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


Tuesday, March 31st, 2015

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Jafari, S. and Alenius, M. (2015). Cis-regulatory mechanisms for robust olfactory sensory neuron class-restricted odorant receptor gene expression in Drosophila. PLoS Genet 11: e1005051. PubMed ID: 25760344
Odor perception requires that each olfactory sensory neuron (OSN) class continuously express a single odorant receptor (OR) regardless of changes in the environment. However, little is known about the control of the robust, class-specific OR expression involved. This study investigates the cis-regulatory mechanisms and components that generate robust and OSN class-specific OR expression in Drosophila. The results demonstrate that the spatial restriction of expression to a single OSN class is directed by clusters of transcription-factor DNA binding motifs. Dissection of motif clusters of differing complexity demonstrated that structural components such as motif overlap and motif order integrates transcription factor combinations and chromatin status to form a spatially restricted pattern. Further, changes in metabolism or temperature perturbed the function of complex clusters. The cooperative regulation between motifs around and within the cluster generated robust, class-specific OR expression.

Nitta, K.R., Jolma, A., Yin, Y., Morgunova, E., Kivioja, T., Akhtar, J., Hens, K., Toivonen, J., Deplancke, B., Furlong, E.E. and Taipale, J. (2015). Conservation of transcription factor binding specificities across 600 million years of bilateria evolution. Elife 4. PubMed ID: 25779349
Divergent morphology of species has largely been ascribed to genetic differences in the tissue-specific expression of proteins, which could be achieved by divergence in cis-regulatory elements or by altering the binding specificity of transcription factors (TFs). The relative importance of the latter has been difficult to assess, as previous systematic analyses of TF binding specificity have been performed using different methods in different species. To address this, this study determined the binding specificities of 242 Drosophila TFs, and compared them to human and mouse data. This analysis revealed that TF binding specificities were highly conserved between Drosophila and mammals, and that for orthologous TFs, the similarity extended even to the level of very subtle dinucleotide binding preferences. The few human TFs with divergent specificities functioned in cell types not found in fruit flies, suggesting that evolution of TF specificities contributes to emergence of novel types of differentiated cells.

Chang, A. T., Liu, Y., Ayyanathan, K., Benner, C., Jiang, Y., Prokop, J. W., Paz, H., Wang, D., Li, H. R., Fu, X. D., Rauscher, F. J. and Yang, J. (2015). An evolutionarily conserved DNA architecture determines target specificity of the TWIST family bHLH transcription factors. Genes Dev [Epub ahead of print]. PubMed ID: 25762439
Basic helix-loop-helix (bHLH) transcription factors recognize the canonical E-box (CANNTG) to regulate gene transcription; however, given the prevalence of E-boxes in a genome, it has been puzzling how individual bHLH proteins selectively recognize E-box sequences on their targets. Twist is a bHLH transcription factor that promotes epithelial-mesenchymal transition (EMT) during development and tumor metastasis. High-resolution mapping of Twist occupancy in human and Drosophila genomes reveals that Twist, but not other bHLH proteins, recognizes a unique double E-box motif with two E-boxes spaced preferentially by 5 nucleotides. Using molecular modeling and binding kinetic analyses, this study found that the strict spatial configuration in the double E-box motif aligns two Twist-E47 (see Drosophila Daughterless) dimers on the same face of DNA, thus providing a high-affinity site for a highly stable intramolecular tetramer. Biochemical analyses showed that the WR domain of Twist dimerizes to mediate tetramer formation, which is functionally required for Twist-induced EMT. These results uncover a novel mechanism for a bHLH transcription factor to recognize a unique spatial configuration of E-boxes to achieve target specificity. The WR-WR domain interaction uncovered here sets an example of target gene specificity of a bHLH protein being controlled allosterically by a domain outside of the bHLH region.

Horvath, A., Batki, J., Henn, L., Lukacsovich, T., Rona, G., Erdelyi, M. and Vertessy, B. G. (2015). dUTPase expression correlates with cell division potential in Drosophila melanogaster. FEBS J [Epub ahead of print]. PubMed ID: 25735890
dUTPase is a dNTP sanitizing enzyme that prevents the appearance of the potentially harmful uracil bases in DNA by hydrolyzing cellular dUTP. This function of dUTPase is found to be essential in many organisms including Drosophila melanogaster. Previously it has been shown that the expression pattern of dUTPase determines the extent of uracil accumulation in the genome of different tissues. This study examined the regulatory mechanism that eventually leaves a set of tissues to have uracil-free and intact genome. The expression pattern established by the promoter of Drosophila dUTPase was found to overlap with mRNA and protein expression pattern, excluding the involvement of other posttranscriptional contribution. This promoter was found to be active in primordial tissues, such as in imaginal discs of the larvae, in the larval brain and in reproductive organs. In the case of brain and imaginal tissues, the promoter activity was found to depend on DRE motifs, the docking site of DREF, which is known as a transcriptional activator of genes involved in replication and proliferation. These results suggest that dUTPase expression is fine-tuned to meet the requirements of DNA synthesis, in tissues where the maintenance of genome integrity is of high importance.

Monday, March 30th

Cugusi, S., Kallappagoudar, S., Ling, H. and Lucchesi, J.C. (2015). The Drosophila helicase MLE is implicated in functions distinct from its role in dosage compensation. Mol Cell Proteomics [Epub ahead of print]. PubMed ID: 25776889
Helicases are ubiquitous enzymes that unwind or remodel single or double-stranded nucleic acids, and that participate in a vast array of metabolic pathways. The ATP-dependent DEXH-box RNA/DNA helicase MLE was first identified as a core member of the chromatin remodeling MSL complex, responsible for dosage compensation in Drosophila males. Although this complex does not assemble in females, MLE is present. Given the multiplicity of functions attributed to its mammalian ortholog RNA helicase A, an analysis was carried out for the purpose of determining whether MLE displays the same diversity. A number of different proteins were identified that associated with MLE, implicating its role in specific pathways. This association was document in selected examples that included the spliceosome complex, heterogeneous Nuclear Ribonucleoproteins involved in RNA Processing and in Heterochromatin Protein 1 deposition, and the NuRD complex.

Lieleg, C., Ketterer, P., Nuebler, J., Ludwigsen, J., Gerland, U., Dietz, H., Mueller-Planitz, F. and Korber, P. (2015). Nucleosome spacing generated by ISWI and CHD1 remodelers is constant regardless of nucleosome density. Mol Cell Biol [Epub ahead of print]. PubMed ID: 25733687
Arrays of regularly spaced nucleosomes are a hallmark of chromatin but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration is experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. This has been called a clamping activity. This study shows in a purified system that ISWI and CHD1 type nucleosome remodelers have such a clamping activity as they not only generate regularly spaced nucleosome arrays, but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila ISWI without HAND-SANT-SLIDE (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding, but that they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms.

Dai, Q., Ren, A., Westholm, J. O., Duan, H., Patel, D. J. and Lai, E. C. (2015). Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family. Genes Dev 29: 48-62. PubMed ID: 25561495
Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain ("BEN-solo" factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, reporter assays were performed that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. This was confirmed by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. It was next shown that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, abundant Insv binding was observed within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, it was observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, these data define an array of common and distinct biochemical and functional properties of this new family of transcription factors.

Verboon, J.M., Rincon-Arano, H., Werwie, T.R., Delrow, J.J., Scalzo, D., Nandakumar, V., Groudine, M. and Parkhurst, S.M. (2015). Wash interacts with Lamin and affects global nuclear organization. Curr Biol [Epub ahead of print]. PubMed ID: 25754639
The cytoplasmic functions of Wiskott-Aldrich syndrome family (WAS) proteins are well established and include roles in cytoskeleton reorganization and membrane-cytoskeletal interactions important for membrane/vesicle trafficking, morphogenesis, immune response, and signal transduction. Misregulation of these proteins is associated with immune deficiency and metastasis. Cytoplasmic WAS proteins act as effectors of Rho family GTPases and polymerize branched actin through the Arp2/3 complex. Previously, Drosophila washout (wash) was identifed as a new member of the WAS family with essential cytoplasmic roles in early development. Studies in mammalian cells and Dictyostelium suggest that WASH functions primarily in a multiprotein complex that regulates endosome shape and trafficking in an Arp2/3-dependent manner. However, roles for classically cytoplasmic proteins in the nucleus are beginning to emerge, in particular, as participants in the regulation of gene expression. This study shows that Drosophila Wash is present in the nucleus, where it plays a key role in global nuclear organization. wash mutant and knockdown nuclei disrupts subnuclear structures/organelles and exhibits the abnormal wrinkled morphology reminiscent of those observed in diverse laminopathies. Nuclear Wash interacts with B-type Lamin (Lamin Dm0), and, like Lamin, Wash associates with constitutive heterochromatin. Wash knockdown increases chromatin accessibility of repressive compartments and resultes in a global redistribution of repressive histone modifications. Thus, these results reveal a novel role for Wash in modulating nucleus morphology and in the organization of both chromatin and non-chromatin nuclear sub-structures.

Sunday, March 29th

Gaspar, P., Holder, M. V., Aerne, B. L., Janody, F. and Tapon, N. (2015). Zyxin antagonizes the FERM protein Expanded to couple F-actin and Yorkie-dependent organ growth. Curr Biol 25: 679-689. PubMed ID: 25728696
Coordinated multicellular growth during development is achieved by the sensing of spatial and nutritional boundaries. The conserved Hippo (Hpo) signaling pathway has been proposed to restrict tissue growth by perceiving mechanical constraints through actin cytoskeleton networks. The actin-associated LIM proteins Zyxin (Zyx) and Ajuba (Jub) have been linked to the control of tissue growth via regulation of Hpo signaling, but the study of Zyx has been hampered by a lack of genetic tools. A zyx mutant was generated in Drosophila using TALEN endonucleases, and this was used to show that Zyx antagonizes the FERM-domain protein Expanded (Ex) to control tissue growth, eye differentiation, and F-actin accumulation. Zyx membrane targeting promotes the interaction between the transcriptional co-activator Yorkie (Yki) and the transcription factor Scalloped (Sd), leading to activation of Yki target gene expression and promoting tissue growth. Finally, this study shows that Zyx's growth-promoting function is dependent on its interaction with the actin-associated protein Enabled (Ena) via a conserved LPPPP motif and is antagonized by Capping Protein (CP). These results show that Zyx is a functional antagonist of Ex in growth control and establish a link between actin filament polymerization and Yki activity.

Diop, S. B., Bisharat-Kernizan, J., Birse, R. T., Oldham, S., Ocorr, K. and Bodmer, R. (2015). PGC-1/Spargel counteracts high-fat-diet-induced obesity and cardiac lipotoxicity downstream of TOR and Brummer ATGL lipase. Cell Rep [Epub ahead of print]. PubMed ID: 25753422
Obesity and metabolic syndrome are associated with an increased risk for lipotoxic cardiomyopathy, which is strongly correlated with excessive accumulation of lipids in the heart. Obesity- and type-2-diabetes-related disorders have been linked to altered expression of the transcriptional cofactor PGC-1alpha, which regulates the expression of genes involved in energy metabolism. Using Drosophila, PGC-1/spargel (PGC-1/srl) was identified as a key antagonist of high-fat diet (HFD)-induced lipotoxic cardiomyopathy. HFD-induced lipid accumulation and cardiac dysfunction are mimicked by reduced PGC-1/srl function and reversed by PGC-1/srl overexpression. Moreover, HFD feeding lowers PGC-1/srl expression by elevating TOR signaling and inhibiting expression of the Drosophila adipocyte triglyceride lipase (ATGL) (Brummer), both of which function as upstream modulators of PGC-1/srl. The lipogenic transcription factor SREBP also contributes to HFD-induced cardiac lipotoxicity, likely in parallel with PGC-1/srl. These results suggest a regulatory network of key metabolic genes that modulates lipotoxic heart dysfunction.

Khaliullina, H., Bilgin, M., Sampaio, J. L., Shevchenko, A. and Eaton, S. (2015). Endocannabinoids are conserved inhibitors of the Hedgehog pathway. Proc Natl Acad Sci U S A 112: 3415-3420. PubMed ID: 25733905
Hedgehog ligands control tissue development and homeostasis by alleviating repression of Smoothened, a seven-pass transmembrane protein. The Hedgehog receptor, Patched, is thought to regulate the availability of small lipophilic Smoothened repressors whose identity is unknown. Lipoproteins contain lipids required to repress Smoothened signaling in vivo. Using biochemical fractionation and lipid mass spectrometry, this study identified these repressors as endocannabinoids. Endocannabinoids circulate in human and Drosophila lipoproteins and act directly on Smoothened at physiological concentrations to repress signaling in Drosophila and mammalian assays. Phytocannabinoids are also potent Smo inhibitors. These findings link organismal metabolism to local Hedgehog signaling and suggest previously unsuspected mechanisms for the physiological activities of cannabinoids.

Zhang. J, Yin, J.C. and Wesley, C.S. (2015). Notch Intracellular Domain (NICD) suppresses long-term memory formation in adult Drosophila flies. Cell Mol Neurobiol [Epub ahead of print]. PubMed ID: 25791355
Notch receptor signaling is evolutionarily conserved and well known for its roles in animal development. Many studies in Drosophila have shown that Notch also performs important functions in memory formation in adult flies. An intriguing observation is that increased expression of the full-length Notch receptor (Nfull) triggers long-term memory (LTM) formation even after very weak training (single training). Canonical Notch signaling is mediated by Notch intracellular domain (NICD), but it is not known whether increased expression of NICD recapitulates the LTM enhancement induced by increased Nfull expression. This study reports that increased NICD expression either has no impact on LTM formation or suppresses it. Furthermore, it either has no impact or decreases both the levels and activity of cAMP response element binding protein, a key factor supporting LTM. These results indicate that NICD signaling is not sufficient to explain Nfull-induced LTM enhancement. These findings may also shed light on the molecular mechanisms of memory loss in neurological diseases associated with increased NICD expression and canonical Notch signaling.

Saturday, March 28th

Mineo, A., Furriols, M. and Casanova, J. (2015). Accumulation of the Drosophila Torso-like protein at the blastoderm plasma membrane suggests that it translocates from the eggshell. Development [Epub ahead of print]. PubMed ID: 25758463
The eggshell serves as a depository for proteins that play an important role in early embryonic development. In particular, the Drosophila eggshell is responsible for transferring asymmetries from the egg chamber to specify the regions at both ends of the embryo through the uneven activation of the Torso (Tor) receptor in its membrane. This process relies on the restricted expression of the gene torso-like (tsl) in subpopulations of follicle cells during oogenesis and its protein accumulation at both poles of the eggshell, but it is not known how this signal is transmitted to the embryo. This study shows that Tsl accumulates at the embryonic plasma membrane, even in the absence of the Tor receptor. However, during oogenesis, Tsl accumulation was detected only at the eggshell. These results suggest that there is a two-step mechanism to transfer the asymmetric positional cues from the egg chamber into the early embryo: initial anchoring of Tsl at the eggshell as it is secreted, followed by its later translocation to the egg plasma membrane, where it enables Tor receptor activation. Translocation of anchored determinants from the eggshell might then regulate the spatial and temporal control of early embryonic developmental processes.

Yoo, H., Roth-Johnson, E.A., Bor, B. and Quinlan, M.E. (2015). Drosophila Cappuccino alleles provide insight into formin mechanism and role in oogenesis. Mol Biol Cell [Epub ahead of print]. PubMed ID: 25788286
During Drosophila development, the formin actin nucleator Cappuccino (Capu) helps build a cytoplasmic actin mesh throughout the oocyte. Loss of Capu leads to female sterility presumably because polarity determinants fail to localize properly in the absence of the mesh. To gain deeper insight into how Capu builds this actin mesh, this study systematically characterized seven capu alleles, which have missense mutations in Capu's formin homology 2 (FH2) domain. It reports that all seven alleles had deleterious effects on fly fertility and the actin mesh in vivo, but had strikingly different effects on Capu's biochemical activity in vitro. Using a combination of bulk and single filament actin-assembly assays, the alleles were found to differentially affected Capu's ability to nucleate and processively elongate actin filaments. They also identified a unique "loop" in the lasso region of Capu's FH2 domain. Removing this loop enhanced Capu's nucleation, elongation, and F-actin bundling activities in vitro. Together, this analysis of the loop and the seven missense mutations provides mechanistic insight into formin function in general and Capu's role in the Drosophila oocyte in particular.

Chow, C.Y., Avila, F.W., Clark, A.G. and Wolfner, M.F. (2015). Induction of excessive endoplasmic reticulum stress in the Drosophila male accessory gland results in infertility. PLoS One 10: e0119386. PubMed ID: 25742606
Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the lumen of the ER. A cell responds to ER stress with the unfolded protein response (UPR), a complex program of transcriptional and translational changes aimed at clearing misfolded proteins. Secretory tissues and cells are particularly well adapted to respond to ER stress because their function requires high protein production and secretory load. The insect male accessory gland (AG) is a secretory tissue involved in male fertility. The AG secretes many seminal fluid proteins (SFPs) essential for male reproduction. Among adult Drosophila tissues, this study finds that genes upregulated by ER stress are most highly expressed in the AG, suggesting that the AG is already undergoing high levels of ER stress due to its normal secretory functions. It was hypothesized that induction of excessive ER stress in the AG above basal levels, would perturb normal function and provide a genetic tool for studying AG and SFP biology. To test this, excessive ER stress was they genetically induced in the AG by conditional 1) expression of a misfolded protein or 2) knockdown of the UPR regulatory protein, BiP (Heat shock 70-kDa protein cognate 3). Both genetic manipulations induced excessive ER stress in the AG, as indicated by the increase in Xbp1 splicing, a marker of ER stress. Both models resulted in a large decrease in or loss of SFP production and male infertility. Sperm production, motility, and transfer appeared unaffected. The induction of strong ER stress in the insect male AG may provide a simple way for studying or manipulating male fertility, as it eliminates AG function while preserving sperm production.

Inaba, M., Venkei, Z.G. and Yamashita, Y.M. (2015). The polarity protein Baz forms a platform for the centrosome orientation during asymmetric stem cell division in the Drosophila male germline. Elife [Epub ahead of print]. PubMed ID: 25793442
Many stem cells divide asymmetrically in order to balance self-renewal with differentiation. The essence of asymmetric cell division (ACD) is the polarization of cells and subsequent division, leading to unequal compartmentalization of cellular/extracellular components that confer distinct cell fates to daughter cells. Because precocious cell division before establishing cell polarity would lead to failure in ACD, these two processes must be tightly coupled; however, the underlying mechanism is poorly understood. In Drosophila male germline stem cells, ACD is prepared by stereotypical centrosome positioning. The centrosome orientation checkpoint (COC) further serves to ensure ACD by preventing mitosis upon centrosome misorientation. This study shows that Bazooka (Baz) provides a platform for the correct centrosome orientation and that Baz-centrosome association is the key event that is monitored by the COC. This work provides a foundation for understanding how the correct cell polarity may be recognized by the cell to ensure productive ACD.

Friday, March 27th

Brás-Pereira, C., Casares, F. and Janody, F. (2015). The retinal determination gene dachshund restricts cell proliferation by limiting the activity of the Homothorax-Yorkie complex. Development [Epub ahead of print]. PubMed ID: 25790852
The Drosophila transcriptional co-activator protein Yorkie and its vertebrate orthologs YAP and TAZ are potent oncogenes, whose activity is normally kept in check by the upstream Hippo kinase module. Upon its translocation into the nucleus, Yorkie forms complexes with several tissue-specific DNA-binding partners, which help to define the tissue-specific target genes of Yorkie. In the progenitor cells of the eye imaginal disc, the DNA-binding transcription factor Homothorax is required for Yorkie-promoted proliferation and survival through regulation of the bantam microRNA (miRNA). The transit from proliferating progenitors to cell cycle quiescent precursors is associated with the progressive loss of Homothorax and gain of Dachshund, a nuclear protein related to the Sno/Ski family of co-repressors. This study has identified Dachshund as an inhibitor of Homothorax-Yorkie-mediated cell proliferation. Loss of dachshund induced Yorkie-dependent tissue overgrowth. Conversely, overexpressing dachshund inhibited tissue growth, prevented Yorkie or Homothorax-mediated cell proliferation of disc epithelia and restricted the transcriptional activity of the Yorkie-Homothorax complex on the bantam enhancer in Drosophila cells. In addition, Dachshund collaborated with the Decapentaplegic receptor Thickveins to repress Homothorax and Cyclin B expression in quiescent precursors. The antagonistic roles of Homothorax and Dachshund in Yorkie activity, together with their mutual repression, ensured that progenitor and precursor cells were under distinct proliferation regimes. Based on the crucial role of the human dachshund homolog DACH1 in tumorigenesis, this work suggests that DACH1 might prevent cellular transformation by limiting the oncogenic activity of YAP and/or TAZ.

Firdaus, H., Mohan, J., Naz, S., Arathi, P., Ramesh, S. R. and Nongthomba, U. (2015). A cis-regulatory mutation in Troponin-I of Drosophila reveals the importance of proper stoichiometry of structural proteins during muscle assembly. Genetics [Epub ahead of print]. PubMed ID: 25747460
Rapid and high wing beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations which have been instrumental in the identification of many proteins and residues, important for muscle assembly, function and disease. In this paper, molecular-genetic characterization is presented of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). fliH is shown to be a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the Troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of Troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms which are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. These results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications in the identification of mutations and disease phenotypes in other species including humans.

Shen, J., Eichinger, F., Michalopoulou, T., Yao, C.K., Chang, C.H., Lin, S.H., Sun, Y.H. and Pflugfelder, G.O. (2015). Optomotor-blind negatively regulates Drosophila eye development by blocking Jak/STAT signaling. PLoS One 10: e0120236. PubMed ID: 25781970
Organ formation requires a delicate balance of positive and negative regulators. In Drosophila eye development, wingless (wg) is expressed at the lateral margins of the eye disc and serves to block retinal development. The T-box gene optomotor-blind (omb) is expressed in a similar pattern and is regulated by Wg. Omb mediates part of Wg activity in blocking eye development. Omb exerts its function primarily by blocking cell proliferation. These effects occurred predominantly in the ventral margin. These results suggest that the primary effect of Omb is the blocking of Jak/STAT signaling by repressing transcription of upd which encodes the Jak receptor ligand Unpaired.

Tsai, Y.C., Grimm, S., Chao, J.L., Wang, S.C., Hofmeyer, K., Grifoni, D., Sollazzo, M., Fontana, E., Froldi, F. and Pession, A. (2015). Multiple strategies of oxygen supply in Drosophila malignancies identify tracheogenesis as a novel cancer hallmark. Sci Rep 5: 9061. PubMed ID: 25762498
Angiogenesis is the term used to describe all the alterations in blood vessel growth induced by a tumour mass following hypoxic stress. The occurrence of multiple strategies of vessel recruitment favours drug resistance, greatly complicating the treatment of certain tumours. In Drosophila, oxygen is conveyed to the internal organs by the tracheal system, a closed tubular network whose role in cancer growth is so far unexplored. This study found that, as observed in human cancers, Drosophila malignant cells suffer from oxygen shortage, release pro-tracheogenic factors, co-opt nearby vessels and get incorporated into the tracheal walls. It was also found that the parallelisms observed in cellular behaviours are supported by genetic and molecular conservation. Finally, this study identified a molecular circuitry associated with the differentiation of cancer cells into tracheal cells. In summary, these findings identify tracheogenesis as a novel cancer hallmark in Drosophila, further expanding the power of the fly model in cancer research.

Thursday, March 26th

Legent, K., Liu, H.H. and Treisman, J.E. (2015). Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation. Development [Epub ahead of print]. PubMed ID: 25790850
Endocytic trafficking of signaling receptors is an important mechanism for limiting signal duration. Components of the Endosomal Sorting Complexes Required for Transport (ESCRT), which target ubiquitylated receptors to intra-lumenal vesicles (ILVs) of multivesicular bodies, are thought to terminate signaling by the epidermal growth factor receptor (EGFR) and direct it for lysosomal degradation. In a genetic screen for mutations that affect Drosophila eye development, this study identified an allele of Vacuolar protein sorting 4 (Vps4), which encodes an AAA ATPase that interacts with the ESCRT-III complex to drive the final step of ILV formation. Photoreceptors were largely absent from Vps4 mutant clones in the eye disc, and even when cell death was genetically prevented, the mutant R8 photoreceptors that developed failed to recruit surrounding cells to differentiate as R1-R7 photoreceptors. This recruitment required EGFR signaling, suggesting that loss of Vps4 disrupted the EGFR pathway. In imaginal disc cells mutant for Vps4, EGFR and other receptors accumulated in endosomes and EGFR target genes were not expressed; epistasis experiments placed the function of Vps4 at the level of the receptor. Surprisingly, Vps4 was required for EGFR signaling even in the absence of Shibire, the Dynamin that internalizes EGFR from the plasma membrane. In ovarian follicle cells, in contrast, Vps4 did not affect EGFR signaling, although it was still essential for receptor degradation. Taken together, these findings indicate that Vps4 can promote EGFR activity through an endocytosis-independent mechanism.

Zalucki, O. H., Menon, H., Kottler, B., Faville, R., Day, R., Bademosi, A. T., Lavidis, N., Karunanithi, S. and van Swinderen, B. (2015) Syntaxin1A-mediated resistance and hypersensitivity to isoflurane in Drosophila melanogaster. Anesthesiology [Epub ahead of print]. PubMed ID: 25738637
Recent evidence suggests that general anesthetics activate endogenous sleep pathways, yet this mechanism cannot explain the entirety of general anesthesia. General anesthetics could disrupt synaptic release processes, as previous work in Caenorhabditis elegans and in vitro cell preparations suggested a role for the soluble NSF attachment protein receptor protein, syntaxin1A, in mediating resistance to several general anesthetics. The authors questioned whether the syntaxin1A-mediated effects found in these reductionist systems reflected a common anesthetic mechanism distinct from sleep-related processes. Using the fruit fly model, Drosophila melanogaster, the relevance of syntaxin1A manipulations to general anesthesia was investigated. Different behavioral and electrophysiological endpoints were used to test the effect of syntaxin1A mutations on sensitivity to isoflurane. Two syntaxin1A mutations were found that confer opposite general anesthesia phenotypes: syxH3-C, a 14-amino acid deletion mutant, is resistant to isoflurane, and syxKARRAA, a strain with two amino acid substitutions, is hypersensitive to the drug. Crucially, these opposing effects are maintained across different behavioral endpoints and life stages. The isoflurane sensitivity of syxH3-C at the larval neuromuscular junction was examined to assess effects on synaptic release. Although isoflurane slightly attenuates synaptic release in wild-type animals, syxH3-C preserves synaptic release in the presence of isoflurane. These results are evidence that volatile general anesthetics target synaptic release mechanisms; in addition to first activating sleep pathways, a major consequence of these drugs may be to decrease the efficacy of neurotransmission.

Kumichel, A., Kapp, K. and Knust, E. (2015) A conserved di-basic motif of Drosophila Crumbs contributes to efficient ER export. Traffic [Epub ahead of print]. PubMed ID: 25753515
The Drosophila type I transmembrane protein Crumbs is an apical determinant required for the maintenance of apico-basal epithelial cell polarity. The level of Crumbs at the plasma membrane is crucial, but how it is regulated is poorly understood. A genetic screen for regulators of Crumbs protein trafficking identified Sar1, the core component of the coat protein complex II (COPII) transport vesicles. sar1 mutant embryos show a reduced plasma membrane localization of Crumbs, a defect similar to that observed in haunted and ghost mutant embryos, which lack Sec23 and Sec24CD, respectively. By pulse-chase assays in Drosophila Schneider cells and analysis of protein transport kinetics based on Endoglycosidase H resistance an RNKR motif was identified in Crumbs that contributes to efficient ER export. The motif identified fits the highly conserved di-basic RxKR motif and mediates interaction with Sar1. The RNKR motif is also required for plasma membrane delivery of transgene-encoded Crumbs in epithelial cells of Drosophila embryos. These data are the first to show that a di-basic motif acts as a signal for ER exit of a type I plasma membrane protein in a metazoan organism.

Fox, R. M. and Andrew, D. J. (2015) Changes in organelle position and epithelial architecture associated with loss of CrebA. Biol Open [Epub ahead of print]. PubMed ID: 25681391
Drosophila CrebA facilitates high-level secretion by transcriptional upregulation of the protein components of the core secretory machinery. In CrebA mutant embryos, both salivary gland (SG) morphology and epidermal cuticle secretion are abnormal, phenotypes similar to those observed with mutations in core secretory pathway component genes. This study examined the cellular defects associated with CrebA loss in the SG epithelium. Apically localized secretory vesicles are smaller and less abundant, consistent with overall reductions in secretion. Unexpectedly, global mislocalization of cellular organelles and excess membrane accumulation in the septate junctions (SJs) are also observed. Whereas mutations in core secretory pathway genes lead to organelle localization defects similar to those of CrebA mutants, they have no effect on SJ-associated membrane. Mutations in tetraspanin genes (see Tsp66E), which are normally repressed by CrebA, have mild defects in SJ morphology that are rescued by simultaneous CrebA loss. Correspondingly, removal of several tetraspanins gives partial rescue of the CrebA SJ phenotype, supporting a role for tetraspanins in SJ organization.

Wednesday, March 25th

Zhu, M., Li, X., Tian, X. and Wu, C. (2015). Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25743185
PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. This study shows that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue could also be achieved if Mask knock-down was induced in parkin adult flies when the mitochondrial dystrophy was already manifested. Mask genetically interacted with Parkin to modulate mitochondrial morphology and negatively regulated the recruitment of Parkin to mitochondria. Loss of Mask activity promoted co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway was required for the rescue of parkin mutant defects by mask loss of function. Together, these data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions.

Tabuchi, M., Lone, S. R., Liu, S., Liu, Q., Zhang, J., Spira, A. P. and Wu, M. N. (2015) Sleep interacts with Aβ to modulate intrinsic neuronal excitability. Curr Biol [Epub ahead of print]. PubMed ID: 25754641
Using a Drosophila model of AD, evidence is provided suggesting that changes in neuronal excitability underlie the effects of sleep loss on AD pathogenesis. β-amyloid (Aβ) accumulation leads to reduced and fragmented sleep, while chronic sleep deprivation increases Aβ burden. Moreover, enhancing sleep reduces Aβ deposition. Increasing neuronal excitability phenocopies the effects of reducing sleep on Aβ, and decreasing neuronal activity blocks the elevated Aβ accumulation induced by sleep deprivation. At the single neuron level, this study found that chronic sleep deprivation, as well as Aβ expression, enhances intrinsic neuronal excitability. Importantly, these data reveal that sleep loss exacerbates Aβ-induced hyperexcitability and suggest that defects in specific K+ currents underlie the hyperexcitability caused by sleep loss and Aβ expression. Finally, this study showed that feeding levetiracetam, an anti-epileptic medication, to Aβ-expressing flies suppresses neuronal excitability and significantly prolongs their lifespan. These findings directly link sleep loss to changes in neuronal excitability and Aβ accumulation and further suggest that neuronal hyperexcitability is an important mediator of Aβ toxicity. Taken together, these data provide a mechanistic framework for a positive feedback loop, whereby sleep loss and neuronal excitation accelerate the accumulation of Aβ, a key pathogenic step in the development of AD.

Besson, M. T., Alegria, K., Garrido-Gerter, P., Barros, L. F. and Lievens, J. C. (2015). Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model. PLoS One 10: e0118765. PubMed ID: 25761110
This study reports the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93). Overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP) impacts HD was investigated. To mimic increased glycolytic flux, phosphofructokinase (PFK), which catalyzes an irreversible step in glycolysis, was overexpressed. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, this study showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. This study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3 protective effects, whereas, in addition, the PPP provides increased protection to oxidative stress.

Yao, Y., Cui, X., Al-Ramahi, I., Sun, X., Li, B., Hou, J., Difiglia, M., Palacino, J., Wu, Z. Y., Ma, L., Botas, J. and Lu, B. (2015) A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity. Elife 4. PubMed ID: 25738228
Huntington's disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. This study reports a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 (knockdown of DopEcR in flies) suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models (flies overexpressing mutant Htt protein). Thus, this discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.

Tuesday, March 24th

Wang, H., Brust-Mascher, I. and Scholey, J. M. (2015). The microtubule crosslinker Feo controls the midzone stability, motor composition and elongation of the anaphase B spindle in Drosophila embryos. Mol Biol Cell [Epub ahead of print]. PubMed ID: 25694445
Chromosome segregation during anaphase depends on chromosome-to-pole motility and pole-to-pole separation. It is proposed that in Drosophila embryos, the latter process (anaphase B) depends on a persistent kinesin-5-generated interpolar (ip) microtubule (MT) sliding filament mechanism that "engages" to push apart the spindle poles when poleward flux is turned off. This study investigated the contribution of the midzonal, anti-parallel MT-crosslinking non-motor MAP, Feo, to this "slide-and-flux-or-elongate" mechanism. While Feo homologues in other systems enhance the midzone localization of the MT-MT crosslinking motors, kinesin-4, -5 and -6, the midzone localization of these motors is respectively enhanced, reduced and unaffected by Feo. Strikingly, kinesin-5 localizes all along ipMTs of the anaphase B spindle in the presence of Feo, including at the midzone, but the antibody-induced dissociation of Feo increases kinesin-5 association with the midzone which becomes abnormally narrow, leading to impaired anaphase B and incomplete chromosome segregation. Thus, although Feo and kinesin-5 both preferentially crosslink MTs into anti-parallel polarity patterns, kinesin-5 cannot substitute for loss of Feo function. It is proposed that Feo controls the organization, stability and motor composition of anti-parallel ipMTs at the midzone, thereby facilitating the kinesin-5-driven sliding filament mechanism underlying proper anaphase B spindle elongation and chromosome segregation.
Beaven, R., Dzhindzhev, N. S., Qu, Y., Hahn, I., Dajas-Bailador, F., Ohkura, H. and Prokop, A. (2015). Drosophila CLIP-190 and mammalian CLIP-170 display reduced microtubule plus end association in the nervous system. Mol Biol Cell [Epub ahead of print]. PubMed ID: 25694447
Axon guidanceAxons act like cables, electrically wiring the nervous system. Polar bundles of microtubules (MTs) form their backbones and drive their growth. Plus-end tracking proteins (+TIPs) regulate MT growth dynamics and directionality at their plus ends. However, current knowledge about +TIP functions, mostly derived from work in vitro and in non-neuronal cells, may not necessarily apply to the very different context of axonal MTs. For example, the CLIP family of +TIPs are known MT polymerization promoters in non-neuronal cells. However, this study shows that neither Drosophila CLIP-190 nor mammalian CLIP-170 are prominent MT plus end trackers in neurons, which it is proposed is due to low plus end affinity of the CAP-Gly domain-containing N-terminus and intramolecular inhibition through the C-terminus. Instead, both CLIP-190 and CLIP-170 form F-actin-dependent patches in growth cones, mediated by binding of the coiled-coil domain to Myosin-VI. Since loss-of-function analyses in vivo and in culture failed to reveal axonal roles for CLIP-190, even in double-mutant combinations with four other +TIPs, it is proposed that CLIP-190 and -170 are not essential axon extension regulators. These findings demonstrate that +TIP functions known from non-neuronal cells do not necessarily apply to the regulation of the very distinct MT networks in axons.

Roth, M., Roubinet, C., Iffländer, N., Ferrand, A. and Cabernard, C. (2015). Asymmetrically dividing Drosophila neuroblasts utilize two spatially and temporally independent cytokinesis pathways. Nat Commun 6: 6551. PubMed ID: 25791062
Precise cleavage furrow positioning is required for faithful chromosome segregation and cell fate determinant distribution. In most metazoan cells, contractile ring placement is regulated by the mitotic spindle through the centralspindlin complex, and potentially also the chromosomal passenger complex (CPC). Drosophila neuroblasts, asymmetrically dividing neural stem cells, but also other cells, utilize both spindle-dependent and spindle-independent cleavage furrow positioning pathways. However, the relative contribution of each pathway towards cytokinesis is currently unclear. This study reports that in Drosophila neuroblasts, the mitotic spindle, but not polarity cues, controls the localization of the CPC component Survivin. They also showed that Survivin and the mitotic spindle were required to stabilize the position of the cleavage furrow in late anaphase and to complete furrow constriction. With ​Survivin this study has thus identified a specific spindle-dependent cytokinesis component, not intersecting with the polarity-dependent pathway. These results support the model that two spatially and temporally separate pathways control different key aspects during asymmetric cell division, ensuring correct cell fate determinant segregation and neuroblast self-renewal.

Okumura, T., et al. (2015). Class I myosins have overlapping and specialized junctions in left-right asymmetric development in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 25659376
The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left-right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. This study generated class I myosin double and triple mutants to address this issue. The triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Theses studies also revealed differential co-localization of the class I myosins with F-actin in the brush border of intestinal enterocytes.

Monday, March 23rd

Le Bourg, E. and Massou, I. (2015) Fasting increases survival to cold in FOXO, DIF, autophagy mutants and in other genotypes of Drosophila melanogaster. Biogerontology [Epub ahead of print]. PubMed ID: 25663303
Fasting increases survival to a severe cold stress in young and middle-aged wild-type flies, this effect being lowered or absent at old age. As an attempt to determine the mechanisms of this effect, genes involved in metabolism (dFOXO), autophagy (Atg7), innate immunity (Dif 1), and resistance to cold (Frost) were studied. The 12 mutant, RNAi and control lines tested in this study displayed an increased survival to cold after fasting. This shows that fasting has a robust effect on survival to cold in many genotypes, but the mechanism of this effect remains unknown. This mechanism does not seem to be linked to metabolic pathways often considered to play a critical role in ageing and longevity determinations (insulin/insulin-like growth factor-1 pathway and autophagy).

Shklover, J., Levy-Adam, F. and Kurant, E. (2015) The role of Drosophila TNF Eiger in developmental and damage-induced neuronal apoptosis. FEBS Lett [Epub ahead of print]. PubMed ID: 25754009
Eiger, the sole Drosophila TNF-alpha homolog, causes ectopic apoptosis through JNK pathway activation. Yet, its role in developmental apoptosis remains unclear. eiger mutant flies are viable and fertile but display compromised elimination of oncogenic cells and extracellular bacteria. This study shows that Eiger, specifically expressed in embryonic neurons and glia, is not involved in developmental neuronal apoptosis or in apoptotic cell clearance. Instead, evidence is provided that Eiger is required for damage-induced apoptosis in the embryonic CNS through regulation of the pro-apoptotic gene hid independently of the JNK pathway. This study thus reveals a new requirement for Eiger in eliminating damaged cells during development.

Tang, H. L., Tang, H. M., Fung, M. C. and Hardwick, J. M. (2015) In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity. Sci Rep 5: 9015. PubMed ID: 25757939
The discovery that mammalian cells can survive late-stage apoptosis challenges the general assumption that active caspases are markers of impending death. However, tools have not been available to track healthy cells that have experienced caspase activity at any time in the past. Therefore, to determine if cells in whole animals can undergo reversal of apoptosis, known as anastasis, a dual color CaspaseTracker system for Drosophila was developed to identify cells with ongoing or past caspase activity. Transient exposure of healthy females to environmental stresses such as cold shock or starvation activated the CaspaseTracker coincident with caspase activity and apoptotic morphologies in multiple cell types of developing egg chambers. Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation. In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons. The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development.

Zirin, J., Nieuwenhuis, J., Samsonova, A., Tao, R. and Perrimon, N. (2015) Regulators of autophagosome formation in Drosophila muscles. PLoS Genet 11: e1005006. PubMed ID: 25692684
Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. This study used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) an autophagic response was induced that was fully suppressible by knockdown of core ATG genes. D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network were screened, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 (CG7112) and Rab3Gap1 (CG31935). The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.

Sunday, March 22nd

Shapiro-Kulnane, L., Smolko, A. E. and Salz, H. K. (2015) Maintenance of Drosophila germline stem cell sexual identity in oogenesis and tumorigenesis. Development 142: 1073-1082. PubMed ID: 25758221
Adult stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. In Drosophila females, germline stem cells (GSCs) require Sex lethal (Sxl) to exit the stem cell state and to enter the differentiation pathway. Without Sxl GSCs do not differentiate and instead form tumors. Previous studies have shown that these tumors are not caused by a failure in the self-renewal/differentiation switch. This study shows that Sxl is also necessary for the cell-autonomous maintenance of germ cell female identity and demonstrates that tumors are caused by the acquisition of male characteristics. Germ cells without Sxl protein exhibit a global derepression of testis genes, including Phf7, a male germline sexual identity gene. Phf7 is a key effector of the tumor-forming pathway, as it is both necessary and sufficient for tumor formation. In the absence of Sxl protein, inappropriate Phf7 expression drives tumor formation through a cell-autonomous mechanism that includes sex-inappropriate activation of Jak/Stat signaling. Remarkably, tumor formation requires a novel response to external signals emanating from the GSC niche, highlighting the importance of interactions between mutant cells and the surrounding normal cells that make up the tumor microenvironment. Derepression of testis genes, and inappropriate Phf7 expression, is also observed in germ cell tumors arising from the loss of bag of marbles (bam), demonstrating that maintenance of female sexual identity requires the concerted actions of Sxl and bam. This work reveals that GSCs must maintain their sexual identity as they are reprogrammed into a differentiated cell, or risk tumorigenesis.

Zeng, X., Han, L., Singh, S. R., Liu, H., Neumuller, R. A., Yan, D., Hu, Y., Liu, Y., Liu, W., Lin, X. and Hou, S. X. (2015) Genome-wide RNAi screen identifies networks involved in intestinal stem cell regulation in Drosophila. Cell Rep 10: 1226-1238. PubMed ID: 25704823
The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs) in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, a genome-wide transgenic RNAi screen was performed in adult Drosophila intestine, and 405 genes were identified that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, functional networks were further developed that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC) lineage differentiation, and ISC-to-enteroendocrine (EE) lineage differentiation. RNAi-mediated knockdowns of genes in the Notch-signaling network resulted in either expansion of both ISCs and EE cells. Osa-containing SWI/SNF chromatin-remodeling complex was found to regulate ISC-to-EC lineage differentiation by controlling Dl transcription and EE cell lineage differentiation by controlling ase transcription. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, factors related to basic stem cell cellular processes were found that are commonly required in all stem cells, and stem-cell-specific, niche-related signals were found that were required only in the unique stem cell type. These findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.

Fuwa, T.J., Kinoshita, T., Nishida, H. and Nishihara, S. (2015). Reduction of T antigen causes loss of hematopoietic progenitors in Drosophila through the inhibition of filopodial extensions from the hematopoietic niche. Dev Biol [Epub ahead of print]. PubMed ID: 25779703
Hematopoietic stem cells (HSCs) are present in hematopoietic organs and differentiate into mature blood cells as required. Defective HSCs have been implicated in the human autoimmune disease Tn syndrome, which results from the failure of the core 1 β1,3-galactosyltransferase 1 enzyme (C1β3GalT1) to synthesize T antigen. In both mice and humans, a reduced level of T antigen is associated with a reduction in blood cell numbers. However, the precise roles of T antigen in hematopoiesis are unknown. This study shows that the Drosophila T antigen, supplied by plasmatocytes, is essential for the regulation of HSCs. T antigen appeared to be an essential factor in maintaining the extracellular environment to support filopodial extensions from niches that are responsible for transmitting signaling molecules to maintain the HSCs. Additionally, the clotting factor, hemolectin, disrupted the hemolymph environment of C1β3GalT1- mutants. This study identified a novel mucin function for the regulation of HSCs that may be conserved in other species

Tian, A., Shi, Q., Jiang, A., Li, S., Wang, B. and Jiang, J. (2015) Injury-stimulated Hedgehog signaling promotes regenerative proliferation of Drosophila intestinal stem cells. J Cell Biol 208: 807-819. PubMed ID: 25753035
Many adult tissues are maintained by resident stem cells that elevate their proliferation in response to injury. The regulatory mechanisms underlying regenerative proliferation are still poorly understood. This study shows that injury induces Hedgehog (Hh) signaling in enteroblasts (EBs) to promote intestinal stem cell (ISC) proliferation in Drosophila melanogaster adult midgut. Elevated Hh signaling by patched (ptc) mutations drove ISC proliferation non-cell autonomously. Inhibition of Hh signaling in the ISC lineage compromised injury-induced ISC proliferation but had little if any effect on homeostatic proliferation. Hh signaling acted in EBs to regulate the production of Upd2, which activated the JAK-STAT pathway to promote ISC proliferation. Furthermore, Hh signaling was shown to be stimulated by dextran sulfate sodium (DSS) through the JNK pathway and that inhibition of Hh signaling in EBs prevented DSS-stimulated ISC proliferation. Hence, this study uncovers a JNK-Hh-JAK-STAT signaling axis in the regulation of regenerative stem cell proliferation.

Saturday, March 21st

Yamada, R., Deshpande, S. A., Bruce, K. D., Mak, E. M. and Ja, W. W. (2015) Microbes promote amino acid harvest to rescue undernutrition in Drosophila. Cell Rep [Epub ahead of print]. PubMed ID: 25683709
Microbes play an important role in the pathogenesis of nutritional disorders such as protein-specific malnutrition. However, the precise contribution of microbes to host energy balance during undernutrition is unclear. This study shows that Issatchenkia orientalis, a fungal microbe isolated from field-caught Drosophila melanogaster, promotes amino acid harvest to rescue the lifespan of undernourished flies. Using radioisotope-labeled dietary components (amino acids, nucleotides, and sucrose) to quantify nutrient transfer from food to microbe to fly, this study demonstrated that I. orientalis extracts amino acids directly from nutrient-poor diets and increases protein flux to the fly. This microbial association restores body mass, protein, glycerol, and ATP levels and phenocopies the metabolic profile of adequately fed flies. This study uncovers amino acid harvest as a fundamental mechanism linking microbial and host metabolism, and highlights Drosophila as a platform for quantitative studies of host-microbe relationships.

Grifoni, D., Sollazzo, M., Fontana, E., Froldi, F. and Pession, A. (2015) Multiple strategies of oxygen supply in Drosophila malignancies identify tracheogenesis as a novel cancer hallmark. Sci Rep 5: 9061. PubMed ID: 25762498
Angiogenesis is the term used to describe all the alterations in blood vessel growth induced by a tumour mass following hypoxic stress. The occurrence of multiple strategies of vessel recruitment favours drug resistance, greatly complicating the treatment of certain tumours. In Drosophila, oxygen is conveyed to the internal organs by the tracheal system, a closed tubular network whose role in cancer growth is so far unexplored. This study found that, as observed in human cancers, Drosophila malignant cells suffer from oxygen shortage, release pro-tracheogenic factors, co-opt nearby vessels and get incorporated into the tracheal walls. It was also found that the parallelisms observed in cellular behaviours are supported by genetic and molecular conservation. Finally, a molecular circuitry was identfied associated with the differentiation of cancer cells into tracheal cells. In summary, these findings identify tracheogenesis as a novel cancer hallmark in Drosophila, further expanding the power of the fly model in cancer research.

Neckameyer, W.S. and R. Nieto-Romero, A. (2015). Response to stress in Drosophila is mediated by gender, age and stress paradigm. Stress [Epub ahead of print]. PubMed ID: 25783197
All living organisms must maintain equilibrium in response to internal and external challenges within their environment. Changes in neural plasticity (alterations in neuronal populations, dendritic remodeling, and synaptic turnover) are critical components of the homeostatic response to stress, which has been strongly implicated in the onset of affective disorders. However, stress is differentially perceived depending on the type of stress and its context, as well as genetic background, age and sex; therefore, an individual's maintenance of neuronal homeostasis must differ depending upon these variables. This study established Drosophila as a model to analyze homeostatic responses to stress. Sexually immature and mature females and males from an isogenic wild-type strain raised under controlled environmental conditions were exposed to four reproducible and high-throughput translatable stressors to facilitate the analysis of a large number of animals for direct comparisons. These animals were assessed in an open-field arena, in a light-dark box, and in a forced swim test, as well as for sensitivity to the sedative effects of ethanol. These studies established that immature and mature females and males represent behaviorally distinct populations under control conditions as well as after exposure to different stressors. Therefore, the neural substrates mediating the stress response must be differentially expressed depending upon the hormonal status of the brain. In addition, an adaptive response to a given stressor in one paradigm was not predictive for outcomes in other paradigms.

MacMillan, H. A., Andersen, J. L., Loeschcke, V. and Overgaard, J. (2015) Sodium distribution predicts the chill tolerance of Drosophila melanogaster raised in different thermal conditions. Am J Physiol Regul Integr Comp Physiol [Epub ahead of print]. PubMed ID: 25761700
Many insects, including the model holometabolous insect, Drosophila melanogaster, display remarkable plasticity in chill tolerance in response to the thermal environment experienced during development or as adults. At low temperatures many insects lose the ability to regulate Na+ balance, which is suggested to cause a secondary loss of hemolymph water to the tissues and gut lumen that concentrates the K+ remaining in the hemolymph. The resultant increase in extracellular [K+] inhibits neuromuscular excitability and is proposed to cause cellular apoptosis and injury. The present study investigates if and how variation in chill tolerance induced through developmental and adult cold acclimation is associated with changes in Na+, water and K+ balance. Developmental and adult cold acclimation improved the chilling tolerance of D. melanogaster in an additive manner. In agreement with the proposed model, these effects were intimately related to differences in Na+ distribution prior to cold exposure, such that chill tolerant flies had low hemolymph [Na+] while intracellular [Na+] was similar among treatment groups. The low hemolymph Na+ of cold acclimated flies allowed them to maintain hemolymph volume, prevent hyperkalemia, and avoid injury following chronic cold exposure. These findings extend earlier observations of hemolymph volume disruption during cold exposure to the most ubiquitous model insect (D. melanogaster), highlight shared mechanisms of developmental and adult thermal plasticity and provide strong support for ionoregulatory failure as a central mechanism of insect chill susceptibility.

Friday, March 20th

Kakugawa, S., Langton, P. F., Zebisch, M., Howell, S. A., Chang, T. H., Liu, Y., Feizi, T., Bineva, G., O'Reilly, N., Snijders, A. P., Jones, E. Y. and Vincent, J. P. (2015) Notum deacylates Wnt proteins to suppress signalling activity. Nature 519: 187-192. PubMed ID: 25731175
Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Genetic evidence is provided in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum that probably help Notum to co-localize with Wnt proteins. At the active site of human and Drosophila Notum, a large hydrophobic pocket was identified that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.

Zhang, X., Cheong, S. M., Amado, N. G., Reis, A. H., MacDonald, B. T., Zebisch, M., Jones, E. Y., Abreu, J. G. and He, X. (2015) Notum is required for neural and head induction via Wnt deacylation, oxidation, and inactivation. Dev Cell 32(6): 719-30. PubMed ID: 25771893
. Secreted Wnt morphogens (see Drosophila Wingless) are essential for embryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/β hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. This study reports that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus, Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naive ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that Notum is a prerequisite for the "default" neural fate and that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development.

Bleichert, F., Botchan, M. R. and Berger, J. M. (2015) Crystal structure of the eukaryotic origin recognition complex. Nature [Epub ahead of print]. PubMed ID: 25762138
Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. This paper describes the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1-5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90 degrees for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, these data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

Klapholz, B., Herbert, S. L., Wellmann, J., Johnson, R., Parsons, M. and Brown, N. H. (2015) Alternative mechanisms for Talin to mediate integrin function. Curr Biol [Epub ahead of print]. PubMed ID: 25754646
Cell-matrix adhesion is essential for building animals, promoting tissue cohesion, and enabling cells to migrate and resist mechanical force. Talin is an intracellular protein that is critical for linking integrin extracellular-matrix receptors (see Myospheroid) to the actin cytoskeleton. A key question raised by structure-function studies is whether talin, which is critical for all integrin-mediated adhesion, acts in the same way in every context. This study shows that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development. The partial function of some mutant talins requires vinculin, indicating that recruitment of vinculin allows talin to duplicate its own activities. The different requirements are best explained by alternative mechanisms of talin function, with talin using one or both of its integrin-binding sites. These alternatives were confirmed by showing that the proximity between the second integrin-binding site and integrins differs, suggesting that talin adopts different orientations relative to integrins. Finally, it was shown that vinculin and actomyosin activity help change talin's orientation. These findings demonstrate that the mechanism of talin function differs in each developmental context examined. The different arrangements of the talin molecule relative to integrins suggest that talin is able to sense different force vectors, either parallel or perpendicular to the membrane. This provides a paradigm for proteins whose apparent uniform function is in fact achieved by a variety of distinct mechanisms involving different molecular architectures.

Thursday, March 19th

Huetteroth, W., Perisse, E., Lin, S., Klappenbach, M., Burke, C. and Waddell, S. (2015). Sweet taste and nutrient value subdivide rewarding dopaminergic neurons in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 25728694
Dopaminergic neurons provide reward learning signals in mammals and insects. Recent work in Drosophila has demonstrated that water-reinforcing dopaminergic neurons are different to those for nutritious sugars. This study tested whether the sweet taste and nutrient properties of sugar reinforcement further subdivide the fly reward system. They found that dopaminergic neurons expressing the OAMB octopamine receptor specifically conveyed the short-term reinforcing effects of sweet taste. These dopaminergic neurons projected to the β'2 and γ4 regions of the mushroom body lobes. In contrast, nutrient-dependent long-term memory required different dopaminergic neurons that project to the γ5b regions, and it could be artificially reinforced by those projecting to the β lobe and adjacent α1 region. Surprisingly, whereas artificial implantation and expression of short-term memory occurred in satiated flies, formation and expression of artificial long-term memory required flies to be hungry. These studies suggest that short-term and long-term sugar memories have different physiological constraints. They also demonstrate further functional heterogeneity within the rewarding dopaminergic neuron population.

Wu, C.L., Fu, T.F., Chou, Y.Y. and Yeh, S.R. (2015). A single pair of neurons modulates egg-laying decisions in Drosophila. PLoS One 10: e0121335. PubMed ID: 25781933
Animals have to judge environmental cues and choose the most suitable option for them from many different options. Female fruit flies selecting an optimum site to deposit their eggs is a biologically important reproductive behavior. When given the direct choice between ovipositing their eggs in a sucrose-containing medium or a caffeine-containing medium, female flies prefer the latter. However, the neural circuits and molecules that regulate this decision-making processes during egg-laying site selection remain poorly understood. The present study found that amnesiac (amn) mutant flies show significant defects in egg-laying decisions, and such defects can be reversed by expressing the wild-type amn transgene in two dorsal paired medial (DPM) neurons in the brain. Silencing neuronal activity with an inward rectifier potassium channel (Kir2.1) in DPM neurons also impaired egg-laying decisions. Finally, the activity in mushroom body αβ neurons was required for the egg-laying behavior, suggesting a possible "DPM-αβ neurons" brain circuit modulating egg-laying decisions. These results highlight the brain circuits and molecular mechanisms of egg-laying decisions in Drosophila.

Ji, F. and Zhu, Y. (2015) A novel assay reveals hygrotactic behavior in Drosophila. PLoS One 10: e0119162. PubMed ID: 25738801
Humidity is one of the most important factors that determines the geographical distribution and survival of terrestrial animals. The ability to detect variation in humidity is conserved across many species. This study established a novel behavioral assay that revealed the thirsty Drosophila exhibits strong hygrotactic behavior, and it can locate water by detecting humidity gradient. In addition, exposure to high levels of moisture was sufficient to elicit proboscis extension reflex behavior in thirsty flies. Furthermore, this study found that the third antennal segment was necessary for hygrotactic behavior in thirsty flies, while the arista, a bristles arising from the third antennal segment, is required for the avoidance of moist air in hydrated flies. These results indicated that two types of hygroreceptor cells exist in Drosophila: one located in the third antennal segment that mediates hygrotactic behavior in thirst status, and the other located in the arista that is responsible for the aversive behavior toward moist air in hydration status. Using a neural silencing screen, this study demonstrated that synaptic output from the mushroom body α/β surface and posterior neurons was required for both hygrotactic behavior and moisture-aversive behavior.

Tschida, K and Bhandawat, V. (2015). Activity in descending dopaminergic neurons represents but is not required for leg movements in the fruit fly Drosophila. Physiol Rep 3: e12322. PubMed ID: 25742959
Modulatory descending neurons (DNs) that link the brain to body motor circuits, including dopaminergic DNs (DA-DNs), are thought to contribute to the flexible control of behavior. Dopamine elicits locomotor-like outputs and influences neuronal excitability in isolated body motor circuits over tens of seconds to minutes, but it remains unknown how and over what time scale DA-DN activity relates to movement in behaving animals. To address this question, this study identified DA-DNs in the Drosophila brain and developed an electrophysiological preparation to record and manipulate the activity of these cells during behavior. They found that DA-DN spike rates were rapidly modulated during a subset of leg movements and scaled with the total speed of ongoing leg movements, whether occurring spontaneously or in response to stimuli. However, activating DA-DNs did not elicit leg movements in intact flies, nor did acute bidirectional manipulations of DA-DN activity affected the probability or speed of leg movements over a time scale of seconds to minutes. These findings indicate that in the context of intact descending control, changes in DA-DN activity are not sufficient to influence ongoing leg movements and open the door to studies investigating how these cells interact with other descending and local neuromodulatory inputs to influence body motor output.

Wednesday, March 18th

Blythe, S. A. and Wieschaus, E. F. (2015) Zygotic genome activation triggers the DNA replication checkpoint at the midblastula transition. Cell 160: 1169-1181. PubMed ID: 25748651
A conserved feature of the midblastula transition (MBT) is a requirement for a functional DNA replication checkpoint to coordinate cell-cycle remodeling and zygotic genome activation (ZGA). This study investigated what triggers this checkpoint during Drosophila embryogenesis.The magnitude of the checkpoint was found to scale with the quantity of transcriptionally engaged DNA. Measuring RNA polymerase II (Pol II) binding at 20 min intervals over the course of ZGA reveals that the checkpoint coincides with widespread de novo recruitment of Pol II that precedes and does not require a functional checkpoint. This recruitment drives slowing or stalling of DNA replication at transcriptionally engaged loci. Reducing Pol II recruitment in zelda mutants both reduces replication stalling and bypasses the requirement for a functional checkpoint. This suggests a model where the checkpoint functions as a feedback mechanism to remodel the cell cycle in response to nascent ZGA.

Srivastava, A. and Dong, Q. (2015). Regulation of a serine protease homolog by the JNK pathway during thoracic development of Drosophila melanogaster. FEBS Open Bio 5: 117-123. PubMed ID: 25737837
The importance of the Jun N-terminal Kinase (JNK) pathway during normal development and tumor invasion has been well documented in Drosophila. This pathway plays important roles in epithelial morphogenesis, wound healing, apoptosis, immunity and regulation of lifespan. However, which downstream molecules facilitate these effects is not very well elucidated. In this study, data are presented on a serine protease homolog (SPH), scarface. These data showed that scarface is under regulatory control of the JNK pathway and that this pathway is both necessary and sufficient for its expression within the context of thoracic development. Consequently, down-regulation of scarface results in a thoracic-cleft phenotype that phenocopies the JNK pathway defect. A possible role of scarface during thoracic development in Drosophila is discussed.

Sopko, R., Lin, Y. B., Makhijani, K., Alexander, B., Perrimon, N. and Bruckner, K. (2015) A systems-level interrogation identifies regulators of Drosophila blood cell number and survival. PLoS Genet 11: e1005056. PubMed ID: 25749252
This paper reports a study of signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR. Using Kc cells, a genome wide RNAi screen for was performed for regulators of cell number in a sensitized, Pvr deficient background. The receptor tyrosine kinase (RTK) Insulin-like receptor (InR) was identified as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp) were identified as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. Common phosphorylation targets of Pvr and InR were defined that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, this analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

Colines, B., Rodríguez, N.C., Hasson, E.R., Carreira, V. and Frankel, N. (2015). Parental age influences developmental stability of the progeny in Drosophila. Proc Biol Sci 282(1803). PubMed ID: 25673675
The stochastic nature of biochemical processes is a source of variability that influences developmental stability. Developmental instability (DI) is often estimated through fluctuating asymmetry (FA), a parameter that deals with within-individual variation in bilateral structures. A relevant goal is to shed light on how environment, physiology and genotype relate to DI, thus providing a more comprehensive view of organismal development. Using Drosophila melanogaster isogenic lines, this study investigated the effect of parental age, parental diet and offspring heterozygosity on DI. The study uncovered a clear relationship between parental age and offspring asymmetry. Asymmetry of the progeny increased concomitantly with parental age. Moreover, enriching the diet of parents mitigated the effect of age on offspring symmetry. Increasing the heterozygosity of the progeny eliminated the effect of parental age on offspring symmetry. Taken together, these results suggest that diet, genotype and age of the parents interact to determine offspring DI in wild populations. These findings provide an avenue to understand the mechanisms underlying DI.

Tuesday, March 17

Soba, P., Han, C., Zheng, Y., Perea, D., Miguel-Aliaga, I., Jan, L. Y. and Jan, Y. N. (2015) The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion. Elife 4. PubMed ID: 25764303
Neurons develop highly stereotyped receptive fields by coordinated growth of their dendrites. Although cell surface cues play a major role in this process, few dendrite specific signals have been identified to date. An in vivo RNAi screen in Drosophila class IV dendritic arborization (C4da) neurons identified the conserved Ret receptor, known to play a role in axon guidance, as an important regulator of dendrite development. The loss of Ret results in severe dendrite defects due to loss of extracellular matrix adhesion, thus impairing growth within a 2D plane. Evidence is provided that Ret interacts with integrins (see Myospheroid) to regulate dendrite adhesion via rac1. In addition, Ret is required for dendrite stability and normal F-actin distribution suggesting it has an essential role in dendrite maintenance. Novel functions are proposed for Ret as a regulator in dendrite patterning and adhesion distinct from its role in axon guidance.

Kanamori, T., Yoshino, J., Yasunaga, K., Dairyo, Y. and Emoto, K. (2015) . Nat Commun 6: 6515. PubMed ID: 25761586
The refinement of neural circuits involves dendrite pruning, a process that removes inappropriate projections that are formed during development. In Drosophila sensory neurons, compartmentalized calcium (Ca(2+)) transients in dendrites act as spatiotemporal cues to trigger pruning, yet how neurons define the dendrites with Ca(2+) transients remains elusive. This study reports that local elevation of endocytic activity contributes to defining dendrites that generate Ca(2+) transients, triggering pruning. In vivo imaging of single dendrites reveals an increase of endocytosis in proximal dendrites that spatially and temporally correlates with dendrite thinning, an early step in pruning tightly coupled with compartmentalized Ca(2+) transients. Two GTPases, Rab5 and dynamin, are required for both the increased endocytic activity and compartmentalized Ca(2+) transients. Further genetic analyses suggest that local endocytosis in proximal dendrites functions cooperatively with global endocytosis-mediated protein degradation pathways to promote dendrite pruning.

Matzat, T., Sieglitz, F., Kottmeier, R., Babatz, F., Engelen, D. and Klambt, C. (2015) Axonal wrapping in the Drosophila PNS is controlled by glia-derived neuregulin homolog Vein. Development. PubMed ID: 25758464
Efficient neuronal conductance requires that axons are insulated by glial cells. For this, glial membranes need to wrap around axons. Invertebrates show a relatively simple extension of glial membranes around the axons, resembling non-myelinated Remak fibers formed by Schwann cells in the mammalian peripheral nervous system. To unravel the molecular pathways underlying differentiation of glial cells that provide axonal wrapping, the genetically amenable Drosophila model is being used. At the end of larval life, the wrapping glia differentiates into very large cells, spanning more than 1 mm of axonal length. The extension around axonal membranes is not influenced by the caliber of the axon or its modality. Using cell type-specific gene knockdown it was shown that the extension of glial membranes around the axons is regulated by an autocrine activation of the EGF receptor through the neuregulin homolog Vein. This resembles the molecular mechanism employed during cell-autonomous reactivation of glial differentiation after injury in mammals. It was further demonstrated that Vein, produced by the wrapping glia, also regulates the formation of septate junctions in the abutting subperineurial glia. Moreover, the wrapping glia indirectly controls the proliferation of the perineurial glia. Thus, the wrapping glia appears center stage to orchestrate the development of the different glial cell layers in a peripheral nerve.

Wolff, T., Iyer, N. A. and Rubin, G. M. (2015). Neuroarchitecture and neuroanatomy of the Drosophila central complex: A GAL4-based dissection of protocerebral bridge neurons and circuits. J Comp Neurol 523: Spc1. PubMed ID: 25764203
Insects exhibit an elaborate repertoire of behaviors in response to environmental stimuli. The central complex plays a key role in combining various modalities of sensory information with an insect's internal state and past experience to select appropriate responses. Progress has been made in understanding the broad spectrum of outputs from the central complex neuropils and circuits involved in numerous behaviors. Many resident neurons have also been identified. However, the specific roles of these intricate structures and the functional connections between them remain largely obscure. Significant gains rely on obtaining a comprehensive catalog of the neurons and associated GAL4 lines that arborize within these brain regions, and on mapping neuronal pathways connecting these structures. To this end, small populations of neurons in the Drosophila melanogaster central complex were stochastically labeled using the multicolor flip-out technique and a catalog was created of the neurons, their morphologies, trajectories, relative arrangements, and corresponding GAL4 lines. This report focuses on one structure of the central complex, the protocerebral bridge, and identifies just 17 morphologically distinct cell types that arborize in this structure. This work also provides new insights into the anatomical structure of the four components of the central complex and its accessory neuropils. Most strikingly, it was found that the protocerebral bridge contains 18 glomeruli, not 16, as previously believed. Revised wiring diagrams that take into account this updated architectural design are presented. This updated map of the Drosophila central complex will facilitate a deeper behavioral and physiological dissection of this sophisticated set of structures.

Monday, March 16

Vensko Ii, S.P. and Stone, E.A. (2015). X-to-autosome expression and msl-2 transcript abundance correlate among Drosophila melanogaster somatic tissues. PeerJ 3: e771. PubMed ID: 25737812
In Drosophila melanogaster, the male-specific lethal (MSL) complex has been studied extensively for its role in upregulating male X-linked genes. Recent advances in high-throughput technologies have improved understanding of how the MSL complex mediates dosage compensation through chromosome-wide chromatin modifications. Most studies, however, have focused on cell line models that cannot reflect any potential heterogeneity of in vivo dosage compensation. Comparisons between cell line and organismal gene-level dosage compensation upregulation suggest the possibility of variation in MSL complex activity among somatic tissues. This study hypothesize the degree, up to but not exceeding 2-fold, to which the MSL complex upregulates male X-linked genes varies quantitatively by tissue type. In this model, MSL complex abundance acted as a rheostat to control the extent of upregulation. Using publicly available expression data, this study provided evidence for a model in Drosophila somatic tissues; specifically X-to-autosome expression correlated with the tissue-specific expression of msl-2 which encodes an essential male-specific component of the MSL complex. This result suggests MSL complex mediated dosage compensation varies quantitatively by tissue type. Furthermore, this result has consequences for models explaining the organismal-scale molecular and evolutionary consequences of MSL-mediated dosage compensation.

Kost, N., Kaiser, S., Ostwal, Y., Riedel, D., Stützer, A., Nikolov, M., Rathke, C., Renkawitz-Pohl, R. and Fischle, W. (2015). Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 25735749
Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, this study analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. They showed that Mst77F on its own caused severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F bound DNA via electrostatic interaction. This binding resulted in structural reorganization of the domain, which induced interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F was induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F resulted in chromatin aggregation in fly sperm. Based on these findings they postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.

Nguyen, H.Q., Nye, J., Buster, D.W., Klebba, J.E., Rogers, G.C. and Bosco G. (2015). Drosophila Casein kinase I alpha regulates homolog pairing and genome organization by modulating Condensin II subunit Cap-H2 levels. PLoS Genet 11: e1005014. PubMed ID: 25723539
The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. This study identifies Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilized Cap-H2 protein and resulted in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells resulted in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppressed condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.

Peng, P. C., Hassan Samee, M. A. and Sinha, S. (2015) Incorporating chromatin accessibility data into sequence-to-expression modeling. Biophys J 108: 1257-1267. PubMed ID: 25762337
Prediction of gene expression levels from regulatory sequences is one of the major challenges of genomic biology today. A particularly promising approach to this problem is that taken by thermodynamics-based models that interpret an enhancer sequence in a given cellular context specified by transcription factor concentration levels and predict precise expression levels driven by that enhancer. Such models have so far not accounted for the effect of chromatin accessibility on interactions between transcription factor and DNA and consequently on gene-expression levels. This paper extends a thermodynamics-based model of gene expression, called GEMSTAT (Gene Expression Modeling Based on Statistical Thermodynamics), to incorporate chromatin accessibility data and quantify its effect on accuracy of expression prediction. In the new model, called GEMSTAT-A, accessibility at a binding site is assumed to affect the transcription factor's binding strength at the site, whereas all other aspects are identical to the GEMSTAT model. This modification results in significantly better fits in a data set of over 30 enhancers regulating spatial expression patterns in the blastoderm-stage Drosophila embryo. It is important to note that the improved fits result not from an overall elevated accessibility in active enhancers but from the variation of accessibility levels within an enhancer. With whole-genome DNA accessibility measurements becoming increasingly popular, this work demonstrates how such data may be useful for sequence-to-expression models. It also calls for future advances in modeling accessibility levels from sequence and the transregulatory context, so as to predict accurately the effect of cis and trans perturbations on gene expression.

Kim, Y.J., Igiesuorobo, O., Ramos, C.I., Bao, H., Zhang, B. and Serpe, M. (2015).Prodomain removal enables Neto to stabilize glutamate receptors at the Drosophila neuromuscular junction. PLoS Genet 11: e1004988. PubMed ID: 25723514
Stabilization of neurotransmitter receptors at postsynaptic specializations is a key step in the assembly of functional synapses. Drosophila Neto (Neuropillin and Tolloid-like protein) is an essential auxiliary subunit of ionotropic glutamate receptor (iGluR) complexes required for the iGluRs clustering at the neuromuscular junction (NMJ). This study shows that optimal levels of Neto are crucial for stabilization of iGluRs at synaptic sites and proper NMJ development. Genetic manipulations of Neto levels shifted iGluRs distribution to extrajunctional locations. Perturbations in Neto levels also produced small NMJs with reduced synaptic transmission, but only Neto-depleted NMJs showed diminished postsynaptic components. Drosophila Neto contains an inhibitory prodomain that is processed by Furin1-mediated limited proteolysis. neto null mutants rescued with a Neto variant that could not be processed had severely impaired NMJs and reduced iGluRs synaptic clusters. Unprocessed Neto retained the ability to engage iGluRs in vivo and to form complexes with normal synaptic transmission. However, Neto prodomain must be removed to enable iGluRs synaptic stabilization and proper postsynaptic differentiation.

Merlo, P., Frost, B., Peng, S., Yang, Y. J., Park, P. J. and Feany, M. (2014) p53 prevents neurodegeneration by regulating synaptic genes. Proc Natl Acad Sci U S A. 111(50):18055-60. PubMed ID: 25453105
DNA damage has been implicated in neurodegenerative disorders, including Alzheimer's disease and other tauopathies, but the consequences of genotoxic stress to postmitotic neurons are poorly understood. This study demonstrates that p53, a key mediator of the DNA damage response, plays a neuroprotective role in a Drosophila model of tauopathy. Further, through a whole-genome ChIP-chip analysis, this study identified genes controlled by p53 in postmitotic neurons. A a specific pathway, synaptic function, was genetically validated in p53-mediated neuroprotection. It was then demonstratce that the control of synaptic genes by p53 is conserved in mammals. Collectively, these results implicate synaptic function as a central target in p53-dependent protection from neurodegeneration (Martin, 2014).

Wang, S., Yoo, S., Kim, H. Y., Wang, M., Zheng, C., Parkhouse, W., Krieger, C. and Harden, N. (2015) Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay. J Vis Exp. PubMed ID: 25650626
Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development. Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Thid study demonstrates how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Paul, M. M., Pauli, M., Ehmann, N., Hallermann, S., Sauer, M., Kittel, R. J. and Heckmann, M. (2015) Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation. Front Cell Neurosci 9: 29. PubMed ID: 25698934
The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants brp69. This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, knock-down of Synaptotagmin-1 (Syt) was performed via RNAi sytKD in wildtype (wt), brp69 and rab3 null mutants rab3rup, where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp(69) synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69 synapses. To gain insight into this surprising role of Syt at brp69 AZs, this study analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At 'tonic' type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.

Saturday, March 14th

<Ward, A., Hong, W., Favaloro, V. and Luo, L. (2015). Toll receptors instruct axon and dendrite targeting and participate in synaptic partner matching in a Drosophila olfactory circuit. Neuron 85: 1013-1028. PubMed ID: 25741726
Understanding of the mechanisms that establish wiring specificity of complex neural circuits is far from complete. During Drosophila olfactory circuit assembly, axons of 50 olfactory receptor neuron (ORN) classes and dendrites of 50 projection neuron (PN) classes precisely target to 50 discrete glomeruli, forming parallel information-processing pathways. This study shows that Toll-6 and Toll-7, members of the Toll receptor family best known for functions in innate immunity and embryonic patterning, cell autonomously instruct the targeting of specific classes of PN dendrites and ORN axons, respectively. The canonical ligands and downstream partners of Toll receptors in embryonic patterning and innate immunity were not required for the function of Toll-6/Toll-7 in wiring specificity, nor were their cytoplasmic domains. Interestingly, both Toll-6 and Toll-7 participated in synaptic partner matching between ORN axons and PN dendrites. These investigations reveal that olfactory circuit assembly involves dynamic and long-range interactions between PN dendrites and ORN axons.

Tavares, L., Pereira, E. Correia, A., Santos, M.A., Amaral, N., Martins, T., Relvas, J.B. and Pereira, P.S. (2015). Drosophila PS2 and PS3 integrins play distinct roles in retinal photoreceptors-glia interactions. Glia [Epub ahead of print]. PubMed ID: 25731761
Cellular migration and differentiation are important developmental processes that require dynamic cellular adhesion. Integrins are heterodimeric transmembrane receptors that play key roles in adhesion plasticity. This study explores the developing visual system of Drosophila to study the roles of integrin heterodimers in glia development. Their data show that αPS2 was essential for retinal glia migration from the brain into the eye disc and that glial cells had a role in the maintenance of the fenestrated membrane (Laminin-rich ECM layer) in the disc. Interestingly, the absence of glial cells in the eye disc did not affect the targeting of retinal axons to the optic stalk. In contrast, αPS3 (Scab) was not required for retinal glia migration, but together with Talin, it functioned in glial cells to allow photoreceptor axons to target the optic stalk. Thus, this study presents evidence that αPS2 and αPS3 integrin have different and specific functions in the development of retinal glia.

Couton, L., Mauss, A. S., Yunusov, T., Diegelmann, S., Evers, J. F. and Landgraf, M. (2015) Development of connectivity in a motoneuronal network in Drosophila larvae. Curr Biol 25: 568-576. PubMed ID: 25702582

Much of understanding of how neural networks develop is based on studies of sensory systems, revealing often highly stereotyped patterns of connections, particularly as these diverge from the presynaptic terminals of sensory neurons. Considerably less is known about the wiring strategies of motor networks, where connections converge onto the dendrites of motoneurons. This study investigated patterns of synaptic connections between identified motoneurons with sensory neurons and interneurons in the motor network of the Drosophila larva and how these change as it develops. As animals grow, motoneurons were found to increase the number of synapses with existing presynaptic partners. Different motoneurons form characteristic cell-type-specific patterns of connections. At the same time, there is considerable variability in the number of synapses formed on motoneuron dendrites, which contrasts with the stereotypy reported for presynaptic terminals of sensory neurons. Where two motoneurons of the same cell type contact a common interneuron partner, each postsynaptic cell can arrive at a different connectivity outcome. Experimentally changing the positioning of motoneuron dendrites shows that the geography of dendritic arbors in relation to presynaptic partner terminals is an important determinant in shaping patterns of connectivity. It is concluded that in the Drosophila larval motor network, the sets of connections that form between identified neurons manifest an unexpected level of variability. Synapse number and the likelihood of forming connections appear to be regulated on a cell-by-cell basis, determined primarily by the postsynaptic dendrites of motoneuron terminals.

Fujii, S., Yavuz, A., Slone, J., Jagge, C., Song, X. and Amrein, H. (2015) Drosophila sugar receptors in sweet taste perception, olfaction, and internal nutrient sensing. Curr Biol 25: 621-627. PubMed ID: 25702577
Identification of nutritious compounds is dependent on expression of specific taste receptors in appropriate taste-cell types. In contrast to mammals, which rely on a single, broadly tuned heterodimeric sugar receptor, the Drosophila genome harbors a small subfamily of eight, closely related gustatory receptor (Gr) genes, Gr5a, Gr61a, and Gr64a-Gr64f, of which three have been proposed to mediate sweet taste. However, expression and function of several of these putative sugar Gr genes are not known. This study presents a comprehensive expression and functional analysis using Gr(LEXA/GAL4) alleles that were generated through homologous recombination. Sugar Gr genes are shown to be expressed in a combinatorial manner to yield at least eight sets of sweet-sensing neurons. Behavioral investigations show that most sugar Gr mutations affect taste responses to only a small number of sugars and that effective detection of most sugars is dependent on more than one Gr gene. Surprisingly, Gr64a, one of three Gr genes previously proposed to play a major role in sweet taste, is not expressed in labellar taste neurons, and Gr64a mutant flies exhibit normal sugar responses elicited from the labellum. This analysis provides a molecular rationale for distinct tuning profiles of sweet taste neurons, and it favors a model whereby all sugar Grs contribute to sweet taste. Furthermore, expression in olfactory organs and the brain implies novel roles for sugar Gr genes in olfaction and internal nutrient sensing, respectively. Thus, sugar receptors may contribute to feeding behavior via multiple sensory systems.

Friday, March 13th

Chan, C., Jayasekera, S., Kao, B., Páramo, M., von Grotthuss, M. and Ranz, J.M. (2015). Remodelling of a homeobox gene cluster by multiple independent gene reunions in Drosophila. Nat Commun 6: 6509. PubMed ID: 25739651
Genome clustering of homeobox genes is often thought to reflect arrangements of tandem gene duplicates maintained by advantageous coordinated gene regulation. This study analyses the chromosomal organization of the NK homeobox genes (see Drosophila tinman, bagpipe, C15, ladybird late, ladybird early, and slouch), presumed to be part of a single cluster in the Bilaterian ancestor, across 20 arthropods. The ProtoNK cluster was extensively fragmented in some lineages, showing that NK clustering in Drosophila species did not reflect selectively maintained gene arrangements. More importantly, the arrangement of NK and neighbouring genes across the phylogeny supported that, in two instances within the Drosophila genus, some cluster remnants became reunited via large-scale chromosomal rearrangements. Simulated scenarios of chromosome evolution indicated that these reunion events were unlikely unless the genome neighbourhoods harbouring the participating genes tended to colocalize in the nucleus. These results underscore how mechanisms other than tandem gene duplication can result in paralogous gene clustering during genome evolution.

Ellison, C. E. and Bachtrog, D. (2015) Non-allelic gene conversion enables rapid evolutionary change at multiple regulatory sites encoded by transposable elements. Elife 4. PubMed ID: 25688566
Transposable elements (TEs) allow rewiring of regulatory networks, and the recent amplification of the ISX-element dispersed 77 functional but suboptimal binding-sites for the dosage-compensation-complex to a newly-formed X-chromosome in Drosophila. This study identified two linked refining-mutations within ISX that interact epistatically to increase binding affinity to the dosage-compensation-complex. Selection has increased the frequency of this derived haplotype in the population, which is fixed at 30% of ISX-insertions and polymorphic among another 41%. Sharing of this haplotype indicates that high levels of gene-conversion among ISX-elements allow them to 'crowd-source' refining-mutations, and a refining-mutation that occurs at any single ISX-element can spread in two dimensions: horizontally across insertion sites by non-allelic gene-conversion, and vertically through the population by natural selection. These describes a novel route how fully functional regulatory elements can arise rapidly from TEs and implicate non-allelic gene-conversion as having an important role in accelerating the evolutionary fine-tuning of regulatory networks.

Katzenberger, R.J., Chtarbanova, S., Rimkus, S.A., Fischer, J.A., Kaur, G., Seppala, J.M., Swanson, L.C., Zajac, J.E., Ganetzky, B. and Wassarman, D.A. (2015). Death following traumatic brain injury in Drosophila is associated with intestinal barrier dysfunction. Elife [Epub ahead of print]. PubMed ID: 25742603
Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Unfavorable TBI outcomes result from primary mechanical injuries to the brain and ensuing secondary non-mechanical injuries that are not limited to the brain. This Genome-wide Association study of Drosophila melanogaster revealed that the probability of death following TBI is associated with single nucleotide polymorphisms in genes involved in tissue barrier function and glucose homeostasis. TBI caused intestinal and blood-brain barrier dysfunction and intestinal barrier dysfunction was highly correlated with the probability of death. Furthermore, ingestion of glucose after a primary injury increased the probability of death through a secondary injury mechanism that exacerbated intestinal barrier dysfunction. These results indicate that natural variation in the probability of death following TBI is due in part to genetic differences that affect intestinal barrier dysfunction.

Colines, B., Rodriguez, N. C., Hasson, E. R., Carreira, V. and Frankel, N. (2015) Parental age influences developmental stability of the progeny in Drosophila. Proc Biol Sci 282. PubMed ID: 25673675
The stochastic nature of biochemical processes is a source of variability that influences developmental stability. Developmental instability (DI) is often estimated through fluctuating asymmetry (FA), a parameter that deals with within-individual variation in bilateral structures. A relevant goal is to shed light on how environment, physiology and genotype relate to DI, thus providing a more comprehensive view of organismal development. Using Drosophila melanogaster isogenic lines, this study investigated the effect of parental age, parental diet and offspring heterozygosity on DI. This work uncovered a clear relationship between parental age and offspring asymmetry. Asymmetry of the progeny increases concomitantly with parental age. Moreover, it was demonstrated that enriching the diet of parents mitigates the effect of age on offspring symmetry. Also, increasing the heterozygosity of the progeny eliminates the effect of parental age on offspring symmetry. Taken together, these results suggest that diet, genotype and age of the parents interact to determine offspring DI in wild populations. These findings provide an avenue to understand the mechanisms underlying DI.

Thursday, March 12th

Hale, R., Brittle, A. L., Fisher, K. H., Monk, N. A. and Strutt, D. (2015) Cellular interpretation of the long-range gradient of Four-jointed activity in the Drosophila wing. Elife 4. PubMed ID: 25707557
To understand how long-range patterning gradients are interpreted at the cellular level, this study investigated how a gradient of expression of the Four-jointed kinase specifies planar polarised distributions of the cadherins Fat and Dachsous in the Drosophila wing. Computational modelling was used to test different scenarios for how Four-jointed might act, and the model predictions were tested by employing fluorescence recovery after photobleaching as an in vivo assay to measure the influence of Four-jointed on Fat-Dachsous binding. It was demonstrated that in vivo, Four-jointed acts both on Fat to promote its binding to Dachsous and on Dachsous to inhibit its binding to Fat, with a bias towards a stronger effect on Fat. Overall, this study showed that opposing gradients of Fat and Dachsous phosphorylation are sufficient to explain the observed pattern of Fat-Dachsous binding and planar polarisation across the wing, and thus demonstrate the mechanism by which a long-range gradient is interpreted.

Spratford, C. M. and Kumar, J. P. (2015) Extramacrochaetae functions in dorsal-ventral patterning of Drosophila imaginal discs. Development 142: 1006-1015. PubMed ID: 25715400
Within the Drosophila retina, D/V axis formation is essential to ensure that each unit eye first adopts the proper chiral form and then rotates precisely 90 degrees in the correct direction. These two steps are important because the photoreceptor array must be correctly aligned with the neurons of the optic lobe. Defects in chirality and/or ommatidial rotation will lead to disorganization of the photoreceptor array, misalignment of retinal and optic lobe neurons, and loss of visual acuity. Loss of the helix-loop-helix protein Extramacrochaetae (Emc) leads to defects in both ommatidial chirality and rotation. This study describes a new role for emc in eye development in patterning the D/V axis. The juxtaposition of dorsal and ventral fated tissue in the eye leads to an enrichment of emc expression at the D/V midline. emc expression at the midline can be eliminated when D/V patterning is disrupted and can be induced in situations in which ectopic boundaries are artificially generated. This study also shows that emc functions downstream of Notch signaling to maintain the expression of four-jointed along the midline.

Wojtowicz, I., Jablonska, J., Zmojdzian, M., Taghli-Lamallem, O., Renaud, Y., Junion, G., Daczewska, M., Huelsmann, S., Jagla, K. and Jagla, T. (2015) Drosophila small heat shock protein CryAB ensures structural integrity of developing muscles, and proper muscle and heart performance. Development 142: 994-1005. PubMed ID: 25715399
Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. This study shows that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). The data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryAB(R120G), which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, this study demonstrates that CryAB ensures myofibrillar integrity in Drosophila muscles during development, and it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture.

Pesch, Y.Y., Riedel, D. and Behr, M. (2015). Obstructor-A organizes matrix assembly at the apical cell surface to promote enzymatic cuticle maturation in Drosophila. J Biol Chem [Epub ahead of print]. PubMed ID: 25737451
Assembly and maturation of the apical extracellular matrix (aECM) is crucial for protecting organisms, but underlying molecular mechanisms remain poorly understood. Epidermal cells secrete proteins and enzymes that assemble at the apical cell surface to provide epithelial integrity and stability during developmental growth and upon tissue damage. This study analyzed molecular mechanisms of aECM assembly and identified the conserved chitin-binding protein Obstructor (Obst)-A as an essential regulator. They show in Drosophila that Obst-A was required to coordinate protein- and chitin-matrix packaging at the apical cell surface during development. Secreted by epidermal cells, the Obst-A protein was specifically enriched in the apical assembly zone where matrix components are packaged into their highly ordered architecture. In obst-A null mutant larvae, the assembly zone was strongly diminished resulting in severe disturbance of matrix-scaffold organization and impaired aECM integrity. Furthermore, enzymes that support aECM stability were mislocalized. As a biological consequence, cuticle architecture, integrity and function were disturbed in obst-A mutants finally resulting in immediate lethality upon wounding. These studies identify a new core-organizing center, the assembly zone that controls aECM assembly at the apical cell surface. They propose a genetically conserved molecular mechanism by which Obst-A forms a matrix-scaffold to coordinate trafficking and localization of proteins and enzymes in the newly deposited aECM. This mechanism is essential for maturation and stabilization of the aECM in a growing and remodeling epithelial tissue as an outermost barrier.

Wednesday, March 11th

Liu, Y., Luo, J. Carlsson, M.A. and Nässel, D.R. (2015). Serotonin and insulin-like peptides modulate leucokinin-producing neurons that affect feeding and water homeostasis in Drosophila. J Comp Neurol [Epub ahead of print]. PubMed ID: 25732325
Metabolic homeostasis and water balance is maintained by tight hormonal and neuronal regulation. In Drosophila, insulin-like peptides (DILPs) are key regulators of metabolism, and the neuropeptide Leucokinin (LK; see Leucokinin receptor) is a diuretic hormone that also modulates feeding. However, it is not known whether LK and DILPs act together to regulate feeding and water homeostasis. Since LK neurons express the insulin receptor (dInR), this study tested functional links between DILP and LK signaling in feeding and water balance. Constitutive and conditional manipulations of activity in LK neurons and insulin producing cells (IPCs) were performed in adult flies, and food intake, responses to desiccation and peptide expression levels were monitored. In vivo changes in LK and DILP levels were measured in neurons in response to desiccation and drinking. The data showed that activated LK cells stimulated diuresis in vivo, and that LK and IPC signaling affected food intake in opposite directions. Overexpression of the dInR in LK neurons decreased the LK peptide levels, but only caused a subtle decrease in feeding, and had no effect on water balance. LK neurons expressed the serotonin receptor 5-HT1B . Knockdown of this receptor in LK neurons diminished LK expression, increased desiccation resistance and diminished food intake. Live calcium imaging indicated that serotonin inhibited spontaneous activity in abdominal LK neurons. These results suggest that serotonin via 5-HT1B diminishes activity in the LK neurons and thereby modulates functions regulated by LK peptide, but the action of the dInR in these neurons remains less clear.

Wang, X., Ma, Y., Zhao, Y., Chen, Y., Hu, Y., Chen, C., Shao, Y. and Xue, L. (2015). APLP1 promotes dFoxO-dependent cell death in Drosophila. Apoptosis [Epub ahead of print]. PubMed ID: 25740230
The amyloid precursor like protein-1 (APLP1) belongs to the amyloid precursor protein family that also includes the amyloid precursor protein (APP) and the amyloid precursor like protein-2 (APLP2). Though the three proteins share similar structures and undergo the same cleavage processing by α-, β- and γ-secretases, APLP1 shows divergent subcellular localization from that of APP and APLP2, and thus, may perform distinct roles in vivo. While extensive studies have been focused on APP, which is implicated in the pathogenesis of Alzheimer's disease, the functions of APLP1 remain largely elusive. This study reports that the expression of APLP1 in Drosophila induces cell death and produces developmental defects in wing and thorax. This function of APLP1 depends on the transcription factor dFoxO, as the depletion of dFoxO abrogated APLP1-induced cell death and adult defects. Consistently, APLP1 up-regulated the transcription of dFoxO target hid and reaper-two well known pro-apoptotic genes. Thus, the present study provides the first in vivo evidence that APLP1 is able to induce cell death, and that FoxO is a crucial downstream mediator of APLP1's activity.

Satoh, T., Ohba, A., Liu, Z., Inagaki, T. and Satoh, A. K. (2015). dPob/EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors. Elife 4. PubMed ID: 25715730
In eukaryotes, most integral membrane proteins are synthesized, integrated into the membrane, and folded properly in the endoplasmic reticulum (ER). Mutants affecting rhabdomeric expression of rhodopsin 1 (Rh1) in the Drosophila photoreceptors were screened and dPob/EMC3, EMC1, and EMC8/9, Drosophila homologs of subunits of ER membrane protein complex (EMC), were found to b essential for stabilization of immature Rh1 in an earlier step than that at which another Rh1-specific chaperone (NinaA) acts. dPob/EMC3 localizes to the ER and associates with EMC1 and calnexin. Moreover, EMC is required for the stable expression of other multi-pass transmembrane proteins such as minor rhodopsins Rh3 and Rh4, transient receptor potential, and Na(+)K(+)-ATPase, but not for a secreted protein or type I single-pass transmembrane proteins. Furthermore, dPob/EMC3 deficiency was found to induce rhabdomere degeneration in a light-independent manner. These results collectively indicate that EMC is a key factor in the biogenesis of multi-pass transmembrane proteins, including Rh1, and its loss causes retinal degeneration.

Charbonnier, E., Fuchs, A., Cheung, L.S., Chayengia, M., Veikkolainen, V., Shvartsman, S.Y. and Pyrowolakis, G. (2015). BMP-dependent gene repression cascade in Drosophila eggshell patterning. Dev Biol [Epub ahead of print]. PubMed ID: 25704512
Bone Morphogenetic Proteins (BMPs) signal by activating Smad transcription factors to control a number of decisions during animal development. In Drosophila, signaling by the BMP ligand Decapentaplegic (Dpp) involves the activity of brinker (brk) which, in most contexts, is repressed by Dpp. Brk encodes a transcription factor which represses BMP signaling output by antagonizing Smad-dependent target gene activation. This report studied BMP-dependent gene regulation during Drosophila oogenesis by following the signal transmission from Dpp to its target broad (br), a gene with a crucial function in eggshell patterning. The study identified regulatory sequences that accounted for expression of both brk and br, and connected these to the transcription factors of the pathway. Dpp directly regulated brk transcription through Smad- and Schnurri (Shn)-dependent repression. Brk was epistatic to Dpp in br expression and activated br indirectly, through removal of a repressor, which is yet to be identified. This work provides first cis-regulatory insights into transcriptional interpretation of BMP signaling in eggshell morphogenesis and defines a transcriptional cascade that connects Dpp to target gene regulation.

Tuesday, March 10th

Kohatsu, S. and Yamamoto, D. (2015). Visually induced initiation of Drosophila innate courtship-like following pursuit is mediated by central excitatory state. Nat Commun 6: 6457. PubMed ID: 25743851
The courtship ritual of male Drosophila represents an innate behaviour that is initiated by female-derived sensory stimuli. This study reports that moving light spots can induce courtship-like following pursuit in tethered wild-type male flies provided the fly is primed by optogenetic stimulation of specific dsx-expressing neuronal clusters in the lateral protocerebrum (LPR). Namely, stimulation of the pC1 neuronal cluster initiated unilateral wing extension and vibration of both sides, whereas stimulation of the pC2l cluster initiated only contralateral wing displays. In addition, stimulation of pC2l but not pC1 neurons induced abdominal bending and proboscis extension. Ca(2+) imaging of the pC1 cluster revealed periodic Ca(2+) rises, each corresponding to a turn of the male fly during courtship. In contrast, group-reared fru mutant males exhibited light spot-induced courtship pursuit without optogenetic priming. Ca(2+) imaging revealed enhanced responses of LPR neurons to visual stimuli in the mutants, suggesting a neural correlate of the light spot-induced courtship behaviour.

Musso, P.Y., Tchenio, P. and Preat, T. (2015). Delayed dopamine signaling of energy level builds appetitive long-term memory in Drosophila. Cell Rep [Epub ahead of print]. PubMed ID: 25704807
Sensory cues relevant to a food source, such as odors, can be associated with post-ingestion signals related either to food energetic value or toxicity. Despite numerous behavioral studies, a global understanding of the mechanisms underlying these long delay associations remains out of reach. This study demonstrates in Drosophila that the long-term association between an odor and a nutritious sugar depends on delayed post-ingestion signaling of energy level. At the neural circuit level the activity of two pairs of dopaminergic neurons was necessary and sufficient to signal energy level to the olfactory memory center. Accordingly, a delayed calcium trace has been identified in these dopaminergic neurons that correlates with appetitive long-term memory formation. Altogether, these findings demonstrate that the Drosophila brain remembers food quality through a two-step mechanism that consists of the integration of olfactory and gustatory sensory information and then post-ingestion energetic value.

French, A.S., Sellier, M.J., Moutaz, A.A., Guigue, A., Chabaud, M.A., Reeb, P.D., Mitra, A., Grau, Y., Soustelle, L. and Marion-Poll, F. (2015). Dual mechanism for bitter avoidance in Drosophila. J Neurosci 35: 3990-4004. PubMed ID: 25740527
In flies and humans, bitter chemicals are known to inhibit sugar detection, but the adaptive role of this inhibition is often overlooked. At best, this inhibition is described as contributing to the rejection of potentially toxic food, but no studies have addressed the relative importance of the direct pathway that involves activating bitter-sensitive cells versus the indirect pathway represented by the inhibition of sugar detection. Using toxins to selectively ablate or inactivate populations of bitter-sensitive cells, this study assessed the behavioral responses of flies to sucrose mixed with strychnine (which activates bitter-sensitive cells and inhibits sugar detection) or with l-canavanine (which only activates bitter-sensitive cells). As expected, flies with ablated bitter-sensitive cells failed to detect l-canavanine mixed with sucrose in three different feeding assays (proboscis extension responses, capillary feeding, and two-choice assays). However, such flies were still able to avoid strychnine mixed with sucrose. By means of electrophysiological recordings, the study established that bitter molecules differed in their potency to inhibit sucrose detection and that sugar-sensing inhibition affected taste cells on the proboscis and the legs. The optogenetic response of sugar-sensitive cells was not reduced by strychnine, thus suggesting that this inhibition is linked directly to sugar transduction. The study postulates that sugar-sensing inhibition represents a mechanism in insects to prevent ingesting harmful substances occurring within mixtures.

Lee, K.M., Daubnerov&aacute;, I., Isaac, R.E., Zhang, C., Choi, S., Chung, J. and Kim, Y.J. (2015). A neuronal pathway that controls sperm ejection and storage in female Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 25702579
In polyandrous females, sperm storage permits competition between sperm of different mates, and in some species females influence the relative fertilization success of competing sperm in favor of a preferred mate. In female Drosophila melanogaster, sperm competition is strongly influenced by the timing of sperm ejection from the uterus. Understanding how female behavior influences sperm competition requires knowledge of the neuronal mechanisms controlling sperm retention and storage, which is currently lacking. This study shows that D. melanogaster females eject male ejaculates from the uterus 1-6 hr after mating with a stereotypic behavior regulated by a brain signaling pathway composed of diuretic hormone 44 (Dh44), a neuropeptide related to vertebrate corticotropin-releasing factor (CRF), and its receptor, Dh44R1. Suppression of Dh44 signals in the brain expedited sperm ejection from the uterus, resulting in marked reduction of sperm in the storage organs and decreased fecundity, whereas enhancement of Dh44 signals delayed sperm expulsion. The Dh44 function was mapped to six neurons located in the pars intercerebralis of the brain together with a small subset of Dh44R1 neurons that expressed the sex-specific transcription factor doublesex. This study identifies a neuronal pathway by which females can control sperm retention and storage and provides new insight into how the female might exercise post-copulatory sexual selection.

Monday, March 9th

Chen, W. Y., Shih, H. T., Liu, K. Y., Shih, Z. S., Chen, L. K., Tsai, T. H., Chen, M. J., Liu, H., Tan, B. C., Chen, C. Y., Lee, H. H., Loppin, B., Ait-Ahmed, O. and Wu, J. T. (2015) Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3. EMBO Rep [Epub ahead of print]. PubMed ID: 25666827
Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. This paper reports that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. This work thus establishes a previously unknown negative regulation of H3.3 and advances understanding of BRWD3-dependent intellectual disability.

Okray, Z., de Esch, C. E., Van Esch, H., Devriendt, K., Claeys, A., Yan, J., Verbeeck, J., Froyen, G., Willemsen, R., de Vrij, F. M. and Hassan, B. A. (2015) A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function. EMBO Mol Med [Epub ahead of print]. PubMed ID: 25693964
Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. This study describes a novel FMR1 mutation and reveals an unexpected nuclear export function for the C-terminus of FMRP. A screen was carried out of a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, a guanine insertion was identified in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. This novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. An evolutionarily conserved nuclear export function was observed that was associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

Lin, W. H., He, M. and Baines, R. A. (2015) Seizure suppression through manipulating splicing of a voltage-gated sodium channel.. Brain [Epub ahead of print]. PubMed ID: 25681415
Seizure can result from increased voltage-gated persistent sodium current expression. Although many clinically-approved antiepileptic drugs target voltage-gated persistent sodium current, none exclusively repress this current without also adversely affecting the transient voltage-gated sodium current. Achieving a more selective block has significant potential for the treatment of epilepsy. Recent studies show that voltage-gated persistent sodium current amplitude is regulated by alternative splicing offering the possibility of a novel route for seizure control. This study identified 291 splicing regulators that, on knockdown, alter splicing of the Drosophila voltage-gated sodium channel (Paralytic) to favour inclusion of exon K, rather than the mutually exclusive exon L. This change is associated with both a significant reduction in voltage-gated persistent sodium current, without change to transient voltage-gated sodium current, and to rescue of seizure in this model insect. RNA interference mediated knock-down, in two different seizure mutants, shows that 95 of these regulators are sufficient to significantly reduce seizure duration. Moreover, most suppress seizure activity in both mutants, indicative that they are part of well conserved pathways and likely, therefore, to be optimal candidates to take forward to mammalian studies. Proof-of-principle is provided for such studies by showing that inhibition of a selection of regulators, using small molecule inhibitors, is similarly effective to reduce seizure. Splicing of the Drosophila sodium channel shows many similarities to its mammalian counterparts, including altering the amplitude of voltage-gated persistent sodium current. This study provides the impetus to investigate whether manipulation of splicing of mammalian voltage-gated sodium channels may be exploitable to provide effective seizure control.

Varadarajan, S., Breda, C., Smalley, J. L., Butterworth, M., Farrow, S. N., Giorgini, F. and Cohen, G. M. (2015) The transrepression arm of glucocorticoid receptor signaling is protective in mutant huntingtin-mediated neurodegeneration. Cell Death Differ [Epub ahead of print]. PubMed ID: 25656655
The unfolded protein response (UPR) occurs following the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and orchestrates an intricate balance between its prosurvival and apoptotic arms to restore cellular homeostasis and integrity. However, in certain neurodegenerative diseases, the apoptotic arm of the UPR is enhanced, resulting in excessive neuronal cell death and disease progression, both of which can be overcome by modulating the UPR. This study describes a novel crosstalk between glucocorticoid receptor signaling and the apoptotic arm of the UPR, thus highlighting the potential of glucocorticoid therapy in treating neurodegenerative diseases. Several glucocorticoids, but not mineralocorticoids, selectively antagonize ER stress-induced apoptosis in a manner that is downstream of and/or independent of the conventional UPR pathways. Using GRT10, a novel selective pharmacological modulator of glucocorticoid signaling, this study describes the importance of the transrepression arm of the glucocorticoid signaling pathway in protection against ER stress-induced apoptosis. Furthermore, the protective effects of glucocorticoids were also observed in vivo in a Drosophila model of Huntington's disease (HD), wherein treatment with different glucocorticoids diminished rhabdomere loss and conferred neuroprotection. Finally, this study found that growth differentiation factor 15 has an important role downstream of glucocorticoid signaling in antagonizing ER stress-induced apoptosis in cells, as well as in preventing HD-mediated neurodegeneration in flies. Thus, these studies demonstrate that this novel crosstalk has the potential to be effectively exploited in alleviating several neurodegenerative disorders.

Sunday, March 8th

Lenz, O., Xiong, J., Nelson, M. D., Raizen, D. M. and Williams, J. A. (2015) FMRFamide signaling promotes stress-induced sleep in Drosophila. Brain Behav Immun [Epub ahead of print]. PubMed ID: 25668617
Enhanced sleep in response to cellular stress is a conserved adaptive behavior across multiple species, but the mechanism of this process is poorly understood. Drosophila melanogaster increases sleep following exposure to septic or aseptic injury, and Caenorhabditis elegans displays sleep-like quiescence following exposure to high temperatures that stress cells. Similar to C. elegans, Drosophila responds to heat stress with an increase in sleep. In contrast to Drosophila infection-induced sleep, heat-induced sleep is not sensitive to the time-of-day of the heat pulse. Moreover, the sleep response to heat stress does not require Relish, the NFκB transcription factor that is necessary for infection-induced sleep, indicating that sleep is induced by multiple mechanisms from different stress modalities. A sleep-regulating role was identified for a signaling pathway involving FMRFamide neuropeptides and their receptor FR. Animals mutant for either FMRFamide or for the FMRFamide receptor (FR) have a reduced recovery sleep in response to heat stress. FR mutants, in addition, show reduced sleep responses following infection with Serratia marcescens, and succumb to infection at a faster rate than wild-type controls. Together, these findings support the hypothesis that FMRFamide and its receptor promote an adaptive increase in sleep following stress. Because an FMRFamide-like neuropeptide plays a similar role in C. elegans, it is proposed that FRMFamide neuropeptide signaling is an ancient regulator of recovery sleep which occurs in response to cellular stress.

Price, J. L., Fan, J. Y., Keightley, A. and Means, J. C. (2015) The role of casein kinase I in the Drosophila circadian clock. Methods Enzymol 551: 175-195. PubMed ID: 25662457
The circadian clock mechanism in organisms as diverse as cyanobacteria and humans involves both transcriptional and posttranslational regulation of key clock components. One of the roles for the posttranslational regulation is to time the degradation of the targeted clock proteins, so that their oscillation profiles are out of phase with respect to those of the mRNAs from which they are translated. In Drosophila, the circadian transcriptional regulator Period (Per) is targeted for degradation by a kinase (Doubletime or Dbt) orthologous to mammalian kinases (CKIvarepsilon and CKIdelta) that also target mammalian Per. Since these kinases are not regulated by second messengers, the mechanism (if any) for their regulation is not known. This study has investigated the possibility that regulation of Ddb is conferred by other proteins that associate with Dbt and Per. The methods employed to identify and analyze these factors are discussed. These methods include expression of wild type and mutant proteins with the GAL4/UAS binary expression approach, analysis of Ddt in Drosophila S2 cells, in vitro kinase assays with Dbt isolated from S2 cells, and proteomic analysis of Dbt-containing complexes and of Ddt phosphorylation with mass spectrometry. The work has led to the discovery of a previously unrecognized circadian rhythm component (Bride of Dbt, a noncanonical FK506-binding protein) and the mapping of autophosphorylation sites within the Ddt C-terminal domain with potential regulatory roles.

Arya, G. H., Magwire, M. M., Huang, W., Serrano-Negron, Y. L., Mackay, T. F. and Anholt, R. R. (2015) The genetic basis for variation in olfactory behavior in Drosophila melanogaster. Chem Senses [Epub ahead of print]. PubMed ID: 25687947
The genetic underpinnings that contribute to variation in olfactory perception are not fully understood. To explore the genetic basis of variation in olfactory perception, behavioral responses were measured to 14 chemically diverse naturally occurring odorants in 260400 flies from 186 lines of the Drosophila melanogaster Genetic Reference Panel, a population of inbred wild-derived lines with sequenced genomes. Variation was observed in olfactory behavior for all odorants. Low to moderate broad-sense heritabilities and the large number of tests for genotype-olfactory phenotype association performed precluded any individual variant from reaching formal significance. However, the top variants (nominal P < 5x10-5) were highly enriched for genes involved in nervous system development and function, as expected for a behavioral trait. Further, pathway enrichment analyses showed that genes tagged by the top variants included components of networks centered on cyclic guanosine monophosphate and inositol triphosphate signaling, growth factor signaling, Rho signaling, axon guidance, and regulation of neural connectivity. Functional validation with RNAi and mutations showed that 15 out of 17 genes tested indeed affect olfactory behavior. The results show that in addition to chemoreceptors, variation in olfactory perception depends on polymorphisms that can result in subtle variations in synaptic connectivity within the nervous system.

Chakraborty, S., Bartussek, J., Fry, S. N. and Zapotocky, M. (2015) Independently controlled wing stroke patterns in the fruit fly Drosophila melanogaster. PLoS One 10: e0116813. PubMed ID: 25710715
Flies achieve supreme flight maneuverability through a small set of miniscule steering muscles attached to the wing base. The fast flight maneuvers arise from precisely timed activation of the steering muscles and the resulting subtle modulation of the wing stroke. In addition, slower modulation of wing kinematics arises from changes in the activity of indirect flight muscles in the thorax. This study investigated if these modulations can be described as a superposition of a limited number of elementary deformations of the wing stroke that are under independent physiological control. Using a high-speed computer vision system, the wing motion was recorded of tethered flying fruit flies for up to 12,000 consecutive wing strokes at a sampling rate of 6250 Hz. The joint motion pattern of both wings was then decomposed into components that had the minimal mutual information (a measure of statistical dependence). In 100 flight segments measured from 10 individual flies, seven distinct types of frequently occurring least-dependent components were identified, each defining a kinematic pattern (a specific deformation of the wing stroke and the sequence of its activation from cycle to cycle). Two of these stroke deformations can be associated with the control of yaw torque and total flight force, respectively. A third deformation involves a change in the downstroke-to-upstroke duration ratio, which is expected to alter the pitch torque. A fourth kinematic pattern consists in the alteration of stroke amplitude with a period of 2 wingbeat cycles, extending for dozens of cycles. This analysis indicates that these four elementary kinematic patterns can be activated mutually independently, and occur both in isolation and in linear superposition. The results strengthen the available evidence for independent control of yaw torque, pitch torque, and total flight force. The computational method facilitates systematic identification of novel patterns in large kinematic datasets.

Saturday, March 7th

Goldman-Huertas, B., Mitchell, R. F., Lapoint, R. T., Faucher, C. P., Hildebrand, J. G. and Whiteman, N. K. (2015) Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors, and ancestral diet. Proc Natl Acad Sci U S A. PubMed ID: 25624509
Herbivory is a key innovation in insects, yet has only evolved in one-third of living orders. The evolution of herbivory likely involves major behavioral changes mediated by remodeling of canonical chemosensory modules. Herbivorous flies in the genus Scaptomyza (Drosophilidae) are compelling species in which to study the genomic architecture linked to the transition to herbivory because they recently evolved from microbe-feeding ancestors and are closely related to Drosophila melanogaster. This study found that Scaptomyza flava, a leaf-mining specialist on plants in the family (Brassicaceae), was not attracted to yeast volatiles in a four-field olfactometer assay, whereas D. melanogaster was strongly attracted to these volatiles. Yeast-associated volatiles, especially short-chain aliphatic esters, elicited strong antennal responses in D. melanogaster, but weak antennal responses in electroantennographic recordings from S. flava. The genome of S. flava was sequenced and this species' odorant receptor repertoire was characterized. Orthologs of odorant receptors, which detect yeast volatiles in D. melanogaster and mediate critical host-choice behavior, were deleted or pseudogenized in the genome of S. flava. These genes were lost step-wise during the evolution of Scaptomyza. Additionally, Scaptomyza has experienced gene duplication and likely positive selection in paralogs of Or67b in D. melanogaster. Olfactory sensory neurons expressing Or67b are sensitive to green-leaf volatiles. Major trophic shifts in insects are associated with chemoreceptor gene loss as recently evolved ecologies shape sensory repertoires.

Garud, N. R., Messer, P. W., Buzbas, E. O. and Petrov, D. A. (2015). Recent selective sweeps in North American Drosophila melanogaster show signatures of soft sweeps. PLoS Genet 11: e1005004. PubMed ID: 25706129
Adaptation from standing genetic variation or recurrent de novo mutation in large populations should commonly generate soft rather than hard selective sweeps. In contrast to a hard selective sweep, in which a single adaptive haplotype rises to high population frequency, in a soft selective sweep multiple adaptive haplotypes sweep through the population simultaneously, producing distinct patterns of genetic variation in the vicinity of the adaptive site. Current statistical methods were expressly designed to detect hard sweeps and most lack power to detect soft sweeps. This is particularly unfortunate for the study of adaptation in species such as Drosophila melanogaster, where all three confirmed cases of recent adaptation resulted in soft selective sweeps and where there is evidence that the effective population size relevant for recent and strong adaptation is large enough to generate soft sweeps even when adaptation requires mutation at a specific single site at a locus. This study developed a statistical test based on a measure of haplotype homozygosity (H12) that is capable of detecting both hard and soft sweeps with similar power. H12 was used to identify multiple genomic regions that have undergone recent and strong adaptation in a large population sample of fully sequenced Drosophila melanogaster strains from the Drosophila Genetic Reference Panel (DGRP). Visual inspection of the top 50 candidates reveals that in all cases multiple haplotypes are present at high frequencies, consistent with signatures of soft sweeps. A second haplotype homozygosity statistic (H2/H1) was then developed that, in combination with H12, is capable of differentiating hard from soft sweeps. Surprisingly, it was found that the H12 and H2/H1 values for all top 50 peaks are much more easily generated by soft rather than hard sweeps. The implications are discussed of these results for the study of adaptation in Drosophila and in species with large census population sizes.

Stocks, M., Dean, R., Rogell, B. and Friberg, U. (2015) Sex-specific Trans-regulatory variation on the Drosophila melanogaster X chromosome. PLoS Genet 11: e1005015. PubMed ID: 25679222
The X chromosome constitutes a unique genomic environment because it is present in one copy in males, but two copies in females. This simple fact has motivated several theoretical predictions with respect to how standing genetic variation on the X chromosome should differ from the autosomes. Unmasked expression of deleterious mutations in males and a lower census size are expected to reduce variation, while allelic variants with sexually antagonistic effects, and potentially those with a sex-specific effect, could accumulate on the X chromosome and contribute to increased genetic variation. In addition, incomplete dosage compensation of the X chromosome could potentially dampen the male-specific effects of random mutations, and promote the accumulation of X-linked alleles with sexually dimorphic phenotypic effects. This study tested both the amount and the type of genetic variation on the X chromosome within a population of Drosophila melanogaster, by comparing the proportion of X linked and autosomal trans-regulatory SNPs with a sexually concordant and discordant effect on gene expression. The X chromosome was found to be depleted for SNPs with a sexually concordant effect, but hosts comparatively more SNPs with a sexually discordant effect. Interestingly, the contrasting results for SNPs with sexually concordant and discordant effects are driven by SNPs with a larger influence on expression in females than expression in males. Furthermore, the distribution of these SNPs is shifted towards regions where dosage compensation is predicted to be less complete. These results suggest that intrinsic properties of dosage compensation influence either the accumulation of different types of trans-factors and/or their propensity to accumulate mutations. These findings document a potential mechanistic basis for sex-specific genetic variation, and identify the X as a reservoir for sexually dimorphic phenotypic variation. These results have general implications for X chromosome evolution, as well as the genetic basis of sex-specific evolutionary change.

Luo, S. D. and Baker, B. S. (2015) Constraints on the evolution of a doublesex target gene arising from doublesex's pleiotropic deployment. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25675536
"Regulatory evolution," that is, changes in a gene's expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex (dsx) gene, which encodes sex-specific transcription factors (DSXF in females and DSXM in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. This study focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. dsx was found to be deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. Two cis-regulatory modules (CRM-p and CRM-d) were identified that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues.

Friday, March 6th

Sajwan, S., Sidorov, R., Staskova, T., Zaloudikova, A., Takasu, Y., Kodrik, D. and Zurovec, M. (2015) Targeted mutagenesis and functional analysis of adipokinetic hormone-encoding gene in Drosophila. Insect Biochem Mol Biol [Epub ahead of print]. PubMed ID: 25641265
Adipokinetic hormones (Akhs) are small peptides (8-10 amino acid [aa] residues long) found in insects that regulate metabolic responses to stress by stimulating catabolic reactions and mobilizing energy stores. This study employed Transcription activator-like effector nuclease (TALEN) mutagenesis and isolated an Akh1 mutant carrying a small deletion in the gene that resulted in a truncated peptide; the second aa (Leu) was missing from the functional octapeptide. This null Dmel/Akh mutant is suitable to study Akh function without any effect on the C-terminal associated peptide encoded by the same gene. The mutant flies were fully viable and compared to the control flies had significantly low levels of hemolymph saccharides including trehalose and were resistant to starvation. These characteristics are similar to those obtained from the flies carrying targeted ablation of Akh-expressing neurons. This study also found that the Akh1 mutants are slightly heavy and had a slow metabolic rate. Furthermore, the ectopic expression of Dmel\Akh reverses the Akh1 phenotype and restores the wild-type characteristics. These results show that Akh is an important regulator of metabolic homeostasis in Drosophila (Sijwan, 2015).

Tsuda-Sakurai, K., Seong, K. H., Horiuchi, J., Aigaki, T. and Tsuda, M. (2015) Identification of a novel role for Drosophila MESR4 in lipid metabolism. Genes Cells [Epub ahead of print]. PubMed ID: 25639854
Drosophila provides a powerful genetic model to analyze lipid metabolism. Drosophila has an adipose-like organ called the fat body, which plays a crucial role in energy homeostasis. Thid dyufy conducted a fat body-specific misexpression screen to identify genes involved in lipid metabolism. Over-expression of a nuclear protein with nine C2 H2 type zinc-finger motifs and a PHD-finger, Misexpression suppressor of ras 4 (MESR4) was found to reduce lipid accumulation in the fat body, whereas MESR4 knockdown increases it. It was further shown that MESR4 up-regulates the expression of major lipases, which may account for the reduction in lipid storage in the fat body and the release of free fatty acids (FFAs) in the body. These results suggest that MESR4 acts as an important upstream regulator of energy homeostasis.

Garrido, D., Rubin, T., Poidevin, M., Maroni, B., Le Rouzic, A., Parvy, J. P. and Montagne, J. (2015) Fatty acid synthase cooperates with Glyoxalase 1 to protect against sugar toxicity. PLoS Genet 11: e1004995. PubMed ID: 25692475
Fatty acid (FA) metabolism is deregulated in several human diseases including metabolic syndrome, type 2 diabetes and cancers. Therefore, FA-metabolic enzymes are potential targets for drug therapy, although the consequence of these treatments must be precisely evaluated at the organismal and cellular levels. In healthy organism, synthesis of triacylglycerols (TAGs)-composed of three FA units esterified to a glycerol backbone-is increased in response to dietary sugar. Saturation in the storage and synthesis capacity of TAGs is associated with type 2 diabetes progression. Sugar toxicity likely depends on advanced-glycation-end-products (AGEs) that form through covalent bounding between amine groups and carbonyl groups of sugar or their derivatives alpha-oxoaldehydes. Methylglyoxal (MG) is a highly reactive alpha-oxoaldehyde that is derived from glycolysis through a non-enzymatic reaction. Glyoxalase 1 (Glo1; see Drosophila Glo1 works to neutralize MG, reducing its deleterious effects. This study used the power of Drosophila genetics to generate Fatty acid synthase (FASN) mutants, allowing an investigation of the consequence of this deficiency upon sugar-supplemented diets. FASN mutants were found to be lethal but can be rescued by an appropriate lipid diet. Rescued animals do not exhibit insulin resistance, are dramatically sensitive to dietary sugar and accumulate AGEs. FASN and Glo1 cooperate at systemic and cell-autonomous levels to protect against sugar toxicity. It was observed that the size of FASN mutant cells decreases as dietary sucrose increases. Genetic interactions at the cell-autonomous level, where glycolytic enzymes or Glo1 were manipulated in FASN mutant cells, revealed that this sugar-dependent size reduction is a direct consequence of MG-derived-AGE accumulation. In summary, these findings indicate that FASN is dispensable for cell growth if extracellular lipids are available. In contrast, FA-synthesis appears to be required to limit a cell-autonomous accumulation of MG-derived-AGEs, supporting the notion that MG is the most deleterious alpha-oxoaldehyde at the intracellular level.

Rinkevich, F. D., Du, Y., Tolinski, J., Ueda, A., Wu, C. F., Zhorov, B. S. and Dong, K. (2015) Distinct roles of the DmNa and DSC1 channels in the action of DDT and pyrethroids. Neurotoxicology [Epub ahead of print]. PubMed ID: 25687544
Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (parats) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, d a DmNav/DSC1 double mutant line was generated by introducing a parats1allele (carrying the I265N mutation) into a DSC1 knockout line. The I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, it was found that I265N-mediated pyrethroid resistance in parats1 mutant flies was almost completely abolished in parats;DSC1-/- double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies, and the parats;DSC1-/- double mutant flies were even more resistant to DDT compared to the DSC1 knockout or parats1 mutant. These findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance.

Thursday, March 5th

Lopes, C.S. and Casares, F. (2015). Eye selector logic for a coordinated cell cycle exit. PLoS Genet 11: e1004981. PubMed ID: 25695251
Organ-selector transcription factors control simultaneously cell differentiation and proliferation, ensuring the development of functional organs and their homeostasis. How this is achieved at the molecular level is still unclear. This study has investigated how the transcriptional pulse of string/cdc25 (stg), the universal mitotic trigger, is regulated during Drosophila retina development as an example of coordinated deployment of differentiation and proliferation programs. The study identified the eye specific stg enhancer, stg-FMW, and showed that Pax6 selector genes, in cooperation with Eya and So, two members of the retinal determination network, activated stg-FMW, establishing a positive feed-forward loop. This loop was negatively modulated by the Meis1 protein, Hth. This regulatory logic was reminiscent of that controlling the expression of differentiation transcription factors. This work shows that subjecting transcription factors and key cell cycle regulators to the same regulatory logic ensures the coupling between differentiation and proliferation programs during organ development.

Bergman, Z. J., McLaurin, J. D., Eritano, A. S., Johnson, B. M., Sims, A. Q. and Riggs, B. (2015) Spatial reorganization of the endoplasmic reticulum during mitosis relies on mitotic kinase Cyclin A in the early Drosophila embryo. PLoS One 10: e0117859. PubMed ID: 25689737
Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. Microscopic observation revealed that the dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. This study shows that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. These results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope.

Defachelles, L., Hainline, S. G., Menant, A., Lee, L. A. and Karess, R. E. (2015) A maternal effect rough deal mutation suggesting multiple pathways regulating Drosophila RZZ kinetochore recruitment.J Cell Sci [Epub ahead of print]. PubMed ID: 25616898
Proper kinetochore recruitment and regulation of Dynein and the Mad1-Mad2 complex requires the Rod-Zw10-Zwilch (RZZ) complex. This study describes rodZ3, a maternal-effect Drosophila mutation changing a single residue in the Rough Deal (Rod) subunit of RZZ. Although RZ3ZZ complex is present in early syncytial stage embryos laid by homozygous rodZ3 mothers, it is not recruited to kinetochores. Consequently, the embryos have no spindle assembly checkpoint (SAC), and syncytial mitoses are profoundly perturbed. The polar body (residual meiotic products) cannot remain in its SAC-dependent metaphase-like state, and decondenses into chromatin. In neuroblasts of homozygous rodZ3 larvae, RZ3ZZ recruitment is only partially reduced, the SAC is functional and mitosis is relatively normal. RZ3ZZ nevertheless behaves abnormally: it does not further accumulate on kinetochores when microtubules are depolymerized; it reduces the rate of Mad1 recruitment; and it dominantly interferes with the dynein-mediated streaming of RZZ from attached kinetochores. These results suggest that the mutated residue of rodZ3 is required for normal RZZ kinetochore recruitment and function and moreover that the RZZ recruitment pathway may differ in syncytial stage embryos and post-embryonic somatic cells.

Klebba, J.E., Galletta, B.J., Nye, J., Plevock, K.M., Buster, D.W., Hollingsworth, N.A., Slep, K.C., Rusan, N.M. and Rogers, G.C. (2015). Two Polo-like kinase 4 binding domains in Asterless perform distinct roles inregulating kinase stability. J Cell Biol 208: 401-414. PubMed ID: 25688134
Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4's tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. This study investigated the Asl-Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl was not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus was required. Mechanistic analyses of the different Asl regions revealed that they acted uniquely during the cell cycle: the Asl N terminus promoted Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilized Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.

Wednesday, March 4th

Kim, G., Pai, C.I., Sato, K., Person, M.D., Nakamura, A. and Macdonald, P.M. (2015). Region-specific activation of oskar mRNA translation by inhibition of Bruno-mediated repression. PLoS Genet 11: e1004992. PubMed ID: 25723530
A complex program of translational repression, mRNA localization, and translational activation ensures that Oskar (Osk) protein accumulates only at the posterior pole of the Drosophila oocyte. Inappropriate expression of Osk disrupts embryonic axial patterning, and is lethal. A key factor in translational repression is Bruno (Bru), which binds to regulatory elements in the osk mRNA 3'UTR. After posterior localization of osk mRNA, repression by Bru must be alleviated. This study describes an in vivo assay system to monitor the spatial pattern of Bru-dependent repression, separate from the full complexity of osk regulation. This assay revealed a form of translational activation-region-specific activation-which acted regionally in the oocyte, was not mechanistically coupled to mRNA localization, and functioned by inhibiting repression by Bru. Bru was also shown to dimerize, and mutations were identified that disrupted this interaction, to test its role in vivo. Loss of dimerization did not disrupt repression, as might have been expected from an existing model for the mechanism of repression. However, loss of dimerization did impair regional activation of translation, suggesting that dimerization might constrain, not promote, repression. This work provides new insight into the question of how localized mRNAs become translationally active, showing that repression of osk mRNA is locally inactivated by a mechanism acting independent of mRNA localization.

Fernando, C., Audibert, A., Simon, F., Tazi, J. and Juge, F. (2015) A role for the Serine/Arginine-Rich (SR) protein B52/SRSF6 in cell growth and Myc expression in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 25680814
Serine/arginine-rich (SR) proteins are RNA-binding proteins that are primarily involved in alternative splicing. Expression of some SR proteins is frequently upregulated in tumors, and previous reports have demonstrated that these proteins can directly participate in cell transformation. Identifying factors that can rescue the effects of SR overexpression in vivo is, therefore, of potential therapeutic interest. This study analyzed phenotypes induced by overexpression of the SR protein B52 during Drosophila development and identified several proteins that can rescue these phenotypes. Using the mechanosensory bristle lineage as a developmental model, this study showed that B52 expression level influences cell growth, but not differentiation, in this lineage. In particular, B52 overexpression increases cell growth, upregulates myc transcription, and gives rise to flies lacking thoracic bristles. Using a genetic screen, several suppressors of the phenotypes induced by overexpression of B52 in vivo were identified in two different organs. Upregulation of brain tumor (brat), a tumor suppressor and post-transcriptional repressor of myc, and downregulation of lilliputian (lilli), a subunit of the superelongation complex involved in transcription elongation, efficiently rescue the phenotypes induced by B52 overexpression. hese results demonstrate a role of this SR protein in cell growth, and identify candidate proteins that may potentially overcome the effects of SR protein overexpression in mammals.

Lakhotia, S. C., Mallik, M., Singh, A. K. and Ray, M. (2012) The large noncoding hsrω-n transcripts are essential for thermotolerance and remobilization of hnRNPs, HP1 and RNA polymerase II during recovery from heat shock in Drosophila. Chromosoma 121: 49-70. PubMed ID: 21913129
The hs-GAL4t-driven expression of the Heat shock RNA ω (hsrω)-RNAi transgene or EP93D allele of the noncoding hsrω resulted in global down- or upregulation, respectively, of the large hsrω transcripts following heat shock. Subsequent to temperature shock, hsrω-null or those expressing hsrω-RNAi or the EP93D allele displayed delayed lethality of most embryos, first or third instar larvae. Three-day-old hsrω-null flies mostly died immediately or within a day after heat shock. Heat-shock-induced RNAi or EP expression in flies caused only a marginal lethality but severely affected oogenesis. EP allele or hsrω-RNAi expression after heat shock did not affect heat shock puffs and Hsp70 synthesis. Both down- and upregulation of hsrω-n transcripts suppressed reappearance of the hsrω-n transcript-dependent nucleoplasmic ω speckles during recovery from heat shock. Hrp36, heterochromatin protein 1, and active RNA pol II in unstressed or heat-shocked wild-type or hsrω-null larvae or those expressing the hs-GAL4t-driven hsrω-RNAi or the EP93D allele were comparably distributed on polytene chromosomes. Redistribution of these proteins to pre-stress locations after a 1- or 2-h recovery was severely compromised in glands with down- or upregulated levels of hsrω-n transcripts after heat shock. The hsrω-null unstressed cells always lacked ω speckles and little Hrp36 moved to any chromosome region following heat shock, and its relocation to chromosome regions during recovery was also incomplete. This study reveals for the first time that the spatial restoration of key regulatory factors like hnRNPs, HP1, or RNA pol II to their pre-stress nuclear targets in cells recovering from thermal stress is dependent upon critical level of the large hsrω-n noncoding RNA. In the absence of their relocation to pre-stress chromosome sites, normal developmental gene activity fails to be restored, which finally results in delayed organismal death.

Abruzzi, K., Chen, X., Nagoshi, E., Zadina, A. and Rosbash, M. (2015) RNA-seq profiling of small numbers of Drosophila neurons. Methods Enzymol 551: 369-386. PubMed ID: 25662465
Drosophila melanogaster has a robust circadian clock, which drives a rhythmic behavior pattern: locomotor activity increases in the morning shortly before lights on (M peak) and in the evening shortly before lights off (E peak). This pattern is controlled by ~75 pairs of circadian neurons in the Drosophila brain. One key group of neurons is the M-cells (PDF+ large and small LNvs), which control the M peak. A second key group is the E-cells, consisting of four LNds and the fifth small LNv, which control the E peak. Recent studies show that the M-cells have a second role in addition to controlling the M peak; they communicate with the E-cells (as well as DN1s) to affect their timing, probably as a function of environmental conditions. To learn about molecules within the M-cells important for their functional roles, methods were adapted to manually sort fluorescent protein-expressing neurons of interest from dissociated Drosophila brains. mRNA and miRNA were isolated from sorted M-cells and the resulting DNAs were amplified to create deep-sequencing libraries. Visual inspection of the libraries illustrates that they are specific to a particular neuronal subgroup; M-cell libraries contain timeless and dopaminergic cell libraries contain pale/TH. Using these data, it is possible to identify cycling transcripts as well as many mRNAs and miRNAs specific to or enriched in particular groups of neurons.

Tuesday, March 3rd

Leitão, A.B. and Sucena, È. (2015). Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation. Elife 4. PubMed ID: 25650737
Virtually all species of coelomate animals contain blood cells that display a division of labor necessary for homeostasis. This functional partition depends upon the balance between proliferation and differentiation mostly accomplished in the hematopoietic organs. In Drosophila melanogaster, the lymph gland produces plasmatocytes and crystal cells that are not released until pupariation. Yet, throughout larval development, both hemocyte types increase in numbers. Mature plasmatocytes can proliferate but it is not known if crystal cell numbers increase by self-renewal or by de novo differentiation. This study shows that new crystal cells in third instar larvae originate through a Notch-dependent process of plasmatocyte transdifferentiation. This process occurred in the sessile clusters and was contingent upon the integrity of these structures. The existence of this hematopoietic tissue, relying on structure-dependent signaling events to promote blood homeostasis, creates a new paradigm for addressing outstanding questions in Drosophila hematopoiesis and establishing further parallels with vertebrate systems.

Zaessinger, S., Zhou, Y., Bray, S.J., Tapon, N. and Djiane, A. (2015). Drosophila MAGI interacts with RASSF8 to regulate E-Cadherin-based adherens junctions in the developing eye. Development [Epub ahead of print]. PubMed ID: 25725070
Morphogenesis is crucial during development to generate organs and tissues of the correct size and shape. During Drosophila late eye development, interommatidial cells (IOCs) rearrange to generate the highly organized pupal lattice, in which hexagonal ommatidial units pack tightly. This process involves the fine regulation of adherens junctions (AJs) and of adhesive E-Cadherin (E-Cad) complexes. Localized accumulation of Bazooka (Baz), the Drosophila PAR3 homolog, has emerged as a critical step to specify where new E-Cad complexes should be deposited during junction remodeling. However, the mechanisms controlling the correct localization of Baz are still only partly understood. This study shows that Drosophila Magi, the sole fly homolog of the mammalian MAGI scaffolds, is an upstream regulator of E-Cad-based AJs during cell rearrangements, and that Magi mutant IOCs fail to reach their correct position. They uncovered a direct physical interaction between Magi and the Ras association domain protein RASSF8 through a WW domain-PPxY motif binding, and showed that apical Magi recruited the RASSF8-ASPP complex during AJ remodeling in IOCs. Further, this Magi complex was required for the cortical recruitment of Baz and of the E-Cad-associated proteins α- and β-catenin. They propose that, by controlling the proper localization of Baz to remodeling junctions, Magi and the RASSF8-ASPP complex promote the recruitment or stabilization of E-Cad complexes at junction sites.

Farkas, R., Benova-Liszekova, D., Mentelova, L., Mahmood, S., Datkova, Z., Beno, M., Pecenova, L., Raska, O., Smigova, J., Chase, B. A., Raska, I. and Mechler, B. M. (2015) Vacuole dynamics in the salivary glands of Drosophila melanogaster during prepupal development. Dev Growth Differ 57: 74-96. PubMed ID: 25611296
A central function of the Drosophila salivary glands (SGs), historically known for their polytene chromosomes, is to produce and then release during pupariation the secretory glue used to affix a newly formed puparium to a substrate. This essential event in the life history of Drosophila is regulated by the steroid hormone ecdysone in the late-larval period. Ecdysone triggers a cascade of sequential gene activation that leads to glue secretion and initiates the developmentally-regulated programmed cell death (PCD) of the larval salivary glands, which culminates 16 h after puparium formation (APF). This study demonstrates that, even after the larval salivary glands have completed what is perceived to be one of their major biological functions - glue secretion during pupariation - they remain dynamic and physiologically active up until the execution phase of PCD. This study used specific metabolic inhibitors and genetic tools, including mutations or transgenes for shi, Rab5, Rab11, vha55, vha68-2, vha36-1, syx1A, syx4, and Vps35 to characterize the dramatic series of cellular changes occurring in the SG cells between pupariation and 7-8 h APF. Early in the prepupal period, they are remarkably active in endocytosis, forming acidic vacuoles. Midway through the prepupal period, there is abundant late endosomal trafficking and vacuole growth, which is followed later by vacuole neutralization and disappearance via membrane consolidation. This work provides new insights into the function of Drosophila SGs during the early- to mid-prepupal period.

Moussian, B., Letizia, A., Martinez-Corrales, G., Rotstein, B., Casali, A. and Llimargas, M. (2015) Deciphering the genetic programme triggering timely and spatially-regulated chitin deposition. PLoS Genet 11: e1004939. PubMed ID: 25617778
Organ and tissue formation requires a finely tuned temporal and spatial regulation of differentiation programmes. This is necessary to balance sufficient plasticity to undergo morphogenesis with the acquisition of the mature traits needed for physiological activity. This study addressed this issue by analysing the deposition of the chitinous extracellular matrix of Drosophila, an essential element of the cuticle (skin) and respiratory system (tracheae) in this insect. Chitin deposition requires the activity of the chitin synthase Krotzkopf verkehrt (Kkv). This process was shown to equally require the activity of two other genes, namely expansion (exp) and rebuf (reb; CG13183). Exp and Reb have interchangeable functions, and in their absence no chitin is produced, in spite of the presence of Kkv. Conversely, when Kkv and Exp/Reb are co-expressed in the ectoderm, they promote chitin deposition, even in tissues normally devoid of this polysaccharide. Therefore, these results indicate that both functions are not only required but also sufficient to trigger chitin accumulation. This mechanism is highly regulated in time and space, ensuring chitin accumulation in the correct tissues and developmental stages. Accordingly, it was observed that unregulated chitin deposition disturbs morphogenesis, thus highlighting the need for tight regulation of this process. In summary, this study has identified the genetic programme that triggers the timely and spatially regulated deposition of chitin and thus provide new insights into the extracellular matrix maturation required for physiological activity

Monday, March 2nd

Deady, L.D., Shen, W., Mosure, S.A., Spradling, A.C. and Sun, J. (2015). Matrix Metalloproteinase 2 is required for ovulation and corpus luteum formation in Drosophila. PLoS Genet. 11: e1004989. PubMed ID: 25695427
Ovulation is critical for successful reproduction and correlates with ovarian cancer risk, yet genetic studies of ovulation have been limited. It has long been thought that the mechanism controlling ovulation is highly divergent due to speciation and fast evolution. Using genetic tools available in Drosophila, this study now reports that ovulation in Drosophila strongly resembles mammalian ovulation at both the cellular and molecular levels. Just one of up to 32 mature follicles per ovary pair lost posterior follicle cells ("trimming") and protruded into the oviduct, showing that a selection process prefigured ovulation. Follicle cells that remained after egg release formed a "corpus luteum (CL)" at the end of the ovariole, developed yellowish pigmentation, and expressed genes encoding steroid hormone biosynthetic enzymes that were required for full fertility. Finally, matrix metalloproteinase 2 (Mmp2), a type of protease thought to facilitate mammalian ovulation, was expressed in mature follicle and CL cells. Mmp2 activity was genetically required for trimming, ovulation and CL formation. These studies provide new insights into the regulation of Drosophila ovulation and establish Drosophila as a model for genetically investigating ovulation in diverse organisms, including mammals.

Hartman, T. R., Ventresca, E. M., Hopkins, A., Zinshteyn, D., Singh, T., O'Brien, J. A., Neubert, B. C., Hartman, M. G., Schofield, H. K., Stavrides, K. P., Talbot, D. E., Riggs, D. J., Pritchard, C. and O'Reilly, A. M. (2015) Novel tools for genetic manipulation of follicle stem cells in the Drosophila ovary reveal an integrin-dependent transition from quiescence to proliferation. Genetics [Epub ahead of print]. PubMed ID: 25680813
In many tissues, the presence of stem cells is inferred by the capacity of the tissue to maintain homeostasis and undergo repair after injury. Isolation of self-renewing cells with the ability to generate the full array of cells within a given tissue strongly supports this idea, but the identification and genetic manipulation of individual stem cells within their niche remains a challenge. This study presents novel methods for marking and genetically altering epithelial Follicle Stem Cells (FSCs) within the Drosophila ovary. Using these new tools, a sequential, multi-step process was defined that comprises transitioning of FSCs from quiescence to proliferation. It was further demonstrated that integrins (see Myospheroid) are cell-autonomously required within FSCs to provide directional signals that are necessary at each step of this process. Thus, these methods may be used to define precise roles for specific genes in the sequential events that occur during FSC division after a period of quiescence.

Kimura, S. and Loppin, B. (2015). Two bromodomain proteins functionally interact to recapitulate an essential BRDT-like function in Drosophila spermatocytes. Open Biol 5. PubMed ID: 25652540
In mammals, the testis-specific bromodomain and extra terminal (BET) protein BRDT is essential for spermatogenesis. In Drosophila, it was recently reported that the tBRD-1 protein is similarly required for male fertility. Interestingly, however, tBRD-1 has two conserved bromodomains in its N-terminus but it lacks an extra terminal (ET) domain characteristic of BET proteins. Using proteomics approaches to search for tBRD-1 interactors, this study identified tBRD-2 as a novel testis-specific bromodomain protein. In contrast to tBRD-1, tBRD-2 contained a single bromodomain, but which was associated with an ET domain in its C-terminus. Strikingly, tbrd-2 knock-out males were sterile and displayed aberrant meiosis in a way highly similar to tbrd-1 mutants. Furthermore, these two factors co-localized and were interdependent in spermatocytes. The study proposes that Drosophila tBRD-1 and tBRD-2 associate into a functional BET complex in spermatocytes, which recapitulates the activity of the single mammalian BRDT-like protein.

Li, L., Li, P. and Xue, L. (2015) The RED domain of Paired is specifically required for Drosophila accessory gland maturation. Open Biol 5. PubMed ID: 25694546
The evolutionarily conserved paired domain consists of the N-terminal PAI and the C-terminal RED domains, each containing a helix-turn-helix motif capable of binding DNA. Despite its conserved sequence, the physiological functions of the RED domain remain elusive. This paper descibes the construction of a prd transgene expressing a truncated Paired (Prd) protein without the RED domain, and examination of its rescue ability in prd mutants. The RED domain was found to be specifically required for the expression of Acp26Aa and Sex Peptide in male accessory glands, and the induction of female post-mating response. These data thus identified an important physiological function for the evolutionarily conserved RED domain.

Sunday, March 1st

Finley, J. K., Miller, A. C. and Herman, T. G. (2015) Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye. Neural Dev 10: 2. PubMed ID: 25636358
Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye. R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp), as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens), as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce, Scm, or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices.

Davie, K., Jacobs, J., Atkins, M., Potier, D., Christiaens, V., Halder, G. and Aerts, S. (2015) Discovery of transcription factors and regulatory regions driving in vivo tumor development by ATAC-seq and FAIRE-seq open chromatin profiling. PLoS Genet 11: e1004994. PubMed ID: 25679813
Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. This study asked whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, two different approaches to detect open chromatin were compared in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. Both methods were found to reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, a screen was carried out for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and 3778 regions were identified that become (over-)activated during tumor development. Next, motif discovery was applied to search for candidate transcription factors that could bind these regions and AP-1 (Fos and Jun) and Stat92E were identified as key regulators. The importance of Stat92E in the development of the tumors was validated by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally tests were performed to see if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs; similar chromatin changes indeed occurred. Finally, it was determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, this study showed that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes.

Hüsken, M., Hufnagel, K., Mende, K., Appel, E., Meyer, H., Peisker, H., Tögel, M., Wang, S., Wolff, J., Gorb, S.N. and Paululat, A. (2015).Adhesive pad differentiation in Drosophila melanogaster depends on the Polycomb group gene Su(z)2. J Exp Biol [Epub ahead of print]. PubMed ID: 25714570
The ability of many insects to walk on vertical smooth surfaces such as glass or even on the ceiling has fascinated biologists for ages and has led to the discovery of highly specialized adhesive organs located at the distal end of the animals' legs. So far, research has primarily focused on structural and ultrastructural investigations leading to a deeper understanding of adhesive organ functionality and to the development of new bioinspired materials. Genetic approaches, e.g. the analysis of mutants, to achieve a better understanding of adhesive organ differentiation, have not been used so far. This study describes the first Drosophila melanogaster mutant that develops malformed adhesive organs, resulting in a complete loss of climbing ability on vertical smooth surfaces. Interestingly, these mutants failed to make close contact between the setal tips and the smooth surface, a crucial condition for wet adhesion mediated by capillary forces. Instead, these flies walked solely on their claws. Moreover, the mutation was caused by a P-element insertion into the Su(z)2 gene locus. Remobilization of the P-element restored climbing ability. Furthermore, the P-element insertion resulted in an artificial Su(z)2 transcript, which most likely caused a gain of function mutation. Presumably, this transcript caused deregulation of yet unknown target genes involved in pulvilli differentiation. These results nicely demonstrate that the genetically treatable model organism Drosophila is highly suitable for future investigations on adhesive organ differentiation.

Terzo, E. A., Lyons, S. M., Poulton, J. S., Temple, B. R., Marzluff, W. F. and Duronio, R. J. (2015) Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body. Mol Biol Cell. PubMed ID: 25694448
Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus, were studied. Biochemical analysis was coupled with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi-Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel SIF (Self Interaction Facilitator) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or SIF domains severely impairs Mxc function in vivo resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Lastly, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA processing factor, to the HLB. It is concluded that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis.

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