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


Tuesday January 31st, 2017

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Beshel, J., Dubnau, J. and Zhong, Y. (2017). A leptin analog locally produced in the brain acts via a conserved neural circuit to modulate obesity-linked behaviors in Drosophila. Cell Metab 25: 208-217. PubMed ID: 28076762
Leptin, a typically adipose-derived "satiety hormone," has a well-established role in weight regulation. This study describes a functionally conserved model of genetically induced obesity in Drosophila by manipulating the fly leptin analog unpaired 1 (upd1). Unexpectedly, cell-type-specific knockdown reveals upd1 in the brain, not the adipose tissue, mediates obesity-related traits. Disrupting brain-derived upd1 in flies leads to all the hallmarks of mammalian obesity: increased attraction to food cues, increased food intake, and increased weight. These effects are mediated by domeless receptors on neurons expressing Drosophila neuropeptide F, the orexigenic mammalian neuropeptide Y homolog. In vivo two-photon imaging reveals upd1 and domeless inhibit this hedonic signal in fed animals. Manipulations along this central circuit also create hypersensitivity to obesogenic conditions, emphasizing the critical interplay between biological predisposition and environment in overweight and obesity prevalence. The study proposes that adipose- and brain-derived upd/leptin may control differing features of weight regulation through distinct neural circuits.

Erclik, T., Li, X., Courgeon, M., Bertet, C., Chen, Z., Baumert, R., Ng, J., Koo, C., Arain, U., Behnia, R., Rodriguez, A.D., Senderowicz, L., Negre, N., White, K.P. and Desplan, C. (2017). Integration of temporal and spatial patterning generates neural diversity. Nature 541: 365-370. PubMed ID: 28077877
In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neurons, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. This study shows that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors (see The OPC neuroepithelium is patterned along its dorsal-ventral axis). Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy.

Horne, M., Krebushevski, K., Wells, A., Tunio, N., Jarvis, C., Francisco, G., Geiss, J., Recknagel, A. and Deitcher, D. L. (2017). julius seizure, a Drosophila mutant, defines a neuronal population underlying epileptogenesis. Genetics [Epub ahead of print]. PubMed ID: 28082408
Epilepsy is a neural disorder characterized by recurrent seizures. Bang-sensitive Drosophila represent an important model for studying epilepsy and neuronal excitability. Previous work identified the bang-sensitive gene slamdance (sda) as an allele of the aminopeptidase N gene. This study shows through extensive genetic analysis, including recombination frequency, deficiency mapping, transposon insertion complementation testing, RNA interference, and genetic rescue that the gene responsible for the seizure-sensitivity is julius seizure (jus), formerly CG14509, which encodes a novel transmembrane domain protein and that the bang-sensitivity had been mislocalized to sda. More severe genetic alleles of jus RNAi-mediated knockdown of jus revealed that it is required only in neurons and not glia, and that partial bang-sensitivity is caused by knockdown in GABAergic or cholinergic but not glutamatergic neurons. RNAi knockdown of jus at the early pupal stages lead to strong seizures in adult animals, implicating that stage as critical for epileptogenesis. A C-terminal tagged version of Jus was generated from a fosmid genomic clone. This fosmid fusion rescued the bang-sensitive phenotype, was expressed in the optic lobes, and the subesophageal and thoracic abdominal ganglia. The protein was primarily localized in axons, especially in the neck connectives, extending into the thoracic abdominal ganglion.
Bonar, N. A. and Petersen, C. P. (2017). Integrin suppresses neurogenesis and regulates brain tissue assembly in planarian regeneration. Development. PubMed ID: 28126842
Evolutionary Homolog Study
Animals capable of adult regeneration require specific signaling to control injury-induced cell proliferation, specification and patterning, but comparatively little is known about how the regeneration blastema assembles differentiating cells into well-structured functional tissues. Using the planarian Schmidtea mediterranea as a model, β1-integrin (see Drosophila Myospheroid) as a critical regulator of blastema architecture. beta1-integrin(RNAi) animals formed small head blastemas with severe tissue disorganization, including ectopic neural spheroids containing differentiated neurons normally found in distinct organs. By mimicking aspects of normal brain architecture but lacking normal cell-type regionalization, these spheroids bore a resemblance to mammalian tissue organoids synthesized in vitro. One of four planarian integrin-α subunits was identified, whose inhibition phenocopied these effects, suggesting a specific receptor controls brain organization through regeneration. Neoblast stem cells and progenitor cells were mislocalized in β1-integrin(RNAi) animals without significantly altered body-wide patterning. Furthermore, tissue disorganization phenotypes were most pronounced in animals undergoing brain regeneration and not homeostatic maintenance or regeneration-induced remodeling of the brain. These results suggest that integrin signaling ensures proper progenitor recruitment after injury, enabling the generation of large-scale tissue organization within the regeneration blastema.

Monday, January 30th

Crocker, J., Tsai, A. and Stern, D. L. (2017). A fully synthetic transcriptional platform for a multicellular eukaryote. Cell Rep 18(1): 287-296. PubMed ID: 28052257
Regions of genomic DNA called enhancers encode binding sites for transcription factor proteins. Binding of activators and repressors increase and reduce transcription, respectively, but it is not understood how combinations of activators and repressors generate precise patterns of transcription during development. This problem was explored using a fully synthetic transcriptional platform in Drosophila consisting of engineered transcription factor gradients and artificial enhancers. First, a gradient of transcription-activator like protein (TALEs) fused to a VP16 activator (TALEA) was engineered. The gradient of TALEA protein was generated by driving TALEA expression with the hunchback promoter (hb-TALEA), resulting in a smooth anterior-to-posterior RNA gradient. The binding site for this TALEA, 5′-CCGGATGCTCCTCTT, is not present in the Drosophila genome and allowed construction of enhancers that would respond only to the TALEA. Binding sites for a transcription factor that makes DNA accessible, Zelda, were found to be required together with binding sites for transcriptional activators to produce a functional enhancer. Only in this context can changes in the number of activator binding sites mediate quantitative control of transcription. Using an engineered transcriptional repressor gradient, it was demonstrated that overlapping repressor and activator binding sites provide more robust repression and sharper expression boundaries than non-overlapping sites. This may explain why this common motif is observed in many developmental enhancers.
Nevil, M., Bondra, E. R., Schulz, K. N., Kaplan, T. and Harrison, M. M. (2016). Stable binding of the conserved transcription factor Grainy head to Its target genes throughout Drosophila melanogaster development. Genetics [Epub ahead of print]. PubMed ID: 28007888
It has been suggested that transcription factor binding is temporally dynamic and that changes in binding determine transcriptional output. Nonetheless, this model is based on relatively few examples in which transcription factor binding has been assayed at multiple developmental stages. The essential transcription factor Grainy head is conserved from fungi to humans and controls epithelial development and barrier formation in numerous tissues. Drosophila melanogaster, which possess a single grainy head gene, provide an excellent system to study this conserved factor. To determine whether temporally distinct binding events allow Grainy head to control cell fate specification in different tissue types, a combination of ChIP-seq and RNA-seq was used to elucidate the gene regulatory network controlled by Grainy head during four stages of embryonic development (spanning stages 5 - 17) and in larval tissue. Contrary to expectations, Grainy head was found to remain bound to at least 1146 genomic loci over days of development. In contrast to this stable DNA occupancy, the subset of genes whose expression is regulated by Grainy head varies. Grainy head transitions from functioning primarily as a transcriptional repressor early in development to functioning predominantly as an activator later. The data reveal that Grainy head binds to target genes well before the Grainy head-dependent transcriptional program commences, suggesting it sets the stage for subsequent recruitment of additional factors that execute stage-specific Grainy head functions.
Koenecke, N., Johnston, J., He, Q., Meier, S. and Zeitlinger, J. (2016). Drosophila poised enhancers are generated during tissue patterning with the help of repression. Genome Res [Epub ahead of print]. PubMed ID: 27979994
Histone modifications are frequently used as markers for enhancer states, but how to interpret enhancer states in the context of embryonic development is not clear. The poised enhancer signature, involving H3K4me1 and low levels of H3K27ac, has been reported to mark inactive enhancers that are poised for future activation. However, future activation is not always observed, and alternative reasons for the widespread occurrence of this enhancer signature have not been investigated. By analyzing enhancers during dorsal-ventral (DV) axis formation in the Drosophila embryo, it was found that the poised enhancer signature is specifically generated during patterning in the tissue where the enhancers are not induced, including at enhancers that are known to be repressed by a transcriptional repressor. These results suggest that, rather than serving exclusively as an intermediate step before future activation, the poised enhancer state may be a mark for spatial regulation during tissue patterning. The possibility is discussed that the poised enhancer state is more generally the result of repression by transcriptional repressors.
Desponds, J., Tran, H., Ferraro, T., Lucas, T., Perez Romero, C., Guillou, A., Fradin, C., Coppey, M., Dostatni, N. and Walczak, A. M. (2016). Precision of readout at the hunchback gene: Analyzing short transcription time traces in living fly embryos. PLoS Comput Biol 12(12): e1005256. PubMed ID: 27942043
The simultaneous expression of the hunchback gene in the numerous nuclei of the developing fly embryo gives a unique opportunity to study how transcription is regulated in living organisms. A recently developed MS2-MCP technique for imaging nascent messenger RNA in living Drosophila embryos allows quantification of the dynamics of the developmental transcription process. The initial measurement of the morphogens by the hunchback promoter takes place during very short cell cycles, not only giving each nucleus little time for a precise readout, but also resulting in short time traces of transcription. Additionally, the relationship between the measured signal and the promoter state depends on the molecular design of the reporting probe. An analysis approach based on tailor made autocorrelation functions was developed that overcomes the short trace problems and quantifies the dynamics of transcription initiation. Based on live imaging data, signatures of bursty transcription initiation from the hunchback promoter were identified. The precision of the expression of the hunchback gene to measure its position along the anterior-posterior axis was show to be low both at the boundary and in the anterior even at cycle 13, suggesting additional post-transcriptional averaging mechanisms to provide the precision observed in fixed embryos.

Sunday, January 29th

Li, Y., Li, S., Li, R., Xu, J., Jin, P., Chen, L. and Ma, F. (2016). Genome-wide miRNA screening reveals miR-310 family members negatively regulate the immune response in Drosophila melanogaster via co-targeting Drosomycin. Dev Comp Immunol 68: 34-45. PubMed ID: 27871832
Although innate immunity mediated by Toll signaling has been extensively studied in Drosophila melanogaster, the role of miRNAs in regulating the Toll-mediated immune response remains largely unknown. Following Gram-positive bacterial challenge, this study identified 93 differentially expressed miRNAs via genome-wide miRNA screening. These miRNAs were regarded as immune response related (IRR). Eight miRNAs were confirmed to be involved in the Toll-mediated immune response upon Gram-positive bacterial infection through genetic screening of 41 UAS-miRNA lines covering 60 miRNAs of the 93 IRR miRNAs. Interestingly, four out of these eight miRNAs, miR-310, miR-311, miR-312 and miR-313, are clustered miRNAs and belong to the miR-310 family. These miR-310 family members were shown to target and regulate the expression of Drosomycin, an antimicrobial peptide produced by Toll signaling. Taken together, this study implies important regulatory roles of miRNAs in the Toll-mediated innate immune response of Drosophila upon Gram-positive bacterial infection.
Dobson, A. J., Chaston, J. M. and Douglas, A. E. (2016). The Drosophila transcriptional network is structured by microbiota. BMC Genomics 17(1): 975. PubMed ID: 27887564
This study investigated the impact of resident microorganisms (microbiota) on gene coexpression in Drosophila. Transcriptomic analysis, of 17 lines representative of the global genetic diversity of Drosophila, yielded a total of 11 transcriptional modules of co-expressed genes. For seven of these modules, the strength of the transcriptional network (defined as gene-gene coexpression) differed significantly between flies bearing a defined gut microbiota (gnotobiotic flies) and flies reared under microbiologically sterile conditions (axenic flies). Furthermore, gene coexpression was uniformly stronger in these microbiota-dependent modules than in both the microbiota-independent modules in gnotobiotic flies and all modules in axenic flies, indicating that the presence of the microbiota directs gene regulation in a subset of the transcriptome. The genes constituting the microbiota-dependent transcriptional modules include regulators of growth, metabolism and neurophysiology, previously implicated in mediating phenotypic effects of microbiota on Drosophila phenotype. Together these results provide the first evidence that the microbiota enhances the coexpression of specific and functionally-related genes. This system-wide analysis demonstrates that the presence of microbiota enhances gene coexpression. This finding has potentially major implications for understanding of the mechanisms by which microbiota affect host health and fitness, and the ways in which hosts and their resident microbiota coevolve.
Li, S., Shen, L., Sun, L., Xu, J., Jin, P., Chen, L. and Ma, F. (2017). Small RNA-Seq analysis reveals microRNA-regulation of the Imd pathway during Escherichia coli infection in Drosophila. Dev Comp Immunol [Epub ahead of print]. PubMed ID: 28069431
Drosophila have served as a model for research on innate immunity for decades. However, knowledge of the post-transcriptional regulation of immune gene expression by microRNAs (miRNAs) remains rudimentary. Using small RNA-seq and bioinformatics analysis, this study identified 67 differentially expressed miRNAs in Drosophila infected with Escherichia coli compared to injured flies at three time-points. Furthermore, 21 of these miRNAs are potentially involved in the regulation of Imd pathway-related genes. Strikingly, based on UAS-miRNAs line screening and Dual-luciferase assay, miR-9a and miR-981 were found to both negatively regulate Drosophila antibacterial defenses and decrease the level of the antibacterial peptide, Diptericin. Taken together, these data support the involvement of miRNAs in the regulation of the Drosophila Imd pathway.

Elya, C., Zhang, V., Ludington, W. B. and Eisen, M. B. (2016). Stable host gene expression in the gut of adult Drosophila melanogaster with different bacterial mono-associations. PLoS One 11(11): e0167357. PubMed ID: 27898741
There is growing evidence that the microbes found in the digestive tracts of animals influence host biology, but how they accomplish this is still not understood. This study evaluated how different microbial species commonly associated with laboratory-reared Drosophila melanogaster impact host biology at the level of gene expression in the dissected adult gut and in the entire adult organism. It was observed that guts from animals associated from the embryonic stage with either zero, one or three bacterial species demonstrated indistinguishable transcriptional profiles. Additionally, it was found that the gut transcriptional profiles of animals reared in the presence of the yeast Saccharomyces cerevisiae alone or in combination with bacteria could recapitulate those of conventionally-reared animals. In contrast, whole body transcriptional profiles of conventionally-reared animals were distinct from all of the treatments tested. These data suggest that adult flies are insensitive to the ingestion of the bacteria found in their gut, but that prior to adulthood, different microbes impact the host in ways that lead to global transcriptional differences observable across the whole adult body.

Saturday, January 28th

Akhund-Zade, J., Bergland, A. O., Crowe, S. O. and Unckless, R. L. (2016). The genetic basis of natural variation in Drosophila (Diptera: Drosophilidae) virgin egg retention. J Insect Sci [Epub ahead of print]. PubMed ID: 28042107
Drosophila melanogaster is able to thrive in harsh northern climates through adaptations in life-history traits and physiological mechanisms that allow for survival through the winter. This examined the genetic basis of natural variation in one such trait, female virgin egg retention, which was previously shown to vary clinally and seasonally. To further understanding of the genetic basis and evolution of virgin egg retention, a genome-wide association study (GWAS) was performed using the previously sequenced Drosophila Genetic Reference Panel (DGRP) mapping population. Twenty-nine single nucleotide polymorphisms (SNPs) associated with virgin egg retention were found, and six available mutant lines, each harboring a mutation in a candidate gene, were examined for effects on egg retention time. Four out of the six mutant lines had defects in egg retention time as compared with the respective controls: mun, T48, Mes-4, and Klp67A Surprisingly, none of these genes has a recognized role in ovulation control, but three of the four genes have known effects on fertility or have high expression in the ovaries. The SNP set associated with egg retention time was enriched for clinal SNPs. The majority of clinal SNPs had alleles associated with longer egg retention present at higher frequencies in higher latitudes. These results support previous studies that show higher frequency of long retention times at higher latitude, providing evidence for the adaptive value of virgin egg-retention.
Corbett-Detig, R. and Nielsen, R. (2017). A hidden markov model approach for simultaneously estimating local ancestry and admixture time using next generation sequence data in samples of arbitrary ploidy. PLoS Genet 13(1): e1006529. PubMed ID: 28045893
Admixture-the mixing of genomes from divergent populations-is increasingly appreciated as a central process in evolution. This study introduced a novel hidden Markov model for estimating local ancestry that models the read pileup data, rather than genotypes, is generalized to arbitrary ploidy, and can estimate the time since admixture during local ancestry inference. This method can simultaneously estimate the time since admixture and local ancestry with good accuracy, and it performs well on samples of high ploidy-i.e. 100 or more chromosomes. As this method is very general, it will be useful for local ancestry inference in a wider variety of populations than what previously has been possible. The method was applied to pooled sequencing data derived from populations of Drosophila melanogaster on an ancestry cline on the east coast of North America. Regions of local recombination rates were found to be negatively correlated with the proportion of African ancestry, suggesting that selection against foreign ancestry is the least efficient in low recombination regions. Finally it was shown that clinal outlier loci are enriched for genes associated with gene regulatory functions, consistent with a role of regulatory evolution in ecological adaptation of admixed D. melanogaster populations. These results illustrate the potential of local ancestry inference for elucidating fundamental evolutionary processes.
Croze, M., Wollstein, A., Bozicevic, V., Zivkovic, D., Stephan, W. and Hutter, S. (2017). A genome-wide scan for genes under balancing selection in Drosophila melanogaster. BMC Evol Biol 17(1): 15. PubMed ID: 28086750
In the history of population genetics balancing selection has been considered as an important evolutionary force, yet until today little is known about its abundance and its effect on patterns of genetic diversity. Several well-known examples of balancing selection have been reported from humans, mice, plants, and parasites. However, only very few systematic studies have been carried out to detect genes under balancing selection. This study carried out a genome scan in Drosophila melanogaster to find signatures of balancing selection in a derived (European) and an ancestral (African) population. A total of 34 genomes were scanned, searching for regions of high genetic diversity and an excess of SNPs with intermediate frequency. In total, 183 candidate genes were found: 141 in the European population and 45 in the African one, with only three genes shared between both populations. Most differences between both populations were observed on the X chromosome, though this might be partly due to false positives. Functionally, an overrepresentation of genes involved in neuronal development and circadian rhythm were found. Furthermore, some of the top genes identified are involved in innate immunity. These results revealed evidence of genes under balancing selection in European and African populations. More candidate genes have been found in the European population. They are involved in several different functions.
Dowling, D., Pauli, T., Donath, A., Meusemann, K., Podsiadlowski, L., Petersen, M., Peters, R. S., Mayer, C., Liu, S., Zhou, X., Misof, B. and Niehuis, O. (2017). Phylogenetic origin and diversification of RNAi pathway genes in insects. Genome Biol Evol. PubMed ID: 28062756
RNA interference (RNAi) refers to the set of molecular processes found in eukaryotic organisms in which small RNA molecules mediate the silencing or down-regulation of target genes. In insects, RNAi serves a number of functions, including regulation of endogenous genes, anti-viral defense, and defense against transposable elements. Despite being well studied in model organisms, such as Drosophila, the distribution of core RNAi pathway genes and their evolution in insects is not well understood. This study presents the most comprehensive overview of the distribution and diversity of core RNAi pathway genes across 100 insect species, encompassing all currently recognized insect orders. The phylogenetic origin of insect-specific RNAi pathway genes was inferred, and also several hitherto unrecorded gene expansions were identified using whole-body transcriptome data from the international 1KITE (1000 Insect Transcriptome Evolution) project as well as other resources such as i5K (5000 Insect Genome Project). Specifically, the origin of the double stranded RNA binding protein R2D2 was traced to the last common ancestor of winged insects (Pterygota), the loss of Sid-1/Tag-130 orthologs in Antliophora (fleas, flies and relatives, and scorpionflies in a broad sense), and confirm previous evidence for the splitting of the Argonaute proteins Aubergine and Piwi in Brachyceran flies (Diptera, Brachycera). This study offers new reference points for future experimental research on RNAi-related pathway genes in insects.

Friday, January 27th

Salzberg, Y., Coleman, A., Celestrin, K., Cohen-Berkman, M., Biederer, T., Henis-Korenblit, S. and Bülow, H.E. (2017). Reduced Insulin/Insulin-like growth factor receptor signaling mitigates defective dendrite morphogenesis in mutants of the ER stress sensor IRE-1. PLoS Genet [Epub ahead of print]. PubMed ID: 28114319
Evolutionary Homolog Study:
Using the multidendritic arbor of PVD somatosensory neurons in the nematode Caenorhabditis elegans, this study identified a mutation in the ER stress sensor IRE-1/Ire1 (inositol requiring enzyme 1) (see Drosophila Ire1) as crucial for proper PVD dendrite arborization in vivo (see Drosophila axonogenesis). Regulation of dendrite growth in cultured rat hippocampal neurons depends on Ire1 function, showing an evolutionarily conserved role for IRE-1/Ire1 in dendrite patterning. PVD neurons of nematodes lacking ire-1 display reduced arbor complexity, whereas mutations in genes encoding other ER stress sensors display normal PVD dendrites, specifying IRE-1 as a selective ER stress sensor (see Drosophila as a model for ER stress and Drosophila UPR) that is essential for PVD dendrite morphogenesis. Although structure function analyses indicates that IRE-1's nuclease activity is necessary for its role in dendrite morphogenesis, mutations in xbp-1 (see Drosophila Xbp1), the best-known target of non-canonical splicing by IRE-1/Ire1, do not exhibit PVD phenotypes. Secretion and distal localization to dendrites of the DMA-1/leucine rich transmembrane receptor (DMA-1/LRR-TM) (see Drosophila rdo) is defective in ire-1 but not xbp-1 mutants, suggesting a block in the secretory pathway. Interestingly, reducing Insulin/IGF1 signaling (see Drosophila insulin signaling) can bypass the secretory block and restore normal targeting of DMA-1, and consequently normal PVD arborization even in the complete absence of functional IRE-1. This bypass of ire-1 requires the DAF-16/FOXO (see Drosophila foxo) transcription factor. In sum, this work identifies a conserved role for ire-1 in neuronal branching, which is independent of xbp-1, and suggests that arborization defects associated with neuronal pathologies may be overcome by reducing Insulin/IGF signaling and improving ER homeostasis and function.

Gerdoe-Kristensen, S., Lund, V. K., Wandall, H. H. and Kjaerulff, O. (2016). Mactosylceramide prevents glial cell overgrowth by inhibiting insulin and fibroblast growth factor receptor signaling. J Cell Physiol. PubMed ID: 28019653
Receptor Tyrosine Kinase (RTK) signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Deregulated RTK signaling also underlies many cancers. Glycosphingolipids (GSL) are essential elements of the plasma membrane. By affecting clustering and activity of membrane receptors, GSL modulate signal transduction, including that mediated by the RTK. GSL are abundant in the nervous system, and glial development in Drosophila is emerging as a useful model for studying how GSL modulate RTK signaling. Drosophila has a simple GSL biosynthetic pathway, in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what extent this effect involves changes in upstream signaling events is unresolved. This study shows that glial overgrowth in egh is strongly linked to increased activation of insulin and Fibroblast Growth Factor receptor (FGFR). Glial hypertrophy is phenocopied when overexpressing gain-of-function mutants of the Drosophila Insulin Receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. It is concluded that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of Insulin and Fibroblast Growth Factor Receptors in Drosophila glia.
Blanchette, C. R., Thackeray, A., Perrat, P. N., Hekimi, S. and Benard, C. Y. (2017). Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans. PLoS Genet 13(1): e1006525. PubMed ID: 28068429
Evolutionary Homolog Study
The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family (see Drosophila tout-velu). Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. This study identified and studied previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. The expression pattern of the HS copolymerase was found to be dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. These findings provide insights into general principles underlying HSPG function in development.
Amarnath, S., Stevens, L. M. and Stein, D. S. (2017). Reconstitution of Torso signaling in cultured cells suggests a role for both Trunk and Torsolike in receptor activation. Development [Epub ahead of print]. PubMed ID: 28087630
Formation of the Drosophila embryonic termini is controlled by the localized activation of the receptor tyrosine kinase, Torso. Both Torso and Torso's presumed ligand, Trunk, are expressed uniformly in the early embryo. Polar activation of Torso requires Torsolike, which is expressed by follicle cells adjacent to the ends of the developing oocyte. This study found that Torso expressed at high levels in cultured Drosophila cells is activated by individual application of Trunk, Torsolike or another known Torso ligand, Prothoracicotropic Hormone. In addition to assays of downstream signaling activity, Torso dimerization was detected using bimolecular fluorescence complementation. Trunk and Torsolike were active when co-transfected with Torso and when presented to Torso-expressing cells in conditioned medium. Trunk and Torsolike were also taken up from conditioned medium specifically by cells expressing Torso. At low levels of Torso, similar to those present in the embryo, Trunk and Torsolike alone were ineffective but acted synergistically to stimulate Torso signaling. These results suggest that Torso interacts with both Trunk and Torsolike, which cooperate to mediate dimerization and activation of Torso at the ends of the Drosophila embryo.

Thursday, January 26th

Tare, M., Sarkar, A., Bedi, S., Kango-Singh, M. and Singh, A. (2016). Cullin-4 regulates Wingless and JNK signaling-mediated cell death in the Drosophila eye. Cell Death Dis 7(12): e2566. PubMed ID: 28032862
In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. In a gain-of-function screen, this study found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, cul-4 has been shown to regulate chromatin remodeling, cell cycle and cell division. Genetic characterization of cul-4 in the developing eye revealed that loss-of-function of cul-4 exhibits a reduced eye phenotype. Analysis of twin-spots showed that in comparison with their wild-type counterparts, the cul-4 loss-of-function clones fail to survive. This study shows that cul-4 clones are eliminated by induction of cell death due to activation of caspases. Aberrant activation of signaling pathways is known to trigger cell death in the developing eye. Wingless (Wg) and c-Jun-amino-terminal-(NH2)-Kinase (JNK) signaling were found to be ectopically induced in cul-4 mutant clones, and these signals co-localize with the dying cells. Modulating levels of Wg and JNK signaling by using agonists and antagonists of these pathways demonstrated that activation of Wg and JNK signaling enhances cul-4 mutant phenotype, whereas downregulation of Wg and JNK signaling rescues the cul-4 mutant phenotypes of reduced eye. This study presents evidences to demonstrate that cul-4 is involved in restricting Wg signaling and downregulation of JNK signaling-mediated cell death during early eye development. Overall, these studies provide insights into a novel role of cul-4 in promoting cell survival in the developing Drosophila eye.
Bailey, D., Basar, M. A., Nag, S., Bondhu, N., Teng, S. and Duttaroy, A. (2017). The essential requirement of an animal heme peroxidase protein during the wing maturation process in Drosophila. BMC Dev Biol 17(1): 1. PubMed ID: 28077066
Thus far, a handful of genes have been shown to be related to the wing maturation process in insects. A novel heme peroxidase enzyme known as curly suppressor (Cysu)(formerly CG5873), have been characterized in this report because it is involved in wing morphogenesis. Using bioinformatics tools it was found that Cysu is remarkably conserved in the genus Drosophila (>95%) as well as in invertebrates (>70%), although its vertebrate orthologs show poor homology. Time-lapse imaging and histochemical analyses have confirmed that the defective wing phenotype of Cysu is not a result of any underlying cellular alterations; instead, its wings fail to expand in mature adults. The precise requirement of Cysu in wings was established by identifying a bona fide mutant of Cysu from the Bloomington Drosophila Stock Centre collection. Its requirement in the wing has also been shown by RNA knockdown of the gene. Subsequent transgenic rescue of the mutant wing phenotype with the wild-type gene confirmed the phenotype resulting from Cysu mutant. With appropriate GAL4 driver like engrailed-GAL4, the Cysu phenotype was compartmentalized, which raises a strong possibility that Cysu is not localized in the extracellular matrix (ECM); hence, Cysu is not engaged in bonding the dorsal and ventral cuticular layers. Finally, shortened lifespan of the Cysu mutant suggests it is functionally essential for other biological processes as well. It is concluded that Cysu a peroxinectin-like gene, is required during the wing maturation process in Drosophila because as a heme peroxidase, Cysu is capable of utilizing H2O2, which plays an essential role in post-eclosion wing morphogenesis.
Vollmer, J. and Iber, D. (2016). An unbiased analysis of candidate mechanisms for the regulation of Drosophila wing disc growth. Sci Rep 6: 39228. PubMed ID: 27995964
The control of organ size presents a fundamental open problem in biology. A declining growth rate is observed in all studied higher animals, and the growth limiting mechanism may therefore be evolutionary conserved. Most studies of organ growth control have been carried out in Drosophila imaginal discs. Previous studies have shown that the area growth rate in the Drosophila eye primordium declines inversely proportional to the increase in its area, which is consistent with a dilution mechanism for growth control. This study shows that a dilution mechanism cannot explain growth control in the Drosophila wing disc. A range of alternative candidate mechanisms were computationally evaluate, and the experimental data can be best explained by a biphasic growth law. However, also logistic growth and an exponentially declining growth rate fit the data very well. The three growth laws correspond to fundamentally different growth mechanisms that are discussed. Since a fit to the available experimental growth kinetics is insufficient to define the underlying mechanism of growth control, future experimental studies must focus on the molecular mechanisms to define the mechanism of growth control.
Dorot, O., Steller, H., Segal, D. and Horowitz, M. (2017). Past1 modulates Drosophila eye development. PLoS One 12(1): e0169639. PubMed ID: 28060904
Endocytosis is a multi-step process involving a large number of proteins, both general factors, such as clathrin and adaptor protein complexes, and unique proteins, which modulate specialized endocytic processes, like the EHD proteins. EHDs are a family of Eps15 Homology Domain containing proteins that consists of four mammalian homologs, one C. elegans, one Drosophila melanogaster and two plants orthologs. These membrane-associated proteins are involved in different steps of endocytic trafficking pathways. The Drosophila EHD ortholog, PAST1, has been shown to associate predominantly with the plasma membrane. Mutations in Past1 result in defects in endocytosis, male sterility, temperature sensitivity and premature death of the flies. Also, Past1 genetically interacts with Notch. The present study investigated the role of PAST1 in the developing fly eye. In mutant flies lacking PAST1, abnormal differentiation of photoreceptors R1, R6 and R7 was evident, with partial penetrance. Likewise, five cone cells were present instead of four. Expression of transgenic PAST1 resulted in a dominant negative effect, with a phenotype similar to that of the deletion mutant, and appearance of additional inter-ommatidial pigment cells. These results strongly suggest a role for PAST1 in differentiation of photoreceptors R1/R6/R7 and cone cells of the fly ommatidia.

Wednesday January 25th

Dominado, N., La Marca, J.E., Siddall, N.A., Heaney, J., Tran, M., Cai, Y., Yu, F., Wang, H., Somers, W.G., Quinn, L.M. and Hime, G.R. (2016). Rbf regulates Drosophila spermatogenesis via control of somatic stem and progenitor cell fate in the larval testis. Stem Cell Reports 7: 1152-1163. PubMed ID: 27974223
The Drosophila testis has been fundamental to understanding how stem cells interact with their endogenous microenvironment, or niche, to control organ growth in vivo. This study reports the identification of two independent alleles for the highly conserved tumor suppressor gene, Retinoblastoma-family protein (Rbf), in a screen for testis phenotypes in X chromosome third-instar lethal alleles. Rbf mutant alleles exhibit overproliferation of spermatogonial cells, which is phenocopied by the molecularly characterized Rbf11 null allele. Rbf promotes cell-cycle exit and differentiation of the somatic and germline stem cells of the testes. Intriguingly, depletion of Rbf specifically in the germline does not disrupt stem cell differentiation, rather Rbf loss of function in the somatic lineage drives overproliferation and differentiation defects in both lineages. Together, these observations suggest that Rbf in the somatic lineage controls germline stem cell renewal and differentiation non-autonomously via essential roles in the microenvironment of the germline lineage.

Knapp, E. and Sun, J. (2017). Steroid signaling in mature follicles is important for Drosophila ovulation. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 28069934
Although ecdysteroid signaling regulates multiple steps in oogenesis, it is not known whether it regulates Drosophila ovulation, a process involving a matrix metalloproteinase-dependent follicle rupture. This study demonstrates that ecdysteroid signaling is operating in mature follicle cells to control ovulation. Moreover, knocking down shade (shd), encoding the monooxygenase that converts ecdysone (E) to the more active 20-hydroxyecdysone (20E), specifically in mature follicle cells, blocks follicle rupture, which is rescued by ectopic expression of shd or exogenous 20E. In addition, disruption of the Ecdysone receptor (EcR) in mature follicle cells mimicks shd-knockdown defects, which are reversed by ectopic expression of EcR.B2 but not by EcR.A or EcR.B1 isoforms. Furthermore, ecdysteroid signaling is essential for the proper activation of matrix metalloproteinase 2 (Mmp2) for follicle rupture. These data strongly suggest that 20E produced in follicle cells before ovulation activates EcR.B2 to prime mature follicles to be responsive to neuronal ovulatory stimuli, thus providing mechanistic insights into steroid signaling in Drosophila ovulation.

Chiang, A.C., Yang, H. and Yamashita, Y.M. (2016). spict, a cyst cell-specific gene, regulates starvation-induced spermatogonial cell death in the Drosophila testis. Sci Rep 7: 40245. PubMed ID: 28071722
Tissues are maintained in a homeostatic state by balancing the constant loss of old cells with the continued production of new cells. Tissue homeostasis can shift between high and low turnover states to cope with environmental changes such as nutrient availability. It has been recently shown that the elimination of transit-amplifying cells plays a critical role in maintaining the stem cell population during protein starvation in the Drosophila testis. This study identifies spict, a gene expressed specifically in differentiating cyst cells, as a regulator of spermatogonial death. Spict is upregulated in cyst cells that phagocytose dying spermatogonia. The study proposes that phagocytosis and subsequent clearance of dead spermatogonia, which is partly promoted by Spict, contribute to stem cell maintenance during prolonged protein starvation.

Tirmarche, S., Kimura, S., Dubruille, R., Horard, B. and Loppin, B. (2016). Unlocking sperm chromatin at fertilization requires a dedicated egg thioredoxin in Drosophila. Nat Commun 7: 13539. PubMed ID: 27876811
In most animals, the extreme compaction of sperm DNA is achieved after the massive replacement of histones with sperm nuclear basic proteins (SNBPs), such as protamines. In some species, the ultracompact sperm chromatin is stabilized by a network of disulfide bonds connecting cysteine residues present in SNBPs. Studies in mammals have established that the reduction of these disulfide crosslinks at fertilization is required for sperm nuclear decondensation and the formation of the male pronucleus. This study shows that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization. In dhd mutant eggs, the sperm nucleus fails to decondense and the replacement of SNBPs with maternally-provided histones is severely delayed, thus preventing the participation of paternal chromosomes in embryo development. DHD localizes to the sperm nucleus to reduce its disulfide targets and is then rapidly degraded after fertilization.

Tuesday, January 24th

Tsou, W. L., Qiblawi, S. H., Hosking, R. R., Gomez, C. M. and Todi, S. V. (2016). Polyglutamine length-dependent toxicity from alpha1ACT in Drosophila models of spinocerebellar ataxia type 6. Biol Open 5(12): 1770-1775. PubMed ID: 27979829
Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease that results from abnormal expansion of a polyglutamine (polyQ) repeat. SCA6 is caused by CAG triplet repeat expansion in the gene CACNA1A, resulting in a polyQ tract of 19-33 in patients. CACNA1A, a bicistronic gene, encodes the α1A calcium channel subunit and the transcription factor, alpha1ACT. PolyQ expansion in α1ACT causes degeneration in mice. The first Drosophila models of SCA6 have been described that express α1ACT with a normal (11Q) or hyper-expanded (70Q) polyQ. This study reports additional alpha1ACT transgenic flies, which express full-length α1ACT with a 33Q repeat. α1ACT33Q is toxic in Drosophila, but less so than the 70Q version. When expressed everywhere, α1ACT33Q-expressing adults die earlier than flies expressing the normal allele. α1ACT33Q causes retinal degeneration and leads to aggregated species in an age-dependent manner, but at a slower pace than the 70Q counterpart. According to western blots, α1ACT33Q localizes less readily in the nucleus than α1ACT70Q, providing clues into the importance of polyQ tract length on α1ACT localization and its site of toxicity. These new lines are expected to be highly valuable for future work on SCA6.
Lehmann, S., Loh, S. H. and Martins, L. M. (2016). Enhancing NAD+ salvage metabolism is neuroprotective in a PINK1 model of Parkinson's disease. Biol Open [Epub ahead of print]. PubMed ID: 28011627
Familial forms of Parkinson's disease (PD) caused by mutations in PINK1 are linked to mitochondrial impairment. Defective mitochondria are also found in Drosophila models of PD with pink1 mutations. The co-enzyme nicotinamide adenine dinucleotide (NAD+) is essential for both generating energy in mitochondria and nuclear DNA repair through NAD+-consuming poly(ADP-ribose) polymerases (PARPs). This study found alterations in NAD+ salvage metabolism in Drosophila pink1 mutants and showed that a diet supplemented with the NAD+ precursor nicotinamide rescued mitochondrial defects and protected neurons from degeneration. Additionally, a mutation of Parp improved mitochondrial function and was neuroprotective in the pink1 mutants. It is concluded that enhancing the availability of NAD+ by either the use of a diet supplemented with NAD+ precursors or the inhibition of NAD+-dependent enzymes, such as PARPs, which compete with mitochondria for NAD+ is a viable approach to preventing neurotoxicity associated with mitochondrial defects.
Merzetti, E. M., Dolomount, L. A. and Staveley, B. E. (2016). The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson's disease. Genome: 1-9. PubMed ID: 27936908
Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson's disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, Proteasome inhibitor 31 kDa (PI31), have been shown to be active in proteasome function. This study shows that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established alpha-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control.
Wu, Q., Lian, T., Fan, X., Song, C., Gaur, U., Mao, X., Yang, D., Piper, M. D. and Yang, M. (2016). 2,5-Dimethyl-Celecoxib extends Drosophila life span via a mechanism that requires insulin and Target of rapamycin signaling. J Gerontol A Biol Sci Med Sci [Epub ahead of print]. PubMed ID: 28025308
The search for antiaging drugs is a key component of gerontology research. A few drugs with positive effects on life span in model organisms have been found. This study reports that 2,5-dimethyl-celecoxib, a derivative of the anti-inflammatory drug celecoxib, can extend Drosophila life span and delay aging by a mechanism involving insulin signaling and target of rapamycin signaling. Importantly, its positive effects were apparent when the treatment window was restricted to the beginning of life or the later half. 2,5-Dimethyl-celecoxib-induced longevity was also associated with improvements in physical activity, intestinal integrity, and increased autophagy. In addition, 2,5-dimethyl-celecoxib exhibited protective effects against several kinds of stress such as starvation and heat. The generally positive effects of 2,5-dimethyl-celecoxib on both health and life span, combined with its mode of action via evolutionarily conserved signaling pathways, indicate that it has the potential to become an effective antiaging drug.

Monday, January 23rd

Urban, J. A., Doherty, C. A., Jordan, W. T., 3rd, Bliss, J. E., Feng, J., Soruco, M. M., Rieder, L. E., Tsiarli, M. A. and Larschan, E. N. (2016). The essential Drosophila CLAMP protein differentially regulates non-coding roX RNAs in male and females. Chromosome Res [Epub ahead of print]. PubMed ID: 27995349
Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the male-specific lethal (MSL) complex, which increases transcript levels of active genes 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X ( roX) RNAs that are transcriptionally activated by the MSL complex. Targeting of the MSL complex to the X-chromosome has been shown to be dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. To better understand CLAMP function, the CRISPR/Cas9 genome editing system was used to generate a frameshift mutation in the clamp gene that eliminates expression of the CLAMP protein. clamp null females were found to die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, it was found that in clamp null females roX gene expression is activated, whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. These results provide new insights into sex-specific gene regulation by an essential transcription factor.
Radion, E., Ryazansky, S., Akulenko, N., Rozovsky, Y., Kwon, D., Morgunova, V., Olovnikov, I. and Kalmykova, A. (2016). Telomeric retrotransposon HeT-A contains a bidirectional promoter that initiates divergent transcription of piRNA precursors in Drosophila germline. J Mol Biol [Epub ahead of print]. PubMed ID: 27939293
PIWI-interacting RNAs (piRNAs) provide the silencing of transposable elements in the germline. Drosophila telomeres are maintained by transpositions of specialized telomeric retroelements. piRNAs generated from sense and antisense transcripts of telomeric elements provide telomere length control in the germline. This study showed that common regulatory elements are shared by sense and antisense promoters of HeT-A. Therefore, the HeT-A promoter is a bidirectional promoter capable of processive sense and antisense transcription. Ovarian small RNA data show that a solo HeT-A promoter within an euchromatic transgene initiates the divergent transcription of transgenic reporter genes and subsequent processing of these transcripts into piRNAs. These events lead to the formation of a divergent unistrand piRNA cluster at solo HeT-A promoters, in contrast to endogenous telomeres that represent strong dual-strand piRNA clusters. Solo HeT-A promoters are not immunoprecipitated with heterochromatin protein 1 (HP1) homolog Rhino, a marker of the dual-strand piRNA clusters, but are associated with HP1 itself, which provides piRNA-mediated transcriptional repression of the reporter genes. Unlike endogenous dual-strand piRNA clusters, the solo HeT-A promoter does not produce overlapping transcripts. In a telomeric context, however, bidirectional promoters of tandem HeT-A repeats provide a read-through transcription of both genomic strands, followed by Rhi binding. These data indicate that Drosophila telomeres share properties of unistrand and dual-strand piRNA clusters.
Ruppert, M., Franz, M., Saratsis, A., Velo Escarcena, L., Hendrich, O., Gooi, L. M., Schwenkert, I., Klebes, A. and Scholz, H. (2017). Hangover Links Nuclear RNA Signaling to cAMP Regulation via the Phosphodiesterase 4d Ortholog dunce. Cell Rep 18(2): 533-544. PubMed ID: 28076795
The hangover gene defines a cellular stress pathway that is required for rapid ethanol tolerance in Drosophila melanogaster. To understand how cellular stress changes neuronal function, Hangover function was analyzed on a cellular and neuronal level. Evidence is provided that Hangover acts as a nuclear RNA binding protein and the phosphodiesterase 4d ortholog dunce as a target RNA. A transcript-specific dunce mutant was generated that is impaired not only in ethanol tolerance but also in the cellular stress response. At the neuronal level, Dunce and Hangover are required in the same neuron pair to regulate experience-dependent motor output. Within these neurons, two cyclic AMP (cAMP)-dependent mechanisms balance the degree of tolerance. The balance is achieved by feedback regulation of Hangover and dunce transcript levels. This study provides insight into how nuclear Hangover/RNA signaling is linked to the cytoplasmic regulation of cAMP levels and results in neuronal adaptation and behavioral changes.
Monsanto-Hearne, V., Tham, A. L., Wong, Z. S., Asgari, S. and Johnson, K. N. (2016). Drosophila miR-956 suppression modulates Ectoderm-expressed 4 and inhibits viral replication. Virology 502: 20-27. PubMed ID: 27960110
Small non-coding microRNAs (miRNAs) can modulate the outcome of virus infection. This study explored the role of miRNAs in insect-virus interactions, in vivo, using the natural Drosophila melanogaster-Drosophila C virus (DCV) model system. Comparison of the miRNA expression profiles in DCV-infected and uninfected flies showed altered miRNA levels due to DCV infection, with the largest change in abundance observed for miR-956-3p. Knockout of miR-956 resulted to delayed DCV-induced mortality and decreased viral accumulation compared to wild-type flies. A screen of 84 putative miR-956-3p target genes identified regulation of Ectoderm-expressed 4 (Ect4), a negative regulator of MYD88- and TRIF-dependent toll-like receptor signaling pathway, in miR-956 knockout flies and, separately, DCV infection. In Ect4 knockdown flies DCV-induced mortality occurred more quickly and virus accumulation was increased. Taken together, results suggest that the host-protective and antiviral consequences of miR-956 suppression during in vivo infection of D. melanogaster with its natural pathogen DCV is conferred through miR-956-3p induction of its target Ect4.

Sunday, December 22nd

Tajiri, R., Ogawa, N., Fujiwara, H. and Kojima, T. (2017). Mechanical control of whole body shape by a single cuticular protein Obstructor-E in Drosophila melanogaster. PLoS Genet 13(1): e1006548. PubMed ID: 28076349
Body shapes are much more variable than body plans. One way to alter body shapes independently of body plans would be to mechanically deform bodies. To what extent body shapes are regulated physically, or molecules involved in physical control of morphogenesis, remain elusive. During fly metamorphosis, the cuticle (exoskeleton) covering the larval body contracts longitudinally and expands laterally to become the ellipsoidal pupal case (puparium). This study shows that Drosophila melanogaster Obstructor-E (Obst-E) is a protein constituent of the larval cuticle that confers the oriented contractility/expandability. In the absence of obst-E function, the larval cuticle fails to undergo metamorphic shape change and finally becomes a twiggy puparium. Results are presented indicating that Obst-E regulates the arrangement of chitin, a long-chain polysaccharide and a central component of the insect cuticle, and directs the formation of supracellular ridges on the larval cuticle. It was further shown that Obst-E is locally required for the oriented shape change of the cuticle during metamorphosis, which is associated with changes in the morphology of those ridges. Thus, Obst-E dramatically affects the body shape in a direct, physical manner by controlling the mechanical property of the exoskeleton.
Pesch, Y. Y., Riedel, D. and Behr, M. (2016). Drosophila Chitinase 2 is expressed in chitin producing organs for cuticle formation. Arthropod Struct Dev [Epub ahead of print]. PubMed ID: 27832982
The architecture of the outer body wall cuticle is fundamental to protect arthropods against invading pathogens and numerous other harmful stresses. Such robust cuticles are formed by parallel running chitin microfibrils. Molting and also local wounding leads to dynamic assembly and disassembly of the chitin-matrix throughout development. However, the underlying molecular mechanisms that organize proper chitin-matrix formation are poorly known. A key region has been identified for cuticle thickening at the apical cell surface, the cuticle assembly zone, where Obstructor-A (Obst-A) coordinates the formation of the chitin-matrix. Obst-A binds chitin and the deacetylase Serpentine (Serp) in a core complex which is required for chitin-matrix maturation and preservation. This study presents evidence that Chitinase 2 (Cht2) could be essential for this molecular machinery. Cht2 is expressed in the chitin-matrix of epidermis, trachea, and the digestive system. There, Cht2 is enriched at the apical cell surface and the dense chitin-matrix. It was further shown that in Cht2 knockdown larvae the assembly zone is rudimentary preventing normal cuticle formation and pore canal organization. As sequence similarities of Cht2 and the core complex proteins indicate evolutionarily conserved molecular mechanisms, these findings suggest that Cht2 is involved in chitin formation also in other insects.
Manning, L. and Doe, C. Q. (2017). Immunofluorescent antibody staining of intact Drosophila larvae. Nat Protoc 12(1): 1-14. PubMed ID: 27906168
Antibody staining is a vital technique for studying the development of many model organisms, including Drosophila. Reliable protocols have long been available for antibody staining of Drosophila whole-mount embryos and dissected larvae. By contrast, methods for staining whole larvae have rarely been reported, are unreliable, and fail to work on large third-instar larvae. This has become a major limitation to understanding the role of multitissue interactions such as neural circuit formation and cell metastasis. This study has modified existing embryo protocols to develop a reliable method for antibody staining of whole Drosophila larvae of any developmental stage. The procedure consists of a bleach wash, enzymatic digestion, first fixation, 'cracking', second fixation, (optional) Proteinase K (Pro-K) or sonication treatment, antibody staining, clearing, and mounting. The method takes longer than typical antibody stains of dissected larval tissues-12 or 16 d, depending on the size of the larvae, compared with 2-3 d for embryos or dissected tissue stains-but time is saved by eliminating the need for larval dissections and by allowing hundreds of larvae to be batch-processed. The method also works well for staining embryos, even late-stage embryos with cuticles, allowing characterization from early embryogenesis to the end of larval development.
Christesen, D., Yang, Y. T., Somers, J., Robin, C., Sztal, T., Batterham, P. and Perry, T. (2016). Transcriptome analysis of Drosophila melanogaster third instar larval ring glands points to novel functions and uncovers a cytochrome p450 required for development.. G3 (Bethesda) [Epub ahead of print] PubMed ID: 27974438
In Drosophila melanogaster larvae the ring gland is a control center that orchestrates major developmental transitions. It is a composite organ, consisting of the prothoracic gland, the corpus allatum and the corpora cardiaca, each of which synthesizes and secretes a different hormone. Until now, the ring gland's broader developmental roles beyond endocrine secretion have not been explored. RNA sequencing and analysis of a new transcriptome resource from D. melanogaster wandering third instar larval ring glands has provided a fascinating insight into the diversity of developmental signalling in this organ. Strong enrichment of expression was found of two gene pathways not previously associated with the ring gland: immune response and fatty acid metabolism. Strong expression was uncovered for many uncharacterized genes. Additionally, RNA interference against ring gland-enriched cytochrome p450s Cyp6u1 and Cyp6g2 produced a lethal ecdysone deficiency and a juvenile hormone deficiency respectively, flagging a critical role for these genes in hormone synthesis. This transcriptome provides a valuable new resource for investigation of roles played by the ring gland in governing insect development.

Saturday, January 21st

Nazario-Yepiz, N. O. and Riesgo-Escovar, J. R. (2016). piragua encodes a zinc finger protein required for development in Drosophila. Mech Dev [Epub ahead of print]. PubMed ID: 28011160
Embryonic lethal mutations in piragua (prg) were isolated and characterized. The prg locus encodes a protein with an amino terminus Zinc Finger-Associated-Domain (ZAD) and nine C2H2 zinc fingers (ZF). prg mRNA and protein expression during embryogenesis is dynamic with widespread maternal contribution, and subsequent expression in epithelial precursors. About a quarter of prg mutant embryos do not develop cuticle, and from those that do a small fraction have cuticular defects. Roughly half of prg mutants die during embryogenesis. prg mutants have an extended phenocritical period encompassing embryogenesis and first instar larval stage, since the other half of prg mutants die as first or second instar larvae. During dorsal closure, time-lapse high-resolution imaging shows defects arising out of sluggishness in closure, resolving at times in failures of closure. prg is expressed in imaginal discs, and is required for imaginal development.prg was identified in imaginal tissue in a cell super competition screen, together with other genes, like flower. flower mutations are also embryonic lethal with a similar phenocritical period and strong embryonic mutant phenotypes (head involution defects, primarily). The two loci interact genetically in the embryo, as they increase embryonic mortality to close to 90% with the same embryonic phenotypes (dorsal closure and head involution defects, plus lack of cuticle). Mutant prg clones generated in developing dorsal thorax and eye imaginal tissue have strong developmental defects (lack of bristles and ommatidial malformations). prg is required in several developmental morphogenetic processes.
Koch, L., Feicht, S., Sun, R., Sen, A. and Krahn, M. P. (2016). Domain-specific functions of Stardust in Drosophila embryonic development. R Soc Open Sci 3(11): 160776. PubMed ID: 28018665
In Drosophila, the adaptor protein Stardust is essential for the stabilization of the polarity determinant Crumbs in various epithelial tissues, including the embryonic epidermis, the follicular epithelium and photoreceptor cells of the compound eye. In turn, Stardust recruits another adaptor protein, PATJ, to the subapical region to support adherens junction formation and morphogenetic events. Moreover, Stardust binds to Lin-7, which is dispensable in epithelial cells but functions in postsynaptic vesicle fusion. Finally, Stardust has been reported to bind directly to PAR-6, thereby linking the Crumbs-Stardust-PATJ complex to the PAR-6/aPKC complex. PAR-6 and aPKC are also capable of directly binding Bazooka (the Drosophila homologue of PAR-3) to form the PAR/aPKC complex, which is essential for apical-basal polarity and cell-cell contact formation in most epithelia. However, little is known about the physiological relevance of these interactions in the embryonic epidermis of Drosophila in vivo. Thus, a structure-function analysis was performed of the annotated domains with GFP-tagged Stardust, and the localization and function of the mutant proteins was evaluated in epithelial cells of the embryonic epidermis. The data confirm a crucial role of the PDZ domain in binding Crumbs and recruiting the protein to the subapical region. However, the isolated PDZ domain is not capable of being recruited to the cortex, and the SH3 domain is essential to support the binding to Crumbs. Notably, the conserved N-terminal regions (ECR1 and ECR2) are not crucial for epithelial polarity. Finally, the GUK domain plays an important role for the protein's function, which is not directly linked to Crumbs stabilization, and the L27N domain is essential for epithelial polarization independently of recruiting PATJ.
Goodwin, K., Ellis, S.J., Lostchuck, E., Zulueta-Coarasa, T., Fernandez-Gonzalez, R. and Tanentzapf, G. (2016). Basal cell-extracellular matrix adhesion regulates force transmission during tissue morphogenesis. Dev Cell 39: 611-625. PubMed ID: 27923121
Tissue morphogenesis requires force-generating mechanisms to organize cells into complex structures. Although many such mechanisms have been characterized, little is known about how forces are integrated across developing tissues. This study provides evidence that integrin-mediated cell-extracellular matrix (ECM) adhesion modulates the transmission of apically generated tension during dorsal closure (DC) in Drosophila. Integrin-containing adhesive structures (see Myospheroid) resembling focal adhesions were identified on the basal surface of the amnioserosa (AS), an extraembryonic epithelium essential for DC. Genetic modulation of integrin-mediated adhesion results in defective DC. Quantitative image analysis and laser ablation experiments reveal that basal cell-ECM adhesions provide resistance to apical cell displacements and force transmission between neighboring cells in the AS. Finally, the study provides evidence for integrin-dependent force transmission to the AS substrate. Overall, these data indicate that integrins regulate force transmission within and between cells, thereby playing an essential role in transmitting tension in developing tissues.

Imai, K. S., Hikawa, H., Kobayashi, K. and Satou, Y. (2016). Tfap2 and Sox1/2/3 cooperatively specify ectodermal fates in ascidian embryos. Development [Epub ahead of print]. PubMed ID: 27888190
Evolutionary Homolog Study
Epidermis and neural tissues differentiate from the ectoderm in animal embryos. While epidermal fate is thought to be induced in vertebrate embryos, embryological evidence has indicated that no intercellular interactions during early stages are required for epidermal fate in ascidian embryos. To test this hypothesis, the gene regulatory circuits were determined for epidermal and neural specification in the ascidian embryo. These circuits started with Tfap2-r.b (AP-2-like2; see Drosophila AP-2) and Sox1/2/3 (see Drosophila Dichaete), which are expressed in the ectodermal lineage immediately after zygotic genome activation. Tfap2-r.b expression was diminished in the neural lineages upon of fibroblast growth factor signaling, which is known to induce neural fate, and sustained only in the epidermal lineage. Tfap2-r.b specified the epidermal fate cooperatively with Dlx.b (see Drosophila Dll), which was activated by Sox1/2/3. This Sox1/2/3-Dlx.b circuit was also required for specification of the anterior neural fate. In the posterior neural lineage, Sox1/2/3 activated Nodal, which is required for specification of the posterior neural fate. These findings support the hypothesis that the epidermal fate is specified autonomously in ascidian embryos.

Friday, January 20th

Ye, Y., Li, M., Gu, L., Chen, X., Shi, J., Zhang, X. and Jiang, C. (2016). Chromatin remodeling during in vivo neural stem cells differentiating to neurons in early Drosophila embryos. Cell Death Differ [Epub ahead of print]. PubMed ID: 27858939
Neurons are a key component of the nervous system and differentiate from multipotent neural stem cells (NSCs). Chromatin remodeling has a critical role in the differentiation process. However, its in vivo epigenetic regulatory role remains unknown. This study shows that nucleosome depletion regions (NDRs) form in both proximal promoters and distal enhancers during NSCs differentiating into neurons in the early Drosophila embryonic development. NDR formation in the regulatory regions involves nucleosome shift and eviction. Nucleosome occupancy in promoter NDRs is inversely proportional to the gene activity. Genes with promoter NDR formation during differentiation are enriched for functions related to neuron development and maturation. Active histone-modification signals (H3K4me3 and H3K9ac) in promoters are gained in neurons in two modes: de novo establishment to high levels or increase from the existing levels in NSCs. The gene sets corresponding to the two modes have different neuron-related functions. Dynamic changes of H3K27ac and H3K9ac signals in enhancers and promoters synergistically repress genes associated with neural stem or progenitor cell-related pluripotency and upregulate genes associated with neuron projection morphogenesis, neuron differentiation, and so on. These results offer new insights into chromatin remodeling during in vivo neuron development and lay a foundation for its epigenetic regulatory mechanism study of other lineage specification.
Cleard, F., Wolle, D., Taverner, A. M., Aoki, T., Deshpande, G., Andolfatto, P., Karch, F. and Schedl, P. (2016). Different evolutionary strategies to conserve chromatin boundary function in the Bithorax complex. Genetics [Epub ahead of print]. PubMed ID: 28007886
Chromatin boundary elements subdivide chromosomes in multicellular organisms into physically independent domains. In addition to this architectural function, these elements also play a critical role in gene regulation. This study has investigated the evolution of a Drosophila Bithorax complex boundary element called Fab-7 which is required for the proper parasegment specific expression of the homeotic Abd-B gene. Using a 'gene' replacement strategy, this study shows that Fab-7 boundaries from two closely related species D.erecta, D.yakuba, and a more distant species D. pseudoobscura are able to substitute for the melanogaster boundary. Consistent with this functional conservation, the two known Fab-7 boundary factors, Elba and LBC ( GAF, Mod(mdg4) and E(y)2), have recognition sequences in the boundaries from all species. However, the strategies used for maintaining binding and function in the face of sequence divergence is different. The first is conventional and depends upon conservation of the 8 bp Elba recognition sequence. The second is unconventional and takes advantage of the unusually large and flexible sequence recognition properties of the LBC boundary factor and the deployment of multiple LBC recognition elements in each boundary. In the former case, binding is lost when the recognition sequence is altered. In later case, sequence divergence is accompanied by changes in the number, relative affinity, and location of the LBC recognition elements.
Liu, M. and Pile, L. A. (2016). The transcriptional corepressor SIN3 directly regulates genes involved in methionine catabolism and affects histone methylation, linking epigenetics and metabolism. J Biol Chem [Epub ahead of print]. PubMed ID: 28028175
Chromatin modification and cellular metabolism are tightly connected. Chromatin modifiers regulate the expression of genes involved in metabolism and, in turn, the levels of metabolites. The generated metabolites are utilized by chromatin modifiers to affect epigenetic modification. The mechanism for this cross-talk, however, remains incompletely understood. The corepressor SIN3 controls histone acetylation through association with the histone deacetylase RPD3. The SIN3 complex is known to regulate genes involved in a number of metabolic processes. This study finds that Drosophila SIN3 binds to the promoter region of genes involved in methionine catabolism, and that this binding affects histone modification, which in turn influences gene expression. Specifically, it was observed that reduced expression of SIN3 leads to an increase in S-adenosylmethionine (SAM), which is the major cellular donor of methyl groups for protein modification. Additionally, Sin3A knockdown results in an increase in global histone H3K4me3 levels. Furthermore, decreased H3K4me3 caused by knockdown of either SAM synthetase (Sam-S) or the histone methyltransferase Set1 is restored to near normal levels when SIN3 is also reduced. Taken together, these results indicate that knockdown of Sin3A directly alters the expression of methionine metabolic genes to increase SAM, which in turn leads to an increase in global H3K4me3. This study reveals that SIN3 is an important epigenetic regulator directly connecting methionine metabolism and histone modification.
Yang, D. and Ioshikhes, I. (2016). Drosophila H2A and H2A.Z nucleosome sequences reveal different nucleosome positioning sequence patterns. J Comput Biol [Epub ahead of print]. PubMed ID: 27992255
Nucleosomes are implicated in transcriptional regulation as well as in packing and stabilizing the DNA. Nucleosome positions affect the transcription by impeding or facilitating the binding of transcription factors. The DNA sequence, especially the periodic occurrences of dinucleotides, is a major factor that affects the nucleosome positioning. This study analyzed the Drosophila DNA sequences bound by H2A and H2A.Z nucleosomes. Periodic patterns of dinucleotides (weak-weak/strong-strong or purine-purine/pyrimidine-pyrimidine) were identified as WW/SS and RR/YY nucleosome positioning sequence (NPS) patterns. The WW/SS NPS pattern of the H2A nucleosome has a 10-bp period of weak-weak/strong-strong (W = A or T; S = G or C) dinucleotides. The 10-bp periodicity, however, is disrupted in the middle of the sequence. At the dyad, the SS dinucleotide is preferred. On the other hand, the RR/YY NPS pattern has an 18-bp periodicity of purine-purine/pyrimidine-pyrimidine (R = A or G; Y = T or C) dinucleotides. The NPS patterns from H2A.Z nucleosomes differ from the NPS patterns from H2A nucleosomes. The RR/YY pattern of H2A.Z nucleosomes has major peaks shifted by 10 bp deviated from the H2A nucleosome pattern. The H2A and H2A.Z nucleosomes have different sequence preferences. The shifted peaks coincide with DNA regions interacting with the histone loops.

Thursday, December 19th

McGinnis, J. P., Jiang, H., Agha, M. A., Perez Sanchez, C., Lange, J. J., Yu, Z., Marion-Poll, F. and Si, K. (2016). Immediate perception of a reward is distinct from the reward's long-term salience. Elife 5. PubMed ID: 28005005
Reward perception guides all aspects of animal behavior. However, the relationship between the perceived value of a reward, the latent value of a reward, and the behavioral response remains unclear. This study report that, given a choice between two sweet and chemically similar sugars-L- and D-arabinose-Drosophila melanogaster prefers D- over L-arabinose, but forms long-term memories of L-arabinose (the isomer present in ripening fruits) more reliably. Behavioral assays indicate that L-arabinose-generated memories require sugar receptor Gr43a, and calcium imaging and electrophysiological recording indicate that L- and D-arabinose differentially activate Gr43a-expressing neurons. It is posited that the immediate valence of a reward is not always predictive of the long-term reinforcement value of that reward, and that a subset of sugar-sensing neurons may generate distinct representations of similar sugars, allowing for rapid assessment of the salient features of various sugar rewards and generation of reward-specific behaviors. However, how sensory neurons communicate information about L-arabinose quality and concentration-features relevant for long-term memory-remains unknown.
Gorostiza, E. A., Colomb, J. and Brembs, B. (2016). A decision underlies phototaxis in an insect.Open Biol 6(12). PubMed ID: 28003472
Like a moth into the flame-phototaxis is an iconic example for innate preferences. Such preferences probably reflect evolutionary adaptations to predictable situations and have traditionally been conceptualized as hard-wired stimulus-response links. Perhaps for that reason, the century-old discovery of flexibility in Drosophila phototaxis has received little attention. This study reports that across several different behavioural tests, light/dark preference tested in walking is dependent on various aspects of flight. If flying ability is temporarily compromised, walking photopreference reverses concomitantly. Neuronal activity in circuits expressing dopamine and octopamine, respectively, plays a differential role in photopreference, suggesting a potential involvement of these biogenic amines in this case of behavioural flexibility. It is concluded that flies monitor their ability to fly, and that flying ability exerts a fundamental effect on action selection in Drosophila. This work suggests that even behaviours which appear simple and hard-wired comprise a value-driven decision-making stage, negotiating the external situation with the animal's internal state, before an action is selected.
Wu, M., Nern, A., Williamson, W. R., Morimoto, M. M., Reiser, M. B., Card, G. M. and Rubin, G. M. (2016). Visual projection neurons in the Drosophila lobula link feature detection to distinct behavioral programs. Elife 5. PubMed ID: 28029094
Visual projection neurons (VPNs) provide an anatomical connection between early visual processing and higher brain regions. This study characterized lobula columnar (LC) cells, a class of Drosophila VPNs that project to distinct central brain structures called optic glomeruli. This study anatomically describes 22 different LC types and show that, for several types, optogenetic activation in freely moving flies evokes specific behaviors. The activation phenotypes of two LC types closely resemble natural avoidance behaviors triggered by a visual loom. In vivo two-photon calcium imaging reveals that these LC types respond to looming stimuli, while another type does not, but instead responds to the motion of a small object. Activation of LC neurons on only one side of the brain can result in attractive or aversive turning behaviors depending on the cell type. These results indicate that LC neurons convey information on the presence and location of visual features relevant for specific behaviors.
Baik, L.S., Fogle, K.J., Roberts, L., Galschiodt, A.M., Chevez, J.A., Recinos, Y., Nguy, V. and Holmes, T.C. (2017). Cryptochrome mediates behavioral choice in response to UV light. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 28062690
Drosophila melanogaster Cryptochrome (CRY) mediates behavioral and electrophysiological responses to blue light coded by circadian and arousal neurons. However, spectroscopic and biochemical assays of heterologously expressed CRY suggest that CRY may mediate functional responses to UV-A (ultraviolet A) light as well. To determine the relative contributions of distinct phototransduction systems, this study tested mutants lacking CRY and mutants with disrupted opsin-based phototransduction for behavioral and electrophysiological responses to UV light. CRY and opsin-based external photoreceptor systems cooperate for UV light-evoked acute responses. CRY mediates behavioral avoidance responses related to executive choice, consistent with its expression in central brain neurons.

Wednesday, January 18th

M'Angale, P. G. and Staveley, B. E. (2016). Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster. Genet Mol Res 15(4). PubMed ID: 28002605
Lifeguard is an integral transmembrane protein that modulates FasL-mediated apoptosis by interfering with the activation of caspase 8. It is evolutionarily conserved, with homologues present in plants, nematodes, zebra fish, frog, chicken, mouse, monkey, and human. The Lifeguard homologue in Drosophila, CG3814, contains the Bax inhibitor-1 family motif of unknown function. Downregulation of Lifeguard disrupts cellular homeostasis and disease by sensitizing neurons to FasL-mediated apoptosis. Bioinformatic analyses was used to identify CG3814, a putative homologue of Lifeguard, and knocked down CG3814/LFG expression under the control of the Dopa decarboxylase (Ddc-Gal4) transgene in Drosophila melanogaster neurons to investigate whether it possesses neuroprotective activity. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons resulted in a shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with the degeneration and loss of dopaminergic neurons. Lifeguard interacts with anti-apoptotic Bcl-2 proteins and possibly pro-apoptotic proteins to exert its neuroprotective function. The co-expression of Buffy, the sole anti-apoptotic Bcl-2 gene family member in Drosophila, and CG3814/LFG by stable inducible RNA interference, suppresses the shortened lifespan and the premature age-dependent loss in climbing ability. Suppression of CG3814/LFG in the Drosophila eye reduces the number of ommatidia and increases disruption of the ommatidial array. Overexpression of Buffy, along with the knockdown of CG3814/LFG, counteracts the eye phenotypes. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons in Drosophila diminishes its neuroprotective ability and results in a shortened lifespan and loss of climbing ability, phenotypes that are improved upon overexpression of the pro-survival Buffy.
Tang, H. L., Tang, H. M., Fung, M. C. and Hardwick, J. M. (2016). In vivo biosensor tracks non-apoptotic caspase activity in Drosophila. J Vis Exp(117). PubMed ID: 27929458
Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. This study illustrates the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.
Fujita, N., Huang, W., Lin, T.H., Groulx, J.F., Jean, S., Kuchitsu, Y., Koyama-Honda, I., Mizushima, N., Fukuda, M. and Kiger, A.A. (2017). Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy. Elife [Epub ahead of print]. PubMed ID: 28063257
Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. This study uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. It was shown that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, the screens uncover an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.

Pavel, M., Imarisio, S., Menzies, F. M., Jimenez-Sanchez, M., Siddiqi, F. H., Wu, X., Renna, M., O'Kane, C. J., Crowther, D. C. and Rubinsztein, D. C. (2016). CCT complex restricts neuropathogenic protein aggregation via autophagy. Nat Commun 7: 13821. PubMed ID: 27929117
Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC (see Drosophila Tcp1-like). Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. This study shows that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, these findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity.

Tuesday, January 17th

Ng, J., Browning, A., Lechner, L., Terada, M., Howard, G. and Jefferis, G. S. (2016). Genetically targeted 3D visualisation of Drosophila neurons under Electron Microscopy and X-Ray Microscopy using miniSOG. Sci Rep 6: 38863. PubMed ID: 27958322
Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. This study developed genetically-encoded, electron-dense markers using miniSOG (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. Their efficacy was demonstrated in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. Non-destructive X-ray imaging was also tested on whole Drosophila brains to evaluate contrast staining. This enabled specific regions to be targeted for EM volume acquisition.
Muroyama, Y., Baba, A., Kitagawa, M. and Saito, T. (2016). Olfactory Sensory Neurons Control Dendritic Complexity of Mitral Cells via Notch Signaling. PLoS Genet 12(12): e1006514. PubMed ID: 28027303
Evolutionary Homolog Study
Mitral cells (MCs) of the mammalian olfactory bulb have a single primary dendrite extending into a single glomerulus, where they receive odor information from olfactory sensory neurons (OSNs). Molecular mechanisms for controlling dendritic arbors of MCs, which dynamically change during development, are largely unknown. This study found that MCs displayed more complex dendritic morphologies in mouse mutants of Maml1 (see Drosophila Mastermind), a crucial gene in Notch signaling. Similar phenotypes were observed by conditionally misexpressing a dominant negative form of MAML1 (dnMAML1) in MCs after their migration. Conversely, conditional misexpression of a constitutively active form of Notch reduced their dendritic complexity. Furthermore, the intracellular domain of Notch1 (NICD1) was localized to nuclei of MCs. These findings suggest that Notch signaling at embryonic stages is involved in the dendritic complexity of MCs. After the embryonic misexpression of dnMAML1, many MCs aberrantly extended dendrites to more than one glomerulus at postnatal stages, suggesting that Notch signaling is essential for proper formation of olfactory circuits. Moreover, dendrites in cultured MCs were shortened by Jag1-expressing cells. Finally, blocking the activity of Notch ligands in OSNs led to an increase in dendritic complexity as well as a decrease in NICD1 signals in MCs. These results demonstrate that the dendritic complexity of MCs is controlled by their presynaptic partners, OSNs.
Zhan, H., Bruckner, J., Zhang, Z. and O'Connor-Giles, K. (2016). Three-dimensional imaging of Drosophila motor synapses reveals ultrastructural organizational patterns. J Neurogenet 30(3-4): 237-246. PubMed ID: 27981875
This study combined cryo-preservation of intact Drosophila larvae and electron tomography with comprehensive segmentation of key features to reconstruct the complete ultrastructure of a model glutamatergic synapse in a near-to-native state. Presynaptically, a complex network of filaments was detailed that connects and organizes synaptic vesicles. The complexity of this synaptic vesicle network was linked to proximity to the active zone cytomatrix, consistent with the model that these protein structures function together to regulate synaptic vesicle pools. A net-shaped network of electron-dense filaments spanning the synaptic cleft was identified that suggests conserved organization of trans-synaptic adhesion complexes at excitatory synapses. Postsynaptically, a regular pattern of macromolecules was characterized that yields structural insights into the scaffolding of neurotransmitter receptors. Together, these analyses reveal an unexpected level of conservation in the nanoscale organization of diverse glutamatergic synapses and provide a structural foundation for understanding the molecular machines that regulate synaptic communication at a powerful model synapse.
Ko, H.R., Kwon, I.S., Hwang, I., Jin, E.J., Shin, J.H., Brennan-Minnella, A.M., Swanson, R., Cho, S.W., Lee, K.H. and Ahn, J.Y. (2016). Akt1-Inhibitor of DNA binding2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration. Elife 5. PubMed ID: 27938661
Evolutionary Homolog Study:
Mechanistic studies of axon growth (see Drosophila axonogenesis) during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. This study discovered that in the developing neuron from rat, Akt signaling (see Drosophila Akt1 and insulin signaling) regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2) (see Drosophila emc). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin (see Drosophila Moe) that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. These results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration.

Sasse, S. and Klambt, C. (2016). Repulsive epithelial cues direct glial migration along the nerve. Dev Cell 39(6): 696-707. PubMed ID: 27997826
Most glial cells show pronounced migratory abilities and generally follow axonal trajectories to reach their final destination. However, the molecular cues controlling their directional migration are largely unknown. To address this, glial migration onto the developing Drosophila leg imaginal disc was developed as a model. Here, CNS-derived glial cells move along nerves containing motoaxons and sensory axons. Along their path, glial cells encounter at least three choice points where directional decisions are needed. Subsequent genetic analyses allowed uncovering mechanisms that escaped previous studies. Most strikingly, it was found that glial cells require the expression of the repulsive guidance receptors PlexinA/B and Robo2 to prevent breaking away from the nerve. Interestingly, the repulsive ligands are presented by the underlying leg imaginal disc epithelium, which appears to push glial cells toward the axon fascicle. In conclusion, nerve formation not only requires neuron-glia interaction but also depends on glial-epithelial communication.
Karmakar, K., Narita, Y., Fadok, J., Ducret, S., Loche, A., Kitazawa, T., Genoud, C., Di Meglio, T., Thierry, R., Bacelo, J., Luthi, A. and Rijli, F. M. (2017). Hox2 genes are required for tonotopic map precision and sound discrimination in the mouse auditory brainstem. Cell Rep 18(1): 185-197. PubMed ID: 28052248
Evolutionary Homolog Study
Tonotopy is a hallmark of auditory pathways and provides the basis for sound discrimination. Little is known about the involvement of transcription factors in brainstem cochlear neurons orchestrating the tonotopic precision of pre-synaptic input. This study found that in the absence of Hoxa2 and Hoxb2 (see Drosophila Proboscipedia) function in Atoh1-derived glutamatergic bushy cells of the anterior ventral cochlear nucleus (see Drosophila Atonal), broad input topography and sound transmission were largely preserved. However, fine-scale synaptic refinement and sharpening of isofrequency bands of cochlear neuron activation upon pure tone stimulation were impaired in Hox2 mutants, resulting in defective sound-frequency discrimination in behavioral tests. These results establish a role for Hox factors in tonotopic refinement of connectivity and in ensuring the precision of sound transmission in the mammalian auditory circuit.

Monday, December 16th

Vaccaro, A., Issa, A. R., Seugnet, L., Birman, S. and Klarsfeld, A. (2017). Drosophila Clock is required in brain pacemaker neurons to prevent premature locomotor aging independently of its circadian function. PLoS Genet 13(1): e1006507. PubMed ID: 28072817
Circadian clocks control many self-sustained rhythms in physiology and behavior with approximately 24-hour periodicity. This study examined the effects of clock disruptions on locomotor aging and longevity in Drosophila. Lifespan was found to be similarly reduced in three arrhythmic mutants (ClkAR, cyc0 and tim0) and in wild-type flies under constant light, which stops the clock. In contrast, ClkAR mutants showed significantly faster age-related locomotor deficits (as monitored by startle-induced climbing) than cyc0 and tim0, or than control flies under constant light. Clk, but not Cyc, inactivation by RNA interference in the pigment-dispersing factor (PDF)-expressing central pacemaker neurons led to similar loss of climbing performance as ClkAR. Conversely, restoring Clk function in these cells was sufficient to rescue the ClkAR locomotor phenotype, independently of behavioral rhythmicity. Accelerated locomotor decline of the ClkAR mutant required expression of the PDF receptor and correlated to an apparent loss of dopaminergic neurons in the posterior protocerebral lateral 1 (PPL1) clusters. This neuronal loss was rescued when the ClkAR mutation was placed in an apoptosis-deficient background. Impairing dopamine synthesis in a single pair of PPL1 neurons that innervate the mushroom bodies accelerated locomotor decline in otherwise wild-type flies. These results therefore reveal a novel circadian-independent requirement for Clk in brain circadian neurons to maintain a subset of dopaminergic cells and avoid premature locomotor aging in Drosophila.
He, Q., Zhang, Y., Zhang, X., Xu, D., Dong, W., Li, S. and Wu, R. (2016). Nucleoporin Nup358 facilitates nuclear import of Methoprene-tolerant (Met) in an importin beta- and Hsp83-dependent manner. Insect Biochem Mol Biol 81: 10-18. PubMed ID: 27979731
The bHLH-PAS transcription factor, Methoprene-tolerant (Met)1, functions as a juvenile hormone (JH) receptor and transduces JH signals by directly binding to E-box like motifs in the regulatory regions of JH response genes. Nuclear localization of Met is crucial for its transcriptional activity. It has been shown that the chaperone protein Hsp83 facilitates JH-induced Met nuclear import in Drosophila. However, the exact molecular mechanisms of Met nuclear transport are not fully elucidated. Using DNA affinity chromatography, binding of the nucleoporin Nup358, in the presence of JH, has been detected to the JH response region (JHRR) sequences isolated from the Kruppel-homolog 1 (Kr-h1) promoter. This study demonstrated that Nup358 regulates JH-Hsp83-induced Met nuclear localization. RNAi-mediated knockdown of Nup358 expression in Drosophila fat body perturbs Met nuclear transport during the 3 h after initiation of wandering, when the JH titer is high. The accompanying reduced expression of the transport receptor importin β in Nup358 RNAi flies could be one of the reasons accounting for Met mislocalization. Furthermore, a tetratricopeptide repeat (TPR) domain at the N-terminal end of Nup358 interacts with Hsp83 and is indispensable for Met nuclear localization. Overexpression of the TPR domain in Drosophila fat body prevents Met nuclear localization resulting in a decrease in JHRR-driven reporter activity and Kr-h1 expression. These data show that Nup358 facilitates JH-induced Met nuclear transport in a manner dependent on importin β and Hsp83.
Moon, S., Kim, W., Kim, S., Kim, Y., Song, Y., Bilousov, O., Kim, J., Lee, T., Cha, B., Kim, M., Kim, H., Katanaev, V. L. and Jho, E. H. (2016). Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization. EMBO Rep [Epub ahead of print]. PubMed ID: 27979972
Hippo signaling controls organ size by regulating cell proliferation and apoptosis. Yes-associated protein (YAP; see Drosophila Yorkie) is a key downstream effector of Hippo signaling, and LATS-mediated phosphorylation of YAP at Ser127 inhibits its nuclear localization and transcriptional activity. This study reports that Nemo-like kinase (NLK; see Drosophila Nemo) phosphorylates YAP at Ser128 both in vitro and in vivo, which blocks interaction with 14-3-3 (see Drosophila 14-3-3) and enhances its nuclear localization. Depletion of NLK increases YAP phosphorylation at Ser127 and reduces YAP-mediated reporter activity. These results suggest that YAP phosphorylation at Ser128 and at Ser127 may be mutually exclusive. It was also found that with the increase in cell density, nuclear localization and the level of NLK are reduced, resulting in reduction in YAP phosphorylation at Ser128. Furthermore, knockdown of Nemo (the Drosophila NLK) in fruit fly wing imaginal discs results in reduced expression of the Yorkie (the Drosophila YAP) target genes expanded and DIAP1, while Nemo overexpression reciprocally increased the expression. Overall, these data suggest that NLK/Nemo acts as an endogenous regulator of Hippo signaling by controlling nuclear localization and activity of YAP/Yorkie.
Plaza-Menacho, I., Barnouin, K., Barry, R., Borg, A., Orme, M., Chauhan, R., Mouilleron, S., Martinez-Torres, R. J., Meier, P. and McDonald, N. Q. (2016). RET functions as a Dual-Specificity Kinase that requires allosteric inputs from juxtamembrane elements. Cell Rep 17(12): 3319-3332. PubMed ID: 28009299
Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. This study shows that the juxtamembrane (JM) segment enhances RET (see Drosophila Ret oncogene) catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. This phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. These findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.

Sunday, January 15th

Kale, A., Rimesso, G. and Baker, N. E. (2016). Local cell death changes the orientation of cell division in the developing Drosophila wing imaginal disc without using Fat or Dachsous as orienting signals. PLoS One 11(12): e0167637. PubMed ID: 28030539
Drosophila imaginal disc cells exhibit preferred cell division orientations according to location within the disc. These orientations are altered if cell death occurs within the epithelium, such as is caused by cell competition or by genotypes affecting cell survival. Both normal cell division orientations, and their orientations after cell death, depend on the Fat-Dachsous pathway of planar cell polarity (PCP). The hypothesis that cell death initiates a planar polarity signal was investigated. When clones homozygous for the pineapple eye (pie) mutation were made to initiate cell death, neither Dachsous nor Fat was required in pie cells for the re-orientation of nearby cells, indicating a distinct signal for this PCP pathway. Dpp and Wg were also not needed for pie clones to re-orient cell division. Cell shapes were evaluated in wild type and mosaic wing discs to assess mechanical consequences of cell loss. Although proximal wing disc cells and cells close to the dorso-ventral boundary were elongated in their preferred cell division axes in wild type discs, cell shapes in much of the wing pouch were symmetrical on average and did not predict their preferred division axis. Cells in pie mutant clones were slightly larger than their normal counterparts, consistent with mechanical stretching following cell loss, but no bias in cell shape was detected in the surrounding cells. These findings indicate that an unidentified signal influences PCP-dependent cell division orientation in imaginal discs.
Lebensohn, A.M., Dubey, R., Neitzel, L.R., Tacchelly-Benites, O., Yang, E., Marceau, C.D., Davis, E.M., Patel, B.B., Bahrami-Nejad, Z., Travaglini, K.J., Ahmed, Y., Lee, E., Carette, J.E. and Rohatgi, R. (2016). Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling. Elife [Epub ahead of print]. PubMed ID: 27996937
Evolutionary Homolog Study:
The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, this study conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, sensitizing and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling, and suppressors of constitutive signaling induced by loss of the tumor suppressor APC (see Drosophila Apc) or casein kinase 1α (see Drosophila CkIα) uncovered new regulatory features at many levels of the pathway. These include a requirement for the transcription factor TFAP4 (see Drosophila crp), a role for the DAX domain of AXIN2 (see Drosophila Axn) in controlling β-catenin activity, a contribution of GPI anchor biosynthetic enzymes and glypicans to R-spondin-potentiated signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost.

Recasens-Alvarez, C., Ferreira, A. and Milán, M. (2017). JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling. Nat Commun 8: 13815. PubMed ID: 28045022
A stable pool of morphogen-producing cells is critical for the development of any organ or tissue. This study presents evidence that JAK/STAT signalling in the Drosophila wing promotes the cycling and survival of Hedgehog-producing cells, thereby allowing the stable localization of the nearby BMP/Dpp-organizing centre in the developing wing appendage. The inhibitor of apoptosis dIAP1 and Cyclin A were identified as two critical genes regulated by JAK/STAT and contributing to the growth of the Hedgehog-expressing cell population. JAK/STAT was found to have an early role in guaranteeing Wingless-mediated appendage specification, and a later one in restricting the Dpp-organizing activity to the appendage itself. These results unveil a fundamental role of the conserved JAK/STAT pathway in limb specification and growth by regulating morphogen production and signalling, and a function of pro-survival cues and mitogenic signals in the regulation of the pool of morphogen-producing cells in a developing organ.

Lee, Y. C., Zhou, Q., Chen, J. and Yuan, J. (2016). RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response. Curr Biol 26(24): 3257-3268. PubMed ID: 27818175
Evolutionary Homolog Study

ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. This study reports the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway (see Drosophila meiotic 41) via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, these data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control.

Saturday, January 14th

Xiao, C. and Robertson, R.M. (2017). White-cGMP interaction promotes fast locomotor recovery from anoxia in adult Drosophila. PLoS One 12: e0168361. PubMed ID: 28060942
Increasing evidence indicates that the white (w) gene in Drosophila possesses extra-retinal functions in addition to its classical role in eye pigmentation. It has been previously shown that w+ promotes fast and consistent locomotor recovery from anoxia, but how w+ modulates locomotor recovery is largely unknown. This study shows that in the absence of w+, several PDE mutants, especially cyclic guanosine monophosphate (cGMP)-specific PDE mutants, display wildtype-like fast locomotor recovery from anoxia, and that during the night time, locomotor recovery is light-sensitive in white-eyed mutant w1118, and light-insensitive in PDE mutants under w1118 background. Data indicate the involvement of cGMP in the modulation of recovery timing and presumably, light-evoked cGMP fluctuation is associated with light sensitivity of locomotor recovery. This is further supported by the observations that w-RNAi-induced delay of locomotor recovery is completely eliminated by upregulation of cGMP through multiple approaches, including PDE mutation, simultaneous overexpression of an atypical soluble guanylyl cyclase Gyc88E, or sildenafil feeding. Lastly, prolonged sildenafil feeding promotes fast locomotor recovery from anoxia in w1118. Taken together, these data suggest that a White-cGMP interaction modulates the timing of locomotor recovery from anoxia.

Colinet, H., Renault, D. and Roussel, D. (2016). Cold acclimation allows Drosophila flies to maintain mitochondrial functioning under cold stress. Insect Biochem Mol Biol 80: 52-60. PubMed ID: 27903433
Environmental stress generally disturbs cellular homeostasis. Researchers have hypothesized that chilling injury is linked to a shortage of ATP. However, previous studies conducted on insects exposed to nonfreezing low temperatures presented conflicting results. This study investigated the mitochondrial bioenergetics of Drosophila melanogaster flies exposed to chronic cold stress (4 degrees C). Mitochondrial oxygen consumption was assessed while monitoring the rate of ATP synthesis at various times (0, 1, 2, and 3 days) during prolonged cold stress and at two assay temperatures (25 and 4 ° C). Organelle responses were compared between cold-susceptible and cold-acclimated phenotypes. Continuous exposure to low temperature provoked temporal declines in the rates of mitochondrial respiration and ATP synthesis. Respiratory control ratios (RCRs) suggested that mitochondria were not critically uncoupled. Nevertheless, after 3 days of continuous cold stress, a sharp decline in the mitochondrial ATP synthesis rate was observed in control flies when they were assayed at low temperature. This change was associated with reduced survival capacity in control flies. In contrast, cold-acclimated flies exhibited high survival and maintained higher rates of mitochondrial ATP synthesis and coupling (i.e., higher RCRs). Adaptive changes due to cold acclimation observed in the whole organism were thus manifested in isolated mitochondria. These observations suggest that cold tolerance is linked to the ability to maintain bioenergetics capacity under cold stress.
Croset, V., Schleyer, M., Arguello, J. R., Gerber, B. and Benton, R. (2016). A molecular and neuronal basis for amino acid sensing in the Drosophila larva. Sci Rep 6: 34871. PubMed ID: 27982028
Amino acids are important nutrients for animals, reflected in conserved internal pathways in vertebrates and invertebrates for monitoring cellular levels of these compounds. In mammals, sensory cells and metabotropic glutamate receptor-related taste receptors that detect environmental sources of amino acids in food are also well-characterised. By contrast, it is unclear how insects perceive this class of molecules through peripheral chemosensory mechanisms. This study investigated amino acid sensing in Drosophila melanogaster larvae, which feed ravenously to support their rapid growth. Larvae were shown to display diverse behaviours (attraction, aversion, neutral) towards different amino acids, which depend upon stimulus concentration. Some of these behaviours require IR76b, a member of the variant ionotropic glutamate receptor repertoire of invertebrate chemoreceptors. IR76b is broadly expressed in larval taste neurons, suggesting a role as a co-receptor. A subpopulation of these neurons were identified that displays physiological activation by some, but not all, amino acids, and which mediate suppression of feeding by high concentrations of at least a subset of these compounds. These data reveal the first elements of a sophisticated neuronal and molecular substrate by which these animals detect and behave towards external sources of amino acids.
Kang, M. J., Vasudevan, D., Kang, K., Kim, K., Park, J. E., Zhang, N., Zeng, X., Neubert, T. A., Marr, M. T., and Don Ryoo, H. (2016). 4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging. J Cell Biol. PubMed ID: 27979906
Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP (Thor) is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. This study shows that GCN2 and its downstream transcription factor, ATF4 (Cryptocephal), mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging.

Friday, January 13th

Lai, Y. W., Chu, S. Y., Wei, J. Y., Cheng, C. Y., Li, J. C., Chen, P. L., Chen, C. H. and Yu, H. H. (2016). Drosophila microRNA-34 impairs axon pruning of mushroom body γ neurons by downregulating the expression of ecdysone receptor. Sci Rep 6: 39141. PubMed ID: 28008974
MicroRNA-34 (miR-34) is crucial for preventing chronic large-scale neurite degeneration in the aged brain of Drosophila melanogaster. This study investigated the role of miR-34 in two other types of large-scale axon degeneration in Drosophila: axotomy-induced axon degeneration in olfactory sensory neurons (OSNs) and developmentally related axon pruning in mushroom body (MB) neurons. Ectopically overexpressed miR-34 did not inhibit axon degeneration in OSNs following axotomy, whereas ectopically overexpressed miR-34 in differentiated MB neurons impaired γ axon pruning. Intriguingly, the miR-34-induced γ axon pruning defect resulted from downregulating the expression of ecdysone receptor B1 (EcR-B1) in differentiated MB γ neurons. Notably, the separate overexpression of EcR-B1 or a transforming growth factor- β receptor Baboon, whose activation can upregulate the EcR-B1 expression, in MB neurons rescued the miR-34-induced gamma axon pruning phenotype. Future investigations of miR-34 targets that regulate the expression of EcR-B1 in MB γ neurons are warranted to elucidate pathways that regulate axon pruning, and to provide insight into mechanisms that control large-scale axon degeneration in the nervous system.
Lin, C. J., Wen, J., Bejarano, F., Hu, F., Bortolamiol-Becet, D., Kan, L., Sanfilippo, P., Kondo, S. and Lai, E. C. (2016). Characterization of a TUTase/nuclease complex required for Drosophila gametogenesis. RNA [Epub ahead of print]. PubMed ID: 27974621
The 3' exoribonuclease Dis3L2 metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of non-coding RNAs. This study performed a structure-function analysis of the Drosophila TUTase Tailor, which inhibits biogenesis of splicing-derived miRNA hairpins. Tailor/Dis3L2 forms a complex via N-terminal domains in the respective proteins that are distinct from their catalytic domains. In vitro, Dis3L2 has nuclease activity but substrate oligouridylation by Tailor stimulates their degradation by Dis3L2, especially for structured substrates. Mutants of Tailor and Dis3L2 are viable and lack overt morphological defects. Instead, these mutants exhibit defects in female and male fertility, implying specific requirements in the germline. Dis3L2 defects are more severe than Tailor, and their requirements appear stronger in males than in females. In particular, loss of Dis3L2 completely blocks productive spermatogenesis causing complete male sterility. RNA-seq analysis from single and double mutant testes reveal aberrant gene expression programs, and suggest that non-coding RNAs may be preferentially affected by Dis3L2. Overall, these studies of a new tailing/trimming complex reveals unexpectedly specific requirements during gametogenesis.
Jones, B. C., Wood, J. G., Chang, C., Tam, A. D., Franklin, M. J., Siegel, E. R. and Helfand, S. L. (2016). A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan. Nat Commun 7: 13856. PubMed ID: 28000665
In gonadal tissues, the Piwi-interacting (piRNA) pathway preserves genomic integrity by employing 23-29 nucleotide (nt) small RNAs complexed with argonaute proteins to suppress parasitic mobile sequences of DNA called transposable elements (TEs). Although recent evidence suggests that the piRNA pathway may be present in select somatic cells outside the gonads, the role of a non-gonadal somatic piRNA pathway is not well characterized. This study reports a functional somatic piRNA pathway in the adult Drosophila fat body including the presence of the piRNA effector protein Piwi and canonical 23-29 nt long TE-mapping piRNAs. The piwi mutants exhibit depletion of fat body piRNAs, increased TE mobilization, increased levels of DNA damage and reduced lipid stores. These mutants are starvation sensitive, immunologically compromised and short-lived, all phenotypes associated with compromised fat body function. These findings demonstrate the presence of a functional non-gonadal somatic piRNA pathway in the adult fat body that affects normal metabolism and overall organismal health.
Kim, K., Hung, R. J. and Perrimon, N. (2016). miR-263a regulates ENaC to maintain osmotic and intestinal stem cell homeostasis in Drosophila. Dev Cell [Epub ahead of print]. PubMed ID: 28017617
Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinal stem cell (ISC) homeostasis. This study found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na+ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Evidence is provided that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC.

Thursday, Friday 12th

Nakajima, E., Shimaji, K., Umegawachi, T., Tomida, S., Yoshida, H., Yoshimoto, N., Izawa, S., Kimura, H. and Yamaguchi, M. (2016). The Histone deacetylase gene Rpd3 is required for starvation stress resistance. PLoS One 11(12): e0167554. PubMed ID: 27907135
Epigenetic regulation in starvation is important but not fully understood yet. This study identified the Rpd3 gene, a Drosophila homolog of histone deacetylase 1, as a critical epigenetic regulator for acquiring starvation stress resistance. Immunostaining analyses of Drosophila fat body revealed that the subcellular localization and levels of Rpd3 dynamically changed responding to starvation stress. In response to starvation stress, the level of Rpd3 rapidly increased, and it accumulated in the nucleolus in what appeared to be foci. These observations suggest that Rpd3 plays a role in regulation of rRNA synthesis in the nucleolus. The RT-qPCR and ChIP-qPCR analyses clarified that Rpd3 binds to the genomic region containing the rRNA promoters and activates rRNA synthesis in response to starvation stress. Polysome analyses revealed that the amount of polysomes was decreased in Rpd3 knockdown flies under starvation stress compared with the control flies. Since the autophagy-related proteins are known to be starvation stress tolerance proteins, autophagy activity was examined, and it was reduced in Rpd3 knockdown flies. Taken together, it is concluded that Rpd3 accumulates in the nucleolus in the early stage of starvation, upregulates rRNA synthesis, maintains the polysome amount for translation, and finally increases stress tolerance proteins, such as autophagy-related proteins, to acquire starvation stress resistance.
Cicconi, A., Micheli, E., Verni, F., Jackson, A., Gradilla, A. C., Cipressa, F., Raimondo, D., Bosso, G., Wakefield, J. G., Ciapponi, L., Cenci, G., Gatti, M., Cacchione, S. and Raffa, G. D. (2016). The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27940556
Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. It has been suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. This study used electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. It was also shown that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, Ver-depleted telomeres were found to form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, these findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin.
Nguyen, S. C., Yu, S., Oberlick, E. and Wu, C. T. (2016). An unexpected regulatory cascade governs a core function of the Drosophila PRC1 chromatin protein Su(z)2. Genetics [Epub ahead of print]. PubMed ID: 27881472
Polycomb group (PcG) proteins are major chromatin-bound factors that can read and modify chromatin states to maintain gene silencing throughout development. This study focused on a close homolog of the PcG protein Posterior sex combs in order to better understand how these proteins affect regulation. This homolog, called Suppressor 2 of zeste, or Su(z)2 is comprised of two regions: the N-terminal "homology region" (HR), which serves as a hub for protein interactions, and the C-terminal region (CTR), which is believed to harbor the core activity of compacting chromatin. This paper describes classical genetic studies to dissect the structure of Su(z)2 Surprisingly, it was found that the CTR is dispensable for viability. Furthermore, the core activity of Su(z)2 seems to reside in the HR instead of the CTR. Remarkably, the data also suggest a regulatory cascade between CTR and HR of Su(z)2, which, in turn, may help prioritize the myriad of PcG interactions that occur with the HR.
Babour, A., Shen, Q., Dos-Santos, J., Murray, S., Gay, A., Challal, D., Fasken, M., Palancade, B., Corbett, A., Libri, D., Mellor, J. and Dargemont, C. (2016). The chromatin remodeler ISW1 is a quality control factor that surveys nuclear mRNP biogenesis. Cell 167(5): 1201-1214 e1215. PubMed ID: 27863241
Evolutionary Homolog Study
Chromatin dynamics play an essential role in regulating DNA transaction processes, but it is unclear whether transcription-associated chromatin modifications control the mRNA ribonucleoparticles (mRNPs) pipeline from synthesis to nuclear exit. This study identified the yeast ISW1 chromatin remodeling complex (see Drosophila ISWI) as an unanticipated mRNP nuclear export surveillance factor that retains export-incompetent transcripts near their transcription site. This tethering activity of ISW1 requires chromatin binding and is independent of nucleosome sliding activity or changes in RNA polymerase II processivity. Combination of in vivo UV-crosslinking and genome-wide RNA immunoprecipitation assays show that Isw1 and its cofactors interact directly with premature mRNPs. These results highlight that the concerted action of Isw1 and the nuclear exosome ensures accurate surveillance mechanism that proofreads the efficiency of mRNA biogenesis.

Wednesday, January 11th

Tokusumi, T., Tokusumi, Y., Brahier, M. S., Lam, V., Stoller-Conrad, J. R., Kroeger, P. T., Jr. and Schulz, R. A. (2016). Screening and analysis of Janelia FlyLight project enhancer-Gal4 strains identifies multiple gene enhancers active during hematopoiesis in normal and wasp-challenged Drosophila larvae. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27913635
A GFP expression screen has been conducted on greater than one thousand Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and Delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process.
Cannavo, E., Koelling, N., Harnett, D., Garfield, D., Casale, F. P., Ciglar, L., Gustafson, H. E., Viales, R. R., Marco-Ferreres, R., Degner, J. F., Zhao, B., Stegle, O., Birney, E. and Furlong, E. E. (2016). Genetic variants regulating expression levels and isoform diversity during embryogenesis. Nature [Epub ahead of print]. PubMed ID: 28024300
Embryonic development is driven by tightly regulated patterns of gene expression, despite extensive genetic variation among individuals. Studies of expression quantitative trait loci (eQTL) indicate that genetic variation frequently alters gene expression in cell-culture models and differentiated tissues. However, the extent and types of genetic variation impacting embryonic gene expression, and their interactions with developmental programs, remain largely unknown. This study assessed the effect of genetic variation on transcriptional (expression levels) and post-transcriptional (3' RNA processing) regulation across multiple stages of metazoan development, using 80 inbred Drosophila wild isolates, identifying thousands of developmental-stage-specific and shared QTL. Given the small blocks of linkage disequilibrium in Drosophila, near base-pair resolution was obtained, resolving causal mutations in developmental enhancers and validated transcription-factor-binding sites and RNA motifs. This fine-grain mapping uncovered extensive allelic interactions within enhancers that have opposite effects, thereby buffering their impact on enhancer activity. QTL affecting 3' RNA processing identify new functional motifs leading to transcript isoform diversity and changes in the lengths of 3' untranslated regions. These results highlight how developmental stage influences the effects of genetic variation and uncover multiple mechanisms that regulate and buffer expression variation during embryogenesis.
Weasner, B. M., Weasner, B. P., Neuman, S. D., Bashirullah, A. and Kumar, J. P. (2016). Retinal expression of the Drosophila eyes absent gene is controlled by several cooperatively acting cis-regulatory elements. PLoS Genet 12(12): e1006462. PubMed ID: 27930646
The eyes absent (eya) gene of the fruit fly, Drosophila melanogaster, is a member of an evolutionarily conserved gene regulatory network that controls eye formation in all seeing animals. The loss of eya leads to the complete elimination of the compound eye while forced expression of eya in non-retinal tissues is sufficient to induce ectopic eye formation. Within the developing retina eya is expressed in a dynamic pattern and is involved in tissue specification/determination, cell proliferation, apoptosis, and cell fate choice. This study explores the mechanisms by which eya expression is spatially and temporally governed in the developing eye. Multiple cis-regulatory elements function cooperatively to control eya transcription and spacing between a pair of enhancer elements is important for maintaining correct gene expression. Lastly, it was shown that the loss of eya expression in sine oculis (so) mutants is the result of massive cell death and a progressive homeotic transformation of retinal progenitor cells into head epidermis.
Arnold, C. D., Zabidi, M. A., Pagani, M., Rath, M., Schernhuber, K., Kazmar, T. and Stark, A. (2016). Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution. Nat Biotechnol. PubMed ID: 28024147
Gene expression is controlled by enhancers that activate transcription from the core promoters of their target genes. Although a key function of core promoters is to convert enhancer activities into gene transcription, whether and how strongly they activate transcription in response to enhancers has not been systematically assessed on a genome-wide level. This study describes self-transcribing active core promoter sequencing (STAP-seq), a method to determine the responsiveness of genomic sequences to enhancers, and apply it to the Drosophila melanogaster genome. Candidate fragments at the position of the core promoter (also called minimal promoter) were cloned in reporter plasmids with or without a strong enhancer, the resulting library was transfected into cells, and the transcripts that initiated from each candidate were quantified for each setup by deep sequencing. In the presence of a single strong enhancer, the enhancer responsiveness of different sequences differs by several orders of magnitude, and different levels of responsiveness are associated with genes of different functions. Sequence features were identified that predict enhancer responsiveness and how different core promoters are employed for the regulation of gene expression is discussed.

Tuesday, January 10th

Jussen, D., von Hilchen, J. and Urbach, R. (2016). Genetic regulation and function of epidermal growth factor receptor signalling in patterning of the embryonic Drosophila brain. Open Biol 6(12). PubMed ID: 27974623
The specification of distinct neural cell types in central nervous system development crucially depends on positional cues conferred to neural stem cells in the neuroectoderm. This study investigated the regulation and function of the epidermal growth factor receptor (EGFR) signalling pathway in early development of the Drosophila brain. Localized EGFR signalling in the brain neuroectoderm was found to rely on a neuromere-specific deployment of activating (Spitz, Vein) and inhibiting (Argos) ligands. Activated EGFR controls the spatially restricted expression of all dorsoventral (DV) patterning genes in a gene- and neuromere-specific manner. Further, a novel role of DV genes-ventral nervous system defective (vnd), intermediate neuroblast defective (ind), Nkx6-in regulating the expression of vein and argos, which feed back on EGFR, indicating that EGFR signalling stands not strictly atop the DV patterning genes. Within this network of genetic interactions, Vnd acts as a positive EGFR feedback regulator. Further, it was shown that EGFR signalling becomes dependent on single-minded-expressing midline cells in the posterior brain (tritocerebrum), but remains midline-independent in the anterior brain (deuto- and protocerebrum). Finally, it was demonstrated that activated EGFR controls the proper formation of brain neuroblasts by regulating the number, survival and proneural gene expression of neuroectodermal progenitor cells. These data demonstrate that EGFR signalling is crucially important for patterning and early neurogenesis of the brain.
Pilaz, L. J., Lennox, A. L., Rouanet, J. P. and Silver, D. L. (2016). Dynamic mRNA transport and local translation in radial glial progenitors of the developing brain. Curr Biol 26(24): 3383-3392. PubMed ID: 27916527
Evolutionary Homolog Study
In the developing brain, neurons are produced from neural stem cells termed radial glia. Radial glial progenitors span the neuroepithelium, extending long basal processes to form endfeet hundreds of micrometers away from the soma. Basal structures influence neuronal migration, tissue integrity, and proliferation. Yet, despite the significance of these distal structures, their cell biology remains poorly characterized, impeding understanding of how basal processes and endfeet influence neurogenesis. This study used live imaging of embryonic brain tissue to visualize, for the first time, rapid mRNA transport in radial glia, revealing that the basal process is a highway for directed molecular transport. RNA- and mRNA-binding proteins, including the syndromic autism protein FMRP (see Drosophila Fmr1), move in basal processes at velocities consistent with microtubule-based transport, accumulating in endfeet. An ex vivo tissue preparation was developed to mechanically isolate radial glia endfeet from the soma, and photoconvertible proteins were used to demonstrate that mRNA is locally translated. Using RNA immunoprecipitation and microarray analyses of endfeet, FMRP-bound transcripts, which encode signaling and cytoskeletal regulators, were discovered including many implicated in autism and neurogenesis. FMRP controls transport and localization of one target, Kif26a. These discoveries reveal a rich, regulated local transcriptome in radial glia, far from the soma, and establish a tractable mammalian model for studying mRNA transport and local translation in vivo. It is concluded that cytoskeletal and signaling events at endfeet may be controlled through translation of specific mRNAs transported from the soma, exposing new mechanistic layers within stem cells of the developing brain.
Barber, A. F., Erion, R., Holmes, T. C. and Sehgal, A. (2016). Circadian and feeding cues integrate to drive rhythms of physiology in Drosophila insulin-producing cells. Genes Dev 30(23): 2596-2606. PubMed ID: 27979876
Circadian clocks regulate much of behavior and physiology, but the mechanisms by which they do so remain poorly understood. While cyclic gene expression is thought to underlie metabolic rhythms, little is known about cycles in cellular physiology. This study found that Drosophila insulin-producing cells (IPCs), which are located in the pars intercerebralis and lack an autonomous circadian clock, are functionally connected to the central circadian clock circuit via DN1 neurons. Insulin mediates circadian output by regulating the rhythmic expression of a metabolic gene (sxe2) in the fat body. Patch clamp electrophysiology reveals that IPCs display circadian clock-regulated daily rhythms in firing event frequency and bursting proportion under light:dark conditions. The activity of IPCs and the rhythmic expression of sxe2 are additionally regulated by feeding, as demonstrated by night feeding-induced changes in IPC firing characteristics and sxe2 levels in the fat body. These findings indicate circuit-level regulation of metabolism by clock cells in Drosophila and support a role for the pars intercerebralis in integrating circadian control of behavior and physiology.
Chang, A. E., Vaughan, A. G. and Wilson, R. I. (2016). A mechanosensory circuit that mixes opponent channels to produce selectivity for complex stimulus features. Neuron 92(4): 888-901. PubMed ID: 27974164
Johnston's organ is the largest mechanosensory organ in Drosophila; it analyzes movements of the antenna due to sound, wind, gravity, and touch. Different Johnston's organ neurons (JONs) encode distinct stimulus features. Certain JONs respond in a sustained manner to steady displacements, and these JONs subdivide into opponent populations that prefer push or pull displacements. This study describes neurons in the brain (aPN3 neurons) that combine excitation and inhibition from push/pull JONs in different ratios. Consequently, different aPN3 neurons are sensitive to movement in different parts of the antenna's range, at different frequencies, or at different amplitude modulation rates. A model was used to show how the tuning of aPN3 neurons can arise from rectification and temporal filtering in JONs, followed by mixing of JON signals in different proportions. These results illustrate how several canonical neural circuit components-rectification, opponency, and filtering-can combine to produce selectivity for complex stimulus features.

Monday, January 9th

Jenny, F. H. and Basler, K. (2016). Drosophila DDX3/Belle exerts its function outside of the Wnt/Wingless signaling pathway. PLoS One 11(12): e0166862. PubMed ID: 28030561
The helicases human DDX3 and Drosophila Belle (Bel) are part of a well-defined subfamily of the DEAD-box helicases. Individual subfamily-members perform a myriad of functions in nuclear and cytosolic RNA metabolism. It has also been reported that DDX3X is involved in cell signaling, including IFN-alpha and IFN-beta inducing pathways upon viral infection as well as in Wnt signaling. This study used a collection of EMS-induced bel alleles recovered from a Wingless (Wg) suppressor screen to analyze the role of the Drosophila homolog of DDX3 in Wg/Wnt signaling. These EMS alleles, as well as a P-element induced null allele and RNAi-mediated knock down of bel, all suppressed the phenotype of ectopic Wg signaling in the eye. However, they did not affect the expression of known Wg target genes like senseless, Distalless or wingful/Notum. Ectopic Wg signaling in eye imaginal discs induces apoptosis by increasing grim expression. Mutations in bel revert grim expression to wild-type levels. Together, these results indicate that Bel does not function as a core component in the Drosophila Wg pathway, and that mutations affecting its helicase function suppress the effects of ectopic Wg signaling downstream of the canonical pathway.
Heintz, C., et al. (2016). Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans. Nature [Epub ahead of print]. PubMed ID: 27919065
Evolutionary Homolog Study
Ageing is driven by a loss of transcriptional and protein homeostasis and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses. However, the role of splicing homeostasis in healthy ageing remains unclear. This study demonstrates that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, this study found defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP; see Drosophila SF1). SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. It was also demonstrated that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.
Takemura, M. and Nakato, H. (2016). Drosophila Sulf1 is required for the termination of intestinal stem cell division during regeneration. J Cell Sci [Epub ahead of print]. PubMed ID: 27888216
Stem cell division is activated to trigger regeneration in response to tissue damage. The molecular mechanisms by which this stem cell mitotic activity is properly repressed at the end of regeneration are poorly understood. This study shows that a specific modification of heparan sulfate (HS) is critical in regulating Drosophila intestinal stem cell (ISC) division during normal midgut homeostasis and regeneration. Loss of the extracellular HS endosulfatase Sulf1 results in increased ISC division during normal homeostasis, which is caused by upregulation of mitogenic signaling including the JAK/STAT, EGFR, and Hedgehog pathways. Using a regeneration model, this study found that ISCs failed to properly halt division at the termination stage in Sulf1 mutants, showing that Sulf1 is required for terminating ISC division at the end of regeneration. It is proposed that post-transcriptional regulation of mitogen signaling by HS structural modifications provides a novel regulatory step for precise temporal control of stem cell activity during regeneration.
Vaughen, J. and Igaki, T. (2016). Slit-Robo repulsive signaling extrudes tumorigenic cells from epithelia. Dev Cell 39: 683-695. PubMed ID: 27997825
Cells dynamically interact throughout animal development to coordinate growth and deter disease. For example, cell-cell competition weeds out aberrant cells to enforce homeostasis. In Drosophila, tumorigenic cells mutant for the cell polarity gene scribble (scrib) are actively eliminated from epithelia when surrounded by wild-type cells. While scrib cell elimination depends critically on JNK signaling, JNK-dependent cell death cannot sufficiently explain scrib cell extirpation. Thus, how JNK executed cell elimination remained elusive. This study shows that repulsive Slit-Robo2-Ena signaling exerts an extrusive force downstream of JNK to eliminate scrib cells from epithelia by disrupting E-cadherin. While loss of Slit-Robo2-Ena in scrib cells potentiates scrib tumor formation within the epithelium, Robo2-Ena hyperactivation surprisingly triggers luminal scrib tumor growth following excess extrusion. This extrusive signaling is amplified by a positive feedback loop between Slit-Robo2-Ena and JNK. These observations provide a potential causal mechanism for Slit-Robo dysregulation in numerous human cancers.

Sunday, January 8th

Phillips, M. A., Long, A. D., Greenspan, Z. S., Greer, L. F., Burke, M. K., Villeponteau, B., Matsagas, K. C., Rizza, C. L., Mueller, L. D. and Rose, M. R. (2016). Genome-wide analysis of long-term evolutionary domestication in Drosophila melanogaster. Sci Rep 6: 39281. PubMed ID: 28004838
Experimental evolutionary genomics now allows biologists to test fundamental theories concerning the genetic basis of adaptation. This laboratory conducted one of the longest laboratory evolution experiments with any sexually-reproducing metazoan, Drosophila melanogaster. Next-generation resequencing data from this experiment was conducted to examine genome-wide patterns of genetic variation over an evolutionary time-scale that approaches 1,000 generations. Measures of variation within and differentiation between populations were compared to simulations based on a variety of evolutionary scenarios. This analysis yielded no clear evidence of hard selective sweeps, whereby natural selection acts to increase the frequency of a newly-arising mutation in a population until it becomes fixed. Evidence was found for selection acting on standing genetic variation, as independent replicate populations exhibit similar population-genetic dynamics, without obvious fixation of candidate alleles under selection. A hidden-Markov model test for selection also found widespread evidence for selection. More genetic variation was found genome-wide, and less differentiation was found between replicate populations genome-wide, than arose in any of the simulated evolutionary scenarios.
Griffin, P. C., Hangartner, S. B., Fournier-Level, A. and Hoffmann, A. A. (2016). Genomic trajectories to desiccation resistance: Convergence and divergence among replicate selected Drosophila lines. Genetics [Epub ahead of print]. PubMed ID: 28007884
Adaptation to environmental stress is critical for long-term species persistence. With climate change and other anthropogenic stressors compounding natural selective pressures, understanding the nature of adaptation is as important as ever in evolutionary biology. This study investigated this issue in a set of replicated Drosophila lines selected for increased desiccation resistance, a classical physiological trait that has been closely linked to Drosophila species distributions. Pooled whole-genome sequencing was used to compare the genetic basis of their selection responses. While selected SNPs in replicates of the same treatment (desiccation-selection or lab adaptation) tended to change frequency in the same direction, suggesting some commonality in the selection response, candidate SNP and gene lists often differed among replicates. Three of the five desiccation-selection replicates showed significant overlap at the gene and network level. All five replicates showed enrichment for ovary-expressed genes, suggesting maternal effects on the selected trait. Divergence between pairs of replicate lines for desiccation-candidate SNPs was greater than between pairs of control lines. This difference also far exceeded the divergence between pairs of replicate lines for neutral SNPs. Overall, while there was overlap in the direction of allele frequency changes and the network and functional categories affected by desiccation selection, replicates showed unique responses at all levels likely reflecting hitchhiking effects, and highlighting the challenges in identifying candidate genes from these types of experiments when traits are likely to be polygenic.
Appel, M., Scholz, C. J., Kocabey, S., Savage, S., Konig, C. and Yarali, A. (2016). Independent natural genetic variation of punishment- versus relief-memory. Biol Lett 12(12). PubMed ID: 28003518
A painful event establishes two opponent memories: cues that are associated with pain onset are remembered negatively, whereas cues that coincide with the relief at pain offset acquire positive valence. Such punishment- versus relief-memories are conserved across species, including humans, and the balance between them is critical for adaptive behaviour with respect to pain and trauma. In the fruit fly, Drosophila melanogaster as a study case, this study found that both punishment- and relief-memories display natural variation across wild-derived inbred strains, but they do not covary, suggesting a considerable level of dissociation in their genetic effectors. This provokes the question whether there may be heritable inter-individual differences in the balance between these opponent memories in man, with potential psycho-clinical implications.
Nozawa, M., Onizuka, K., Fujimi, M., Ikeo, K. and Gojobori, T. (2016). Accelerated pseudogenization on the neo-X chromosome in Drosophila miranda. Nat Commun 7: 13659. PubMed ID: 27897175
Y chromosomes often degenerate via the accumulation of pseudogenes and transposable elements. By contrast, little is known about X-chromosome degeneration. This study compared the pseudogenization process between genes on the neo-sex chromosomes in Drosophila miranda and their autosomal orthologues in closely related species. The pseudogenization rate on the neo-X is much lower than the rate on the neo-Y, but appears to be higher than the rate on the orthologous autosome in D. pseudoobscura. Genes under less functional constraint and/or genes with male-biased expression tend to become pseudogenes on the neo-X, indicating the accumulation of slightly deleterious mutations and the feminization of the neo-X. A weak trend was found that the genes with female-benefit/male-detriment effects identified in D. melanogaster are pseudogenized on the neo-X, implying the masculinization of the neo-X. These observations suggest that both X and Y chromosomes can degenerate due to a complex suite of evolutionary forces.

Saturday, January 7th

Zhang, B., Li, Q., Chu, X., Sun, S. and Chen, S. (2016). Salidroside reduces tau hyperphosphorylation via up-regulating GSK-3β phosphorylation in a tau transgenic Drosophila model of Alzheimer's disease. Transl Neurodegener 5: 21. PubMed ID: 27933142
Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease that causes substantial public health care burdens. Intensive efforts have been made to find effective and safe treatment against AD. The plant product Salidroside (Sal) is the main effective component of Rhodiola rosea L., which has several pharmacological activities. The objective of this study was to investigate the efficacy of Sal in the treatment of AD transgenic Drosophila and the associated mechanisms. Microtubule associated protein tau transgenic Drosophila line (TAU) was used in which tau protein is expressed in the central nervous system and eyes by the Gal4/UAS system. After feeding flies with Sal, the lifespan and locomotor activity were recorded. The appearance of vacuoles in the mushroom body was examined using immunohistochemistry, and the levels of total glycogen synthase kinase 3β (t-GSK-3β), phosphorylated GSK-3β (p-GSK-3β), t-tau and p-tau was detected in the brain by western blot analysis. The results showed that the longevity was improved in salidroside-fed Drosophila groups as well as the locomotor activity. Less vacuoles in the mushroom body, upregulated level of p-GSK-3β and downregulated p-tau were detected following Sal treatment. These data presented the evidence that Sal was capable of reducing the neurodegeneration in tau transgenic Drosophila and inhibiting neuronal loss. The neuroprotective effects of Sal were associated with its up-regulation of the p-GSK-3β and down-regulation of the p-tau.
Hatkevich, T., Kohl, K. P., McMahan, S., Hartmann, M. A., Williams, A. M. and Sekelsky, J. (2016). Bloom syndrome helicase promotes meiotic crossover patterning and homolog disjunction. Curr Biol [Epub ahead of print]. PubMed ID: 27989672
In most sexually reproducing organisms, crossover formation between homologous chromosomes is necessary for proper chromosome disjunction during meiosis I. During meiotic recombination, a subset of programmed DNA double-strand breaks (DSBs) are repaired as crossovers, with the remainder becoming noncrossovers. Whether a repair intermediate is designated to become a crossover is a highly regulated decision that integrates several crossover patterning processes, both along chromosome arms (interference and the centromere effect) and between chromosomes (crossover assurance). Because the mechanisms that generate crossover patterning have remained elusive for over a century, it has been difficult to assess the relationship between crossover patterning and meiotic chromosome behavior. This study showed that meiotic crossover patterning is lost in Drosophila melanogaster mutants that lack the Bloom syndrome helicase. In the absence of interference and the centromere effect, crossovers are distributed more uniformly along chromosomes. Crossovers even occur on the small chromosome 4, which normally never has meiotic crossovers. Regulated distribution of crossovers between chromosome pairs is also lost, resulting in an elevated frequency of homologs that do not receive a crossover, which in turn leads to elevated nondisjunction.
Xu, S., Stern, M. and McNew, J. A. (2016). Beneficial effects of rapamycin in a Drosophila model for hereditary spastic paraplegia. J Cell Sci [Epub ahead of print]. PubMed ID: 27909242
The locomotor deficits in the hereditary spastic paraplegias (HSPs) reflect degeneration of upper motor neurons, but the mechanisms underlying this neurodegeneration are unknown. This study established a Drosophila model for the HSP atlastin (atl), which encodes an ER fusion protein. Neuronal atl loss causes degeneration of specific thoracic muscles that is preceded by other pathologies including accumulation of aggregates containing poly-ubiquitin (poly-UB), increased generation of reactive oxygen species, and activation of the JNK/Foxo stress response pathway. Inhibiting the Tor kinase, either genetically or by administering rapamycin, at least partially reversed many of these pathologies. atl loss from muscle also triggers muscle degeneration and rapamycin-sensitive locomotor deficits and poly-UB aggregate accumulation. These results indicate that atl loss triggers muscle degeneration both cell autonomously and nonautonomously.
Delabaere, L., et al. (2016). Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells. Aging Cell [Epub ahead of print]. PubMed ID: 28000382
Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability. This study shows that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, it is proposed that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals.

Friday, January 6th

Allen, A. M., Anreiter, I., Neville, M. C. and Sokolowski, M. B. (2016). Feeding-related traits are affected by dosage of the foraging gene in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28007892
Nutrient acquisition and energy storage are critical parts of achieving metabolic homeostasis. The foraging gene in Drosophila melanogaster has previously been implicated in multiple feeding-related and metabolic traits. Before foraging's functions can be further dissected, a precise genetic null mutant is needed to definitively map its amorphic phenotypes. This study used homologous recombination to precisely delete foraging, generating the for0 null allele, and used recombineering to re-integrate a full copy of the gene, generating the {forBAC} rescue allele. Total loss of foraging expression in larvae results in reduced larval path length and food intake behavior, while conversely showing an increase in triglyceride levels. Furthermore, varying foraging gene dosage demonstrates a linear dose-response on these phenotypes in relation to foraging gene expression levels. These experiments have unequivocally proven a causal, dose-dependent relationship between the foraging gene and its pleiotropic influence on these feeding-related traits. In that regard, this analysis of foraging's transcription start sites, termination sites, and splicing patterns using RACE and full length cDNA sequencing, revealed 4 independent promoters, pr1-4, that produce 21 transcripts with 9 distinct ORFs. The use of alternative promoters and alternative splicing at the foraging locus creates diversity and flexibility in the regulation of gene expression, and ultimately function. Future studies will exploit these genetic tools to precisely dissect the isoform- and tissue-specific requirements of foraging's functions and shed light on the genetic control of feeding-related traits involved in energy homeostasis.
Kubrak, O. I., Kucerova, L., Theopold, U., Nylin, S. and Nassel, D. R. (2016). Characterization of reproductive dormancy in male Drosophila melanogaster. Front Physiol 7: 572. PubMed ID: 27932997
Insects are known to respond to seasonal and adverse environmental changes by entering dormancy, also known as diapause. In some insect species, including Drosophila melanogaster, dormancy occurs in the adult organism and postpones reproduction. This adult dormancy has been studied in female flies where it is characterized by arrested development of ovaries, altered nutrient stores, lowered metabolism, increased stress and immune resistance and drastically extended lifespan. Male dormancy, however, has not been investigated in D. melanogaster, and its physiology is poorly known in most insects. This study shows that unmated 3-6 h old male flies placed at low temperature (11 ° C) and short photoperiod (10 Light:14 Dark) enter a state of dormancy with arrested spermatogenesis and development of testes and male accessory glands. Over 3 weeks of diapause a dynamic increase is seen in stored carbohydrates and an initial increase and then a decrease in lipids. An up-regulated expression of genes involved in metabolism, stress responses and innate immunity is also noted. Interestingly, it was found that male flies that entered reproductive dormancy do not attempt to mate females kept under non-diapause conditions (25 ° C, 12L:12D), and conversely non-diapausing males do not mate females in dormancy. In summary, this study shows that male D. melanogaster can enter reproductive dormancy. However, these data suggest that dormant male flies deplete stored nutrients faster than females, studied earlier, and that males take longer to recover reproductive capacity after reintroduction to non-diapause conditions.
Zhan, Y. P., Liu, L. and Zhu, Y. (2016). Taotie neurons regulate appetite in Drosophila. Nat Commun 7: 13633. PubMed ID: 27924813
The brain has an essential role in maintaining a balance between energy intake and expenditure of the body. Deciphering the processes underlying the decision-making for timely feeding of appropriate amounts may improve our understanding of physiological and psychological disorders related to feeding control. This study identified a group of appetite-enhancing neurons in a behavioural screen for flies with increased appetite. Manipulating the activity of these neurons, which were name Taotie neurons, induces bidirectional changes in feeding motivation. Long-term stimulation of Taotie neurons results in flies with highly obese phenotypes. Furthermore, it was shown that the in vivo activity of Taotie neurons in the neuroendocrine region reflects the hunger/satiety states of un-manipulated animals, and that appetitive-enhancing Taotie neurons control the secretion of insulin, a known regulator of feeding behaviour. Thus, this study reveals a new set of neurons regulating feeding behaviour in the high brain regions that represents physiological hunger states and control feeding behaviour in Drosophila.
Zhang, R., Wang, B., Grossi, G., Falabella, P., Liu, Y., Yan, S., Lu, J., Xi, J. and Wang, G. (2016). Molecular Basis of Alarm Pheromone Detection in Aphids. Curr Biol. PubMed ID: 27916525
Evolutionary Homolog Study
The sesquiterpene (E)-beta-farnesene (EBF) is the alarm pheromone for many species of aphids. When released from aphids attacked by parasitoids or predators, it alerts nearby conspecifics to escape by walking away and dropping off the host plan. The reception of alarm pheromone in aphids is accomplished through a highly sensitive chemosensory system. This study demonstrates that ApisOR5, a member of the large superfamily of odorant receptors, is expressed in large placoid sensillum neurons on the sixth antennal segment and confers response to EBF when co-expressed with Orco, an obligate odorant receptor co-receptor, in parallel heterologous expression systems. In addition, the repellent behavior of Acyrthosiphon pisum to EBF disappears after knocking down ApisOR5 by RNAi as well as two A. pisum odorant-binding proteins known to bind EBF (ApisOBP3 and ApisOBP7). Furthermore, other odorants that can also activate ApisOR5, such as geranyl acetate, significantly repel A. pisum, as does EBF. Taken together, these data lead to the conclusion that ApisOR5 is essential to EBF reception in A. pisum. The characterization of the EBF receptor allows high-throughput screening of aphid repellents, providing the necessary information to develop new strategies for aphid control.

Thursday, January 5th

Sengupta, S., Rath, U., Yao, C., Zavortink, M., Wang, C., Girton, J., Johansen, K.M. and Johansen, J. (2016). Digitor/dASCIZ has multiple roles in Drosophila development. PLoS One 11: e0166829. PubMed ID: 27861562
This study provides evidence that the spindle matrix protein Skeletor in Drosophila interacts with the human ASCIZ (also known as ATMIN and ZNF822) ortholog, Digitor. This interaction was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays. The study also confirms a previously documented function of Digitor as a regulator of Dynein light chain/Cut up expression. Digitor was shown to be a nuclear protein that localizes to interband and developmental puff chromosomal regions during interphase but redistributes to the spindle region during mitosis. Its mitotic localization and physical interaction with Skeletor suggests the possibility that Digitor plays a direct role in mitotic progression as a member of the spindle matrix complex. Furthermore, a true null Digitor allele results in complete pupal lethality when homozygous, indicating that Digitor is an essential gene. Digitor plays critical roles in regulation of metamorphosis and organogenesis as well as in the DNA damage response. In the Digitor null mutant larvae there are greatly elevated levels of γH2Av, indicating accumulation of DNA double-strand breaks. Furthermore, reduced levels of Digitor decrease the resistance to paraquat-induced oxidative stress resulting in increased mortality in a stress test paradigm. It was shown that an early developmental consequence of the absence of Digitor is reduced third instar larval brain size although overall larval development appears otherwise normal at this stage. Altogether the data shows that Digitor is a nuclear protein that performs multiple roles in Drosophila larval and pupal development.

Kulkarni, A., Khan, Y. and Ray, K. (2016). Heterotrimeric kinesin-2, together with kinesin-1, steers vesicular acetylcholinesterase movements toward the synapse. Faseb j. [Epub ahead of print]. PubMed ID: 27920150
Acetylcholinesterase (AChE), which is implicated in the pathophysiology of neurological disorders, is distributed along the axon and enriched at the presynaptic basal lamina. It hydrolyses the neurotransmitter acetylcholine, which inhibits synaptic transmission. Aberrant AChE activity and ectopic axonal accumulation of the enzyme are associated with neurodegenerative disorders, such as Alzheimer's disease. The molecular mechanism that underlies AChE transport is still unclear. This study shows that expression of Drosophila AChE tagged with photoactivable green fluorescent protein and m-Cherry (GPAC) in cholinergic neurons compensates for the RNA interference-mediated knockdown of endogenous AChE activity. GPAC-AChE, which is enriched in the neuropil region of the brain, moves in the apparently vesicular form in axons with an anterograde bias in Drosophila larvae. Two anterograde motors, kinesin-1 and kinesin-2, propel distinct aspects of GPAC-AChE movements. Total loss of kinesin-2 reduces the density of anterograde traffic and increases bidirectional movements of GPAC-AChE vesicles without altering their speed. A partial loss of kinesin-1 reduces both the density and speed of anterograde GPAC-AChE traffic and enhances the pool of stationary vesicles. Together, these results suggest that combining activity of a relatively weak kinesin-2 with that of a stronger kinesin-1 motor could steer AChE-containing vesicles toward synapse, and provides a molecular basis for the observed subcellular distribution of the enzyme.
Qu, Y., Hahn, I., Webb, S., Pearce, S. P. and Prokop, A. (2016). Periodic actin structures in neuronal axons are required to maintain microtubules. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27881663
Axons are the cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly-spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily-conserved, ubiquitous, highly-ordered feature of axons, the function of PMS is unknown. This paper examined PMS abundance, organisation and function, combining versatile Drosophila genetics with super-resolution microscopy and various functional readouts. Analyses with 11 different actin regulators and 3 actin-targeting drugs suggest PMS to contain short actin filaments which are depolymerisation resistant and sensitive to spectrin, adducin and nucleator deficiency - consistent with microscopy-derived models proposing PMS as specialised cortical actin. Upon actin removal gaps were observed in microtubule bundles, reduced microtubule polymerisation and reduced axon numbers suggesting a role of PMS in microtubule organisation. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilising protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerisation contributes to their maintenance in axons. Potential implications are discussed of this novel PMS function along axon shafts for axon maintenance and regeneration.
Radford, S. J., Go, A. M. and McKim, K. S. (2016). Cooperation between kinesin motors promotes spindle symmetry and chromosome organization in oocytes. Genetics [Epub ahead of print]. PubMed ID: 27932541
The oocyte spindle in most animal species is assembled in the absence of the microtubule-organizing centers called centrosomes. Without the organization provided by centrosomes, acentrosomal meiotic spindle organization may rely heavily on the bundling of microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-14 NCD and the plus-end directed microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-6 Subito are known to be required for oocyte spindle organization in Drosophila melanogaster How multiple microtubule-bundling kinesins interact to produce a functional acentrosomal spindle is not known. In addition, there have been few studies on the meiotic function of one of the most important microtubule-bundlers in mitotic cells, the kinesin-5 KLP61F. This study found that the kinesin-5 KLP61F is required for spindle and centromere symmetry in oocytes. The asymmetry observed in the absence of KLP61F depends on NCD, the kinesin-12 KLP54D, and the microcephaly protein ASP. In contrast, KLP61F and Subito work together in maintaining a bipolar spindle. It is proposed that the prominent central spindle, stabilized by Subito, provides the framework for the coordination of multiple microtubule-bundling activities. The activities of several proteins, including NCD, KLP54D, and ASP, generate asymmetries within the acentrosomal spindle, while KLP61F and Subito balance these forces resulting in the capacity to accurately segregate chromosomes.

Wednesday, January 4th

Marie, P.P., Ronsseray, S. and Boivin, A. (2016). From embryo to adult: piRNA-mediated silencing throughout germline development in Drosophila. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27932388
In metazoan germ cells, transposable element activity is repressed by small noncoding PIWI-associated RNAs (piRNAs). Numerous studies in Drosophila have elucidated the mechanism of this repression in the adult germline. However, when and how transposable element repression is established during germline development, has not been addressed. This study shows that homology-dependent trans silencing is active in female primordial germ cells from late embryogenesis through pupal stages, and that genes related to the adult piRNA pathway are required for silencing during development. In larval gonads, rhino-dependent piRNAs are detected indicating de novo biogenesis of functional piRNAs during development. Those piRNAs exhibit the molecular signature of the "ping-pong" amplification step. Moreover, Heterochromatin Protein 1a (HP1a) is required for the production of piRNAs coming from telomeric transposable elements. Furthermore, as in adult ovaries, incomplete, bimodal and stochastic repression resembling variegation can occur at all developmental stages. Clonal analysis indicates that the repression status established in embryonic germ cells is maintained until the adult stage, suggesting the implication of a cellular memory mechanism. Taken together, these data show that piRNAs and their associated proteins are epigenetic components of a continuous repression system throughout germ cell development.

Li, S., Li, Y., Shen, L., Jin, P., Chen, L. and Ma, F. (2016). miR-958 inhibits Toll signaling and Drosomycin expression via directly targeting Toll and Dif in Drosophila melanogaster. Am J Physiol Cell Physiol [Epub ahead of print]. PubMed ID: 27974298
Drosophila melanogaster is widely used as a model system to study innate immunity and signaling pathways related to innate immunity, including the Toll signaling pathway. Although this pathway is well-studied, the precise mechanisms of post-transcriptional regulation of key components of the Toll signaling pathway by microRNAs (miRNAs) remain obscure. This study used an in silico strategy in combination with the Gal80ts-Gal4 driver system and identified microRNA-958 (miR-958) as a candidate Toll pathway regulating miRNA in Drosophila. Overexpression of miR-958 significantly reduces the expression of Drosomycin, a key antimicrobial peptide involved in Toll signaling and the innate immune response. It was demonstrated in vitro and in vivo that miR-958 targets the Toll and Dif genes, key components of the Toll signaling pathway, to negatively regulate Drosomycin expression. In addition, a miR-958-sponge rescues the expression of Toll and Dif, resulting in increased expression of Drosomycin. These results not only reveal a novel function and modulation pattern of miR-958, but also provide a new insight into the underlying molecular mechanisms of Toll signaling in regulation of innate immunity.

Lence, T., Akhtar, J., Bayer, M., Schmid, K., Spindler, L., Ho, C. H., Kreim, N., Andrade-Navarro, M. A., Poeck, B., Helm, M. and Roignant, J. Y. (2016). m6A modulates neuronal functions and sex determination in Drosophila. Nature 540(7632): 242-247. PubMed ID: 27919077
N6-methyladenosine RNA (m6A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. This study carried out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. The member of the split ends protein family, Spenito, was identified as a novel bona fide subunit of the methyltransferase complex. Important roles of this complex were demonstrated in neuronal functions and sex determination, and the nuclear YT521-B protein was implicated as a main m6A effector in these processes. Altogether, this work substantially extends knowledge of m6A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal.
Gaspar, I., Sysoev, V., Komissarov, A. and Ephrussi, A. (2016). An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs. Embo J [Epub ahead of print]. PubMed ID: 28028052
Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport-competent mRNPs. The posterior-targeting kinesin-1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein-dependent transport from the nurse cells into the oocyte. Kinesin-1 recruitment requires the DmTropomyosin1-I/C isoform, an atypical RNA-binding tropomyosin that binds directly to dimerizing oskar 3'UTRs. The isoform is on of 17 predicted mRNA isoforms and 13 distinct polypeptides encoded by the TM1 gene. Finally, a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin-1, and that the motor is activated during mid-oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo-dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo-transport processes and between the cargo-associated dynein and kinesin-1.

Tuesday, January 3rd

Yuan, I., Leontiou, I., Amin, P., May, K. M., Soper Ni Chafraidh, S., Zlamalova, E. and Hardwick, K. G. (2016). Generation of a spindle checkpoint arrest from synthetic signaling assemblies. Curr Biol [Epub ahead of print]. PubMed ID: 28017606
Evolutionary Homolog Study

The spindle checkpoint acts as a mitotic surveillance system, monitoring interactions between kinetochores and spindle microtubules and ensuring high-fidelity chromosome segregation. The checkpoint is activated by unattached kinetochores, and Mps1 kinase phosphorylates KNL1 on conserved MELT motifs to generate a binding site for the Bub3-Bub1 complex (see Drosophila Bub3). This leads to dynamic kinetochore recruitment of Mad proteins, a conformational change in Mad2, and formation of the mitotic checkpoint complex (MCC: Cdc20-Mad3-Mad2: see Drosophila Cdc20). MCC formation inhibits the anaphase-promoting complex/cyclosome (Cdc20-APC/C), thereby preventing the proteolytic destruction of securin and cyclin and delaying anaphase onset. What happens at kinetochores after Mps1-dependent Bub3-Bub1 recruitment remains mechanistically unclear, and it is not known whether kinetochore proteins other than KNL1 have significant roles to play in checkpoint signaling and MCC generation. This study took a reductionist approach, avoiding the complexities of kinetochores, and demonstrate that co-recruitment of KNL1Spc7 and Mps1Mph1 is sufficient to generate a robust checkpoint signal and prolonged mitotic arrest in fission yeast. A Mad1-Bub1 complex is formed during synthetic checkpoint signaling. Analysis of bub3Δ mutants demonstrates that Bub3 acts to suppress premature checkpoint signaling. This synthetic system will enable detailed, mechanistic dissection of MCC generation and checkpoint silencing. After analyzing several mutants that affect localization of checkpoint complexes, it is concluded that spindle checkpoint arrest can be independent of their kinetochore, spindle pole, and nuclear envelope localization.

Mochida, S., Rata, S., Hino, H., Nagai, T. and Novak, B. (2016). Two bistable switches govern M phase entry. Curr Biol 26(24): 3361-3367. PubMed ID: 27889260
Evolutionary Homolog Study

The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex (see Drosophila Cdk1). Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible. Cdk1 auto-activation was found to be dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). This study shows, in Xenopus egg extracts, that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway (see Drosophila Greatwall) is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, a sharp Cdk1 threshold was found for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations-evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. These results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry.

Duranteau, M., Montagne, J. J. and Rahmani, Z. (2016). A novel mutation in the N-terminal domain of Drosophila BubR1 affects the spindle assembly checkpoint function of BubR1. Biol Open [Epub ahead of print]. PubMed ID: 27742609
The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism that ensures accurate segregation of chromosomes into two daughter cells. BubR1, a key component of the SAC, play also a role in the mitotic timing since depletion of BubR1 leads to an accelerated mitosis. Unlike what was reported in mammalian cells, mutation of the KEN1-box domain of Drosophila BubR1 (bubR1-KEN1 mutant) that affects the binding of BubR1 to Cdc20, the activating co-factor of the APC/C, did not accelerate the mitotic timing despite resulting in a defective SAC. This study shows that a mutation in a novel Drosophila short sequence (bubR1-KAN mutant) leads to an accelerated mitotic timing as well as SAC failure. Moreover, the data indicate that the level of Fzy, the Drosophila homolog of Cdc20, recruited to kinetochores is diminished in bubR1-KEN1 mutant cells and further diminished in bubR1-KAN mutant cells. Altogether, these data show that this newly identified Drosophila BubR1 KAN motif is required for a functional SAC and suggest that it may play an important role on Cdc20/Fzy kinetochore recruitment.
Vo, N., Anh Suong, D. N., Yoshino, N., Yoshida, H., Cotterill, S. and Yamaguchi, M. (2016). Novel roles of HP1a and Mcm10 in DNA replication, genome maintenance and photoreceptor cell differentiation. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27903903
Both Mcm10 and HP1a are known to be required for DNA replication. However, underlying mechanism is not clarified yet especially for HP1. Knockdown of both HP1a and Mcm10 genes inhibited the progression of S phase in Drosophila eye imaginal discs. Proximity Ligation Assay (PLA) demonstrated that HP1a is in close proximity to DNA replication proteins including Mcm10, RFC140 and DNA polymerase 255 kDa subunit in S-phase. This was further confirmed by co-immunoprecipitation assay. The PLA signals between Mcm10 and HP1a are specifically observed in the mitotic cycling cells, but not in the endocycling cells. Interestingly, many cells in the posterior regions of eye imaginal discs carrying a double knockdown of Mcm10 and HP1a induced ectopic DNA synthesis and DNA damage without much of ectopic apoptosis. Therefore, the G1-S checkpoint may be affected by knockdown of both proteins. This event was also the case with other HP family proteins such as HP4 and HP6. In addition, both Mcm10 and HP1a are required for differentiation of photoreceptor cells R1, R6 and R7. Further analyses on several developmental genes involved in the photoreceptor cell differentiation suggest that a role of both proteins is mediated by regulation of the lozenge gene.

Monday, January 2nd

Şahin, A., Held, A., Bredvik, K., Major, P., Achilli, T.M., Kerson, A.G., Wharton, K., Stilwell, G. and Reenan, R. (2016). Human SOD1 ALS mutations in a Drosophila knock-in model cause severe phenotypes and reveal dosage-sensitive gain and loss of function components. Genetics [Epub ahead of print]. PubMed ID: 27974499
Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc Superoxide Dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, this study genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical and behavioral phenotypes. Contrary to previous transgenic models, ALS-like phenotypes recapitulate without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R and H71Y) exhibits neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) is phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes are recessive, a gain of function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which fail to show severe locomotor defects or nerve degeneration. The study concludes that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies.

Ramaker, J. M., Cargill, R. S., Swanson, T. L., Quirindongo, H., Cassar, M., Kretzschmar, D. and Copenhaver, P. F. (2016). Amyloid precursor proteins are dynamically trafficked and processed during neuronal development. Front Mol Neurosci 9: 130. PubMed ID: 27932950
Proteolytic processing of the Amyloid Precursor Protein (APP) produces β-amyloid (Aβ) peptide fragments that accumulate in Alzheimer's Disease (AD), but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. Insects express only a single APP-related protein (APP-Like, or APPL) that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aβ-like fragment that can induce neurotoxic responses in the brain. This study investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, it was shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, it was found that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate APPL-dependent responses within the nervous system. Lastly, targeted expression of the double-tagged constructs (combined with time-lapse imaging) revealed that APP family proteins are subject to complex patterns of trafficking and processing that vary dramatically between different neuronal subtypes. In combination, these results provide a new perspective on how the regulation of APP family proteins can be modulated to accommodate a variety of cell type-specific responses within the embryonic and adult nervous system.
Pomatto, L. C., Carney, C., Shen, B., Wong, S., Halaszynski, K., Salomon, M. P., Davies, K. J. and Tower, J. (2016). The mitochondrial Lon protease is required for age-specific and sex-specific adaptation to oxidative stress. J Curr Biol [Epub ahead of print]. PubMed ID: 27916526
Multiple human diseases involving chronic oxidative stress show a significant sex bias, including neurodegenerative diseases, cancer, immune dysfunction, diabetes, and cardiovascular disease. This study reports that Drosophila females but not males adapt to hydrogen peroxide stress, whereas males but not females adapt to paraquat (superoxide) stress. Stress adaptation in each sex requires the conserved mitochondrial Lon protease and is associated with sex-specific expression of Lon protein isoforms and proteolytic activity. Adaptation to oxidative stress is lost with age in both sexes. Transgenic expression of transformer gene during development transforms chromosomal males into pseudo-females and confers the female-specific pattern of Lon isoform expression, Lon proteolytic activity induction, and H2O2 stress adaptation; these effects were also observed using adult-specific transformation. Conversely, knockdown of transformer in chromosomal females eliminates the female-specific Lon isoform expression, Lon proteolytic activity induction, and H2O2 stress adaptation and produces the male-specific paraquat (superoxide) stress adaptation. Sex-specific expression of alternative Lon isoforms was also observed in mouse tissues. The results develop Drosophila melanogaster as a model for sex-specific stress adaptation regulated by the Lon protease, with potential implications for understanding sexual dimorphism in human disease.
Yedlapudi, D., Joshi, G. S., Luo, D., Todi, S. V. and Dutta, A. K. (2016). Inhibition of α-synuclein aggregation by multifunctional dopamine agonists assessed by a novel in vitro assay and an in vivo Drosophila synucleinopathy model. Sci Rep 6: 38510. PubMed ID: 27917933
Aggregation of α synuclein (α-syn) leading to dopaminergic neuronal death has been recognized as one of the main pathogenic factors in the initiation and progression of Parkinson's disease (PD). Consequently, α-syn has been targeted for the development of therapeutics for PD. This study developed a novel assay to screen compounds with α-syn modulating properties by mimicking recent findings from in vivo animal studies involving intrastriatal administration of pre-formed fibrils in mice, resulting in increased α-syn pathology accompanying the formation of Lewy-body (LB) type inclusions. In vitro generated α-syn pre-formed fibrils induce seeding of α-syn monomers to produce aggregates in a dose-and time-dependent manner under static conditions in vitro. These aggregates were toxic towards rat pheochromocytoma cells (PC12). Multifunctional dopamine agonists D-519 and D-520 exhibited significant neuroprotection in this assay, while their parent molecules did not. The neuroprotective properties of these compounds were further evaluated in a Drosophila model of synucleinopathy. Both of the compounds showed protective properties in fly eyes against the toxicity caused by α-syn. Thus, the in vitro results on modulation of aggregation and toxicity of α-syn by a novel assay were further validated with the in vivo experiments.

Sunday, January 1st

Sabeva, N., Cho, R. W., Vasin, A., Gonzalez, A., Littleton, J. T. and Bykhovskaia, M. (2016). Complexin mutants reveal partial segregation between recycling pathways that drive evoked and spontaneous neurotransmission. J Neurosci [Epub ahead of print]. PubMed ID: 27913592
Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. Synaptic vesicle recycling pathways were examined at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. Loading of the lipophilic dye FM1-43 was combined with photoconversion, electron microscopy (EM), and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. The total number of recycling vesicles was found to be equal to those retrieved through spontaneous and evoked pools, suggesting retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1-43 destaining and synaptic depression measured in the presence of the vesicle refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1-43 photoconversion combined with EM analysis indicated spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, while vesicles recycled following evoked release involve a larger intra-terminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion.
Gokhale, A., Hartwig, C., Freeman, A. H., Das, R., Zlatic, S. A., Vistein, R., Burch, A., Carrot, G., Lewis, A. F., Nelms, S., Dickman, D. K., Puthenveedu, M. A., Cox, D. N. and Faundez, V. (2016). The proteome of BLOC-1 genetic defects identifies the Arp2/3 actin polymerization complex to function downstream of the schizophrenia susceptibility factor Dysbindin at the synapse. J Neurosci 36(49): 12393-12411. PubMed ID: 27927957
Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. This idea was tested by determining the proteome sensitive to genetic defects in a locus encoding dysbindin (see Drosophila Dysbindin), a protein required for synapse biology and implicated in schizophrenia risk. Quantitative mass spectrometry was applied to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). 491 proteins sensitive to dysbindin and BLOC-1 loss of function were found. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex (see Drosophila Arpc1), as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, it was demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. These results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects.
Laugks, U., Hieke, M. and Wagner, N. (2016). MAN1 restricts BMP signaling during synaptic growth in Drosophila. Cell Mol Neurobiol [Epub ahead of print]. PubMed ID: 27848060
Bone morphogenic protein (BMP) signaling is crucial for coordinated synaptic growth and plasticity. This study shows that the nuclear LEM-domain protein MAN1 is a negative regulator of synaptic growth at Drosophila larval and adult neuromuscular junctions (NMJs). Loss of MAN1 is associated with synaptic structural defects, including floating T-bars, membrane attachment defects, and accumulation of vesicles between perisynaptic membranes and membranes of the subsynaptic reticulum. In addition, MAN1 mutants accumulate more heterogeneously sized vesicles and multivesicular bodies in larval and adult synapses, the latter indicating that MAN1 may function in synaptic vesicle recycling and endosome-to-lysosome trafficking. Synaptic overgrowth in MAN1 is sensitive to BMP signaling levels, and loss of key BMP components attenuate BMP-induced synaptic overgrowth. Based on these observations, it is proposed that MAN1 negatively regulates accumulation and distribution of BMP signaling components to ensure proper synaptic growth and integrity at larval and adult NMJs.
Yoon, E. J., Jeong, Y. T., Lee, J. E., Moon, S. J. and Kim, C. H. (2016). Tubby domain superfamily protein is required for the formation of the 7S SNARE complex in Drosophila. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 27888110
Tubby domain superfamily protein (TUSP) is a distant member of the Tubby-like protein (TULP) family. Although other TULPs play important roles in sensation, metabolism, and development, the molecular functions of TUSP are completely unknown. This study explored the function of TUSP in the Drosophila nervous system where it is expressed in all neurons. Tusp mutant flies exhibit a temperature-sensitive paralysis. This paralysis can be rescued by tissue-specific expression of Tusp in the giant fibers and peripherally synapsing interneurons of the giant fiber system, a well-characterized neuronal circuit that mediates rapid escape behavior in flies. Consistent with this paralytic phenotype, a profound reduction was observed in the assembly of the ternary 7S SNARE complex (see synaptobrevin, syntaxin, and SNAP-25) that is required for neurotransmitter release despite seeing no changes in the expression of each individual SNARE complex component. Together, these data suggest TUSP is a novel regulator of SNARE assembly and, therefore, of neurotransmitter release.

Kauwe, G., Tsurudome, K., Penney, J., Mori, M., Gray, L., Calderon, M. R., Elazouzzi, F., Chicoine, N., Sonenberg, N. and Haghighi, A. P. (2016). Acute fasting regulates retrograde synaptic enhancement through a 4E-BP-dependent mechanism.Neuron [Epub ahead of print]. PubMed ID: 27916456
While beneficial effects of fasting on organismal function and health are well appreciated, little is known about the molecular details of how fasting influences synaptic function and plasticity. Genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, the findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. These results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits.
Nagel, B. M., Bechtold, M., Rodriguez, L. G. and Bogdan, S. (2016). Drosophila WASH is required for integrin-mediated cell adhesion, cell motility and lysosomal neutralization. J Cell Sci [Epub ahead of print]. PubMed ID: 27884932
The Wiskott-Aldrich Syndrome Protein and SCAR Homologue (WASH) is a conserved actin nucleation promoting factor controlling Arp2/3 complex activity in endosomal sorting and recycling. Previous studies have identified WASH as an essential regulator in Drosophila development. This study shows that homozygous wash mutant flies are viable and fertile. Drosophila WASH has conserved functions in integrin receptor recycling and lysosome neutralization. WASH generates actin patches on endosomes and lysosomes mediating both functions. Consistently, loss of WASH function results in cell spreading and cell migration defects of macrophages, and an increased lysosomal acidification that affects efficient phagocytic and autophagic clearance. WASH physically interacts with the vacuolar ATPase subunit Vha55 that is crucial to establish and maintain lysosome acidification. As a consequence, starved flies lacking WASH function show a dramatic increase in acidic autolysosomes causing a reduced lifespan. Thus, these data highlight a conserved role for WASH in the endocytic sorting and recycling of membrane proteins like integrins and the V-ATPase that increase the likelihood of survival under nutrient deprivation.

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