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


Wednesday, August 31st, 2016

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Otto, N., Risse, B., Berh, D., Bittern, J., Jiang, X. and Klämbt, C. (2016). Interactions among Drosophila larvae before and during collision. Sci Rep 6: 31564. PubMed ID: 27511760
In populations of Drosophila larvae, both, an aggregation and a dispersal behavior can be observed. However, the mechanisms coordinating larval locomotion in respect to other animals, especially in close proximity and during/after physical contacts are currently only little understood. This study tested whether relevant information is perceived before or during larva-larva contacts, analyze its influence on behavior and ask whether larvae avoid or pursue collisions. Employing frustrated total internal reflection-based imaging (FIM) it was found that larvae visually detect other moving larvae in a narrow perceptive field and respond with characteristic escape reactions. To decipher larval locomotion not only before but also during the collision, a two color FIM approach (FIM2c) was utilized, allowing faithful extraction of the posture and motion of colliding animals. It was found that during collision, larval locomotion freezes and sensory information is sampled during a KISS phase (german: Kollisions Induziertes Stopp Syndrom or english: collision induced stop syndrome). Interestingly, larvae react differently to living, dead or artificial larvae, discriminate other Drosophila species and have an increased bending probability for a short period after the collision terminates. Thus, Drosophila larvae evolved means to specify behaviors in response to other larvae. 

Buehlmann, C., Woodgate, J. L. and Collett, T. S. (2016). On the encoding of panoramic visual scenes in navigating wood ants. Curr Biol [Epub ahead of print]. PubMed ID: 27476601
A natural visual panorama is a complex stimulus formed of many component shapes. It gives an animal a sense of place and supplies guiding signals for controlling the animal's direction of travel. Insects with their economical neural processing are good subjects for analyzing the encoding and memory of such scenes. Honeybees and ants foraging from their nest can follow habitual routes guided only by visual cues within a natural panorama. This study analyzed the headings that ants adopt when a familiar panorama composed of two or three shapes is manipulated by removing a shape or by replacing training shapes with unfamiliar ones. Ants were shown to recognize a component shape not only through its particular visual features, but also by its spatial relation to other shapes in the scene, and that (2) each segmented shape contributes its own directional signal to generating the ant's chosen heading. It was found earlier that ants trained to a feeder placed to one side of a single shape and tested with shapes of different widths learn the retinal position of the training shape's center of mass (CoM) when heading toward the feeder. They then guide themselves by placing the shape's CoM in the remembered retinal position. This use of CoM in a one-shape panorama combined with the results here suggests that the ants' memory of a multi-shape panorama comprises the retinal positions of the horizontal CoMs of each major component shape within the scene, bolstered by local descriptors of that shape.
Klein, A., Schultner, E., Lowak, H., Schrader, L., Heinze, J., Holman, L. and Oettler, J. (2016). Evolution of social insect polyphenism facilitated by the sex differentiation cascade. PLoS Genet 12: e1005952. PubMed ID: 27031240
The major transition to eusociality required the evolution of a switch to canalize development into either a reproductive or a helper, the nature of which is currently unknown. Following predictions from the 'theory of facilitated variation', this study identified sex differentiation pathways as promising candidates because of their pre-adaptation to regulating development of complex phenotypes. Conserved core genes, including the juvenile hormone-sensitive master sex differentiation gene doublesex (dsx) and a kruppel homolog 2 (kr-h2) with putative regulatory function, exhibit both sex and morph-specific expression across life stages in the ant Cardiocondyla obscurior. It is hypothesized that genes in the sex differentiation cascade evolved perception of alternative input signals for caste differentiation (i.e. environmental or genetic cues), and that their inherent switch-like and epistatic behavior facilitated signal transfer to downstream targets, thus allowing them to control differential development into morphological castes.
Ding, Y., Berrocal, A., Morita, T., Longden, K. D. and Stern, D. L. (2016). Natural courtship song variation caused by an intronic retroelement in an ion channel gene. Nature 536: 329-332. PubMed ID: 27509856
Animal species display enormous variation for innate behaviours, but little is known about how this diversity arose. Using an unbiased genetic approach, this study mapped a courtship song difference between wild isolates of Drosophila simulans and Drosophila mauritiana to a 966 base pair region within the slowpoke (slo) locus, which encodes a calcium-activated potassium channel. Using the reciprocal hemizygosity test, it was confirmed that slo is the causal locus, and the causal mutation was resolved to the evolutionarily recent insertion of a retroelement in a slo intron within D. simulans. Targeted deletion of this retroelement reverts the song phenotype and alters slo splicing. Like many ion channel genes, slo is expressed widely in the nervous system and influences a variety of behaviours; slo-null males sing little song with severely disrupted features. By contrast, the natural variant of slo alters a specific component of courtship song, illustrating that regulatory evolution of a highly pleiotropic ion channel gene can cause modular changes in behaviour.

Tuesday, August 30th

Takashima, S., Aghajanian, P., Younossi-Hartenstein, A. and Hartenstein, V. (2016). Origin and dynamic lineage characteristics of the developing Drosophila midgut stem cells. Dev Biol [Epub ahead of print]. PubMed ID: 27321560
Proliferating intestinal stem cells (ISCs) generate all cell types of the Drosophila midgut, including enterocytes, endocrine cells, and gland cells (e.g., copper cells), throughout the lifetime of the animal. Among the signaling mechanisms controlling the balance between ISC self-renewal and the production of different cell types, Notch (N) plays a pivotal role. This study investigates the emergence of ISCs during metamorphosis and the role of N in this process. Precursors of the Drosophila adult intestinal stem cells (pISCs) can be first detected within the pupal midgut during the first hours after onset of metamorphosis as motile mesenchymal cells. pISCs perform 2-3 rounds of parasynchronous divisions. The first mitosis yields only an increase in pISC number. During the following rounds of mitosis, dividing pISCs give rise to more pISCs, as well as the endocrine cells that populate the midgut of the eclosing fly. Enterocytes do not appear among the pISC progeny until around the time of eclosion. The "proendocrine" gene prospero (pros), expressed from mid-pupal stages onward in pISCs, is responsible to advance the endocrine fate in these cells; following removal of pros, pISCs continue to proliferate, but endocrine cells do not form. Conversely, the onset of N activity that occurs around the stage when pros comes on restricts pros expression among pISCs. Loss of N abrogates proliferation and switches on an endocrine fate among all pISCs. These results suggest that a switch depending on the activity of N and pros acts at the level of the pISC to decide between continued proliferation and endocrine differentiation.

Giordani, G., Barraco, M., Giangrande, A., Martinelli, G., Guadagnuolo, V., Simonetti, G., Perini, G. and Bernardoni, R. (2016). The human Smoothened inhibitor PF-04449913 induces exit from quiescence and loss of multipotent Drosophila hematopoietic progenitor cells. Oncotarget [Epub ahead of print]. PubMed ID: 27486815
The efficient treatment of hematological malignancies as Acute Myeloid Leukemia, myelofibrosis and Chronic Myeloid Leukemia, requires the elimination of cancer-initiating cells. In mammals, inhibition of Smoothened, the key mediator of the Hedgehog signaling pathway, reduces Chronic Myeloid Leukemia progression and propagation. These findings make Smo a candidate target to inhibit maintenance of leukemia-initiating cells. In Drosophila the same pathway maintains the hematopoietic precursor cells of the lymph gland, the hematopoietic organ that develops in the larva. This study investigated in Drosophila the mode of action of PF-04449913, a small-molecule inhibitor of the human Smo protein. Drosophila larvae fed with PF-04449913 showed traits of altered hematopoietic homeostasis. These include the development of melanotic nodules, increase of circulating hemocytes, the size increase of the lymph gland and accelerated differentiation of blood cells likely due to the exit of multi-potent precursors from quiescence. Importantly, the Smo inhibition can lead to the complete loss of hematopoietic precursors. It is concluded that PF-04449913 inhibits Drosophila Smo blocking the Hh signaling pathway and causing the loss of hematopoietic precursor cells. Interestingly, this is the effect expected in patients treated with PF-04449913: number decrease of cancer initiating cells in the bone marrow to reduce the risk of leukemia relapse. Altogether these results indicate that Drosophila comprises a model system for the in vivo study of molecules that target evolutionary conserved pathways implicated in human hematological malignancies.
Jiang, K., Liu, Y., Fan, J., Zhang, J., Li, X. A., Evers, B. M., Zhu, H. and Jia, J. (2016). PI(4)P promotes phosphorylation and conformational change of Smoothened through interaction with its C-terminal tail. PLoS Biol 14: e1002375. PubMed ID: 26863604
In Hedgehog (Hh) signaling, binding of Hh to the Patched-Interference Hh (Ptc-Ihog) receptor complex relieves Ptc inhibition on Smoothened (Smo). A longstanding question is how Ptc inhibits Smo and how such inhibition is relieved by Hh stimulation. This study found that Hh elevates production of phosphatidylinositol 4-phosphate (PI(4)P). Increased levels of PI(4)P promote, whereas decreased levels of PI(4)P inhibit, Hh signaling activity. It was further found that PI(4)P directly binds Smo through an arginine motif, which then triggers Smo phosphorylation and activation. Moreover, the pleckstrin homology (PH) domain of G protein-coupled receptor kinase 2 (Gprk2) was identified as an essential component for enriching PI(4)P and facilitating Smo activation. PI(4)P also binds mouse Smo (mSmo) and promotes its phosphorylation and ciliary accumulation. Finally, Hh treatment increases the interaction between Smo and PI(4)P but decreases the interaction between Ptc and PI(4)P, indicating that, in addition to promoting PI(4)P production, Hh regulates the pool of PI(4)P associated with Ptc and Smo.
Simon, C. M., Janas, A. M., Lotti, F., Tapia, J. C., Pellizzoni, L. and Mentis, G. Z. (2016). A stem cell model of the motor circuit uncouples motor neuron death from hyperexcitability induced by SMN deficiency. Cell Rep. PubMed ID: 27452470
Evolutionary Homolog Study:
In spinal muscular atrophy, a neurodegenerative disease caused by ubiquitous deficiency in the survival motor neuron (SMN) protein, sensory-motor synaptic dysfunction and increased excitability precede motor neuron (MN) loss. Whether central synaptic dysfunction and MN hyperexcitability are cell-autonomous events or they contribute to MN death is unknown. These issues were addressed using a stem-cell-based model of the motor circuit consisting of MNs and both excitatory and inhibitory interneurons (INs) in which SMN protein levels are selectively depleted. SMN deficiency was shown to induce selective MN death through cell-autonomous mechanisms, while hyperexcitability is a non-cell-autonomous response of MNs to defects in pre-motor INs, leading to loss of glutamatergic synapses and reduced excitation. Findings from this in vitro model suggest that dysfunction and loss of MNs result from differential effects of SMN deficiency in distinct neurons of the motor circuit and that hyperexcitability does not trigger MN death.

Monday, August 29th

Jahanshahi, M., Hsiao, K., Jenny, A. and Pfleger, C. M. (2016). The Hippo pathway targets Rae1 to regulate mitosis and organ size and to feed back to regulate upstream components Merlin, Hippo, and Warts. PLoS Genet 12: e1006198. PubMed ID: 27494403
Hippo signaling acts as a master regulatory pathway controlling growth, proliferation, and apoptosis and also ensures that variations in proliferation do not alter organ size. How the pathway coordinates restricting proliferation with organ size control remains a major unanswered question. This study identifies Rae1 as a highly-conserved target of the Hippo Pathway integrating proliferation and organ size. Genetic and biochemical studies in Drosophila cells and tissues and in mammalian cells indicate that Hippo signaling promotes Rae1 degradation downstream of Warts/Lats. In proliferating cells, Rae1 loss restricts Cyclin B levels and organ size while Rae1 over-expression increases Cyclin B levels and organ size, similar to Hippo Pathway over-activation or loss-of-function, respectively. Importantly, Rae1 regulation by the Hippo Pathway is crucial for its regulation of Cyclin B and organ size; reducing Rae1 blocks Cyclin B accumulation and suppresses overgrowth caused by Hippo pathway loss. Surprisingly, in addition to suppressing overgrowth, reducing Rae1 also compromises survival of epithelial tissue overgrowing due to loss of Hippo signaling leading to a tissue 'synthetic lethality' phenotype. Excitingly, Rae1 plays a highly conserved role to reduce the levels and activity of the Yki/YAP oncogene. Rae1 increases activation of the core kinases Hippo and Warts and plays a post-transcriptional role to increase the protein levels of the Merlin, Hippo, and Warts components of the pathway; therefore, in addition to Rae1 coordinating organ size regulation with proliferative control, it is proposed that Rae1 also acts in a feedback circuit to regulate pathway homeostasis.
Ashton-Beaucage, D., Lemieux, C., Udell, C. M., Sahmi, M., Rochette, S. and Therrien, M. (2016). The deubiquitinase USP47 stabilizes MAPK by counteracting the function of the N-end rule ligase POE/UBR4 in Drosophila. PLoS Biol 14: e1002539. PubMed ID: 27552662
RAS-induced MAPK signaling is a central driver of the cell proliferation apparatus. Disruption of this pathway is widely observed in cancer and other pathologies. Consequently, considerable effort has been devoted to understanding the mechanistic aspects of RAS-MAPK signal transmission and regulation. While much information has been garnered on the steps leading up to the activation and inactivation of core pathway components, comparatively little is known on the mechanisms controlling their expression and turnover. Several factors have been identified that dictate Drosophila MAPK levels. This study describes the function of one of these, the deubiquitinase (DUB) USP47. USP47 was shown to act post-translationally to counteract a proteasome-mediated event that reduces MAPK half-life and thereby dampens signaling output. Using an RNAi-based genetic interaction screening strategy, UBC6, POE/UBR4, and UFD4 (CG5604) were identified, respectively, as E2 and E3 enzymes that oppose USP47 activity. Further characterization of POE-associated factors uncovered KCMF1 (CG11984) as another key component modulating MAPK levels. Together, these results identify a novel protein degradation module that governs MAPK levels. Given the role of UBR4 as an N-recognin ubiquitin ligase, these findings suggest that RAS-MAPK signaling in Drosophila is controlled by the N-end rule pathway and that USP47 counteracts its activity.
Jayakumar, S., Richhariya, S., Reddy, O. V., Texada, M. J. and Hasan, G. (2016). Drosophila larval to pupal switch under nutrient stress requires IP3R/Ca2+ signalling in glutamatergic interneurons. Elife 5 [Epub ahead of print] PubMed ID: 27494275
Neuronal circuits are known to integrate nutritional information, but the identity of the circuit components is not completely understood. Amino acids are a class of nutrients that are vital for the growth and function of an organism. This study reports a neuronal circuit that allows Drosophila larvae to overcome amino acid deprivation and pupariate. Nutrient stress is sensed by the class IV multidendritic cholinergic neurons. Through live calcium imaging experiments, this study shows that these cholinergic stimuli are conveyed to glutamatergic neurons in the ventral ganglion through mAChR. IP3R-dependent calcium transients in the glutamatergic neurons convey this signal to downstream medial neurosecretory cells (mNSCs). The circuit ultimately converges at the ring gland and regulates expression of ecdysteroid biosynthetic genes. Activity in this circuit is thus likely to be an adaptation that provides a layer of regulation to help surpass nutritional stress during development.
Roberts, B., Casillas, C., Alfaro, A. C., Jagers, C. and Roelink, H. (2016). Patched1 and Patched2 inhibit Smoothened non-cell autonomously. Elife 5. PubMed ID: 27552050
Evolutionary Homolog Study:
Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non cell-autonomous Smo inhibition, genetically mosaic neuralized embryoid bodies (nEBs) were generated from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing measurement of non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor.

Sunday, August 28th

Soriano, E. V., Ivanova, M. E., Fletcher, G., Riou, P., Knowles, P. P., Barnouin, K., Purkiss, A., Kostelecky, B., Saiu, P., Linch, M., Elbediwy, A., Kjaer, S., O'Reilly, N., Snijders, A. P., Parker, P. J., Thompson, B. J. and McDonald, N. Q. (2016). aPKC inhibition by Par3 CR3 flanking regions controls substrate access and underpins apical-junctional polarization. Dev Cell 38: 384-398. PubMed ID: 27554858
Atypical protein kinase C (aPKC) is a key a apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). This study shows that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/alphaB/alphaC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. These results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.
Cinnamon, E., Makki, R., Sawala, A., Wickenberg, L.P., Blomquist, G.J., Tittiger, C., Paroush, Z. and Gould, A.P. (2016). Drosophila Spidey/Kar regulates oenocyte growth via PI3-kinase signaling. PLoS Genet 12: e1006154. PubMed ID: 27500738
Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. This study focused on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. It was shown that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, it was found that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, these findings show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes.

Losada-Perez, M., Harrison, N. and Hidalgo, A. (2016). Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki. J Cell Biol [Epub ahead of print]. PubMed ID: 27551055
Neuron glia antigen 2 (NG2)-positive glia are repair cells that proliferate upon central nervous system (CNS) damage, promoting functional recovery. However, repair is limited because of the failure of the newly produced glial cells to differentiate. It is a key goal to discover how to regulate NG2 to enable glial proliferation and differentiation conducive to repair. Drosophila has an NG2 homologue called kon-tiki (kon), of unknown CNS function. This study shows that kon promotes repair and identifies the underlying mechanism. Crush injury up-regulates kon expression downstream of Notch. Kon in turn induces glial proliferation and initiates glial differentiation by activating glial genes and prospero (pros). Two negative feedback loops with Notch and Pros allow Kon to drive the homeostatic regulation required for repair. By modulating Kon levels in glia, CNS repair could be prevented or promoted. Thus, the functional links between Kon, Notch, and Pros are essential for, and can drive, repair. Analogous mechanisms could promote CNS repair in mammals.

Musashe, D.T., Purice, M.D., Speese, S.D., Doherty, J. and Logan, M.A. (2016). Insulin-like signaling promotes glial phagocytic clearance of degenerating axons through regulation of Draper. Cell Rep [Epub ahead of print]. PubMed ID: 27498858
Neuronal injury triggers robust responses from glial cells, including altered gene expression and enhanced phagocytic activity to ensure prompt removal of damaged neurons. The molecular underpinnings of glial responses to trauma remain unclear. This study shows that the evolutionarily conserved insulin-like signaling (ILS) pathway promotes glial phagocytic clearance of degenerating axons in adult Drosophila. It was found that the insulin-like receptor (InR) and downstream effector Akt1 are acutely activated in local ensheathing glia after axotomy and are required for proper clearance of axonal debris. InR/Akt1 activity is also essential for injury-induced activation of STAT92E and its transcriptional target draper, which encodes a conserved receptor essential for glial engulfment of degenerating axons. Increasing Draper levels in adult glia partially rescues delayed clearance of severed axons in glial InR-inhibited flies. The study proposes that ILS functions as a key post-injury communication relay to activate glial responses, including phagocytic activity.

Saturday, August 27th

Chawla, G., Deosthale, P., Childress, S., Wu, Y. C. and Sokol, N. S. (2016). A let-7-to-miR-125 MicroRNA Switch Regulates Neuronal Integrity and Lifespan in Drosophila. PLoS Genet 12: e1006247. PubMed ID: 27508495
Messenger RNAs (mRNAs) often contain binding sites for multiple, different microRNAs (miRNAs). However, the biological significance of this feature is unclear, since such co-targeting miRNAs could function coordinately, independently, or redundantly with one another. This study shows that two co-transcribed Drosophila miRNAs, let-7 and miR-125, non-redundantly regulate a common target, the transcription factor Chronologically Inappropriate Morphogenesis (Chinmo). Novel adult phenotypes were characterized that were associated with loss of both let-7 and miR-125, which are derived from a common, polycistronic transcript that also encodes a third miRNA, miR-100. Consistent with the coordinate upregulation of all three miRNAs in aging flies, these phenotypes include brain degeneration and shortened lifespan. However, transgenic rescue analysis reveal separable roles for these miRNAs: adult miR-125 but not let-7 mutant phenotypes are associated with ectopic Chinmo expression in adult brains and are suppressed by chinmo reduction. In contrast, let-7 is predominantly responsible for regulating chinmo during nervous system formation. These results indicate that let-7 and miR-125 function during two distinct stages, development and adulthood, rather than acting at the same time. These different activities are facilitated by an increased rate of processing of let-7 during development and a lower rate of decay of the accumulated miR-125 in the adult nervous system. Thus, this work not only establishes a key role for the highly conserved miR-125 in aging, it also demonstrates that two co-transcribed miRNAs function independently during distinct stages to regulate a common target, raising the possibility that such biphasic control may be a general feature of clustered miRNAs.
Yang, M., Wang, Y., Jiang, F., Song, T., Wang, H., Liu, Q., Zhang, J., Zhang, J. and Kang, L. (2016). miR-71 and miR-263 jointly regulate target genes Chitin synthase and Chitinase to control locust molting. PLoS Genet 12: e1006257. PubMed ID: 27532544
Evolutionary Homolog Study
Chitin synthase (see Drosophila Chitin synthase)  and chitinase (see Drosophila chitinase) play crucial roles in chitin biosynthesis and degradation during insect molting (see Drosophila molting). Silencing of Dicer-1 (see Drosophila Dicer-1) results in reduced levels of mature miRNAs and severely blocks molting in the migratory locust. However, the regulatory mechanism of miRNAs in the molting process of locusts has remained elusive. This study found that in chitin metabolism, two crucial enzymes, chitin synthase (CHS) and chitinase (CHT) are regulated by miR-71 and miR-263 (see Drosophila bft) during nymph molting. The coding sequence of CHS1 and the 3'-untranslated region of CHT10 contain functional binding sites for miR-71 and miR-263, respectively. miR-71/miR-263 display cellular co-localization with their target genes in epidermal cells and directly interact with CHS1 and CHT10 in the locust integument, respectively. Injections of miR-71 and miR-263 agomirs suppresses the expression of CHS1 and CHT10, which consequently alters chitin production of new and old cuticles and results in a molting-defective phenotype in locusts. Unexpectedly, reduced expression of miR-71 and miR-263 increases CHS1 and CHT10 mRNA expression and leads to molting defects similar to those induced by miRNA delivery. This study reveals a novel function and balancing modulation pattern of two miRNAs in chitin biosynthesis and degradation, and it provides insight into the underlying molecular mechanisms of the molting process in locusts. 

Rijal, K. and Maraia, R.J. (2016). Active center control of termination by RNA Polymerase III and tRNA gene transcription levels in vivo. PLoS Genet 12: e1006253. PubMed ID: 27518095
Evolutionary Homolog Study
The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. This study used two tRNA-mediated suppression systems to screen for Rpc1 (see Drosophila CG17209) mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, a tRNA reporter with a processing defect and a strong terminator was used. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1 (see Drosophila Maf1). The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.

Niinuma, S., Fukaya, T. and Tomari, Y. (2016). CCR4 and CAF1 deadenylases have an intrinsic activity to remove the post-poly(A) sequence. RNA. PubMed ID: 27484313
MicroRNAs (miRNAs) recruit the CCR4-NOT complex, which contains two deadenylases, CCR4 and CAF1, to promote shortening of the poly(A) tail. Although both CCR4 and CAF1 generally have a strong preference for poly(A) RNA substrates, it has been reported from yeast to humans that they can also remove non-A residues in vitro to various degrees. However, it remains unknown how CCR4 and CAF1 remove non-A sequences. This study shows that Drosophila miRNAs can promote the removal of 3'-terminal non-A residues in an exonucleolytic manner, but only if an upstream poly(A) sequence exists. This non-A removing reaction is directly catalyzed by both CCR4 and CAF1 and depends on the balance between the length of the internal poly(A) sequence and that of the downstream non-A sequence. These results suggest that the CCR4-NOT complex has an intrinsic activity to remove the 3'-terminal non-A modifications downstream from the poly(A) tail

Sumiyoshi, T., Sato, K., Yamamoto, H., Iwasaki, Y. W., Siomi, H. and Siomi, M. C. (2016). Loss of l(3)mbt leads to acquisition of the ping-pong cycle in Drosophila ovarian somatic cells. Genes Dev 30: 1617-1622. PubMed ID: 27474440
In Drosophila germ cells, PIWI-interacting RNAs (piRNAs) are amplified through a PIWI slicer-dependent feed-forward loop termed the ping-pong cycle, yielding secondary piRNAs. However, the detailed mechanism remains poorly understood, largely because an ex vivo model system amenable to biochemical analyses has not been available. This study shows that CRISPR-mediated loss of function of lethal (3) malignant brain tumor [l(3)mbt] leads to ectopic activation of the germ-specific ping-pong cycle in ovarian somatic cells. Perinuclear foci resembling nuage, the ping-pong center, appeared following l(3)mbt mutation. This activation of the ping-pong machinery in cultured cells will greatly facilitate elucidation of the mechanism underlying secondary piRNA biogenesis in Drosophila.

Friday, August 26th

Hamp, J., Löwer, A., Dottermusch-Heidel, C., Beck, L., Moussian, B., Flötenmeyer, M. and Önel, S.F. (2016). Drosophila Kette/Nap1/Hem-2 coordinates myoblast junction dissolution and the Scar-WASp ratio during myoblast fusion. J Cell Sci [Epub ahead of print]. PubMed ID: 27521427
The fusion of founder (FCs) and fusion-competent myoblasts (FCMs) is crucial for muscle formation in Drosophila. Characteristic events of myoblast fusion are the recognition and adhesion of myoblasts and the formation of branched F-actin by the Arp2/3 complex at the site of cell-cell contact. At the ultrastructural level, these events are reflected by the appearance of finger-like protrusions and electron-dense plaques that appear prior to fusion. Severe defects in myoblast fusion are caused by the loss of Kette, a member of the regulatory Scar/WAVE complex. kette mutants form finger-like protrusions, but the electron-dense plaques are extended. This study shows that the electron-dense plaques in wild-type and kette mutant myoblasts resemble other electron-dense structures that are known to function as cellular junctions. Furthermore, analysis of double mutants and attempts to rescue the kette mutant phenotype with N-cadherin, wasp and genes of members of the regulatory Scar/WAVE complex reveals that Kette has two functions during myoblast fusion. First, Kette controls the dissolution of electron-dense plaques. Second, Kette controls the ratio of the Arp2/3 activators Scar/WAVE and WASp in FCMs.

Hara, Y., Shagirov, M. and Toyama, Y. (2016). Cell boundary elongation by non-autonomous contractility in cell oscillation. Curr Biol [Epub ahead of print]. PubMed ID: 27524484
This study explored the dynamics of the amnioserosa, which is known to exhibit cell shape oscillation, as a model system to study the subcellular-level mechanics that spatiotemporally evolve during Drosophila dorsal closure. It was shown that cell boundary elongation occurs through a combination of a non-autonomous active process and an autonomous process. The former is driven by a transient change in the level of non-muscle myosin II in the neighboring cells that pull the vertices, whereas the latter is governed by the relaxation of junctional tension. By monitoring cell boundary deformation during live imaging, junctional tension at the specific phase of cell boundary oscillation, e.g., contraction or elongation, was probed by laser ablation. Junctional tension during boundary elongation is lower than during the other phase of oscillation. The tension measurements were extended to non-invasively estimate a tension map across the tissue, and a correlation between junctional tension and vinculin dynamics at the cell junction was found. The study proposes that the medial actomyosin network is used as an entity to both contract and elongate the cell boundary. Moreover, these findings raise a possibility that the level of vinculin at the cell boundary could be used to approximate junctional tension in vivo.

Grintsevich, E. E., Yesilyurt, H. G., Rich, S. K., Hung, R. J., Terman, J. R. and Reisler, E. (2016). F-actin dismantling through a redox-driven synergy between Mical and cofilin. Nat Cell Biol 18: 876-885. PubMed ID: 27454820
Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF (actin-depolymerizing factor)/cofilins (encoded by the twinstar gene in Drosophila), sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. This study uncovered a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared with either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodelling, axon guidance and Semaphorin-Plexin repulsion. Mical and cofilin, therefore, form a redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties.
Xie, S., Mason, F. M. and Martin, A. C. (2016). Loss of Galpha12/13 exacerbates apical area-dependence of actomyosin contractility. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27489340
During development, coordinated cell shape changes alter tissue shape. In the Drosophila ventral furrow and other epithelia, apical constriction of hundreds of epithelial cells folds the tissue. Genes in the Galpha12/13 pathway coordinate collective apical constriction, but the mechanism of coordination is poorly understood. Coupling live-cell imaging with a computational approach to identify contractile events, this study discovered that differences in constriction behavior are biased by initial cell shape. Disrupting Galpha12/13 exacerbates this relationship. Larger apical area is associated with delayed initiation of contractile pulses, lower apical E-cadherin and F-actin levels, and aberrantly mobile Rho-Kinase structures. These results suggest that loss of Galpha12/13 disrupts apical actin cortex organization and pulse initiation in a size-dependent manner. It is proposed that Galpha12/13 robustly organizes the apical cortex despite variation in apical area to ensure the timely initiation of contractile pulses in a tissue with heterogeneity in starting cell shape.

Thursday, August 25th

Janssen, A., Breuer, G. A., Brinkman, E. K., van der Meulen, A. I., Borden, S. V., van Steensel, B., Bindra, R. S., LaRocque, J. R. and Karpen, G. H. (2016). A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin. Genes Dev 30: 1645-1657. PubMed ID: 27474442
Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. This study developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.
Gupta, T., Morgan, H. R., Bailey, J. A. and Certel, S. J. (2016). Functional conservation of MBD proteins: MeCP2 and Drosophila MBD proteins alter sleep. Genes Brain Behav [Epub ahead of print]. PubMed ID: 27489246d
Proteins containing a methyl-CpG-binding domain (MBD) bind 5-hydroxymethylcytosine and convert the methylation pattern information into appropriate functional cellular states. Recent evidence indicates the genome of Drosophila melanogaster is methylated and two MBD proteins, dMBD2/3 and dMBD-R2, are present. Are Drosophila MBD proteins required for neuronal function, and as MBD-containing proteins have diverged and evolved, does the MBD domain retain the molecular properties required for conserved cellular function across species? To address these questions, the human MBD-containing protein, hMeCP2, was expressed in distinct amine neurons and functional changes were quantified in sleep circuitry output using a high throughput assay in Drosophila. hMeCP2 expression resulted in phase-specific sleep loss and sleep fragmentation with the hMeCP2-mediated sleep deficits requiring an intact MBD-domain. Reducing endogenous dMBD2/3 and dMBD-R2 levels also generated sleep fragmentation, with an increase in sleep occurring upon dMBD-R2 reduction. To examine if hMeCP2 and dMBD-R2 are targeting common neuronal functions, dMBD-R2 levels were reduced in combination with hMeCP2 expression and a complete rescue of sleep deficits was observed. Furthermore, chromosomal binding experiments indicate MBD-R2 and MeCP2 associate on shared genomic loci. These results provide the first demonstration that Drosophila MBD-containing family members are required for neuronal function and suggest the MBD domain retains considerable functional conservation at the whole organism level across species.
Swenson, J.M., Colmenares, S.U., Strom, A.R., Costes, S.V. and Karpen, G.H. (2016). The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic. Elife 5 [Epub ahead of print]. PubMed ID: 27514026
Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, this study purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. It was found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. The study concludes that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and proposes that a network of subdomains regulates diverse heterochromatin functions.

Wood, C. D., Veenstra, H., Khasnis, S., Gunnell, A., Webb, H. M., Shannon-Lowe, C., Andrews, S., Osborne, C. S. and West, M. J. (2016). MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs. Elife 5 [Epub ahead of print]. PubMed ID: 27490482
Evolutionary Homolog Study
Lymphomagenesis in the presence of deregulated MYC (see Drosophila Myc) requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim; see Drosophila Death executioner Bcl-2). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. This study shows that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller (see Drosophila Brahma) to MYC enhancers, and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, a haematopoietic enhancer hub was identified that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2 (see Drosophila Enhancer of zeste). Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma.

Wednesday, August 24th

Landeen, E. L., Muirhead, C. A., Wright, L., Meiklejohn, C. D. and Presgraves, D. C. (2016). Sex chromosome-wide transcriptional suppression and compensatory cis-regulatory evolution mediate gene expression in the Drosophila male germline. PLoS Biol 14: e1002499. PubMed ID: 27404402
In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower-approximately 3-fold or more-for transgenes inserted into X chromosome versus autosome locations. This study characterized transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, most endogenous X-linked genes, not just testis-specific ones, were shown to be transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. A promoter element sequence motif was was identified and experimentally validated that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarily conserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes.
Trost, M., Blattner, A. C., Leo, S. and Lehner, C. F. (2016). Drosophila dany is essential for transcriptional control and nuclear architecture in spermatocytes. Development 143: 2664-2676. PubMed ID: 27436041
The terminal differentiation of adult stem cell progeny depends on transcriptional control. A dramatic change in gene expression programs accompanies the transition from proliferating spermatogonia to postmitotic spermatocytes, which prepare for meiosis and subsequent spermiogenesis. More than a thousand spermatocyte-specific genes are transcriptionally activated in early Drosophila spermatocytes. This study describes the identification and initial characterization of dany (CG30401), a gene required in spermatocytes for the large-scale change in gene expression. Similar to tMAC and tTAFs, the known major activators of spermatocyte-specific genes, dany has a recent evolutionary origin, but it functions independently. Like dan and danr, its primordial relatives with functions in somatic tissues, dany encodes a nuclear Psq domain protein. Dany associates preferentially with euchromatic genome regions. In dany mutant spermatocytes, activation of spermatocyte-specific genes and silencing of non-spermatocyte-specific genes are severely compromised and the chromatin no longer associates intimately with the nuclear envelope. Therefore, as suggested recently for Dan/Danr, it is proposed that Dany is essential for the coordination of change in cell type-specific expression programs and large-scale spatial chromatin reorganization.
Yu, J., Lan, X., Chen, X., Yu, C., Xu, Y., Liu, Y., Xu, L., Fan, H. Y. and Tong, C. (2016). Protein synthesis and degradation are critical to regulate germline stem cell homeostasis in Drosophila testes. Development [Epub ahead of print]. PubMed ID: 27471256
The homeostasis of self-renewal and differentiation in stem cells is strictly controlled by intrinsic signals and their niche. A large-scale RNA interference (RNAi) screen was conducted in Drosophila testes and 221 genes required for germline stem cell (GSC) maintenance or differentiation were identified. Knockdown of these genes in transit-amplifying spermatogonia and cyst cells further revealed various phenotypes. Complex analysis uncovered that many of the identified genes are involved in key steps of protein synthesis and degradation. A group of genes that are required for mRNA splicing and protein translation contributes to both GSC self-renewal and early germ cell differentiation. Loss of genes in protein degradation pathway in cyst cells leads to testis tumor with overproliferated germ cells. Importantly, in the Cullin 4 - Ring E3 ubiquitin ligase (CRL4) complex, multiple proteins were identified that are critical to GSC self-renewal. pic/DDB1, the linker protein of CRL4, is not only required for GSC self-renewal in flies but also for maintenance of spermatogonial stem cells (SSCs) in mice.
Liu, Y., Ge, Q., Chan, B., Liu, H., Singh, S. R., Manley, J., Lee, J., Weideman, A. M., Hou, G. and Hou, S. X. (2016). Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila. Nat Commun 7: [Epub ahead of print] 12149. PubMed ID: 27484291
Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, a genome-wide transgenic RNAi screen was performed through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). This study identified 530 genes that regulate GSC maintenance and differentiation. Of these, 113 selected genes were further knocked down using cell-type-specific Gal4s, and more than half were found to be external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a Myb/SANT-like DNA-binding domain-containing heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. It was also found that mitosis/cytokinesis proteins are especially important for male GSC maintenance. These findings provide valuable insights and resources for studying stem cell regulation at the organismal level.

Tuesday, August 23rd

Hong, S. H., Kang, M., Lee, K. S. and Yu, K. (2016). High fat diet-induced TGF-beta/Gbb signaling provokes insulin resistance through the tribbles expression. Sci Rep 6: 30265. PubMed ID: 27484164
Hyperglycemia, hyperlipidemia, and insulin resistance are hallmarks of obesity-induced type 2 diabetes, which is often caused by a high-fat diet (HFD). However, the molecular mechanisms underlying HFD-induced insulin resistance have not been elucidated in detail. This study established a Drosophila model to investigate the molecular mechanisms of HFD-induced diabetes. HFD model flies recapitulate mammalian diabetic phenotypes including elevated triglyceride and circulating glucose levels, as well as insulin resistance. Expression of glass bottom boat (gbb), a Drosophila homolog of mammalian transforming growth factor-β (TGF-β), is elevated under HFD conditions. Furthermore, overexpression of gbb in the fat body produced obese and insulin-resistant phenotypes similar to those of HFD-fed flies, whereas inhibition of Gbb signaling significantly ameliorated HFD-induced metabolic phenotypes. tribbles, a negative regulator of AKT, is a target gene of Gbb signaling in the fat body. Overexpression of tribbles in flies in the fat body phenocopied the metabolic defects associated with HFD conditions or Gbb overexpression, whereas tribbles knockdown rescued these metabolic phenotypes. These results indicate that HFD-induced TGF-β/Gbb signaling provokes insulin resistance by increasing tribbles expression.
Sørensen, J.G., Schou, M.F., Kristensen, T.N. and Loeschcke, V. (2016). Thermal fluctuations affect the transcriptome through mechanisms independent of average temperature. Sci Rep 6: 30975. PubMed ID: 27487917
Terrestrial ectotherms are challenged by variation in both mean and variance of temperature. Phenotypic plasticity (thermal acclimation) might mitigate adverse effects, however, there is lack in fundamental understanding of the molecular mechanisms of thermal acclimation and how they are affected by fluctuating temperature. This study investigated the effect of thermal acclimation in Drosophila melanogaster on critical thermal maxima (CTmax) and associated global gene expression profiles as induced by two constant and two ecologically relevant (non-stressful) diurnally fluctuating temperature regimes. Both mean and fluctuation of temperature contribute to thermal acclimation and affect the transcriptome. The transcriptomic response to mean temperatures comprises modification of a major part of the transcriptome, while the response to fluctuations affects a much smaller set of genes, which is highly independent of both the response to a change in mean temperature and to the classic heat shock response. Although the independent transcriptional effects caused by fluctuations are relatively small, they are likely to contribute to the understanding of thermal adaptation. It was also found that environmental sensing, particularly phototransduction, is a central mechanism underlying the regulation of thermal acclimation to fluctuating temperatures. Thus, genes and pathways involved in phototransduction are likely of importance in fluctuating climates.

von Heckel, K., Stephan, W. and Hutter, S. (2016). Canalization of gene expression is a major signature of regulatory cold adaptation in temperate Drosophila melanogaster. BMC Genomics 17: 574. PubMed ID: 27502401
Transcriptome analysis may provide means to investigate the underlying genetic causes of shared and divergent phenotypes in different populations and help to identify potential targets of adaptive evolution. Applying RNA sequencing to whole male Drosophila melanogaster from the ancestral tropical African environment and a very recently colonized cold-temperate European environment at both standard laboratory conditions and following a cold shock, this study sought to uncover the transcriptional basis of cold adaptation. In both the ancestral and the derived populations, the predominant characteristic of the cold shock response is the swift and massive upregulation of heat shock proteins and other chaperones. Although ~25 % of the genome was found to be differentially expressed following a cold shock, only relatively few genes (n = 16) are up- or down-regulated in a population-specific way. Intriguingly, 14 of these 16 genes show a greater degree of differential expression in the African population. Likewise, there is an excess of genes with particularly strong cold-induced changes in expression in Africa on a genome-wide scale. The analysis of the transcriptional cold shock response most prominently reveals an upregulation of components of a general stress response, which is conserved over many taxa and triggered by a plethora of stressors. Despite the overall response being fairly similar in both populations, there is a definite excess of genes with a strong cold-induced fold-change in Africa. This is consistent with a detrimental deregulation or an overshooting stress response. Thus, the canalization of European gene expression might be responsible for the increased cold tolerance of European flies.

Wen, D. T., Zheng, L., Ni, L., Wang, H., Feng, Y. and Zhang, M. (2016). The expression of CG9940 affects the adaptation of cardiac function, mobility, and lifespan to exercise in aging Drosophila. Exp Gerontol 83: 6-14. PubMed ID: 27448710
The CG9940 gene, which encodes the NAD+ synthase protein in Drosophila, is conserved in human, zebra fish, and mosquito. NAD+ synthase is a homodimer, which catalyzes the final step in de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis, an amide transfer from either ammonia or glutamine to nicotinic acid adenine dinucleotide (NaAD). Both the CG9940 and exercise are closely relative to NAD+ level, and NAD+ plays important roles not only in energy metabolism and mitochondrial functions but also in aging. This study changed expression of CG9940 by UAS/GAL4 system in Drosophila. Flies were trained by a training device. Cardiac function was analyzed by M-mode traces, climbing index was measured through negative geotaxis assay, and lifespan was measured via lifespan assays. The important new findings from this study included the following: (1) the expression of the CG9940 could affect cardiac function, mobility, and lifespan in Drosophila. Over-expression of the CG9940 gene had positive effects on Drosophila, such as enhanced aging cardiac output, reduced heart failure, delayed age-related mobility decline, and prolonged lifespan, but lower-expression of the CG9940 had negative effects on them. (2) Different expressions of the CG9940 resulted in different influences on the adaptation of cardiac function, mobility, and lifespan to exercise in aging Drosophila. Both normal-expression and over-expression of the CG9940 resulted in positive influences on the adaptation of cardiac functions, mobility, and lifespan to exercise in aging Drosophila such as exercise slowed age-related decline of cardiac function, mobility and extent of lifespan in these flies, while lower-expression of the CG9940 led to negative impacts on the adaptation of mobility and lifespan to exercise in Drosophila.

Monday, August 22nd

Anderl, I., Vesala, L., Ihalainen, T.O., Vanha-Aho, L.M., Andó, I., Rämet, M. and Hultmark, D. (2016). Transdifferentiation and proliferation in two distinct hemocyte lineages in Drosophila melanogaster larvae after wasp infection. PLoS Pathog 12: e1005746. PubMed ID: 27414410
Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, this study developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. It was found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which were named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. These data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail.
Ji, Y., Thomas, C., Tulin, N., Lodhi, N., Boamah, E., Kolenko, V. and Tulin, A. V. (2016). Charon mediates immune deficiency-driven PARP-1-dependent immune responses in Drosophila. J Immunol [Epub ahead of print]. PubMed ID: 27527593
Regulation of NF-κB nuclear translocation and stability is central to mounting an effective innate immune response. This article describes a novel molecular mechanism controlling NF-κB-dependent innate immune response. A previously unknown protein, termed as Charon, functions as a regulator of antibacterial and antifungal immune defense in Drosophila. Charon is an ankyrin repeat-containing protein that mediates poly(ADP-ribose) polymerase-1 (PARP-1)-dependent transcriptional responses downstream of the innate immune pathway. The results demonstrate that Charon interacts with the NF-κB ortholog Relish inside perinuclear particles and delivers active Relish to PARP-1-bearing promoters, thus triggering NF-κB/PARP-1-dependent transcription of antimicrobial peptides. Ablating the expression of Charon prevents Relish from targeting promoters of antimicrobial genes and effectively suppresses the innate immune transcriptional response. Taken together, these results implicate Charon as an essential mediator of PARP-1-dependent transcription in the innate immune pathway. Thus, these results are the first to describe the molecular mechanism regulating translocation of the NF-κB subunit from cytoplasm to chromatin.
Eleftherianos, I., Castillo, J.C. and Patrnogic, J. (2016). TGF-β signaling regulates resistance to parasitic nematode infection in Drosophila melanogaster. Immunobiology [Epub ahead of print]. PubMed ID: 27473342
Over the past decade important advances have been made in the field of innate immunity; however, appreciation of the signaling pathways and molecules that participate in host immune responses to parasitic nematode infections lags behind that of responses to microbial challenges. This study examined the regulation and immune activity of Transforming Growth Factor-beta (TGF-β) signaling in the model host Drosophila melanogaster upon infection with the nematode parasites Heterorhabditis gerrardi and H. bacteriophora containing their mutualistic bacteria Photorhabdus. It was found that the genes encoding the Activin and Bone Morphogenic Protein (BMP) ligands Dawdle (Daw) and Decapentaplegic (Dpp) are transcriptionally induced in flies responding to infection with the nematode parasites, containing or lacking their associated bacteria. It was also shown that deficient Daw or Dpp regulates the survival of D. melanogaster adults to the pathogens, whereas inactivation of Daw reduces the persistence of the nematodes in the mutant flies. These findings demonstrate a novel role for the TGF-β signaling pathways in the host anti-nematode immune response. Understanding the molecular mechanisms of host anti-nematode processes will potentially lead to the development of novel means for the efficient control of parasitic nematodes.

Forbes-Beadle, L., Crossman, T., Johnson, T. K., Burke, R., Warr, C. G. and Whisstock, J. C. (2016). Development of the cellular immune system of Drosophila requires the membrane attack complex/perforin-like protein Torso-like. Genetics [Epub ahead of print]. PubMed ID: 27535927
Pore-forming members of the Membrane attack complex/perforin-like (MACPF) protein superfamily perform well-characterised roles as mammalian immune effectors. For example, complement component 9 and perforin function to directly form pores in the membrane of Gram-negative pathogens or virally infected / transformed cells respectively. In contrast, the only known MACPF protein in Drosophila melanogaster, Torso-like, plays crucial roles during development in embryo patterning and larval growth. This study reports that in addition to these functions, Torso-like plays an important role in Drosophila immunity. However, in contrast to a hypothesised effector function in, for example, elimination of Gram-negative pathogens, torso-like null mutants instead show increased susceptibility to certain Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis. This deficit is due to a severely reduced number of circulating immune cells and, as a consequence, an impaired ability to phagocytose bacterial particles. Together these data suggest that Torso-like plays an important role in controlling the development of the Drosophila cellular immune system.

Sunday, August 21st

Carreira, V. P., Mensch, J., Hasson, E. and Fanara, J. J. (2016). Natural genetic variation and candidate genes for morphological traits in Drosophila melanogaster. PLoS One 11: e0160069. PubMed ID: 27459710
Body size is a complex character associated to several fitness related traits that vary within and between species as a consequence of environmental and genetic factors. This study investigated genetic variation for different morphological traits associated to the second chromosome in natural populations of D. melanogaster along latitudinal and altitudinal gradients in Argentina. The results revealed weak clinal signals and a strong population effect on morphological variation. Moreover, most pairwise comparisons between populations were significant. The study also showed important within-population genetic variation, which must be associated to the second chromosome, as the lines are otherwise genetically identical. Next, the contribution was examined of different candidate genes to natural variation for these traits. Quantitative complementation tests were performed using a battery of lines bearing mutated alleles at candidate genes located in the second chromosome and six second chromosome substitution lines derived from natural populations which exhibited divergent phenotypes. Results of complementation tests revealed that natural variation at all candidate genes studied, invected, Fasciclin 3, toucan, Reticulon-like1, jing and CG14478, affects the studied characters, suggesting that they are Quantitative Trait Genes for morphological traits. Finally, the phenotypic patterns observed suggest that different alleles of each gene might contribute to natural variation for morphological traits.
Wolff, J. N., Tompkins, D. M., Gemmell, N. J. and Dowling, D. K. (2016). Mitonuclear interactions, mtDNA-mediated thermal plasticity, and implications for the Trojan Female Technique for pest control. Sci Rep 6: 30016. PubMed ID: 27443488
Pest species pose major challenges to global economies, ecosystems, and health. Unfortunately, most conventional approaches to pest control remain costly, and temporary in effect. As such, a heritable variant of the Sterile Insect Technique (SIT) was proposed, based on the introduction of mitochondrial DNA mutations into pest populations, which impair male fertility but have no effects on females. Evidence for this "Trojan Female Technique" (TFT) was recently provided, in the form of a mutation in the mitochondrial cytochrome b gene (mt:Cyt-b) of Drosophila melanogaster which reduces male fertility across diverse nuclear backgrounds. However, recent studies have shown that the magnitude of mitochondrial genetic effects on the phenotype can vary greatly across environments, with mtDNA polymorphisms commonly entwined in genotype-by-environment (G x E) interactions. This study tested whether the male-sterilizing effects previously associated with the mt:Cyt-b mutation are consistent across three thermal and three nuclear genomic contexts. The effects of this mutation were indeed moderated by the nuclear background and thermal environment, but crucially the fertility of males carrying the mutation was invariably reduced relative to controls. This mutation thus constitutes a promising candidate for the further development of the TFT.
Gibert, J. M., Mouchel-Vielh, E., De Castro, S. and Peronnet, F. (2016). Phenotypic plasticity through transcriptional regulation of the evolutionary hotspot gene tan in Drosophila melanogaster. PLoS Genet 12: e1006218. PubMed ID: 27508387
Phenotypic plasticity is the ability of a given genotype to produce different phenotypes in response to distinct environmental conditions. Phenotypic plasticity can be adaptive. Furthermore, it is thought to facilitate evolution. Although phenotypic plasticity is a widespread phenomenon, its molecular mechanisms are only beginning to be unravelled. Environmental conditions can affect gene expression through modification of chromatin structure, mainly via histone modifications, nucleosome remodelling or DNA methylation, suggesting that phenotypic plasticity might partly be due to chromatin plasticity. As a model of phenotypic plasticity, abdominal pigmentation was studied of Drosophila melanogaster females, which is temperature sensitive. Abdominal pigmentation is indeed darker in females grown at 18 degrees C than at 29 degrees C. This phenomenon is thought to be adaptive as the dark pigmentation produced at lower temperature increases body temperature. This study showed that temperature modulates the expression of tan (t), a pigmentation gene involved in melanin production. t is expressed 7 times more at 18 ° C than at 29 &176; C in female abdominal epidermis. Genetic experiments show that modulation of t expression by temperature is essential for female abdominal pigmentation plasticity. Temperature modulates the activity of an enhancer of t without modifying compaction of its chromatin or level of the active histone mark H3K27ac. By contrast, the active mark H3K4me3 on the t promoter is strongly modulated by temperature. The H3K4 methyl-transferase involved in this process is likely Trithorax, since it regulates t expression and the H3K4me3 level on the t promoter and also participates in female pigmentation and its plasticity. Interestingly, t was previously shown to be involved in inter-individual variation of female abdominal pigmentation in Drosophila melanogaster, and in abdominal pigmentation divergence between Drosophila species. Sensitivity of t expression to environmental conditions might therefore give more substrate for selection, explaining why this gene has frequently been involved in evolution of pigmentation.
Caizzi, R., Moschetti, R., Piacentini, L., Fanti, L., Marsano, R. M. and Dimitri, P. (2016). Comparative genomic analyses provide new insights into the evolutionary dynamics of heterochromatin in Drosophila. PLoS Genet 12: e1006212. PubMed ID: 27513559
The term heterochromatin has been long considered synonymous with gene silencing, but it is now clear that the presence of transcribed genes embedded in pericentromeric heterochromatin is a conserved feature in the evolution of eukaryotic genomes. Several studies have addressed the epigenetic changes that enable the expression of genes in pericentric heterochromatin, yet little is known about the evolutionary processes through which this has occurred. By combining genome annotation analysis and high-resolution cytology, this study has identified and mapped 53 orthologs of D. melanogaster heterochromatic genes in the genomes of two evolutionarily distant species, D. pseudoobscura and D. virilis. The results show that the orthologs of the D. melanogaster heterochromatic genes are clustered at three main genomic regions in D. virilis and D. pseudoobscura. In D. virilis, the clusters lie in the middle of euchromatin, while those in D. pseudoobscura are located in the proximal portion of the chromosome arms. Some orthologs map to the corresponding Muller C element in D. pseudoobscura and D. virilis, while others localize on the Muller B element, suggesting that chromosomal rearrangements that have been instrumental in the fusion of two separate elements involved the progenitors of genes currently located in D. melanogaster heterochromatin. These results demonstrate an evolutionary repositioning of gene clusters from ancestral locations in euchromatin to the pericentromeric heterochromatin of descendent D. melanogaster chromosomes. Remarkably, in both D. virilis and D. pseudoobscura the gene clusters show a conserved association with the HP1a protein, one of the most highly evolutionarily conserved epigenetic marks. In light of these results, a new scenario is suggested whereby ancestral HP1-like proteins (and possibly other epigenetic marks) may have contributed to the evolutionary repositioning of gene clusters into heterochromatin.

Saturday, August 20th

Winding, M., Kelliher, M.T., Lu, W., Wildonger, J. and Gelfand, V.I. (2016). Role of kinesin-1-based microtubule sliding in Drosophila nervous system development. Proc Natl Acad Sci [Epub ahead of print]. PubMed ID: 27512046
The plus-end microtubule (MT) motor kinesin-1 is essential for normal development, with key roles in the nervous system. Kinesin-1 drives axonal transport of membrane cargoes to fulfill the metabolic needs of neurons and maintain synapses. It has been previously demonstrated that kinesin-1, in addition to its well-established role in organelle transport, can drive MT-MT sliding by transporting "cargo" MTs along "track" MTs, resulting in dramatic cell shape changes. The mechanism and physiological relevance of this MT sliding are unclear. In addition to its motor domain, kinesin-1 contains a second MT-binding site, located at the C terminus of the heavy chain. In this study, the C-terminal MT-binding site was mutated such that the ability of kinesin-1 to slide MTs is significantly compromised, whereas cargo transport is unaffected. This mutation was introduced into the genomic locus of kinesin-1 heavy chain (KHC), generating the KhcmutA allele. KhcmutA neurons display significant MT sliding defects while maintaining normal transport of many cargoes. Using this mutant, it was demonstrated that MT sliding is required for axon and dendrite outgrowth in vivo. Consistent with these results, KhcmutA flies display severe locomotion and viability defects. To test the role of MT sliding further, a chimeric motor that actively slides MTs but cannot transport organelles was engineered. Activation of MT sliding in KhcmutA neurons using this chimeric motor rescues axon outgrowth in cultured neurons and in vivo, firmly establishing the role of sliding in axon outgrowth. These results demonstrate that MT sliding by kinesin-1 is an essential biological phenomenon required for neuronal morphogenesis and normal nervous system development.
Lu, W., Winding, M., Lakonishok, M., Wildonger, J. and Gelfand, V.I. (2016). Microtubule-microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27512034
Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. It has been shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, this study used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. It was demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, two populations of microtubules in fast-streaming oocytes were identified: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that move in the ooplasm. It was further demonstrated that the reduced streaming in sliding-deficient oocytes results in posterior determination defects. Together, the study proposes that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.
Kowanda, M., Bergalet, J., Wieczorek, M., Brouhard, G., Lecuyer, E. and Lasko, P. (2016). Loss of function of the Drosophila Ninein-related centrosomal protein Bsg25D causes mitotic defects and impairs embryonic development. Biol Open [Epub ahead of print]. PubMed ID: 27422905
The centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) play significant roles in microtubule stability, nucleation and anchoring at the centrosome in mammalian cells. This study investigated Blastoderm specific gene 25D (Bsg25D), which encodes the only Drosophila protein that is closely related to Nin and Nlp. In early embryos, Bsg25D mRNA and Bsg25D protein are closely associated with centrosomes and astral microtubules. Sequences within the coding region and 3'UTR of Bsg25D mRNAs are important for proper localization of this transcript in oogenesis and embryogenesis. Ectopic expression of eGFP-Bsg25D from an unlocalized mRNA disrupts microtubule polarity in mid-oogenesis and compromises the distribution of the axis polarity determinant Gurken. Using total internal reflection fluorescence microscopy,an N-terminal fragment of Bsg25D was shown to bind microtubules in vitro and can move along them, predominantly toward minus-ends. While flies homozygous for a Bsg25D null mutation are viable and fertile, 70% of embryos lacking maternal and zygotic Bsg25D do not hatch and exhibit chromosome segregation defects, as well as detachment of centrosomes from mitotic spindles. It is concluded that Bsg25D is a centrosomal protein that, while dispensable for viability, nevertheless helps ensure the integrity of mitotic divisions in Drosophila.
Aw, W. Y., Heck, B. W., Joyce, B. and Devenport, D. (2016). Transient tissue-scale deformation coordinates alignment of planar cell polarity junctions in the mammalian skin. Curr Biol [Epub ahead of print]. PubMed ID: 27451904
Evolutionary Homolog Study
Planar cell polarity (PCP) refers to the collective alignment of polarity along the tissue plane. In skin, the largest mammalian organ, PCP aligns over extremely long distances, but the global cues that orient tissue polarity are unknown. This study shows that Celsr1 (homolog of Drosophila Starry night) asymmetry arises concomitant with a gradient of tissue deformation oriented along the medial-lateral axis. This uniaxial tissue tension, whose origin remains unknown, transiently transforms basal epithelial cells from initially isotropic and disordered states into highly elongated and aligned morphologies. Reorienting tissue deformation is sufficient to shift the global axis of polarity, suggesting that uniaxial tissue strain can act as a long-range polarizing cue. Observations both in vivo and in vitro suggest that the effect of tissue anisotropy on Celsr1 polarity is not a direct consequence of cell shape but rather reflects the restructuring of cell-cell interfaces during oriented cell divisions and cell rearrangements that serve to relax tissue strain. Cell intercalations remodel intercellular junctions predominantly between the mediolateral interfaces of neighboring cells. This restructuring of the cell surface polarizes Celsr1, which is slow to accumulate at nascent junctions yet stably associates with persistent junctions. It is proposed that tissue anisotropy globally aligns Celsr1 polarity by creating a directional bias in the formation of new cell interfaces while simultaneously aligning the persistent interfaces at which Celsr1 prefers to accumulate.

Friday, August 19th

Neto, M., Aguilar-Hidalgo, D. and Casares, F. (2016). Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye. Dev Biol [Epub ahead of print]. PubMed ID: 27502436
During organ development, the progenitor state is transient, and depends on specific combinations of transcription factors and extracellular signals. Not surprisingly, abnormal maintenance of progenitor transcription factors may lead to tissue overgrowth, and the concurrence of signals from the local environment is often critical to trigger this overgrowth. Therefore, identifying specific combinations of transcription factors/signals promoting -or opposing- proliferation in progenitors is essential to understand normal development and disease. This study used the Drosophila eye as a model where the transcription factors hth and tsh are transiently expressed in eye progenitors causing the expansion of the progenitor pool. However, if their co-expression is maintained experimentally, cell proliferation continues and differentiation is halted. It was shown that Hth+Tsh-induced tissue overgrowth requires the BMP2 Dpp and the abnormal hyperactivation of its pathway. Rather than using autocrine Dpp expression, Hth+Tsh cells increase their avidity for Dpp, produced locally, by upregulating extracellular matrix components. During normal development, Dpp represses hth and tsh ensuring that the progenitor state is transient. However, cells in which Hth+Tsh expression is forcibly maintained use Dpp to enhance their proliferation.

Tang, W., Wang, D. and Shen, J. (2016). Asymmetric distribution of Spalt in Drosophila wing squamous and columnar epithelia ensures correct cell morphogenesis.Sci Rep 6: 30236. PubMed ID: 27452716
The Drosophila wing imaginal disc is a sac-like structure that is composed of two opposing cell layers: peripodial epithelium (PE, also known as squamous epithelia) and disc proper (DP, also known as pseudostratified columnar epithelia). The molecular mechanism of cell morphogenesis has been well studied in the DP but not in the PE. Although proper Dpp signalling activity is required for proper PE formation, the detailed regulation mechanism is poorly understood. This study found that the Dpp target gene spalt (sal) is only expressed in DP cells, not in PE cells, although pMad is present in the PE. Increasing Dpp signalling activity cannot activate Sal in PE cells. The absence of Sal in the PE is essential for PE formation. The ectopic expression of sal in PE cells is sufficient to increase the PE cell height. Down-regulation of sal in the DP reduced DP cell height. It was further demonstrated that the known PE cell height regulator Lines, which can convert PE into a DP cell fate, is mediated by sal mis-activation in PE because sal-RNAi and lines co-expression largely restores PE cell morphology. By revealing the microtubule distribution, it was demonstrated that Lines- and Sal-heightened PE cells are morphologically similar to the intermediate cell with cuboidal morphology.
Gokhale, R. H., Hayashi, T., Mirque, C. D. and Shingleton, A. W. (2016). Intra-organ growth coordination in Drosophila is mediated by systemic ecdysone signaling. Dev Biol [Epub ahead of print]. PubMed ID: 27452628
In developing Drosophila, perturbing the growth of one imaginal disc - the parts of a holometabolous larva that become the external adult organs - has been shown to retard growth of other discs and delays development, resulting in tight inter-organ growth coordination and the generation of a correctly proportioned adult. This study used the wing imaginal disc in Drosophila to study and identify mechanisms of intra-organ growth coordination. Larvae were generated in which the two compartments of the wing imaginal disc have ostensibly different growth rates (wild-type or growth-perturbed). It was found that there is tightly coordinated growth between the wild-type and growth-perturbed compartments, where growth of the wild-type compartment is retarded to match that of the growth-perturbed compartment. Crucially, this coordination is disrupted by application of exogenous 20-hydroxyecdysone (20E), which accelerates growth of the wild-type compartment. The role of 20E signaling in growth coordination was further elucidate by showing that in wild-type discs, compartment-autonomous up-regulation of 20E signaling accelerates compartment growth and disrupts coordination. Interestingly, growth acceleration through exogenous application of 20E is inhibited with suppression of the Insulin/Insulin-like Growth Factor Signaling (IIS) pathway. This suggests that an active IIS pathway is necessary for ecdysone to accelerate compartment growth. Collectively, these data indicate that discs utilize systemic mechanisms, specifically ecdysone signaling, to coordinate intra-organ growth.
Santos, C. G., Fernandez-Nicolas, A. and Belles, X. (2016). Smads and insect hemimetabolan metamorphosis. Dev Biol [Epub ahead of print]. PubMed ID: 27452629
In contrast with Drosophila melanogaster, practically nothing is known about the involvement of the TGF-β signaling pathway in the metamorphosis of hemimetabolan insects. To partially fill this gap, the role of Smad factors in the metamorphosis of the German cockroach, Blattella germanica, was studied. In Drosophila, Mad is the canonical R-Smad of the BMP branch of the TGF-β signaling pathway, Smox is the canonical R-Smad of the TGF-β/Activin branch and Medea participates in both branches. In insects, metamorphosis is regulated by the MEKRE93 pathway, which starts with juvenile hormone (JH), whose signal is transduced by Methoprene-tolerant (Met), which stimulates the expression of Kruppel homolog 1 (Kr-h1) that acts to repress E93, the metamorphosis trigger. In B. germanica, metamorphosis is determined at the beginning of the sixth (final) nymphal instar (N6), when JH production ceases, the expression of Kr-h1 declines, and the transcription of E93 begins to increase. The RNAi of Mad, Smox and Medea in N6 of B. germanica reveals that the BMP branch of the TGF-beta signaling pathway regulates adult ecdysis and wing extension, mainly through regulating the expression of Bursicon, whereas the TGF-beta/Activin branch contributes to increasing E93 and decreasing Kr-h1 at the beginning of N6, crucial for triggering adult morphogenesis, as well as to regulating the imaginal molt timing.

Thursday, August 18th

Pimentel, D., Donlea, J.M., Talbot, C.B., Song, S.M., Thurston, A.J. and Miesenböck, G. (2016). Operation of a homeostatic sleep switch. Nature [Epub ahead of print]. PubMed ID: 27487216
In Drosophila, a crucial component of the machinery for sleep homeostasis is a cluster of neurons innervating the dorsal fan-shaped body (dFB) of the central complex. dFB neurons in sleep-deprived flies tend to be electrically active, with high input resistances and long membrane time constants, while neurons in rested flies tend to be electrically silent. This study demonstrates state switching by dFB neurons, identifies dopamine as a neuromodulator that operates the switch, and delineates the switching mechanism. Arousing dopamine causes transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects are transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involves the downregulation of voltage-gated A-type currents carried by Shaker and Shab, and the upregulation of voltage-independent leak currents through a two-pore-domain potassium channel that was termed Sandman. Sandman is encoded by the CG8713 gene and translocates to the plasma membrane in response to dopamine. dFB-restricted interference with the expression of Shaker or Sandman decreases or increases sleep, respectively, by slowing the repetitive discharge of dFB neurons in the ON state or blocking their entry into the OFF state. Biophysical changes in a small population of neurons are thus linked to the control of sleep-wake state.

Clovis, Y. M., Seo, S. Y., Kwon, J. S., Rhee, J. C., Yeo, S., Lee, J. W., Lee, S. and Lee, S. K. (2016). Chx10 consolidates V2a interneuron identity through two distinct gene repression modes. Cell Rep [Epub ahead of print]. PubMed ID: 27477290
Evolutionary Homolog Study
During development, two cell types born from closely related progenitor pools often express identical transcriptional regulators despite their completely distinct characteristics. This phenomenon implies the need for a mechanism that operates to segregate the identities of the two cell types throughout differentiation after initial fate commitment. To understand this mechanism, this study investigated the fate specification of spinal V2a interneurons, which share important developmental genes with motor neurons (MNs). The paired homeodomain factor Chx10 (see Drosophila Vsx1 and Vsx2) was shown to function as a critical determinant for V2a fate and is required to consolidate V2a identity in postmitotic neurons. Chx10 actively promotes V2a fate, downstream of the LIM-homeodomain factor Lhx3, while concomitantly suppressing the MN developmental program by preventing the MN-specific transcription complex from binding and activating MN genes. This dual activity enables Chx10 to effectively separate the V2a and MN pathways. This study uncovers a widely applicable gene regulatory principle for segregating related cell fates.

Huang, J., Liu, W., Qi, Y.X., Luo, J. and Montell, C. (2016). Neuromodulation of courtship drive through tyramine-responsive neurons in the Drosophila brain. Curr Biol [Epub ahead of print]. PubMed ID: 27498566
Neuromodulators influence the activities of collections of neurons and have profound impacts on animal behavior. Male courtship drive is complex and subject to neuromodulatory control. Using the fruit fly Drosophila melanogaster, this study identified neurons in the brain (inferior posterior slope; IPS) that impact courtship drive and are controlled by tyramine-a biogenic amine related to dopamine, whose roles in most animals are enigmatic. A tyramine-specific receptor, TyrR, which is expressed in IPS neurons, was knocked out. Loss of TyrR leads to a striking elevation in courtship activity between males. This effect occurrs only in the absence of females, as TyrRGal4 mutant males exhibit a wild-type preference for females. Artificial hyperactivation of IPS neurons causes a large increase in male-male courtship, whereas suppression of IPS activity decreases male-female courtship. The study concludes that TyrR is a receptor for tyramine, and suggests that it serves to curb high levels of courtship activity through functioning as an inhibitory neuromodulator.

Haag, J., Arenz, A., Serbe, E., Gabbiani, F. and Borst, A. (2016). Complementary mechanisms create direction selectivity in the fly. JElife 5 [Epub ahead of print] PubMed ID: 27502554
How neurons become sensitive to the direction of visual motion represents a classic example of neural computation. Two alternative mechanisms have been discussed in the literature so far: preferred direction enhancement, by which responses are amplified when stimuli move along the preferred direction of the cell, and null direction suppression, where one signal inhibits the response to the subsequent one when stimuli move along the opposite, i.e. null direction. Along the processing chain in the Drosophila optic lobe, directional responses first appear in T4 and T5 cells. Visually stimulating sequences of individual columns in the optic lobe with a telescope while recording from single T4 neurons, this study found both mechanisms at work implemented in different sub-regions of the receptive field. This finding explains the high degree of directional selectivity found already in the fly's primary motion-sensing neurons and marks an important step in understanding of elementary motion detection.

Wednesday, August 17th

Weidmann, C.A., Qiu, C., Arvola, R.M., Lou, T.F., Killingsworth, J., Campbell, Z.T., Tanaka Hall, T.M. and Goldstrohm, A.C. (2016). Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio. Elife [Epub ahead of print]. PubMed ID: 27482653
Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet understanding of these key regulatory complexes has been limited to single RBPs. This study investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. The obtained results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. These results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.
Nimura, K., Yamamoto, M., Takeichi, M., Saga, K., Takaoka, K., Kawamura, N., Nitta, H., Nagano, H., Ishino, S., Tanaka, T., Schwartz, R.J., Aburatani, H. and Kaneda, Y. (2016). Regulation of alternative polyadenylation by Nkx2-5 and Xrn2 during mouse heart development. Elife [Epub ahead of print]. PubMed ID: 27331609
Evolutionary Homolog Study
Transcription factors organize gene expression profiles by regulating promoter activity. However, the role of transcription factors after transcription initiation is poorly understood. This study shows that the homeoprotein Nkx2-5 (see Drosophila tin) and the 5'-3' exonuclease Xrn2 (see Drosophila Rat1) are involved in the regulation of alternative polyadenylation (APA) during mouse heart development (see Drosophila dorsal vessel). Nkx2-5 occupies not only the transcription start sites (TSSs) but also the downstream regions of genes, serving to connect these regions in primary embryonic cardiomyocytes (eCMs). Nkx2-5 deficiency affects Xrn2 binding to target loci and results in increases in RNA polymerase II (RNAPII) occupancy and in the expression of mRNAs with long 3'untranslated regions (3' UTRs) from genes related to heart development. siRNA-mediated suppression of Nkx2-5 and Xrn2 leads to heart looping anomaly. Moreover, Nkx2-5 genetically interacts with Xrn2 because Nkx2-5+/-Xrn2+/-, but neither Nkx2-5+/- nor Xrn2+/-, newborns exhibit a defect in ventricular septum formation, suggesting that the association between Nkx2-5 and Xrn2 is essential for heart development. These results indicate that Nkx2-5 regulates not only the initiation but also the usage of poly(A) sites during heart development and suggest that tissue-specific transcription factors are involved in the regulation of APA.

Eichhorn, S. W., Subtelny, A. O., Kronja, I., Kwasnieski, J. C., Orr-Weaver, T. L. and Bartel, D. P. (2016). mRNA poly(A)-tail changes specified by deadenylation broadly reshape translation in Drosophila oocytes and early embryos. Elife 5. PubMed ID: 27474798
Because maturing oocytes and early embryos lack appreciable transcription, posttranscriptional regulatory processes control their development. To better understand this control, this study profiled translational efficiencies and poly(A)-tail lengths throughout Drosophila oocyte maturation and early embryonic development. The correspondence between translational-efficiency changes and tail-length changes indicated that tail-length changes broadly regulate translation until gastrulation, when this coupling disappears. During egg activation, relative changes in poly(A)-tail length, and thus translational efficiency, were largely retained in the absence of cytoplasmic polyadenylation, which indicated that selective poly(A)-tail shortening primarily specifies these changes. Many translational changes depended on Pan Gu and Smaug, and both acted primarily through tail-length changes. These results also revealed the presence of tail-length-independent mechanisms that maintained translation despite tail-length shortening during oocyte maturation, and prevented essentially all translation of bicoid and several other mRNAs before egg activation. In addition to these fundamental insights, the results provide valuable resources for future studies.
Fagegaltier, D., Falciatori, I., Czech, B., Castel, S., Perrimon, N., Simcox, A. and Hannon, G. J. (2016). Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway. Genes Dev 30: 1623-1635. PubMed ID: 27474441
Germline genes often become re-expressed in soma-derived human cancers as 'cancer/testis antigens' (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. This study found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer.

Tuesday, August 16th

Sepil, I., Carazo, P., Perry, J. C. and Wigby, S. (2016). Insulin signalling mediates the response to male-induced harm in female Drosophila melanogaster. Sci Rep 6: 30205. PubMed ID: 27457757
Genetic manipulations in nutrient-sensing pathways are known to both extend lifespan and modify responses to environmental stressors (e.g., starvation, oxidative and thermal stresses), suggesting that similar mechanisms regulate lifespan and stress resistance. However, despite being a key factor reducing female lifespan and affecting female fitness, male-induced harm has rarely been considered as a stressor mediated by nutrient sensing pathways. This study explored whether a lifespan-extending manipulation also modifies female resistance to male-induced harm. To do so, long-lived female Drosophila melanogaster were used that had their insulin signalling pathway downregulated by genetically ablating the median neurosecretory cells (mNSC). The level of exposure to males was varied for control and ablated females, and tests were performed for interacting effects on female lifespan and fitness. As expected, lifespan significantly declined with exposure to males. However, mNSC-ablated females maintained significantly increased lifespan across all male exposure treatments. Furthermore, lifespan extension and relative fitness of mNSC-ablated females were maximized under intermediate exposure to males, and minimized under low and high exposure to males. Overall, these results suggest that wild-type levels of insulin signalling reduce female susceptibility to male-induced harm under intense sexual conflict, and may also protect females when mating opportunities are sub-optimally low.
Essers, P., Tain, L. S., Nespital, T., Goncalves, J., Froehlich, J. and Partridge, L. (2016). Reduced insulin/insulin-like growth factor signaling decreases translation in Drosophila and mice. Sci Rep 6: 30290. PubMed ID: 27452396
Down-regulation of insulin/insulin-like growth factor signaling (IIS) can increase lifespan in C. elegans, Drosophila and mice. In C. elegans, reduced IIS results in down-regulation of translation, which itself can extend lifespan. However, the effect of reduced IIS on translation has yet to be determined in other multicellular organisms. Using two long-lived IIS models, namely Drosophila lacking three insulin-like peptides (dilp2-3,5-/-) and mice lacking insulin receptor substrate 1 (Irs1-/-), and two independent translation assays, polysome profiling and radiolabeled amino acid incorporation, it was shown that reduced IIS lowers translation in these organisms. In Drosophila, reduced IIS decreased polysome levels in fat body and gut, but reduced the rate of protein synthesis only in the fat body. Reduced IIS in mice decreased protein synthesis rate only in skeletal muscle, without reducing polysomes in any tissue. This lowered translation in muscle was independent of Irs1 loss in the muscle itself, but a secondary effect of Irs1 loss in the liver. In conclusion, down-regulation of translation is an evolutionarily conserved response to reduced IIS, but the tissues in which it occurs can vary between organisms. Furthermore, the mechanisms underlying lowered translation may differ in mice, possibly associated with the complexity of the regulatory processes.
Schimizzi, G. V., Maher, M. T., Loza, A. J. and Longmore, G. D. (2016). Disruption of the Cdc42/Par6/aPKC or Dlg/Scrib/Lgl polarity complex promotes epithelial proliferation via overlapping mechanisms. PLoS One 11: e0159881. PubMed ID: 27454609
The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Using the proliferating Drosophila wing disc epithelium, this study demonstrates that disruption of the junctional [Cdc42/Par6/Par3/Atypical PKC (aPKC)] complex vs. basolateral polarity complex [Scribble (Scrib)/Discs Large (Dlg)/Lethal Giant Larvae (Lgl)] complex results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basolateral polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF; Eiger) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally it was demonstrated that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin.
Minakhina, S., Naryshkina, T., Changela, N., Tan, W. and Steward, R. (2016). Zfrp8/PDCD2 interacts with RpS2 connecting ribosome maturation and gene-specific translation. PLoS One 11: e0147631. PubMed ID: 26807849
Zfrp8/PDCD2 is a highly conserved protein essential for stem cell maintenance in both flies and mammals. It is also required in fast proliferating cells such as cancer cells. Previous studies suggested that Zfrp8 functions in the formation of mRNP (mRNA ribonucleoprotein) complexes and also controls RNA of select Transposable Elements (TEs). This study shows that in Zfrp8/PDCD2 knock down (KD) ovaries, specific mRNAs and TE transcripts show increased nuclear accumulation. Zfrp8/PDCD2 was also shown to interact with the (40S) small ribosomal subunit through direct interaction with RpS2 (uS5). By studying the distribution of endogenous and transgenic fluorescently tagged ribosomal proteins, it was demonstrated that Zfrp8/PDCD2 regulates the cytoplasmic levels of components of the small (40S) ribosomal subunit, but does not control nuclear/nucleolar localization of ribosomal proteins. These results suggest that Zfrp8/PDCD2 functions at late stages of ribosome assembly and may regulate the binding of specific mRNA-RNPs to the small ribosomal subunit ultimately controlling their cytoplasmic localization and translation.

Monday, August 15th

Zee, B. M., Alekseyenko, A. A., McElroy, K. A. and Kuroda, M. I. (2016). Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry. Proc Natl Acad Sci U S A 113: 1784-1789. PubMed ID: 26831069
Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. A cross-linked affinity technique, BioTAP-XL, was introduced to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. This study advances BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore it was demonstrated that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. Hydrophilic interaction chromatography was integrated to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, this study greatly extends the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.
Rickels, R., Hu, D., Collings, C. K., Woodfin, A. R., Piunti, A., Mohan, M., Herz, H. M., Kvon, E. and Shilatifard, A. (2016). An evolutionary conserved epigenetic mark of Polycomb response elements implemented by Trx/MLL/COMPASS. Mol Cell 63: 318-328. PubMed ID: 27447986
Evolutionary Homolog Study
Polycomb response elements (PREs) are specific DNA sequences that stably maintain the developmental pattern of gene expression. Drosophila PREs are well characterized, whereas the existence of PREs in mammals remains debated. Accumulating evidence supports a model in which CpG islands recruit Polycomb group (PcG) complexes; however, which subset of CGIs is selected to serve as PREs is unclear. Trithorax (Trx) positively regulates gene expression in Drosophila and co-occupies PREs to antagonize Polycomb-dependent silencing. This study demonstrated that Trx-dependent H3K4 dimethylation (H3K4me2) marks Drosophila PREs and maintains the developmental expression pattern of nearby genes. Similarly, the mammalian Trx homolog, MLL1, deposits H3K4me2 at CpG-dense regions that could serve as PREs. In the absence of MLL1 and H3K4me2, H3K27me3 levels, a mark of Polycomb repressive complex 2 (PRC2), increase at these loci. By inhibiting PRC2-dependent H3K27me3 in the absence of MLL1, expression of these loci can be rescued, demonstrating a functional balance between MLL1 and PRC2 activities at these sites. Thus, this study provides rules for identifying cell-type-specific functional mammalian PREs within the human genome.
De, S., Mitra, A., Cheng, Y., Pfeifer, K. and Kassis, J. A. (2016). Formation of a Polycomb-domain in the absence of strong Polycomb response elements. PLoS Genet 12: e1006200. PubMed ID: 27466807
Polycomb group response elements (PREs) in Drosophila are DNA-elements that recruit Polycomb proteins (PcG) to chromatin and regulate gene expression. PREs are easily recognizable in the Drosophila genome as strong peaks of PcG-protein binding over discrete DNA fragments; many small but statistically significant PcG peaks are also observed in PcG domains. Surprisingly, in vivo deletion of the four characterized strong PREs from the PcG regulated invected-engrailed (inv-en) gene complex did not disrupt the formation of the H3K27me3 domain and did not affect inv-en expression in embryos or larvae suggesting the presence of redundant PcG recruitment mechanism. Further, the 3D-structure of the inv-en domain was only minimally altered by the deletion of the strong PREs. A reporter construct containing a 7.5kb en fragment that contains three weak peaks but no large PcG peaks forms an H3K27me3 domain and is PcG-regulated. These data suggests a model for the recruitment of PcG-complexes to Drosophila genes via interactions with multiple, weak PREs spread throughout an H3K27me3 domain.
Xu, S., Panikker, P., Iqbal, S. and Elefant, F. (2016). Tip60 HAT action mediates environmental enrichment induced cognitive restoration. PLoS One 11: e0159623. PubMed ID: 27454757
Environmental enrichment (EE) conditions have beneficial effects for reinstating cognitive ability in neuropathological disorders like Alzheimer's disease (AD). While EE benefits involve epigenetic gene control mechanisms that comprise histone acetylation, the histone acetyltransferases (HATs) involved remain largely unknown. This study examine a role for Tip60 HAT action in mediating activity- dependent beneficial neuroadaptations to EE using the Drosophila CNS mushroom body (MB) as a well-characterized cognition model. Flies raised under EE conditions were shown to display enhanced MB axonal outgrowth, synaptic marker protein production, histone acetylation induction and transcriptional activation of cognition linked genes when compared to their genotypically identical siblings raised under isolated conditions. Further, these beneficial changes are impaired in both Tip60 HAT mutant flies and APP neurodegenerative flies. While EE conditions provide some beneficial neuroadaptive changes in the APP neurodegenerative fly MB, such positive changes are significantly enhanced by increasing MB Tip60 HAT levels. These results implicate Tip60 as a critical mediator of EE-induced benefits, and provide broad insights into synergistic behavioral and epigenetic based therapeutic approaches for treatment of cognitive disorder.

Sunday, August 14th

Byrne, E. F., Sircar, R., Miller, P. S., Hedger, G., Luchetti, G., Nachtergaele, S., Tully, M. D., Mydock-McGrane, L., Covey, D. F., Rambo, R. P., Sansom, M. S., Newstead, S., Rohatgi, R. and Siebold, C. (2016). Structural basis of Smoothened regulation by its extracellular domains. Nature 535: 517-522. PubMed ID: 27437577
Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs; see Drosophila Frizzled) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. This study presents crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, a cholesterol molecule was found bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD–linker domain–TMD interface. These results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.
Lin, X. W., Tang, L., Yang, J. and Xu, W. H. (2016). HIF-1 regulates insect lifespan extension by inhibiting c-Myc-TFAM signaling and mitochondrial biogenesis. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 27469241
Diapause (developmental arrest) is characterized by dramatic depression of metabolic activity and profoundly extends insect lifespan, similar to the Caenorhabditis elegans dauer stage and Drosophila longevity; however, the molecular mechanism of low metabolism in insect diapause is unclear. This study showed that HIF-1α expression is significantly increased in diapause-destined pupal brains compared to nondiapause-destined pupal brains and that HIF-1α negatively regulates mitochondrial biogenesis. HIF-1α mediates this effect by inhibiting c-Myc activity via proteasome-dependent degradation of c-Myc. The mitochondrial transcription factor A (TFAM), which encodes a key factor involved in mitochondrial transcription and mitochondrial DNA replication, is activated by the binding of c-Myc to the TFAM promoter, thereby inducing transcription. Loss of TFAM expression is a major factor contributing to reducing the mitochondrial activity. Thus, the HIF-1alpha-c-Myc-TFAM signaling pathway participates in the regulation of mitochondrial activity for insect diapause or lifespan extension.
Hu, L., Xu, J., Yin, M. X., Zhang, L., Lu, Y., Wu, W., Xue, Z., Ho, M. S., Gao, G., Zhao, Y. and Zhang, L. (2016). Ack promotes tissue growth via phosphorylation and suppression of the Hippo pathway component Expanded. Cell Discov 2: 15047. PubMed ID: 27462444
Non-receptor tyrosine kinase Activated cdc42 kinase (see Drosophila Ack) was reported to participate in several types of cancers in mammals. It is also believed to have an anti-apoptotic function in Drosophila. This study reports the identification of Drosophila Activated cdc42 kinase as a growth promoter and a novel Hippo signaling pathway regulator. Activated cdc42 kinase promotes tissue growth through modulating Yorkie activity. Furthermore, Activated cdc42 kinase interacts with Expanded and induces tyrosine phosphorylation of Expanded on multiple sites. A model is proposed that activated cdc42 kinase negatively regulates Expanded by changing its phosphorylation status to promote tissue growth. Moreover, ack genetically interacts with merlin and expanded. Thus, this study identifies Drosophila Activated cdc42 kinase as a Hippo pathway regulator.
Tan, G. C., Mazzoni, E. O. and Wichterle, H. (2016). Iterative role of Notch signaling in spinal motor neuron diversification. Cell Rep 16: 907-916. PubMed ID: 27425621
Evolutionary Homolog Study
The motor neuron progenitor domain in the ventral spinal cord gives rise to multiple subtypes of motor neurons and glial cells. This study examined whether progenitors found in this domain are multipotent and which signals contribute to their cell-type-specific differentiation. Using an in vitro neural differentiation model, motor neuron progenitor differentiation was shown to be iteratively controlled by Notch signaling. First, Notch controls the timing of motor neuron genesis by repressing Neurogenin 2 (Ngn2) and maintaining Olig2-positive progenitors in a proliferative state. Second, in an Ngn2-independent manner, Notch contributes to the specification of median versus hypaxial motor column identity and lateral versus medial divisional identity of limb-innervating motor neurons. Thus, motor neuron progenitors are multipotent, and their diversification is controlled by Notch signaling that iteratively increases cellular diversity arising from a single neural progenitor domain.

Saturday, July 13th

Zwart, M.F., Pulver, S,R., Truman, J.W., Fushiki, A., Cardona, A. and Landgraf, M. (2016). Selective inhibition mediates the sequential recruitment of motor pools. Neuron [Epub ahead of print]. PubMed ID: 27427461
Locomotor systems generate diverse motor patterns to produce the movements underlying behavior, requiring that motor neurons be recruited at various phases of the locomotor cycle. Reciprocal inhibition produces alternating motor patterns; however, the mechanisms that generate other phasic relationships between intrasegmental motor pools, all of the motor neurons that innervate single muscles, are unknown. This study investigated one such motor pattern in the Drosophila larva, using a multidisciplinary approach including electrophysiology and ssTEM-based circuit reconstruction. It was found that two motor pools that are sequentially recruited during locomotion have identical excitable properties. In contrast, they receive input from divergent premotor circuits. It was also found that this motor pattern is not orchestrated by differential excitatory input but by a GABAergic interneuron acting as a delay line to the later-recruited motor pool. These findings show how a motor pattern is generated as a function of the modular organization of locomotor networks through segregation of inhibition, a potentially general mechanism for sequential motor patterns.
Morton, A., Murawski, C., Pulver, S.R. and Gather, M.C. (2016). High-brightness organic light-emitting diodes for optogenetic control of Drosophila locomotor behaviour. Sci Rep 6: 31117. PubMed ID: 27484401
Organic light emitting diodes (OLEDs) are in widespread use in today's mobile phones and are likely to drive the next generation of large area displays and solid-state lighting. This study shows steps towards their utility as a platform technology for biophotonics, by demonstrating devices capable of optically controlling behaviour in live animals. Using devices with a pin OLED architecture, sufficient illumination intensity (0.3 mW.mm-2) to activate channelrhodopsins (ChRs) in vivo was reliably achieved at low operating voltages (5 V). In Drosophila melanogaster third instar larvae expressing ChR2H134R in motor neurons, it was found that pulsed illumination from blue and green OLEDs triggers robust and reversible contractions in animals. This response is temporally coupled to the timing of OLED illumination. With blue OLED illumination, the initial rate and overall size of the behavioural response is strongest. Green OLEDs achieve roughly 70% of the response observed with blue OLEDs. Orange OLEDs do not produce contractions in larvae, in agreement with the spectral response of ChR2H134R. The device configuration presented here could be modified to accommodate other small model organisms, cell cultures or tissue slices and the ability of OLEDs to provide patterned illumination and spectral tuning can further broaden their utility in optogenetics experiments. 

Jourjine, N., Mullaney, B.C., Mann, K. and Scott, K. (2016). Coupled sensing of hunger and thirst signals balances sugar and water consumption. Cell [Epub ahead of print]. PubMed ID: 27477513
Hunger and thirst are ancient homeostatic drives for food and water consumption. Although molecular and neural mechanisms underlying these drives are currently being uncovered, less is known about how hunger and thirst interact. This study used molecular genetic, behavioral, and anatomical studies in Drosophila to identify four neurons that modulate food and water consumption. Activation of these neurons promotes sugar consumption and restricts water consumption, whereas inactivation promotes water consumption and restricts sugar consumption. By calcium imaging studies, it was shown that these neurons are directly regulated by a hormone signal of nutrient levels and by osmolality. Finally, a hormone receptor and an osmolality-sensitive ion channel that underlie this regulation were identified. Thus, a small population of neurons senses internal signals of nutrient and water availability to balance sugar and water consumption. These results suggest an elegant mechanism by which interoceptive neurons oppositely regulate homeostatic drives to eat and drink.

Laturney, M. and Billeter, J.C. (2016). Drosophila melanogaster females restore their attractiveness after mating by removing male anti-aphrodisiac pheromones. Nat Commun 7: 12322. PubMed ID: 27484362
Males from many species ensure paternity by preventing their mates from copulating with other males. One mate-guarding strategy involves marking females with anti-aphrodisiac pheromones (AAPs), which reduces the females' attractiveness and dissuades other males from courting. Since females benefit from polyandry, sexual conflict theory predicts that females should develop mechanisms to counteract AAPs to achieve additional copulations, but no such mechanisms have been documented. This study shows that during copulation Drosophila melanogaster males transfer two AAPs: cis-Vaccenyl Acetate (cVA) to the females' reproductive tract, and 7-Tricosene (7-T) to the females' cuticle. A few hours after copulation, females actively eject cVA from their reproductive tract, which results in increased attractiveness and re-mating. Although 7-T remains on those females, it was shown that it is the combination of the two chemicals that reduces attractiveness. Thus, female AAP ejection provides the first example of a female mechanism that counter-acts chemical mate-guarding.

Friday, August 12th

Farkas, R., Pecenova, L., Mentelova, L., Beno, M., Benova-Liszekova, D., Mahmoodova, S., Tejnecky, V., Raska, O., Juda, P., Svidenska, S., Hornacek, M., Chase, B. A. and Raska, I. (2016). Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial-like anion transport. Dev Growth Differ 58: 562-574. PubMed ID: 27397870
The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well-documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally-regulated programmed cell death (PCD), as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, it has been shown that for the first 3-4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. This study shows that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. Genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A1 or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD.
Paredes, J. C., Herren, J. K., Schupfer, F. and Lemaitre, B. (2016). The role of lipid competition for endosymbiont-mediated protection against parasitoid wasps in Drosophila. MBio 7 [Epub ahead of print]. PubMed ID: 27406568
Insects commonly harbor facultative bacterial endosymbionts, such as Wolbachia and Spiroplasma species, that are vertically transmitted from mothers to their offspring. These endosymbiontic bacteria increase their propagation by manipulating host reproduction or by protecting their hosts against natural enemies. While an increasing number of studies have reported endosymbiont-mediated protection, little is known about the mechanisms underlying this protection. This study analyze the mechanisms underlying protection from parasitoid wasps in Drosophila mediated by its facultative endosymbiont Spiroplasma poulsonii. The results indicate that S. poulsonii exerts protection against two distantly related wasp species, Leptopilina boulardi and Asobara tabida. S. poulsonii-mediated protection against parasitoid wasps takes place at the pupal stage and is not associated with an increased cellular immune response. This work provides three important observations that support the notion that S. poulsonii bacteria and wasp larvae compete for host lipids and that this competition underlies symbiont-mediated protection. First, lipid quantification shows that both S. poulsonii and parasitoid wasps deplete Drosophila hemolymph lipids. Second, the depletion of hemolymphatic lipids using the Lpp RNA interference (Lpp RNAi) construct reduces wasp success in larvae that are not infected with S. poulsonii and blocks S. poulsonii growth. Third, the growth of S. poulsonii bacteria is not affected by the presence of the wasps, indicating that when S. poulsonii is present, larval wasps will develop in a lipid-depleted environment. It is proposed that competition for host lipids may be relevant to endosymbiont-mediated protection in other systems and could explain the broad spectrum of protection provided.
Yoshida, M., Matsuda, H., Kubo, H. and Nishimura, T. (2016). Molecular characterization of Tps1 and Treh genes in Drosophila and their role in body water homeostasis. Sci Rep 6: 30582. PubMed ID: 27469628
In insects, trehalose serves as the main sugar component of haemolymph. Trehalose is also recognized as a mediator of desiccation survival due to its proposed ability to stabilize membranes and proteins. Although the physiological role of trehalose in insects has been documented for decades, genetic evidence to support the importance of trehalose metabolism remains incomplete. This study shows on the basis of genetic and biochemical evidence that the trehalose synthesis enzyme Tps1 is solely responsible for the de novo synthesis of trehalose in Drosophila. Conversely, a lack of the gene for the trehalose hydrolyzing enzyme Treh causes an accumulation of trehalose that is lethal during the pupal period, as is observed with Tps1 mutants. Lack of either Tps1 or Treh results in a significant reduction in circulating glucose, suggesting that the maintenance of glucose levels requires a continuous turnover of trehalose. Furthermore, changes in trehalose levels are positively correlated with the haemolymph water volume. In addition, both Tps1 and Treh mutant larvae exhibit a high lethality after desiccation stress. These results demonstrate that the regulation of trehalose metabolism is essential for normal development, body water homeostasis, and desiccation tolerance in Drosophila.
Grillet, M., Dominguez Gonzalez, B., Sicart, A., Pottler, M., Cascalho, A., Billion, K., Hernandez Diaz, S., Swerts, J., Naismith, T. V., Gounko, N. V., Verstreken, P., Hanson, P. I. and Goodchild, R. E. (2016). Torsins are essential regulators of cellular lipid metabolism. Dev Cell. PubMed ID: 27453503
Torsins are developmentally essential AAA+ proteins, and mutation of human torsinA causes the neurological disease DYT1 dystonia. They localize in the ER membranes, but their cellular function remains unclear. This study shows that dTorsin is required in Drosophila adipose tissue, where it suppresses triglyceride levels, promotes cell growth, and elevates membrane lipid content. Human torsinA at the inner nuclear membrane is associated with membrane expansion and elevated cellular lipid content. Furthermore, the key lipid metabolizing enzyme, lipin, is mislocalized in dTorsin-KO cells, and dTorsin increases levels of the lipin substrate, phosphatidate, and reduces the product, diacylglycerol. Finally, genetic suppression of dLipin rescues dTorsin-KO defects, including adipose cell size, animal growth, and survival. These findings identify that torsins are essential regulators of cellular lipid metabolism and implicate disturbed lipid biology in childhood-onset DYT1 dystonia.

Thursday, August 11th

Li, Y., Hassinger, L., Thomson, T., Ding, B., Ashley, J., Hassinger, W. and Budnik, V. (2016). Lamin mutations accelerate aging via defective export of mitochondrial mRNAs through nuclear envelope budding. Curr Biol [Epub ahead of print]. PubMed ID: 27451905
Defective RNA metabolism and transport are implicated in aging and degeneration, but the underlying mechanisms remain poorly understood. A prevalent feature of aging is mitochondrial deterioration. This study links a novel mechanism for RNA export through nuclear envelope (NE) budding that requires A-type lamin, an inner nuclear membrane-associated protein, to accelerated aging observed in Drosophila LaminC (LamC) mutations. These LamC mutations were modeled after A-lamin (LMNA) mutations causing progeroid syndromes (PSs) in humans. Mitochondrial assembly regulatory factor (Marf), a mitochondrial fusion factor (mitofusin) as well as other transcripts required for mitochondrial integrity and function, were identified in a screen for RNAs that exit the nucleus through NE budding. PS-modeled LamC mutations induced premature aging in adult flight muscles, including decreased levels of specific mitochondrial protein transcripts (RNA) and progressive mitochondrial degradation. PS-modeled LamC mutations also induced the accelerated appearance of other phenotypes associated with aging, including a progressive accumulation of polyubiquitin aggregates and myofibril disorganization. Consistent with these observations, the mutants had progressive jumping and flight defects. Downregulating marf alone induced the above aging defects. Nevertheless, restoring marf was insufficient for rescuing the aging phenotypes in PS-modeled LamC mutations, as other mitochondrial RNAs are affected by inhibition of NE budding. Analysis of NE budding in dominant and recessive PS-modeled LamC mutations suggests a mechanism by which abnormal lamina organization prevents the egress of these RNAs via NE budding. These studies connect defects in RNA export through NE budding to progressive loss of mitochondrial integrity and premature aging.
Giorgetti, L., Lajoie, B. R., Carter, A. C., Attia, M., Zhan, Y., Xu, J., Chen, C. J., Kaplan, N., Chang, H. Y., Heard, E. and Dekker, J. (2016). Structural organization of the inactive X chromosome in the mouse.Nature 535: 575-579. PubMed ID: 27437574 Evolutionary Homolog Study
X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite. This study investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. A crucial role for Xist and the DXZ4-containing boundary was demonstrated in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. It was also shown that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes in different neural progenitor clones are associated with the presence of different TAD-like structures after XCI. These findings suggest a key role for transcription and CTCF in the formation of TADs in the context of the Xi chromosome in neural progenitors.
Ji, Y. and Tulin, A. V. (2016). Poly(ADP-ribosyl)ation of hnRNP A1 protein controls translational repression in Drosophila. Mol Cell Biol [Epub ahead of print]. PubMed ID: 27402862
Poly(ADP-ribosyl)ation of heterogeneous nuclear RNA binding proteins (hnRNP) regulates the post-transcriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germline stem cell maintenance and oocyte localization. The mRNA-targets regulated by Hrp38 are mostly unknown. This study identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. Hrp38 binds to the 3' untranslated region (3' UTR) of Nanos mRNA, which contains a translation control element. Translation of the luciferase reporter bearing Nanos 3' UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. These data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to Nanos 3' UTR, increasing the translation in vivo and in vitro Hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. It is concluded that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis.
Jaksic, A. M. and Schlotterer, C. (2016). The interplay of temperature and genotype on patterns of alternative splicing in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 27440867
Alternative splicing is the highly regulated process of variation in the removal of introns from pre-mRNA transcripts. Variation in alternative splicing among four different temperatures, 13°, 18°, 23° and 29° was studied in two Drosophila genotypes. Plasticity of alternative splicing was shown, with up to 10% of the expressed genes being differentially spliced between the most extreme temperatures for a given genotype. Comparing the two genotypes at different temperatures, less than 1% of the genes were found being differentially spliced at 18°. At extreme temperatures, however, substantial differences were detected in alternative splicing - with almost 10% of the genes having differential splicing between the genotypes: a magnitude similar to between species differences. Genes with differential alternative splicing between genotypes frequently exhibit dominant inheritance. Remarkably, the pattern of surplus of differences in alternative splicing at extreme temperatures resembled the pattern seen for gene expression intensity. Since different sets of genes were involved for the two phenotypes, it is proposed that purifying selection results in the reduction of differences at benign temperatures. Relaxed purifying selection at temperature extremes, on the other hand may cause the divergence in gene expression and alternative splicing between the two strains in rarely encountered environments.

Wednesday, August 10th

Ryazansky, S. S., Kotov, A. A., Kibanov, M. V., Akulenko, N. V., Korbut, A. P., Lavrov, S. A., Gvozdev, V. A. and Olenina, L. V. (2016). RNA helicase Spn-E is required to maintain Aub and AGO3 protein levels for piRNA silencing in the germline of Drosophila. Eur J Cell Biol [Epub ahead of print]. PubMed ID: 27320195
Germline-specific RNA helicase Spindle-E (Spn-E) is known to be essential for piRNA silencing in Drosophila that takes place mainly in the perinuclear nuage granules. Loss-of-function spn-E mutations lead to tandem Stellate genes derepression in the testes and retrotransposon mobilization in the ovaries. However, Spn-E functions in the piRNA pathway are still obscure. Analysis of total library of short RNAs from the testes of spn-E heterozygous flies revealed the presence of abundant piRNA ping-pong pairs originating from Su(Ste) transcripts. The abundance of these ping-pong pairs were sharply reduced in the library from the testes of spn-E mutants. Thus the ping-pong mechanism contributes to Su(Ste) piRNA generation in the testes. The lack of Spn-E caused a significant drop of protein levels of key ping-pong participants, Aubergine (Aub) and AGO3 proteins of PIWI subfamily, in the germline of both males and females, but did not disrupt of their assembly in nuage granules. Observed decline of the protein expression was not caused by suppression of aub and ago3 transcription as well as total transcription, indicating possible contribution of Spn-E to post-transcriptional regulation.
Lim, J., Lee, M., Son, A., Chang, H. and Kim, V. N. (2016). mTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development. Genes Dev 30: 1671-1682. PubMed ID: 27445395
Eukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. TAIL-seq has been developed to investigate RNA tails at the genomic scale, but its low sensitivity precluded its application to biological materials of minute quantity. This study reports a new version of TAIL-seq (mRNA TAIL-seq [mTAIL-seq]) with enhanced sequencing depth for mRNAs (by approximately 1000-fold compared with the previous version). The improved method allows investigation of the regulation of poly(A) tails in Drosophila oocytes and embryos. Maternal mRNAs were found to be polyadenylated mainly during late oogenesis, prior to fertilization, and further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs, with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAIL-seq data with ribosome profiling data, a strong coupling was found between poly(A) tail length and translational efficiency during egg activation. The data suggest that regulation of poly(A) tails in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve understanding of mRNA tailing in diverse biological systems.
Jain, R., Iglesias, N. and Moazed, D. (2016). Distinct functions of Argonaute slicer in siRNA, maturation and heterochromatin formation. Mol Cell 63: 191-205. PubMed ID: 27397687
Evolutionary Homolog Study
Small-RNA (sRNA)-guided transcriptional gene silencing by Argonaute (Ago)-containing complexes is fundamental to genome integrity and epigenetic inheritance. The RNA cleavage ("Slicer") activity of Argonaute has been implicated in both sRNA maturation and target RNA cleavage. Typically, Argonaute slices and releases the passenger strand of duplex sRNA to generate active silencing complexes, but it remains unclear whether slicing of target nascent RNAs, or other RNAi components, also contributes to downstream transcriptional silencing. This study developd a strategy for loading the fission yeast Ago1 with a single-stranded sRNA guide, which bypasses the requirement for slicer activity in generation of active silencing complexes. Slicer-defective Ago1 was shown to mediate secondary sRNA generation, H3K9 methylation, and silencing similar to or better than wild-type and associates with chromatin more efficiently. The results define an ancient and minimal sRNA-mediated chromatin silencing mechanism, which resembles the germline-specific sRNA-dependent transcriptional silencing pathways in Drosophila and mammals.
Gao, Z., Putnam, A.A., Bowers, H.A., Guenther, U.P., Ye, X., Kindsfather, A., Hilliker, A.K. and Jankowsky, E. (2016). Coupling between the DEAD-box RNA helicases Ded1p and eIF4A. Elife [Epub ahead of print]. PubMed ID: 27494274
Evolutionary Homolog Study
Eukaryotic translation initiation involves two conserved DEAD-box RNA helicases, eIF4A and Ded1p. This study shows that S. cerevisiae eIF4A (see Drosophila eIF4A) and Ded1p (see Drosophila bel)  directly interact with each other and simultaneously with the scaffolding protein eIF4G (see Drosophila eIF4G). The study delineates a comprehensive thermodynamic framework for the interactions between Ded1p, eIF4A, eIF4G, RNA and ATP, which indicates that eIF4A, with and without eIF4G, acts as modulator for activity and substrate preferences of Ded1p, which is the RNA remodeling unit in all complexes. These results reveal and characterize an unexpected interdependence between the two RNA helicases and eIF4G, and suggest that Ded1p is an integral part of eIF4F, the complex comprising eIF4G, eIF4A, and eIF4E.

Tuesday, August 9th

Andres, M., Seifert, M., Spalthoff, C., Warren, B., Weiss, L., Giraldo, D., Winkler, M., Pauls, S. and Gopfert, M. C. (2016). Auditory efferent system modulates mosquito hearing. Curr Biol [Epub ahead of print]. PubMed ID: 27476597
The performance of vertebrate ears is controlled by auditory efferents that originate in the brain and innervate the ear, synapsing onto hair cell somata and auditory afferent fibers. Efferent activity can provide protection from noise and facilitate the detection and discrimination of sound by modulating mechanical amplification by hair cells and transmitter release as well as auditory afferent action potential firing. Insect auditory organs are thought to lack efferent control, but when this study inspected mosquito ears, evidence was found for its existence. Antibodies against synaptic proteins recognized rows of bouton-like puncta running along the dendrites and axons of mosquito auditory sensory neurons. Electron microscopy identified synaptic and non-synaptic sites of vesicle release, and some of the innervating fibers co-labeled with somata in the CNS. Octopamine, GABA, and serotonin were identified as efferent neurotransmitters or neuromodulators that affect auditory frequency tuning, mechanical amplification, and sound-evoked potentials. Mosquito brains thus modulate mosquito ears, extending the use of auditory efferent systems from vertebrates to invertebrates and adding new levels of complexity to mosquito sound detection and communication.
Jin, X., Jeon, H. M., Jin, X., Kim, E. J., Yin, J., Jeon, H. Y., Sohn, Y. W., Oh, S. Y., Kim, J. K., Kim, S. H., Jung, J. E., Kwak, S., Tang, K. F., Xu, Y., Rich, J. N. and Kim, H. (2016). The ID1-CULLIN3 axis regulates intracellular SHH and WNT signaling in glioblastoma stem cells. Cell Rep. PubMed ID: 27477274
Evolutionary Homolog Study
Inhibitor of differentiation 1 (ID1; see Drosophila Extra macrochaetae) is highly expressed in glioblastoma stem cells (GSCs). However, the regulatory mechanism responsible for its role in GSCs is poorly understood. This study reports that ID1 activates GSC proliferation, self-renewal, and tumorigenicity by suppressing CULLIN3 ubiquitin ligase (see Drosophila Cullin-3). ID1 induces cell proliferation through increase of CYCLIN E, a target molecule of CULLIN3. ID1 overexpression or CULLIN3 knockdown confers GSC features and tumorigenicity to murine Ink4a/Arf-deficient astrocytes. Proteomics analysis revealed that CULLIN3 interacts with GLI2 (see Drosophila Cubitus interruptus) and DVL2 (see Drosophila Dishevelled) and induces their degradation via ubiquitination. Consistent with ID1 knockdown or CULLIN3 overexpression in human GSCs, pharmacologically combined control of GLI2 and beta-CATENIN (see Drosophila Armadillo) effectively diminishes GSC properties. A ID1-high/CULLIN3-low expression signature correlates with a poor patient prognosis, supporting the clinical relevance of this signaling axis. Taken together, a loss of CULLIN3 represents a common signaling node for controlling the activity of intracellular WNT and SHH signaling pathways mediated by ID1.
Guo, F., Yu, J., Jung, H.J., Abruzzi, K.C., Luo, W., Griffith, L.C. and Rosbash, M. (2016). Circadian neuron feedback controls the Drosophila sleep-activity profile. Nature [Epub ahead of print]. PubMed ID: 27479324
Little is known about the ability of Drosophila circadian neurons to promote sleep. This study, using optogenetic manipulation and video recording, shows that a subset of dorsal clock neurons (DN1s) are potent sleep-promoting cells that release glutamate to directly inhibit key pacemaker neurons. The pacemakers promote morning arousal by activating these DN1s, implying that a late-day feedback circuit drives midday siesta and night-time sleep. To investigate more plastic aspects of the sleep program, the study used a calcium assay to monitor and compare the real-time activity of DN1 neurons in freely behaving males and females. It was found that DN1 neurons are more active in males than in females, consistent with the finding that male flies sleep more during the day. DN1 activity is also enhanced by elevated temperature, consistent with the ability of higher temperatures to increase sleep. These new approaches indicate that DN1s have a major effect on the fly sleep-wake profile and integrate environmental information with the circadian molecular program.

Zhang, Y.V., Aikin, T.J., Li, Z. and Montell, C. (2016). The basis of food texture sensation in Drosophila. Neuron [Epub ahead of print]. PubMed ID: 27478019
Food texture has enormous effects on food preferences. However, the mechanosensory cells and key molecules responsible for sensing the physical properties of food are unknown. This study shows that akin to mammals, the fruit fly, Drosophila melanogaster, prefers food with a specific hardness or viscosity. This food texture discrimination depends upon a previously unknown multidendritic (md-L) neuron, which extends elaborate dendritic arbors innervating the bases of taste hairs. The md-L neurons exhibit directional selectivity in response to mechanical stimuli. Moreover, these neurons orchestrate different feeding behaviors depending on the magnitude of the stimulus. It was demonstrated that the single Drosophila transmembrane channel-like (TMC) protein is expressed in md-L neurons, where it is required for sensing two key textural features of food-hardness and viscosity. The study proposes that md-L neurons are long sought after mechanoreceptor cells through which food mechanics are perceived and encoded by a taste organ, and that this sensation depends on TMC.

Monday, August 8th

Marta, M. (2016). Dpr-DIP matching expression in Drosophila synaptic pairs. Fly (Austin): 1-8. PubMed ID: 27450981
Neurons form precise patterns of connections. The cellular recognition mechanisms regulating the selection of synaptic partners are poorly understood. As final mediators of cell-cell interactions, cell surface and secreted molecules (CSMs) are expected to play important roles in this process. To gain insight into how neurons discriminate synaptic partners, the transcriptomes were profiled of 7 closely related neurons forming distinct synaptic connections in discrete layers in the medulla neuropil of the fly visual system. The sequencing data revealed that each one of these neurons expresses a unique combination of hundreds of CSMs at the onset of synapse formation. 21 Ig domain paralogs of the defective proboscis extension response (see Drosophila Piecing Together the Extracellular Puzzle) family were shown to be expressed in a unique cell-type-specific fashion, consistent with the distinct connectivity pattern of each neuron profiled. Expression analysis of their cognate binding partners, the 9 members of the Dpr interacting protein (DIP) family, revealed complementary layer-specific expression in the medulla, suggestive of interactions between neurons expressing Dpr and those expressing DIP in the same layer. Through coexpression analysis and correlation to connectome data, neurons expressing DIP were identified as a subset of the synaptic partners of the neurons expressing Dpr. It is proposed that Dpr-DIP interactions regulate patterns of connectivity between the neurons expressing them.
Nguyen, C. T. and Stewart, B. A. (2016). The influence of postsynaptic structure on missing quanta at the Drosophila neuromuscular junction. BMC Neurosci 17: 53. PubMed ID: 27459966
Synaptic transmission requires both pre- and post-synaptic elements for neural communication. The postsynaptic structure contributes to the ability of synaptic currents to induce voltage changes in postsynaptic cells. At the Drosophila neuromuscular junction (NMJ), the postsynaptic structure, known as the subsynaptic reticulum (SSR), consists of elaborate membrane folds that link the synaptic contacts to the muscle, but its role in synaptic physiology is poorly understood. This study investigated the role of the SSR with simultaneous intra- and extra-cellular recordings that allow identification of the origin of spontaneously occurring synaptic events. Data from Type 1b and 1s synaptic boutons, which have naturally occurring variations of the SSR, were compared with genetic mutants that up or down-regulate SSR complexity. Some synaptic currents do not result in postsynaptic voltage changes, events that were called 'missing quanta'. The frequency of missing quanta is positively correlated with SSR complexity in both natural and genetically-induced variants. Rise-time and amplitude data suggest that passive membrane properties contribute to the observed differences in synaptic effectiveness. It is concluded that electrotonic decay within the postsynaptic structure contributes to the phenomenon of missing quanta. Further studies directed at understanding the role of the SSR in synaptic transmission and the potential for regulating 'missing quanta' will yield important information about synaptic transmission at the Drosophila NMJ.
McMillan, B. J., Tibbe, C., Jeon, H., Drabek, A. A., Klein, T. and Blacklow, S. C. (2016). Electrostatic interactions between elongated monomers drive filamentation of Drosophila Shrub, a metazoan ESCRT-III protein. Cell Rep 16: 1211-1217. PubMed ID: 27452459
The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. This study reports the structural basis for polymerization of Shrub and defines a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins.
Isabella, A.J. and Horne-Badovinac, S. (2016). Rab10-mediated secretion synergizes with tissue movement to build a polarized basement membrane architecture for organ morphogenesis. Dev Cell 38: 47-60. PubMed ID: 27404358
Basement membranes (BMs) are planar protein networks that support epithelial function. Regulated changes to BM architecture can also contribute to tissue morphogenesis, but how epithelia dynamically remodel their BMs is unknown. In Drosophila, elongation of the initially spherical egg chamber correlates with the generation of a polarized network of fibrils in its surrounding BM. This study used live imaging and genetic manipulations to determine how these fibrils form. BM fibrils are assembled from newly synthesized proteins in the pericellular spaces between the egg chamber's epithelial cells and undergo oriented insertion into the BM by directed epithelial migration. It was found that a Rab10-based secretion pathway promotes pericellular BM protein accumulation and fibril formation. Finally, by manipulating this pathway, it was shown that BM fibrillar structure influences egg chamber morphogenesis. This work highlights how regulated protein secretion can synergize with tissue movement to build a polarized BM architecture that controls tissue shape.

Sunday, August 7th

Burke, M.K., Barter, T.T., Cabral, L.G., Kezos, J.N., Phillips, M.A., Rutledge, G.A., Phung, K.H., Chen, R.H., Nguyen, H.D., Mueller, L.D. and Rose, M.R. (2016). Rapid divergence and convergence of life-history in experimentally evolved Drosophila melanogaster. Evolution [Epub ahead of print]. PubMed ID: 27431916
Laboratory selection experiments are alluring in their simplicity, power, and ability to inform about how evolution works. A longstanding challenge facing evolution experiments with metazoans is that significant generational turnover takes a long time. This study presents data from a unique system of experimentally evolved laboratory populations of Drosophila melanogaster that have experienced three distinct life-history selection regimes. The goal of the study was to determine how quickly populations of a certain selection regime diverge phenotypically from their ancestors, and how quickly they converge with independently derived populations that share a selection regime. Results indicate that phenotypic divergence from an ancestral population occurs rapidly, within dozens of generations, regardless of that population's evolutionary history. Similarly, populations sharing a selection treatment converge on common phenotypes in this same time frame, regardless of selection pressures those populations may have experienced in the past. These patterns of convergence and divergence were found to emerge much faster than expected, suggesting that intermediate evolutionary history has transient effects in this system. These results are applicable to other experimental evolution projects, and suggest that many relevant questions can be sufficiently tested on shorter timescales than previously thought.

Nishiguchi, S., Yagi, A., Sakai, N. and Oda, H. (2016). Divergence of structural strategies for E-cadherin homophilic binding among bilaterians. J Cell Sci [Epub ahead of print]. PubMed ID: 27422100
Homophilic binding of E-cadherins through their ectodomains is fundamental to epithelial cell-cell adhesion. Despite this, E-cadherin ectodomains have evolved differently in the vertebrate and hexapod lineages. Of the five rod-like, tandemly aligned extracellular cadherin domains (ECs) of vertebrate E-cadherin, the tip EC plays a pivotal role in binding interactions. Comparatively, the N-terminal six consecutive ECs of Drosophila E-cadherin, DE-cadherin, can mediate adhesion; however, the underlying mechanism is unknown. This study reports atomic force microscopy imaging of DE-cadherin ECs. A tightly folded globular structure formed by the four N-terminal-most ECs stabilized by the subsequent two ECs was identified. Analysis of hybrid cadherins of different hexapods indicated association of the E-cadherin globular portion with the determinants of homophilic binding specificity. The second to fourth ECs were identified as the minimal portion capable of mediating exclusive homophilic binding specificity. These findings suggested that the N-terminal-most four ECs of hexapod E-cadherin are functionally comparable with the N-terminal-most single EC of vertebrate E-cadherin, but that their mechanisms might significantly differ. This work illuminates the divergence of structural strategies for E-cadherin homophilic binding among bilaterians.
Fuller, Z. L., Haynes, G. D., Richards, S. and Schaeffer, S. W. (2016). Genomics of natural populations: How differentially expressed genes shape the evolution of chromosomal inversions in Drosophila pseudoobscura. Genetics [Epub ahead of print]. PubMed ID: 27401754
The third chromosome of Drosophila pseudoobscura is a model system to test hypotheses about how rearrangements are established in populations because its third chromosome is polymorphic for > 30 gene arrangements that were generated by a series of overlapping inversion mutations. Circumstantial evidence has suggested that these gene arrangements are selected. Despite the expected homogenizing effects of extensive gene flow, the frequencies of arrangements form gradients or clines in nature, which have been stable since the system was first described more than 80 years ago. Furthermore, multiple arrangements exist at appreciable frequencies across several ecological niches providing the opportunity for heterokaryotypes to form. This study tested whether genes are differentially expressed among chromosome arrangements in first instar larvae, adult females and males. In addition, it wass asked whether transcriptional patterns in heterokaryotypes are dominant, semidominant, overdominant, or underdominant. Evidence was found for a significant abundance of differentially expressed genes across the inverted regions of the third chromosome, including an enrichment of genes involved in sensory perception for males. The majority of loci show additivity in heterokaryotypes. These results suggest that multiple genes have expression differences among arrangements that were either captured by the original inversion mutation or accumulated after it reached polymorphic frequencies, providing a potential source of genetic variation for selection to act upon. These data suggest that the inversions are favored because of their indirect effect of recombination suppression that has held different combinations of differentially expressed genes together in the various gene arrangement backgrounds.
Puerma, E., Orengo, D. J. and Aguade, M. (2016). The origin of chromosomal inversions as a source of segmental duplications in the Sophophora subgenus of Drosophila. Sci Rep 6: 30715. PubMed ID: 27470196
Chromosomal inversions can contribute to the adaptation of organisms to their environment by capturing particular advantageous allelic combinations of a set of genes included in the inverted fragment and also by advantageous functional changes due to the inversion process itself that might affect not only the expression of flanking genes but also their dose and structure. Of the two mechanisms originating inversions -ectopic recombination, and staggered double-strand breaks and subsequent repair- only the latter confers the inversion the potential to have dosage effects and/or to generate advantageous chimeric genes. In Drosophila subobscura, there is ample evidence for the adaptive character of its chromosomal polymorphism, with an important contribution of some warm-climate arrangements such as E1+2+9+12. This study has characterized the breakpoints of inversion E12 and established that it originated through the staggered-break mechanism like four of the five inversions of D. subobscura previously studied. This mechanism that also predominates in the D. melanogaster lineage might be prevalent in the Sophophora subgenus and contribute to the adaptive character of the polymorphic and fixed inversions of its species. Finally, the D. subobscura inversion breakpoint regions were shown to have generally been disrupted by additional structural changes that occurred at different time scales.
Yu, Y., Zhou, H., Kong, Y., Pan, B., Chen, L., Wang, H., Hao, P. and Li, X. (2016). The landscape of A-to-I RNA editome is shaped by both positive and purifying selection. PLoS Genet 12: e1006191. PubMed ID: 27467689
The hydrolytic deamination of adenosine to inosine (A-to-I editing) in precursor mRNA induces variable gene products at the post-transcription level. How and to what extent A-to-I RNA editing diversifies transcriptome is not fully characterized in the evolution, and very little is known about the selective constraints that drive the evolution of RNA editing events. This study on A-to-I RNA editing, by generating a global profile of A-to-I editing for a phylogeny of seven Drosophila species, presents a model system spanning an evolutionary timeframe of approximately 45 million years. Of totally 9281 editing events identified, 5150 (55.5%) are located in the coding sequences (CDS) of 2734 genes. Phylogenetic analysis places these genes into 1,526 homologous families, about 5% of total gene families in the fly lineages. Based on conservation of the editing sites, the editing events in CDS are categorized into three distinct types, representing events on singleton genes (type I), and events not conserved (type II) or conserved (type III) within multi-gene families. While both type I and II events are subject to purifying selection, notably type III events are positively selected, and highly enriched in the components and functions of the nervous system. The tissue profiles are documented for three editing types, and their critical roles are further implicated by their shifting patterns during holometabolous development and in post-mating response. In conclusion, three A-to-I RNA editing types are found to have distinct evolutionary dynamics. It appears that nervous system functions are mainly tested to determine if an A-to-I editing is beneficial for an organism. The coding plasticity enabled by A-to-I editing creates a new class of binary variations, which is a superior alternative to maintain heterozygosity of expressed genes in a diploid mating system.
Izumitani, H. F., Kusaka, Y., Koshikawa, S., Toda, M. J. and Katoh, T. (2016). Phylogeography of the Subgenus Drosophila (Diptera: Drosophilidae): Evolutionary history of faunal divergence between the old and the new worlds. PLoS One 11: e0160051. PubMed ID: 27462734
The current subgenus Drosophila (the traditional immigrans-tripunctata radiation) includes major elements of temperate drosophilid faunas in the northern hemisphere. Despite previous molecular phylogenetic analyses, the phylogeny of the subgenus Drosophila has not fully been resolved: the resulting trees have more or less varied in topology. One possible factor for such ambiguous results is taxon-sampling that has been biased towards New World species in previous studies. In this study, taxon sampling was balanced between Old and New World species, and phylogenetic relationships among 45 ingroup species selected from ten core species groups of the subgenus Drosophila were analyzed using nucleotide sequences of three nuclear and two mitochondrial genes. Based on the resulting phylogenetic tree, ancestral distributions and divergence times were estimated for each clade to test Throckmorton's hypothesis that there was a primary, early-Oligocene disjunction of tropical faunas and a subsequent mid-Miocene disjunction of temperate faunas between the Old and the New Worlds that occurred in parallel in separate lineages of the Drosophilidae. The results of this study substantially support Throckmorton's hypothesis of ancestral migrations via the Bering Land Bridge mainly from the Old to the New World, and subsequent vicariant divergence of descendants between the two Worlds occurred in parallel among different lineages of the subgenus Drosophila. However, the results also indicate that these events took place multiple times over a wider time range than Throckmorton proposed, from the late Oligocene to the Pliocene.

Saturday, August 6th

Parinejad, N., Peco, E., Ferreira, T., Stacey, S.M. and van Meyel, D.J. (2016). Disruption of an EAAT-mediated chloride channel in a Drosophila model of Ataxia. J Neurosci 36: 7640-7647. PubMed ID: 27445142
Patients with Type 6 episodic ataxia (EA6) have mutations of the excitatory amino acid transporter EAAT1 (also known as GLAST), but the underlying pathophysiological mechanism for EA6 is not known. EAAT1 is a glutamate transporter expressed by astrocytes and other glia, and it serves dual function as an anion channel. One EA6-associated mutation is a P>R substitution (EAAT1(P>R)) that in transfected cells has a reduced rate of glutamate transport and an abnormal anion conductance. This study expressed this EAAT1(P>R) mutation in glial cells of Drosophila larvae and found that these larvae exhibit episodic paralysis, and their astrocytes poorly infiltrate the CNS neuropil. These defects are not seen in Eaat1-null mutants, and so they cannot be explained by loss of glutamate transport. To explore the role of the abnormal anion conductance of the EAAT1(P>R) mutation, chloride cotransporters were expressed in astrocytes. Like the EAAT1(P>R) mutation, the chloride-extruding K(+)-Cl(-) cotransporter KccB also causes astroglial malformation and paralysis, supporting the idea that the EAAT1(P>R) mutation causes abnormal chloride flow from CNS glia. In contrast, the Na(+)-K(+)-Cl(-) cotransporter Ncc69, which normally allows chloride into cells, rescues the effects of the EAAT1(P>R) mutation. Together, these results indicate that the cytopathology and episodic paralysis in the Drosophila EA6 model stem from a gain-of-function chloride channelopathy of glial cells.

Hjerrild, K.A., et al. (2016). Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system. Sci Rep 6: 30357. PubMed ID: 27457156
The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). This study reports the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS(2), based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that differed in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identified quantitative and qualitative differences in terms of the functional IgG antibody response against the P. falciparum parasite. The antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifies Drosophila S2 cells as a clinically-relevant platform suited for the production of 'difficult-to-make' proteins from Plasmodium parasites, and identifies a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen. 

Crippa, V., et al. (2016). The chaperone HSPB8 reduces the accumulation of truncated TDP-43 species in cells and protects against TDP-43-mediated toxicity. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27466192
Aggregation of TAR-DNA binding protein 43 (TDP-43; see Drosophila TBPH) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragment of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. This study reports that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster. Overexpression of HSP67Bc, the Drosophila functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, a novel Drosophila model expressing TDP-35 is reported, and it was shown that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively these data demonstrate that HSPB8 upregulation mitigates TDP fragment mediated toxicity, in mammalian neuronal cells and flies (Crippa, 2016).
Jumbo-Lucioni, P. P., Parkinson, W. M., Kopke, D. L. and Broadie, K. (2016). Coordinated movement, neuromuscular synaptogenesis and trans-synaptic signaling defects in Drosophila galactosemia models. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27466186
The multiple galactosemia disease states manifest long-term neurological symptoms. Galactosemia I results from loss of galactose-1-phosphate uridyltransferase (GALT), which converts galactose-1-phosphate + UDP-glucose to glucose-1-phosphate + UDP-galactose. Galactosemia II results from loss of galactokinase (GALK), phosphorylating galactose to galactose-1-phosphate. Galactosemia III results from the loss of UDP-galactose 4'-epimerase (GALE), which interconverts UDP-galactose and UDP-glucose, as well as UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. UDP-glucose pyrophosphorylase (UGP) alternatively makes UDP-galactose from uridine triphosphate and galactose-1-phosphate. All four UDP-sugars are essential donors for glycoprotein biosynthesis with critical roles at the developing neuromuscular synapse. Drosophila galactosemia I (dGALT) and II (dGALK) disease models genetically interact; manifesting deficits in coordinated movement, neuromuscular junction (NMJ) development, synaptic glycosylation, and Wnt trans-synaptic signaling. Similarly, dGALE and dUGP mutants display striking locomotor and NMJ formation defects, including expanded synaptic arbors, glycosylation losses, and differential changes in Wnt trans-synaptic signaling. In combination with dGALT loss, both dGALE and dUGP mutants compromise the synaptomatrix glycan environment that regulates Wnt trans-synaptic signaling that drives 1) presynaptic Futsch/MAP1b microtubule dynamics and 2) postsynaptic Frizzled nuclear import (FNI). Taken together, these findings indicate UDP-sugar balance is a key modifier of neurological outcomes in all three interacting galactosemia disease models, suggest that Futsch homolog MAP1B and the Wnt Frizzled receptor may be disease-relevant targets in epimerase and transferase galactosemias, and identify UGP as promising new potential therapeutic target for galactosemia neuropathology.

Friday, August 5th

Dominguez, C., Zuniga, A., Hanna, P., Hodar, C., Gonzalez, M. and Cambiazo, V. (2016). Target genes of Dpp/BMP signaling pathway revealed by transcriptome profiling in the early D.melanogaster embryo. Gene [Epub ahead of print]. PubMed ID: 27397649
In the early Drosophila melanogaster embryo, the gene regulatory network controlled by Dpp signaling is involved in the subdivision of dorsal ectoderm into the presumptive dorsal epidermis and amnioserosa. This study identified new Dpp downstream targets involved in dorsal ectoderm patterning. Oligonucleotide Drosophila microarrays were used to identify the set of genes that are differential expressed between wild type embryos and embryos that overexpress Dpp (nos-Gal4>UAS-dpp) during early stages of embryo development. By using this approach, 358 genes were identified whose relative abundance significantly increased in response to Dpp overexpression. Among them, the entire set of known Dpp target genes that function in dorsal ectoderm patterning (zen, doc, hnt, pnr, ush, tup, and others) were identified, in addition to several up-regulated genes of unknown functions. One of the candidate genes, CG13653, which is expressed at the dorsal-most cells of the embryo during a restricted period of time was further analyzed. CG13653 orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa. The enhancer region of CG13653 was characterized, and CG13653 was shown to be directly regulated by Dpp signaling pathway.
Yang, S., Ben-Shalom, R., Ahn, M., Liptak, A. T., van Rijn, R. M., Whistler, J. L. and Bender, K. J. (2016). β-Arrestin-dependent dopaminergic regulation of Calcium channel activity in the axon initial segment. Cell Rep. PubMed ID: 27452469
Evolutionary Homolog Study:
G-protein-coupled receptors (GPCRs) initiate a variety of signaling cascades, depending on effector coupling. β-arrestins, which were initially characterized by their ability to "arrest" GPCR signaling by uncoupling receptor and G protein, have recently emerged as important signaling effectors for GPCRs. β-arrestins (see Drosophila Kurtz) engage signaling pathways that are distinct from those mediated by G protein. As such, arrestin-dependent signaling can play a unique role in regulating cell function, but whether neuromodulatory GPCRs utilize β-arrestin-dependent signaling to regulate neuronal excitability remains unclear. In a study of cartwheel cells, a class of glycinergic interneuron in the auditory brainstem dorsal cochlear nucleus (DCN), D3 dopamine receptors (D3R) were found to regulate axon initial segment (AIS) excitability through β-arrestin-dependent signaling, modifying CaV3 voltage dependence to suppress high-frequency action potential generation. This non-canonical D3R signaling thereby gates AIS excitability via pathways distinct from classical GPCR signaling pathways.
Atkins, M., Potier, D., Romanelli, L., Jacobs, J., Mach, J., Hamaratoglu, F., Aerts, S. and Halder, G. (2016). An ectopic network of transcription factors regulated by Hippo signaling drives growth and invasion of a malignant tumor model. Curr Biol [Epub ahead of print]. PubMed ID: 27476594
Cancer cells have abnormal gene expression profiles; however, to what degree these are chaotic or driven by structured gene regulatory networks is often not known. This study focused on a model of Ras-driven invasive tumorigenesis in Drosophila epithelial tissues and combined in vivo genetics with next-generation sequencing and computational modeling to decipher the regulatory logic of tumor cells. Surprisingly, it was discovered that the bulk of the tumor-specific gene expression is controlled by an ectopic network of a few transcription factors that are overexpressed and/or hyperactivated in tumor cells. These factors are Stat, AP-1, the bHLH proteins Myc and AP-4, the nuclear hormone receptor Ftz-f1, the nuclear receptor coactivator Taiman/SRC3, and Mef2. Notably, many of these transcription factors also are hyperactivated in human tumors. Bioinformatic analysis predicted that these factors directly regulate the majority of the tumor-specific gene expression, that they are interconnected by extensive cross-regulation, and that they show a high degree of co-regulation of target genes. Indeed, the factors of this network were required in multiple epithelia for tumor growth and invasiveness, and knockdown of several factors caused a reversion of the tumor-specific expression profile but had no observable effect on normal tissues. It was further found that the Hippo pathway effector Yorkie is strongly activated in tumor cells and initiates cellular reprogramming by activating several transcription factors of this network. Thus, modeling regulatory networks identifies an ectopic and ordered network of master regulators that control a large part of tumor cell-specific gene expression.

Schmid, M. R., Anderl, I., Vo, H. T., Valanne, S., Yang, H., Kronhamn, J., Ramet, M., Rusten, T. E. and Hultmark, D. (2016). Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility. PLoS One 11: e0159473. PubMed ID: 27467079
To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), a genetic modifier screen was carried out, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). This study describes the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. The specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 were identified as suppressors, and the role of ird1 was studied in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, it is concluded that the vesicular transport system may be of particular importance during an immune response.

Thursday, August 4th

Gyuricza, M. R., Manheimer, K. B., Apte, V., Krishnan, B., Joyce, E. F., McKee, B. D. and McKim, K. S. (2016). Dynamic and stable cohesins regulate synaptonemal complex assembly and chromosome segregation. Curr Biol [Epub ahead of print]. PubMed ID: 27291057
Assembly of the synaptonemal complex (SC) in Drosophila depends on two independent pathways defined by the chromosome axis proteins C(2)M and ORD. Because C(2)M encodes a Kleisin-like protein and ORD is required for sister-chromatid cohesion, the hypothesis was tested that these two SC assembly pathways depend on two cohesin complexes. Through single- and double-mutant analysis to study the mitotic cohesion proteins Stromalin (SA) and Nipped-B (SCC2) in meiosis, evidence was provided that there are at least two meiosis-specific cohesin complexes. One complex depends on C(2)M, SA, and Nipped-B. Despite the presence of mitotic cohesins SA and Nipped-B, this pathway has only a minor role in meiotic sister-centromere cohesion and is primarily required for homolog interactions. C(2)M is continuously incorporated into pachytene chromosomes even though SC assembly is complete. In contrast, the second complex, which depends on meiosis-specific proteins SOLO, SUNN, and ORD is required for sister-chromatid cohesion, localizes to the centromeres and is not incorporated during prophase. Multiple cohesin complexes may provide the diversity of activities required by the meiotic cell.
Romero, N. E., Matson, S. W. and Sekelsky, J. (2016). Biochemical activities and genetic functions of the Drosophila melanogaster Fancm helicase in DNA repair. Genetics [Epub ahead of print]. PubMed ID: 27466228
Repair of DNA damage is essential to the preservation of genomic stability. During repair of double-strand breaks, several helicases function to promote accurate repair and prevent the formation of crossovers through homologous recombination. Among these helicases is the Fanconi anemia group M (FANCM) protein. FANCM is important in the response to various types of DNA damage and has been suggested to prevent mitotic crossovers during double-strand break repair. The helicase activity of FANCM is believed to be important in these functions, but no helicase activity has been detected in vitro. This paper reports a genetic and biochemical study of Drosophila melanogaster Fancm. Purified Fancm is a 3' to 5' ATP-dependent helicase that can disassemble recombination intermediates, but only through limited lengths of duplex DNA. Using transgenic flies expressing full-length or truncated Fancm, each with either a wild-type or mutated helicase domain, it was found that there are helicase-independent and C-terminus-independent functions in responding to DNA damage and in preventing mitotic crossovers.
Vieira, V., Cardoso, M. A. and Araujo, H. (2016). Calpain A controls mitotic synchrony in the Drosophila blastoderm embryo. Mech Dev [Epub ahead of print]. PubMed ID: 27264536
The beautiful mitotic waves that characterize nuclear divisions in the early Drosophila embryo have been the subject of intense research to identify the elements that control mitosis. Calcium waves in phase with mitotic waves suggest that calcium signals control this synchronized pattern of nuclear divisions. However, protein targets that would translate these signals into mitotic control have not been described. This study investigated the role of the calcium-dependent protease Calpain A in mitosis. Impaired Calpain A function was shown to result in loss of mitotic synchrony and ultimately halted embryonic development. The presence of defective microtubules and chromosomal architecture at the mitotic spindle during metaphase and anaphase and perturbed levels of Cyclin B indicate that Calpain A is required for the metaphase-to-anaphase transition. The results suggest that Calpain A functions as part of a timing module in mitosis, at the interface between calcium signals and mitotic cycles of the Drosophila embryo.
Cappell, S. D., Chung, M., Jaimovich, A., Spencer, S. L. and Meyer, T. (2016). Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry. Cell 166: 167-180. PubMed ID: 27368103
Evolutionary Homolog Study:
Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb; see Drosophila Rb) protein. This study shows that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1); see Drosophila Cdh1/Fzr) in a cultured mammalian epithelial cell line has the necessary characteristics to be the point of no return for cell-cycle entry. APC(Cdh1) inactivation is shown to be a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 (see Drosophila Cyclin E) and made irreversible by Emi1 (see Drosophila Rca1). Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle.

Wednesday, August 3

Gabilondo, H., Stratmann, J., Rubio-Ferrera, I., Millán-Crespo, I., Contero-García, P., Bahrampour, S., Thor, S. and Benito-Sipos, J. (2016). Neuronal cell fate specification by the convergence of different spatiotemporal cues on a common terminal selector cascade. PLoS Biol 14: e1002450. PubMed ID: 27148744
Specification of the myriad of unique neuronal subtypes found in the nervous system depends upon spatiotemporal cues and terminal selector gene cascades, often acting in sequential combinatorial codes to determine final cell fate. However, a specific neuronal cell subtype can often be generated in different parts of the nervous system and at different stages, indicating that different spatiotemporal cues can converge on the same terminal selectors to thereby generate a similar cell fate. However, the regulatory mechanisms underlying such convergence are poorly understood. The Nplp1 neuropeptide neurons in the Drosophila ventral nerve cord can be subdivided into the thoracic-ventral Tv1 neurons and the dorsal-medial dAp neurons. The activation of Nplp1 in Tv1 and dAp neurons depends upon the same terminal selector cascade: col->ap/eya->dimm->Nplp1. However, Tv1 and dAp neurons are generated by different neural progenitors (neuroblasts) with different spatiotemporal appearance. It was found that the same terminal selector cascade is triggered by Kr/pdm->grn in dAp neurons, but by Antp/hth/exd/lbe/cas in Tv1 neurons. Hence, two different spatiotemporal combinations can funnel into a common downstream terminal selector cascade to determine a highly related cell fate.
Kraft, K.F., Massey, E.M., Kolb, D., Walldorf, U. and Urbach, R. (2016). Retinal homeobox promotes cell growth, proliferation and survival of mushroom body neuroblasts in the Drosophila brain. Mech Dev [Epub ahead of print]. PubMed ID: 27455861
The Drosophila mushroom bodies, centers of olfactory learning and memory in the fly 'forebrain', develop from a set of neural stem cells (neuroblasts) that generate a large number of Kenyon cells (KCs) during sustained cell divisions from embryonic to late pupal stage. This study shows that retinal homeobox (rx), encoding for an evolutionarily conserved transcription factor, is required for proper development of the mushroom bodies. Throughout development rx is expressed in mushroom body neuroblasts (MBNBs), their ganglion mother cells (MB-GMCs) and young KCs. In the absence of rx function, MBNBs form correctly but exhibit a reduction in cell size and mitotic activity, whereas overexpression of rx increases growth of MBNBs. These data suggest that Rx is involved in the control of MBNB growth and proliferation. Rx also promotes cell cycling of MB-GMCs. Moreover, it was found that Rx is important for the survival of MBNBs and Kenyon cells which undergo premature cell death in the absence of rx function. Simultaneous blocking of cell death restores the normal set of MBNBs and part of the KCs, demonstrating that both, impaired proliferation and premature cell death (of MBNBs and KCs) account for the observed defects in mushroom body development. It was shown that Rx controls proliferation within the MBNB clones independently of Tailless (Tll) and Prospero (Pros), and does not regulate the expression of other key regulators of MB development, Eyeless (Ey) and Dachshund (Dac). These data support that the role of Rx in forebrain development is conserved between vertebrates and fly.

Mino, R. E., Rogers, S. L., Risinger, A. L., Rohena, C., Banerjee, S. and Bhat, M. A. (2016). Drosophila Ringmaker regulates microtubule stabilization and axonal extension during embryonic development. J Cell Sci [Epub ahead of print]. PubMed ID: 27422099
Axonal growth and targeting are fundamental to the organization of the nervous system, and require active engagement of the cytoskeleton. Polymerization and stabilization of axonal microtubules is central to axonal growth and maturation of neuronal connectivity. Studies have suggested that members of the Tubulin Polymerization Promoting Protein (P25alpha/TPPP) family are involved in cellular process extension. However, no in vivo knockout data exists regarding its role in axonal growth during development. This study reports the characterization of Ringmaker (Ringer; CG45057), the only Drosophila homolog of long p25alpha proteins. Immunohistochemical analyses indicate that Ringer expression is dynamically regulated in the embryonic CNS. ringer null mutants show cell misplacement, and errors in axonal extension and targeting. Ultrastructural examination of ringer mutants revealed defective microtubule morphology and organization. Primary neuronal cultures of ringer mutants exhibit defective axonal extension, and Ringer expression in cells induced microtubule stabilization and bundling into rings. In vitro assays showed that Ringer directly affects tubulin, and promotes microtubule bundling and polymerization. Together these studies uncover an essential function of Ringer in axonal extension and targeting through proper microtubule organization.
Mestres, I., Chuang, J. Z., Calegari, F., Conde, C. and Sung, C. H. (2016). SARA regulates neuronal migration during neocortical development through L1 trafficking. Development [Epub ahead of print]. PubMed ID: 27471254
Evolutionary Homolog Study:
Emerging evidence suggests that endocytic trafficking of adhesion proteins plays a critical role in neuronal migration during neocortical development. However, the molecular insights of these processes remain elusive. This paper examines an early endosomal protein Smad Anchor for Receptor Activation (SARA) (see Drosophila Sara) in the developing mouse brain. SARA is enriched at the apical endfeet of radial glia of mouse neocortex. While silencing SARA did not lead to detectable neurogenic phenotypes, SARA-suppressed neurons exhibit impaired orientation and migration across the intermediate zone. Mechanistically, SARA-silenced neurons were shown to exhibit increased surface expression of L1, a cell adhesion molecule (see Drosophila Neuroglian). Neurons ectopically expressing L1 phenocopy the migration and orientation defects caused by SARA silencing, and display increased contact with neighboring neurites. L1 knockdown effectively rescues SARA suppression-caused phenotypes. SARA-silenced neurons eventually overcome their migration defect and enter later into the cortical plate. Nevertheless, these neurons localized at more superficial cortical layers compared to their controls counterparts. These results suggest that SARA regulates the orientation, multipolar-to-bipolar transition, and positioning of cortical neurons via modulating surface L1 expression.

Tuesday, August 2nd

Aso, Y. and Rubin, G.M. (2016). Dopaminergic neurons write and update memories with cell-type-specific rules. Elife [Epub ahead of print]. PubMed ID: 27441388
Associative learning is thought to involve parallel and distributed mechanisms of memory formation and storage. In Drosophila, the mushroom body (MB) is the major site of associative odor memory formation. The anatomy of the adult MB and 20 types of dopaminergic neurons (DANs) that each innervate distinct MB compartments have been previously defined and described. This study compared the properties of memories formed by optogenetic activation of individual DAN cell types. Extensive differences were found in training requirements for memory formation, decay dynamics, storage capacity and flexibility to learn new associations. Even a single DAN cell type can either write or reduce an aversive memory, or write an appetitive memory, depending on when it is activated relative to odor delivery. These results show that different learning rules are executed in seemingly parallel memory systems, providing multiple distinct circuit-based strategies to predict future events from past experiences. The mechanisms that generate these distinct learning rules are unknown. They could arise from differences in the dopamine release properties of different DAN cell types or from local differences in the biochemical response to dopamine signaling in each MB compartment. For example, KCs express four distinct dopamine receptors, which might be deployed differently in each compartment. Or they could originate from circuit properties.
Diaz-Balzac, C. A., Rahman, M., Lazaro-Pena, M. I., Martin Hernandez, L. A., Salzberg, Y., Aguirre-Chen, C., Kaprielian, Z. and Bulow, H. E. (2016). Muscle- and skin-derived cues jointly orchestrate patterning of somatosensory dendrites. Curr Biol [Epub ahead of print]. PubMed ID: 27451901
Evolutionary Homolog Study:
Sensory dendrite arbors are patterned through cell-autonomously and non-cell-autonomously functioning factors. The conserved MNR-1/Menorin-SAX-7/L1CAM (see Drosophila Neuroglian) complex acts from the skin to pattern the stereotypic dendritic arbors of PVD and FLP somatosensory neurons in C. elegans through the leucine-rich transmembrane receptor DMA-1/LRR-TM expressed on PVD neurons. This study describes a role for the diffusible C. elegans protein LECT-2, which is homologous to vertebrate leukocyte cell-derived chemotaxin 2 (LECT2)/Chondromodulin II. LECT2/Chondromodulin II has been implicated in a variety of pathological conditions, but the developmental functions of LECT2 have remained elusive. LECT-2/Chondromodulin II is required for development of PVD and FLP dendritic arbors and can act as a diffusible cue from a distance to shape dendritic arbors. Expressed in body-wall muscles, LECT-2 decorates neuronal processes and hypodermal cells in a pattern similar to the cell adhesion molecule SAX-7/L1CAM. LECT-2 functions genetically downstream of the MNR-1/Menorin-SAX-7/L1CAM adhesion complex and upstream of the DMA-1 receptor. LECT-2 localization is dependent on SAX-7/L1CAM, but not on MNR-1/Menorin or DMA-1/LRR-TM, suggesting that LECT-2 functions as part of the skin-derived MNR-1/Menorin-SAX-7/L1CAM adhesion complex. Collectively, these findings suggest that LECT-2/Chondromodulin II acts as a muscle-derived, diffusible cofactor together with a skin-derived cell adhesion complex to orchestrate the molecular interactions of three tissues during patterning of somatosensory dendrites.
Scholz-Kornehl, S. and Schwarzel, M. (2016). Circuit analysis of a Drosophila Dopamine type 2 receptor that supports anesthesia-resistant memory. J Neurosci 36: 7936-7945. PubMed ID: 27466338
Dopamine is central to reinforcement processing and exerts this function in species ranging from humans to fruit flies. It can do so via two different types of receptors (i.e., D1 or D2) that mediate either augmentation or abatement of cellular cAMP levels. Whereas D1 receptors are known to contribute to Drosophila aversive odor learning per se, this study shows that D2 receptors are specific for support of a consolidated form of odor memory known as anesthesia-resistant memory. By means of genetic mosaicism, this function was localized to Kenyon cells, the mushroom body intrinsic neurons, as well as GABAergic APL neurons and local interneurons of the antennal lobes, suggesting that consolidated anesthesia-resistant memory requires widespread dopaminergic modulation within the olfactory circuit. Additionally, dopaminergic neurons themselves require D2R, suggesting a critical role in dopamine release via its recognized autoreceptor function. Considering the dual role of dopamine in balancing memory acquisition (proactive function of dopamine) and its 'forgetting' (retroactive function of dopamine), this analysis suggests D2R as central player of either process.
Kage-Nakadai, E., Ohta, A., Ujisawa, T., Sun, S., Nishikawa, Y., Kuhara, A. and Mitani, S. (2016). Caenorhabditis elegans homologue of Prox1/Prospero is expressed in the glia and is required for sensory behavior and cold tolerance. Genes Cells. PubMed ID: 27402188
The Caenorhabditis elegans (C. elegans) amphid sensory organ contains only 4 glia-like cells and 24 sensory neurons, providing a simple model for analyzing glia or neuron-glia interactions. To better characterize glial development and function, RNA interference screening was carried out for transcription factors that regulate the expression of an amphid sheath glial cell marker. pros-1, which encodes a homeodomain transcription factor homologous to Drosophila Prospero/mammalian Prox1, was identified as a positive regulator. The functional PROS-1::EGFP fusion protein was localized in the nuclei of the glia and the excretory cell but not in the amphid sensory neurons. pros-1, deletion mutants exhibited larval lethality, and rescue experiments showed that pros-1, and human Prox1 transgenes were able to rescue the larval lethal phenotype, suggesting that pros-1, is a functional homologue of mammalian Prox1, at least partially. It was further found that the structure and functions of sensory neurons, such as the morphology of sensory endings, sensory behavior and sensory-mediated cold tolerance, appeared to be affected by the pros-1, RNAi. Together, these results show that the C. elegans PROS-1 is a transcriptional regulator in the glia but is involved not only in sensory behavior but also in sensory-mediated physiological tolerance.

Monday, August 1st

Dresch, J. M., Zellers, R. G., Bork, D. K. and Drewell, R. A. (2016). Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome. Gene Regul Syst Bio 10: 21-33. PubMed ID: 27330274
Relatively little is known about the sequence-specific binding preferences of many transcription factors (TFs), especially with respect to the possible interdependencies between the nucleotides that make up binding sites. A particular limitation of many existing algorithms that aim to predict binding site sequences is that they do not allow for dependencies between nonadjacent nucleotides. This study used a recently developed computational algorithm, MARZ, to compare binding site sequences using 32 distinct models in a systematic and unbiased approach to explore nucleotide dependencies within binding sites for 15 distinct TFs known to be critical to Drosophila development. The results indicate that many of these proteins have varying levels of nucleotide interdependencies within their DNA recognition sequences, and that, in some cases, models that account for these dependencies greatly outperform traditional models that are used to predict binding sites. The ability of different models to identify the known Kruppel TF binding sites in cis-regulatory modules (CRMs) was directly compared, and a more complex model that accounts for nucleotide interdependencies performs better when compared with simple models. This ability to identify TFs with critical nucleotide interdependencies in their binding sites will lead to a deeper understanding of how these molecular characteristics contribute to the architecture of CRMs and the precise regulation of transcription during organismal development.
He, X., Ohba, S., Hojo, H. and McMahon, A. P. (2016). AP-1 family members act with Sox9 to promote chondrocyte hypertrophy. Development [Epub ahead of print]. PubMed ID: 27471255
Evolutionary Homolog Study
An analysis of mammalian Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif. This study has explored the functional interplay between Sox9 and AP-1 (see Drosophila Fos and Jun) in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun- and Sox9-bound regions throughout the chondrocyte genome. The similar profiles reflect direct binding of each factor to the same enhancers and a potential for protein-protein interactions within AP-1 and Sox9 containing complexes. In vitro reporter analysis indicated that direct co-binding of Sox9 and AP-1 at target motifs promoted gene activity. In contrast, where only one factor can engage its DNA target, the presence of the other factor suppresses target activation consistent with protein-protein interactions attenuating transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 confirmed the requirement of Sox9 for hypertrophic chondrocyte development, while in vitro and ex vivo analyses showed AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model where AP-1-family members contribute to Sox9-action in the transition of chondrocytes to the hypertrophic program.
Esposito, E., Lim, B., Guessous, G., Falahati, H. and Levine, M. (2016). Mitosis-associated repression in development. Genes Dev 30: 1503-1508. PubMed ID: 27401553
Transcriptional repression is a pervasive feature of animal development. This study employed live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. The study proposes that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail.

Kuzu, G., et al. (2016). Expansion of GA dinucleotide repeats increases the density of CLAMP binding sites on the X-Chromosome to promote Drosophila dosage compensation. PLoS Genet 12: e1006120. PubMed ID: 27414415
Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females. In Drosophila melanogaster, the Chromatin-linked adaptor for MSL proteins (CLAMP) zinc finger protein links the dosage compensation complex to the X-chromosome. However, the mechanism for X-chromosome identification has remained unknown. This study combine biochemical, genomic and evolutionary approaches to reveal that expansion of GA-dinucleotide repeats likely accumulated on the X-chromosome over evolutionary time to increase the density of CLAMP binding sites, thereby driving the evolution of dosage compensation. Overall, this study presents new insight into how subtle changes in genomic architecture, such as expansions of a simple sequence repeat, promote the evolution of coordinated gene expression.

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