What's hot today: Current papers in developmental biology and gene function Follow @interactivefly Tweet

Monday, June 15th

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

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

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

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

Sunday, June 14th

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

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

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

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

Saturday, June 13th

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

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

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

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

Friday, June 12th

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

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

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

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

Thursday, June 11th

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

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

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

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

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

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

Wednesday, June 10th

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

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

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

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

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

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

Tuesday, June 9th

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

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

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

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

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

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

Monday, June 8th

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

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

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

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

Sunday, June 7th

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

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

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

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

Saturday, June 6th

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

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

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

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

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

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

Friday, June 5th

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

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

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

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

Thursday, June 4th

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

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

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

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

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

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

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

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

Wednesday, June 3rd

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

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

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

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

Tuesday, June 2nd

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

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

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

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

Monday, June 1st

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

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

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

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

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