June 25th

Koshikawa, S., Giorgianni, M. W., Vaccaro, K., Kassner, V. A., Yoder, J. H., Werner, T. and Carroll, S. B. (2015) Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila. Proc Natl Acad Sci U S A 112: 7524-7529. PubMed ID: 26034272
Changes in gene expression during animal development are largely responsible for the evolution of morphological diversity. However, the genetic and molecular mechanisms responsible for the origins of new gene-expression domains have been difficult to elucidate. This study sought to identify molecular events underlying the origins of three novel features of wingless (wg) gene expression that are associated with distinct pigmentation patterns in Drosophila guttifera. The activity of cis-regulatory sequences (enhancers) across the wg locus in D. guttifera and Drosophila melanogaster were compared, and strong functional conservation was found among the enhancers that control similar patterns of wg expression in larval imaginal discs that are essential for appendage development. For pupal tissues, however, three novel wg enhancer activities were found in D. guttifera associated with novel domains of wg expression, including two enhancers located surprisingly far away in an intron of the distant Wnt10 gene. Detailed analysis of one enhancer (the vein-tip enhancer) revealed that it overlapped with a region controlling wg expression in wing crossveins (crossvein enhancer) in D. guttifera and other species. These results indicate that one novel domain of wg expression in D. guttifera wings evolved by co-opting pre-existing regulatory sequences governing gene activity in the developing wing. It is suggested that the modification of existing enhancers is a common path to the evolution of new gene-expression domains and enhancers.

Wei, Y., Mondal, S. S., Mouawad, R., Wilczynski, B., Henry, R. W. and Arnosti, D. N. (2015) Genome-wide analysis of Drosophila Rbf2 protein highlights diversity of RB family targets and possible role in regulation of ribosome biosynthesis. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 25999584
Rbf2 is a recently evolved Retinoblastoma family member in Drosophila, differing from Rbf1 especially in the C-terminus. To investigate whether the unique features of Rbf2 contribute to diverse roles in gene regulation, ChIP-Seq analysis was performed for both Rbf2 and Rbf1 in embryos. A previous model for Rb-E2F interactions suggested that Rbf1 binds dE2F1 or dE2F2, while Rbf2 is restricted to binding to dE2F2, however, this study found that Rbf2 targets approximately twice as many genes as Rbf1. Highly enriched among the Rbf2 targets were ribosomal protein genes. The functional significance of this finding was tested by assessing Rbf activity on ribosomal protein promoters and the endogenous genes. Rbf1 and Rbf2 significantly repressed expression of some ribosomal protein genes, although not all bound genes showed transcriptional effects. Interestingly, many ribosomal protein genes are similarly targeted in human cells, indicating that these interactions may be relevant for control of ribosome biosynthesis and growth. Bioinformatic analysis was carried out to investigate the basis for differential targeting by these two proteins, and Rbf2-specific promoters were found to have distinct sequence motifs, suggesting unique targeting mechanisms. Association of Rbf2 with these promoters appears to be independent of dE2F2/dDP, although promoters bound by both Rbf1 and Rbf2 require dE2F2/dDP. The presence of unique Rbf2 targets suggest that evolutionary appearance of this corepressor represents the acquisition of potentially novel roles in gene regulation for the RB family.

Shir-Shapira, H., Sharabany, J., Filderman, M., Ideses, D., Ovadia-Shochat, A., Mannervik, M. and Juven-Gershon, T. (2015). Structure-function analysis of the Drosophila melanogaster Caudal provides insights into core promoter-preferential activation. J Biol Chem [Epub ahead of print]. PubMed ID: 26018075
Regulation of RNA polymerase II transcription is critical for the proper development, differentiation and growth of an organism. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters encompass the RNA start site and consist of functional elements such as the TATA box, initiator and downstream core promoter element (DPE), which confer specific properties to the core promoter. Previous studies have discovered that Drosophila Caudal, which is a master regulator of genes involved in development and differentiation, is a DPE-specific transcriptional activator. This study shows that the mouse Caudal-related Cdx proteins (mCdx1, mCdx2 and mCdx4) are also preferential core promoter transcriptional activators. To elucidate the mechanism that enables Caudal to preferentially activate DPE transcription, structure-function analysis was performed. Using a systematic series of deletion mutants (all containing the intact DNA-binding homeodomain) it was discovered that the C-terminal region of Caudal contributes to the preferential activation of the fushi tarazu (ftz). Caudal target gene. Furthermore, the region containing both the homeodomain and the C-terminus of Caudal is sufficient to confer core promoter-preferential activation to the heterologous GAL4 DNA-binding domain. Importantly, it was discovered that Drosophila CBP (dCBP) is a co-activator for Caudal-regulated activation of ftz. Strikingly, dCBP confers the ability to preferentially activate the DPE-dependent ftz reporter to mini-Caudal proteins that are unable to preferentially activate ftz transcription themselves. Taken together, it is the unique combination of dCBP and Caudal that enables the co-activation of ftz in a core promoter-preferential manner.

Li, X., Ghezzi, A., Krishnan, H. R., Pohl, J. B., Bohm, A. Y. and Atkinson, N. S. (2015). A histone modification identifies a DNA element controlling slo BK channel gene expression in muscle. J Neurogenet: 1-32. PubMed ID: 25967280
The slowpoke (slo) gene encodes BK type Ca2+-activated K+ channels. In Drosophila, expression of slo is induced by organic solvent sedation (benzyl alcohol and ethanol) and this increase in neural slo expression contributes to the production of functional behavioral tolerance (inducible resistance) to these drugs. Within the slo promoter region, it was observed that benzyl alcohol sedation produces a localized spike of histone acetylation over a 65 n conserved DNA element called 55b. Changes in histone acetylation are commonly the consequence of transcription factor activity and previously, a localized histone acetylation spike was used to successfully map a DNA element involved in benzyl alcohol-induced slo expression. To determine whether the 55b element was also involved in benzyl alcohol-induced neural expression of slo, it was deleted from the endogenous slo gene by homologous recombination. Flies lacking the 55b element were normal with respect to basal and benzyl alcohol-induced neural slo expression, the capacity to acquire and maintain functional tolerance, their threshold for electrically-induced seizures and most slo-related behaviors. Removal of the 55b element did however increase the level of basal expression from the muscle/tracheal cell-specific slo core promoter and produced a slight increase in overall locomotor activity. It is concluded that the 55b element is involved in control of slo expression from the muscle and tracheal-cell promoter but is not involved in the production of functional benzyl alcohol tolerance.

Wednesday, June 24th

Jaszczak, J.S., Wolpe, J.B., Dao, A.Q. and Halme, A. (2015). Nitric oxide synthase regulates growth coordination during Drosophila melanogaster imaginal disc regeneration. Genetics [Epub ahead of print]. PubMed ID: 26081194
Mechanisms that coordinate growth during development are essential for producing animals with proper organ proportion. This study describes a pathway through which tissues communicate to coordinate growth. During Drosophila melanogaster larval development, damage to imaginal discs activates a regeneration checkpoint through expression of Dilp8. This produces both a delay in developmental timing and slows the growth of undamaged tissues, coordinating regeneration of the damaged tissue with developmental progression and overall growth. It was demonstrated that Dilp8-dependent growth coordination between regenerating and undamaged tissues, but not developmental delay, requires the activity of nitric oxide synthase (NOS) in the prothoracic gland. NOS limits the growth of undamaged tissues by reducing ecdysone biosynthesis, a requirement for imaginal disc growth during both the regenerative checkpoint and normal development. Therefore, NOS activity in the prothoracic gland coordinates tissue growth through regulation of endocrine signals.

Di Cara, F., et al. (2015). The Hippo pathway promotes cell survival in response to chemical stress. Cell Death Differ [Epub ahead of print]. PubMed ID: 26021298
Cellular stress defense mechanisms have evolved to maintain homeostasis in response to a broad variety of environmental challenges. To identify novel players acting in stress response pathways, a cell culture RNA interference (RNAi) screen was conducted using caffeine as a xenobiotic stress-inducing agent, as this compound is a well-established inducer of detoxification response pathways. Specifically, how caffeine affects cell survival was evaluated when Drosophila kinases and phosphatases were depleted via RNAi. Using this approach, ten kinases and 4 phosphatases that are essential for cell survival were identified. Remarkably, the screen yielded an enrichment of Hippo pathway components, indicating that this pathway regulates cellular stress responses. Indeed, it was shown that the Hippo pathway acts as a potent repressor of stress-induced cell death. Further, it was demonstrate that Hippo activation is necessary to inhibit a pro-apoptotic program triggered by the interaction of the transcriptional co-activator Yki with the transcription factor p53 in response to a range of stress stimuli. These in vitro and in vivo loss-of-function data therefore implicate Hippo signaling in the transduction of cellular survival signals in response to chemical stress.

Zang, Y., et al. (2015). Plasma membrane overgrowth causes fibrotic collagen accumulation and immune activation in Drosophila adipocytes. Elife [Epub ahead of print]. PubMed ID: 26090908
Many chronic diseases are associated with fibrotic deposition of Collagen and other matrix proteins. This study shows that plasma membrane overgrowth causes pericellular Collagen accumulation in Drosophila adipocytes. It was found that loss of Dynamin and other endocytic components causes pericellular trapping of outgoing Collagen IV due to dramatic cortex expansion when endocytic removal of plasma membrane is prevented. Deposits also form in the absence of negative Toll immune regulator Cactus, excess plasma membrane being caused in this case by increased secretion. Finally, it was shown that trimeric Collagen accumulation, downstream of Toll or endocytic defects, activates a tissue damage response. This work indicates that traffic imbalances and plasma membrane topology may contribute to fibrosis. It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases.

Kuo, Y., Huang, H., Cai, T. and Wang, T. (2015). Target of Rapamycin Complex 2 regulates cell growth via Myc in Drosophila. Sci Rep 5: 10339. PubMed ID: 25999153
Target of rapamycin (TOR) is an evolutionarily conserved serine/threonine protein kinase that functions as a central regulator of cellular growth and metabolism by forming two distinct complexes: TOR complex 1 (TORC1) and TORC2. As well as TORC1, TORC2 plays a key role in regulation of cell growth. But little is known about how TORC2 regulates cell growth. The transcription factor Myc also plays a critical role in cell proliferation and growth. This study reports that TORC2 and Myc regulate cell growth via a common pathway. Expression of Myc fully rescues growth defects associated with lst8 and rictor mutations, both of which encode essential components of TORC2. Furthermore, loss of TORC2 disrupted the nuclear localization of Myc, and inhibited Myc-dependent transcription. Together, these results reveal a Myc-dependent pathway by which TORC2 regulates cell growth.

Tuesday, June 23rd

Berry, J.A., Cervantes-Sandoval, I., Chakraborty, M. and Davis, R.L. (2015). Sleep facilitates memory by blocking dopamine neuron-mediated forgetting. Cell [Epub ahead of print]. PubMed ID: 26073942
Early studies from psychology suggest that sleep facilitates memory retention by stopping ongoing retroactive interference caused by mental activity or external sensory stimuli. Neuroscience research with animal models, on the other hand, suggests that sleep facilitates retention by enhancing memory consolidation. Recently, in Drosophila, the ongoing activity of specific dopamine neurons was shown to regulate the forgetting of olfactory memories. This study shows that this ongoing dopaminergic activity is modulated with behavioral state, increasing robustly with locomotor activity and decreasing with rest. Increasing sleep-drive, with either the sleep-promoting agent Gaboxadol or by genetic stimulation of the neural circuit for sleep, decreases ongoing dopaminergic activity, while enhancing memory retention. Conversely, increasing arousal stimulates ongoing dopaminergic activity and accelerates dopaminergic-based forgetting. Therefore, forgetting is regulated by the behavioral state modulation of dopaminergic-based plasticity. These findings integrate psychological and neuroscience research on sleep and forgetting.

Busto, G.U., Guven-Ozkan, T., Fulga, T.A., Van Vactor, D. and Davis, R.L. (2015). microRNAs that promote or inhibit memory formation in Drosophila melanogaster. Genetics 200: 569-580. PubMed ID: 26088433
microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Prior studies have shown that they regulate numerous physiological processes critical for normal development, cellular growth control, and organismal behavior. This study systematically surveyed 134 different miRNAs for roles in olfactory learning and memory formation using "sponge" technology to titrate their activity broadly in the Drosophila melanogaster central nervous system. At least five different miRNAs involved in memory formation or retention were identified from this large screen, including miR-9c, miR-31a, miR-305, miR-974, and miR-980. Surprisingly, the titration of some miRNAs increases memory, while the titration of others decreases memory. More detailed experiments were performed on two miRNAs, miR-974 and miR-31a, by mapping their roles to subpopulations of brain neurons and testing the functional involvement in memory of potential mRNA targets through bioinformatics and a RNA interference knockdown approach. This screen offers an important first step toward the comprehensive identification of all miRNAs and their potential targets that serve in gene regulatory networks important for normal learning and memory.

Jaumouille, E., Machado Almeida, P., Stahli, P., Koch, R. and Nagoshi, E. (2015). Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock. Curr Biol 25: 1502-1508. PubMed ID: 26004759
Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and Clock/Cycle (Clk/Cyc) (in Drosophila) drive the expression of the period (per) family genes. The Per-containing complexes inhibit the activity of CLOCK/BMAL1 or Clk/Cyc, thereby forming a negative feedback loop. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. This study shows that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor Unfulfilled (Hr51) are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and Unf bind to per regulatory sequences and act together to enhance the Clk/Cyc-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. These results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks (Jaumouille, 2015).

Miyazaki, T., Lin, T. Y., Ito, K., Lee, C. H. and Stopfer, M. (2015). A gustatory second-order neuron that connects sucrose-sensitive primary neurons and a distinct region of the gnathal ganglion in the Drosophila brain. J Neurogenet: 1-26. PubMed ID: 26004543
This study identified and characterized a bilateral pair of gustatory second-order neurons in Drosophila. GRASP (GFP reconstitution across synaptic partners) was combined with presynaptic labeling to visualize potential synaptic contacts between the dendrites of the candidate gustatory second-order neurons (G2Ns) and the axonal terminals of Gr5a-expressing gustatory sensory neurons GSNs, which are known to respond to sucrose. Results of this analysis revealed a pair of neurons that contact Gr5a axon terminals in both brain hemispheres and send axonal arborizations to a distinct region outside the PGC but within the GNG. To characterize the input and output branches, respectively, fluorescence-tagged acetylcholine receptor subunit (Dalpha7) and active-zone marker (Brp) were expressed in the G2Ns. G2N input sites were found to overlay GRASP-labeled synaptic contacts to Gr5a neurons, while presynaptic sites were broadly distributed throughout the neurons' arborizations. The identified G2Ns were found to receive synaptic inputs from Gr5a-expressing GSNs, but not Gr66a-expressing GSNs, which respond to caffeine. The identified G2Ns relay information from Gr5a-expressing GSNs to distinct regions in the gnathal ganglia (GNG), and are distinct from other, recently identified gustatory projection neurons, which relay information about sugars to a brain region called the antennal mechanosensory and motor center (AMMC). These findings suggest unexpected complexity for taste information processing in the first relay of the gustatory system.

Ha, A.1, Polyanovsky, A. and Avidor-Reiss, T. (2015). DrosophilaHook-Related Protein (Girdin) is essential for sensory dendrite formation. Genetics [Epub ahead of print]. PubMed ID: 26058848
The dendrite of the sensory neuron is surrounded by support cells and is composed of two specialized compartments: the inner segment and the sensory cilium. How the sensory dendrite is formed and maintained is not well understood. Hook-related proteins (HkRP) like Girdin, DAPLE, and Gipie are actin-binding proteins, implicated in actin organization and in cell motility. This study shows that the Drosophila melanogaster single member of the Hook-related protein family, Girdin, is essential for sensory dendrite formation and function. Mutations in girdin were identified during a screen for fly mutants with no mechanosensory function. Physiological, morphological, and ultra-structural studies of girdin mutant flies indicate that the mechanosensory neurons innervating external sensory organs (bristles) initially form a ciliated dendrite that degenerates shortly after, followed by the clustering of their cell bodies. Importantly, it was observed that Girdin is expressed transiently during dendrite morphogenesis in three previously unidentified actin-based structures surrounding the inner segment tip and the sensory cilium. These actin structures are largely missing in girdin. Defects in cilia are observed in other sensory organs such as those mediating olfaction and taste, suggesting that Girdin has a general role in forming sensory dendrites in Drosophila. These suggest that Girdin functions temporarily within the sensory organ and that this function is essential for the formation of the sensory dendrites via actin structures.

Wang, Y., Wang, H., Li, X. and Li, Y. (2015). Epithelial microRNA-9a regulates dendrite growth through Fmi-Gq signaling in Drosophila sensory neurons. Dev Neurobiol [Epub ahead of print]. PubMed ID: 26016469
microRNA-9 (miR-9) is highly expressed in the nervous system across species and plays essential roles in neurogenesis and axon growth; however, little is known about the mechanisms that link miR-9a with dendrite growth. Using an in vivo model of Drosophila class I dendrite arborization (da) neurons, miR-9a, a Drosophila homolog of mammalian miR-9a, was shown to downregulate the cadherin protein Flamingo (Fmi) thereby attenuating dendrite development in a non-cell autonomous manner. In miR-9a knockout mutants, the dendrite length of a sensory neuron ddaE was significantly increased. Intriguingly, miR-9a is specifically expressed in epithelial cells but not in neurons, thus the expression of epithelial but not neuronal Fmi is greatly elevated in miR-9a mutants. In contrast, overexpression of Fmi in the neuron resulted in a reduction in dendrite growth, suggesting that neuronal Fmi plays a suppressive role in dendrite growth, and that increased epithelial Fmi might promote dendrite growth by competitively binding to neuronal Fmi. Fmi has been proposed as a G protein-coupled receptor (GPCR). Neuronal G protein Galphaq (Gq), but not Go, may function downstream of Fmi to negatively regulate dendrite growth. Taken together, these results reveal a novel function of miR-9a in dendrite morphogenesis. Moreover, it is suggested that Gq might mediate the intercellular signal of Fmi in neurons to suppress dendrite growth. These findings provide novel insights into the complex regulatory mechanisms of microRNAs in dendrite development, and further reveal the interplay between the different components of Fmi, functioning in cadherin adhesion and GPCR signalling.

Monday, June 22nd

González-Morales, .N, Géminard, C., Lebreton, G., Cerezo, D., Coutelis, J.B. and Noselli, S. (2015). The atypical cadherin Dachsous controls left-right asymmetry in Drosophila. Dev Cell [Epub ahead of print]. PubMed ID: 26073018
Left-right (LR) asymmetry is essential for organ development and function in metazoans, but how initial LR cue is relayed to tissues still remains unclear. This study proposes a mechanism by which the Drosophila LR determinant Myosin ID (MyoID) transfers LR information to neighboring cells through the planar cell polarity (PCP) atypical cadherin Dachsous (Ds). Molecular interaction between MyoID and Ds in a specific LR organizer controls dextral cell polarity of adjoining hindgut progenitors and is required for organ looping in adults. Loss of Ds blocks hindgut tissue polarization and looping, indicating that Ds is a crucial factor for both LR cue transmission and asymmetric morphogenesis. It was further shown that the Ds/Fat and Frizzled PCP pathways are required for the spreading of LR asymmetry throughout the hindgut progenitor tissue. These results identify a direct functional coupling between the LR determinant MyoID and PCP, essential for non-autonomous propagation of early LR asymmetry.

Xie, S. and Martin, A. C. (2015) Intracellular signalling and intercellular coupling coordinate heterogeneous contractile events to facilitate tissue folding. Nat Commun 6: 7161. PubMed ID: 26006267
Cellular forces generated in the apical domain of epithelial cells reshape tissues. Recent studies highlighted an important role for dynamic actomyosin contractions, called pulses, that change cell and tissue shape. Net cell shape change depends on whether cell shape is stabilized, or ratcheted, between pulses. Whether there are different classes of contractile pulses in wild-type embryos and how pulses are spatiotemporally coordinated is unknown. This study developed a computational framework to identify and classify pulses and determine how pulses are coordinated during invagination of the Drosophila ventral furrow. Biased transitions in pulse behaviour were demonstrated, where weak or unratcheted pulses transition to ratcheted pulses. The transcription factor Twist directs this transition, with cells in Twist-depleted embryos exhibiting abnormal reversed transitions in pulse behaviour. It was demonstrated that ratcheted pulses have higher probability of having neighbouring contractions, and that ratcheting of pulses prevents competition between neighbouring contractions, allowing collective behaviour.

Francis, D. and Ghabrial, A. S. (2015). Compensatory branching morphogenesis of stalk cells in the Drosophila trachea. Development 142: 2048-2057. PubMed ID: 25977367
Tubes are essential for nutrient transport and gas exchange in multicellular eukaryotes, but how connections between different tube types are maintained over time is unknown. In the Drosophila tracheal system, mutations in oak gall (okg) and conjoined (cnj) confer identical defects, including late onset blockage near the terminal cell-stalk cell junction and the ectopic extension of autocellular, seamed tubes into the terminal cell. It was determined that okg and cnj encode the E and G subunits of the vacuolar ATPase (vATPase); both the V0 and V1 domains are required for terminal cell morphogenesis. Remarkably, the ectopic seamed tubes running along vATPase-deficient terminal cells belonged to the neighboring stalk cells. All vATPase-deficient tracheal cells had reduced apical domains and terminal cells displayed mislocalized apical proteins. Consistent with recent reports that the mTOR and vATPase pathways intersect, this study found that mTOR pathway mutants phenocopied okg and cnj. Furthermore, terminal cells depleted for the apical determinants Par6 or aPKC had identical ectopic seamed tube defects. This study has thus identified a novel mechanism of compensatory branching in which stalk cells extend autocellular tubes into neighboring terminal cells with undersized apical domains. This compensatory branching also occurs in response to injury, with damaged terminal cells being rapidly invaded by their stalk cell neighbor.

Laver, J. D., et al. (2015). Brain tumor is a sequence-specific RNA-binding protein that directs maternal mRNA clearance during the Drosophila maternal-to-zygotic transition. Genome Biol 16: 94. PubMed ID: 25962635
Brain tumor (BRAT) is a Drosophila member of the protein family. This family is conserved among metazoan and its members function as post-transcriptional regulators. BRAT was thought to be recruited to mRNAs indirectly through interaction with the RNA-binding protein Pumilio (PUM). However, it has recently been demonstrated that BRAT directly binds to RNA. The precise sequence recognized by BRAT, the extent of BRAT-mediated regulation, and the exact roles of PUM and BRAT in post-transcriptional regulation are unknown. Genome-wide identification of transcripts associated with BRAT or with PUM in Drosophila embryos shows that they bind largely non-overlapping sets of mRNAs. BRAT binds mRNAs that encode proteins associated with a variety of functions, many of which are distinct from those implemented by PUM-associated transcripts. Computational analysis of in vitro and in vivo data identified a novel RNA motif recognized by BRAT that confers BRAT-mediated regulation in tissue culture cells. Transcriptomic analysis of embryos lacking functional BRAT reveals an important role in mediating the decay of hundreds of maternal mRNAs during the maternal-to-zygotic transition. These results represent the first genome-wide analysis of the mRNAs associated with a TRIM-NHL protein and the first identification of an RNA motif bound by this protein family. BRAT is a prominent post-transcriptional regulator in the early embryo through mechanisms that are largely independent of PUM.

Sunday, June 21st

Gee, H. Y., et al. (2015) KANK deficiency leads to podocyte dysfunction and nephrotic syndromeJ Clin Invest 125: 2375-2384. PubMed ID: 25961457
Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of progressive renal function decline and affects millions of people. This study identified recessive mutations in kidney ankyrin repeat-containing protein 1 (KANK1), KANK2, and KANK4 in individuals with nephrotic syndrome. In an independent functional genetic screen of Drosophila cardiac nephrocytes, which are equivalents of mammalian podocytes, it was determined that the Drosophila KANK homolog (dKank) is essential for nephrocyte function. RNAi-mediated knockdown of dKank in nephrocytes disrupted slit diaphragm filtration structures and lacuna channel structures. In rats, KANK1, KANK2, and KANK4 all localized to podocytes in glomeruli, and KANK1 partially colocalized with synaptopodin. Knockdown of kank2 in zebrafish recapitulated a nephrotic syndrome phenotype, resulting in proteinuria and podocyte foot process effacement. In rat glomeruli and cultured human podocytes, KANK2 interacted with ARHGDIA, a known regulator of RHO GTPases in podocytes that is dysfunctional in some types of nephrotic syndrome. Knockdown of KANK2 in cultured podocytes increased active GTP-bound RHOA and decreased migration. Together, these data suggest that KANK family genes play evolutionarily conserved roles in podocyte function, likely through regulating RHO GTPase signaling.

Tsou, W. L., Ouyang, M., Hosking, R. R., Sutton, J. R., Blount, J. R., Burr, A. A. and Todi, S. V. (2015) The deubiquitinase ataxin-3 requires Rad23 and DnaJ-1 for its neuroprotective role in Drosophila melanogasterNeurobiol Dis 82: 12-21. PubMed ID: 26007638
Ataxin-3 is a deubiquitinase and polyglutamine (polyQ) disease protein with a protective role in Drosophila melanogaster models of neurodegeneration. In the fruit fly, wild-type ataxin-3 suppresses toxicity from several polyQ disease proteins, including a pathogenic version of itself that causes spinocerebellar ataxia type 3 and pathogenic huntingtin, which causes Huntington's disease. The molecular partners of ataxin-3 in this protective function are unclear. This study reports that ataxin-3 requires its direct interaction with the ubiquitin-binding and proteasome-associated protein, Rad23 (known as hHR23A/B in mammals) in order to suppress toxicity from polyQ species in Drosophila. According to additional studies, ataxin-3 does not rely on autophagy or the proteasome to suppress polyQ-dependent toxicity in fly eyes. Instead this deubiquitinase, through its interaction with Rad23, leads to increased protein levels of the co-chaperone DnaJ-1 and depends on it to protect against degeneration. Through DnaJ-1, these data connect ataxin-3 and Rad23 to protective processes involved with protein folding rather than increased turnover of toxic polyQ species.

Dialynas, G., Shrestha, O. K., Ponce, J. M., Zwerger, M., Thiemann, D. A., Young, G. H., Moore, S. A., Yu, L., Lammerding, J. and Wallrath, L. L. (2015). Myopathic lamin mutations cause reductive stress and activate the nrf2/keap-1 pathway. PLoS Genet 11: e1005231. PubMed ID: 25996830
Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, a structural and functional analysis was performed on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, these mutations were modeled in Drosophila Lamin C, and the mutant lamins were expressed in muscle. The structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of the Drosophila model. Thus, novel connections were made between mutant lamins and the Nrf2 signaling pathway, suggesting new avenues of therapeutic intervention that include regulation of protein folding and metabolism, as well as maintenance of redox homoeostasis.

Kinghorn, K. J., Castillo-Quan, J. I., Bartolome, F., Angelova, P. R., Li, L., Pope, S., Cocheme, H. M., Khan, S., Asghari, S., Bhatia, K. P., Hardy, J., Abramov, A. Y. and Partridge, L. (2015). Loss of PLA2G6 leads to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction. Brain [Epub ahead of print]. PubMed ID: 26001724
The PLA2G6 gene encodes a group VIA calcium-independent phospholipase A2 beta enzyme that selectively hydrolyses glycerophospholipids to release free fatty acids. Mutations in PLA2G6 have been associated with disorders such as infantile neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type II and Karak syndrome. More recently, PLA2G6 was identified as the causative gene in a subgroup of patients with autosomal recessive early-onset dystonia-parkinsonism. Neuropathological examination revealed widespread Lewy body pathology and the accumulation of hyperphosphorylated tau, supporting a link between PLA2G6 mutations and parkinsonian disorders. This study shows that knockout of the Drosophila homologue of the PLA2G6 gene, iPLA2-VIA, results in reduced survival, locomotor deficits and organismal hypersensitivity to oxidative stress. Furthermore, it was demonstrated that loss of iPLA2-VIA function leads to a number of mitochondrial abnormalities, including mitochondrial respiratory chain dysfunction, reduced ATP synthesis and abnormal mitochondrial morphology. Moreover, it was shown that loss of iPLA2-VIA is strongly associated with increased lipid peroxidation levels. These findings were confirmed using cultured fibroblasts taken from two patients with mutations in the PLA2G6 gene. Similar abnormalities were seen including elevated mitochondrial lipid peroxidation and mitochondrial membrane defects, as well as raised levels of cytoplasmic and mitochondrial reactive oxygen species. Finally, it was demonstrated that deuterated polyunsaturated fatty acids, which inhibit lipid peroxidation, were able to partially rescue the locomotor abnormalities seen in aged flies lacking iPLA2-VIA gene function, and restore mitochondrial membrane potential in fibroblasts from patients with PLA2G6 mutations. Taken together, these findings demonstrate that loss of normal PLA2G6 gene activity leads to lipid peroxidation, mitochondrial dysfunction and subsequent mitochondrial membrane abnormalities. Furthermore it was shown that the iPLA2-VIA knockout fly model provides a useful platform for the further study of PLA2G6-associated neurodegeneration.

Saturday, June 20th

Ayyaz, A., Li, H. and Jasper, H. (2015). Haemocytes control stem cell activity in the Drosophila intestine. Nat Cell Biol 17: 736-748. PubMed ID: 26005834
Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes were recruited to the intestine and secreted the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switched their response to DPP by inducing expression of Thickveins, a second type I receptor that had previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promoted infection resistance, but also contributed to the development of intestinal dysplasia in ageing flies. The study proposes that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans.

Xing, Y., Su, T. T. and Ruohola-Baker, H. (2015). Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster. Nat Commun 6: 7058. PubMed ID: 25959206
Induction of cell death by a variety of means in wing imaginal discs of Drosophila larvae resulted in the activation of an anti-apoptotic microRNA, bantam. Cells in the vicinity of dying cells also become harder to kill by ionizing radiation (IR)-induced apoptosis. Both ban activation and increased protection from IR required receptor tyrosine kinase Tie, which was identified in a genetic screen for modifiers of ban. tie mutants are hypersensitive to radiation, and radiation sensitivity of tie mutants was rescued by increased ban gene dosage. It is proposed that dying cells activate ban in surviving cells through Tie to make the latter cells harder to kill, thereby preserving tissues and ensuring organism survival. The protective effect reported in this study differs from classical radiation bystander effect in which neighbors of irradiated cells become more prone to death. The protective effect also differs from the previously described effect of dying cells that results in proliferation of nearby cells in Drosophila larval discs. If conserved in mammals, a phenomenon in which dying cells make the rest harder to kill by IR could have implications for treatments that involve the sequential use of cytotoxic agents and radiation therapy.

Antonello, Z.A., Reiff, T., Ballesta-Illan, E. and Dominguez, M. (2015). Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch. EMBO J [Epub ahead of print]. PubMed ID: 26077448
The intestinal epithelium is remarkably robust despite perturbations and demand uncertainty. This study investigates the basis of such robustness using novel tracing methods that allow simultaneously capturing the dynamics of stem and committed progenitor cells (called enteroblasts) and intestinal cell turnover with spatiotemporal resolution. It was found that intestinal stem cells (ISCs) divide "ahead" of demand during Drosophila midgut homeostasis. Their newborn enteroblasts, on the other hand, take on a highly polarized shape, acquire invasive properties and motility. Such enteroblasts also extend long membrane protrusions that make cell-cell contact with mature cells, while exercising a capacity to delay their final differentiation until a local demand materializes. This cellular plasticity is mechanistically linked to the epithelial-mesenchymal transition (EMT) programme mediated by escargot, a snail family gene. Activation of the conserved microRNA miR-8/miR-200 in "pausing" enteroblasts in response to a local cell loss promotes timely terminal differentiation via a reverse MET by antagonizing escargot. These findings unveil that robust intestinal renewal relies on hitherto unrecognized plasticity in enteroblasts and reveal their active role in sensing and/or responding to local demand.

Park, J. S., Na, H. J., Pyo, J. H., Jeon, H. J., Kim, Y. S. and Yoo, M. A. (2015). Requirement of ATR for maintenance of intestinal stem cells in aging Drosophila. Aging (Albany NY) [Epub ahead of print]. PubMed ID: 26000719
The stem cell genomic stability forms the basis for robust tissue homeostasis, particularly in high-turnover tissues. For the genomic stability, DNA damage response (DDR) is essential. This study focused on the role of two major DDR-related factors, ataxia telangiectasia-mutated (ATM) and ATM- and RAD3-related (ATR) kinases, in the maintenance of intestinal stem cells (ISCs) in the adult Drosophila midgut. ATM and ATR phosphorylate their substrates, including H2AX and p53, preferentially on a serine or threonine preceding a glutamine (pS/TQ). The role of ATM and ATR was explored utilizing immunostaining with an anti-pS/TQ antibody as an indicator of ATM/ATR activation, gamma-irradiation as a DNA damage inducer, and the UAS/GAL4 system for cell type-specific knockdown of ATM, ATR, or both during adulthood. The results showed that the pS/TQ signals got stronger with age and after oxidative stress. The pS/TQ signals were found to be more dependent on ATR rather than on ATM in ISCs/enteroblasts (EBs). Furthermore, an ISC/EB-specific knockdown of ATR, ATM, or both decreased the number of ISCs and oxidative stress-induced ISC proliferation. The phenotypic changes that were caused by the ATR knockdown were more pronounced than those caused by the ATM knockdown; however, the data indicate that ATR and ATM are both needed for ISC maintenance and proliferation; ATR seems to play a bigger role than does ATM.

Friday, June 19th

Karim, M.R., Taniguchi, H. and Kobayashi, A. (2015). Constitutive activation of Drosophila CncC transcription factor reduces lipid formation in the fat body. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 26049108
Accumulating evidence indicates that the vertebrate stress-response transcription factors Nrf1 and Nrf2 are involved in hepatic lipid metabolism. To elucidate the precise roles of Nrfs in this process, this study analyzed the physiological role of CncC in lipid metabolism as a Drosophila model for vertebrate Nrf1 and Nrf2. It was examined whether CncC activity was repressed under physiological conditions through a species-conserved NHB (N-terminal homology box 1) domain, similar to that observed for Nrf1. Deletion of the NHB1 domain (CncCΔN) led to CncC-mediated rough-eye phenotypes and the induced expression of the CncC target gene gstD1 both in vivo and in vitro. Thus, the affect of CncCΔN overexpression on the formation of the fat body, which is the major lipid storage organ, was explored. Intriguingly, CncCΔN caused a significant reduction in lipid droplet size and triglyceride (TG) levels in the fat body compared to wild type. It was found that CncCΔN induced a number of genes related to innate immunity that might have an effect on the regulation of cellular lipid storage. This study provides new insights into the regulatory mechanism of CncC and its role in lipid homeostasis.

Dus, M., Lai, J.S., Gunapala, K.M., Min, S., Tayler, T.D., Hergarden, A.C., Geraud, E., Joseph, C.M. and Suh, G.S. (2015). Nutrient sensor in the brain directs the action of the brain-gut axis in Drosophila. Neuron [Epub ahead of print]. PubMed ID: 26074004
Animals can detect and consume nutritive sugars without the influence of taste. However, the identity of the taste-independent nutrient sensor and the mechanism by which animals respond to the nutritional value of sugar are unclear. This study reports that six neurosecretory cells in the Drosophila brain that produce Diuretic hormone 44 (Dh44), a homolog of the mammalian corticotropin-releasing hormone (CRH), are specifically activated by nutritive sugars. Flies in which the activity of these neurons or the expression of Dh44 is disrupted fail to select nutritive sugars. Manipulation of the function of Dh44 receptors has a similar effect. Notably, artificial activation of Dh44 receptor-1 neurons results in proboscis extensions and frequent episodes of excretion. Conversely, reduced Dh44 activity leads to decreased excretion. Together, these actions facilitate ingestion and digestion of nutritive foods. The study proposes that the Dh44 system directs the detection and consumption of nutritive sugars through a positive feedback loop.

Chen, J., Nolte, V. and Schlotterer, C. (2015). Temperature related reaction norms of gene expression: regulatory architecture and functional implications. Mol Biol Evol [Epub ahead of print]. PubMed ID: 25976350
The environment has profound effects on the expression of many traits and reaction norms describe the expression dynamics of a trait across a broad range of environmental conditions. This study analyzed gene expression in Drosophila melanogaster across four different developmental temperatures (13 ° C-29 ° C). Gene expression is highly plastic with 83.3% of the genes being differentially expressed. Three components of plasticity were distinguished: 1) dynamics of gene expression intensity (sum of change, SOC), 2) direction of change and 3) curvature of the reaction norm (linear vs. quadratic). All three plasticity components were found to be most strongly affected by the number of different transcription factors binding to the target gene. More TFs were found in genes with less expression changes across temperatures. Although the effect of microRNAs was weaker, a trend was consistently noted in the opposite direction. The most plastic genes were regulated by fewer transcription factors and more microRNAs than less plastic genes. Different patterns of plasticity were also reflected by their functional characterization based on gene ontology. These results suggest that reaction norms provide an important key to understand the functional requirements of natural populations exposed to variable environmental conditions.

Schou, M.F., Loeschcke, V. and Kristensen, T.N. (2015). Strong costs and benefits of winter acclimatization in Drosophila melanogaster. PLoS One 10: e0130307. PubMed ID: 26075607
This study estimates costs and benefits in thermal tolerance limits in relation to winter acclimatization of Drosophila melanogaster. The flies were sampled from a natural habitat during winter in Denmark (field flies) and heat and cold tolerance of these was compared to that of flies collected from the same natural population, but acclimated to 25 °C or 13 °C in the laboratory (laboratory flies). Thermal performance curves for egg-to-adult viability of field and laboratory (25 °C) flies were obtained to estimate possible cross-generational effects of acclimation. Much higher cold tolerance and a lowered heat tolerance in field flies was found as compared to laboratory flies reared at 25 °C. Flies reared in the laboratory at 13 °C exhibit the same thermal cost-benefit relations as the winter acclimatized flies. A cost of winter acclimatization in terms of decreased egg-to-adult viability at high temperatures of eggs laid by winter acclimatized flies was also found. Based on these findings it is suggested that winter acclimatization in nature can induce strong benefits in terms of increased cold tolerance. These benefits can be reproduced in the laboratory under ecologically relevant rearing and testing conditions, and should be incorporated in species distribution modelling. Winter acclimatization also leads to decreased heat tolerance. This may create a mismatch between acclimation responses and the thermal environment, e.g. if temperatures suddenly increase during spring, under current and expected more variable future climatic conditions.

Thursday, June 18th

Baumbach, J., Novak, Z.A., Raff, J.W. and Wainman, A. (2015). Dissecting the function and assembly of acentriolar microtubule organizing centers in Drosophila cells in vivo. PLoS Genet 11: e1005261. PubMed ID: 26020779
Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. This study shows that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2-the same proteins that appear to drive mitotic centrosome assembly in flies. This finding was used to ablate aMTOC formation in acentriolar cells, and perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. It was shown that although aMTOCs could nucleate microtubules, these microtubules did not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. However, they were found to be required for robust microtubule array assembly in cells without centrioles that also lacked microtubule nucleation from around the chromatin. Importantly, aMTOCs were also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). The study proposes an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.

Cavolo, S. L., Zhou, C., Ketcham, S. A., Suzuki, M. M., Ukalovic, K., Silverman, M. A., Schroer, T. A. and Levitan, E. S. (2015). Mycalolide B dissociates dynactin and abolishes retrograde axonal transport of dense-core vesicles. Mol Biol Cell [Epub ahead of print]. PubMed ID: 26023088
Axonal transport is critical for maintaining synaptic transmission. Interestingly, anterograde and retrograde axonal transport appear to be interdependent, as perturbing one directional motor often impairs movement in the opposite direction. In this study, live imaging of Drosophila and hippocampal neuron dense-core vesicles (DCVs) containing a neuropeptide or brain-derived neurotrophic factor (BDNF) shows that the F-actin depolymerizing macrolide toxin mycalolide B (MB) rapidly and selectively abolishes retrograde, but not anterograde, transport in the axon and the nerve terminal. Latrunculin A does not mimic MB, demonstrating that F-actin depolymerization is not responsible for unidirectional transport inhibition. As dynactin initiates retrograde transport and amino acids sequences implicated in macrolide toxin binding are found in the dynactin component actin-related protein 1 (Arp1), dynactin integrity was examined. Remarkably, cell extract and purified protein experiments show that MB induces disassembly of the dynactin complex. Thus, imaging selective retrograde transport inhibition led to the discovery of a small molecule dynactin disruptor. The rapid unidirectional inhibition by MB suggests that dynactin is absolutely required for retrograde DCV transport, but does not directly facilitate ongoing anterograde DCV transport in the axon or nerve terminal. More generally, MB's effects bolster the conclusion that anterograde and retrograde axonal transport are not necessarily interdependent.

Li, Y.C., Yang, W.T., Cheng, L.C., Lin, C.M., Ho, Y.H., Lin, P.Y., Chen, B.C., Rickoll, W.L. and Hsu, J.C. (2015). Novel transport function of adherens junction revealed by live imaging in Drosophila. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 26047695
Adherens junctions are known for their role in mediating cell-cell adhesion. DE-cadherin and Echinoid are the principle adhesion molecules of adherens junctions in Drosophila epithelia. This study uses live imaging to trace the movement of endocytosed Echinoid vesicles in the epithelial cells of Drosophila embryos. Echinoid vesicles co-localized and moved with Rab5- or Rab11-positive endosomes. Surprisingly, these Echinoid-containing endosomes underwent directional cell-to-cell movement, through adherens junctions. Consistent with this, cell-to-cell movement of Echinoid vesicles required the presence of DE-cadherin at adherens junctions. Live imaging further revealed that Echinoid vesicles moved along adherens junction-associated microtubules into adjacent cells, a process requiring a kinesin motor. Importantly, DE-cadherin- and EGFR-containing vesicles also exhibited intercellular movement. Together, these results unveil a transport function of adherens junctions. Furthermore, this adherens junctions-based intercellular transport provides a platform for the exchange of junctional proteins and signaling receptors between neighboring cells.

Dopie, J., Rajakyla, E. K., Joensuu, M. S., Huet, G., Ferrantelli, E., Xie, T., Jaalinoja, H., Jokitalo, E. and Vartiainen, M. K. (2015) Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulatorsJ Cell Sci [Epub ahead of print]. PubMed ID: 26021350
Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. This study consisted of a genome-wide RNAi screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. Nineteen factors were validated as specific hits, and it was shown that Chinmo/Bach2, SNF4Agamma/Prkag1 and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. This study identified several novel regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active twinstar/cofilin by repressing Cdi/Tes kinase expression. Finally, Nup98 and Lam were shown to be candidates for regulating nuclear actin polymerization. This screen therefore reveals novel aspects of actin regulation and links nuclear actin to many cellular processes.

Wednesday, June 17th

Apte, M.S. and Meller, V.H. (2015). Sex differences in Drosophila melanogaster heterochromatin are regulated by non-sex specific factors. PLoS One 10: e0128114. PubMed ID: 26053165
The eukaryotic genome is assembled into distinct types of chromatin. Gene-rich euchromatin has active chromatin marks, while heterochromatin is gene-poor and enriched for silencing marks. In spite of this, genes native to heterochromatic regions are dependent on their normal environment for full expression. Expression of genes in autosomal heterochromatin is reduced in male flies mutated for the noncoding roX RNAs, but not in females. roX mutations also disrupt silencing of reporter genes in male, but not female, heterochromatin, revealing a sex difference in heterochromatin. This study adopted a genetic approach to determine how this difference is regulated, and found no evidence that known X chromosome counting elements, or the sex determination pathway that these control, are involved. This suggested that the sex chromosome karyotype regulated autosomal heterochromatin by a different mechanism. To address this, candidate genes that regulate chromosome organization were examined. In XX flies mutation of Topoisomerase II (Top2), a gene involved in chromatin organization and homolog pairing, made heterochromatic silencing dependent on roX, and thus male-like. Interestingly, Top2 also bound to a large block of pericentromeric satellite repeats (359 bp repeats) that are unique to the X chromosome. Deletion of X heterochromatin also made autosomal heterochromatin in XX flies dependent on roX and enhanced the effect of Top2 mutations, suggesting a combinatorial action. The study postulates that Top2 and X heterochromatin in Drosophila comprise a novel karyotype-sensing pathway that determines the sensitivity of autosomal heterochromatin to loss of roX RNA.

Kang, H., McElroy, K.A., Jung, Y.L., Alekseyenko, A.A., Zee, B.M., Park, P.J. and Kuroda, M.I. (2015). Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila. Genes Dev 29: 1136-1150. PubMed ID: 26063573
The Polycomb group (PcG) proteins are key regulators of development in Drosophila and are strongly implicated in human health and disease. How PcG complexes form repressive chromatin domains remains unclear. Using cross-linked affinity purifications of BioTAP-Polycomb (Pc) or BioTAP-Enhancer of zeste [E(z)], this study captured all PcG-repressive complex 1 (PRC1) or PRC2 core components and Sex comb on midleg (Scm) as the only protein strongly enriched with both complexes. Although previously not linked to PRC2, the direct binding of Scm and PRC2 was confirmed using recombinant protein expression and colocalization of Scm with PRC1, PRC2, and H3K27me3 in embryos and cultured cells using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing). Furthermore, it was found that RNAi knockdown of Scm and overexpression of the dominant-negative Scm-SAM (sterile α motif) domain both affect the binding pattern of E(z) on polytene chromosomes. Aberrant localization of the Scm-SAM domain in long contiguous regions on polytene chromosomes revealed its independent ability to spread on chromatin, consistent with its previously described ability to oligomerize in vitro. Pull-downs of BioTAP-Scm captured PRC1 and PRC2 and additional repressive complexes, including PhoRC, LINT, and CtBP. The study proposes that Scm is a key mediator connecting PRC1, PRC2, and transcriptional silencing. Combined with previous structural and genetic analyses, these results strongly suggest that Scm coordinates PcG complexes and polymerizes to produce broad domains of PcG silencing.

Yung, P. Y., Stuetzer, A., Fischle, W., Martinez, A. M. and Cavalli, G. (2015). Histone H3 serine 28 is essential for efficient Polycomb-mediated gene repression in Drosophila. Cell Rep [Epub ahead of print]. PubMed ID: 26004180
Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, a Drosophila H3 histone mutant was generated with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2, suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, these data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis.

Dupont, C. A., Dardalhon-Cumenal, D., Kyba, M., Brock, H. W., Randsholt, N. B. and Peronnet, F. (2015) Drosophila Cyclin G and epigenetic maintenance of gene expression during development. Epigenetics Chromatin 8: 18. PubMed ID: 25995770
PcG genes act as repressors, counteracted by trxG genes that maintain gene activation, while Enhancer of Trithorax and Polycomb (ETP) proteins interact with both, behaving alternatively as repressors or activators. Drosophila Cyclin G negatively regulates cell growth and cell cycle progression, binds and co-localizes with the ETP Corto on chromatin, and participates with Corto in Abdominal-B Hox gene regulation. This study addressed further implications of Cyclin G in epigenetic maintenance of gene expression. Cyclin G was shown to physically interact and extensively co-localize on chromatin with the conserved ETP Additional sex combs (ASX), belonging to the repressive PR-DUB complex that participates in H2A deubiquitination and Hox gene silencing. Furthermore, Cyclin G mainly co-localizes with RNA polymerase II phosphorylated on serine 2 that is specific to productive transcription. CycG interacts with Asx, PcG, and trxG genes in Hox gene maintenance, and behaves as a PcG gene. These interactions correlate with modified ectopic Hox protein domains in imaginal discs, consistent with a role for Cyclin G in PcG-mediated Hox gene repression. This study shows that Drosophila CycG is a Polycomb-group gene enhancer, acting in epigenetic maintenance of the Hox genes Sex combs reduced (Scr) and Ultrabithorax (Ubx). However, the data suggest that Cyclin G acts alternatively as a transcriptional activator or repressor depending on the developmental stage, the tissue or the target gene.

Tuesday, June 16th

Harris, D.T., Kallman, B.R., Mullaney, B.C. and Scott, K. (2015). Representations of taste modality in the Drosophila brain. Neuron [Epub ahead of print]. PubMed ID: 26051423
Gustatory receptors and peripheral taste cells have been identified in flies and mammals, revealing that sensory cells are tuned to taste modality across species. How taste modalities are processed in higher brain centers to guide feeding decisions is unresolved. This study developed a large-scale calcium-imaging approach coupled with cell labeling to examine how different taste modalities are processed in the fly brain. These studies revealed that sweet, bitter, and water sensory cells activated different cell populations throughout the subesophageal zone, with most cells responding to a single taste modality. Pathways for sweet and bitter tastes were segregated from sensory input to motor output, and this segregation was maintained in higher brain areas, including regions implicated in learning and neuromodulation. The study reveals independent processing of appetitive and aversive tastes, suggesting that flies and mammals use a similar coding strategy to ensure innate responses to salient compounds.

Berni, J. (2015) Genetic dissection of a regionally differentiated network for exploratory behavior in Drosophila larvae Curr Biol 25: 1319-1326. PubMed ID: 25959962
An efficient strategy to explore the environment for available resources involves the execution of random walks where straight line locomotion alternates with changes of direction. This strategy is highly conserved in the animal kingdom, from zooplankton to human hunter-gatherers. Drosophila larvae execute a routine of this kind, performing straight line crawling interrupted at intervals by pause turns that halt crawling and redirect the trajectory of movement. The execution of this routine depends solely on the activity of networks located in the thoracic and abdominal segments of the nervous system, while descending input from the brain serves to modify it in a context-dependent fashion. This study used a genetic method to investigate the location and function of the circuitry required for the different elements of exploratory crawling. By using the Slit-Robo axon guidance pathway to target neuronal midline crossing defects selectively to particular regions of the thoracic and abdominal networks, it has been possible to define at least three functions required for the performance of the exploratory routine: (1) symmetrical outputs in thoracic and abdominal segments that generate the crawls; (2) asymmetrical output that is uniquely initiated in the thoracic segments and generates the turns; and (3) an intermittent interruption to crawling that determines the time-dependent transition between crawls and turns.

Mamiya, A. and Dickinson, M. H. (2015). Antennal mechanosensory neurons mediate wing motor reflexes in flying Drosophila. J Neurosci 35: 7977-7991. PubMed ID: 25995481
Although many behavioral studies have shown the importance of antennal mechanosensation in various aspects of insect flight control, the identities of the mechanosensory neurons responsible for these functions are still unknown. One candidate is the Johnston's organ (JO) neurons that are located in the second antennal segment and detect phasic and tonic rotations of the third antennal segment relative to the second segment. To investigate how different classes of JO neurons respond to different types of antennal movement during flight, 2-photon calcium imaging was combined with a machine vision system to simultaneously record JO neuron activity and the antennal movement from tethered flying fruit flies (Drosophila melanogaster). Most classes of JO neurons were found to respond strongly to antennal oscillation at the wing beat frequency, but not to the tonic deflections of the antennae. To study how flies use input from the JO neurons during flight, specific classes of JO neurons were genetically ablated, and their effect on the wing motion was examined. Tethered flies flying in the dark require JO neurons to generate slow antiphasic oscillation of the left and right wing stroke amplitudes. However, JO neurons are not necessary for this antiphasic oscillation when visual feedback is available, indicating that there are multiple pathways for generating antiphasic movement of the wings. Collectively, these results are consistent with a model in which flying flies use JO neurons to detect increases in the wing-induced airflow and that JO neurons are involved in a response that decreases contralateral wing stoke amplitude.

Tsuda, M., Peyre, J. B., Asano, T. and Aigaki, T. (2015). Visualizing molecular functions and cross-species activity of Sex-peptide in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 26022240
The Drosophila melanogaster sex-peptide (melSP) is a seminal fluid component that induces post-mating responses (PMR) of females via the sex peptide receptor (SPR). Although SP orthologs are found in many Drosophila species, their functions remain poorly characterized. It is unknown whether SP functions are conserved across species or rather specific to each species. This study developed a GFP-tagged melSP (G-SP) and used it to visualize cross-species binding activity to the female reproductive system of various species. First it was demonstrated that ectopically expressed G-SP induced PMR in D. melanogaster females and bound to the female reproductive system, most notably to the common oviduct. No binding occurred in the females lacking SPR, indicating that G-SP binding was dependent on SPR. Next it was tested whether G-SP binds to the common oviducts from 11 Drosophila species using dissected reproductive tracts. The binding was observed in six species belonging to the D. melanogaster species group, but not to those outside the group. Injection of melSP reduced the receptivity of females belonging to the D. melanogaster species group, but not of those outside the group, being consistent with the ability to bind G-SP. Thus the SP-mediated PMR appears to be limited to this species group. SPR was expressed in the oviducts at high levels in this group, therefore it is speculated that an enhanced expression of SPR in the oviduct was critical to establish the SP-mediated PMR during evolution.

Monday, June 15th

Sano, H., et al. (2015). The nutrient-responsive hormone CCHamide-2 controls growth by regulating Insulin-like peptides in the brain of Drosophila melanogasterPLoS Genet 11: e1005209. PubMed ID: 26020940
In Drosophila melanogaster, the fat body (adipose tissue) has been suggested to play an important role in coupling growth with nutritional status. This study shows that the peripheral tissue-derived peptide hormone CCHamide-2 (CCHa2) acts as a nutrient-dependent regulator of Drosophila insulin-like peptides (Dilps). A BAC-based transgenic reporter revealed strong expression of CCHa2 receptor (CCHa2-R) in insulin-producing cells (IPCs) in the brain. Calcium imaging of brain explants and IPC-specific CCHa2-R knockdown demonstrated that peripheral-tissue derived CCHa2 directly activates IPCs. Interestingly, genetic disruption of either CCHa2 or CCHa2-R caused almost identical defects in larval growth and developmental timing. Consistent with these phenotypes, the expression of dilp5, and the release of both Dilp2 and Dilp5, were severely reduced. Furthermore, transcription of CCHa2 is altered in response to nutritional levels, particularly of glucose. These findings demonstrate that CCHa2 and CCHa2-R form a direct link between peripheral tissues and the brain, and that this pathway is essential for the coordination of systemic growth with nutritional availability. A mammalian homologue of CCHa2-R, Bombesin receptor subtype-3 (Brs3), is an orphan receptor that is expressed in the islet beta-cells; however, the role of Brs3 in insulin regulation remains elusive. This genetic approach in Drosophila melanogaster provides the first evidence that bombesin receptor signaling with its endogenous ligand promotes insulin production.

Denes, A. S., Kanca, O. and Affolter, M. (2015) A cellular process that includes asymmetric cytokinesis remodels the dorsal tracheal branches in Drosophila larvae Development 142: 1794-1805. PubMed ID: 25968315
Tubular networks are central to the structure and function of many organs, such as the vertebrate lungs or the Drosophila tracheal system. Their component epithelial cells are able to proliferate and to undergo complex morphogenetic movements, while maintaining their barrier function. Little is known about the details of the mitotic process in tubular epithelia. This study presents a comprehensive model of cellular remodeling and proliferation in the dorsal branches of third-instar Drosophila larvae. Through a combination of immunostaining and novel live imaging techniques, this study identified the key steps in the transition from a unicellular to a multicellular tube. Junctional remodeling precedes mitosis and, as the cells divide, new junctions are formed through several variations of what is refered to as 'asymmetric cytokinesis'. Depending on the spacing of cells along the dorsal branch, mitosis can occur either before or after the transition to a multicellular tube. In both instances, cell separation is accomplished through asymmetric cytokinesis, a process that is initiated by the ingression of the cytokinetic ring. Unequal cell compartments are a possible but rare outcome of completing mitosis through this mechanism. The Dpp signaling pathway was found to be required but not sufficient for cell division in the dorsal branches.

Wurmbach, E. and Preiss, A. (2014). Deletion mapping in the Enhancer of split complex. Hereditas 151: 159-168. PubMed ID: 25588303
The Enhancer of split complex [E(spl)-C] comprises twelve genes of different classes. Seven genes encode proteins of with a basic-helix-loop-helix-orange (bHLH-O) domain that function as transcriptional repressors and serve as effectors of the Notch signalling pathway. They have been named E(spl)m8-, m7-, m5-, m3-, mbeta-, mgamma- and mdelta-HLH. Four genes, E(spl)m6-, m4-, m2- and malpha-BFM are intermingled and encode Notch repressor proteins of the Bearded-family (BFM). The complex is split by a single gene of unrelated function, encoding a Kazal-type protease inhibitor (Kaz-m1). All members within a family, bHLH-O or BFM, are very similar in structure and in function. In an attempt to generate specific mutants, P-element constructs residing next to E(spl)m7-HLH and E(spl)mgamma-HLH, respectively, were mobilized. The resulting deletions were mapped molecularly and by cytology. Two small deletions affected only E(spl)m7-HLH and E(spl)mdelta. The deficient flies were viable without apparent phenotype. Larger deletions, generated also by X-ray mutagenesis, uncover most of the E(spl)-C. The phenotypes of homozygous deficient embryos were analysed to characterize the respective loss of Notch signalling activity.

Wang, S., Reuveny, A. and Volk, T. (2015). Nesprin provides elastic properties to muscle nuclei by cooperating with spectraplakin and EB1. J Cell Biol 209: 529-538. PubMed ID: 26008743
Muscle nuclei are exposed to variable cytoplasmic strain produced by muscle contraction and relaxation, but their morphology remains stable. Still, the mechanism responsible for maintaining myonuclear architecture, and its importance, is currently elusive. This study uncovered a unique myonuclear scaffold in Drosophila melanogaster larval muscles, exhibiting both elastic features contributed by the stretching capacity of MSP300 (nesprin) and rigidity provided by a perinuclear network of microtubules stabilized by Shot (spectraplakin) and EB1. Together, they form a flexible perinuclear shield that protects myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and subsequently reduced levels of essential nuclear factors such as lamin A/C, lamin B, and HP1. Overall, a novel mechanism is proposed for maintaining myonuclear morphology, and its critical link to correct levels of nuclear factors in differentiated muscle fibers is revealed. These findings may shed light on the underlying mechanism of various muscular dystrophies.

Sunday, June 14th

Ayyaz, A., Li, H. and Jasper, H. (2015). Haemocytes control stem cell activity in the Drosophila intestine. Nat Cell Biol 17: 736-748. PubMed ID: 26005834
Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes were recruited to the intestine and secreted the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switched their response to DPP by inducing expression of Thickveins, a second type I receptor that had previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promoted infection resistance, but also contributed to the development of intestinal dysplasia in ageing flies. The study proposes that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans.

Ghosh, S., Singh, A., Mandal, S. and Mandal, L. (2015). Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response. Dev Cell 33(4):478-88. PubMed ID: 25959225
Blood cell development in Drosophila shares significant similarities with vertebrate. The conservation ranges from biphasic mode of hematopoiesis to signaling molecules crucial for progenitor cell formation, maintenance, and differentiation. Primitive hematopoiesis in Drosophila ensues in embryonic head mesoderm, whereas definitive hematopoiesis happens in larval hematopoietic organ, the lymph gland. This organ, with the onset of pupation, ruptures to release hemocytes into circulation. It is believed that the adult lacks a hematopoietic organ and survives on the contribution of both embryonic and larval hematopoiesis. However, these studies revealed a surge of blood cell development in the dorsal abdominal hemocyte clusters of adult fly. These active hematopoietic hubs are capable of blood cell specification and can respond to bacterial challenges. The presence of progenitors and differentiated hemocytes embedded in a functional network of Laminin A and Pericardin within this hematopoietic hub projects it as a simple version of the vertebrate bone marrow.

Panda, D., Gold, B., Tartell, M. A., Rausch, K., Casas-Tinto, S. and Cherry, S. (2015) The transcription factor FoxK participates with Nup98 to regulate antiviral gene expression MB 6 [Epub ahead of print]. PubMed ID: 25852164
Nup98 regulates the expression of antiviral genes that restrict RNA virus infections in Drosophila by promoting RNA polymerase occupancy at the promoters of these antiviral genes. It was reasoned that additional transcription factors facilitate the Nup98-dependent expression of antiviral genes. In a genome-wide RNA interference (RNAi) screen, a forkhead transcription factor, FoxK, was identified as active against Sindbis virus (SINV) in Drosophila. FoxK is active against the panel of viruses that are restricted by Nup98, including vesicular stomatitis virus (VSV). FoxK was shown to coordinately regulate the Nup98-dependent expression of antiviral genes. Depletion of FoxK significantly reduces Nup98-dependent induction of antiviral genes and reduces the expression of a forkhead response element-containing luciferase reporter. Together, these data show that FoxK-mediated activation of gene expression is Nup98 dependent. The mammalian ortholog FOXK1 was also found to antiviral against two disparate RNA viruses, SINV and VSV, in human cells. FOXK1 was found to attenuate virus-inducible interferon-stimulated response element (ISRE) reporter expression. Overall, these results demonstrate a novel role for FOXK1 in regulating the expression of antiviral genes, from insects to humans.

Beebe, K., Park, D., Taghert, P. H. and Micchelli, C. A. (2015). The Drosophila pro-secretory transcription factor dimmed is dynamically regulated in adult enteroendocrine cells and protects against gram-negative infection. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 25999585
The endocrine system employs peptide hormone signals to translate environmental changes into physiological responses. The diffuse endocrine system embedded in the gastrointestinal barrier epithelium is one of the largest and most diverse endocrine tissues. Furthermore, it is the only endocrine tissue in direct physical contact with the microbial environment of the gut lumen. However, it remains unclear how this sensory epithelium responds to specific pathogenic challenges in a dynamic and regulated manner.This study demonstrates that the enteroendocrine cells of the adult Drosophila melanogaster midgut display a transient, sensitive, and systemic induction of the pro-secretory factor dimmed (dimm) in response to the Gram-negative pathogen Pseudomonas entomophila (Pe). In enteroendocrine cells, dimm controls the levels of the targets phantom, cat-4 and the peptide hormone, Allatostatin A. Finally, dimm was identified as a host factor that protects against Pe infection and controls the expression of antimicrobial peptides. It is proposed that dimm provides "gain" in enteroendocrine output during the adaptive response to episodic pathogen exposure.

Saturday, June 13th

Karg, T., Warecki, B. and Sullivan, W. (2015) Aurora B mediated localized delays in nuclear envelope formation facilitates inclusion of late segregating chromosome fragments. Mol Biol Cell [Epub ahead of print]. PubMed ID: 25877868
To determine how mitotic chromosome segregation is coordinated with nuclear envelope formation (NEF), the dynamics of NEF was examined in the presence of lagging acentric chromosomes in Drosophila neuroblasts. Acentric chromosomes often exhibit delayed but ultimately successful segregation and incorporation into daughter nuclei. However, it is unknown whether these late segregating acentric fragments influence NEF to ensure their inclusion in daughter nuclei. Through live analysis, this study showed that acentric chromosomes induce highly localized delays in the reassembly of the nuclear envelope. These delays result in a gap in the nuclear envelope that facilitates the inclusion of lagging acentrics into telophase daughter nuclei. Localized delays of nuclear envelope reassembly require Aurora B kinase activity. In cells with reduced Aurora B activity, there is a decrease in the frequency of local nuclear envelope reassembly delays, resulting in an increase in the frequency of acentric-bearing lamin-coated micronuclei. These studies reveal a novel role of Aurora B for maintaining genomic integrity by promoting the formation of a passageway in the nuclear envelope through which late segregating acentric chromosomes enter the telophase daughter nucleus.

Rosa, A., Vlassaks, E., Pichaud, F. and Baum, B. (2015). Ect2/Pbl acts via Rho and polarity proteins to direct the assembly of an isotropic actomyosin cortex upon mitotic entry. Dev Cell 32: 604-616. PubMed ID: 25703349
Entry into mitosis is accompanied by profound changes in cortical actomyosin organization. This study delineate a pathway downstream of the RhoGEF Pbl/Ect2 that directs this process in Drosophila notum epithelial cells. The data suggest that the release of Pbl/Ect2 from the nucleus at mitotic entry drives Rho-dependent activation of Myosin-II and, in parallel, induces a switch from Arp2/3 (see Actin-related protein 2/3 complex, subunit 1) to Diaphanous-mediated cortical actin nucleation that depends on Cdc42, aPKC, and Par6. At the same time, the mitotic relocalization of these apical protein complexes to more lateral cell surfaces enables Cdc42/aPKC/Par6 to take on a mitosis-specific function-aiding the assembly of a relatively isotropic metaphase cortex. Together, these data reveal how the repolarization and remodeling of the actomyosin cortex are coordinated upon entry into mitosis to provide cells with the isotropic and rigid form they need to undergo faithful chromosome segregation and division in a crowded tissue environment.

Bang, C. and Cheng, J. (2015) Dynamic interplay of spectrosome and centrosome organelles in asymmetric stem cell divisionsPLoS One 10: e0123294. PubMed ID: 25849996
Stem cells have remarkable self-renewal ability and differentiation potency, which are critical for tissue repair and tissue homeostasis. Recently it has been found, in many systems (e.g. gut, neurons, and hematopoietic stem cells), that the self-renewal and differentiation balance is maintained when the stem cells divide asymmetrically. Drosophila male germline stem cells (GSCs), one of the best characterized model systems with well-defined stem cell niches, were reported to divide asymmetrically, where centrosome plays an important role. Utilizing time-lapse live cell imaging, customized tracking, and image processing programs, this study found that most acentrosomal GSCs have the spectrosomes reposition from the basal end (wild type) to the apical end close to hub-GSC interface (acentrosomal GSCs). In addition, these apically positioned spectrosomes were mostly stationary while the basally positioned spectrosomes were mobile. For acentrosomal GSCs, their mitotic spindles were still highly oriented and divided asymmetrically with longer mitosis duration, resulting in asymmetric divisions. Moreover, when the spectrosome was knocked out, the centrosomes velocity decreased and centrosomes located closer to hub-GSC interface. It is proposed that in male GSCs, the spectrosome recruited to the apical end plays a complimentary role in ensuring proper spindle orientation when centrosome function is compromised.

Gupte, T. M. (2015). Mitochondrial fragmentation due to inhibition of fusion increases Cyclin B through mitochondrial superoxide radicals. PLoS One 10: e0126829. PubMed ID: 26000631
During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G1-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G2- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. This study bypassed the 'mitotic mitochondrial fragmentation' checkpoint by inducing fragmented mitochondrial morphology and then measuring the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R+ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae it was shown that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. These experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. Evidence is provided for the importance of mitochondrial superoxide in this process. These results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.

Friday, June 12th

Chouhan, N.S., Wolf, R., Helfrich-Förster, C. and Heisenberg, M. (2015). Flies remember the time of day. Curr Biol [Epub ahead of print]. PubMed ID: 26028434
The circadian clock enables organisms to anticipate daily environmental cycles and drives corresponding changes in behavior. Such endogenous oscillators also enable animals to display time-specific memory. For instance, mice and honeybees associate the location of a stimulus (like food or mate) with a certain time of day (time-place learning). However, the mechanism underlying time-related learning and memory is not known. The present study investigated time-specific odor learning by using a genetically tractable animal, the fly Drosophila melanogaster. Starved flies were trained in the morning and afternoon to associate distinct odors with sucrose reward. The training was repeated the next day, and their time-dependent odor preference was tested on the third day. Drosophila were found to express appetitive memory at the relevant time of day if the two conditioning events were separated by more than 4 hr. Flies could form time-odor associations in constant darkness (DD) as well as in a daily light-dark (LD) cycle, but not when kept under constant light (LL) conditions. Circadian clock mutants, period01 (per01) and clockAR (clkAR), learned to associate sucrose reward with a certain odor but were unable to form time-odor associations. The findings of this study show that flies can utilize temporal information as an additional cue in appetitive learning. Time-odor learning in flies depends on a per- and clk-dependent endogenous mechanism that is independent of environmental light cues.

Docherty, J.E., Manno, J.E., McDermott, J.E. and DiAngelo, J.R. (2015). Mio acts in the Drosophila brain to control nutrient storage and feeding. Gene [Epub ahead of print]. PubMed ID: 26024590
Mlx interactor (Mio), the Drosophila homolog of carbohydrate response element binding protein (ChREBP), functions as a transcription factor in the fat body of the fly to control triglyceride storage as well as feeding, suggesting that Mio may act in a nutrient-sensing pathway to coordinate food consumption and metabolism. This study shows that Mio functions in neurons in Drosophila to regulate feeding and nutrient storage. Pan-neuronal disruption of Mio function lead to increased triglyceride and glycogen storage, and this phenotype was not due to increased food consumption. Interestingly, targeted disruption of Mio specifically in the insulin-producing cells (IPCs) had little effect on nutrient storage, but increased food consumption suggesting that Mio acts in these neurons to control feeding behavior. Since Mio is a transcription factor, one possible way Mio may act in the IPCs to control feeding is through regulating the expression of Drosophila insulin-like peptides (dilps) or drosulfakinin (dsk), neuropeptides produced in the IPCs. Consistent with this hypothesis, IPC-specific knockdown of Mio lead to an increase in dilp3 expression, while not affecting dilp2, 5 or dsk levels. Together, this study indicates a new function for Mio in the Drosophila brain and specifically in the IPCs, controlling neuropeptide gene expression, feeding and metabolism in accordance with nutrient availability.

Tomita, J., Ueno, T., Mitsuyoshi, M., Kume, S. and Kume, K. (2015). The NMDA receptor promotes sleep in the fruit fly, Drosophila melanogaster. PLoS One 10: e0128101. PubMed ID: 26023770
A short sleeper mutant of Drosophila, fumin (fmn), was previously identified as mutation in the Dopamine transporter gene. In aversive olfactory learning tasks, fmn mutants demonstrate defective memory retention, which suggests an association between sleep and memory. In an attempt to discover additional sleep related genes in Drosophila, this study carried out a microarray analysis comparing mRNA expression in heads of fmn and control flies and found that 563 genes were differentially expressed. Next, using the pan-neuronal Gal4 driver elav-Gal4 and UAS-RNA interference (RNAi) to knockdown individual genes, a functional screen was performed. It was found that knockdown of the NMDA type glutamate receptor channel gene (Nmdar1) (also known as dNR1) reduced sleep. The NMDA receptor (NMDAR) plays an important role in learning and memory both in Drosophila and mammals. The application of the NMDAR antagonist, MK-801, reduced sleep in control flies, but not in fmn. These results suggest that NMDAR promotes sleep regulation in Drosophila.

Akitake, B., Ren, Q., Boiko, N., Ni, J., Sokabe, T., Stockand, J.D., Eaton, B.A. and Montell, C. (2015). Coordination and fine motor control depend on Drosophila TRPγ. Nat Commun 6: 7288. PubMed ID: 26028119
Motor coordination is broadly divided into gross and fine motor control, both of which depend on proprioceptive organs. However, the channels that function specifically in fine motor control are unknown. This study shows that mutations in trpγ disrupt fine motor control while leaving gross motor proficiency intact. The mutants were unable to coordinate precise leg movements during walking, and were ineffective in traversing large gaps due to an inability in making subtle postural adaptations that are requisite for this task. TRPγ was expressed in proprioceptive organs, and was required in both neurons and glia for gap crossing. By expressing TRPγ in vitro, it was found that its activity was promoted by membrane stretch. A mutation eliminating the Na(+)/Ca(2+) exchanger suppressed the gap-crossing phenotype of trpγflies. These findings indicate that TRPγ contributes to fine motor control through mechanical activation in proprioceptive organs, thereby promoting Ca(2+) influx, which is required for function.

Thursday, June 11th

Packard, M., Jokhi, V., Ding, B., Ruiz-Canada, C., Ashley, J. and Budnik, V. (2015) Nucleus to synapse Nesprin1 railroad tracks direct synapse maturation through RNA localizationNeuron [Epub ahead of print]. PubMed ID: 25959729
An important mechanism underlying synapse development and plasticity is the localization of mRNAs that travel from the nucleus to synaptic sites. This study demonstrates that the giant nuclear-associated Nesprin1 (dNesp1) forms striated F-actin-based filaments, which were dubbed "railroad tracks," that span from muscle nuclei to postsynaptic sites at the neuromuscular junction in Drosophila. These railroad tracks specifically wrap around immature boutons formed during development and in response to electrical activity. In the absence of dNesp1, mRNAs normally localized at postsynaptic sites are lacking and synaptic maturation is inhibited. This dNesp1 function does not depend on direct association of dNesp1 isoforms with the nuclear envelope. It was also show that dNesp1 functions with an unconventional myosin, Myo1D (Myosin 31DF), and that both dNesp1 and Myo1D are mutually required for their localization to immature boutons. These studies unravel a novel pathway directing the transport of mRNAs from the nucleus to postsynaptic sites during synaptic maturation.

Enriquez, J., Venkatasubramanian, L., Baek, M., Peterson, M., Aghayeva, U. and Mann, R. S. (2015) Specification of individual adult motor neuron morphologies by combinatorial transcription factor codes. Neuron [Epub ahead of print]. PubMed ID: 25959734
How the highly stereotyped morphologies of individual neurons are genetically specified is not well understood. This study identified six transcription factors (TFs) expressed in a combinatorial manner in seven post-mitotic adult leg motor neurons (MNs) that are derived from a single neuroblast in Drosophila. Unlike TFs expressed in mitotically active neuroblasts, these TFs do not regulate each other’s expression. Removing the activity of a single TF resulted in specific morphological defects, including muscle targeting and dendritic arborization, and in a highly specific walking defect in adult flies. In contrast, when the expression of multiple TFs was modified, nearly complete transformations in MN morphologies were generated. These results show that the morphological characteristics of a single neuron are dictated by a combinatorial code of morphology TFs (mTFs including Zfh1, Ems, Pb, Zfh2 and Toy). mTFs function at a previously unidentified regulatory tier downstream of factors acting in the NB but independently of factors that act in terminally differentiated neurons (Enriquez, 2015).

Bauke, A.C., Sasse, S., Matzat, T. and Klämbt, C. (2015). A transcriptional network controlling glial development in the Drosophila visual system. Development [Epub ahead of print]. PubMed ID: 26015542
In the nervous system, glial cells need to be specified from a set of progenitor cells. In the developing Drosophila eye, perineurial glia proliferate and differentiate as wrapping glia in response to a neuronal signal conveyed by the FGF receptor pathway. To unravel the underlying transcriptional network, this study silenced all genes encoding predicted DNA-binding proteins in glial cells using RNAi. Dref and other factors of the TATA box-binding protein-related factor 2 (TRF2) complex were previously predicted to be involved in cellular metabolism and cell growth. Silencing of these genes impaired early glia proliferation and subsequent differentiation. Dref was found to control proliferation via activation of the Pdm3 transcription factor, whereas glial differentiation was regulated via Dref and the homeodomain protein Cut. Cut expression was controlled independently of Dref by FGF receptor activity. Loss- and gain-of-function studies showed that Cut was required for glial differentiation and was sufficient to instruct the formation of membrane protrusions, a hallmark of wrapping glial morphology. This work discloses a network of transcriptional regulators controlling the progression of a naïve perineurial glia towards the fully differentiated wrapping glia.

Chakraborty, M., Paul, B. K., Nayak, T., Das, A., Jana, N. R. and Bhutani, S. (2015). The E3 ligase ube3a is required for learning in Drosophila melanogaster. Biochem Biophys Res Commun 462: 71-77. PubMed ID: 25935478
Angelman syndrome and autism are neurodevelopmental disorders linked to mutations and duplications of an E3 ligase called ube3a respectively. Since cognitive deficits and learning disabilities are hallmark symptoms of both these disorders, this study investigated a role for dube3a in the learning ability of flies using the aversive phototaxis suppression assay. Down and up-regulation of dube3a were shown to both be detrimental to learning in larvae and adults. Using conditional gene expression it was found that dube3a is required for normal brain development and during adulthood. Furthermore, it is suggested that dube3a could be interacting with other learning and memory genes such as derailed. Along with firmly establishing dube3a as a gene that is required for learning, this work also opens avenues for further understanding the role played by this gene in brain development and behavior.

Lone, S.R., Venkataraman, A., Srivastava, M., Potdar, S. and Sharma, V.K. (2015). Or47b-neurons promote male-mating success in Drosophila. Biol Lett 11(5) [Epub ahead of print]. PubMed ID: 26018835
Drosophila performs elaborate well-defined rituals of courtship, which involve several types of sensory inputs. This study reports that Or47b-neurons promote male-mating success. Males with Or47b-neurons silenced/ablated exhibit reduced copulation frequency and increased copulation latency. Copulation latency of Or47b-manipulated flies increased proportionately with size of the assay arena, whereas in controls it remained unchanged. While competing for mates, Or47b-ablated males were outperformed by intact controls. Or47b-neurons express fruM, which is activated by both male and female odours, and they therefore are believed to play a crucial role in male courtship behaviour. Of the three olfactory neurons, Or67d projects to DA1, Or47b to VA1v and IR84a to VL2a. These three glomeruli are larger in males than females, which could be the reason behind their greater role in males than in females. Furthermore, Or67d and Or47b genes show higher expression in males than females. Previous studies suggest that these two receptors are involved in promoting male reproductive fitness-related behaviours including male-male aggression and male-female courtship. Furthermore, Or47b along with Or88a receptors promote mating by responding to both male and female odours. Although the ligand recognized by Or47b receptors is yet unknown, there is enough evidence suggesting its role in male courtship

Song, Y., Sretavan, D., Salegio, E. A., Berg, J., Huang, X., Cheng, T., Xiong, X., Meltzer, S., Han, C., Nguyen, T. T., Bresnahan, J. C., Beattie, M. S., Jan, L. Y. and Jan, Y. N. (2015). Regulation of axon regeneration by the RNA repair and splicing pathway. Nat Neurosci 18: 817-825. PubMed ID: 25961792
Mechanisms governing a neuron's regenerative ability are important but not well understood. This study has identified Rtca (RNA 3'-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease (CG6353) operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. This study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma (Song 2015).

Wednesday, June 10th

Sreesankar, E., Bharathi, V., Mishra, R.K. and Mishra, K. (2015). DrosophilaRif1 is an essential gene and controls late developmental events by direct interaction with PP1-87B. Sci Rep 5: 10679. PubMed ID: 26022086
Rif1, identified as a regulator of telomerase in yeast, is an evolutionarily conserved protein and functions in diverse processes including telomere length regulation, epigenetic gene regulation, anti-checkpoint activity, DNA repair and establishing timing of firing at replication origins. All Rif1 homologues have been shown to have a PP1 interacting SILK-RVxF motif. This study shows that Drosophila Rif1 is essential for normal fly development and loss of dRif1 impairs temporal regulation of initiation of DNA replication. In multiple tissues dRif1 colocalized with HP1, a protein known to orchestrate timing of replication in fly. dRif1 associated with chromosomes in a mitotic stage-dependent manner coinciding with dephosphorylation of histones. Ectopic expression of dRif1 caused enlarged larval imaginal discs and early pupal lethality which was completely reversed by co-expression of PP1 87B, the major protein phosphatase in Drosophila, indicating genetic and functional interaction. These findings suggest that dRif1 is an adaptor for PP1 and functions by recruiting PP1 to multiple sites on the chromosome.

Fan, J. Y., Means, J. C., Bjes, E. S. and Price, J. L. (2015) Drosophila DBT autophosphorylation of its C terminal domain antagonized by SPAG and involved in UV-induced apoptosis Mol Cell Biol [Epub ahead of print] PubMed ID: 25939385
Drosophila DBT and vertebrate CKI/δ phosphorylate Per to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. This study identified sites of C terminal autophosphorylation. Mutation of 6 serines and threonines in the C terminus (DBTC/ala) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIdelta, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBTC/ala did not affect circadian behavior differently from DBTWT, and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBTWT protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBTC/ala did not protect and was not degraded. Finally, it was shown that the HSP-90 cochaperone spaghetti (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.

Ji, H. H., Zhang, H. M., Shen, M., Yao, L. L. and Li, X. D. (2015). The motor function of Drosophila melanogaster myosin-5 is activated by calcium and cargo-binding protein dRab11. Biochem J [Epub ahead of print]. PubMed ID: 25940004
Myosin-5 (DmM5; Didum) plays two distinct roles in response to light stimulation: transport of pigment granules to the rhabdomere base to decrease light exposure and transport of rhodopsin-bearing vesicles to the rhabdomere base to compensate for the rhodopsin loss during light exposure. This study overexpressed DmM5 in Sf9 cells and investigated its regulation using purified proteins. The actin-activated ATPase activity of DmM5 is significantly lower than that of the truncated DmM5 having the C-terminal globular tail domain (GTD) deleted, indicating that the GTD is the inhibitory domain. The actin-activated ATPase activity of DmM5 is significantly activated by micromolar levels of calcium. DmM5 associates with pigment granules and rhodopsin-bearing vesicles through cargo-binding proteins Lightoid and dRab11 respectively. GTP-bound dRab11, but not Lightoid, significantly activates DmM5 actin-activated ATPase activity. Moreover, Gln1689 in the GTD was identified as the critical residue for the interaction with dRab11. Based on those results, it is proposed that DmM5-dependent transport of pigment granules is directly activated by light-induced calcium influx and the DmM5-dependent transport of rhodopsin-bearing vesicle is activated by active GTP-bound dRab11, whose formation is stimulated by light-induced calcium influx.

Klebba, J. E., Buster, D. W., McLamarrah, T. A., Rusan, N. M. and Rogers, G. C. (2015). Autoinhibition and relief mechanism for Polo-like kinase 4. Proc Natl Acad Sci U S A 112: E657-666. PubMed ID: 25646492
Polo-like kinase 4 (Plk4) is a master regulator of centriole duplication, and its hyperactivity induces centriole amplification. Homodimeric Plk4 has been shown to be ubiquitinated as a result of autophosphorylation, thus promoting its own degradation and preventing centriole amplification. Unlike other Plks, Plk4 contains three rather than two Polo box domains, and the function of its third Polo box (PB3) is unclear. A functional analysis of Plk4's structural domains was performed in this study. Like other Plks, Plk4 possesses a previously unidentified autoinhibitory mechanism mediated by a linker (L1) near the kinase domain. Thus, autoinhibition is a conserved feature of Plks. In the case of Plk4, autoinhibition is relieved after homodimerization and is accomplished by PB3 and by autophosphorylation of L1. In contrast, autophosphorylation of the second linker promotes separation of the Plk4 homodimer. Therefore, autoinhibition delays the multiple consequences of activation until Plk4 dimerizes. These findings reveal a complex mechanism of Plk4 regulation and activation which govern the process of centriole duplication.

Ejeskar, K., Vickes, O., Kuchipudi, A., Wettergren, Y., Uv, A. and Rotter Sopasakis, V. (2015). The unique non-catalytic C-Terminus of P37delta-PI3K adds proliferative properties in vitro and in vivo. PLoS One 10: e0127497. PubMed ID: 26024481
The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. p37delta, an alternatively spliced product of human PI3K p110delta, has been shown to display strong proliferation-promoting properties despite lacking the catalytic domain completely. This study functionally evaluated the different domains of human p37delta in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and the unique C-terminal region of human p37delta further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37delta both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37delta prevents this effect. Furthermore, it was found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. These results indicate a novel kinase-independent function of the PI3K catalytic subunit.

Ren, G. R., Folke, J., Hauser, F., Li, S. and Grimmelikhuijzen, C. J. (2015). The A- and B-type muscarinic acetylcholine receptors from Drosophila melanogaster couple to different second messenger pathways. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 25964087
Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are activated by the agonists acetylcholine and muscarine and blocked by several antagonists, among them atropine. In mammals five mAChRs (m1-m5) exist of which m1, m3, and m5 are coupled to members of the Gq/11 family and m2 and m4 to members of the Gi/0 family. Drosophila melanogaster and other arthropods have two mAChRs, named mAChR-A and mAChR-B, where the A-type has the same pharmacology as the mammalian mAChRs, while the B-type has a very low affinity to muscarine and no affinity to classical antagonists such as atropine. This study found that the D. melanogaster A-type mAChR is coupled to Gq/11 and D. melanogaster B-type mAChR to Gi/0. Furthermore, by comparing the second and third intracellular loops of all animal mAChRs for which the G protein coupling has been established, it was possible to identify several amino acid residues likely to be specific for either Gq/11 or Gi/0 coupling. Using these hallmarks for specific mAChR G protein interaction it was found that all protostomes with a sequenced genome have one mAChR coupled to Gq/11 and one to four mAChRs coupled to Gi/0. Furthermore, in protostomes, probably all A-type mAChRs are coupled to Gq/11 and all B-type mAChRs to G0/i.

Tuesday, June 9th

Ismail, M.Z., Hodges, M.D., Boylan, M., Achall, R., Shirras, A. and Broughton, S.J. (2015). The Drosophila insulin receptor independently modulates lifespan and locomotor senescence. PLoS One 10: e0125312. PubMed ID: 26020640
The Insulin/IGF-like signalling (IIS) pathway plays an evolutionarily conserved role in ageing. In model organisms reduced IIS extends lifespan and ameliorates some forms of functional senescence. However, little is known about IIS in nervous system ageing and behavioural senescence. To investigate this role in Drosophila melanogaster, the effect of reduced IIS on senescence of two locomotor behaviours, negative geotaxis and exploratory walking, was measured in this study. Two long-lived fly models with systemic IIS reductions (daGAL4/UAS-InRDN (ubiquitous expression of a dominant negative insulin receptor) and d2GAL/UAS-rpr (ablation of insulin-like peptide producing cells)) showed an amelioration of negative geotaxis senescence similar to that previously reported for the long-lived IIS mutant chico. In contrast, exploratory walking in daGAL4/UAS-InRDN and d2GAL/UAS-rpr flies declined with age similarly to controls. To determine the contribution of IIS in the nervous system to these altered senescence patterns and lifespan, the InRDN was targeted to neurons (elavGAL4/UAS-InRDN), which resulted in extension of lifespan in females, normal negative geotaxis senescence in males and females, and detrimental effects on age-specific exploratory walking behaviour in males and females. These data indicate that the Drosophila insulin receptor independently modulates lifespan and age-specific function of different types of locomotor behaviour. The data suggest that ameliorated negative geotaxis senescence of long-lived flies with systemic IIS reductions is due to ageing related effects of reduced IIS outside the nervous system. The lifespan extension and coincident detrimental or neutral effects on locomotor function with a neuron specific reduction (elavGAL4/UAS-InRDN) indicates that reduced IIS is not beneficial to the neural circuitry underlying the behaviours despite increasing lifespan

Srivastav, S., Singh, S. K., Yadav, A. K. and Srikrishna, S. (2015) Folic acid supplementation ameliorates oxidative stress, metabolic functions and developmental anomalies in a novel fly model of Parkinson's disease. Neurochem Res [Epub ahead of print]. PubMed ID: 25963948
Mutations in parkin cause early-onset Parkinson's disease. Studies involving Drosophila model have emphasised mitochondrial dysfunction as a critical event in disease pathogenesis. In this context, a novel recessive allele of parkin, parkc00062, was used for the current study. The piggyBac insertion at 3rd intron of parkc00062, was confirmed by PCR. Homozygous parkc00062 has diminished levels of truncated parkin transcript with no detectable protein as confirmed by qRT-PCR and western blot analysis, respectively. The homozygous parkc00062 displayed severe developmental anomalies involving reduced body size, ~45 % pupal lethality, high mortality with locomotory defect, elevated oxidative stress, low metabolic active cell status with low mitochondrial respiration as reflected from reduced ATP levels. Further, folic acid therapeutic potential was analysed in parkc00062. This study shows that dietary folic acid provided protection against disparities involving pupal lethality, high mortality, locomotory defect, elevated oxidative stress and low metabolic active cell status associated with parkc00062 . Further mitochondrial respiration was enhanced as reflected from improved ATP levels in folate supplemented parkc00062. To corroborate mitochondrial functioning further analysis regarding transcript status of p53 and spargel by qRT-PCR, revealed down regulation of p53 and up regulation of spargel in folate supplemented parkc00062, which was originally vice a versa. Thee data thus support the potential of FA in alleviating the disparities associated with parkin loss of function in fly model. Further, FA role in alleviating mitochondrial dysfunction is encouraging to further explore FA mechanistic role to be utilized as potential therapeutics for parkin mediated neurodegenerative diseases.

Lim, J. Y., Reighard, C. P. and Crowther, D. C. (2015). The pro-domains of neurotrophins, including BDNF, are linked to Alzheimer's disease through a toxic synergy with Abeta. Hum Mol Genet. PubMed ID: 25954034
Brain-derived neurotrophic factor (BDNF) has a crucial role in learning and memory by promoting neuronal survival and modulating synaptic connectivity. BDNF levels are lower in the brains of individuals with Alzheimer's disease (AD), suggesting a pathogenic involvement. The Drosophila orthologue of BDNF is the highly conserved Neurotrophin 1 (DNT1). BDNF and DNT1 have the same overall protein structure and can be cleaved, resulting in the conversion of a full-length polypeptide into separate pro- and mature-domains. While the BDNF mature-domain is neuroprotective, the role of the pro-domain is less clear. In flies and mammalian cells, this study has identified a synergistic toxic interaction between the amyloid-beta peptide (Aβ1-42; see Drosophila Appl) and the pro-domains of both DNT1 and BDNF. Specifically, DNT1 pro-domain acquires a neurotoxic activity in the presence of Aβ1-42. In contrast, DNT1 mature-domain is protective against Aβ1-42 toxicity. Likewise, in SH-SY5Y cell culture, BDNF pro-domain is toxic only in the presence of Aβ1-42. Western blots indicate that this synergistic interaction likely results from the Aβ1-42-induced upregulation of the BDNF pro-domain receptor p75NTR. The clinical relevance of these findings is underlined by a greater than thirty fold increase in the ratio of BDNF pro- to mature-domains in the brains of individuals with AD. This unbalanced BDNF pro:mature-domain ratio in patients represents a possible biomarker of AD and may offer a target for therapeutic intervention.

Ma, X., Chen, Y., Zhang, S., Xu, W., Shao, Y., Yang, Y., Li, W., Li, M. and Xue, L. (2015). Rho1-Wnd signaling regulates loss-of-cell polarity-induced cell invasion in Drosophila. Oncogene [Epub ahead of print]. PubMed ID: 25961917
Both cell polarity and c-Jun N-terminal kinase (JNK) activity are essential to the maintenance of tissue homeostasis, and disruption of either is commonly seen in cancer progression. Despite the established connection between loss-of-cell polarity and JNK activation, much less is known about the molecular mechanism by which aberrant cell polarity induces JNK-mediated cell migration and tumor invasion. This study presents results from a genetic screen using an in vivo invasion model via knocking down cell polarity gene in Drosophila wing discs, and identifies Rho1-Wnd signaling as an important molecular link that mediates loss-of-cell polarity-triggered JNK activation and cell invasion. Wallenda (Wnd), a protein kinase of the mitogen-activated protein kinase kinase kinase family, by forming a complex with the GTPase Rho1, is both necessary and sufficient for Rho1-induced JNK-dependent cell invasion, MMP1 activation and epithelial-mesenchymal transition. Furthermore, Wnd promotes cell proliferation and tissue growth through Wingless production when apoptosis is inhibited by p35. Finally, Wnd shows oncogenic cooperation with RasV12 to trigger tumor growth in eye discs and causes invasion into the ventral nerve cord. Together, these data not only provides a novel mechanistic insight on how cell polarity loss contributes to cell invasion, but also highlights the value of the Drosophila model system to explore human cancer biology.

Grice, S. J., Sleigh, J. N., Motley, W. W., Liu, J. L., Burgess, R. W., Talbot, K. and Cader, M. Z. (2015). Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25972375
Charcot-Marie-Tooth (CMT) neuropathies are collectively the most common hereditary neurological condition and a major health burden for society. Dominant mutations in the gene GARS, encoding the ubiquitous enzyme, glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and lead to CMT disease type 2D (CMT2D). This genetic disorder exemplifies a recurring motif in neurodegeneration, whereby mutations in essential, widely expressed genes have selective deleterious consequences for the nervous system. Using novel Drosophila models, this study shows a potential solution to this phenomenon. Ubiquitous expression of mutant GlyRS leads to motor deficits, progressive neuromuscular junction (NMJ) denervation, and pre-synaptic build-up of mutant GlyRS. Intriguingly, neuronal toxicity is, at least in part, non-cell autonomous, as expression of mutant GlyRS in mesoderm or muscle alone results in similar pathology. This mutant GlyRS toxic gain-of-function, which is WHEP domain-dependent, coincides with abnormal NMJ assembly, leading to synaptic degeneration, and, ultimately, reduced viability. These findings suggest that mutant GlyRS gains access to ectopic sub-compartments of the motor neuron, providing a possible explanation for the selective neuropathology caused by mutations in a widely expressed gene.

Chi, T., et al. (2015). A Drosophila model identifies a critical role for zinc in mineralization for kidney stone disease. PLoS One 10: e0124150. PubMed ID: 25970330
Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. This study establishes a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (muXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall's plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, zinc transporter genes in the ZnT family were inhibited, and suppression of Drosophila stone formation was observed. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. These findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

Monday, June 8th

Houssin, E., Tepass, U. and Laprise, P. (2015) Girdin-mediated interactions between cadherin and the actin cytoskeleton are required for epithelial morphogenesis in Drosophila Development 142: 1777-1784. PubMed ID: 25968313
E-cadherin-mediated cell-cell adhesion is fundamental for epithelial tissue morphogenesis, physiology and repair. E-cadherin is a core transmembrane constituent of the zonula adherens (ZA), a belt-like adherens junction located at the apicolateral border in epithelial cells. The anchorage of ZA components to cortical actin filaments strengthens cell-cell cohesion and allows for junction contractility, which shapes epithelial tissues during development. This study reports that the cytoskeletal adaptor protein Girdin physically and functionally interacts with components of the cadherin-catenin complex during Drosophila embryogenesis. Fly Girdin is broadly expressed throughout embryonic development and enriched at the ZA in epithelial tissues. Girdin associates with the cytoskeleton and co-precipitates with the cadherin-catenin complex protein alpha-Catenin (alpha-Cat). Girdin mutations strongly enhance adhesion defects associated with reduced DE-cadherin (DE-Cad) expression. Moreover, the fraction of DE-Cad molecules associated with the cytoskeleton decreases in the absence of Girdin, thereby identifying Girdin as a positive regulator of adherens junction function. Girdin mutant embryos display isolated epithelial cell cysts and rupture of the ventral midline, consistent with defects in cell-cell cohesion. In addition, loss of Girdin impairs the collective migration of epithelial cells, resulting in dorsal closure defects. It is proposed that Girdin stabilizes epithelial cell adhesion and promotes morphogenesis by regulating the linkage of the cadherin-catenin complex to the cytoskeleton.

Zhang, P., Dai, W., Hahn, J. and Gilbert, S. P. (2015) Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s Proc Natl Acad Sci U S A 112: 6359-6364. PubMed ID: 25941402
Drosophila kinesin-14 Ncd cross-links parallel microtubules at the spindle poles and antiparallel microtubules within the spindle midzone to play roles in bipolar spindle assembly and proper chromosome distribution. As observed for Saccharomyces cerevisiae kinesin-14 Kar3Vik1 and Kar3Cik1, Ncd binds adjacent microtubule protofilaments in a novel microtubule binding configuration and uses an ATP-promoted powerstroke mechanism. The hypothesis tested in this study is that Kar3Vik1 and Kar3Cik1, as well as Ncd, use a common ATPase mechanism for force generationß. The presteady-state kinetics and computational modeling establish an ATPase mechanism for a powerstroke model of Ncd that is very similar to those determined for Kar3Vik1 and Kar3Cik1, although these heterodimers have one Kar3 catalytic motor domain and a Vik1/Cik1 partner motor homology domain whose interactions with microtubules are not modulated by nucleotide state but by strain. The results indicate that both Ncd motor heads bind the microtubule lattice; two ATP binding and hydrolysis events are required for each powerstroke; and a slow step occurs after microtubule collision and before the ATP-promoted powerstroke. Note that unlike conventional myosin-II or other processive molecular motors, Ncd requires two ATP turnovers rather than one for a single powerstroke-driven displacement or step. These results are significant because all metazoan kinesin-14s are homodimers, and the results presented show that despite their structural and functional differences, the heterodimeric and homodimeric kinesin-14s share a common evolutionary structural and mechanochemical mechanism for force generation.

Goins, L. M. and Mullins, R. D. (2015). A novel tropomyosin isoform functions at the mitotic spindle and Golgi in Drosophila. Mol Biol Cell [Epub ahead of print]. PubMed ID: 25971803
Most eukaryotic cells express multiple isoforms of the actin-binding protein tropomyosin that help construct a variety of cytoskeletal networks. Using biochemical and molecular genetic approaches, this study identified three tropomyosins expressed in Drosophila S2 cells: Tm1A, Tm1J (both coded for by the Tm1 gene), and Tm2A (coded for by Tm2). The Tm1A isoform localizes to the cell cortex, lamellar actin networks, and the cleavage furrow of dividing cells- always together with myosin-II. Isoforms Tm1J and Tm2A colocalize around the Golgi apparatus with the formin-family protein Diaphanous and loss of either isoform perturbs cell cycle progression. During mitosis, Tm1J localizes to the mitotic spindle where it promotes chromosome segregation. Using chimeras, this study identified the determinants of tropomyosin localization near the C-terminus. This work: (1) identifies and characterizes previously unknown non-muscle tropomyosins in Drosophila; (2) reveals a function for tropomyosin in the mitotic spindle; and (3) uncovers sequence elements that specify isoform-specific localizations and functions of tropomyosin.

Cottee, M.A., Muschalik, N., Johnson, S., Leveson, J., Raff, J.W. and Lea, S.M. (2015). The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies. Elife [Epub ahead of print]. PubMed ID: 26002084
Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the nine-fold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). This study shows that the Drosophila Ana2 CCCD forms a tetramer, and solves its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. The structure of the Drosophila Sas-6 N-terminal domain was also solved to 2.9 Å revealing that it formed higher-order oligomers through canonical interactions. Point mutations that perturbed Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturbed centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

Sunday, June 7th

Zhang, B., Rotelli, M., Dixon, M. and Calvi, B. R. (2015) The function of Drosophila p53 isoforms in apoptosis. Cell Death Differ [Epub ahead of print]. PubMed ID: 25882045
The p53 protein is a major mediator of the cellular response to genotoxic stress and is a crucial suppressor of tumor formation. In a variety of organisms, p53 and its paralogs, p63 and p73, each encode multiple protein isoforms through alternative splicing, promoters, and translation start sites. The function of these isoforms in development and disease are still being defined. This study evaluated the apoptotic potential of multiple isoforms of the single p53 gene in Drosophila. Most previous studies have focused on the p53A isoform, but it has been recently shown that a larger p53B isoform can induce apoptosis when overexpressed. It has remained unclear, however, whether one or both isoforms are required for the apoptotic response to genotoxic stress. This study shows that p53B is a much more potent inducer of apoptosis than p53A when overexpressed. Overexpression of two newly identified short isoforms perturbed development and inhibited the apoptotic response to ionizing radiation. Analysis of physiological protein expression indicated that p53A is the most abundant isoform, and that both p53A and p53B can form a complex and co-localize to sub-nuclear compartments. In contrast to the overexpression results, new isoform-specific loss-of-function mutants indicated that it is the shorter p53A isoform, not full-length p53B, that is the primary mediator of pro-apoptotic gene transcription and apoptosis after ionizing radiation. Together, these data show that it is the shorter p53A isoform that mediates the apoptotic response to DNA damage, and further suggest that p53B and shorter isoforms have specialized functions.

Jain, A., et al. (2015) p62/sequestosome-1, autophagy-related gene 8 and autophagy in Drosophila are regulated by Nuclear factor erythroid 2-related factor 2 (NRF2), independent of transcription factor TFEBJ Biol Chem [Epub ahead of print]. PubMed ID: 25931115
The selective autophagy receptor p62/sequestosome 1 (SQSTM1) interacts directly with LC3 and is involved in oxidative stress signaling in two ways in mammals. First, p62 is transcriptionally induced upon oxidative stress by the NF-E2-related factor 2 (NRF2) by direct binding to an antioxidant response element (ARE) in the p62 promoter. Secondly, p62 accumulation, occurring when autophagy is impaired, lead to increased p62 binding to the NRF2 inhibitor KEAP1 resulting in reduced proteasomal turnover of NRF2. This gives chronic oxidative stress signaling through a feed forward loop. This study shows that the Drosophila p62/SQSTM1 orthologue, Ref(2)P, interacts directly with DmAtg8a via a LC3-interacting region (LIR) motif, supporting a role for Ref(2)P in selective autophagy. The ref(2)P promoter also contains a functional ARE that is directly bound by the NRF2 orthologue, CncC which can induce ref(2)P expression along with the oxidative stress associated gene gstD1. However, distinct from the situation in mammals, Ref(2)P does not interact directly with DmKeap1 via a KEAP1-interacting region (KIR) motif. Neither does ectopically expressed Ref(2)P, nor autophagy deficiency, activate the oxidative stress response. Instead, DmAtg8a interacts directly with DmKeap1, and DmKeap1 is removed upon programmed autophagy in Drosophila gut cells. Strikingly, CncC induced increased Atg8a levels and autophagy independent of TFEB/Mitf in fat body and larval gut tissues. Thus, these results extend the intimate relationship between oxidative stress sensing NRF2/CncC transcription factors and autophagy, and suggests that NRF2/CncC may regulate autophagic activity in other organisms too.

Ma, X., Xu, W., Zhang, D., Yang, Y., Li, W. and Xue, L. (2015). Wallenda regulates JNK-mediated cell death in Drosophila. Cell Death Dis 6: e1737. PubMed ID: 25950467
The c-Jun N-terminal kinase (JNK) pathway plays essential roles in regulating a variety of cellular processes including proliferation, migration and survival. Previous genetic studies in Drosophila have identified numerous cell death regulating genes, providing new insights into the mechanisms for related diseases. Despite the known role of the small GTPase Rac1 in regulating cell death, the downstream components and underlying mechanism remain largely elusive. This study shows that Rac1 promotes JNK-dependent cell death through Wallenda (Wnd). In addition, Wnd triggers JNK activation and cell death via its kinase domain. Moreover, both MKK4 and Hep are critical for Wnd-induced cell death. Furthermore, Wnd is essential for ectopic Egr- or Rho1-induced JNK activation and cell death. Finally, Wnd is physiologically required for loss of Scribble-induced JNK-dependent cell death. Thus, these data suggest that wnd encodes a novel essential cell death regulator in Drosophila.

Guntermann, S., Fraser, B., Hazes, B. and Foley, E. (2015). Independent proteolytic activities control the stability and size of Drosophila Inhibitor of apoptosis 2 Protein. J Innate Immun [Epub ahead of print]. PubMed ID: 25968339
The Drosophila immune deficiency pathway defends many bacterial pathogens and bears striking molecular similarities to the mammalian tumor necrosis factor signal transduction pathway. Orthologous inhibitors of apoptosis ubiquitin ligases act at a proximal stage of both responses to coordinate the assembly of signal transduction platforms that shape host immune responses. This study examined the molecular basis for inhibitor of apoptosis 2 protein regulation in the immune deficiency pathway. These studies identified two distinct proteolytic events that determine the stability and composition of cellular inhibitor of apoptosis 2 protein pools. Apoptotic caspase activity cleaves inhibitor of apoptosis 2 at an N-terminal aspartate to generate a truncated protein that retains the ability to interact with immune deficiency pathway members. It was also showed that a C-terminal ubiquitin ligase activity within inhibitor of apoptosis 2 directs the proteasomal destruction of full-length and truncated inhibitor of apoptosis 2 isoforms. These studies add to the appreciation of the regulation of innate immunity and suggest potential links between apoptotic caspases and innate defenses.

Saturday, June 6th

Morgante, F., Sorensen, P., Sorensen, D. A., Maltecca, C. and Mackay, T. F. (2015). Genetic architecture of micro-environmental plasticity in Drosophila melanogasterSci Rep 5: 9785. PubMed ID: 25943032
Individuals of the same genotype do not have the same phenotype for quantitative traits when reared under common macro-environmental conditions, a phenomenon called micro-environmental plasticity. Genetic variation in micro-environmental plasticity is assumed in models of the evolution of phenotypic variance, and is important in applied breeding and personalized medicine. This study quantified genetic variation for micro-environmental plasticity for three quantitative traits in the inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel. Substantial genetic variation was found for micro-environmental plasticity for all traits, with broad sense heritabilities of the same magnitude or greater than those of trait means. Micro-environmental plasticity is not correlated with residual segregating variation, is trait-specific, and has genetic correlations with trait means ranging from zero to near unity. Several candidate genes were identified that were associated with micro-environmental plasticity of startle response, including Drosophila Hsp90, setting the stage for future genetic dissection of this phenomenon.

Jing, X., White, T. A., Yang, X. and Douglas, A. E. (2015) The molecular correlates of organ loss: the case of insect Malpighian tubulesBiol Lett 11 [Epub ahead of print]. PubMed ID: 25972400
Malpighian tubules play an essential role in excretion, osmoregulation and immunity of most insects. Exceptionally, aphids lack Malpighian tubules, providing the opportunity to investigate the fate of genes expressed in an organ that has undergone evolutionary reduction and loss. Making use of the sequenced genomes of Drosophila melanogaster and the pea aphid Acyrthosiphon pisum, this study demonstrates that more than 50% of Drosophila genes expressed specifically in the Malpighian tubules had orthologues in the pea aphid genome and that most of the pea aphid orthologues with detectable expression were identified in the gut transcriptome. Relative to the whole genome, genes functioning in amino acid metabolism are significantly over-represented among the pea aphid orthologues of Malpighian tubule genes, likely reflecting the central importance of amino acid acquisition and metabolism in aphids. This study demonstrates that the evolutionary loss of a key insect organ, the Malpighian tubules, has not been associated with the coupled loss of molecular functions.

Kofler, R., Hill, T., Nolte, V., Betancourt, A. J. and Schlotterer, C. (2015). The recent invasion of natural Drosophila simulans populations by the P-element. Proc Natl Acad Sci U S A 112: 6659-6663. PubMed ID: 25964349
The P-element is one of the best understood eukaryotic transposable elements. It invaded Drosophila melanogaster populations within a few decades but was thought to be absent from close relatives, including Drosophila simulans. Five decades after the spread in D. melanogaster, evidence is provided that the P-element has also invaded D. simulans. P-elements in D. simulans appear to have been acquired recently from D. melanogaster probably via a single horizontal transfer event. Expression data indicate that the P-element is processed in the germ line of D. simulans, and genomic data show an enrichment of P-element insertions in putative origins of replication, similar to that seen in D. melanogaster. This ongoing spread of the P-element in natural populations provides a unique opportunity to understand the dynamics of transposable element spread and the associated piwi-interacting RNAs defense mechanisms.

Sachs, L., Chen, Y. T., Drechsler, A., Lynch, J. A., Panfilio, K. A., Lassig, M., Berg, J. and Roth, S. (2015). Dynamic BMP signaling polarized by Toll patterns the dorsoventral axis in a hemimetabolous insect. Elife 4 [Epub ahead of print]. PubMed ID: 25962855
Toll-dependent patterning of the dorsoventral axis in Drosophila represents one of the best understood gene regulatory networks. However, its evolutionary origin has remained elusive. Outside the insects Toll is not known for a patterning function, but rather for a role in pathogen defense. This study shows that in the milkweed bug Oncopeltus fasciatus, whose lineage split from Drosophila's more than 350 million years ago, Toll is only required to polarize a dynamic BMP signaling network. A theoretical model reveals that this network has self-regulatory properties and that shallow Toll signaling gradients are sufficient to initiate axis formation. Such gradients can account for the experimentally observed twinning of insect embryos upon egg fragmentation and might have evolved from a state of uniform Toll activity associated with protecting insect eggs against pathogens.

Assaf, Z. J., Petrov, D. A. and Blundell, J. R. (2015) Obstruction of adaptation in diploids by recessive, strongly deleterious alleles Proc Natl Acad Sci U S A 112: E2658-2666. PubMed ID: 25941393
Recessive deleterious mutations are common, causing many genetic disorders in humans and producing inbreeding depression in the majority of sexually reproducing diploids. The abundance of recessive deleterious mutations in natural populations suggests they are likely to be present on a chromosome when a new adaptive mutation occurs, yet the dynamics of recessive deleterious hitchhikers and their impact on adaptation remains poorly understood. This study modeled how a recessive deleterious mutation impacts the fate of a genetically linked dominant beneficial mutation. The frequency trajectory of the adaptive mutation in this case is dramatically altered and results in what is termed in this study a "staggered sweep." It is named for its three-phased trajectory: (1) Initially, the two linked mutations have a selective advantage while rare and will increase in frequency together, then (2), at higher frequencies, the recessive hitchhiker is exposed to selection and can cause a balanced state via heterozygote advantage (the staggered phase), and (3) finally, if recombination unlinks the two mutations, then the beneficial mutation can complete the sweep to fixation. Using both analytics and simulations, it was shown that strongly deleterious recessive mutations can substantially decrease the probability of fixation for nearby beneficial mutations, thus creating zones in the genome where adaptation is suppressed. These mutations can also significantly prolong the number of generations a beneficial mutation takes to sweep to fixation, and cause the genomic signature of selection to resemble that of soft or partial sweeps. This study shows that recessive deleterious variation could impact adaptation in humans and Drosophila.

Tang, S. and Presgraves, D. C. (2015) Lineage-specific evolution of the complex Nup160 hybrid incompatibility between Drosophila melanogaster and its sister species. Genetics [Epub ahead of print]. PubMed ID: 26022241
Two genes encoding protein components of the nuclear pore complex, Nup160 and Nup96, cause lethality in F2-like hybrid genotypes between Drosophila simulans and D. melanogaster. In particular, D. simulans Nup160 and Nup96 each cause inviability when hemizygous or homozygous in species hybrids that are also hemizygous (or homozygous) for the D. melanogaster X chromosome. The hybrid lethality of Nup160 is however genetically complex, depending on one or more unknown additional factors in the autosomal background. The genetics and evolution of Nup160-mediated hybrid lethality was studied in three ways. First, variability in Nup160-mediated hybrid lethality was tested within and among the three species of the Drosophila simulans clade- D. simulans, D. sechellia, and D. mauritiana. The hybrid lethality of Nup160 was shown to be fixed in D. simulans and in D. sechellia but absent in D. mauritiana. Second, how the hybrid lethality of Nup160 depends on other loci in the autosomal background was explored. D, simulans Nup160-mediated hybrid lethality was found not to depend on the presence of D. melanogaster Nup96, and D. simulans and D. mauritiana were found to be functionally differentiated at Nup160 as well as the other autosomal factor(s). Finally, population genetics data were used to show that Nup160 has experienced histories of recurrent positive selection both before and after the split of the three D. simulans clade species, ~240,000 years ago. These genetic results suggest that a hybrid lethal Nup160 allele evolved before the split of the three D. simulans clade species, whereas the other autosomal factor(s) evolved more recently.

Friday, June 5th

Matsuoka, Y., Bando, T., Watanabe, T., Ishimaru, Y., Noji, S., Popadic, A. and Mito, T. (2015) Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes Biol Open. PubMed ID: 25948756
In insect species that undergo long germ segmentation, such as Drosophila, all segments are specified simultaneously at the early blastoderm stage. As embryogenesis progresses, the expression boundaries of Hox genes are established by repression of gap genes, which is subsequently replaced by Polycomb group (PcG) silencing. At present, however, it is not known whether patterning occurs this way in a more ancestral (short germ) mode of embryogenesis, where segments are added gradually during posterior elongation. In this study, two members of the PcG family, Enhancer of zeste (E(z)) and Suppressor of zeste 12 (Su(z)12), were analyzed in the short germ cricket, Gryllus bimaculatus. Results suggest that although stepwise negative regulation by gap and PcG genes is present in anterior members of the Hox cluster, it does not account for regulation of two posterior Hox genes, abdominal-A (abd-A) and Abdominal-B (Abd-B). Instead, abd-A and Abd-B are predominantly regulated by PcG genes, which is the mode present in vertebrates. These findings suggest that PcG-mediated silencing of Hox genes may have occurred during animal evolution. The ancestral bilaterian state may have resembled the current vertebrate mode of regulation, where PcG-mediated silencing of Hox genes occurs before their expression is initiated and is responsible for the establishment of individual expression domains. Then, during insect evolution, the repression by transcription factors may have been acquired in anterior Hox genes of short germ insects, while PcG silencing was maintained in posterior Hox genes.

Naval-Sanchez, M., Potier, D., Hulselmans, G., Christiaens, V. and Aerts, S. (2015) Identification of lineage-specific cis-regulatory modules associated with variation in transcription factor binding and chromatin activity using Ornstein-Uhlenbeck models Mol Biol Evol [Epub ahead of print]. PubMed ID: 25944915
Scoring the impact of non-coding variation on the function of cis-regulatory regions, on their chromatin state, and on the qualitative and quantitative expression levels of target genes is a fundamental problem in evolutionary genomics. A particular challenge is how to model the divergence of quantitative traits and to identify relationships between the changes across the different levels of the genome, the chromatin activity landscape, and the transcriptome. This study examined the use of the Ornstein-Uhlenbeck (OU) model to infer selection at the level of predicted cis-regulatory modules, and link these with changes in transcription factor binding and chromatin activity. Using publicly available cross-species ChIP-seq and STARR-seq data it was shown how OU can be applied genome-wide to identify candidate transcription factors for which binding site and CRM turnover is correlated with changes in regulatory activity. Next, open chromatin was profiled in the developing eye across three Drosophila species. The recognition motifs of the chromatin remodelers, Trithorax-like and Grainyhead, were identified as mostly correlating with species-specific changes in open chromatin. In conclusion, this study that CRM scores can be used as quantitative traits and that motif discovery approaches can be extended towards more complex models of divergence.

Fresán, U., Cuartero, S., O'Connor, M.B. and Espinás, M.L. (2015). The insulator protein CTCF regulates Drosophila steroidogenesis. Biol Open [Epub ahead of print]. PubMed ID: 25979705
The steroid hormone ecdysone is a central regulator of insect development. This report shows that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF resulted in delayed and less synchronized larval development that could only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells lead to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis.

Zhang, G., et al. (2015). N(6)-methyladenine DNA modification in Drosophila. Cell 161: 893-906. PubMed ID: 25936838
DNA N(6)-methyladenine (6mA) modification is commonly found in microbial genomes and plays important functions in regulating numerous biological processes in bacteria. However, whether 6mA occurs and what its potential roles are in higher-eukaryote cells remain unknown. This study shows that 6mA is present in Drosophila genome and that the 6mA modification is dynamic and is regulated by the Drosophila Tet homolog, DNA 6mA demethylase (DMAD), during embryogenesis. Importantly, biochemical assays demonstrate that DMAD directly catalyzes 6mA demethylation in vitro. Further genetic and sequencing analyses reveal that DMAD is essential for development and that DMAD removes 6mA primarily from transposon regions, which correlates with transposon suppression in Drosophila ovary. Collectively, this study has uncovered a DNA modification in Drosophila and describe a potential role of the DMAD-6mA regulatory axis in controlling development in higher eukaryotes.

Thursday, June 4th

Kidd, S., Struhl, G. and Lieber, T. (2015). Notch is required in adult Drosophila sensory neurons for morphological and functional plasticity of the olfactory circuit. PLoS Genet 11: e1005244. PubMed ID: 26011623
Olfactory receptor neurons (ORNs) convey odor information to the central brain, but like other sensory neurons were thought to play a passive role in memory formation and storage. This study show that Notch, part of an evolutionarily conserved intercellular signaling pathway, is required in adult Drosophila ORNs for the structural and functional plasticity of olfactory glomeruli that is induced by chronic odor exposure. Specifically, it was shown that Notch activity in ORNs is necessary for the odor specific increase in the volume of glomeruli that occur as a consequence of prolonged odor exposure. Calcium imaging experiments indicated that Notch in ORNs is also required for the chronic odor induced changes in the physiology of ORNs and the ensuing changes in the physiological response of their second order projection neurons (PNs). It was further shown that Notch in ORNs acts by both canonical cleavage-dependent and non-canonical cleavage-independent pathways. The Notch ligand Delta (Dl) in PNs switches the balance between the pathways. These data define a circuit whereby, in conjunction with odor, N activity in the periphery regulates the activity of neurons in the central brain and Dl in the central brain regulates N activity in the periphery. This study highlights the importance of experience dependent plasticity at the first olfactory synapse.

Spratford, C. M. and Kumar, J. P. (2015) Inhibition of Daughterless by Extramacrochaetae mediates Notch-induced cell proliferationDevelopment 142: 2058-2068. PubMed ID: 25977368
During development, the rate of cell proliferation must be constantly monitored so that an individual tissue achieves its correct size. Mutations in genes that normally promote tissue growth often result in undersized, disorganized and non-functional organs. However, mutations in genes that encode growth inhibitors can trigger the onset of tumorigenesis and cancer. The developing eye of the fruit fly, Drosophila melanogaster, has become a premier model system for studies that are focused on identifying the molecular mechanisms that underpin growth control. This study examine the mechanism by which the Notch pathway, a major contributor to growth, promotes cell proliferation in the developing eye. Current models propose that the Notch pathway directly influences cell proliferation by regulating growth-promoting genes such as four-jointed, cyclin D1 and E2f1. This study shows that, in addition to these mechanisms, some Notch signaling is devoted to blocking the growth-suppressing activity of the bHLH DNA-binding protein Daughterless (Da). Notch signaling activates the expression of extramacrochaetae (emc), which encodes a helix-loop-helix (HLH) transcription factor. Emc, in turn, then forms a biochemical complex with Da. As Emc lacks a basic DNA-binding domain, the Emc-Da heterodimer cannot bind to and regulate genomic targets. One effect of Da sequestration is to relieve the repression on growth. Data is presented supporting the model that Notch-induced cell proliferation in the developing eye is mediated in part by the activity of Emc.

Scholz, N., Gehring, J., Guan, C., Ljaschenko, D., Fischer, R., Lakshmanan, V., Kittel, R. J. and Langenhan, T. (2015). The adhesion GPCR Latrophilin/CIRL shapes mechanosensation. Cell Rep 11: 866-874. PubMed ID: 25937282
G-protein-coupled receptors (GPCRs) are typically regarded as chemosensors that control cellular states in response to soluble extracellular cues. However, the modality of stimuli recognized through adhesion GPCR (aGPCR), the second largest class of the GPCR superfamily, is unresolved. This study characterizes the Drosophila aGPCR Latrophilin/dCirl, a prototype member of this enigmatic receptor class. dCirl was shown to shape the perception of tactile, proprioceptive, and auditory stimuli through chordotonal neurons, the principal mechanosensors of Drosophila. dCirl sensitizes these neurons for the detection of mechanical stimulation by amplifying their input-output function. Larval chordotonal organs respond to tactile stimuli arising through gentle touch, mechanical deformation of the larval body wall and musculature during the locomotion cycle, and vibrational cues elicited through sound. All these mechanical qualities are reduced in the absence of dCirl. Behavioral defects can be rescued by re-expression of dCirl in chordotonal neurons, one of several cell types with endogenous dCirl expression. Mechanically stimulated lch5 neurons lacking dCirl respond with action currents at approximately half the control rate across a broad spectrum of stimulation frequencies, providing direct functional evidence for a role of dCirl in chordotonal dendrites. Combining dCirlKO with strong hypomorphs of trp homologs, ion channels that are directly responsible for the conversion of mechanical stimulation into electrical signals within chordotonal neurons, implies that dCirl operates upstream of them. Finally, the dCirl promoter contains a RFX/Fd3F transcription factor signature that implicates dCirl in the mechanosensitive specialization of sensory cilia. On the basis of these results, it is proposed that dCirl partakes in the process of mechanotransduction or spike initiation and transmission to promote sensory encoding.

Niewalda, T., Michels, B., Jungnickel, R., Diegelmann, S., Kleber, J., Kahne, T. and Gerber, B. (2015). Synapsin determines memory strength after punishment- and relief-learning. J Neurosci 35: 7487-7502. PubMed ID: 25972175
Adverse life events can induce two kinds of memory with opposite valence, dependent on timing: "negative" memories for stimuli preceding them and "positive" memories for stimuli experienced at the moment of "relief." Such punishment memory and relief memory are found in insects, rats, and man. For example, fruit flies (Drosophila melanogaster) avoid an odor after odor-shock training ("forward conditioning" of the odor), whereas after shock-odor training ("backward conditioning" of the odor) they approach it. Do these timing-dependent associative processes share molecular determinants? This study focused on the role of Synapsin, a conserved presynaptic phosphoprotein regulating the balance between the reserve pool and the readily releasable pool of synaptic vesicles. A lack of Synapsin leaves task-relevant sensory and motor faculties unaffected. In contrast, both punishment memory and relief memory scores are reduced. These defects reflect a true lessening of associative memory strength, as distortions in nonassociative processing (e.g., susceptibility to handling, adaptation, habituation, sensitization), discrimination ability, and changes in the time course of coincidence detection can be ruled out as alternative explanations. Reductions in punishment- and relief-memory strength are also observed upon an RNAi-mediated knock-down of Synapsin, and are rescued both by acutely restoring Synapsin and by locally restoring it in the mushroom bodies of mutant flies. Thus, both punishment memory and relief memory require the Synapsin protein and in this sense share genetic and molecular determinants. It is noted that corresponding molecular commonalities between punishment memory and relief memory in humans would constrain pharmacological attempts to selectively interfere with excessive associative punishment memories, e.g., after traumatic experiences.

Savall, J., Ho, E.T., Huang, C., Maxey, J.R. and Schnitzer, M.J. (2015). Dexterous robotic manipulation of alert adult Drosophila for high-content experimentation. Nat Methods [Epub ahead of print]. PubMed ID: 26005812
A robot has been constructed that enables high-content studies of alert adult Drosophila by combining operations including gentle picking; translations and rotations; characterizations of fly phenotypes and behaviors; microdissection; or release. To illustrate, the study assessed fly morphology, tracked odor-evoked locomotion, sorted flies by sex, and dissected the cuticle to image neural activity. The robot's tireless capacity for precise manipulations enables a scalable platform for screening flies' complex attributes and behavioral patterns. Overall, this programmable system flexibly combines automated handling, surgical maneuvers, machine vision and behavioral assessments-without using anesthesia and while providing greater statistical power than humans can easily muster. A key virtue is the possibility of performing multiple analyses of individual flies, such as of morphological, behavioral and neurophysiological traits, for studies of how attributes interrelate. The capacity to catch and release individual flies will also enable time-lapse experiments involving repeated examinations of phenotypes across days or weeks, for studies of development, aging or disease. Future implementations might include additional mechanical capabilities or multiple picking units. As with any new technology, users will need time to explore the possibilities, but it is expected that a diverse library of programs will develop.

Nern, A., Pfeiffer, B. D. and Rubin, G. M. (2015). Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system. Proc Natl Acad Sci U S A 112(22):E2967-76. PubMed ID: 25964354
The development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy is described in this study. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. The utility of MCFO is demonstrated in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. Within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns.

Shih, C.T., Sporns, O., Yuan, S.L., Su, T.S., Lin, Y.J., Chuang, C.C., Wang, T.Y., Lo, C.C., Greenspan, R.J. and Chiang, A.S. (2015). Connectomics-based analysis of information flow in the Drosophila brain. Curr Biol 25: 1249-1258. PubMed ID: 25866397
Understanding the overall patterns of information flow within the brain has become a major goal of neuroscience. The current study has produced a first draft of the Drosophila connectome at the mesoscopic scale, reconstructed from 12,995 images of neuron projections collected in FlyCircuit (version 1.1). Neuron polarities were predicted according to morphological criteria, with nodes of the network corresponding to brain regions designated as local processing units (LPUs). The weight of each directed edge linking a pair of LPUs was determined by the number of neuron terminals that connected one LPU to the other. The resulting network showed hierarchical structure and small-world characteristics and consisted of five functional modules that corresponded to sensory modalities (olfactory, mechanoauditory, and two visual) and the pre-motor center. Rich-club organization was present in this network and involved LPUs in all sensory centers, and rich-club members formed a putative motor center of the brain. Major intra- and inter-modular loops were also identified that could play important roles for recurrent and reverberant information flow. It is proposed that the overall organizational scheme shows fundamental similarities to the network structure of the mammalian brain.

Gao, X. J., Riabinina, O., Li, J., Potter, C. J., Clandinin, T. R. and Luo, L. (2015) A transcriptional reporter of intracellular Ca(2+) in Drosophila. Nat Neurosci 18: 917-925 PubMed ID: 25961791
Intracellular Ca(2+) is a widely used neuronal activity indicator. This study describes a transcriptional reporter of intracellular Ca(2+) (TRIC) in Drosophila that uses a binary expression system to report Ca(2+)-dependent interactions between calmodulin and its target peptide. In vitro assays predicted in vivo properties of TRIC. TRIC signals in sensory systems were show to depend on neuronal activity. TRIC was able to quantitatively monitor neuronal responses that changed slowly, such as those of neuropeptide F-expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis cells to food and arousal. Furthermore, TRIC-induced expression of a neuronal silencer in nutrient-activated cells enhanced stress resistance, providing a proof of principle that TRIC can be used for circuit manipulation. Thus, TRIC facilitates the monitoring and manipulation of neuronal activity, especially those reflecting slow changes in physiological states that are poorly captured by existing methods. TRIC's modular design should enable optimization and adaptation to other organisms.

Wednesday, June 3rd

Yang, H. and Yamashita, Y.M. (2015). The regulated elimination of transit-amplifying cells preserves tissue homeostasis during protein starvation in Drosophila testis. Development 142: 1756-1766. PubMed ID: 25968311
How tissues adapt to varying nutrient conditions is of fundamental importance for robust tissue homeostasis throughout the life of an organism, but the underlying mechanisms are poorly understood. This study shows that Drosophila testis responds to protein starvation by eliminating transit-amplifying spermatogonia (SG) while maintaining a reduced pool of actively proliferating germline stem cells (GSCs). During protein starvation, SG died in a manner that was mediated by the apoptosis of somatic cyst cells (CCs) that encapsulated SG and regulated their development. Strikingly, GSCs could not be maintained during protein starvation when CC-mediated SG death was inhibited, leading to an irreversible collapse of tissue homeostasis. The study proposes that the regulated elimination of transit-amplifying cells is essential to preserve stem cell function and tissue homeostasis during protein starvation.

Luo, L., Wang, H., Fan, C., Liu, S. and Cai, Y. (2015). Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche. J Cell Biol 209: 595-608. PubMed ID: 26008746
Stem cell self-renewal versus differentiation is regulated by the niche, which provides localized molecules that favor self-renewal. In the Drosophila melanogaster female germline stem cell (GSC) niche, Decapentaplegic (Dpp), a fly transforming growth factor β molecule and well-established long-range morphogen, acts over one cell diameter to maintain the GSCs. This study shows that Thickveins (Tkv; a type I receptor of Dpp) is highly expressed in stromal cells next to Dpp-producing cells and functions to remove excess Dpp outside the niche, thereby spatially restricting its activity. Interestingly, Tkv expression in these stromal cells was regulated by multiple Wnt ligands that were produced by the niche. These data demonstrate a self-restraining mechanism by which the Drosophila ovarian GSC niche acts to define its own boundary.

Yeh, T.H., Huang, S.Y., Lan, W.Y., Liaw, G.J. and Yu, J.Y. (2015). Modulation of cell morphogenesis by Tousled-like kinase in the Drosophila follicle cell. Dev Dyn [Epub ahead of print]. PubMed ID: 25981356
Tousled-like kinase (Tlk) is a conserved Serine/Threonine kinase regulating DNA replication, chromatin assembly, and DNA repair. Previous studies have suggested that Tlk is involved in cell morphogenesis in vitro. In addition, tlk genetically interact with Rho1, which encodes a key regulator of the cytoskeleton. However, whether Tlk plays a physiological role in cell morphogenesis and cytoskeleton rearrangement remains unknown. This study found that in tlk mutant follicle cells, area of the apical domain was reduced and the density of microtubules was increased. The density of actin filaments was increased in the apical region and decreased in the basal region. Since area of the apical domain was reduced, the levels of proteins located in the apical region were examined by using immunofluorescence. The fluorescence intensities of two adherens junction proteins Armadillo (Arm) and DE-cadherin (DE-cad), atypical protein kinase C (aPKC), and Notch, were all increased in tlk mutant cells. The basolateral localized Discs large (Dlg) shifted apically in tlk mutant cells. Increase of protein densities in the apical region might be resulting from disruption of the cytoskeleton and shrinkage of the apical domain. Together, these data suggest a novel role of Tlk in maintaining cell morphology, possibly through modulating the cytoskeleton

Lee, D. M., Wilk, R., Hu, J., Krause, H. M. and Harris, T. J. (2015). Germ cell segregation from the Drosophila soma is controlled by an inhibitory threshold set by the Arf-GEF Steppke. Genetics [Epub ahead of print]. PubMed ID: 25971667
Germline cells segregate from the soma to maintain their totipotency, but the cellular mechanisms of this segregation are unclear. The Drosophila melanogaster embryo forms a posterior group of primordial germline cells (PGCs) by their division from the syncytial soma. Extended plasma membrane furrows enclose the PGCs in response to the germ plasm protein Germ cell-less (Gcl) and Rho1-actomyosin activity. Recently, it was found that loss of the Arf-GEF Steppke (Step) leads to similar Rho1-dependent plasma membrane extensions but from pseudocleavage furrows of the soma. This study reports that the loss of step also leads to premature formation of a large cell group at the anterior pole of the embryo. These anterior cells lacked germ plasm, but budded and formed at the same time as posterior PGCs, and then divided asynchronously as PGCs also do. With genetic analyses it was found that Step normally activates Arf small G proteins and antagonizes Rho1-actomyosin pathways to inhibit anterior cell formation. A uniform distribution of step mRNA around the one-cell embryo cortex suggested that Step restricts cell formation through a global control mechanism. Thus, the effect of Step on PGC formation at the posterior pole was examined. Reducing Gcl or Rho1 levels decreased PGC numbers, but additional step RNAi restored their numbers. Reciprocally, GFP-Step overexpression induced dosage- and Arf-GEF-dependent loss of PGCs, an effect worsened by reducing Gcl or actomyosin pathway activity. It is proposed that a global distribution of Step normally sets an inhibitory threshold for Rho1 activity to restrict early cell formation to the posterior.

Tuesday, June 2nd

Kanke, M. and Macdonald, P. M. (2015). Translational activation of Oskar mRNA: Reevaluation of the role and importance of a 5' regulatory element. PLoS One 10: e0125849. PubMed ID: 25938537
Local translation of oskar (osk) mRNA at the posterior pole of the Drosophila oocyte is essential for axial patterning of the embryo, and is achieved by a program of translational repression, mRNA localization, and translational activation. Multiple forms of repression are used to prevent Oskar protein from accumulating at sites other than the oocyte posterior. Activation is mediated by several types of cis-acting elements, which presumably control different forms of activation. This study characterized a 5' element, positioned in the coding region for the Long Osk isoform and in the extended 5' UTR for translation of the Short Osk isoform. This element was previously thought to be essential for osk mRNA translation, with a role in posterior-specific release from repression. From this work, which includes assays which separate the effects of mutations on RNA regulatory elements and protein coding capacity, it was found that the element is not essential, and the study concludes that there is no evidence supporting a role for the element only at the posterior of the oocyte. The 5' element has a redundant role, and is only required when Long Osk is not translated from the same mRNA. Mutations in the element do disrupt the anchoring function of Long Osk protein through their effects on the amino acid sequence, a confounding influence on interpretation of previous experiments.

Mugat, B., Akkouche, A., Serrano, V., Armenise, C., Li, B., Brun, C., Fulga, T.A., Van Vactor, D., Pélisson, A. and Chambeyron, S. (2015). MicroRNA-dependent transcriptional silencing of transposable elements in Drosophila follicle cells. PLoS Genet 11: e1005194. PubMed ID: 25993106
In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. This study shows that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. This is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

Yuva-Aydemir, Y., Xu, X.L., Aydemir, O., Gascon, E., Sayin, S., Zhou, W., Hong, Y. and Gao, F.B. (2015). Downregulation of the host gene jigr1 by miR-92 is essential for neuroblast self-renewal in Drosophila. PLoS Genet 11: e1005264. PubMed ID: 26000445
Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. This study shows that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3'UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b were highly expressed in neuroblasts of larval brain where Jigr1 expression was low. Genetic deletion of both miR-92a and miR-92b demonstrated an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. It was also shown that miR-92a and miR-92b directly targeted jigr1 in vivo and that some phenotypes due to the absence of these miRNAs were partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development.

Mohn, F., Handler, D. and Brennecke, J. (2015). Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis Science 348: 812-817. PubMed ID: 25977553
In animal gonads, PIWI-clade Argonaute proteins repress transposons sequence-specifically via bound Piwi-interacting RNAs (piRNAs). These are processed from single-stranded precursor RNAs by largely unknown mechanisms. This study shows that primary piRNA biogenesis is a 3'-directed and phased process that, in the Drosophila germ line, is initiated by secondary piRNA-guided transcript cleavage. Phasing results from consecutive endonucleolytic cleavages catalyzed by Zucchini, implying coupled formation of 3' and 5' ends of flanking piRNAs. Unexpectedly, Zucchini also participates in 3' end formation of secondary piRNAs. Its function can, however, be bypassed by downstream piRNA-guided precursor cleavages coupled to exonucleolytic trimming. These data uncover an evolutionarily conserved piRNA biogenesis mechanism in which Zucchini plays a central role in defining piRNA 5' and 3' ends.

Monday, June 1st

Dissel, S., Angadi, V., Kirszenblat, L., Suzuki, Y., Donlea, J., Klose, M., Koch, Z., English, D., Winsky-Sommerer, R., van Swinderen, B. and Shaw, P.J. (2015). Sleep restores behavioral plasticity to Drosophila mutants. Curr Biol 25: 1270-1281. PubMed ID: 25913403
Given the role that sleep plays in modulating plasticity, this study hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist THIP. Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reversed memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders.

Rohrscheib, C. E., Bondy, E., Josh, P., Riegler, M., Eyles, D., van Swinderen, B., Weible, M. W. and Brownlie, J. C. (2015). Wolbachia influences the production of octopamine and affects Drosophila male aggression. Appl Environ Microbiol [Epub ahead of print]. PubMed ID: 25934616
Wolbachia are endosymbionts that infect approximately 40% of all insect species and are best known for their ability to manipulate host reproductive systems. Though the effect Wolbachia infection has on somatic tissues is less well understood, when present in cells of the adult Drosophila melanogaster brain, it exerts an influence over behaviors related to olfaction. This study shows that a strain of Wolbachia influences male aggression in flies, which is critically important in mate competition. A specific strain of Wolbachia was observed to reduce the initiation of aggressive encounters in Drosophila males when compared against their uninfected controls. To determine how Wolbachia was able to alter aggressive behavior, the role of octopamine, a neurotransmitter known to influence male aggressive behavior in many insect species, was investigated. Transcriptional analysis of the octopamine biosynthesis pathway revealed that two essential genes, tryrosine decarboxylase and tyramine beta-hydroxylase, were significantly down regulated in Wolbachia infected flies. Quantitative chemical analysis also showed that total octopamine levels were significantly reduced in the adult heads.

Appel, M., Scholz, C.J., Müller, T., Dittrich, M., König, C., Bockstaller, M., Oguz, T., Khalili, A., Antwi-Adjei, E., Schauer, T., Margulies, C., Tanimoto, H. and Yarali, A. (2015). Genome-wide association analyses point to candidate genes for electric shock avoidance in Drosophila melanogaster. PLoS One 10: e0126986. PubMed ID: 25992709
Electric shock is a common stimulus for nociception-research and the most widely used reinforcement in aversive associative learning experiments. Yet, nothing is known about the mechanisms it recruits at the periphery. To help fill this gap, this study undertook a genome-wide association analysis using 38 inbred Drosophila melanogaster strains, which avoided shock to varying extents. 514 genes whose expression levels and/ or sequences co-varied with shock avoidance scores were identified. 14 of these genes were independently scrutinized using mutants, validating the effect of 7 of them on shock avoidance. This emphasized the value of the study's candidate gene list as a guide for follow-up research. In addition, by integrating association results with external protein-protein interaction data, a shock avoidance-associated network of 38 genes was obtained. Both this network and the original candidate list contained a substantial number of genes that affected mechanosensory bristles, which are hair-like organs distributed across the fly's body. These results may point to a potential role for mechanosensory bristles in shock sensation. Thus, this study not only provides a first list of candidate genes for shock avoidance, but also points to an interesting new hypothesis on nociceptive mechanisms.

Hernandez-Nunez, L., Belina, J., Klein, M., Si, G., Claus, L., Carlson, J. R. and Samuel, A. D. (2015). Reverse-correlation analysis of navigation dynamics in larva using optogenetics. Elife 4. PubMed ID: 25942453
Neural circuits for behavior transform sensory inputs into motor outputs in patterns with strategic value. Determining how neurons along a sensorimotor circuit contribute to this transformation is central to understanding behavior. To do this, a quantitative framework to describe behavioral dynamics is needed. A high-throughput optogenetic system for Drosophila larva was buil to quantify the sensorimotor transformations underlying navigational behavior. CsChrimson, a red-shifted variant of Channelrhodopsin, was expressed in specific chemosensory neurons, and large numbers of freely moving animals were exposed to random optogenetic activation patterns. Their behavioral responses were quantified, and reverse correlation analysis was used to uncover the linear and static nonlinear components of navigation dynamics as functions of optogenetic activation patterns of specific sensory neurons. Linear-nonlinear (LN) models were found to accurately predict navigational decision-making for different optogenetic activation waveforms.This method was used to establish the valence and dynamics of navigation driven by optogenetic activation of different combinations of bitter sensing gustatory neurons. This method captures the dynamics of optogenetically-induced behavior in compact, quantitative transformations that can be used to characterize circuits for sensorimotor processing and their contribution to navigational decision making.

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