What's hot today
Tuesday, September 30th, 2014
Chen, X. J., Squarr, A. J., Stephan, R., Chen, B., Higgins, T. E., Barry, D. J., Martin, M. C., Rosen, M. K., Bogdan, S. and Way, M. (2014). Ena/VASP proteins cooperate with the WAVE complex to regulate the actin cytoskeleton. Dev Cell 30: 569-584. PubMed ID: 25203209
Ena/VASP proteins and the WAVE regulatory complex (WRC) regulate cell motility by virtue of their ability to independently promote actin polymerization. This study demonstrates that Ena/VASP and the WRC control actin polymerization in a cooperative manner through the interaction of the Ena/VASP EVH1 domain with an extended proline rich motif in Abelson interacting protein (Abi). This interaction increases cell migration and enables VASP to cooperatively enhance WRC stimulation of Arp2/3 complex-mediated actin assembly in vitro in the presence of Rac. Loss of this interaction in Drosophila macrophages results in defects in lamellipodia formation, cell spreading, and redistribution of Ena to the tips of filopodia-like extensions. Rescue experiments of abi mutants also reveals a physiological requirement for the Abi:Ena interaction in photoreceptor axon targeting and oogenesis. These data demonstrate that the activities of Ena/VASP and the WRC are intimately linked to ensure optimal control of actin polymerization during cell migration and development.
Sailem, H., Bousgouni, V., Cooper, S. and Bakal, C. (2014). Cross-talk between Rho and Rac GTPases drives deterministic exploration of cellular shape space and morphological heterogeneity. Open Biol 4: 130132. PubMed ID: 24451547
One goal of cell biology is to understand how cells adopt different shapes in response to varying environmental and cellular conditions. Achieving a comprehensive understanding of the relationship between cell shape and environment requires a systems-level understanding of the signalling networks that respond to external cues and regulate the cytoskeleton. Classical biochemical and genetic approaches have identified thousands of individual components that contribute to cell shape, but it remains difficult to predict how cell shape is generated by the activity of these components using bottom-up approaches because of the complex nature of their interactions in space and time. This study describes the regulation of cellular shape by signalling systems using a top-down approach. The shape diversity generated by systematic RNAi screening was exploited, and the shape space a migratory cell explores was comprehensively defined. A simple Boolean model, involving the activation of Rac and Rho GTPases in two compartments is suggested to explain the basis for all cell shapes in the dataset (see Boolean model - Figure 4). Critically, a probabilistic graphical model was generated to show how cells explore this space in a deterministic, rather than a stochastic, fashion. The predictions made by the model were evaluated using live-cell imaging. This work explains how cross-talk between Rho and Rac can generate different cell shapes, and thus morphological heterogeneity, in genetically identical populations (Sailem, 2014).
Nie, J., Mahato, S. and Zelhof, A. C. (2014). The actomyosin machinery is required for Drosophila retinal lumen formation. PLoS Genet 10: e1004608. PubMed ID: 25233220
Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, Actin5C was identifed as a key molecule. The results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter (see Model for Drosophila retinal lumen formation). Together, these results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen.
Diggle, C. P., et al. (2014). HEATR2 plays a conserved role in assembly of the ciliary motile apparatus. PLoS Genet 10: e1004577. PubMed ID: 25232951
Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, a recently elucidated cilia transcriptional RFX-FOX code was used to identify novel PCD candidate genes. This study reports characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with outer arm dynein intermediate chain (DNAI2; see dynein, axonemal, intermediate chain 2), but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.
Monday, September 29th
Pekkurnaz, G., Trinidad, J. C., Wang, X., Kong, D. and Schwarz, T. L. (2014). Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase. Cell 158: 54-68. PubMed ID: 24995978
Cells allocate substantial resources toward monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. This study reports a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT; termed super sex combs by FlyBase), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. The mitochondrial motor-adaptor protein Milton was established as a required substrate for OGT to arrest mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. The GlcNAcylation state of Milton was found to be altered by extracellular glucose, and OGT was found to alter mitochondrial motility in vivo. These findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability.
Rauschenbach, I. Y., Karpova, E. K., Adonyeva, N. V., Andreenkova, O. V., Faddeeva, N. V., Burdina, E. K., Alekseev, A. A., Menshanov, P. N. and Gruntenko, N. E. (2014). Disruption of insulin signalling affects the neuroendocrine stress reaction in Drosophila females. J Exp Biol [Epub ahead of print]. PubMed ID: 25214494
Juvenile hormone (JH) and dopamine are involved in the stress response in insects. The insulin/insulin-like growth factor signalling pathway has also recently been found to be involved in the regulation of various processes, including stress tolerance. However, the relationships among the JH, dopamine and insulin signalling pathways remain unclear. The role of insulin signalling in the regulation of JH and dopamine metabolism under normal and heat stress conditions was investigated in Drosophila melanogaster females. Suppression of the insulin-like receptor (InR) in the corpus allatum, a specialised endocrine gland that synthesises JH, causes an increase in dopamine level and JH-hydrolysing activity and alters the activities of enzymes that produce as well as those that degrade dopamine (alkaline phosphatase (ALP), tyrosine hydroxylase (TH) and dopamine-dependent arylalkylamine N-acetyltransferase (DAT)). It was also found that InR suppression in the corpus allatum modulates dopamine, ALP, TH and JH-hydrolysing activity in response to heat stress and that it decreases the fecundity of the flies. JH application restores dopamine metabolism and fecundity in females with decreased InR expression in the corpus allatum. These data provide evidence that the insulin/insulin-like growth factor signalling pathway regulates dopamine metabolism in females of D. melanogaster via the system of JH metabolism and that it affects the development of the neuroendocrine stress reaction and interacts with JH in the control of reproduction in this species.
Tian, L., Chen, J., Chen, M., Gui, C., Zhong, C. Q., Hong, L., Xie, C., Wu, X., Yang, L., Ahmad, V. and Han, J. (2014). The p38 pathway regulates oxidative stress tolerance by phosphorylation of mitochondrial protein IscU. J Biol Chem [Epub ahead of print]. PubMed ID: 25204651
The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However the underlying mechanisms are largely unknown. This study demonstrates that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. The importance of MK2 was shown as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, the iron-sulfur cluster scaffold protein IscU was identified as a new substrate of MK2 both in Drosophila and mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondria iron-sulfur clusters.
Mondal, B. C., Shim, J., Evans, C. J. and Banerjee, U. (2014). Pvr expression regulators in equilibrium signal control and maintenance of Drosophila blood progenitors. Elife: e03626. PubMed ID: 25201876
Blood progenitors within the lymph gland, a larval organ that supports hematopoiesis in Drosophila melanogaster, are maintained by integrating signals emanating from niche-like cells and those from differentiating blood cells. The signal from differentiating cells has been termed the 'equilibrium signal' in order to distinguish it from the 'niche signal'. Earlier work showed that Equilibrium signaling utilizes Pvr (the Drosophila PDGF/VEGF receptor), STAT92E, and Adenosine deaminase-related growth factor A (ADGF-A). Little is known about how this signal initiates during hematopoietic development. To identify new genes involved in lymph gland blood progenitor maintenance, particularly those involved in equilibrium signaling, a genetic screen was performed that identified bip1 (bric a brac interacting protein 1) and Nucleoporin 98 (Nup98) as additional regulators of the equilibrium signal. The products of these genes along with the Bip1-interacting protein RpS8 (Ribosomal protein S8) are required for the proper expression of Pvr.
Sunday, September 28th
Devergne, O., Tsung, K., Barcelo, G. and Schupbach, T. (2014). Polarized deposition of basement membrane proteins depends on Phosphatidylinositol synthase and the levels of Phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci U S A 111: 7689-7694. PubMed ID: 24828534
The basement membrane (BM), a specialized sheet of the extracellular matrix contacting the basal side of epithelial tissues, plays an important role in the control of the polarized structure of epithelial cells. However, little is known about how BM proteins themselves achieve a polarized distribution. This study identifies phosphatidylinositol 4,5-bisphosphate (PIP2) as a critical regulator of the polarized secretion of BM proteins. A decrease of PIP2 levels, in particular through mutations in Phosphatidylinositol synthase (Pis) and other members of the phosphoinositide pathway, leads to the aberrant accumulation of BM components at the apical side of the cell without primarily affecting the distribution of apical and basolateral polarity proteins. In addition, PIP2 controls the apical and lateral localization of Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a factor specifically required to prevent aberrant apical secretion of BM. It is proposed that PIP2, through the control of Crag's subcellular localization, restricts the secretion of BM proteins to the basal side.
Sing, A., Tsatskis, Y., Fabian, L., Hester, I., Rosenfeld, R., Serricchio, M., Yau, N., Bietenhader, M., Shanbhag, R., Jurisicova, A., Brill, J. A., McQuibban, G. A. and McNeill, H. (2014). The atypical cadherin fat directly regulates mitochondrial function and metabolic state. Cell 158: 1293-1308. PubMed ID: 25215488
Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. This study shows Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ftmito) that is imported into mitochondria. Ftmito binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect.
Chen, Y., Wang, Z., Wang, P., Li, D., Zhou, J. and Wu, S. (2014). CYLD negatively regulates Hippo signaling by limiting Hpo phosphorylation in Drosophila. Biochem Biophys Res Commun. PubMed ID: 25201729
Cylindromatosis (CYLD), a deubiquitinase and regulator of microtubule dynamics, has important roles in the regulation of inflammation, immune response, apoptosis, mitosis, cell migration and tumorigenesis. Although great progress has been made in the biochemical and cellular functions of CYLD, its role in animal development remains elusive. This study identified Drosophila CYLD (dCYLD) as a negative regulator of the Hippo pathway in vivo. dCYLD associates and colocalizes with Hpo, a core component of the Hippo pathway, in the cytoplasm, and decreases Hpo activity through limiting its phosphorylation at T195. dCYLD also limits Hippo signal transduction as evidenced by decreasing phosphorylation and thereby increasing activity of Yki, the key downstream effector of the Hippo pathway. These findings uncover dCYLD as a negative regulator of the Hippo pathway and provide new insights into the physiological function of dCYLD in animal development.
Merkel, M., Sagner, A., Gruber, F. S., Etournay, R., Blasse, C., Myers, E., Eaton, S., Julicher, F. (2014). The balance of Prickle/Spiny-Legs isoforms controls the amount of coupling between core and Fat PCP Systems. Curr Biol [Epub ahead of print]. PubMed ID: 25201685
The conserved Fat and Core planar cell polarity (PCP) pathways work together to specify tissue-wide orientation of hairs and ridges in the Drosophila wing. Their components form intracellularly polarized complexes at adherens junctions that couple the polarity of adjacent cells and form global patterns. How Fat and Core PCP systems interact is not understood. Some studies suggest that Fat PCP directly orients patterns formed by Core PCP components. Others implicate oriented tissue remodeling in specifying Core PCP patterns. This study used genetics, quantitative image analysis, and physical modeling to study Fat and Core PCP interactions during wing development. Their patterns were shown to change during morphogenesis, undergoing phases of coupling and uncoupling that are regulated by antagonistic Core PCP protein isoforms Prickle and Spiny-legs. Evolving patterns of Core PCP are hysteretic: the early Core PCP pattern is modified by tissue flows and then by coupling to Fat PCP, producing sequential patterns that guide hairs and then ridges. These data quantitatively account for altered hair and ridge polarity patterns in PCP mutants. Premature coupling between Fat and Core PCP explains altered polarity patterns in pk mutants. In other Core PCP mutants, hair polarity patterns are guided directly by Fat PCP. When both systems fail, hairs still align locally and obey signals associated with veins. It is concluded that temporally regulated coupling between the Fat and Core PCP systems enables a single tissue to develop sequential polarity patterns that orient distinct morphological structures.
Saturday, September 27th
Gomez-Lamarca, M. J., Cobreros-Reguera, L., Ibanez-Jimenez, B., Palacios, I. M. and Martin-Bermudo, M. D. (2014). Integrins regulate epithelial cell differentiation by modulating Notch activity. J Cell Sci [Epub ahead of print]. PubMed ID: 25179603
Coordinating exit from the cell cycle with differentiation is critical for proper development and tissue homeostasis. Failure to do so can lead to aberrant organogenesis and tumorigenesis. However, little is known about the developmental signals that regulate the cell cycle exit-to-differentiation switch. Signals downstream of two key developmental pathways, Notch and Salvador-Warts-Hippo (SWH), and of myosin activity regulate this switch during the development of the follicle cell epithelium of the Drosophila ovary. This study identified a fourth player, the integrin signaling pathway (see Myospheroid). Elimination of integrin function blocks mitosis-to-endocycle switch and differentiation in posterior follicle cells (PFCs), via regulation of the CDK inhibitor Dacapo. In addition, integrin mutant PFCs show defective Notch signalling and endocytosis. Furthermore, integrins act in PFCs by modulating the activity of the Notch pathway, as reducing the amount of Hairless, the major antagonist of Notch, or misexpressing Notch intracellular domain rescues the cell cycle and differentiation defects. Altogether, these findings reveal a direct involvement of integrin signalling on the spatial and temporal regulation of epithelial cell differentiation during development.
Kunttas-Tatli, E., Roberts, D. M. and McCartney, B. M. (2014). Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex. Mol Biol Cell [Epub ahead of print]. PubMed ID: 25208568
The tumor suppressor Adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3beta and CK1 that targets β-catenin/Armadillo (β-cat/Arm) for proteosomal degradation. The destruction complex forms macromolecular particles termed the destructosome. While APC functions in the complex through its ability to bind both β-cat and Axin, it is hypothesized that APC proteins play an additional role in destructosome assembly through self-association. This study shows that a novel N-terminal coil, the APC Self-Association Domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells. Consistent with this, removal of the ASAD from the Drosophila embryo results in β-cat/Arm accumulation and aberrant Wnt pathway activation. These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine.
Abeysundara, N., Leung, A. C., Primrose, D. A. and Hughes, S. C. (2014). Regulation of cell proliferation and adhesion via a novel region of Drosophila Merlin interacting with Sip1. Dev Dyn [Epub ahead of print]. PubMed ID: 25204795
The tumour suppressor protein Merlin is thought to regulate cell proliferation and cell adhesion through interaction with protein partners. Loss of Merlin is associated with Neurofibromatosis Type 2 (NF2) tumours. NHERF1 or EBP50 is a scaffolding protein that functions in apical organization of polarized cells. Merlin and NHERF1 have been shown to interact in vitro in vertebrates. This study investigated how the Drosophila NHERF1 orthologue, Sip1, and Merlin function to regulate cell proliferation and adhesion. Two conserved arginine residues (R325 and R335) were identified in Merlin that, in addition to the FERM domain, are required for interaction with Sip1. Mutation of the arginine residues result in reduced Sip1 binding to Merlin and loss of Merlin growth suppressor function. Over-expression of MerlinR325A and/or MerlinR335L in Drosophila wings result in increased proliferation in the adult wing (increase in size), which is rescued by co-over-expression of constitutively active Merlin protein. Reduced Sip1 binding to Merlin also produces defects in adhesion in follicle epithelial cells. It is concluded that Sip1 facilitates the activation of Merlin as a tumour suppressor protein. Thus, this work provides insight into how Merlin functions as a tumour suppressor and in adhesion and this provides insight into the mechanism of NF2 pathogenesis.
Liu, C., Lin, C., Gao, C., May-Simera, H., Swaroop, A. and Li, T. (2014). Null and hypomorph Prickle1 alleles in mice phenocopy human Robinow syndrome and disrupt signaling downstream of Wnt5a. Biol Open 3(9):861-70. PubMed ID: 25190059
Planar cell polarity (PCP) signaling plays a critical role in tissue morphogenesis. In mammals, disruption of three of the six 'core PCP' components results in polarity-dependent defects with rotated cochlear hair cell stereocilia and open neural tube. Recent studies have demonstrated a role of Prickle1, a core PCP molecule Prickle in Drosophila, in mammalian neuronal development. To examine Prickle1 function along a broader developmental window, three mutant alleles were generated in mice. The complete loss of Prickle1 leads to systemic tissue outgrowth defects, aberrant cell organization and disruption of polarity machinery. Curiously, Prickle1 mutants recapitulate the characteristic features of human Robinow syndrome and phenocopy mouse mutants with Wnt5a (see Drosophila Wingless) or Ror2 gene defects, prompting an exploration of an association of Prickle1 with the Wnt pathway. This study shows that Prickle1 is a proteasomal target of Wnt5a signaling and that Dvl2 (see Drosophila Disheveled), a target of Wnt5a signaling, is misregulated in Prickle1 mutants. These studies implicate Prickle1 as a key component of the Wnt-signaling pathway and suggest that Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome.
Friday, September 26th
Archbold, H. C., Broussard, C., Chang, M. V. and Cadigan, K. M. (2014). Bipartite recognition of DNA by TCF/Pangolin is remarkably flexible and contributes to transcriptional responsiveness and tissue specificity of Wingless signaling. PLoS Genet 10: e1004591. PubMed ID: 25188465
The T-cell factor (TCF) family of transcription factors are major mediators of Wnt/beta-catenin signaling in metazoans. All TCFs contain a High Mobility Group (HMG) domain that possesses specific DNA binding activity. In addition, many TCFs contain a second DNA binding domain, the C-clamp, which binds to DNA motifs referred to as Helper sites. While HMG and Helper sites are both important for the activation of several Wnt dependent cis-regulatory modules (W-CRMs), the rules of what constitutes a functional HMG-Helper site pair are unknown. This study employed a combination of in vitro binding, reporter gene analysis and bioinformatics to address this question, using the Drosophila family member TCF/Pangolin (TCF/Pan) as a model. It was found that while there are constraints for the orientation and spacing of HMG-Helper pairs, the presence of a Helper site near a HMG site in any orientation increases binding and transcriptional response, with some orientations displaying tissue-specific patterns. Altering an HMG-Helper site pair from a sub-optimal to optimal orientation/spacing dramatically increases the responsiveness of a W-CRM in several fly tissues. In addition, the knowledge gained was used to bioinformatically identify two novel W-CRMs, one that was activated by Wnt/beta-catenin signaling in the prothoracic gland, a tissue not previously connected to this pathway. In sum, this work extends the importance of Helper sites in fly W-CRMs and suggests that the type of HMG-Helper pair is a major factor in setting the threshold for Wnt activation and tissue-responsiveness.
Esteves, F. F., Springhorn, A., Kague, E., Taylor, E., Pyrowolakis, G., Fisher, S. and Bier, E. (2014). BMPs regulate msx gene expression in the dorsal neuroectoderm of Drosophila and vertebrates by distinct mechanisms. PLoS Genet 10: e1004625. PubMed ID: 25210771
In a broad variety of bilaterian species the trunk central nervous system (CNS) derives from three primary rows of neuroblasts. The fates of these neural progenitor cells are determined in part by three conserved transcription factors: vnd/nkx2.2, ind/gsh and msh/msx in Drosophila melanogaster/vertebrates, which are expressed in corresponding non-overlapping patterns along the dorsal-ventral axis. While this conserved suite of 'neural identity' gene expression strongly suggests a common ancestral origin for the patterning systems, it is unclear whether the original regulatory mechanisms establishing these patterns have been similarly conserved during evolution. In Drosophila, genetic evidence suggests that Bone Morphogenetic Proteins (BMPs; see Drosophila Dpp) act in a dosage-dependent fashion to repress expression of neural identity genes. BMPs also play a dose-dependent role in patterning the dorsal and lateral regions of the vertebrate CNS, however, the mechanism by which they achieve such patterning has not yet been clearly established. This report examined the mechanisms by which BMPs act on cis-regulatory modules (CRMs) that control localized expression of the Drosophila msh and zebrafish (Danio rerio) msxB in the dorsal central nervous system (CNS). This analysis suggests that BMPs act differently in these organisms to regulate similar patterns of gene expression in the neuroectoderm: repressing msh expression in Drosophila, while activating msxB expression in the zebrafish. These findings suggest that the mechanisms by which the BMP gradient patterns the dorsal neuroectoderm have reversed since the divergence of these two ancient lineages.
Cheng, Y., Brunner, A. L., Kremer, S., DeVido, S. K., Stefaniuk, C. M. and Kassis, J. A. (2014). Co-regulation of invected and engrailed by a complex array of regulatory sequences in Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 25172431
invected (inv) and engrailed (en) form a gene complex that extends about 115kb. These two genes encode highly related homeodomain proteins that are co-regulated in a complex manner throughout development. Dissection of inv/en regulatory DNA shows that most enhancers are spread throughout a 62kb region. Two types of constructs were used to analyze the function of this DNA: P-element based reporter constructs with small pieces of DNA fused to the en promoter driving lacZ expression and large constructs with HA-tagged en and inv inserted in the genome with the phiC31 system. In addition, deletions of inv and en DNA were generated in situ, and their effects on inv/en expression were assayed. The results support and extend knowledge of inv/en regulation. First, inv and en share regulatory DNA, most of which is flanking the en transcription unit. In support of this, a 79-kb HA-en transgene can rescue inv en double mutants to viable, fertile adults. In contrast, an 84-kb HA-inv transgene lacks most of the enhancers for inv/en expression. Second, there are multiple enhancers for inv/en stripes in embryos; some of these may be redundant but others play discrete roles at different stages of embryonic development. Finally, no small reporter construct gave expression in the posterior compartment of imaginal discs, a hallmark of inv/en expression. Robust expression of HA-en in the posterior compartment of imaginal discs is evident from the 79-kb HA-en transgene, while a 45-kb HA-en transgene gives weaker, variable imaginal disc expression. It is suggested that the activity of the imaginal disc enhancer(s) is dependent on the chromatin structure of the inv/en domain.
Bing, X., Rzezniczak, T. Z., Bateman, J. R. and Merritt, T. J. (2014). Transvection-based gene regulation in Drosophila is a complex and plastic trait. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 25213691
Transvection, a chromosome pairing-dependent form of trans-based gene regulation, is potentially widespread in the Drosophila melanogaster genome, and varies across cell types and within tissues in D. melanogaster, characteristics of a complex trait. This study demonstrate that the trans-interactions at the Malic enzyme (Men) locus are, in fact, transvection as classically defined, and are plastic with respect to both genetic background and environment. Using chromosomal inversions, trans-interactions at the Men locus were eliminated by changes in chromosomal architecture that presumably disrupt somatic pairing. It was further shown that the magnitude of transvection at the Men locus is modified by both genetic background and environment (temperature), demonstrating that transvection is a plastic phenotype. These results suggest that transvection effects in Drosophila are shaped by a dynamic interplay between environment and genetic background. Interestingly, cis-based regulation of the Men gene was found to be more robust to genetic background and environment than trans-based. Finally, this study begins to uncover the non-local factors that may contribute to variation in transvection overall, implicating Abd-B in the regulation of Men in cis and in trans in an allele-specific and tissue-specific manner, driven by differences in expression of the two genes across genetic backgrounds and environmental conditions.
Meyer, S. and Beslon, G. (2014). Torsion-mediated interaction between adjacent genes. PLoS Comput Biol 10: e1003785. PubMed ID: 25188032
DNA torsional stress is generated by virtually all biomolecular processes involving the double helix, in particular transcription where a significant level of stress propagates over several kilobases. If another promoter is located in this range, this stress may strongly modify its opening properties, and hence facilitate or hinder its transcription. This mechanism implies that transcribed genes distant of a few kilobases are not independent, but coupled by torsional stress, an effect for which this paper proposes the first quantitative and systematic model. In contrast to previously proposed mechanisms of transcriptional interference, the suggested coupling is not mediated by the transcription machineries, but results from the universal mechanical features of the double-helix. The model shows that the effect likely affects prokaryotes as well as eukaryotes, but with different consequences owing to their different basal levels of torsion. It also depends crucially on the relative orientation of the genes, enhancing the expression of eukaryotic divergent pairs while reducing that of prokaryotic convergent ones. To test the in vivo influence of the torsional coupling, the expression of isolated gene pairs was analyzed in the Drosophila melanogaster genome. Their orientation and distance dependence is fully consistent with the model, suggesting that torsional gene coupling may constitute a widespread mechanism of (co)regulation in eukaryotes.
Thursday, September 25th
Boukhatmi, H., Schaub, C., Bataille, L., Reim, I., Frendo, J. L., Frasch, M. and Vincent, A. (2014). An Org-1-Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila. Development 141(19):3761-71. PubMed ID: 25209244
The T-box transcription factor Tbx1 and the LIM-homeodomain
transcription factor Islet1 are key components in regulatory circuits
that generate myogenic and cardiogenic lineage diversity in
chordates. This study shows that Optomotor-blind-related-gene-1 (Org-1) and Tup, the Drosophila
orthologs of Tbx1 and Islet1, are co-expressed and required for
formation of the heart-associated alary muscles (AMs) in the
abdomen. The same holds true for lineage-related muscles in
the thorax that have not been described previously, which were named
thoracic alary-related muscles (TARMs). Lineage analyses identified
the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut,
respectively, and surround-specific tracheal branches, pointing to an
architectural role in the internal anatomy of the larva. Org-1 controls
tup expression in the AM/TARM lineage by direct binding to two
regulatory sites within an AM/TARM-specific cis-regulatory module,
tupAME. The contributions of Org-1 and Tup to the specification of
Drosophila AMs and TARMs provide new insights into the
transcriptional control of Drosophila larval muscle diversification
and highlight new parallels with gene regulatory networks involved in
the specification of cardiopharyngeal mesodermal derivatives in
Fagan, J. K., Dollar, G., Lu, Q., Barnett, A., Pechuan Jorge, J., Schlosser, A., Pfleger, C., Adler, P. and Jenny, A. (2014). Combover/CG10732, a novel PCP effector for Drosophila wing hair formation. PLoS One 9: e107311. PubMed ID: 25207969
The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, Combover (Cmb)/CG10732 was identified as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. These data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh.
Vandersmissen, H. P., Hiel, M. B., Loy, T. V., Vleugels, R. and Broeck, J. V. (2014). Silencing D. melanogaster lgr1 impairs transition from larval to pupal stage. Gen Comp Endocrinol. PubMed ID: 25157788
G protein-coupled receptors (GPCRs) play key roles in a wide diversity of physiological processes and signalling pathways. The leucine-rich repeats containing GPCRs (LGRs) are a subfamily that is well-conserved through[Epub ahead of print]out most metazoan phyla and have important regulatory roles in vertebrates. This study reports on the critical role of Drosophila melanogaster LGR1, the fruit fly homologue of the vertebrate glycoprotein hormone receptors, in development as a factor involved in the regulation of pupariation. Transcript profiling revealed that lgr1 transcripts are most abundant in third instar larvae and adult flies. The tissues displaying the highest transcript levels were the hindgut, the rectum and the salivary glands. Knockdown using RNA interference (RNAi) demonstrated that white pupa formation was severely suppressed in D. melanogaster lgr1 RNAi larvae. Associated with this developmental defect was a reduced ecdysteroid titer, which is in line with significantly reduced transcript levels detected for the Halloween genes shadow (sad) and spookier (spok) in the third instar lgr1 RNAi larvae compared to the control condition.
Smith, F. W. and Jockusch, E. L. (2014). Hox genes require homothorax and extradenticle for body wall identity specification but not for appendage identity specification during metamorphosis of Tribolium castaneum. Dev Biol [Epub ahead of print]. PubMed ID: 25195194
The establishment of segment identity is a key developmental process that allows for divergence along the anteroposterior body axis in arthropods. In Drosophila, the identity of a segment is determined by the complement of Hox genes it expresses. In many contexts, Hox transcription factors require the protein products of extradenticle (exd) and homothorax (hth) as cofactors to perform their identity specification functions. In holometabolous insects, segment identity may be specified twice, during embryogenesis and metamorphosis. To glean insight into the relationship between embryonic and metamorphic segmental identity specification, these processes were compared in the flour beetle Tribolium castaneum, which develop ventral appendages during embryogenesis that later metamorphose into adult appendages with distinct morphologies. At metamorphosis, comparisons of RNAi phenotypes indicate that Hox genes function jointly with Tc-hth and Tc-exd to specify several region-specific aspects of the adult body wall. In contrast, Hox genes specify appendage identities along the anteroposterior axis independently of Tc-hth/Tc-exd and Tc-hth/Tc-exd specify proximal vs. distal identity within appendages independently of Hox genes during this stage. During embryogenesis, Tc-hth and Tc-exd play a broad role in the segmentation process and are required for specification of body wall identities in the thorax; however, contrasting with results from other species, no homeotic transformations of embryonic appendages were obtained in response to Tc-hth or Tc-exd RNAi. In general, the homeotic effects of interference with the function of Hox genes and Tc-hth/Tc-exd during metamorphosis did not match predictions based on embryonic roles of these genes. Comparing metamorphic patterning in T. castaneum to embryonic and post-embryonic development in hemimetabolous insects suggests that holometabolous metamorphosis combines patterning processes of both late embryogenesis and metamorphosis of the hemimetabolous life cycle.
Wednesday, September 24th
Meyer, R. E., Algazeery, A., Capri, M., Brazier, H., Ferry, C. and Ait-Ahmed, O. (2014). Drosophila Yemanuclein is a cohesin and synaptonemal complex associated protein. J Cell Sci [Epub ahead of print]. PubMed ID: 25189620
Meiosis is characterized by two chromosome segregation rounds (Meiosis I and II), which follow a single round of DNA replication, resulting in haploid genome formation. Chromosome reduction occurs at meiosis I. It relies on key structures, such as chiasma, which is formed by repair between homologous chromatids of a double-strand break (DSB) in one of them; to function for segregation of homologues chiasma in turn relies on maintenance of sister chromatid cohesion. In most species, chiasma formation requires the prior synapsis of homologous chromosome axes, which is signaled by the Synaptonemal Complex (SC), a tripartite proteinaceous structure specific to prophase I of meiosis. Yemanuclein (YEM) is a maternal factor that is crucial for sexual reproduction. It is required in the zygote for chromatin assembly of the male pronucleus as a histone H3.3 chaperone in complex with HIRA. This study reports YEM association to the SC and the cohesin complex. A genetic interaction between yem1 (V478E) and the Spo11 homologue mei-W68, added to a yem1 dominant effect on crossover distribution suggest an early role in meiotic recombination. This is further supported by the impact of yem mutations on DSB kinetics. Hira mutant showed a similar effect presumably through disruption of HIRA-YEM complex.
Krishnan, B., Thomas, S. E., Yan, R., Yamada, H., Zhulin, I. B. and McKee, B. D. (2014). Sisters Unbound Is Required for Meiotic Centromeric Cohesion in Drosophila melanogaster. Genetics. PubMed ID: 25194162
Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two Structural Maintenance of Chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. This study identified a novel Drosophila gene called sisters unbound (sunn) (CG32088), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of nondisjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO (Sisters on the loose/Vasa) in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit Stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, these data show that SUNN is an essential meiosis-specific Drosophila cohesion protein.
Nerusheva, O. O., Galander, S., Fernius, J., Kelly, D. and Marston, A. L. (2014). Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation. Genes Dev 28: 1291-1309. PubMed ID: 24939933
During mitosis and meiosis, sister chromatid cohesion resists the pulling forces of microtubules, enabling the generation of tension at kinetochores upon chromosome biorientation. How tension is read to signal the bioriented state remains unclear. Shugoshins form a pericentromeric platform that integrates multiple functions to ensure proper chromosome biorientation. This study shows that budding yeast shugoshin Sgo1 (see Drosophila Mei-S332) dissociates from the pericentromere reversibly in response to tension. The antagonistic activities of the kinetochore-associated Bub1 kinase (see Drosophila Bub1-related kinase) and the Sgo1-bound phosphatase protein phosphatase 2A (PP2A)-Rts1 (see Drosophila Twins) underlie a tension-dependent circuitry that enables Sgo1 removal upon sister kinetochore biorientation. Sgo1 dissociation from the pericentromere triggers dissociation of condensin and Aurora B (see Drosophila Aurora B) from the centromere, thereby stabilizing the bioriented state. Conversely, forcing sister kinetochores to be under tension during meiosis I leads to premature Sgo1 removal and precocious loss of pericentromeric cohesion. Overall, this study shows that the pivotal role of shugoshin is to build a platform at the pericentromere that attracts activities that respond to the absence of tension between sister kinetochores. Disassembly of this platform in response to intersister kinetochore tension signals the bioriented state. Therefore, tension sensing by shugoshin is a central mechanism by which the bioriented state is read.
Crown, K. N., McMahan, S. and Sekelsky, J. (2014). Eliminating both canonical and short-patch mismatch repair in Drosophila melanogaster suggests a new meiotic recombination model. PLoS Genet 10: e1004583. PubMed ID: 25188408
In most meiotic systems, recombination is essential to form connections between homologs that ensure their accurate segregation from one another. Meiotic recombination is initiated by DNA double-strand breaks that are repaired using the homologous chromosome as a template. Studies of recombination in budding yeast have led to a model in which most early repair intermediates are disassembled to produce noncrossovers. Selected repair events are stabilized so they can proceed to form double-Holliday junction (dHJ) intermediates, which are subsequently resolved into crossovers. This model is supported in yeast by physical isolation of recombination intermediates, but the extent to which it pertains to animals is unknown. This study sought to test this model in Drosophila melanogaster by analyzing patterns of heteroduplex DNA (hDNA) in recombination products. Previous attempts to do this have relied on knocking out the canonical mismatch repair (MMR) pathway, but in both yeast and Drosophila the resulting recombination products are complex and difficult to interpret. In Drosophila, this complexity results from a secondary, short-patch MMR pathway that requires nucleotide excision repair. Knocking out both canonical and short-patch MMR reveals hDNA patterns that reveal that many noncrossovers arise after both ends of the break have engaged with the homolog. Patterns of hDNA in crossovers could be explained by biased resolution of a dHJ; however, considering the noncrossover and crossover results together suggests a model in which a two-end engagement intermediate with unligated HJs can be disassembled by a helicase to a produce noncrossover or nicked by a nuclease to produce a crossover. While some aspects of this model are similar to the model from budding yeast, production of both noncrossovers and crossovers from a single, late intermediate is a fundamental difference that has important implications for crossover control.
Tuesday, September 23rd
Zhang, B., Mehrotra, S., Ng, W. L., Calvi, B. R. (2014). Low Levels of p53 Protein and Chromatin Silencing of p53 Target Genes Repress Apoptosis in Drosophila Endocycling Cells. PLoS Genet 10: e1004581. PubMed ID: 25211335
Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. This study shows that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform does not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain does, suggesting that dampened p53B protein levels are crucial for apoptotic repression. It was also found that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A is the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, the data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, these results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals.
Perez, E., Das, G., Bergmann, A. and Baehrecke, E. H. (2014). . Oncogene [Epub ahead of print]. PubMed ID: 25174403
Autophagy is a catabolic process that has been implicated both as a tumor suppressor and in tumor progression. This study investigated this dichotomy in cancer biology by studying the influence of altered autophagy in Drosophila models of tissue overgrowth. The impact of altered autophagy was found to depend on both genotype and cell type. As previously observed in mammals, decreased autophagy suppresses Ras-induced eye epithelial overgrowth. In contrast, autophagy restricts epithelial overgrowth in a Notch-dependent eye model. Even though decreased autophagy did not influence Hippo pathway-triggered overgrowth, activation of autophagy strongly suppresses this eye epithelial overgrowth. Surprisingly, activation of autophagy enhances Hippo pathway-driven overgrowth in glia cells. These results indicate that autophagy has different influences on tissue growth in distinct contexts, and highlight the importance of understanding the influence of autophagy on growth to augment a rationale therapeutic strategy.
Clavier, A., Baillet, A., Rincheval-Arnold, A., Coleno-Costes, A., Lasbleiz, C., Mignotte, B. and Guenal, I. (2014). The pro-apoptotic activity of Drosophila Rbf1 involves dE2F2-dependent downregulation of diap1 and buffy mRNA. Cell Death Dis 5: e1405. PubMed ID: 25188515
The retinoblastoma gene, rb, ensures at least its tumor suppressor function by inhibiting cell proliferation. Its role in apoptosis is more complex and less described than its role in cell cycle regulation. Rbf1, the Drosophila homolog of Rb, has been found to be pro-apoptotic in proliferative tissue. However, the way it induces apoptosis at the molecular level is still unknown. To decipher this mechanism, rbf1 expression was induced in wing proliferative tissue. It was found that Rbf1-induced apoptosis depends on dE2F2/dDP heterodimer, whereas dE2F1 transcriptional activity is not required. Furthermore, Rbf1 and dE2F2 downregulate two major anti-apoptotic genes in Drosophila: buffy, an anti-apoptotic member of Bcl-2 family and diap1, a gene encoding a caspase inhibitor. On the one hand, Rbf1/dE2F2 repress buffy at the transcriptional level, which contributes to cell death. On the other hand, Rbf1 and dE2F2 upregulate how expression. How is a RNA binding protein involved in diap1 mRNA degradation. By this way, Rbf1 downregulates diap1 at a post-transcriptional level. Moreover, the dREAM complex (see Rbf) has a part in these transcriptional regulations. Taken together, these data show that Rbf1, in cooperation with dE2F2 and some members of the dREAM complex, can downregulate the anti-apoptotic genes buffy and diap1, and thus promote cell death in a proliferative tissue.
Huang, Q., Tang, X., Wang, G., Fan, Y., Ray, L., Bergmann, A., Belenkaya, T. Y., Ling, X., Yan, D., Lin, Y., Ye, X., Shi, W., Zhou, X., Lu, F., Qu, J. and Lin, X. (2014). Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila. Cell Death Differ. PubMed ID: 25146930
Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. This study demonstrates that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. Loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, through the UBR-box domain, Ubr3 physically interacts with the neo-epitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, these data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway.
Monday September 22nd
Pegoraro, M., Gesto, J. S., Kyriacou, C. P. and Tauber, E. (2014). Role for circadian clock genes in seasonal timing: testing the bunning hypothesis. PLoS Genet 10: e1004603. PubMed ID: 25188283
A major question in chronobiology focuses around the 'Bunning hypothesis' which implicates the circadian clock in photoperiodic (day-length) measurement and is supported in some systems (e.g. plants) but disputed in others. This study used the seasonally-regulated thermotolerance of Drosophila melanogaster to test the role of various clock genes in day-length measurement. In Drosophila, freezing temperatures induce reversible chill coma, a narcosis-like state. Previous observations were have corroborated that wild-type flies developing under short photoperiods (winter-like) exhibit significantly shorter chill-coma recovery times (CCRt) than flies that were raised under long (summer-like) photoperiods. Arrhythmic mutant strains, per01, tim01 and ClkJrk, as well as variants that speed up or slow down the circadian period, disrupt the photoperiodic component of CCRt. The results support an underlying circadian function mediating seasonal daylength measurement and indicate that clock genes are tightly involved in photo- and thermo-periodic measurements.
Williams, M. J., Goergen, P., Rajendran, J., Zheleznyakova, G., Hagglund, M. G., Perland, E., Bagchi, S., Kalogeropoulou, A., Khan, Z., Fredriksson, R. and Schioth, H. B. (2014). Obesity-linked homologues TfAP-2 and Twz establish meal Frequency in Drosophila melanogaster. PLoS Genet 10: e1004499. PubMed ID: 25187989
In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. The current study demonstrates that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, this study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, it was found that the mouse TfAP-2 and Twz homologues, AP-2beta and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2beta and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, it was show that in this mouse hypothalamic cell line AP-2beta and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2beta may itself be sumoylated. This study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior.
Thoma, M., Hansson, B. S. and Knaden, M. (2014). Compound valence is conserved in binary odor mixtures in Drosophila melanogaster. J Exp Biol [Epub ahead of print]. PubMed ID: 25189369
Most naturally occurring olfactory signals do not consist of monomolecular odorants but, rather, are mixtures whose composition and concentration ratios vary. While there is ample evidence for the relevance of complex odor blends in ecological interactions and for interactions of chemicals in both peripheral and central neuronal processing, a fine-scale analysis of rules governing the innate behavioral responses of Drosophila melanogaster towards odor mixtures is lacking. This study examined whether the innate valence of odors is conserved in binary odor mixtures. Binary mixtures of attractants are more attractive than individual mixture constituents. In contrast, mixing attractants with repellents elicits responses which are lower than the responses towards the corresponding attractants. This decrease in attraction is repellent-specific, independent of the identity of the attractant and more stereotyped across individuals than responses towards the repellent alone. Mixtures of repellents are either less attractive than the individual mixture constituents or these mixtures represent an intermediate. Within the limits of the data set, most mixture responses are quantitatively predictable on the basis of constituent responses. In summary, the valence of binary odor mixtures is predictable on the basis of valences of mixture constituents. These findings will further understanding of innate behavior towards ecologically relevant odor blends and will serve as a powerful tool for deciphering the olfactory valence code.
Dawydow, A., Gueta, R., Ljaschenko, D., Ullrich, S., Hermann, M., Ehmann, N., Gao, S., Fiala, A., Langenhan, T., Nagel, G. and Kittel, R. J. (2014). Channelrhodopsin-2-XXL, a powerful optogenetic tool for low-light applications. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25201989
Channelrhodopsin-2 (ChR2) has provided a breakthrough for the optogenetic control of neuronal activity. In adult Drosophila melanogaster, however, its applications are severely constrained. This limitation in a powerful model system has curtailed unfolding the full potential of ChR2 for behavioral neuroscience. This study describes the D156C mutant, termed ChR2-XXL (extra high expression and long open state), which displays increased expression, improved subcellular localization, elevated retinal affinity, an extended open-state lifetime, and photocurrent amplitudes greatly exceeding those of all heretofore published ChR variants. As a result, neuronal activity could be efficiently evoked with ambient light and even without retinal supplementation. The benefits of the variant was validated in intact flies by eliciting simple and complex behaviors. Efficient and prolonged photostimulation was demonstrated of monosynaptic transmission at the neuromuscular junction and reliable activation of a gustatory reflex pathway. Innate male courtship was triggered in male and female flies, and olfactory memories were written through light-induced associative training.
Sunday, September 21st
Schmid, M. R., Anderl, I., Vesala, L., Vanha-Aho, L. M., Deng, X. J., Ramet, M. and Hultmark, D. (2014). Control of Drosophila blood cell activation via Toll signaling in the fat body. PLoS One 9: e102568. PubMed ID: 25102059
The Toll signaling pathway, first discovered in Drosophila, has a well-established role in immune responses in insects as well as in mammals. In Drosophila, the Toll-dependent induction of antimicrobial peptide production has been intensely studied as a model for innate immune responses in general. Besides this humoral immune response, Toll signaling is also known to activate blood cells in a reaction that is similar to the cellular immune response to parasite infections, but the mechanisms of this response are poorly understood. This paper describes a study of this response in detail and found that Toll signaling in several different tissues can activate a cellular immune defense and that this response does not require Toll signaling in the blood cells themselves. As in the humoral immune response, Toll signaling in the fat body (analogous to the liver in vertebrates) is of major importance in the Toll-dependent activation of blood cells. However, this Toll-dependent mechanism of blood cell activation contributes very little to the immune response against the parasitoid wasp, Leptopilina boulardi, probably because the wasp is able to suppress Toll induction. Other redundant pathways may be more important in the defense against this pathogen.
Tsuzuki, S., Matsumoto, H., Furihata, S., Ryuda, M., Tanaka, H., Jae Sung, E., Bird, G. S., Zhou, Y., Shears, S. B. and Hayakawa, Y. (2014). Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP. Nat Commun 5: 4628. PubMed ID: 25130174
Insects combat infection through carefully measured cellular (for example, phagocytosis) and humoral (for example, secretion of antimicrobial peptides (AMPs)) innate immune responses. Little is known concerning how these different defense mechanisms are coordinated. This study used insect plasmatocytes and hemocyte-like Drosophila S2 cells to characterize mechanisms of immunity that operate in the haemocoel. A Drosophila cytokine, growth-blocking peptides (GBP), acts through the phospholipase C (PLC)/Ca(2+) signalling cascade (see Small wing) to mediate the secretion of Pvf, a ligand for platelet-derived growth factor- and vascular endothelial growth factor-receptor (Pvr) homologue. Activated Pvr recruits extracellular signal-regulated protein kinase to inhibit humoral immune responses, while stimulating cell 'spreading', an initiating event in cellular immunity. The double-stranded RNA (dsRNA)-targeted knockdown of either Pvf2 or Pvr inhibits GBP-mediated cell spreading and activates AMP expression. Conversely, Pvf2 overexpression enhances cell spreading but inhibits AMP expression. Thus, this study describes mechanisms to initiate immune programs that are either humoral or cellular in nature, but not both; such immunophysiological polarization may minimize homeostatic imbalance during infection.
Bonnay, F., Nguyen, X. H., Cohen-Berros, E., Troxler, L., Batsche, E., Camonis, J., Takeuchi, O., Reichhart, J. M. and Matt, N. (2014). Akirin specifies NF-kappaB selectivity of Drosophila innate immune response via chromatin remodeling. EMBO J [Epub ahead of print]. PubMed ID: 25180232
The network of NF-kappaB-dependent transcription that activates both pro- and anti-inflammatory genes in mammals is still unclear. As NF-kappaB factors are evolutionarily conserved, Drosophila was used to understand this network. The NF-kappaB transcription factor Relish activates effector gene expression following Gram-negative bacterial immune challenge. This study shows, using a genome-wide approach, that the conserved nuclear protein Akirin is a NF-kappaB co-factor required for the activation of a subset of Relish-dependent genes correlating with the presence of H3K4ac epigenetic marks. A large-scale unbiased proteomic analysis revealed that Akirin orchestrates NF-kappaB transcriptional selectivity through the recruitment of the Osa-containing-SWI/SNF-like Brahma complex (BAP). Immune challenge in Drosophila shows that Akirin is required for the transcription of a subset of effector genes, but dispensable for the transcription of genes that are negative regulators of the innate immune response. Therefore, Akirins act as molecular selectors specifying the choice between subsets of NF-kappaB target genes. The discovery of this mechanism, conserved in mammals, paves the way for the establishment of more specific and less toxic anti-inflammatory drugs targeting pro-inflammatory genes.
Perrin, J., Mortier, M., Jacomin, A. C., Viargues, P., Thevenon, D. and Fauvarque, M. O. (2014). The Nonaspanins TM9SF2 and TM9SF4 Regulate the Plasma Membrane Localization and Signalling Activity of the Peptidoglycan Recognition Protein PGRP-LC in Drosophila. J Innate Immun [Epub ahead of print]. PubMed ID: 25139117
Transmembrane 9 (TM9) proteins, or nonaspanins, are a family of proteins conserved throughout evolution and characterized by 9 transmembrane domains. In Drosophila, TM9 superfamily protein member 4 (TM9SF4) and its closest paralogue, TM9SF2 (CG9318), contribute to phagocytosis of various types of particles, while TM9SF4 displays non-redundant requirement in Gram-negative bacteria engulfment. In addition, the two TM9 proteins control the actin cytoskeleton in larval haemocytes and in Drosophila S2 cells. This study show that TM9SF4 and TM9SF2 co-immunoprecipitate with the peptidoglycan recognition protein (PGRP)-LC, which triggers the Drosophila immune response to bacterial infection. Furthermore, both TM9 proteins co-localize with this receptor in intracellular vesicles and at the plasma membrane in Drosophila S2 cells in culture and in the fly fat body. Silencing TM9SF4 prevents plasma membrane localization of PGRP-LC, whereas silencing TM9SF2 does not, which may account for the non-redundant role of TM9SF4 in phagocytosis of Gram-negative bacteria. Finally, a set of data is provided suggesting that TM9 proteins can prevent inappropriate signalling from the unstimulated receptor.
Saturday, September 20th
Xing, G., Gan, G., Chen, D., Sun, M., Yi, J., Lv, H., Han, J. and Xie, W. (2014) . Drosophila Neuroligin3 regulates neuromuscular junction development and synaptic differentiation. J Biol Chem. [Epub ahead of print]. PubMed ID: 25228693
Summary: Neuroligins (Nlgs) are a family of cell adhesion molecules thought to be important for synapse maturation and function. Studies in mammals have shown that different Nlgs have different roles in synaptic maturation and function. The functions of Drosophila Neuroligin1 (DNlg1), DNlg2, and DNlg4 have also been examined. This study reports the role of DNlg3 in synaptic development and function by using Drosophila neuromuscular junctions (NMJs) as a model system. DNlg3 was found to be expressed in both CNS and NMJs where it was largely restricted to the postsynaptic site. By generating and examining dnlg3 mutants, the mutants mutants were found to exhibit an increased bouton number and reduced bouton size compared to the wild-type. Consistent with alterations in bouton properties, pre- and postsynaptic differentiations were also affected including abnormal synaptic vesicle endocytosis, increased PSD length and reduced GluRIIA recruitment. Additionally, synaptic transmission was reduced. Altogether, this study shows that DNlg3 is required for NMJ development, synaptic differentiation and function.
Halstead, J. M., Lin, Y. Q., Durraine, L., Hamilton, R. S., Ball, G., Neely, G. G., Bellen, H. J. and Davis, I. (2014). Syncrip/hnRNP Q influences synaptic transmission and regulates BMP signaling at the Drosophila neuromuscular synapse. Biol Open. 3(9): 839-49. PubMed ID: 25171887
Synaptic plasticity involves the modulation of synaptic connections in response to neuronal activity via multiple pathways. One mechanism modulates synaptic transmission by retrograde signals from the post-synapse that influence the probability of vesicle release in the pre-synapse. Despite its importance, very few factors required for the expression of retrograde signals, and proper synaptic transmission, have been identified. This study identified the conserved RNA binding protein Syncrip as a new factor that modulates the efficiency of vesicle release from the motoneuron and is required for correct synapse structure. syncrip is required genetically and its protein product is detected only in the muscle and not in the motoneuron itself. This unexpected non-autonomy is at least partly explained by the fact that Syncrip modulates retrograde BMP signals from the muscle back to the motoneuron. Syncrip influences the levels of the Bone Morphogenic Protein ligand Glass Bottom Boat from the post-synapse and regulates the pre-synapse. These results highlight the RNA-binding protein Syncrip as a novel regulator of synaptic output. Given its known role in regulating translation, it is proposed that Syncrip is important for maintaining a balance between the strength of presynaptic vesicle release and postsynaptic translation.
McDermott, S. M., Yang, L., Halstead, J. M., Hamilton, R. S., Meignin, C. and Davis, I. (2014).. Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction. RNA 20(10): 1593-606. PubMed ID: 25171822
Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function.
Muthukumar, A. K., Stork, T. and Freeman, M. R. (2014). Activity-dependent regulation of astrocyte GAT levels during synaptogenesis. Nat Neurosci [Epub ahead of print]. PubMed ID: 25151265
Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system; however, mechanisms by which astrocytes regulate GAT levels are undefined. This study found that at mid-pupal stages the Drosophila melanogaster CNS neuropil was devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrated the neuropil coordinately with synaptogenesis, and astrocyte ablation reduced synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses formed in earnest, GAT was upregulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor 1 and 2 (GABABR1/2) signaling in astrocytes led to a decrease in astrocytic GAT. Notably, developmental depletion of astrocytic GABABR1/2 signaling suppressed mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal that astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity.
Ehmann, N., van de Linde, S., Alon, A., Ljaschenko, D., Keung, X. Z., Holm, T., Rings, A., DiAntonio, A., Hallermann, S., Ashery, U., Heckmann, M., Sauer, M. and Kittel, R. J. (2014). Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states. Nat Commun 5: 4650. PubMed ID: 25130366
The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. This study introduces new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), the AZ cytomatrix (CAZ) was found to be composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. Tests were performed for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. The results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.
Friday, September 19th
Militti, C., Maenner, S., Becker, P. B., Gebauer, F. (2014). UNR facilitates the interaction of MLE with the lncRNA roX2 during Drosophila dosage compensation. Nat Commun 5: 4762. PubMed ID: 25158899
Dosage compensation is a regulatory process that balances the expression of X-chromosomal genes between males (XY) and females (XX). In Drosophila, this requires non-coding RNAs and RNA-binding proteins (RBPs) whose specific functions remain elusive. This study shows that the Drosophila RBP UNR promotes the targeting of the activating male-specific-lethal complex to the X-chromosome by facilitating the interaction of two crucial subunits: the RNA helicase MLE and the long non-coding RNA roX2.
Hennig, J., Militti, C., Popowicz, G. M., Wang, I., Sonntag, M., Geerlof, A., Gabel, F., Gebauer, F., Sattler, M. (2014). Structural basis for the assembly of the Sxl-Unr translation regulatory complex. Nature [Epub ahead of print]. PubMed ID: 25209665
Genetic equality between males and females is established by chromosome-wide dosage-compensation mechanisms. In the fruitfly Drosophila melanogaster, the dosage-compensation complex promotes twofold hypertranscription of the single male X-chromosome and is silenced in females by inhibition of the translation of msl2, which codes for the limiting component of the dosage-compensation complex. The female-specific protein Sex-lethal (Sxl) recruits Upstream-of-N-ras (Unr) to the 3' untranslated region of msl2 messenger RNA, preventing the engagement of the small ribosomal subunit3. This study reports the 2.8 Å crystal structure, NMR and small-angle X-ray and neutron scattering data of the ternary Sxl–Unr–msl2 ribonucleoprotein complex featuring unprecedented intertwined interactions of two Sxl RNA recognition motifs, a Unr cold-shock domain and RNA. Cooperative complex formation is associated with a 1,000-fold increase of RNA binding affinity for the Unr cold-shock domain and involves novel ternary interactions, as well as non-canonical RNA contacts by the α1 helix of Sxl RNA recognition motif 1. These results suggest that repression of dosage compensation, necessary for female viability, is triggered by specific, cooperative molecular interactions that lock a ribonucleoprotein switch to regulate translation. The structure serves as a paradigm for how a combination of general and widespread RNA binding domains expands the code for specific single-stranded RNA recognition in the regulation of gene expression.
Igreja, C., Peter, D., Weiler, C. and Izaurralde, E. (2014). 4E-BPs require non-canonical 4E-binding motifs and a lateral surface of eIF4E to repress translation. Nat Commun 5: 4790. PubMed ID: 25179781
eIF4E-binding proteins (4E-BPs; see Drosophila Thor) are a widespread class of translational regulators that share a canonical (C) eIF4E-binding motif (4E-BM) with eIF4G. Consequently, 4E-BPs compete with eIF4G for binding to the dorsal surface on eIF4E to inhibit translation initiation. Some 4E-BPs contain non-canonical 4E-BMs (NC 4E-BMs), but the contribution of these motifs to the repressive mechanism-and whether these motifs are present in all 4E-BPs-remains unknown. This study shows that the three annotated Drosophila melanogaster 4E-BPs contain NC 4E-BMs. These motifs bind to a lateral surface on eIF4E that is not used by eIF4G. This distinct molecular recognition mode is exploited by 4E-BPs to dock onto eIF4E-eIF4G complexes and effectively displace eIF4G from the dorsal surface of eIF4E. These data reveal a hitherto unrecognized role for the NC4E-BMs and the lateral surface of eIF4E in 4E-BP-mediated translational repression, and suggest that bipartite 4E-BP mimics might represent efficient therapeutic tools to dampen translation during oncogenic transformation.
Simmons, M. J., Thorp, M. W., Buschette, J. T. and Becker, J. R. (2014). Transposon regulation in Drosophila: piRNA-producing P elements facilitate repression of hybrid dysgenesis by a P element that encodes a repressor polypeptide. Mol Genet Genomics. PubMed ID: 25159111
The transposons of Drosophila melanogaster are regulated by small RNAs that interact with the Piwi family of proteins. These piRNAs are generated from transposons inserted in special loci such as the telomere-associated sequences at the left end of the X chromosome. Drosophila's P transposons can also be regulated by a polypeptide encoded by the KP element (see DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro), a 1.15-kb-long member of the P family. Using piRNA-generating telomeric P elements (TPs) and repressor-producing transgenic KP elements, this study demonstrates a functional connection between these two modes of regulation. By themselves, the TPs partially repress gonadal dysgenesis, a trait caused by rampant P-element activity in the germ line. This repression is manifested as a strictly maternal effect arising from the cytoplasmic transmission of P-specific piRNAs from mother to offspring. The repression is enhanced by genetic interactions between the TPs and other, non-telomeric P elements-a phenomenon attributable to ping-pong amplification of maternal piRNAs. KP elements, like other kinds of non-telomeric P elements, enhance regulation anchored in the TPs. However, with some TPs, the enhanced regulation is manifested as a strictly zygotic effect of the KP element. This effect is seen when the TP has few sequences in common with the KP element, a condition not conducive to ping-pong amplification of piRNAs (see Properties and biogenesis of piRNA); it can be attributed to the action of the KP repressor polypeptide. Because the effect is seen only when a TP was present in the mother's genotype, maternally generated P-element piRNAs could facilitate regulation by the KP repressor polypeptide.
Thursday, September 18th
Bagley, J. A., Yan, Z., Zhang, W., Wildonger, J., Jan, L. Y. and Jan, Y. N. (2014). Double-bromo and extraterminal (BET) domain proteins regulate dendrite morphology and mechanosensory function. Genes Dev 28: 1940-1956. PubMed ID: 25184680
A complex array of genetic factors regulates neuronal dendrite morphology. Epigenetic regulation of gene expression represents a plausible mechanism to control pathways responsible for specific dendritic arbor shapes. By studying the Drosophila dendritic arborization (da) neurons, this study discovered a role of the double-bromodomain and extraterminal (BET) family proteins in regulating dendrite arbor complexity. A loss-of-function mutation in the single Drosophila BET protein encoded by female sterile 1 homeotic [fs(1)h] causes loss of fine, terminal dendritic branches. Moreover, fs(1)h is necessary for the induction of branching caused by a previously identified transcription factor, Cut (Ct), which regulates subtype-specific dendrite morphology. Finally, disrupting fs(1)h function impairs the mechanosensory response of class III da sensory neurons without compromising the expression of the ion channel NompC, which mediates the mechanosensitive response. Thus, these results identify a novel role for BET family proteins in regulating dendrite morphology and a possible separation of developmental pathways specifying neural cell morphology and ion channel expression. Since the BET proteins are known to bind acetylated histone tails, these results also suggest a role of epigenetic histone modifications and the 'histone code,' in regulating dendrite morphology.
Yakulov, T., Gunesdogan, U., Jackle, H. and Herzig, A. (2014). Ballchen participates in proliferation control and prevents the differentiation of Drosophila melanogaster neuronal stem cells. Biol Open [Epub ahead of print]. PubMed ID: 25190057
Stem cells continuously generate differentiating daughter cells and are essential for tissue homeostasis and development. Their capacity to self-renew as undifferentiated and actively dividing cells is controlled by either external signals from a cellular environment, the stem cell niche, or asymmetric distribution of cell fate determinants during cell division. This study reports that the protein kinase Ballchen (Ball) is required to prevent differentiation as well as to maintain normal proliferation of neuronal stem cells of Drosophila melanogaster, called neuroblasts. These results show that the brains of ball mutant larvae are severely reduced in size, which is caused by a reduced proliferation rate of the neuroblasts. Moreover, ball mutant neuroblasts gradually lose the expression of the neuroblast determinants Miranda and aPKC, suggesting their premature differentiation. These results indicate that Ball represents a novel cell intrinsic factor with a dual function regulating the proliferative capacity and the differentiation status of neuronal stem cells during development.
Pezier, A., Jezzini, S. H., Marie, B. and Blagburn, J. M. (2014). Engrailed alters the specificity of synaptic connections of Drosophila auditory neurons with the giant fiber. J Neurosci 34: 11691-11704. PubMed ID: 25164665
A subset of sound-detecting Johnston's Organ neurons (JONs) in Drosophila melanogaster that express the transcription factors Engrailed (En) and Invected (Inv) form mixed electrical and chemical synaptic inputs onto the giant fiber (GF) dendrite. These synaptic connections are detected by trans-synaptic Neurobiotin (NB) transfer and by colocalization of Bruchpilot-short puncta. Misexpressing En postmitotically in a second subset of sound-responsive JONs causes them to form ectopic electrical and chemical synapses with the GF, in turn causing that postsynaptic neuron to redistribute its dendritic branches into the vicinity of these afferents. A simple electrophysiological recording paradigm was introduced for quantifying the presynaptic and postsynaptic electrical activity at this synapse, by measuring the extracellular sound-evoked potentials (SEPs) from the antennal nerve while monitoring the likelihood of the GF firing an action potential in response to simultaneous subthreshold sound and voltage stimuli. Ectopic presynaptic expression of En strengthens the synaptic connection, consistent with there being more synaptic contacts formed. Finally, RNAi-mediated knockdown of En and Inv in postmitotic neurons reduces SEP amplitude but also reduces synaptic strength at the JON-GF synapse. Overall, these results suggest that En and Inv in JONs regulate both neuronal excitability and synaptic connectivity.
Iyengar, A. and Wu, C. F. (2014). Flight and seizure motor patterns in Drosophila mutants: Simultaneous acoustic and electrophysiological recordings of wing beats and flight muscle activity. J Neurogenet: 1-22. PubMed ID: 25159538
Tethered flies allow studies of biomechanics and electrophysiology of flight control. Microelectrode recordings were performed of spikes in an indirect flight muscle (the dorsal longitudinal muscle, DLMa) coupled with acoustic analysis of wing beat frequency (WBF) via microphone signals. Simultaneous electrophysiological recording of direct and indirect flight muscles has been technically challenging; however, the WBF is thought to reflect in a one-to-one relationship with spiking in a subset of direct flight muscles, including muscle m1b. Therefore, this approach enables systematic mutational analysis for changes in temporal features of electrical activity of motor neurons innervating subsets of direct and indirect flight muscles. This paper reports the consequences of specific ion channel disruptions on the spiking activity of myogenic DLMs (firing at approximately 5 Hz) and the corresponding wing beat frequency (approximately 200 Hz). Mutants were examined of: 1) voltage-gated Ca2+ channels (cacophony, cac), 2) Ca2+-activated K+ channels (slowpoke, slo), and 3) voltage-gated K+ channels (Shaker, Sh) and their auxiliary subunits (Hyperkinetic, Hk and quiver, qvr). Flight initiation in response to an air puff was severely disrupted in both cac and slo mutants. However, once initiated, slo flight was largely unaltered, whereas cac displayed disrupted DLM firing rates and WBF. Sh, Hk, and qvr mutants were able to maintain normal DLM firing rates, despite increased WBF. Notably, defects in the auxiliary subunits Hk and qvr could lead to distinct consequences, i.e. disrupted DLM firing rhythmicity, not observed in Sh. This mutant analysis of direct and indirect flight muscle activities indicates that the two motor activity patterns may be independently modified by specific ion channel mutations, and that this approach can be extended to other dipteran species and additional motor programs, such as electroconvulsive stimulation-induced seizures.
Wednesday, September 17th
Flici, H., Cattenoz, P. B., Komonyi, O., Laneve, P., Erkosar, B., Karatas, O. F., Reichert, H., Berzsenyi, S. and Giangrande, A. (2014). Interlocked loops trigger lineage specification and stable fates in the Drosophila nervous system. Nat Commun 5: 4484. PubMed ID: 25066644
Multipotent precursors are plastic cells that generate different, stable fates at the correct number, place and time, to allow tissue and organ formation. While fate determinants are known to trigger specific transcriptional programs, the molecular pathway driving the progression from multipotent precursors towards stable and specific identities remains poorly understood. This study demonstrates that, in Drosophila neural precursors, the glial determinant Glial cell missing (Gcm) acts as a 'time bomb' and triggers its own degradation once the glial programme is stably activated. This requires a sequence of transcriptional and posttranscriptional loops, whereby a Gcm target first affects the expression and then acetylation of the fate determinant, thus controlling Gcm levels and stability over time. Defective homeostasis between the loops alters the neuron:glia ratio and freezes cells in an intermediate glial/neuronal phenotype. In sum, this study identified an efficient strategy triggering cell identity, a process altered in pathological conditions such as cancer.
Kim, S. N., Jeibmann, A., Halama, K., Witte, H. T., Walte, M., Matzat, T., Schillers, H., Faber, C., Senner, V., Paulus, W. and Klambt, C. (2014). ECM stiffness regulates glial migration in Drosophila and mammalian glioma models. Development 141: 3233-3242. PubMed ID: 25063458
Cell migration is an important feature of glial cells. This study used the Drosophila eye disc to decipher the molecular network controlling glial migration. Glial motility was stimulated by pan-glial PDGF receptor (PVR) activation, and several genes acting downstream of PVR were identified. Drosophila lox is a non-essential gene encoding a secreted protein that stiffens the extracellular matrix (ECM). Glial-specific knockdown of integrin (see Myospheroid) results in ECM softening. Moreover, it was shown that lox expression is regulated by integrin signaling and vice versa, suggesting that a positive-feedback loop ensures a rigid ECM in the vicinity of migrating cells. The general implication of this model was tested in a mammalian glioma model, where a Lox-specific inhibitor unraveled a clear impact of ECM rigidity in glioma cell migration.
Mauri, F., Reichardt, I., Mummery-Widmer, J. L., Yamazaki, M., Knoblich, J. A. (2014) . The conserved Discs-large binding partner Banderuola regulates asymmetric cell division in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 25088559
Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood. To identify additional proteins required for ACD, an in vivo live imaging RNAi screen was carried out for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. Banderuola (Bnd) was identified an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms.
It is concluded that Bnd is a novel regulator of ACD in different types of cells. The data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution.
Zhang, H., Wang, Y., Wong, J. J., Lim, K. L., Liou, Y. C., Wang, H., Yu, F. (2014). Endocytic pathways downregulate the L1-type cell adhesion molecule Neuroglian to oromote dendrite pruning in Drosophila. Dev Cell 30: 463-478. PubMed ID: 25158855
Pruning of unnecessary axons and/or dendrites is crucial for maturation of the nervous system. However, little is known about cell adhesion molecules (CAMs) that control neuronal pruning. In Drosophila, dendritic arborization neurons, ddaCs, selectively prune their larval dendrites. This study reports that Rab5/ESCRT-mediated endocytic pathways are critical for dendrite pruning. Loss of Rab5 or ESCRT function (see Hrs) leads to robust accumulation of the L1-type CAM Neuroglian (Nrg) on enlarged endosomes in ddaC neurons. Nrg is localized on endosomes in wild-type ddaC neurons and downregulated prior to dendrite pruning. Overexpression of Nrg alone is sufficient to inhibit dendrite pruning, whereas removal of Nrg causes precocious dendrite pruning. Epistasis experiments indicate that Rab5 and ESCRT restrain the inhibitory role of Nrg during dendrite pruning. Thus, this study demonstrates the cell-surface molecule that controls dendrite pruning and defines an important mechanism whereby sensory neurons, via endolysosomal pathway, downregulate the cell-surface molecule to trigger dendrite pruning.
Tuesday, September 16th
Gorostiza, E. A., Depetris-Chauvin, A., Frenkel, L., Pirez, N., Ceriani, M. F. (2014). Circadian pacemaker neurons change synaptic contacts across the day. Curr Biol. PubMed ID: 25155512
Daily cycles of rest and activity are a common example of circadian control of physiology. In Drosophila, rhythmic locomotor cycles rely on the activity of 150-200 neurons grouped in seven clusters. Work from many laboratories points to the small ventral lateral neurons (sLNvs) as essential for circadian control of locomotor rhythmicity. sLNv neurons undergo circadian remodeling of their axonal projections, opening the possibility for a circadian control of connectivity of these relevant circadian pacemakers. This study shows that circadian plasticity of the sLNv axonal projections has further implications than mere structural changes. First, it was found that the degree of daily structural plasticity exceeds that originally described, underscoring that changes in the degree of fasciculation as well as extension or pruning of axonal terminals could be involved. Interestingly, the quantity of active zones changes along the day, lending support to the attractive hypothesis that new synapses are formed while others are dismantled between late night and the following morning. More remarkably, taking full advantage of the GFP reconstitution across synaptic partners (GRASP) technique, this study showed that, in addition to new synapses being added or removed, sLNv neurons contact different synaptic partners at different times along the day. These results lead to a proposal that the circadian network, and in particular the sLNv neurons, orchestrates some of the physiological and behavioral differences between day and night by changing the path through which information travels.
Djiane, A., Zaessinger, S., Babaoglan, A. B., Bray, S. J. (2014). Notch inhibits yorkie activity in Drosophila wing discs. PLoS One 9: e106211. PubMed ID: 25157415
During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. This study shows that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. This cross-talk between the Notch and SWH pathways is shown to be mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). It is proposed that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs.
Wang, C. H., Chen, G. C. and Chien, C. T. (2014). The deubiquitinase Leon/USP5 regulates ubiquitin homeostasis during Drosophila development. Biochem Biophys Res Commun. PubMed ID: 25152394
Ubiquitination and the reverse process deubiquitination regulate protein stability and function during animal development. The Drosophila USP5 homolog Leon functions as other family members of unconventional deubiquitinases, disassembling free, substrate-unconjugated polyubiquitin chains to replenish the pool of mono-ubiquitin, and maintaining cellular ubiquitin homeostasis. However, the significance of Leon/USP5 in animal development is still unexplored. This study generated leon mutants to show that Leon is essential for animal viability and tissue integrity during development. Both free and substrate-conjugated polyubiquitin chains accumulate in leon mutants, suggesting that abnormal ubiquitin homeostasis caused tissue disorder and lethality in leon mutants. Further analysis of protein expression profiles in leon mutants shows that the levels of all proteasomal subunits were elevated. Also, proteasomal enzymatic activities were elevated in leon mutants. However, proteasomal degradation of ubiquitinated substrates was impaired. Thus, aberrant ubiquitin homeostasis in leon mutants disrupts normal proteasomal degradation, which is compensated by elevating the levels of proteasomal subunits and activities. Ultimately, the failure to fully compensate the dysfunctional proteasome in leon mutants leads to animal lethality and tissue disorder.
Wang, L., Zeng, X., Ryoo, H. D. and Jasper, H. (2014). Integration of UPRER and oxidative stress signaling in the control of intestinal stem cell proliferation. PLoS Genet 10: e1004568. PubMed ID: 25166757
The Unfolded Protein Response of the endoplasmic reticulum (UPRER: see Drosophila Inositol-requiring enzyme-1) controls proteostasis by adjusting the protein folding capacity of the ER to environmental and cell-intrinsic conditions. In metazoans, loss of proteostasis results in degenerative and proliferative diseases and cancers. The cellular and molecular mechanisms causing these phenotypes remain poorly understood. This study shows that the UPRER is a critical regulator of intestinal stem cell (ISC) quiescence in Drosophila melanogaster. ISCs were found to require activation of the UPRER for regenerative responses, but a tissue-wide increase in ER stress was found to trigger ISC hyperproliferation and epithelial dysplasia in aging animals. These effects are mediated by ISC-specific redox signaling through Jun-N-terminal Kinase (JNK) and the transcription factor CncC. The results identify a signaling network of proteostatic and oxidative stress responses that regulates ISC function and regenerative homeostasis in the intestinal epithelium.
Huang, X., Shi, L., Cao, J., He, F., Li, R., Zhang, Y., Miao, S., Jin, L., Qu, J., Li, Z. and Lin, X. (2014). The sterile 20-like kinase tao controls tissue homeostasis by regulating the hippo pathway in Drosophila adult midgut. J Genet Genomics 41: 429-438. PubMed ID: 25160975
The proliferation and differentiation of adult stem cells must be tightly controlled in order to maintain resident tissue homeostasis. Dysfunction of stem cells is implicated in many human diseases, including cancer. However, the regulation of stem cell proliferation and differentiation is not fully understood. This study shows that the sterile-like 20 kinase, Tao, controls tissue homeostasis by regulating the Hippo pathway in the Drosophila adult midgut. Depletion of Tao in the progenitors leads to rapid intestinal stem cell (ISC) proliferation and midgut homeostasis loss. Meanwhile, it was find that the STAT signaling activity and cytokine production are significantly increased, resulting in stimulated ISC proliferation. Furthermore, expression of the Hippo pathway downstream targets, Diap1 and bantam, is dramatically increased in Tao knockdown intestines. Consistently, it was shown that the Yorkie (Yki) acts downstream of Tao to regulate ISC proliferation. Together, these results provide insights into understanding of the mechanisms of stem cell proliferation and tissue homeostasis control.
Monday, September 15th
Bertet, C., Li, X., Erclik, T., Cavey, M., Wells, B., Desplan, C. (2014). Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper. Cell 158: 1173-1186. PubMed ID: 25171415
Temporal patterning of neural progenitors is one of the core mechanisms generating neuronal diversity in the central nervous system. This study shows that, in the tips of the outer proliferation center (tOPC) of the developing Drosophila optic lobes, a unique temporal series of transcription factors not only governs the sequential production of distinct neuronal subtypes but also controls the mode of progenitor division, as well as the selective apoptosis of NotchOFF or NotchON neurons during binary cell fate decisions. Within a single lineage, intermediate precursors initially do not divide and generate only one neuron; subsequently, precursors divide, but their NotchON progeny systematically die through Reaper activity, whereas later, their NotchOFF progeny die through Hid activity. These mechanisms dictate how the tOPC produces neurons for three different optic ganglia. It is concluded that temporal patterning generates neuronal diversity by specifying both the identity and survival/death of each unique neuronal subtype.
Chu, B., Chui, V., Mann, K. and Gordon, M. D. (2014). Presynaptic gain control drives sweet and bitter taste integration in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 25131672
The sense of taste is critical in determining the nutritional suitability of foods. Sweet and bitter are primary taste modalities in mammals, and their behavioral relevance is similar in flies. Sweet taste drives the appetitive response to energy sources, whereas bitter taste drives avoidance of potential toxins and also suppresses the sweet response. Despite their importance to survival, little is known about the neural circuit mechanisms underlying integration of sweet and bitter taste. This study describes a presynaptic gain control mechanism in Drosophila that differentially affects sweet and bitter taste channels and mediates integration of these opposing stimuli. Gain control is known to play an important role in fly olfaction, where GABAB receptor (GABABR) mediates intra- and interglomerular presynaptic inhibition of sensory neuron output. In the taste system, gustatory receptor neurons (GRNs) responding to sweet compounds were found to express GABABR, whereas those that respond to bitter do not. GABABR mediates presynaptic inhibition of calcium responses in sweet GRNs, and both sweet and bitter stimuli evoke GABAergic neuron activity in the vicinity of GRN axon terminals. Pharmacological blockade and genetic reduction of GABABR both lead to increased sugar responses and decreased suppression of the sweet response by bitter compounds. A model is proposed in which GABA acts via GABABR to expand the dynamic range of sweet GRNs through presynaptic gain control and suppress the output of sweet GRNs in the presence of opposing bitter stimuli.
Chen, Y. and Amrein, H. (2014). Enhancing perception of contaminated food through acid-mediated modulation of taste neuron responses. Curr Biol [Epub ahead of print]. PubMed ID: 25131671
Natural foods contain not only nutrients, but also nonnutritious and potentially harmful chemicals. Thus, animals need to evaluate food content in order to make adequate feeding decisions. This study investigated the effects of acids on the taste neuron responses and on taste behavior of desirable, nutritious sugars and sugar/bitter compound mixtures in Drosophila melanogaster. Using Ca2+ imaging, acids were shown to activate neither sweet nor bitter taste neurons in tarsal taste sensilla. However, they suppress responses to bitter compounds in bitter-sensing neurons. Moreover, acids reverse suppression of bitter compounds exerted on sweet-sensing neurons. Consistent with these observations, behavioral analyses show that bitter-compound-mediated inhibition on feeding behavior is alleviated by acids. To investigate the cellular mechanism by which acids modulate these effects, bitter-sensing gustatory neurons were silenced. Surprisingly, this intervention had little effect on acid-mediated derepression of sweet neuron or feeding responses to either sugar/bitter compound mixtures or sugar/bitter compound/acid mixtures, suggesting that there are two independent pathways by which bitter compounds are sensed. These investigations reveal that acids, when presented in dietary relevant concentrations, enhance the perception of sugar/bitter compound mixtures. Drosophila's natural food sources - fruits and cohabitating yeast - are rich in sugars and acids but are rapidly colonized by microorganisms, such as fungi, protozoan parasites, and bacteria, many of which produce bitter compounds. It is proposed that the acids present in most fruits counteract the inhibitory effects of these bitter compounds during feeding.
Gan, G., Lv, H. and Xie, W. (2014). Morphological identification and development of neurite in Drosophila ventral nerve cord neuropil. PLoS One 9: e105497. PubMed ID: 25166897
In Drosophila, ventral nerve cord (VNC) occupies most of the larval central nervous system (CNS). However, there is little literature elaborating upon the specific types and growth of neurites as defined by their structural appearance in Drosophila larval VNC neuropil. This study reports the ultrastructural development of different types VNC neurites in ten selected time points in embryonic and larval stages utilizing transmission electron microscopy. There are four types of axonal neurites as classified by the type of vesicular content: clear vesicle (CV) neurites have clear vesicles and some T-bar structures; Dense-core vesicle (DV) neurites have dense-core vesicles and without T-bar structures; Mixed vesicle (MV) neurites have mixed vesicles and some T-bar structures; Large vesicle (LV) neurites are dominated by large, translucent spherical vesicles but rarely display T-bar structures. We found dramatic remodeling in CV neurites which can be divided into five developmental phases. The neurite is vacuolated in primary (P) phase, they have mitochondria, microtubules or big dark vesicles in the second (S) phase, and they contain immature synaptic features in the third (T) phase. The subsequent bifurcate (B) phase appears to undergo major remodeling with the appearance of the bifurcation or dendritic growth. In the final mature (M) phase, high density of commensurate synaptic vesicles are distributed around T-bar structures. There are four kinds of morphological elaboration of the CVI neurite sub-types. First, new neurite produces at the end of axon. Second, new neurite bubbles along the axon. Third, the preexisting neurite buds and develops into several neurites. The last, the bundled axons form irregularly shape neurites. Most CVI neurites in M phase have about 1.5-3 microm diameter, they could be suitable to analyze their morphology and subcellular localization of specific proteins by light microscopy, and they could serve as a potential model in CNS in vivo development.
Sunday, September 14th
Dietz, K. N., Di Stefano, L., Maher, R. C., Zhu, H., MacDonald, M. E., Gusella, J. F. and Walker, J. A. (2014). The Drosophila Huntington's disease gene ortholog dhtt influences chromatin regulation during development. Hum Mol Genet. PubMed ID: 25168387
Huntington's disease is an autosomal dominant neurodegenerative disorder caused by a CAG expansion mutation in HTT, the gene encoding huntingtin. Evidence from both human genotype-phenotype relationships and mouse model systems suggests that the mutation acts by dysregulating some normal activity of huntingtin. Recent work in the mouse has revealed a role for huntingtin in epigenetic regulation during development. This study examined the role of the Drosophila huntingtin ortholog (dhtt) in chromatin regulation in the development of the fly. Although null dhtt mutants display no overt phenotype, dhtt was found to act as a suppressor of position effect variegation (PEV), suggesting that it influences chromatin organization. dhtt affects heterochromatin spreading in a PEV model by modulating histone H3K9 methylation levels at the heterochromatin-euchromatin boundary. To gain mechanistic insights into how dhtt influences chromatin function, a candidate genetic screen was constructed using RNAi lines targeting known PEV modifier genes. dhtt was found to modify phenotypes caused by knockdown of a number of key epigenetic regulators, including chromatin-associated proteins, histone demethylases and methyltransferases. Notably, dhtt strongly modifies phenotypes resulting from loss of the histone demethylase dLsd1, in both the ovary and wing, and dhtt appears to act as a facilitator of dLsd1 function in regulating global histone H3K4 methylation levels. These findings suggest that a fundamental aspect of huntingtin function in heterochromatin/euchromatin organization is evolutionarily conserved across phyla.
Da-Re, C., von Stockum, S., Biscontin, A., Millino, C., Cisotto, P., Zordan, M., Zeviani, M., Bernardi, P., De Pitta, C. and Costa, R. (2014). Leigh syndrome in Drosophila melanogaster: Morphological and biochemical characterization of Surf1 post-transcriptional silencing. J Biol Chem [Epub ahead of print]. PubMed ID: 25164807
Leigh Syndrome (LS) is the most common early-onset, progressive mitochondrial encephalopathy usually leading to early death. The single most prevalent cause of LS is occurrence of mutations in the SURF1 gene, and LSSurf1 patients show a ubiquitous and specific decrease in the activity of mitochondrial respiratory chain complex IV (cytochrome c oxidase, COX). SURF1 encodes an inner membrane mitochondrial protein involved in COX assembly. A D. melanogaster model of LS was establised based on the post-transcriptional silencing of Surfeit 1 (CG9943), the Drosophila homolog of SURF1. Knock down of Surf1 was induced ubiquitously in larvae and adults, which led to lethality; in the mesodermal derivatives, which led to pupal lethality; or in the central nervous system, which allowed survival. A biochemical characterization was carried out in knock down individuals, which revealed that larvae unexpectedly displayed defects in all complexes of the mitochondrial respiratory chain and in the F-ATP synthase, while adults had a COX-selective impairment. Silencing of Surf1 expression in Drosophila S2R+ cells led to selective loss of COX activity associated with decreased oxygen consumption and respiratory reserve. It is concluded that Surf1 is essential for COX activity and mitochondrial function in D. melanogaster, thus providing a new tool that may help clarify the pathogenic mechanisms of LS.
Long, D. M., Blake, M. R., Dutta, S., Holbrook, S. D., Kotwica-Rolinska, J., Kretzschmar, D. and Giebultowicz, J. M. (2014). Relationships between the circadian system and Alzheimer's disease-Like symptoms in Drosophila. PLoS One 9: e106068. PubMed ID: 25171136
Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer's disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, this study used transgenic Drosophila expressing various Amyloid-beta (Abeta; see Drosophila Appl) peptides, which are prone to form aggregates characteristic of AD pathology in humans. Development of AD-like symptoms were compared in adult flies expressing Abeta peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per01). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Abeta peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. Rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Abeta peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Abeta peptides causes rhythm degradation downstream from the central clock mechanism.
Cassar, M., Issa, A. R., Riemensperger, T., Petitgas, C., Rival, T., Coulom, H., Iche-Torres, M., Han, K. A. and Birman, S. (2014). A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila. Hum Mol Genet [Epub ahead of print]. PubMed ID: 25158689
Long-term exposure to environmental oxidative stressors, like the herbicide paraquat (PQ), has been linked to the development of Parkinson's disease (PD), the most frequent neurodegenerative movement disorder. PQ is thus frequently used in the fruit fly Drosophila melanogaster and other animal models to study PD and the degeneration of dopaminergic neurons (DNs) that characterizes this disease. This study shows that a D1-like dopamine (DA) receptor, DAMB (Dopamine 1-like receptor 2), actively contributes to the fast central nervous system (CNS) failure induced by PQ in the fly. Firstly, it was found that a long-term increase in neuronal DA synthesis reduced DAMB expression and protected against PQ neurotoxicity. Secondly, a striking age-related decrease in PQ resistance correlated to an augmentation of DAMB expression. This aging-induced increase in oxidative stress vulnerability not observed in a DAMB-deficient mutant. Thirdly, targeted inactivation of this receptor in glutamatergic neurons (GNs) markedly enhanced the survival of Drosophila exposed to either PQ or neurotoxic levels of DA, while, conversely, DAMB overexpression in these cells made the flies more vulnerable to both compounds. Fourthly, a mutation in the Drosophila ryanodine receptor (RyR), which inhibits activity-induced increase in cytosolic Ca2+, also strongly enhanced PQ resistance. Finally, this study found that DAMB overexpression in specific neuronal populations arrested development of the fly and that in vivo stimulation of either DNs or GNs increased PQ susceptibility. This suggests a model for DA receptor-mediated potentiation of PQ-induced neurotoxicity. Further studies of DAMB signaling in Drosophila could have implications for better understanding DA-related neurodegenerative disorders in humans.
Martin, C. A., Barajas, A., Lawless, G., Lawal, H. O., Assani, K., Lumintang, Y. P., Nunez, V. and Krantz, D. E. (2014). Synergistic effects on dopamine cell death in a Drosophila model of chronic toxin exposure. Neurotoxicology [Epub ahead of print]. PubMed ID: 25160001
The neurodegenerative effects of Parkinson's disease (PD) are marked by a selective loss of dopaminergic (DA) neurons. Epidemiological studies suggest that chronic exposure to the pesticide paraquat may increase the risk for PD and DA cell loss. However, combined exposure with additional fungicide(s) including maneb and/or ziram may be required for pathogenesis. To explore potential pathogenic mechanisms, a Drosophila model of chronic paraquat exposure was developed. While chronic paraquat exposure alone decreased organismal survival and motor function, combined chronic exposure to both paraquat and maneb was required for DA cell death in the fly. To initiate mechanistic studies of this interaction, additional genetic reagents were used to target the ubiquitin proteasome system, implicated in some rare familial forms of PD and the toxic effects of ziram. Genetic inhibition of E1 ubiquitin ligase, but not the proteasome itself, increased DA cell death in combination with maneb but not paraquat. These studies establish a model for long-term exposure to multiple pesticides, and support the idea that pesticide interactions relevant to PD may involve inhibition of protein ubiquitination.
Saturday, September 13th
Singh, N. D., Koerich, L. B., Carvalho, A. B. and Clark, A. G. (2014). Positive and purifying selection on the Drosophila Y chromosome. Mol Biol Evol [Epub ahead of print]. PubMed ID: 24974375
Y chromosomes, with their reduced effective population size, lack of recombination and male-limited transmission, present a unique collection of constraints for the operation of natural selection. Male-limited transmission may greatly increase the efficacy of selection for male-beneficial mutations, but the reduced effective size also inflates the role of random drift. Together, these defining features of the Y chromosome are expected to influence rates and patterns of molecular evolution on the Y as compared with X-linked or autosomal loci. This study used sequence data from eleven genes in nine Drosophila species to gain insight into the efficacy of natural selection on the Drosophila Y relative to the rest of the genome. Drosophila is an ideal system for assessing the consequences of Y-linkage for molecular evolution in part because the gene content of Drosophila Y chromosomes is highly dynamic, with orthologous genes being Y-linked in some species while autosomal in others. The results confirm the expectation that the efficacy of natural selection at weakly selected sites is reduced on the Y chromosome. In contrast, purifying selection on the Y chromosome for strongly deleterious mutations does not appear to be compromised. Finally, evidence was found of recurrent positive selection for four of the 11 genes in this study. The results thus highlight the variable nature of the mode and impact of natural selection on the Drosophila Y chromosome.
Kim, Y. B., Oh, J. H., McIver, L. J., Rashkovetsky, E., Michalak, K., Garner, H. R., Kang, L., Nevo, E., Korol, A. B. and Michalak, P. (2014). Divergence of Drosophila melanogaster repeatomes in response to a sharp microclimate contrast in Evolution Canyon, Israel. Proc Natl Acad Sci U S A 111(29): 10630-5. PubMed ID: 25006263
Repeat sequences, especially mobile elements, make up large portions of most eukaryotic genomes and provide enormous, albeit commonly underappreciated, evolutionary potential. This study analyzed repeatomes of Drosophila melanogaster that have been diverging in response to a microclimate contrast in Evolution Canyon (Mount Carmel, Israel), a natural evolutionary laboratory with two abutting slopes at an average distance of only 200 m, which pose a constant ecological challenge to their local biotas. Flies inhabiting the colder and more humid north-facing slope carried about 6% more transposable elements than those from the hot and dry south-facing slope, in parallel to a suite of other genetic and phenotypic differences between the two populations. Nearly 50% of all mobile element insertions were slope unique, with many of them disrupting coding sequences of genes critical for cognition, olfaction, and thermotolerance, consistent with the observed patterns of thermotolerance differences and assortative mating (Kim, 2014).
Puerma, E., Orengo, D. J. and Aguade, M. (2014). Evidence for a gene involved in multiple and diverse rearrangements in the Drosophila genus. Mol Biol Evol [Epub ahead of print]. PubMed ID: 25135946
In Drosophila, chromosomes have been extensively reorganized during evolution, with most rearrangements affecting the gene order in chromosomal elements but not their gene content. The level of reorganization and the evidence for breakpoint reuse vary both between and within elements. The subito gene stands out as a gene involved in multiple rearrangements both because of its active single-gene transposition and because it is the nearest gene to diverse rearrangements breakpoints. Indeed, subito has undergone three single-gene transpositions and it is the nearest gene to the breakpoints of other single-gene transpositions and of two chromosomal inversions. Given that subito is involved in meiosis and therefore active in the female germ line, the high number of nearby fixed breakages might be related among others to the presumed high accessibility of the subito region to the machinery associated with double-strand breaks repair. A second important contributor would be the reduced and simple regulatory region of subito, which would imply that a fraction of the rearrangements originating from subito nearby breakages would have not affected either its pattern or timing of expression and would have, thus, not resulted in reduced fitness.
Ambrosi, P., Chahda, J. S., Koslen, H. R., Chiel, H. J. and Mizutani, C. M. (2014). Modeling of the Dorsal gradient across species reveals interaction between embryo morphology and Toll signaling pathway during evolution. PLoS Comput Biol 10: e1003807. PubMed ID: 25165818
Morphogenetic gradients are essential to allocate cell fates in embryos of varying sizes within and across closely related species. The maternal NF-kappaB/Dorsal (Dl) gradient has acquired different shapes in Drosophila species, which result in unequally scaled germ layers along the dorso-ventral axis and the repositioning of the neuroectodermal borders. This study combined experimentation and mathematical modeling to investigate which factors might have contributed to the fast evolutionary changes of this gradient. To this end, a previously developed model was developed that employs differential equations of the main biochemical interactions of the Toll (Tl) signaling pathway, which regulates Dl nuclear transport. The original model simulations fit well the D. melanogaster wild type, but not mutant conditions. To broaden the applicability of this model and probe evolutionary changes in gradient distributions, a set of 19 independent parameters was adjusted to reproduce three quantified experimental conditions (i.e. Dl levels lowered, nuclear size and density increased or decreased). Next, the most relevant parameters were sought that reproduce the species-specific Dl gradients. Adjusting parameters relative to morphological traits (i.e. embryo diameter, nuclear size and density) alone is not sufficient to reproduce the species Dl gradients. Since components of the Tl pathway simulated by the model are fast-evolving, it was next asked which parameters related to Tl would most effectively reproduce these gradients, and a particular subset was identified. A sensitivity analysis reveals the existence of nonlinear interactions between the two fast-evolving traits tested above, namely the embryonic morphological changes and Tl pathway components. The modeling further suggests that distinct Dl gradient shapes observed in closely related melanogaster sub-group lineages may be caused by similar sequence modifications in Tl pathway components, which are in agreement with their phylogenetic relationships.
Wang, G. Z., Marini, S., Ma, X., Yang, Q., Zhang, X. and Zhu, Y. (2014). Improvement of Dscam homophilic binding affinity throughout Drosophila evolution. BMC Evol Biol 14: 186. PubMed ID: 25158691
Drosophila Dscam1 is a cell-surface protein that plays important roles in neural development and axon tiling of neurons. It is known that thousands of isoforms bind themselves through specific homophilic interactions, a process which provides the basis for cellular self-recognition. Detailed biochemical studies of specific isoforms strongly suggest that homophilic binding, i.e. the formation of homodimers by identical Dscam1 isomers, is of great importance for the self-avoidance of neurons. Due to experimental limitations, it is currently impossible to measure the homophilic binding affinities for all 19,000 potential isoforms. This study reconstructed the DNA sequences of an ancestral Dscam form (which likely existed approximately 40 ~ 50 million years ago) using a comparative genomic approach. On the basis of this sequence, a working model was established to predict the self-binding affinities of all isoforms in both the current and the ancestral genome, using machine-learning methods. Detailed computational analysis was performed to compare the self-binding affinities of all isoforms present in these two genomes. The results revealed that 1) isoforms containing newly derived variable domains exhibit higher self-binding affinities than those with conserved domains, and 2) current isoforms display higher self-binding affinities than their counterparts in the ancient genome. As thousands of Dscam isoforms are needed for the self-avoidance of the neuron, it is proposed that an increase in self-binding affinity provides the basis for the successful evolution of the arthropod brain. These data provide an excellent model for future experimental studies of the binding behavior of Dscam isoforms. The results of this analysis indicate that evolution favored the rise of novel variable domains thanks to their higher self-binding affinities, rather than selection merely on the basis of simple expansion of isoform diversity; this particular selection process would established a powerful mechanism required for neuronal self-avoidance. Thus, this study reveals a new molecular mechanism for the successful evolution of arthropod brains.
Friday, September 12th
Zhang, C. U., Blauwkamp, T. A., Burby, P. E. and Cadigan, K. M. (2014). Wnt-Mediated Repression via Bipartite DNA Recognition by TCF in the Drosophila Hematopoietic System. PLoS Genet 10: e1004509. PubMed ID: 25144371
The Wnt/beta-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previous work has demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. This study reports that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/beta-catenin signaling controls prohemocytic differentiation. This study provides a paradigm for direct repression of target gene expression by Wnt/beta-catenin signaling and allosteric regulation of a transcription factor by DNA.
Bernardo, T. J., Dubrovskaya, V. A., Xie, X. and Dubrovsky, E. B. (2014). A view through a chromatin loop: insights into the ecdysone activation of early genes in Drosophila. Nucleic Acids Res. PubMed ID: 25143532
The early genes are a key group of ecdysone targets that function at the top of the signaling hierarchy. In the presence of ecdysone, early genes exhibit a highly characteristic rapid and powerful induction that represents a primary response. Multiple isoforms encoded by early genes then coordinate the activation of a larger group of late genes. While the general mechanism of ecdysone-dependent transcription is well characterized, it is not known whether a distinct mechanism governs the hormonal response of early genes. Previous work has found that one of the Drosophila early genes, E75, harbors multiple functional ecdysone response elements (EcREs). This study extends the analysis to Broad and E74 and has found that EcRE multiplicity is a general feature of the early genes. Since most of the EcREs within early gene loci are situated distantly from promoters, the chromosome conformation capture method was used to determine whether higher order chromatin structure facilitates hormonal activation. For each early gene chromatin loops were detected that juxtapose their promoters and multiple distant EcREs prior to ecdysone activation. These findings suggest that higher order chromatin structure may serve as an important mechanism underlying the distinct response of early genes to ecdysone.
Krivega, I., Dale, R. K. and Dean, A. (2014). Role of LDB1 in the transition from chromatin looping to transcription activation. JOURNAL
Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, lim domain binding 1 (LDB1; see Drosophila Chip) protein is recruited to the beta-globin locus via LMO2 (see Drosophila Beadex) and is required for looping of the beta-globin locus control region (LCR) to the active beta-globin promoter. This study shows that the LDB1 dimerization domain (DD) is necessary and, when fused to LMO2, sufficient to completely restore LCR-promoter looping and transcription in LDB1-depleted cells. The looping function of the DD is unique and irreplaceable by heterologous DDs. Dissection of the DD revealed distinct functional properties of conserved subdomains. Notably, a conserved helical region (DD4/5) is dispensable for LDB1 dimerization and chromatin looping but essential for transcriptional activation. DD4/5 is required for the recruitment of the coregulators FOG1 (U-shaped in Drosophila) and the nucleosome remodeling and deacetylating (NuRD) complex. Lack of DD4/5 alters histone acetylation and RNA polymerase II recruitment and results in failure of the locus to migrate to the nuclear interior, as normally occurs during erythroid maturation. These results uncouple enhancer-promoter looping from nuclear migration and transcription activation and reveal new roles for LDB1 in these processes.
Lopez, Y., Vandenbon, A. and Nakai, K. (2014). A set of structural features defines the cis-regulatory modules of antenna-expressed genes in Drosophila melanogaster. PLoS One 9: e104342. PubMed ID: 25153327
Unraveling the biological information within the regulatory region (RR) of genes has become one of the major focuses of current genomic research. It has been hypothesized that RRs of co-expressed genes share similar architecture, but to the best of our knowledge, no studies have simultaneously examined multiple structural features, such as positioning of cis-regulatory elements relative to transcription start sites and to each other, and the order and orientation of regulatory motifs, to accurately describe overall cis-regulatory structure. This work present an improved computational method that builds a feature collection based on all of these structural features. The utility of this approach was demonstrated by modeling the cis-regulatory modules of antenna-expressed genes in Drosophila melanogaster. Six potential antenna-related motifs were predicted initially, including three that appeared to be novel. A feature set was created with the predicted motifs, where a correlation-based filter was used to remove irrelevant features, and a genetic algorithm was designed to optimize the feature set. Finally, a set of eight highly informative structural features was obtained for the RRs of antenna-expressed genes, achieving an area under the curve of 0.841. These features were used to score all D. melanogaster RRs for potentially unknown antenna-expressed genes sharing a similar regulatory structure. Validation of these predictions with an independent RNA sequencing dataset showed that 76.7% of genes with high scoring RRs were expressed in antenna. In addition, it was found that the structural features that were identified are highly conserved in RRs of orthologs in other Drosophila sibling species. This approach to identify tissue-specific regulatory structures showed comparable performance to previous approaches, but also uncovered additional interesting features because it also considered the order and orientation of motifs.
Thursday, September 11th
Kaul, A., Schuster, E., Jennings, B. H. (2014). The Groucho co-repressor is primarily recruited to local target sites in active chromatin to attenuate transcription. PLoS Genet 10: e1004595. PubMed ID: 25165826
Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. This study used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. Gro was found to predominantly bind at discrete peaks (<1 kilobase). It was also demonstrated that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in 'active' chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. It was also found that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. These results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation and polymerase pausing.
Kalb, R., Latwiel, S., Baymaz, H. I., Jansen, P. W., Muller, C. W., Vermeulen, M. and Muller, J. (2014). Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression. Nat Struct Mol Biol 21: 569-571. PubMed ID: 24837194
A key step in gene repression by Polycomb is trimethylation of histone H3 K27 by PCR2 to form H3K27me3. H3K27me3 provides a binding surface for PRC1. This study shows that monoubiquitination of histone H2A by PRC1-type complexes to form H2Aub creates a binding site for Jarid2-Aebp2-containing PRC2 and promotes H3K27 trimethylation on H2Aub nucleosomes. Jarid2, Aebp2 and H2Aub thus constitute components of a positive feedback loop establishing H3K27me3 chromatin domains.
Maksimenko, O., Kyrchanova, O., Bonchuk, A., Stakhov, V., Parshikov, A. and Georgiev, P. (2014). Highly conserved ENY2/Sus1 protein binds to Drosophila CTCF and is required for barrier activity. Epigenetics 9 [Epub ahead of print]. PubMed ID: 25147918
Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers for the spreading of repressive chromatin. Drosophila insulator protein dCTCF marks active promoters and boundaries of many histone H3K27 trimethylation domains associated with repressed chromatin. In particular, dCTCF binds to such boundaries between the parasegment-specific regulatory domains of the Bithorax complex. This study demonstrate that the evolutionarily conserved protein ENY2 is recruited to the zinc-finger domain of dCTCF and is required for the barrier activity of dCTCF-dependent insulators in transgenic lines. Inactivation of ENY2 by RNAi in BG3 cells leads to the spreading of H3K27 trimethylation and Pc protein at several dCTCF boundaries. The results suggest that evolutionarily conserved ENY2 is responsible for barrier activity mediated by the dCTCF protein.
Agelopoulos, M., McKay, D. J. and Mann, R. S. (2014). cgChIP: A cell type- and gene-specific method for chromatin analysis. Methods Mol Biol 1196: 291-306. PubMed ID: 25151171
Hox and other homeobox-containing genes encode critical transcriptional regulators of animal development. Although these genes are well known for their roles in the body axis and appendage development, little is known regarding the mechanisms by which these factors influence chromatin landscapes. Chromatin structure can have a profound influence on gene expression during animal body formation. However, when applied to developing embryos, conventional chromatin analysis of genes and cis-regulatory modules (CRMs) typically lacks the required cell type-specific resolution due to the heterogeneous nature of animal bodies. This study presents a strategy to analyze both the composition and conformation of in vivo-tagged CRM sequences in a cell type-specific manner, using as a system Drosophila embryos. This method is termed cgChIP (cell- and gene-specific Chromatin Immunoprecipitation) by which regulatory chromatin is accessed and analyzed in specific cell types. The cgChIP approach utilizes the operator sequence from the E. coli lac operon to 'tag' a DNA sequence of choice. Expression of an epitope-tagged Lac repressor protein under control of a Gal4 driver results in a tissue-specific DNA binding interaction. After formaldehyde fixation and chromatin preparation, antibodies recognizing the epitope-tagged Lac repressor protein are used to pulldown the lac operator and surrounding chromatin. This process results in the enrichment of the tagged chromatin specifically from the desired cells. cgChIP is an in vivo method designed to analyze genetic elements derived from limited cell populations. cgChIP can be used for both the analysis of chromatin structure (e.g., long-distance interactions between DNA elements) and the composition of histones and histone modifications and the occupancy of transcription factors and chromatin modifiers. This method was applied to the Hox target gene Distalless (Dll), which encodes for a homeodomain-containing transcription factor critical for the formation of appendages in Drosophila. However, cgChIP can be applied in diverse animal models to better dissect CRM-dependent gene regulation and body formation in developing animals.
Wednesday, September 10th
Lim, J., Sabandal, P. R., Fernandez, A., Sabandal, J. M., Lee, H. G., Evans, P. and Han, K. A. (2014). The Octopamine receptor Octβ2R regulates ovulation in Drosophila melanogaster. PLoS One 9: e104441. PubMed ID: 25099506
Oviposition is induced upon mating in most insects. Ovulation is a primary step in oviposition, representing an important target to control insect pests and vectors, but limited information is available on the underlying mechanism. This study reports that the beta adrenergic-like octopamine receptor Octβ2R serves as a key signaling molecule for ovulation and recruits Protein kinase A and Ca2+/calmodulin-sensitive kinase II as downstream effectors for this activity. The octβ2r homozygous mutant females are sterile. They displayed normal courtship, copulation, sperm storage and post-mating rejection behavior but are unable to lay eggs. It has been shown previously that octopamine neurons in the abdominal ganglion innervate the oviduct epithelium. Consistently, restored expression of Octβ2R in oviduct epithelial cells is sufficient to reinstate ovulation and full fecundity in the octβ2r mutant females, demonstrating that the oviduct epithelium is a major site of Octβ2R's function in oviposition. It was also found that overexpression of the protein kinase A catalytic subunit or Ca2+/calmodulin-sensitive protein kinase II leads to partial rescue of octβ2r's sterility. This suggests that Octβ2R activates cAMP as well as additional effectors including Ca2+/calmodulin-sensitive protein kinase II for oviposition. All three known β adrenergic-like octopamine receptors stimulate cAMP production in vitro. Octβ1R, when ectopically expressed in the octβ2r's oviduct epithelium, fully reinstated ovulation and fecundity. Ectopically expressed Octβ3R, on the other hand, partly restores ovulation and fecundity while OAMB-K3 and OAMB-AS that increase Ca2+ levels yielded partial rescue of ovulation but not fecundity deficit. These observations suggest that Octβ2R have distinct signaling capacities in vivo and activate multiple signaling pathways to induce egg laying. The findings reported in this study narrow the knowledge gap and offer insight into novel strategies for insect control (Lim, 2014).
Ferreira, T., Prudencio, P. and Goncalo Martinho, R. (2014). Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis. Dev Biol [Epub ahead of print]. PubMed ID: 25131196
Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of any given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. This study isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis it was observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. This work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. Pkn was shown to negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. It is proposed that Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase (Ferreira, 2014).
Lin, T. H., Yeh, T. H., Wang, T. W. and Yu, J. Y. (2014). The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary. Genetics [Epub ahead of print]. PubMed ID: 25161211
The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator Yorkie (Yki). YAP (a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. This study used border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. This study found that hpo and wts are required for migration of the border cell cluster. In outer border cells, over-expression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo, wts, or over-expression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced-expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, these data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: 1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; 2) in polar cells, it regulates upd expression to control border cell induction and migration (Lin, 2014).
Maimon, I., Popliker, M. and Gilboa, L. (2014). Without children is required for Stat-mediated zfh1 transcription and for germline stem cell differentiation. Development 141: 2602-2610. PubMed ID: 24903753
Tissue homeostasis is maintained by balancing stem cell self-renewal and differentiation. How surrounding cells support this process has not been entirely resolved. This study shows that the chromatin and telomere-binding factor Without children (Woc) is required for maintaining the association of escort cells (ECs) with germ cells in adult ovaries. This tight association is essential for germline stem cell (GSC) differentiation into cysts. Woc is also required in larval ovaries for the association of intermingled cells (ICs) with primordial germ cells. Reduction in the levels of two other proteins, Stat92E and its target Zfh1, produce phenotypes similar to woc in both larval and adult ovaries, suggesting a molecular connection between these three proteins. Antibody staining and RT-qPCR demonstrate that Zfh1 levels are increased in somatic cells that contact germ cells, and that Woc is required for a Stat92E-mediated upregulation of zfh1 transcription. These results further demonstrate that overexpression of Zfh1 in ECs can rescue GSC differentiation in woc-deficient ovaries. Thus, Zfh1 is a major Woc target in ECs. Stat signalling in niche cells has been previously shown to maintain GSCs non-autonomously. This study now shows that Stat92E also promotes GSC differentiation. The results highlight the Woc-Stat-Zfh1 module as promoting somatic encapsulation of germ cells throughout their development. Each somatic cell type can then provide the germline with the support it requires at that particular stage. Stat is thus a permissive factor, which explains its apparently opposite roles in GSC maintenance and differentiation (Maimon, 2014).
Tuesday, September 9th
Conduit, P. T., Richens, J. H., Wainman, A., Holder, J., Vicente, C. C., Pratt, M. B., Dix, C. I., Novak, Z. A., Dobbie, I. M., Schermelleh, L. and Raff, J. W. (2014). A molecular mechanism of mitotic centrosome assembly in Drosophila. Elife: e03399. PubMed ID: 25149451
Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. This study shows that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn mitotic PCM assembly is diminished; in the absence of both proteins it appears to be abolished. DSpd-2 helps incorporate Cnn into the PCM, and Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies.
Cunha-Ferreira, I., Bento, I., Pimenta-Marques, A., Jana, S. C., Lince-Faria, M., Duarte, P., Borrego-Pinto, J., Gilberto, S., Amado, T., Brito, D., Rodrigues-Martins, A., Debski, J., Dzhindzhev, N. and Bettencourt-Dias, M. (2013). Regulation of autophosphorylation controls PLK4 self-destruction and centriole number. Curr Biol 23: 2245-2254. PubMed ID: 24184099
Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/betaTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/betaTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. This study shows that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/betaTrCP binding. A multisite trans-autophosphorylation mechanism is demonstrated that is likely to ensure that both a threshold of PLK4 concentration is attained, and a sequence of events is observed before PLK4 can autodestruct. First, it was shown that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, the importance was shown of PLK4-Slimb/betaTrCP regulation as it operates in both soma and germline. As betaTrCP, PLK4, and centriole number are deregulated in several cancers, this work provides novel links between centriole number control and tumorigenesis.
Schoenfelder, K. P., Montague, R. A., Paramore, S. V., Lennox, A. L., Mahowald, A. P. and Fox, D. T. (2014). Indispensable pre-mitotic endocycles promote aneuploidy in the Drosophila rectum. Development [Epub ahead of print]. PubMed ID: 25142462
The endocycle is a modified cell cycle that lacks M phase. Endocycles are well known for enabling continued growth of post-mitotic tissues. By contrast, this study discovered pre-mitotic endocycles in precursors of Drosophila rectal papillae (papillar cells). Unlike all known proliferative Drosophila adult precursors, papillar cells endocycle before dividing. Furthermore, unlike diploid mitotic divisions, these polyploid papillar divisions are frequently error prone, suggesting papillar structures may accumulate long-term aneuploidy. This study demonstrates an indispensable requirement for pre-mitotic endocycles during papillar development and also demonstrates that such cycles seed papillar aneuploidy. Blocking pre-mitotic endocycles disrupts papillar morphogenesis and causes organismal lethality under high-salt dietary stress. It was further shown that pre-mitotic endocycles differ from post-mitotic endocycles, since only the M-phase-capable polyploid cells of the papillae and female germline can retain centrioles. In papillae, this centriole retention contributes to aneuploidy, as centrioles amplify during papillar endocycles, causing multipolar anaphase. Such aneuploidy is well tolerated in papillae, as it does not significantly impair cell viability, organ formation or organ function. Together, these results demonstrate that pre-mitotic endocycles can enable specific organ construction and are a mechanism that promotes highly tolerated aneuploidy.
Petkau, K., Parsons, B. D., Duggal, A. and Foley, E. (2014). A deregulated intestinal cell cycle program disrupts tissue homeostasis, without affecting longevity in Drosophila. J Biol Chem [Epub ahead of print]. PubMed ID: 25170078
Recent studies illuminate a complex relationship between the control of stem cell division and intestinal tissue organization in the model system Drosophila melanogaster. Host and microbial signals drive intestinal proliferation to maintain an effective epithelial barrier. While it is widely assumed that proliferation induces dysplasia and shortens the lifespan of the host, the phenotypic consequences of deregulated intestinal proliferation for an otherwise healthy host remain unexplored. To address this question, the cell cycle programs of adult stem cells and enterocytes were genetically isolated and manipulated. These studies revealed that cell cycle alterations led to extensive cell death and morphological disruptions. Despite the extensive tissue damage, no impact was observed on longevity, suggesting a remarkable degree of plasticity in intestinal function.
Lim, R. S., Eyjolfsdottir, E., Shin, E., Perona, P. and Anderson, D. J. (2014). How food controls aggression in Drosophila. PLoS One 9: e105626. PubMed ID: 25162609
How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. For instance, it is unclear whether food increases aggression directly, or as a secondary consequence of increased social interactions caused by attraction to food. This study used the vinegar fly, Drosophila melanogaster, to investigate the manner by which food influences aggression. Food was shown to promote aggression in flies, and it does so independently of any effect on frequency of contact between males, increase in locomotor activity or general enhancement of social interactions. Importantly, the level of aggression depends on the absolute amount of food, rather than on its surface area or concentration. When food resources exceed a certain level, aggression is diminished, suggestive of reduced competition. Finally, it was shown that detection of sugar via Gr5a+ gustatory receptor neurons (GRNs) is necessary for food-promoted aggression. These data demonstrate that food exerts a specific effect to promote aggression in male flies, and that this effect is mediated, at least in part, by sweet-sensing GRNs.
Corrigan, L., Redhai, S., Leiblich, A., Fan, S. J., Perera, S. M., Patel, R., Gandy, C., Wainwright, S. M., Morris, J. F., Hamdy, F., Goberdhan, D. C., Wilson, C. (2014). BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior. J Cell Biol. PubMed ID: 25154396
Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success. In Drosophila melanogaster, these signals are generated by a variety of seminal peptides, many produced by the accessory glands (AGs). One epithelial cell type in the adult male AGs, the secondary cell (SC), grows selectively in response to bone morphogenetic protein (BMP) signaling. This signaling is involved in blocking the rapid remating of mated females, which contributes to the reproductive advantage of the first male to mate. This paper shows that SCs secrete exosomes, membrane-bound vesicles generated inside late endosomal multivesicular bodies (MVBs). After mating, exosomes fuse with sperm (as also seen in vitro for human prostate-derived exosomes and sperm) and interact with female reproductive tract epithelia. Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling. Indeed, when BMP signaling was reduced in SCs, vesicles were still formed in MVBs but not secreted as exosomes. These results demonstrate a new function for the MVB-exosome pathway in the reproductive tract that appears to be conserved across evolution.
Liu, Z. and Zhao, Z. (2014). Effects of light interruption on sleep and viability of Drosophila melanogaster. PLoS One 9: e105678. PubMed ID: 25148297
Light is a very important regulator of the daily sleep rhythm. This study investigated the influence of nocturnal light stimulation on Drosophila sleep. Results showed that total daytime sleep was reduced due to a decrease in daytime sleep episode duration caused by discontinuous light stimulation, but sleep was not strongly impacted at nighttime although the discontinuous light stimulation occurred during the dark phase of the cycle, the scotophase. During a subsequent recovery period without light interruption, the sleep quality of nighttime sleep was improved and of daytime sleep reduced, indicating flies have a persistent response to nocturnal light stimulation. Further studies showed that the discontinuous light stimulation damped the daily rhythm of a circadian light-sensitive protein Cryptochrome both at the mRNA and protein levels, which subsequently caused disappearance of circadian rhythm of the core oscillator timeless and decrease of Timeless protein at nighttime. These data indicate that the nocturnal light interruption plays an important role in sleep through core proteins Cryptochrome and Timeless, Moreover, interruption of sleep further impacted reproduction and viability.
Goda, T., Sharp, B. and Wijnen, H. (2014). Temperature-dependent resetting of the molecular circadian oscillator in Drosophila. Proc Biol Sci 281 [Epub ahead of print]. PubMed ID: 25165772
Circadian clocks responsible for daily time keeping in a wide range of organisms synchronize to daily temperature cycles via pathways that remain poorly understood. To address this problem from the perspective of the molecular oscillator, temperature-dependent resetting of four of its core components was monitored in Drosophila: the transcripts and proteins for the clock genes period (per) and timeless (tim). The molecular circadian cycle in adult heads exhibited parallel responses to temperature-mediated resetting at the levels of per transcript, tim transcript and Tim protein. Early phase adjustment specific to per transcript rhythms was explained by clock-independent temperature-driven transcription of per. The cold-induced expression of Drosophila per contrasts with the previously reported heat-induced regulation of mammalian Period 2. An altered and more readily re-entrainable temperature-synchronized circadian oscillator that featured temperature-driven per transcript rhythms and phase-shifted Tim and Per protein rhythms was found for flies of the 'Tim 4' genotype, which lacked daily tim transcript oscillations but maintained post-transcriptional temperature entrainment of tim expression. The accelerated molecular and behavioural temperature entrainment observed for Tim 4 flies indicates that clock-controlled tim expression constrains the rate of temperature cycle-mediated circadian resetting.
Sunday, September 7th
Herz, H. M., Morgan, M., Gao, X., Jackson, J., Rickels, R., Swanson, S. K., Florens, L., Washburn, M. P., Eissenberg, J. C. and Shilatifard, A. (2014). Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling. Science 345: 1065-1070. PubMed ID: 25170156
Histone H3 lysine(27)-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. This study established a Drosophila animal model for the pathogenic histone H3K27M mutation and shows that its overexpression resembles polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing derepression of PRC2 target genes and developmental perturbations. Similarly, an H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M-containing nucleosomes, and its misregulation in Drosophila results in changes of H3K9 methylation levels and heterochromatic silencing defects. This study has established histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin signaling pathways.
Vogelmann, J., Le Gall, A., Dejardin, S., Allemand, F., Gamot, A., Labesse, G., Cuvier, O., Negre, N., Cohen-Gonsaud, M., Margeat, E. and Nollmann, M. (2014). Chromatin insulator factors involved in long-range DNA interactions and their role in the folding of the Drosophila genome. PLoS Genet 10: e1004544. PubMed ID: 25165871
Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. This study investigated the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and developed a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. BEAF32 was shown to be able to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. A general model is proposed for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division.
Cheng, Z., Cheung, P., Kuo, A. J., Yukl, E. T., Wilmot, C. M., Gozani, O. and Patel, D. J. (2014). A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36. Genes Dev 28: 1758-1771. PubMed ID: 25128496
The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Structural studies reveal that H3K36 specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni(2+) and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue), and Y41 (inserts into its own pocket). Given that KDM2A was found to also bind the H3K36me3 peptide, it was postulated that steric constraints could prevent alpha-ketoglutarate from undergoing an 'off-line'-to-'in-line' transition necessary for the demethylation reaction. Furthermore, structure-guided substitutions of residues in the KDM2A catalytic pocket abrogate KDM2A-mediated functions important for suppression of cancer cell phenotypes. Together, these results deduce insights into the molecular basis underlying KDM2A regulation of the biologically important methylated H3K36 mark.
Wozniak, G. G. and Strahl, B. D. (2014). Catalysis-dependent stabilization of Bre1 fine-tunes histone H2B ubiquitylation to regulate gene transcription. Genes Dev 28: 1647-1652. PubMed ID: 25085417
Monoubiquitylation of histone H2B on Lys123 (H2BK123ub1) plays a multifaceted role in diverse DNA-templated processes, yet the mechanistic details by which this modification is regulated are not fully elucidated. This study shows, in yeast, that H2BK123ub1 is regulated in part through the protein stability of the E3 ubiquitin ligase Bre1. Bre1 stability is controlled by the Rtf1 subunit of the polymerase-associated factor (PAF) complex and through the ability of Bre1 to catalyze H2BK123ub1. Using a domain in Rtf1 that stabilizes Bre1, it was shown that inappropriate Bre1 levels lead to defects in gene regulation. Collectively, these data uncover a novel quality control mechanism used by the cell to maintain proper Bre1 and H2BK123ub1 levels, thereby ensuring proper control of gene expression.
Saturday, September 6th
Bosch, J. A., Sumabat, T. M., Hafezi, Y., Pellock, B. J., Gandhi, K. D. and Hariharan, I. (2014). The Drosophila F-box protein Fbxl7 binds to the protocadherin Fat and regulates Dachs localization and Hippo signaling. Elife: e03383. PubMed ID: 25107277
The Drosophila protocadherin, Fat (Ft) regulates growth, planar cell polarity (PCP) and proximodistal patterning. A key downstream component of Ft signaling is the atypical myosin Dachs (D). Multiple regions of the intracellular domain of Ft have been implicated in regulating growth and PCP but how Ft regulates D is not known. Mutations in Fbxl7 (CG4221), which encodes an F-box protein, result in tissue overgrowth and abnormalities in proximodistal patterning that phenocopy deleting a specific portion of the intracellular domain (ICD) of Ft that regulates both growth and PCP. Fbxl7 binds to this same portion of the Ft ICD, co-localizes with Ft to the proximal edge of cells and regulates the levels and asymmetry of D at the apical membrane. Fbxl7 can also regulate the trafficking of proteins between the apical membrane and intracellular vesicles. Thus Fbxl7 functions in a subset of pathways downstream of Ft and links Ft to D localization.
Luchtenborg, A. M., Solis, G. P., Egger-Adam, D., Koval, A., Lin, C., Blanchard, M. G., Kellenberger, S. and Katanaev, V. L. (2014). Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton. Development 141: 3399-3409. PubMed ID: 25139856
Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. This study demonstrates that the heterotrimeric G protein Go functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. Ankyrin 2 was identified as a target of Go signaling required for NMJ formation. Moreover, the Go-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the Go-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. This work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function.
Qian, Y., Dominado, N., Zoller, R., Ng, C., Kudyba, K., Siddall, N. A., Hime, G. G. and Schulz, C. (2014). Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 25169192
The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, this study showed that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. It is proposed that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells.
Milet, C., Rincheval-Arnold, A., Morieras, A., Clavier, A., Garrigue, A., Mignotte, B. and Guenal, I. (2014). Mutating RBF Can Enhance Its Pro-Apoptotic Activity and Uncovers a New Role in Tissue Homeostasis. PLoS One 9: e102902. PubMed ID: 25089524
The tumor suppressor retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb (RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, this study generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBFD253A, conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. This overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo.
Friday, September 5th
Coelho, D. S., Gaspar, C. J. and Domingos, P. M. (2014). Ire1 mediated mRNA splicing in a C-terminus deletion mutant of Drosophila xbp1. PLoS One 9: e105588. PubMed ID: 25136861
The Unfolded Protein Response is a homeostatic mechanism that permits eukaryotic cells to cope with Endoplasmic Reticulum (ER) stress caused by excessive accumulation of misfolded proteins in the ER lumen. The more conserved branch of the UPR relies on an ER transmembrane enzyme, Ire1, which, upon ER stress, promotes the unconventional splicing of a small intron from the mRNA encoding the transcription factor Xbp1. In mammals, two specific regions (the hydrophobic region 2 - HR2 - and the C-terminal translational pausing site) present in the Xbp1unspliced protein mediate the recruitment of the Xbp1 mRNA-ribosome-nascent chain complex to the ER membrane, so that Xbp1 mRNA can be spliced by Ire1. This study generated a Drosophila Xbp1 deletion mutant (Excision101) lacking both HR2 and C-terminal region, but not the Ire1 splicing site. Ire1-dependent splicing of Xbp1 mRNA is reduced, but not abolished in Excision101. The results suggest the existence of additional mechanisms for ER membrane targeting of Xbp1 mRNA that are independent of the C-terminal domain of Drosophila Xbp1unspliced.
Wang, C., Feng, T., Wan, Q., Kong, Y. and Yuan, L. (2014). miR-124 controls Drosophila behavior and is required for neural development. Int J Dev Neurosci [Epub ahead of print]. PubMed ID: 25169673
MicroRNA-124 (miR-124) is an evolutionarily conserved, small, noncoding RNA molecule that participates in the central nervous system (CNS) developmental control of gene expression. This study found that Drosophila embryos lacking the miR-124 gene did not exhibit detectable defects in axon growth or CNS development. In contrast, adult mutants showed severe problems in locomotion, flight, and female fertility. Furthermore, the deficits that were observed in the adult stage could be marginally rescued with elav-GAL4 driven expression of miR-124, making elav-GAL4 an excellently simulated driver to induce entopic over-expression of miR-124. Further developmental assessment in the third instar larval neuromuscular junction (NMJ) and dendritic arborization (DA) neurons was performed with miR-124 knock outs, flies over-expressing miR-124, and rescue models. Typically, the absence and over-abundance of a molecular signal exerts opposite effects on development or phenotype. However, it was determined that both miR-124 knock-outs and over-expressing flies displayed reduced NMJ 6/7 bouton number and branch length. Similarly, reduced ddaE branching numbers were observed between the two mutant lines. As to ddaF, branching number was not influenced by miR-124 knock out, but was statistically reduced by miR-124 over-expression. While it was not possible determine any causal relationship between behavioral defects and dysplasia of NMJs or DA neurons, there were some accompanying relationships among behavioral phenotypes, NMJ abnormalities, and ddaE/ddaF dendritic branching which were all controlled by miR-124.
Ninova, M., Ronshaugen, M. and Griffiths-Jones, S. (2014). Conserved temporal patterns of microRNA expression in Drosophila support a developmental hourglass model.. Genome Biol Evol [Epub ahead of print]. PubMed ID: 25169982
The spatiotemporal control of gene expression is crucial for the successful completion of animal development. The evolutionary constraints on development are particularly strong for the mid-embryonic stage when body segments are specified, as evidenced by a high degree of morphological and protein-coding gene conservation during this period - a phenomenon known as the developmental hourglass. The discovery of microRNA-mediated gene control revealed an entirely new layer of complexity of the molecular networks that orchestrate development. However, the constraints on microRNA developmental expression and evolution, and the implications for animal evolution are less well understood. To systematically explore the conservation of microRNAs during development, a genome-wide comparative study was carried out of microRNA expression levels throughout the ontogenesis of two divergent fruit flies, Drosophila melanogaster and Drosophila virilis. Orthologous microRNAs were shown to display highly similar temporal profiles regardless of their mutation rates, suggesting that the timely expression of microRNA genes can be more constrained than their sequence. Furthermore, transitions between key developmental events in the different species are accompanied by conserved shifts in microRNA expression profiles, with the mid-embryonic period between gastrulation and segmentation characterized with the highest similarity of microRNA expression. The conservation of microRNA expression therefore displays an hourglass pattern similar to that observed for protein-coding genes.
Kozomara, A., Hunt, S., Ninova, M., Griffiths-Jones, S. and Ronshaugen, M. (2014). Target repression induced by endogenous microRNAs: Large differences, small effects. PLoS One 9: e104286. PubMed ID: 25141277
MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity - that is, highly expressed microRNAs will repress their target mRNAs more. This study investigated the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, more abundant microRNAs were found to repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, examples were found of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. The association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.
Aspden, J. L., Eyre-Walker, Y. C., Philips, R. J., Amin, U., Mumtaz, M. A., Brocard, M. and Couso, J. P. (2014). Extensive translation of small ORFs revealed by Poly-Ribo-Seq. Elife: e03528. PubMed ID: 25144939
Thousands of small Open Reading Frames (smORFs) with the potential to encode small peptides of fewer than 100 amino acids exist in our genomes. However, the number of smORFs actually translated, and their molecular and functional roles are still unclear. This study presents a genome-wide assessment of smORF translation by ribosomal profiling of polysomal fractions in Drosophila. Two types of smORFs bound by multiple ribosomes and thus undergoing productive translation were detected. The 'longer' smORFs of around 80 amino-acids resemble canonical proteins in translational metrics and conservation, and display a propensity to contain transmembrane motifs. The 'dwarf' smORFs are in general shorter (around 20 amino-acid long), are mostly found in 5'-UTRs and non-coding RNAs, are less well conserved and have no bioinformatic indicators of peptide function. These findings indicate that thousands of smORFs are translated in metazoan genomes, reinforcing the idea that smORFs are an abundant and fundamental genome component.
Thursday, September 4th
Gunage, R. D., Reichert, H. and VijayRaghavan, K. (2014). Identification of a new stem cell population which generates Drosophila flight muscles. Elife: e03126. PubMed ID: 25135939
How myoblast populations are regulated for the formation of muscles of different sizes is an essentially unanswered question. The large flight muscles of Drosophila develop from adult muscle progenitor (AMP) cells set-aside embryonically. The thoracic segments are all allotted the same small AMP number, while those associated with the wing-disc proliferate extensively to give rise to over 2500 myoblasts. An initial amplification occurs through symmetric divisions and is followed by a switch to asymmetric divisions in which the AMPs self-renew and generate post-mitotic myoblasts. Notch signaling controls the initial amplification of AMPs, while the switch to asymmetric division additionally requires Wingless, which regulates Numb expression in the AMP lineage. In both cases, the epidermal tissue of the wing imaginal disc acts as a niche expressing the ligands Serrate and Wingless. The disc-associated AMPs are a novel muscle stem cell population that orchestrates the early phases of adult flight muscle development.
Pan, L., Wang, S., Lu, T., Weng, C., Song, X., Park, J. K., Sun, J., Yang, Z. H., Yu, J., Tang, H., McKearin, D. M., Chamovitz, D. A., Ni, J. and Xie, T. (2014). Protein competition switches the function of COP9 from self-renewal to differentiation. Nature [Epub ahead of print]. PubMed ID: 25119050
The balance between stem cell self-renewal and differentiation is controlled by intrinsic factors and niche signals. In the Drosophila melanogaster ovary, some intrinsic factors promote germline stem cell (GSC) self-renewal, whereas others stimulate differentiation. However, it remains poorly understood how the balance between self-renewal and differentiation is controlled. This study used D. melanogaster ovarian GSCs to demonstrate that the differentiation factor Bam controls the functional switch of the COP9 complex (see CSN5) from self-renewal to differentiation via protein competition. The COP9 complex is composed of eight Csn subunits, Csn1-8, and removes Nedd8 modifications from target proteins. Genetic results indicated that the COP9 complex is required intrinsically for GSC self-renewal, whereas other Csn proteins, with the exception of Csn4, were also required for GSC progeny differentiation. Bam-mediated Csn4 sequestration from the COP9 complex via protein competition inactivated the self-renewing function of COP9 and allowed other Csn proteins to promote GSC differentiation. Therefore, this study reveals a protein-competition-based mechanism for controlling the balance between stem cell self-renewal and differentiation. Because numerous self-renewal factors are ubiquitously expressed throughout the stem cell lineage in various systems, protein competition may function as an important mechanism for controlling the self-renewal-to-differentiation switch.
Homem, C. C., Steinmann, V., Burkard, T. R., Jais, A., Esterbauer, H. and Knoblich, J. A. (2014). Ecdysone and mediator change energy metabolism to terminate proliferation in Drosophila neural stem cells. Cell 158: 874-888. PubMed ID: 25126791
Stem cells are highly abundant during early development but become a rare population in most adult organs. The molecular mechanisms causing stem cells to exit proliferation at a specific time are not well understood. This study shows that changes in energy metabolism induced by the steroid hormone ecdysone and the Mediator (see Med19) initiate an irreversible cascade of events leading to cell-cycle exit in Drosophila neural stem cells. The timely induction of oxidative phosphorylation and the mitochondrial respiratory chain are required in neuroblasts to uncouple the cell cycle from cell growth. This results in a progressive reduction in neuroblast cell size and ultimately in terminal differentiation. Brain tumor mutant neuroblasts fail to undergo this shrinkage process and continue to proliferate until adulthood. These findings show that cell size control can be modified by systemic hormonal signaling and reveal a unique connection between metabolism and proliferation in stem cells.
Zander, M. A., Cancino, G. I., Gridley, T., Kaplan, D. R. and Miller, F. D. (2014). The Snail transcription factor regulates the numbers of neural precursor cells and newborn neurons throughout mammalian life. PLoS One 9: e104767. PubMed ID: 25136812
The Snail transcription factor regulates diverse aspects of stem cell biology in organisms ranging from Drosophila to mammals. This study asked whether it regulates the biology of neural precursor cells (NPCs) in the forebrain of postnatal and adult mice, taking advantage of a mouse containing a floxed Snail allele (Snailfl/fl mice). Inducibly ablating Snail in the embryonic cortex has long-term consequences for cortical organization. In particular, when Snailfl/fl mice are crossed to Nestin-cre mice that express Cre recombinase in embryonic neural precursors, this causes inducible ablation of Snail expression throughout the postnatal cortex. This loss of Snail causes a decrease in proliferation of neonatal cortical neural precursors and mislocalization and misspecification of cortical neurons. Moreover, these precursor phenotypes persist into adulthood. Adult neural precursor cell proliferation is decreased in the forebrain subventricular zone and in the hippocampal dentate gyrus, coincident with a decrease in the number of adult-born olfactory and hippocampal neurons. Thus, Snail is a key regulator of the numbers of neural precursors and newborn neurons throughout life.
Wednesday, September 3rd
Vogt, K., Schnaitmann, C., Dylla, K. V., Knapek, S., Aso, Y., Rubin, G. M. and Tanimoto, H. (2014). Shared mushroom body circuits underlie visual and olfactory memories in Drosophila. Elife 3: e02395. PubMed ID: 25139953
In nature, animals form memories associating reward or punishment with stimuli from different sensory modalities, such as smells and colors. It is unclear, however, how distinct sensory memories are processed in the brain. This study established appetitive and aversive visual learning assays for Drosophila that are comparable to the widely used olfactory learning assays. These assays share critical features, such as reinforcing stimuli (sugar reward and electric shock punishment), and allow direct comparison of the cellular requirements for visual and olfactory memories. It was found that the same subsets of dopamine neurons drive formation of both sensory memories. Furthermore, distinct yet partially overlapping subsets of mushroom body intrinsic neurons are required for visual and olfactory memories. Thus, these results suggest that distinct sensory memories are processed in a common brain center. Such centralization of related brain functions is an economical design that avoids the repetition of similar circuit motifs.
Koh, T. W., He, Z., Gorur-Shandilya, S., Menuz, K., Larter, N. K., Stewart, S. and Carlson, J. R. (2014). The Drosophila IR20a Clade of Ionotropic Receptors Are Candidate Taste and Pheromone Receptors. Neuron 83(4):850-65. PubMed ID: 25123314
Insects use taste to evaluate food, hosts, and mates. Drosophila has many "orphan" taste neurons that express no known taste receptors. The Ionotropic Receptor (IR) superfamily is best known for its role in olfaction, but virtually nothing is known about a clade of approximately 35 members, the IR20a clade. Here, a comprehensive analysis of this clade reveals expression in all taste organs of the fly. Some members are expressed in orphan taste neurons, whereas others are coexpressed with bitter- or sugar-sensing Gustatory receptor (Gr) genes. Analysis of the closely related IR52c and IR52d genes reveals signatures of adaptive evolution, roles in male mating behavior, and sexually dimorphic expression in neurons of the male foreleg, which contacts females during courtship. These neurons are activated by conspecific females and contact a neural circuit for sexual behavior. Together, these results greatly expand the repertoire of candidate taste and pheromone receptors in the fly.
Ray, A., van Naters, W. G. and Carlson, J. R. (2014). Molecular determinants of odorant receptor function in insects. J Biosci 39: 555-563. PubMed ID: 25116610
The olfactory system of Drosophila melanogaster provides a powerful model to study molecular and cellular mechanisms underlying function of a sensory system. In the 1970s Siddiqi and colleagues pioneered the application of genetics to olfactory research and isolated several mutant Drosophila with odorant-specific defects in olfactory behaviour, suggesting that odorants are detected differentially by the olfactory system. Since then basic principles of olfactory system function and development have emerged using Drosophila as a model. Nearly four decades later computational methods can be added to further understanding of how specific odorants are detected by receptors. Using a comparative approach this study identified two categories of short amino acid sequence motifs: ones that are conserved family-wide predominantly in the C-terminal half of most receptors, and ones that are present in receptors that detect a specific odorant, 4-methylphenol, found predominantly in the N-terminal half. The odorant-specific sequence motifs are predictors of phenol detection in Anopheles gambiae and other insects, suggesting they are likely to participate in odorant binding. Conversely, the family-wide motifs are expected to participate in shared functions across all receptors and a mutation in the most conserved motif leads to a reduction in odor response. These findings lay a foundation for investigating functional domains within odorant receptors that can lead to a molecular understanding of odor detection.
Petzoldt, A. G., Lee, Y. H., Khorramshahi, O., Reynolds, E., Plested, A. J., Herzel, H. and Sigrist, S. J. (2014). Gating Characteristics Control Glutamate Receptor Distribution and Trafficking In Vivo. Curr Biol. PubMed ID: 25131677
Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking. In recent years, structural and electrophysiological analyses have improved understanding of GluR gating mechanism. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking in vivo, this study provides evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity.
Tuesday, September 2nd
Lin, R., Angelin, A., Da Settimo, F., Martini, C., Taliani, S., Zhu, S. and Wallace, D. C. (2014). Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Ab42-induced neurodegeneration. Aging Cell 13: 507-518. PubMed ID: 24977274
The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, the Drosophila homolog, CG2789/dTSPO, was identified, and the effects of its inactivation was studied by P-element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5-4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to gamma-irradiation or H2O2 exposure, as well as extended male fly lifespan and inhibited Aβ42-induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target.
DeVorkin, L. and Gorski, S. M. (2014). A mitochondrial-associated link between an effector caspase and autophagic flux. Autophagy 10 [Epub ahead of print]. PubMed ID: 25126735
It has become evident that caspases function in nonapoptotic cellular processes in addition to the canonical role for caspases in apoptotic cell death. It has been demonstrated that the Drosophila effector caspase Dcp-1 localizes to the mitochondria and positively regulates starvation-induced autophagic flux during mid-oogenesis. Loss of Dcp-1 leads to elongation of the mitochondrial network, increased levels of the adenine nucleotide translocase sesB, increased ATP levels, and a reduction in autophagy. sesB is a negative regulator of autophagic flux, and Dcp-1 interacts with sesB in a nonproteolytic manner to regulate its stability, uncovering a novel mechanism of mitochondrial associated, caspase-mediated regulation of autophagy in vivo.
Choi, Y. J., Saez, B., Anders, L., Hydbring, P., Stefano, J., Bacon, N. A., Cook, C., Kalaszczynska, I., Signoretti, S., Young, R. A., Scadden, D. T. and Sicinski, P. (2014). D-Cyclins Repress Apoptosis in Hematopoietic Cells by Controlling Death Receptor Fas and Its Ligand FasL. Dev Cell [Epub ahead of print]. PubMed ID: 25087893
D-type cyclins (D1, D2, and D3; see Drosophila Cyclin D) are components of the mammalian core cell-cycle machinery and function to drive cell proliferation. This study reports that D-cyclins perform a rate-limiting antiapoptotic function in vivo. Acute shutdown of all three D-cyclins in bone marrow of adult mice resulted in massive apoptosis of all hematopoietic cell types. Adult hematopoietic stem cells are particularly dependent on D-cyclins for survival, and they are especially sensitive to cyclin D loss. Surprisingly, it was found that the antiapoptotic function of D-cyclins also operates in quiescent hematopoietic stem and progenitor cells. This analyses revealed that D-cyclins repress the expression of the death receptor Fas and its ligand, FasL. Acute ablation of D-cyclins upregulated these proapoptotic genes and led to Fas- and caspase 8-dependent apoptosis. These results reveal an unexpected function of cell-cycle proteins in controlling apoptosis.
Seo, T. W., Lee, J. S. and Yoo, S. J. (2014). Cellular Inhibitor of Apoptosis Protein 1 ubiquitinates endonuclease G but does not affect endonuclease G-mediated cell death. Biochem Biophys Res Commun. PubMed ID: 25139236
Inhibitors of Apoptosis Proteins (IAPs) are evolutionarily well conserved and have been recognized as the key negative regulators of apoptosis. Recently, the role of IAPs as E3 ligases through the Ring domain was revealed. Using proteomic analysis to explore potential target proteins of DIAP1, Drosophila Endonuclease G (dEndoG), which is known as an effector of caspase-independent cell death, was identified. This study demonstrates that human EndoG interacts with IAPs, including human cellular Inhibitor of Apoptosis Protein 1 (cIAP1). EndoG was ubiquitinated by IAPs in vitro and in human cell lines. Interestingly, cIAP1 was capable of ubiquitinating EndoG in the presence of wild-type and mutant Ubiquitin, in which all lysines except K63 were mutated to arginine. cIAP1 expression did not change the half-life of EndoG and cIAP1 depletion did not alter its levels. Expression of dEndoG 54310, in which the mitochondrial localization sequence was deleted, led to cell death that could not be suppressed by DIAP1 in S2 cells. Moreover, EndoG-mediated cell death induced by oxidative stress in HeLa cells was not affected by cIAP1. Therefore, these results indicate that IAPs interact and ubiquitinate EndoG via K63-mediated isopeptide linkages without affecting EndoG levels and EndoG-mediated cell death, suggesting that EndoG ubiquitination by IAPs may serve as a regulatory signal independent of proteasomal degradation.
Monday, September 1st
Donlea, J. M., Ramanan, N., Silverman, N. and Shaw, P. J. (2014). Genetic rescue of functional senescence in synaptic and behavioral plasticity. Sleep 37 [Epub ahead of print]. PubMed ID: 25142573
Aging has been linked with decreased neural plasticity and memory formation in humans and in laboratory model species such as the fruit fly, Drosophila melanogaster. This study examined plastic responses following social experience in Drosophila as a high-throughput method to identify interventions that prevent these impairments. Young (5-day old) or aged (20-day old) adult female Drosophila were housed in socially enriched or isolated environments, then assayed for changes in sleep and for structural markers of synaptic terminal growth in the ventral lateral neurons (LNVs) of the circadian clock. When young flies are housed in a socially enriched environment, they exhibit synaptic elaboration within a component of the circadian circuitry, the LNVs, which is followed by increased sleep. Aged flies, however, no longer exhibit either of these plastic changes. Because of the tight correlation between neural plasticity and ensuing increases in sleep, sleep after enrichment was used as a high-throughput marker for neural plasticity to identify interventions that prolong youthful plasticity in aged flies. To validate this strategy, three independent genetic manipulations were used that delay age-related losses in plasticity: (1) elevation of dopaminergic signaling, (2) over-expression of the serum response factor transcription factor blistered (bs) in the LNVs, and (3) reduction of the Imd immune signaling pathway. These findings provide proof-of-principle evidence that measuring changes in sleep in flies after social enrichment may provide a highly scalable assay for the study of age-related deficits in synaptic plasticity. These studies demonstrate that Drosophila provides a promising model for the study of age-related loss of neural plasticity and begin to identify genes that might be manipulated to delay the onset of functional senescence.
Singh, K., Ju, J. Y., Walsh, M. B., DiIorio, M. A. and Hart, A. C. (2014). Deep Conservation of Genes Required for Both Drosophila melanogaster and Caenorhabditis elegans Sleep Includes a Role for Dopaminergic Signaling. Sleep 37 [Epub ahead of print]. PubMed ID: 25142568
Cross-species conservation of sleep-like behaviors predicts the presence of conserved molecular mechanisms underlying sleep. However, limited experimental evidence of conservation exists. This prediction is tested directly in this study. During lethargus, Caenorhabditis elegans spontaneously sleep in short bouts that are interspersed with bouts of spontaneous locomotion. Twenty-six genes required for Drosophila melanogaster sleep wee identified. Twenty orthologous C. elegans genes were selected based on similarity. Their effect on C. elegans sleep and arousal during the last larval lethargus was assessed. The 20 most similar genes altered both the quantity of sleep and arousal thresholds. In 18 cases, the direction of change was concordant with Drosophila studies published previously. Additionally, a conserved genetic pathway was delineated by which dopamine regulates sleep and arousal. In C. elegans neurons, G-alpha S, adenylyl cyclase, and protein kinase A act downstream of D1 dopamine receptors to regulate these behaviors. Finally, a quantitative analysis of genes examined herein revealed that C. elegans arousal thresholds were directly correlated with amount of sleep during lethargus. However, bout duration varies little and was not correlated with arousal thresholds. The comprehensive analysis presented in this study suggests that conserved genes and pathways are required for sleep in invertebrates and, likely, across the entire animal kingdom. The genetic pathway delineated in this study implicates G-alpha S and previously known genes downstream of dopamine signaling in sleep. Quantitative analysis of various components of quiescence suggests that interdependent or identical cellular and molecular mechanisms are likely to regulate both arousal and sleep entry.
Seeds, A. M., Ravbar, P., Chung, P., Hampel, S., Midgley, F. M., Jr., Mensh, B. D. and Simpson, J. H. (2014). A suppression hierarchy among competing motor programs drives sequential grooming in Drosophila. Elife 3: e02951. PubMed ID: 25139955
Motor sequences are formed through the serial execution of different movements, but how nervous systems implement this process remains largely unknown. This study determined the organizational principles governing how dirty fruit flies groom their bodies with sequential movements. Using genetically targeted activation of neural subsets, distinct motor programs were driven that clean individual body parts. This enabled competition experiments revealing that the motor programs are organized into a suppression hierarchy; motor programs that occur first suppress those that occur later. Cleaning one body part reduces the sensory drive to its motor program, which relieves suppression of the next movement, allowing the grooming sequence to progress down the hierarchy. A model featuring independently evoked cleaning movements activated in parallel, but selected serially through hierarchical suppression, was successful in reproducing the grooming sequence. This provides the first example of an innate motor sequence implemented by the prevailing model for generating human action sequences.
Dunlap, A. S. and Stephens, D. W. (2014). Experimental evolution of prepared learning. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 25071167
Animals learn some things more easily than others. To explain this so-called prepared learning, investigators commonly appeal to the evolutionary history of stimulus-consequence relationships experienced by a population or species. This study offers a simple model that formalizes this long-standing hypothesis. The key variable in the model is the statistical reliability of the association between stimulus, action, and consequence. Experimental evolution was used to test this hypothesis in populations of Drosophila. The reliability of two types of experience (the pairing of the aversive chemical quinine with color or with odor) was systematically manipulated. Following 40 generations of evolution, data from learning assays support the basic prediction: Changes in learning abilities track the reliability of associations during a population's selective history. In populations where, for example, quinine-color pairings were unreliable but quinine-odor pairings were reliable, increased sensitivity to learning the quinine-odor experience was found and reduced sensitivity to learning quinine-color. This is the first experimental demonstration of the evolution of prepared learning.
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